Cortical and Trabecular Histomorphometry of the Rib, Clavicle and Iliac Crest of Individuals from the Chiribaya Polity of Ancient Southern Coastal Peru

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

In the Graduate School of The Ohio State University

Lara Elizabeth McCormick, M.A.

Graduate Program in Anthropology

The Ohio State University

2013

Dissertation Committee:

Dr. Sam D. Stout, Advisor

Dr. Clark Spencer Larsen

Dr. Paul W. Sciulli

Dr. Mark Hubbe

Lara Elizabeth McCormick

2013

Abstract

This study examined microstructural properties of bone in individuals from the ancient Peruvian polity of the Chiribaya. The three sites include San Geronimo, El Yaral, and Chiribaya Alta which represent fishing, agricultural and elite lifestyles, respectively.

The purpose of the study was to test four hypotheses: 1.Variability in relative area measurements due to differential loading throughout the skeleton exist, 2. Differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age, 3. Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral and San Geronimo due to variation of diet and general subsistence level activities, 4. Sex differences exist in histomorphometric areal and perimeter/length measurements in the

Chiribaya sample. Sections were removed from the rib and clavicle at midshaft, and from the standardized site of clinical biopsy in the iliac crest. Data collection included areal and perimeter/length measurements on cortical bone in the rib and clavicle and a combination of cortical and trabecular bone in iliac crest specimens. Variables examined include total area (Tt.Ar.), cortical area (Ct.Ar.), endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.) in rib and clavicle samples. In iliac crest sections, measurements of were taken of: total area of section (Tt.Ar.), the area containing trabecular bone (Es.Ar.), cortical area (Ct.Ar.), ii

relative cortical area (Rel.Ct.Ar.), area of trabecular bone (Tb.Ar.), relative trabecular area (Rel.Tb.Ar.), mean trabecular width (Tb.Wi.), total section diameter (Tt.Dm.), total cortical diameter (Ct.Dm.) and marrow cavity diameter (Es.Dm.). This study included variables measured on 62 rib sections, 54 clavicle section and 62 iliac crest sections of males and females of varying ages. Intra-skeletal variability was examined using 43 individuals who had all three skeletal sampling sites intact. Histomorphometric data was collected using compiled photomicrographs and ImageJ® software on a PC Tablet.

Hypotheses were tested using a combination of t-tests, one-way and two-way ANOVAs, and a randomized block ANOVA. Sex differences were significant in areal and perimeter measurements in rib, clavicle and iliac crest sections, supporting Hypothesis 1.

Hypothesis 2 was supported and statistically significant with every variable examined in the clavicle when comparing two sites, and with some, but not all, variables demonstrating statistical significance in the rib and iliac crest. When data from San

Geronimo and El Yaral were pooled together and compared to the Chiribaya Alta, the results were less significant, supporting the thought of genetic homogeneity within the

Chiribaya population. Hypothesis 3 was not supported in the rib, clavicle, or iliac crest, suggesting that the number of older adults in the sample was not truly indicative of the

Chiribaya population, or that Chiribaya individuals did not experience the degree of cortical and trabecular bone loss seen in modern populations. Intra-skeletal variability was significant with the use of a randomized block ANOVA. This study describes a general pattern for skeletal microstructural changes exhibited in the rib, clavicle and iliac crest with increasing age in an ancient Andean archaeological that can be used for comparison in future studies of past and present Andean and other skeletal populations.

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Dedication

Dedicated to the Memory of Bernice Jensen:

I wish you had lived long enough to see me finish this task

And

To my parents, James and Elizabeth McCormick:

For teaching me that life is about independence as much as connectedness, but most importantly, for showing me how to laugh through the hard times

Acknowledgments

First and foremost, I would like to thank my advisor, Dr. Sam Stout, for guiding me through my graduate career, teaching me invaluable skills and helping me become a better writer. My dissertation committee, Dr. Sam Stout, Dr. Paul Sciulli, Dr. Clark S.

Larsen, and Dr. Mark Hubbe provided me with guidance on this project. I would like to thank Dr. Paul Sciulli for your statistical knowledge and for challenging me to see the world from different angles. Dr. Clark Larsen served as a mentor and role model for a future career in physical anthropology. Thank you to Dr. Hubbe for your thorough reading of this document and your many suggestions for improvement and future directions. I would also like to thank Dr. Jane Buikstra for providing the Chiribaya samples used in this project.

This project would not have been possible without the financial and intellectual support of a number of individuals and institutions. Financial assistance was provided by the Coca-Cola Critical Difference for Women Grant. The Bioarchaeology Laboratory provided facilities and equipment. A huge thank you is owed to Amanda Agnew, Corey

Maggiano, Steve Schlecht, and Jesse Goliath for your many impromptu discussions regarding sample preparation and data collection. Several undergraduate students volunteered time and energy to assist with sample procurement and preparation, and I am indebted to them for their commitment: Brittney Stacey, Alexis Goots, Jennifer Hauck,

Ashley Cook, Leslie O’Donnell, and Matt Adkins. I thank Alexandra Keenan-Krilevich v

for providing several drawings for this document. Sara Becker, Leslie Williams, Sarah

Martin, and Matt Senn provided humor and emotional support through the writing phase of this dissertation.

I would like to thank my cohort for helping me through the beginning phases of my graduate career: Amanda Agnew, Erica Chambers, Meghan-Tomasita Cosgriff-

Hernandez, Jim Gosman, Amelia Hubbard, Gabriela Jakubowska, Heather Jarrell, Sarah

Martin, Deborrah Pinto, Micah Soltz and Dan Tyree. Thanks to Vida Devic and Barry

Gross for regular daily doses of dreams, reality, laughter, encouragement, and brought me down to earth when necessary. I also owe a thank you to other Anthropology Department graduate students who had a hand in shaping my experience at OSU: Lori Critcher, Ana

Casado, Karen Royce, Adam Kolatorowicz, Josh Sadvari, Dave Rose, Tim Gocha,

Samantha Blatt, Lesley Gregoricka, Thank you to David Sweasey, for the cheese balls, trivia, daily debates, laughter and helping me see the light through the darker days.

Thank you to the faculty and staff at CSCC for preparing me for academia.

To my family members, the consanguineal and affinal crew, you all had a hand in helping me through this journey: Mom, Dad, Grandma, Bryan, Jon, Erin, Robert, Sarah,

Daniel, Kelly, Marina, Becky, Valerie, Nate, Beth, Madelyn, Ayla, Camilla, Andrew and

Krystofer. Many thanks are owed to my neighborhood family: Nancy, Betty, Tom, Mary,

Liz, Jules, Cindy, Dave, Theresia and Micah for ensuring I spent time away from my computer. Thanks to Loki and Lexus for getting me out of bed every morning. I thank the faculty, staff and students at Columbus Torah Academy, especially Eliza Delman, Rabbi

Kahn, Dr. Kennedy and my brilliant 8th grade students.

Last, but definitely not least, I would like to thank Dr. John Tew of the Mayfield

Clinic and University Hospital of Cincinnati; his compassion, skills and training gave me the capability of completing this project, my degree, and gave me hope for the future.

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Vita

1995 Diploma, Pulaski High School; Pulaski, WI

1995-1998 BA, Arizona State University

2001-2004 MA, California State University, Los Angeles

2004-2010 Graduate Teaching Associate, Department of Anthropology, The Ohio State University

2007-2008 Graduate Teaching Fellow, Department of Anthropology, The Ohio State University

2008 Graduate Research Associate, Department of Anthropology, The Ohio State University

Publications

Stewart MC, McCormick LE, Goliath JR, Sciulli PW, and Stout SD. 2013. A Comparison of Histomorphometric Data Collection Methods. Journal of Forensic Sciences 58(1).

Fields of Study

Major Field: Anthropology

Area of Emphasis: Biological Anthropology

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Table of Contents

Abstract ...... ii Dedication ...... iv Acknowledgments ...... v Vita ...... viii List of Figures ...... xiii List of Tables ...... xvii Chapter 1: Dissertation Outline ...... 1 1.1 Overview of Histomorphometry ...... 1 1.2 Organization of Study ...... 5 1.3 Purpose ...... 8 1.4 Hypotheses Examined ...... 9 1.5 Chapter Summary ...... 11

Chapter 2: Overview of Bone Components and Bone Remodeling ...... 12 2.1 Types of Bone ...... 13 2.2 Bone Envelopes ...... 14 2.3 Bone Cells and Cell Communication ...... 15 2.3.1 Osteoclasts ...... 18 2.3.2 Osteoblasts...... 18 2.3.3 Osteocytes ...... 19 2.3.4 Bone Lining Cells ...... 20 2.4 Modeling and Remodeling ...... 21 2.5 Chapter Summary ...... 29

Chapter 3: Age-Associated Bone Loss ...... 31 3.1 Osteopenia and Osteoporosis ...... 31 3.2 Diagnoses of Osteopenia and Osteoporosis ...... 33 3.3 General Factors Relating to Age-Associated Bone Loss ...... 38 3.3.1 Genetics ...... 38 3.3.2 Nutritional Considerations and Diet ...... 39 3.3.3 The Role of Androgens and Estrogens in Age-Associated Bone Loss ...... 46 3.3.4 Mechanical Loading and Physical Activity ...... 50 3.4 General Skeletal Trends Associated with Age-Associated Bone Loss ...... 58 ix

3.4.1 General Age-Associated Trends with Bone Loss in Cortical Bone ...... 60 3.4.2 General Age-Associated Trends with Bone Loss in Trabecular Bone ...... 61 3.5. The Issue of Intra-skeletal Variability ...... 63 3.6 Sex Differences in Age-Associated Bone Loss...... 64 3.7 Population-Based Differences in Age-Associated Bone Loss ...... 71 3.8 Chapter Summary ...... 78

Chapter 4: The Chiribaya ...... 80 4.1 General Overview ...... 82 4.2 Chiribaya Timeframe ...... 83 4.3 The Socio-political Environment ...... 85 4.4 Chiribaya Subsamples ...... 87 4.4.1 Chiribaya Alta: Mercaderes ...... 88 4.4.2 San Geronimo: Pescadores ...... 90 4.4.3 El Yaral: Labradores ...... 91 4.4.4 Community Membership ...... 93 4.5 Chiribaya: General Health Indicators ...... 94 4.6 Chiribaya: Genetic Relatedness ...... 97 4.7 Social Interactions ...... 99 4.9 Chapter Summary ...... 100

Chapter 5: Materials and Methods ...... 105 5.1 Skeletal Sampling Sites ...... 105 5.1.1 Iliac Crest ...... 105 5.1.2 Rib...... 108 5.1.3 Clavicle ...... 109 5.2 Sample Procurement/Preparation ...... 111 5.3 Microscopic Analysis ...... 112 5.4 Variables Examined ...... 113 5.5 Sample Demographics...... 118 5.6 Hypotheses ...... 121 5.7 Analyses Employed ...... 130 5.8 Chapter Summary ...... 131

Chapter 6: Results ...... 134 6.1 Intra-Observer Error ...... 134 6.2 Normality Tests ...... 135 6.2.1 Normality Measures by Bone for Examination of Sex ...... 136 6.2.2 Normality Measures by Bone for Examination of Subsistence by Site Affiliation ...... 139 6.2.3 Normality Measures by Bone for Examination of Age ...... 142 6.2.4 Normality Measures for Examination of Intra-Skeletal Variability ...... 145 6.3 Examination of Sex ...... 145 x

6.3.1 Sex Comparisons Using Rib Variables ...... 145 6.3.2 Sex Comparisons Using Clavicle Variables ...... 147 6.3.3 Sex Comparisons Using Iliac Crest Variables ...... 149 6.3.4 Summation of Sex-Related Differences ...... 152 6.4 Examination of Subsistence as Indicated by Cultural Site Affiliation ...... 153 6.4.1 Examination of Subsistence Differences Demonstrated in Rib Data ...... 154 6.4.2 Examination of Subsistence Differences Demonstrated in Clavicle Data ...... 162 6.4.3 Examination of Subsistence Differences Demonstrated in Iliac Crest Data...... 170 6.4.4 Summary of Findings Regarding Subsistence-Level Differences ...... 180 6.5 Examination of Age ...... 181 6.5.1 Examination of Age in Rib Variables ...... 181 6.5.1.1. Descriptive Statistics of Rib Data ...... 184 6.5.2 Examination of Age in Clavicle Data ...... 191 6.5.2.2 Descriptive Statistics of Clavicle Data ...... 191 6.5.3 Examination of Age in Iliac Crest Data ...... 198 6.5.3.1 Descriptive Statistics of Iliac Crest Data ...... 203 6.6 Examination of Intra-skeletal Variability ...... 214 6.7 Chapter Summary ...... 215

Chapter 7: Discussion ...... 220 7.1 Examination and Discussion of Sex Differences ...... 220 7.1.1. Examination of Sex Differences in the Rib ...... 221 7.1.2 Examination of Sex Differences in the Clavicle ...... 222 7.1.3 Examination of Sex Differences in the Iliac Crest ...... 222 7.1.4 Summary of Sex Examination in Rib, Clavicle, and Iliac Crest ...... 223 7.2 Examination and Discussion of Subsistence by Site Affiliation ...... 226 7.2.1 Examination of Site and Subsistence Level Differences in Rib Data ...... 227 7.2.2 Examination of Site and Subsistence Level Differences in Clavicle Data ...... 228 7.2.3 Examination of Site and Subsistence Level Differences in Iliac Crest Data...... 229 7.2.5 Summary of Site and Subsistence Level Differences in the Chiribaya Population ...... 230 7.3 Examination and Discussion of Age Differences ...... 233 7.3.1 Examination of Age Differences in the Rib ...... 234 7.3.2 Examination of Age Differences in the Clavicle ...... 236 7.3.3 Examination of Age Differences in the Iliac Crest ...... 237 7.3.5 Summary of Age Examination in the Chiribaya Population ...... 239 7.4 Discussion of Intra-skeletal Variability Results ...... 243 7.5 General Chiribaya Histomorphometrics in Comparison to Other Populations ..... 244 7.5.1 Chiribaya Rib and Clavicle Histomorphometry ...... 244 7.5.2 Chiribaya Iliac Crest Histomorphometry ...... 247 7.6 Anthropological Considerations ...... 251 7.7 Limitations ...... 252 7.8 Directions for Future Research ...... 253 7.9 Chapter Summary ...... 254 xi

Chapter 8: Research Summary and Conclusions ...... 259 Bibliography ...... 264 Appendix A: Histograms from T-Test Analyses ...... 310 Appendix B: Box and Whisker Plots for ANOVA Tests ...... 333 Appendix C: Raw Data ...... 340

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List of Figures

Figure 1. Bone Envelopes...... 15 Figure 2. Gap junctions located between dendrites of osteons...... 16 Figure 3. Overview of cell signaling ...... 17 Figure 4. Osteocyte lacunae (dark spots) of a bone thin section (100x) ...... 20 Figure 5. Bone remodeling cycle ...... 23 Figure 6. Activation, resorption, formation sequence of bone formation ...... 25 Figure 7. Hemi-osteonal BMU ...... 27 Figure 8. Map of Chiribaya Sites ...... 88 Figure 9. Site of procurement of wedge from iliac crest specimens ...... 107 Figure 10. Demonstration of region of rib section removal ...... 109 Figure 11. Clavicle demarcating section removed for analysis ...... 111 Figure 12. Compiled photomicrograph of rib and indication of variables measured ...... 115 Figure 13. Example of a compiled image of an iliac crest sample demarcating normal biopsy site (superior border is to the right; anterior is facing the top)...... 117 Figure 14. Variables collected from iliac crest specimens ...... 118 Figure 15. Boxplot of Rib Total Area (Tt.Ar). by Site ...... 158 Figure 16. Boxplot of Rib Cortical Area (Ct.Ar.) by Site ...... 159 Figure 17. Boxplot of Rib Endosteal Area (Es.Ar.) by Site ...... 160 Figure 18. Boxplot of Rib Relative Cortical Area (Rel.Ct.Ar.) by Site ...... 160 Figure 19. Boxplot of Rib Periosteal Perimeter (Ps.Pm.) by Site ...... 161 Figure 20. Boxplot of Rib Endosteal Perimeter (Es.Pm). by Site ...... 161 Figure 21. Boxplot of Clavicle Total Area (Tt.Ar.) by Site ...... 167 Figure 22. Boxplot of Clavicle Cortical Area (Ct.Ar.) by Site ...... 167 Figure 23. Boxplot of Clavicle Endosteal Area (Es.Ar.) by Site...... 168 Figure 24. Boxplot of Clavicle Relative Cortical Area (Rel.Ct.Ar.) by Site ...... 168 Figure 25. Boxplot of Clavicle Periosteal Perimeter (Ps.Pm.) by Site ...... 169 Figure 26. Boxplot of Clavicle Endosteal Perimeter (Es.Pm.) by Site ...... 169 Figure 27. Boxplot of Iliac Crest Total Area (Tt.Ar.) by Site ...... 176 Figure 28. Boxplot of Iliac Crest Cortical Area (Ct.Ar.) by Site ...... 176 Figure 29. Boxplot of Iliac Crest Marrow Cavity Area (Es.Ar.) by Site ...... 177 Figure 30. Boxplot of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) by Site ...... 177 Figure 31. Boxplot of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) by Site ...... 178

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Figure 32. Boxplot of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) by Site ...... 178 Figure 33. Boxplot of Iliac Crest Total Diameter (Tt.Dm.) by Site ...... 179 Figure 34. Boxplot of Iliac Crest Cortical Diameter (Ct.Dm.) by Site ...... 179 Figure 35. Boxplot of Iliac Crest Marrow Cavity Diameter (Es.Dm.) by Site ...... 180 Figure 36. Boxplot of Rib Total Area (Tt.Ar.) by Age and Sex ...... 188 Figure 37. Boxplot of Rib Cortical Area (Ct.Ar.) by Sex and Age Category ...... 188 Figure 38. Boxplot of Rib Endosteal Area (Es.Ar.) by Age and Sex ...... 189 Figure 39. Boxplot of Rib Relative Cortical Area (Rel.Ct.Ar.) by Age and Sex ...... 189 Figure 40. Boxplot of Rib Periosteal Perimeter (Ps.Pm.) by Age and Sex ...... 190 Figure 41. Boxplot of Rib Endosteal Perimeter (Es.Pm.) of Age and Sex ...... 190 Figure 42. Boxplot of Clavicle Total Area (Tt.Ar.) by Age and Sex ...... 195 Figure 43. Boxplot of Clavicle Cortical Area (Ct.Ar.) by Age and Sex ...... 195 Figure 44. Boxplot of Clavicle Endosteal Area (Es.Ar.) by Age and Sex ...... 196 Figure 45. Boxplot of Clavicle Relative Cortical Area (Rel.Ct.Ar.) by Age and Sex ...... 196 Figure 46. Boxplot of Clavicle Periosteal Perimeter (Ps.Pm.) by Age and Sex ...... 197 Figure 47. Boxplot of Clavicle Endosteal Perimeter (Es.Pm.) by Age and Sex ...... 197 Figure 48. Distribution of Iliac Crest Total Area (Tt.Ar.) by Sex and Age ...... 209 Figure 49. Distribution of Iliac Crest Cortical Area (Ct.Ar.) by Sex and Age ...... 209 Figure 50. Distribution of Iliac Crest Area Containing Trabecular Bone (Es.Ar.) by Sex and Age ...... 210 Figure 51. Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) by Sex and Age . 210 Figure 52. Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) by Sex and Age ... 211 Figure 53. Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) by Sex and Age ...... 211 Figure 54. Distribution of Iliac Crest Mean Trabecular Width ...... 212 Figure 55. Distribution of Iliac Crest Total Diameter/Thickness (Tt.Dm.) by Sex and Age 212 Figure 56. Distribution of Iliac Crest Marrow Cavity Diameter/Thickness (Es.Dm.) by Sex and Age ...... 213 Figure 57. Distribution of Iliac Crest Cortical Diameter/Thickness (Ct.Dm.) by Sex and Age ...... 213 Figure 58. Summary of Statistical Significance Found in Sex Examinations ...... 216 Figure 59. Site Affiliation/Subsistence Examination: Summary of Statistical Significance. 217 Figure 60. Sex Distribution of Rib Total Area (Tt.Ar.) ...... 311 Figure 61. Sex Distribution of Rib Cortical Area (Ct.Ar.) ...... 311 Figure 62. Sex Distribution of Rib Relative Cortical Area (Rel.Ct.Ar.) ...... 312 Figure 63. Sex Distribution of Rib Endosteal Area (Es.Ar.) ...... 312 Figure 64. Sex Distribution of Rib Periosteal Perimeter (Ps.Pm.) ...... 313 Figure 65. Sex Distribution of Rib Endosteal Perimeter (Es.Pm.) ...... 313 Figure 66. Sex Distribution of Clavicle Total Area (Tt.Ar.) ...... 314 Figure 67. Sex Distribution of Clavicle Cortical Area (Ct.Ar.) ...... 314 Figure 68. Sex Distribution of Clavicle Endosteal Area (Es.Ar.) ...... 315

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Figure 69. Sex Distribution of Clavicle Relative Cortical Area (Rel.Ct.Ar.) ...... 315 Figure 70. Sex Distribution of Clavicle Periosteal Perimeter (Ps.Pm.) ...... 316 Figure 71. Sex Distribution of Clavicle Endosteal Perimeter (Es.Pm.) ...... 316 Figure 72. Sex Distribution of Iliac Crest Total Area (Tt.Ar.) ...... 317 Figure 73. Sex Distribution of Iliac Crest Cortical Area (Ct.Ar.) ...... 317 Figure 74. Sex Distribution of Iliac Crest Marrow Cavity Area (Es.Ar.) ...... 318 Figure 75. Sex Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) ...... 318 Figure 76. Sex Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) ...... 319 Figure 77. Sex Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) ...... 319 Figure 78. Sex Distribution of Iliac Crest Mean Trabecular Width (Tb.Wi.) ...... 320 Figure 79.Sex Distribution of Iliac Crest Cortical Diameter (Ct.Dm.) ...... 320 Figure 80. Sex Distribution of Iliac Crest Marrow Cavity Diameter (Es.Dm.) ...... 321 Figure 81.Site Distribution of Rib Total Area (Tt.Ar.) ...... 322 Figure 82. Site Distribution of Rib Cortical Area (Ct.Ar.) ...... 322 Figure 83. Site Distribution of Rib Endosteal Area (Es.Ar.) ...... 323 Figure 84. Site Distribution of Rib Relative Cortical Area (Rel.Ct.Ar.) ...... 323 Figure 85. Site Distribution of Rib Periosteal Perimeter (Ps.Pm.) ...... 324 Figure 86.Site Distribution of Rib Endosteal Perimeter (Es.Pm.) ...... 324 Figure 87. Site Distribution of Clavicle Total Area (Tt.Ar.) ...... 325 Figure 88. Site Distribution of Clavicle Cortical Area (Ct.Ar.) ...... 325 Figure 89. Site Distribution of Clavicle Endosteal Area (Es.Ar.) ...... 326 Figure 90. Site Distribution of Clavicle Relative Cortical Area (Rel.Ct.Ar.) ...... 326 Figure 91. Site Distribution of Clavicle Periosteal Perimeter (Ps.Pm.) ...... 327 Figure 92. Site Distribution of Clavicle Endosteal Perimeter (Es.Pm.) ...... 327 Figure 93. Site Distribution of Iliac Crest Total Area (Tt.Ar.) ...... 328 Figure 94. Site Distribution of Iliac Crest Cortical Area (Ct.Ar.) ...... 328 Figure 95. Site Distribution of Iliac Crest Endosteal Area (Es.Ar.) ...... 329 Figure 96. Site Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) ...... 329 Figure 97. Site Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) ...... 330 Figure 98. Site Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) ...... 330 Figure 99. Site Distribution of Iliac Crest Mean Trabecular Width (Tb.Wi.) ...... 331 Figure 100.Site Distribution of Iliac Crest Total Diameter (Tt.Dm.) ...... 331 Figure 101. Site Distribution of Iliac Crest Cortical Diameter/Thickness (Ct.Dm.) ...... 332 Figure 102. Site Distribution of Iliac Crest Marrow Cavity Diameter (Es.Dm.) ...... 332 Figure 103. One-Way ANOVA Box and Whisker Plot of Age and Rib Cortical Area (Ct.Ar.) ...... 334 Figure 104. One-Way ANOVA Box and Whisker Plot of Age and Rib Relative Cortical Area (Rel.Ct.Ar.) ...... 334 Figure 105. Two-Way ANOVA Interaction Plot for Age, Sex and Rib Cortical Area (Ct.Ar.) ...... 335

Figure 106. Two-Way ANOVA Interaction Plot for Age, Sex and Rib Relative Cortical Area (Rel.Ct.Ar.) ...... 335 Figure 107. One-Way ANOVA Box and Whisker Plot for Age and Iliac Crest Cortical Area (Ct.Ar.) ...... 336 Figure 108. One-Way ANOVA Box and Whisker Plot for Age and Iliac Crest Relative Trabecular Area ( Rel.Tb.Ar.) ...... 336 Figure 109. One-Way ANOVA Box and Whisker Plot of Age and Iliac Crest Cortical Thickness/Diameter (Ct.Dm.) ...... 337 Figure 110. One-Way ANOVA Box and Whisker Plot of Age and Iliac Crest Mean Trabecular Width (Tb.Wi.) ...... 337 Figure 111. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Mean Trabecular Width (Tb.Wi.) ...... 338 Figure 112. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Cortical Diameter (Ct.Dm.) ...... 338 Figure 113. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Cortical Area (Ct.Ar.) ...... 339 Figure 114. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) ...... 339

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List of Tables

Table 1. Cultural periods of Peru ...... 85 Table 2. Summary of Chiribaya Subsample Characteristics ...... 102 Table 3. Demographics of sample used in rib analysis ...... 120 Table 4. Demographics of sample used in clavicle analysis ...... 120 Table 5. Demographics of sample used in iliac crest analysis ...... 121 Table 6. Statistical Hypotheses Examining Sex Differences ...... 123 Table 7. Statistical Hypotheses Examining Subsistence Differences ...... 126 Table 8. Statistical Hypotheses Examining Age Differences ...... 128 Table 9. Statistical Hypothesis Examining Intra-skeletal Variability ...... 129 Table 10. Intra-observer Error Rates for Each Variable ...... 135 Table 11. Rib Normality Measures of Males and Females ...... 136 Table 12. Clavicle Normality Measures of Males and Females ...... 137 Table 13. Iliac Crest Normality Measures of Males and Females ...... 138 Table 14. Rib Normality Measures by Site ...... 139 Table 15. Clavicle Normality Measures by Site ...... 140 Table 16. Iliac Crest Normality Measures by Site ...... 141 Table 17. Rib Normality Measures for Each Age Category ...... 142 Table 18. Clavicle Normality Measures for Each Age Category ...... 143 Table 19. Iliac Crest Test Normality Measures for Each Age Category ...... 144 Table 20. Intra-skeletal Variability Normality Measures ...... 145 Table 21. T-test Output of Rib Variables Analyzed for Sex Comparisons ...... 146 Table 22. Summary of T-Test Results for Rib Analysis by Sex ...... 146 Table 23. Descriptive Statistics of Clavicle Dataset Analyzed for Sex Comparison ...... 148 Table 24. Summary of T-Test Results for Clavicle Analysis by Sex ...... 148 Table 25. Output Values for Iliac Crest Analysis by Sex ...... 150 Table 26. Summary of T-Test Results for Iliac Crest Analysis by Sex ...... 150 Table 27. Descriptive Statistics of Tt.Dm. of Iliac Crest by Sex ...... 152 Table 28. Output Values of Rib Dataset Analyzed for Site Comparisons ...... 154 Table 29. Summary of T-Test Results for Rib Analysis by Site ...... 154 Table 30. Descriptive Statistics for Rib Data from El Yaral Sample ...... 156 Table 31. T-Test Output Values for Combined Site Rib Comparisons ...... 157 Table 32. T-Test Results for Combined Site Rib Comparisons ...... 157 Table 33. Output Values of Clavicle Dataset Analyzed for Site Comparison ...... 162 Table 34. Summary of T-Test Results for Clavicle Analysis by Site ...... 163 Table 35. Descriptive Statistics for Clavicle Data for El Yaral Sample ...... 164 xvii

Table 36. T-Test Output Values for Combined Site Data Set of Clavicle ...... 165 Table 37. T-Test Results for Combined Site Data Set of Clavicle ...... 165 Table 38. Output Values for Site Comparisons of Iliac Crest Data ...... 171 Table 39. T-Test Results of Iliac Crest Data for Site Comparisons ...... 171 Table 40. Descriptive Statistics for Iliac Crest Variables from El Yaral Sample ...... 173 Table 41. Output Values for T-Test Results of Grouped Site Data Set for Iliac Crest ...... 174 Table 42. T-Test Results for Combined Site Data Set of Iliac Crest Data ...... 175 Table 43. One-Way ANOVA Table for Rib Cortical Area (Ct.Ar.) ...... 182 Table 44. Two-Way ANOVA Table of Rib Cortical Area (Ct.Ar.) ...... 183 Table 45. Descriptive Statistics of Female Rib Data Categorized by Age and Sex ...... 184 Table 46. Descriptive Statistics of Male Rib Data by Age Category ...... 186 Table 47. Descriptive Statistics of Female Rib Data by Age Category ...... 187 Table 48. Descriptive Statistics of Clavicle Data by Age Category ...... 192 Table 49. Descriptive Statistics of Clavicle Variables for Males by Age Category ...... 193 Table 50. Descriptive Statistics of Female Clavicles by Age Category ...... 194 Table 51. One-Way ANOVA Table for Iliac Crest Cortical Area (Ct.Ar.) for Age ...... 198 Table 52. Two-way ANOVA Table for Iliac Crest Cortical Area (Ct.Ar.) ...... 199 Table 53. One-Way ANOVA Table of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) ...... 200 Table 54. Two-Way ANOVA Table of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) ...... 200 Table 55. One-Way ANOVA Table of Iliac Crest Cortical Thickness (Ct.Dm.) ...... 201 Table 56. Two-Way ANOVA Table of Iliac Crest Cortical Thickness (Ct.Dm.) ...... 201 Table 57. One-Way ANOVA Table of Iliac Crest Mean Trabecular Thickness (Tb.Wi.) ...... 202 Table 58. Two-Way ANOVA Table of Iliac Crest Average Mean Trabecular Thickness (Tb.Wi.) ...... 202 Table 59. Descriptive Statistics of Iliac Crest Data by Age Category ...... 203 Table 60. Descriptive Statistics of Male Iliac Crest by Age Category ...... 206 Table 61. Descriptive Statistics of Female Iliac Crest by Age Category ...... 208 Table 62. Randomized Block ANOVA Table Testing Intra-skeletal Variability ...... 214 Table 63. Raw Data for Rib ...... 341 Table 64. Raw Data for Clavicle ...... 344 Table 65. Raw Data for Iliac Crest ...... 346

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Chapter 1: Dissertation Outline

The skeleton is a dynamic system that creates the strong, internal framework of vertebrates. The skeletal system provides support for the heart and lungs, protection for the brain and also acts as a mineral reservoir. Skeletal biology is the area of study concerned with the skeleton as a biological system; bone histomorphology refers to the study of the form and structure of bone tissue as seen through a microscope.

1.1 Overview of Histomorphometry

Histomorphology is stereologic in that one is viewing the three-dimensional structures of bone in a two-dimensional manner. In dealing with past populations, one is limited to variables taken at one point in time (after death) rather than having the capability of measuring variables throughout the life of an individual. Within histomorphology, a quantitative approach is referred to as histomorphometry and can address issues related to overall bone health and bone disease.

Bone constantly changes throughout an individual’s life (Frost 1966) and can be used to reveal information about the smaller phases of bone turnover in addition to being used to make inferences about the health status of populations, estimate the age of an individual, estimate activity types and levels and examine pathological conditions. Bone diseases are typically characterized by quantitative abnormalities and this quantitative 1

approach to bone tissue analysis is often advantageous for analyses into age-related bone changes as well as the pathophysiology of bone diseases (Boivin et al. 1990).

This analysis takes a histomorphometric approach and examines microscopic bony changes in males and females of varying ages in three communities differing in subsistence in ancient southern Peru. This analysis uses a combination of methods utilized by both clinical and anthropological researchers to examine how members of this population are impacted by non-genetic components of bone activity.

Age-associated bone loss is globally ubiquitous and impacts both males and females of all socioeconomic statuses and ancestries. It is a major health care threat for many Americans with over 10 million people already diagnosed with osteoporosis (a propensity toward bone fracture) and 34 million with osteopenia (a decrease in bone mass) (NOF 2011; USDHHS 2004b). More than 1.5 million osteoporotic fractures occur in the United States each year. This results in more than half a million hospitalizations, more than 800,000 emergency room visits, more than 2.6 million physician office visits and nearly 180,000 individuals placed in elder care facilities (USDHHS 2004b). Skeletal research on past and present populations has shed light on these diseases; however, the precise mechanisms and etiology remain incompletely understood.

In anthropological literature, age-associated bone loss has been indicated in past populations (Agarwal and Stout 2003). Archaeological studies are typically limited to the analysis of cortical bone, which is more likely to preserve in a variety of burial conditions, of non-Western hunters and gatherers or agriculturalists (Agarwal and

Grynpas 1996). Within biomedical research publications, the primary focus has been on trabecular bone which is thought to be more dynamic due to its proximity to bone

marrow (Barger-Lux and Recker 2006). This projects aims at closing the disparity that exists between the study of microscopic bone research between past and present populations in terms of comparability of findings, variables and the bony envelopes investigated.

A difficulty in using archaeological skeletal remains for microscopic analysis lies in the fact that soft tissues and cells do not remain; which are of primary importance in biomedical and paleopathological diagnoses (Schultz 2001). Therefore, no true cytological investigation may be carried out and a true histological examination of archaeological skeletal remains in the primary sense of the term ‘histology’ is not really possible in all aspects. Microscopic analysis of bone from past populations is further complicated by factors that can affect the structure of bone from the time it is deposited and the time it is excavated. This may include damage due to roots, fungi, algae, bacteria, insects and their larvae, heat and fire (Schultz 1996). Due to these processes, skeletal materials from archaeological contexts tend to be much more fragile and often unsuitable for histological analyses. However, past studies have found (Stout 1978; Stout 1983;

Stout and Teitelbaum 1976) that the histomorphology of archaeological bone samples are often well preserved with osteons, Haversian canals and cement lines, and osteocyte lacunae clearly identifiable. Features of micro-architectural deterioration of trabecular bone have also been studied in archaeological bone (Brickley 1998; Roberts and Wakely

1992). In skeletal populations, individuals with low bone mass such as the very young and very old (Lyman 1994) may not be well-represented. In addition, diagenesis, the addition and removal of elements in soil due to chemical exchanges and reactions, needs to be assessed before histological analyses may be undertaken (Grynpas 2003).

Oftentimes, chemical changes may occur within the bony envelopes that do not impact the macroscopic shape/size of the bones. The factors mentioned above pose a minimal limitation on the focal population of this study being that there are representatives of both young and old individuals present and microscopic investigations have not indicated large amounts of diagenesis.

At the microstructural level, histological features in archaeological bone samples can provide information about remodeling rates (Burr et al. 1990; Frost and Wu 1967;

Mulhern 2000; Stout and Lueck 1995) bone quantity and quality (Agarwal et al. 2004;

Grynpas 2003), bone loss (Brickley and Agarwal 2003; Cho and Stout 2003; Laughlin et al. 1979; Martin and Armelagos 1979; Martin and Armelagos 1985; Martin et al. 1981) physical activity levels (Burr et al. 1990; Pfeiffer et al. 2006; Robling 1998; Ruff and

Hayes 1983a; Ruff and Hayes 1983b) and remodeling rates due to dietary factors

(Richman et al. 1979) of past populations. As stated above, histological examinations of archaeological populations tend to focus on cortical bone because it is more dense and easily preserved; however, in adults hematopoiesis occurs in regions of bone that have large surface areas for the production of red blood cells and regions comprised of larger areas of trabecular bone are good sources of red marrow throughout life and are largely responsible for rapid bone turnover and play an important role in long-term control of calcium balance (Miller and Jee 1980). Therefore, the endosteal and trabecular envelopes have the potential to reveal more information about bone health of the past.

Although it is possible to utilize non-invasive and/or three-dimensional techniques such as MRI or CT scanning in biomedical research (Kuhn et al. 2007; Rühli et al. 2007), histologic analysis allows for an in-depth view of the bone remodeling

processes that occur at the level of the tissue and is more diagnostic in terms of distinguishing pathological conditions. Thomsen et al. (2005a) report in a study of human tibial bone that with proper care, 3D μCT can be used as a substitute for section-based stereological methods for describing trabecular bone architecture but not for dynamical bone assessments.

This project presents a comprehensive histomorphometric protocol for analyzing all four bone surfaces (osteonal, endosteal, periosteal and trabecular) and examines intra- skeletal variability in bone mass variables in a past population in relation to age- associated bone loss. This project also utilizes minimal technology (transmitted light microscope with attached camera, inexpensive eyepiece grids and free downloadable software) in the data collection methods and analyses as a way to create a protocol of techniques that are available and affordable for anthropological research facilities which typically have limited funding sources. Although more advanced technologies exist (e.g.

μCT, DEXA, MRI) and are commonly used in clinical research, evidence has shown that histological examinations remain the ‘gold standard’ in examining bone at the tissue level

(Barger-Lux and Recker 2006) and with a trained observer, the results can offer abundant information. This study also utilizes bone from three areas of the skeleton (rib, clavicle, and iliac crest) to investigate intra-skeletal variability in bone loss across the three subpopulations.

1.2 Organization of Study

This dissertation is organized into eight chapters. The following details the organization of the material contained within this document to facilitate reading.

Chapter one contains the introduction, significance, purpose and expectations of the study. The goal of this project is to document and interpret microscopic bone remodeling responses of both cortical and trabecular bone of a prehistoric Peruvian population as impacted by subsistence differences. Histological analyses of ancient human remains are fewer in number and traditionally focused on the dense cortical bone, such as that found in the shafts of long bones, than those conducted on modern skeletal material in both clinical trials which primarily focus upon trabecular bone found in the vertebral column and iliac crest. This chapter gives a brief overview of the importance of studying bone in archaeological populations; in particular, how research of the past can influence research on present and future populations. General research questions are introduced in this chapter with a brief description of their importance.

Chapter two gives an overview of the current theoretical understanding of bone biology and its relationship to the study at hand. Within this chapter, the types of bone, bone envelopes, bone cells and communication between cells are discussed. In addition, the processes that create and shape bone are described.

Chapter three defines osteoporosis and osteopenia, factors leading to each condition, and describes how the two conditions are diagnosed in clinical and past settings. The chapter also describes how bone loss is seen differently in both cortical and trabecular bone and how physical activity, diet and nutrition, biological sex and ethnicity play into risk factors. Chapter three also discusses histomorphometric analyses of bone microstructure and the pattern of age-associated bone loss in various regions of the skeleton.

Chapter four introduces the Chiribaya population of ancient southern coastal Peru, which is the sample utilized in this study. The contents of this dissertation focus on the human remains of three Chiribaya subpopulations: Chiribaya Alta, El Yaral, and San

Geronimo. These communities, known as parcialidades, served a specific socio- economic function within the larger community, the señorío. San Geronimo consists of a community of fisherman, pescadores; El Yaral is comprised of a community of agriculturalists, labradores; Chiribaya Alta was composed of individuals who belonged to an elite, merchant class who received tribute from the communities of San Geronimo and El Yaral and performed administrative functions of the señorío. Chapter 4 reviews literature regarding the Chiribaya in regards to their biocultural context including general health status, isotopic analysis of dietary patterns, biodistance analysis, socio-political history and relationships with neighboring peoples.

Chapter five describes the skeletal sampling sites used (rib, clavicle, iliac crest), methods of procurement and preparation, methods of microscopic analysis, variables examined, sample demographics, hypotheses, and statistical analyses employed in this study. The methods involved with sectioning and microscopically examining these samples in addition to the data collection methods is detailed. Chapter five also discusses the statistical methods employed and lists the specific statistical hypotheses that were used to examine each general hypothesis.

Chapter six presents the statistical and non-statistical results generated to test each of the hypotheses of the study. Chapter six also reports results of intra-observer error rates, normality testing procedures, and the results of t-tests and ANOVA tests conducted to examine each statistical hypothesis. Results are categorized by hypothesis examined

and further by skeletal sampling site. SAS® statistical software was employed for all analyses.

Chapter seven further discusses the results of each hypothesis examined and places the findings within the context of previous studies in past and present populations.

Chapter seven also addresses general trends found within the Chiribaya population and addresses anthropological considerations of the study, limitations of the study, and directions for future research.

Chapter eight concludes the dissertation summarizing the findings of this study and concludes the research.

1.3 Purpose

The study examines the differences subsistence patterns and nutrition play on age- associated bone loss in skeletons of ancient males and females. Three skeletal sampling sites (rib, clavicle and iliac crest) were examined in each individual from an ancient

Peruvian population were sectioned and examined microscopically. These sites were chosen for the following reasons: a) the iliac crest is a standard biopsy site in clinical analyses because of the high content of trabecular bone allowing for a comparison with clinically-based standards; b) the ribs consist primarily of cortical bone and allow comparisons with data from other prehistoric samples and biomedical studies; c) the clavicle was selected as a third area for comparison to examine intra-skeletal variability and because it represents a bone derived from both intramembranous and endochondral ossification. Each of these skeletal elements functions within a unique biomechanical environment which is further discussed in Chapter five.

1.4 Hypotheses Examined This research project examines the following hypotheses:

1. Sex differences exist in histomorphometric areal and perimeter/length

measurements in the Chiribaya sample.

It is expected that similar patterns of sex variation of bone microstructure exist in the Chiribaya as demonstrated in other populations. Males are known to exhibit larger amounts of endosteal resorption with a compensatory response of periosteal apposition, leading to larger perimeter lengths on the external and internal cortex of the rib and clavicle (Seeman 1999; Stini 2003), a differential pattern expected in Chiribaya males and females. Areal measurements are also expected to differ between males and females due to a propensity toward larger sizes in males. Relative areal measurements are expected to be larger in females, a pattern visible in most populations until the fifth decade, as due to the effects of hormonal action associated with pregnancy and childbirth in females (Drusini et al. 2000).

2. Differences in histomorphometric areal and length/perimeter measurements

exist among individuals from Chiribaya Alta, El Yaral and San Geronimo due

to variation of diet and general subsistence level activities.

It is expected that both cortical and trabecular variables will differ significantly between individuals at the three Chiribaya sites in this analysis due to subsistence variation. It is expected that San Geronimo will demonstrate indications of higher bone remodeling due to a high intake of marine-based resources in comparison to individuals from El Yaral who subsisted in a higher amount of carbohydrates due to their agricultural reliance. The individuals from Chiribaya Alta are expected to either be intermediary 9

between San Geronimo and El Yaral as it is thought to be composed of elites drawn from other Chiribaya sites, or exhibit larger relative bone areal measurements due to having access to premiere dietary resources.

3. Differences in histomorphometric areal and length measurements exist among

different age groups due to the effects of increasing age.

It is expected the periosteal and endosteal perimeters of the rib and clavicle exhibit differences with relation to increasing age. Past research on modern populations indicates a general trend with an increase in the periosteal perimeter due to apposition with age, with an accompanying increase in the endosteal perimeter due to resorption

(Epker and Frost 1965; Epker et al. 1965). In relation to the expansion of the periosteal and endosteal perimeters, it is expected that the medullary and total areas will be larger with relationship to increasing age due to expansion of the cortex, while cortical area and trabecular area will be reduced. Research on modern populations indicates a general decrease in bone mass with age (Epker et al. 1965; Sedlin et al. 1963b) in both the cortical and trabecular envelopes (Mellish et al. 1987; Ostertag et al. 2009). It is expected the same pattern will be found in the Chiribaya population with respect to increasing age.

Research on modern samples demonstrates that the majority of trabeculae decrease in thickness with increasing age (Dalle Carbonare et al. 2005; Ding and Hvid

2000; Guo and Kim 2002; Parfitt et al. 1983) which should be evident in a comparison of mean trabecular width of all trabeculae measured. It is also expected that a decrease in cortical thickness at the iliac crest and a reduction in cortical bone mass will be evidence

in the Chiribaya population with increasing age category (Christiansen et al. 1993; Vedi et al. 2011).

4. Variability in relative area measurements due to differential loading throughout

the skeleton exist.

1.5 Chapter Summary

This chapter offers an overview of histomorphometry and its usefulness in examining bone tissue for health and bone diseases. Chapter one also introduces the study of osteopenia and osteorporosis and its importance in examining bone loss in populations of the past. This chapter describes the various complications in studying bone in past populations in comparison to clinical samples and the usefulness of the added element of bone microstructure to skeletal analyses of past peoples. Chapter one also describes the purpose of this project, which is to present a comprehensive documentation and interpretation of the microscopic bone remodeling responses in multiple bone envelopes among individuals of a prehistoric Peruvian population as impacted by differences in sex, subsistence strategy, and age category. Chapter one also introduces the hypotheses addressed in this study and the expectations of each. An outline of the presentation of the study is given in addition to a concise overview of each chapter’s contents.

Chapter 2: Overview of Bone Components and Bone Remodeling

Bone is a complex, yet highly organized, connective tissue. It is rigid, strong, responsible for protection of organs, acts as a lever arm for joints and muscles and serves as a mineral reservoir. Bone is comprised of both organic and inorganic matter. Similar to most connective tissues, approximately 35% of the dry weight of bone is organic, of which 90% is collagen, with the remaining 10% is ground substance consisting of a variety of compounds, such as proteoglycans (Behari 2009). Of the some 20 basic types of collagen, type 1 collagen is found in bone tissue. Collagen fibers, which are organized into bundles, play an important role in determining bone strength, as they serve to resist pulling forces (Behari 2009). The inorganic component of bone is primarily hydroxyapatite (an analog of calcium phosphate) which stiffens the bone to resist the forces of bending and compression (Nather et al. 2005). A group of specialized cells, including osteoclasts, osteoblasts, and osteocytes create, adapt, and maintain bone tissues through the life of an individual through the processes of modeling and remodeling

(Crockett et al. 2011). During growth, the bone modeling process determines the size and shape of bones, while the lifelong process of bone remodeling maintains bone tissue through focused renewal or bone turnover. During the life of an individual, these two processes respond to a variety of factors, such as the mechanical loading environment, hormonal fluctuations, disease, nutrition, and the individuals’ age and sex. 12

2.1 Types of Bone

At a higher level, bone tissues are organized into two larger categories of bone types based upon structure. Cortical (compact bone) forms the outer shell of the shafts of bones, whereas cancellous (trabecular) bone consists of a network of plates approximately 200 μm in diameter contained within a cortical shell (Martin et al. 1998).

Cortical bone is designed to resist bending forces and trabecular bone is designed to resist compression. The network of plates in trabecular bone creates a lattice-like structure whereby 30-90% of the enclosed area is open space (Stini 1995). The strength of the latticework depends on the density of the structural components and their connectedness

(Stini 1995) in addition to its architecture (Odgaard 1997).

In mammals, each of these types of bone tissue can exist at a smaller scale as either primary bone tissue (non-lamellar or ‘woven’ bone) or secondary bone tissue

(lamellar bone). Woven bone, normally found in juveniles and fetal skeletons, is formed relatively quickly, and is poorly organized. In contrast, lamellar bone is formed through a slower process and has an organized ‘plywood-like’ structure composed of multiple layers of lamellae (Martin et al. 1998). Lamellar bone can be further broken down into two types: primary and secondary. Primary lamellar bone is formed during growth and modeling processes. Secondary lamellar bone is the result of the resorption and replacement of primary bone that occurs by the process known as remodeling (Crockett et al. 2011). Another type of mammalian bone is plexiform, or what may also be referred to as laminar or fibrolamellar bone (Currey 2002). In larger mammals that grow rapidly, there is a need for bone to grow faster than lamellar bone can be laid down. To

compensate for this difference, parallel-fibered bone is laid down rapidly to be later replaced later with lamellar bone (Currey 2002).

2.2 Bone Envelopes

Bone has four envelopes: periosteal, endosteal, trabecular and osteonal (Gosman et al. 2011), which are surfaces that have differences regarding behavioral and functional purposes. The periosteal envelope covers the outside surface of bone, the endosteal envelopes is divided between the endocortical and trabecular, and the osteonal

(Haversian) envelope which includes the Volkmann’s canal surfaces (further described in section 2.4). The envelopes describe osteogenic surfaces covered by a layer of bone- lining cells and connective tissue (Figure 1). The outer surface of cortical bone is covered by the periosteum, which is a thin, vascular-like membrane that blends with the fibers of ligaments and muscle insertions (Nather et al. 2005). It has two distinct layers:

1) an outer layer that can be subdivided into two parts and 2) an inner cambium layer

(Dwek 2010). The superficial portion of the outer layer is inelastic and contains few cells.

The deep portion of the outer layer is the fibroelastic layer because it contains many elastic fibers and is therefore, elastic. The inner cambium layer contains “retired” osteoblasts (bone lining cells) that lie in contact with the cortical surface as well as small compact cells resembling fibroblasts (Dwek 2010). Bone lining cells sense chemical and mechanical signals and may be involved with the activation of bone remodeling processes (Burr 2002; Martin 2000). After sensing strain, they communicate with underlying osteocytes through the canalicular network (described in the following subsection).

Figure 1. Bone Envelopes (Adopted from Freeman 2012)

2.3 Bone Cells and Cell Communication

Bone is a dynamic tissue in that it is constantly undergoing transformation in response to factors such as growth, mechanical strain, disease, trauma and disuse. Like other eukaryotic animal cells, the cells involved with the resorption and addition of bone communicate with one another by local and long distance communication via signaling.

Local communication between bone cells occurs through gap junctions made possible by the lacuna-canalicular network (Figure 2). An osteocyte lies within a small cavity in the bone matrix known as a lacuna. From the lacunae, microscopic canals called canaliculi radiate and penetrate the lamellae and anastomose with the canaliuli of neighboring lacunae, thus forming a network between bone cells which allow molecules to pass through.

Figure 2. Gap junctions located between dendrites of osteons

(Adapted from Donahue 2000)

The process of long distance signaling (Figure 3) involves several steps and is referred to as a signal transduction pathway. With a signal transduction pathway, messenger molecules, such as growth factors, are secreted by signaling cells. The receiving portion of the signal transduction pathway involves three stages: reception, 16

transduction and response. Reception refers to the detection of a signaling molecule by the target cell, such as an osteoblast or bone lining cell, when the signaling molecule binds to a receptor protein located at the cell’s surface. The transduction phase converts the signal to a form that can bring about a cellular response. The binding of the signaling molecule to its receptor protein initiates cellular response, such as the secretion of a functional cytokine. The triggering of a specific cellular response by the transduced signal marks the last phase: response. This three stage process of cell-signaling ensures the right cells are involved at the right time in the right places. The cells involved with the bony tissue response are described below.

Figure 3. Overview of cell signaling (Adapted from Reece et al. 2010) 17

2.3.1 Osteoclasts

The cells that model and remodel bone fall into the categories of those that resorb bone and those that form, or have already formed, bone. Osteoclasts are the cells responsible for resorbing bone. They are multi-nuclear, typically contain around 15-20 nuclei and are approximately 40 μm in breadth. When active, these cells have a ruffled border that faces the bone surface (Cohen 2006). Osteoclasts arise from mononuclear cells which originate in the hematopoietic portion of the bone marrow and spleen (Martin et al. 1998; Robling et al. 2006).

Mononuclear cells become pre-osteoclasts which are introduced into the bloodstream via the actions of several transcription and stimulating factors. The precursor cells circulate near an area of bone that is to be resorbed and fuse with each other, creating a multi-nucleated yet immature, osteoclast. The fusion of these cells requires the expression of activator factors which determine the cell’s involvement in resorbing activities. Once they have matured, multi-nucleated osteoclasts employ cell-surface proteins to create a ‘pocket’ in the ruffled border of the osteoclasts and the bone matrix.

Hydrogen cations are sent into this pocket to dissolve the mineral component of bone while the collagen is broken down by protease enzymes. The result of this activity is the creation of a resorption bay known as a Howship’s lacunae.

2.3.2 Osteoblasts

Osteoblasts are mononuclear cells that create the unmineralized organic portion of the bone matrix known as osteoid, and are also involved with the mineralization of this

matrix. Osteoblasts differentiate from mesenchymal cells which can either come from the deep layer of the periosteum or of the bone marrow (Martin et al. 1998). Essentially, osteoblasts are a specialized form of fibroblast, a type of cell that creates and maintains connective tissues of animals.

Mature osteoblasts express Type I collagen along with osteocalcin and alkaline phosphotase, which are needed for the mineralization process. Osteiod, a combination of collagen, noncollagenous proteins, proteoglycans and water, is secreted by a row of activated osteoblasts (Martin et al. 1998). Long cytoplasmic processes are also created so cells can remain in communication with surrounding cells through gap junctions.

Hydroxyapatite, calcium carbonate and calcium phosphate are deposited within the osteoid secreted by the osteoblasts. After production and mineralization events osteoblasts undergo one of several fates: apoptosis or continuing their existence as either osteocytes or bone lining cells.

2.3.3 Osteocytes

Osteocytes are former osteoblasts that have become entombed in the bone matrix.

They are also the most numerous type of bone cell. Osteocytes have two options for their life span: 1) scheduled cell death via apoptosis or 2) removal by osteoclasts during the resorption process (Parfitt 2002a). As described earlier, osteocytes lie in small cavities called lacunae and communicate with one another via dendritic extensions contained within canaliculi (Figure 4). Any area of bone is never more than a few micrometers away from an osteocyte allowing information about their surroundings to be communicated between osteocytes via the network of canaliculi (Seeman 2006).

Figure 4. Osteocyte lacunae (dark spots) of a bone thin section (100x) (Original image by author)

2.3.4 Bone Lining Cells

Bone lining cells are also former osteoblasts that remain on the surface when bone formation was complete (Martin et al. 1998). As bone matrix production stops, the bone lining cells become flattened against the bone matrix as they become quiescent, although they do not form a continuous barrier along the bone surface (Martin et al. 1998). Bone lining cells also communicate with one another and with osteocytes through gap-

junctions. They also maintain receptors for parathyroid hormone, estrogen and other chemical messengers (Martin et al. 1998). Bone lining cells are also thought to be responsible for the transfer of mineral in and out of bone and sensing mechanical strain

(Miller and Jee 1987; Parfitt 1987).

2.4 Modeling and Remodeling

Modeling and remodeling describe the two processes that create the size and shape of bones or modify its material characteristics. Both processes require the use of the cells described above; modeling involves the independent action of osteoclasts and osteoblasts while remodeling involves the coupled action by the two cell types. In addition, modeling will change the shape and size of a bone while remodeling does not have a significant impact on the overall size or shape of bones. The rate of modeling is greatly reduced after skeletal maturity whereas remodeling occurs throughout the life of an individual (Martin et al. 1998). Because the focus of this particular research is centered around an understanding of the factors at play on adult human bone, this chapter focuses on the description of bone remodeling.

Remodeling occurs both while the skeleton is developing and throughout the remainder of an individual’s life as a replacement process for previously created bone.

According to Burr (2002) bone remodeling fulfills three purposes: 1) maintaining mineral balance in the body by increasing or decreasing amounts in the serum, 2) adapting to mechanical demands and 3) repair of microdamage to maintain bone integrity. The actions of bone repair involve a concerted effort of both osteoclasts and osteoblasts to remove and replace bone at the same location.

Bone remodeling occurs on all four skeletal ‘envelopes’ (periosteal, Haversian, cortical-endosteal and trabecular) throughout the life of an individual and is a phenomenon that is especially active on the endosteal surfaces of the adult skeleton

(Frost 1987; Parfitt 2003). Remodeling is accomplished by the concerted efforts of osteoclasts and osteoblasts that work together as a unit termed by (Frost 1966) as a basic multicellular unit (BMU). A BMU has a beginning (initiation), a middle (progression) and an end (termination) in which origination occurs close to a blood vessel and progression moves toward a targeted site (Parfitt 2003) (Figure 5). In the BMU, a team of multi-nucleated osteoclasts form the front known as a cutting cone (Frost 1966). The front end of the BMU contains a capillary bud and surrounding loose connective tissue that follows the cutting cone, that supplies nutrients (Martin et al. 1998) and circulating monocytes (to become pre-osteoclasts)(Frost 1969). A team of osteoblasts follows to form what is known as the closing cone (Frost 1969). Only osteoclasts are employed during the phase of initiation and only osteoblasts are required during termination; however, both types of cells are required during progression, only at different locations

(Parfitt 2003).

Figure 5. Bone remodeling cycle (Adopted from LaJeunesse and Martel-Pelletier 2010)

There are three main phases to the lifespan of a BMU: activation, resorption and formation (ARF) (Figure 6) (Martin and Burr 1989). The ARF sequence can be broken down into six stages of an osteon’s lifespan and are always sequential in normal bone remodeling: activation, bone resorption, reversal, bone formation, mineralization and quiescence (Martin and Burr 1989). Activation refers to the process by which a region of quiescent surface covered by bone lining cells becomes transformed to a resorbing surface. During activation, osteocytes communicate with one another through the canaliculi to lining cells on the closest bone surface which then send signals to nearby capillaries after which the capillaries move toward the initiation site of the BMU and penetrate the retracting lining cells (Parfitt 2002b). Osteocytes detect mechanical stimuli

and send signals to other cells that regulate bone formation and resorption. It was originally proposed (Frost 1960) that microcracks disrupted the canalicular connections between osteocytes and stimulated repair. An alternate view is presented by Burr (2002) in which the osteocyte-canalicular system acts as more of an inhibitor of osteoclastic activity. Since neither bone lining cells or osteocytes actively form or resorb bone, they signal the effector cells: osteoblasts or osteoclasts. The relationship of stimulus to response remains unclear; however, remodeling may be initiated when an osteocyte undergoes apoptosis or fails with its normal functions (Stout and Crowder 2012). The most likely intermediaries for cell-to-cell communication between sensor cells and effector cells are prostaglandins (hormone-like, fat-soluble, regulatory molecules made from fatty acids) and nitric oxide (Bergmann et al. 2010).

Osteoclasts are only activated after the activation of lining cells (Rodan and

Martin 1981) after a target for bone remodeling has been identified. Osteoclasts either resorb bone by creating a tunnel (in compact bone) or trench (on the surface of endosteal or trabecular bone). These resorptive areas are characterized by a scalloped surface referred to as a reversal line produced by the creation of Howship’s lacunae (Schenk et al.

1969). The reversal period, during which the transition from resorption to formation occurs, can take several days and results in a cylindrical-shaped space between the resorptive region and the refilling region (Martin and Burr 1989). The creation of a cement line coincides with the location of the bone surface during the reversal period

(Martin and Burr 1989).

During the formation period, osteoblasts appear around the periphery of the tunnel that was created by the osteoclasts and lay down concentric lamellae leaving a pathway

for capillaries to support the BMU (Martin and Burr 1989). Following the formation of the organic bone matrix (osteoid), inorganic bone mineral is deposited between the collagen fibers (Martin and Burr 1989). After the sequence of tunneling and refilling has been completed, the osteoclasts disappear and osteoblasts that have not become osteocytes, become lining cells or apoptose. In the average adult of good health, the resorption period takes about three weeks whereas the formation period requires about three months to complete (Martin and Burr 1989).

Figure 6. Activation, resorption, formation sequence of bone formation (Adopted from Baron 2008)

In compact bone, the end result of the BMU is a histomorphological feature known as an osteon or Haversian system, a term first introduced by Biedermann in 1914 and was named for Clopton Havers who described in detail the longitudinal and transverse ‘pores’ in compact bone (Havers 1861). Osteonal BMUs are isolated within the cortex of bone so they must maintain a vascular supply; this is accomplished by their network of Haversian canals. Each 50 to 90 µm in diameter Haversian canal contains two capillaries (one for supply and one for return). These connect with the vasculature in the medullary cavity or on the periosteal surface. The entire secondary osteon or secondary

Haversian system is approximately 200-300 μm in diameter (Martin and Burr 1989).

Secondary osteons are distinguished by the presence of a cement line that is about 1 to 5

μm thick and separates the osteon from the rest of the bone matrix (Martin and Burr

1989). Osteons are connected to one another through tunnels called Volkmann’s canals that are about 20 μm in diameter (Martin et al. 1998). Remnants of un-remodeled primary lamellar bone remain in between Haversian systems and are referred to as interstitial lamellae, while lamellae that are arranged parallel with the periosteal and endosteal surfaces form circumferential lamellae.

In trabecular bone, BMUs operate in a similar manner; however, they work on the surface of the bone, rather than being buried in the cortex by digging and refilling trenches (Figure 7) (Martin et al. 1998). The end result of the BMU in trabecular bone is referred to as hemiosteonal because the BMU excavates a trench rather than a tunnel through the bone, leaving a semicircular hemiosteon in its wake (Parfitt 2003; van Oers et al. 2008a). The front of the BMU is then referred to as an osteoclastic cutting hemicone

and the team of osteoblasts is referred to as a closing hemicone (Parfitt 2003). Because trabecular bone has a greater accessibility for resorption, it is usually the first element of the skeleton to experience loss of mass with aging, illness or other factors (Stini 1995) and is thus a type of bone that should potentially be more informative for interpreting bone quality and disease in past populations.

Figure 7. Hemi-osteonal BMU

(original sketch by the author)

Bone remodeling is regulated by a series of checks and balances. Osteoblasts can initiate fully functional osteoclasts capable of resorption yet can also prevent osteoclasts from becoming fully functional (Cohen 2006). The body appears to be able to regulate

bone remodeling through varying activation rates more than by controlling the number of osteoblasts or osteoclasts within a BMU or the rates at which they resorb or form bone

(Martin and Burr 1989). Cortical bone remodeling rates are determined by several factors. The rate at which new foci of remodeling area created (activation frequency) can increase or decrease, or the amount of bone remodeled per remodeling unit (osteon size) can vary (Stout and Lueck 1995).

There is also evidence that two kinds of remodeling may exist: one that is stochastic (not site-dependent) and non-deterministic whereas another is targeted toward specific sites (site-dependent) (Burr 2002; Parfitt 2002b) to repair and replace microfractures within bone to maintain bone integrity. Burr (2002) hypothesizes that approximately 30% of bone is targeted toward repair of microdamage, while 70% is not.

The concept known as the mechanostat was presented by Frost (1987; 1996; 1998;

2003b) to explain the relationship of stress and strain to bone remodeling. With this model, disuse activates the remodeling process, while an overload inhibits remodeling but activates modeling. Turner (1999a) offers a mathematical interpretation of bone biology coined as the principle of cellular accommodation whereby bone mass changes are explained mathematically in terms of external stimuli. With this principle, final bone mass is the result of temporal sequence of preceding mechanical loading events in addition to hormonal stimuli, especially parathyroid hormone. This interpretation draws upon the finding that glutamate, which is a key neurotransmitter involved with learning and memory may play a role in cell-cell communication (Mason et al. 1997). With the principle of cellular accommodation, bones acquire a long-term memory of a mechanical

loading environment that will influence its responsiveness to external stimuli (Turner et al. 2002).

2.5 Chapter Summary

Chapter two offers an overview of bone structure, bone components, and bone remodeling. At a higher level, bone tissues are organized into two larger categories of bone types based upon structure. Cortical (compact bone) forms the outer shell of the shafts of bones, whereas cancellous (trabecular) bone consists of a network of plates approximately 200 μm in diameter contained within a cortical shell (Martin et al. 1998).

Bone has four envelopes: periosteal, endosteal, trabecular and osteonal.

The cells that model and remodel bone fall into the categories of those that resorb bone and those that form bone. Osteoclasts are the cells responsible for resorbing bone.

Osteoblasts are mononuclear cells that create the unmineralized organic portion of the bone matrix known as osteoid, and are also involved with the mineralization of this matrix. Osteocytes are former osteoblasts that have become entombed in the bone matrix and are the most numerous type of bone cell. Bone lining cells are also former osteoblasts that remain on the surface when bone formation was complete. As bone matrix production stops, the bone lining cells become flattened against the bone matrix as they become quiescent, although they do not form a continuous barrier along the bone surface.

Local communication between bone cells occurs through gap junctions made possible by the lacuna-canalicular network.

Modeling and remodeling describe the two processes that create the size and shape of bones or modify its material characteristics. Both processes require the use of

the cells described above; modeling involves the independent action of osteoclasts and osteoblasts while remodeling involves the coupled action by the two cell types.

Remodeling is accomplished by the concerted efforts of osteoclasts and osteoblasts that work together as a unit termed by as a basic multicellular unit (BMU). There are three main phases to the lifespan of a BMU: activation, resorption and formation (ARF). The

ARF sequence can be broken down into six stages of an osteon’s lifespan and are always sequential in normal bone remodeling: activation, bone resorption, reversal, bone formation, mineralization and quiescence. In compact bone, the end result of the BMU is a histomorphological feature known as an osteon or Haversian system. Bone remodeling is regulated by a series of checks and balances. There is also evidence that two kinds of remodeling may exist: one that is stochastic (not site-dependent) and non-deterministic whereas another is targeted toward specific sites (site-dependent)

The goals of this project are directed toward non-deterministic, stochastic remodeling events as influenced by subsistence differences and their influence on age- associated bone loss; therefore, directed remodeling events will not be addressed further.

The following chapter address remodeling as it relates to age-associated bone loss and the factors that lead to bone loss in the various bony envelopes in terms of differences among the sexes, population affinity, hormonal activity, physical activity and diet.

Chapter 3: Age-Associated Bone Loss

Bone is in a continual state of turnover with approximately 10% of bone replaced each year and a complete renewal of bone every ten years (Cohen 2006). Normally, there is a balance between osteoclast resorption and osteoblast production, but an imbalance can lead to serious consequences. This balance can be disrupted for a variety of reasons.

If osteoclast production is increased or osteoblast production is decreased, bone mass will decrease and lead to osteopenia or osteoporosis where the bones become brittle and fragile.

3.1 Osteopenia and Osteoporosis

Osteopenia refers to the condition of having lower bone mass and less whole-bone strength than average in comparison to other healthy people of the same age, height, weight, sex and race (Frost 2004). Osteopenia weakens bone so that fracturing becomes more likely to occur.

Osteoporosis is similar to osteopenia is that it is characterized by a decrease in bone mass but is typically diagnosed by having a propensity toward non-trauma induced fracturing. It has been noted that a decrease in bone mineral density by one standard deviation increases the risk of osteoporotic fractures by about 2.4 times (Cohen 2006).

Osteoporosis is characterized by reduced bone mass, variation in microarchitecture, reduced bone strength and increased fracture risk. Fractures associated with osteoporosis

generally occur under conditions, and in areas where a normal, healthy person would not break a bone such as the hip, vertebral column, wrist or rib and are referred to as fragility fractures. Bone quality, then, can be understood as resistance to failure (Barger-Lux and

Recker 2006).

Bone loss can occur in all four bone envelopes: periosteal, Haversian, endosteal and trabecular, although most age-related bone loss comes occurs where bone exists next to marrow (Frost 2003a). Individuals with osteopenia and osteoporosis also demonstrate macroscopic changes to bone by which the marrow space becomes enlarged due to relative increase in resorption at the inner cortex.

Subperiosteal expansion of the femur with age was first noted by Smith and

Walker (1964) and since described in other skeletal sites (Garn 1972; Garn et al. 1969;

Ruff and Hayes 1982). Bone apposition at the periosteum may serve as a compensatory mechanism for bone resorption that occurs with age at the endosteum (Garn 1972; Smith and Walker 1964). Osteopenia and osteoporosis, then, refer to conditions of greater relative bone loss e.g. relative cortical area. Ruff and Hayes (1982) found this process to be modified by localized bone site differences in mechanical loadings in that less mechanically stressed areas led to a higher risk of fracture in later life. Loss of trabecular bone with age also occurs as increased net bone loss on bone trabeculae in the marrow cavity leads to thinner and few trabeculae and a loss in connectivity between trabeculae.

3.2 Diagnoses of Osteopenia and Osteoporosis

Different forms of osteoporosis have been distinguished: primary type I, primary type II, and secondary osteoporosis. Primary type I osteoporosis occurs more commonly in women and is associated with the endocrine changes that occur with menopause and is often referred to as ‘postmenopausal osteoporosis.’ Primary type II osteoporosis is associated with a later onset and known to occur after the age of 75 and occurs in both males and females (Riggs and Melton 1983) and often referred to as ‘senile osteoporosis.’

Secondary osteoporosis can occur at any age and is associated with chronic medical conditions such as hyperparathyroidism and/or extensive use of medications such as glucocorticoids (Vedi et al. 2005). All three types can be observed in both modern and archaeological bone (Schultz 2003) and all types can be studied by microscopic techniques.

Among living individuals, osteoporosis is generally diagnosed in one of three ways: radiographic measurement of bone density, biochemical markers, or making a pathological assessment of a bone sample (Bonnick and Shulman 2006). With radiography, a measurement of bone mineral density (BMD) is taken, generally with the use of Dual X-Ray Absorptiometry (DEXA). BMD refers to the amount of mineral matter within one square inch of bone, which is slightly different than the true physical density of bone which is determined as bone mass per volume. BMD is, essentially, the ratio of bone mineral content (BMC) to bone size (Deng et al. 2002). DEXA results in a measurement of ‘areal’ BMD in gm/cm2 which is then converted into either a ‘T-score’ or a ‘Z-score’ by the manufacturer’s software (Carey and Delaney 2010).

When ratio scale data exhibit a ‘normal’ or ‘Gaussian’ distribution, it can be transferred to a standard normal distribution with a mean of ‘0’ and a standard deviation of ‘1’ (Zar 1999). A z-score can be obtained for any value within a data set by dividing the value of its difference from the mean and dividing by the standard deviation value

(Zar 1999). A ‘t’-distribution is present when the data are symmetric and the mean is ‘0’, as in a z-distribution, but the standard deviations differ, depending on the data sample

(Zar 1999). In young, healthy, white women, BMD has a Gaussian distribution, making the derivation of a ‘z-score’ appropriate for that set of data (USDHHS 2004a; USDHHS

2004b; WHO 2003; Zar 1999).

In bone densitometry, values similar to the ‘z-score’ and ‘t-score’ are calculated to allow for standardization of data; these values are referred to as Z-scores and T-scores

(ISCD 2007). The terms are used to reflect values obtained from the Third United States

National Health and Nutrition Examination Survey (NHANES III) reference population and an age, sex and ethnicity-matched reference population (ISCD 2007).

Z-score = (measured BMD)–( mean BMD of age – matched reference group) Standard Deviation

T-score = (measured BMD) –( mean BMD of young healthy reference group)

Standard Deviation

Although variations in interpretations exist in the literature (Bonnick 2009; Carey et al. 2007; ISCD 2007) it is generally thought that Z-scores are ‘age-matched’ value.

Peak bone mass is achieved in the spine and proximal femur by the age of 20-25 in males and females but declines with age (Bates et al. 2002; Bonnick and Shulman 2006;

USDHHS 2004a). As BMD decreases, one would expect an individual’s T-score to decline relative to bone mass, whereas the Z-score may not since it reflects their BMD in relation to their age-matched peers (Carey and Delaney 2010). With the use of T-scores, osteoporosis is diagnosed when an individual has a BMD level that is less than or equal to 2.5 standard deviations below that of a reference population which is translated to a ‘T- score’. A T-score of -1.0 or greater is ‘normal’, a T-score between -1.0 and -2.5 is

‘osteopenic’ and a T-score of -2.5 or below is ‘osteoporotic’(WHO 2003). The results are dependent on user training in addition to proper calibration of equipment to avoid misinterpretation of DEXA scans (Gafni and Baron 2004) but despite the risk of false positives, the threshold criteria of DEXA scans simplifies things for clinicians and researchers and is enhanced when used in comparison with the most appropriate populations.

In San Francisco, the Study of Osteoporotic Fractures (SOF 2011) began in 1986 by the National Institutes of Health as a longitudinal study of 9,704 primarily European

American females with an additional 662 African American women added in 1997.

Research projects from this study have demonstrated correlations between decreases in

BMD and increasing risk of fracture among a population composed of individuals of

European and African ancestries.

Other diagnostic radiographic tools included Quantitative Computed Tomography

(QCT), Peripheral QCT, Micro Computed Tomography (µCT), and Quantitative

Ultrasound. Barger-Lux and Recker (2006) offer a comparison of technological devices along with the benefits and drawbacks of each that can be used to assess sources of bone

fragility. It is important to note that bone densitometry based on DEXA results in a measurement of ‘areal’ BMD in gm/cm2 while QCT provides BMD in gm/cm3.

Biochemical markers for bone loss include bone alkaline phosphatase, osteocalcin in serum and deoxypyridinoline in urine (Barger-Lux and Recker 2006). Alkaline phosphatase will reflect osteoblast activity in bone, but can be at an elevated or decreased level with many diseases (Bonnick and Shulman 2006). Osteocalcin is synthesized by osteoblasts and incorporated into bone cells’ extracelullar matrix and is released into circulating blood where it can be measured (Bonnick and Shulman 2006; Vanderschueren et al. 1990). Osteocalcin reflects osteoblast activity and correlates with bone mineralization but can also be influenced by age, sex and seasonal variation (Bonnick and

Shulman 2006; Vanderschueren et al. 1990). Bone resorption markers in urine are breakdown products of Type I collagen reflect bone remodeling, and therefore not necessarily only bone mineral density.

With bone biopsy, the individual undergoes double in vivo labeling so that the amount of appositional growth can be directly measured from a bone sample. Labeling is accomplished by the administration of Tetracycline, a member of a family of polyketide antibiotics. Tetracycline binds with newly formed bone at the mineralization front and fluoresces under UV light microscopy. A trephine is used to remove a small core of bone and thin sections of the sample are created to histomorphometrically determine the amount of appositional growth between the two times the tetracycline was administered

(Barger-Lux and Recker 2006). The most typical sample site for a bone biopsy is the iliac crest due to its proximity to the surface of the skin. A bone biopsy also allows for direct

observation of bone cells and their activities in situ, making it a crucial research tool

(Barger-Lux and Recker 2006). The limitations of iliac crest biopsies lie in the inability to sample other areas of the ilium as they are often taken from living individuals and the biopsies are often limited to the iliac crest itself (Chalmers and Weaver 1966). Significant intra-skeletal variability in measures of bone mass and osteopenia has been reported

(Frost 1963b; Hansen et al. 1990; Harris et al. 1968; Malluche et al. 1982; Marotti 1976;

Peck and Stout 2007; Wronski et al. 1981). This study is unique in that it incorporates histomorphological assessments from multiple skeletal sites.

Bone histomorphometry makes it possible to analyze bony changes in both cortical and trabecular bone. Although clinical studies tend to use non-invasive radiologic methods such as μCT, histological analysis is still considered the ‘gold standard’ (Kuhn et al. 2007; Rühli et al. 2007). Kruse et al. (1975) demonstrated the benefit of using multiple methods of diagnosis (histomorphometric, biochemical and densitometry) in determining osteoporosis. Osteoporosis has a higher prevalence in women especially with respect to age and age-related endocrine changes such as the decline in estrogen production occurring at menopause. There are many environmental factors that affect an individual’s susceptibility to osteoporosis such as diet, physical activity, medication use and co-existing diseases. Genetic variations can also affect a proper diagnosis of osteoporosis by altering the ultrasound properties of bone, femoral neck geometry, bone turnover markers and body mass index.

3.3 General Factors Relating to Age-Associated Bone Loss

There are multiple factors that can interplay and lead to age-associated bone loss including genetic markers, nutrition, physical activity, sex and hormonal activities. The impact these factors play into age-associated bone loss is described below.

3.3.1 Genetics

Osteoporosis has a polygenic inheritance in that the disorder is determined by the effects of several genes, with each one said to have modest effects on bone mass and other determinants of fracture risk (Stewart and Ralston 2000). In rare occasions it can occur as the result of a mutation in a single gene (Gueguen et al. 1995). Population studies and case studies have identified polymorphisms in candidate genes that are associated with bone mass and/or osteoporotic fracture including the vitamin D receptor, estrogen receptor and collagen type IαI gene (Dundar et al. 2009; Stewart and Ralston

2000). It is noted that the individual contribution of each of these genes to the pathogenesis of osteoporosis is relatively small given that the relationship between candidate genes and osteoporosis has been inconsistent in past studies (Stewart and

Ralston 2000).

The SOST gene, which encodes sclerostin, has recently been suggested to regulate bone mineral density and susceptibility for osteoporosis (Huang et al. 2009; Kim et al. 2008; Liu et al. 2010; Nielsen et al. 2012; Richards et al. 2009; Sims et al. 2008;

Styrkarsdottir et al. 2008; Styrkarsdottir et al. 2010; Yerges et al. 2009). Sclerostin is expressed by osteocytes and is a known regulator of bone formation. Mutations of this gene have been associated with Van Buchem disease and sclerosteosis which are both

associated with an enormous increase in bone mass (Balemans et al. 2001; Balemans et al. 2002; Brunkow et al. 2001; Kim et al. 2008; Staehling‐Hampton et al. 2002). Piters et al. (2010) found an association between one of the SOST polymorphisms and body composition; another polymorphism was found to be in association with hip geometry and hip BMD but is likely to be site-specific among males.

A list of 24 single gene mutations that are known to cause changes with an aspect of bone modeling and remodeling is available (Ferrari 2008). Some of these affect bone matrix composition while others impact bone cell functions in addition to the estrogen pathway and phosphate metabolism.

3.3.2 Nutritional Considerations and Diet

Vitamin D and calcium are the key nutrients favoring bone growth and health maintenance (Vieth 2003). Having an adequate calcium intake is important to maintain mineral homeostasis. From population to population, calcium intake varies, and few of today’s populations achieve a dose of 1000 mg per day of calcium that is recommended by the Food and Nutrition Board (Food and Nutrition Board 2010). In the United States, mean calcium intake levels for males range from 871 to 1,266 mg/day with females averaging 748 to 968 mg/day with older age groups for both sexes, and young females falling short (Food and Nutrition Board 2010). The diets of all mammals, aside from humans, are abundant in calcium. Research suggests that the diet of early Homo sapiens probably contained 2000 to 3000 mg of calcium per day (Heaney and Barger-Lux 1994); however, the normal human serum calcium concentration for modern humans is 10% lower than other organisms (Stini 1995).This may represent an evolutionary mechanism

whereby avoidance of hypercalcemia was created in past environments where dietary calcium was more abundant (Stini 1995).

Daily calcium requirements can be achieved in most people by consuming half a liter of milk and 25 g of cheese (Michaelsen et al. 1994). Other good sources of calcium include dark, leafy vegetables such as broccoli, beans and cabbage. If fish is consumed with the bones, individuals can obtain dietary calcium without much fat, although diets that are higher in protein increase the amount of calcium that is excreted because of the increased acidity of the urine associated with high protein diets. Loss of calcium through the urine stimulates release of calcium through bone resorption which results in a linear relationship between animal protein intake and urinary calcium loss (Abelow et al. 1992).

Animal proteins are high in the amino acids methionine and cysteine which increase acid secretion by the kidney. Having more acidic urine can lead to greater excretion of calcium because the high acid level requires buffering and calcium is an important buffer (Thorpe and Evans 2011). In many Western countries, individuals eat large amounts of animal protein throughout their lives which is thought to be an important risk factor for osteoporosis (Remer and Manz 1994; Remer and Manz 1995;

Riond 2001). Vegetarians in Western countries experience a lower demand for calcium buffering which has been thought to create higher bone densities among vegetarians

(Abelow et al. 1992); however, BMD has been shown to be lower in vegetarians, especially vegans, than in non-vegetarians (Ho-Pham et al. 2009). Despite the discrepancy in BMD among vegetarians and omnivores, the risk of fracture in vegetarians has been shown to be similar to that of non-vegetarians (Appleby et al. 2007;

Ho-Pham et al. 2009). A more complete understanding of the relationship between protein intake and its influence has been developed which describes the influence of protein intake through both negative and beneficial pathways simultaneously (Sebastian

2005). It is suggested that any negative influence of protein-related dietary acid load is opposed by increased calcium absorption with a net influence being either positive, negative or null depending on the additional dietary considerations (Thorpe and Evans

2011). It is suggested that the combination of moderate increases in protein intake with ample dietary calcium, along with alkalizing nutrients such as fruits and vegetables, may uncouple the negative and positive effects (Thorpe and Evans 2011).

Ethnic differences in calcium absorption and excretion may also affect overall calcium balance. Bell et al. (1985) demonstrated that African Americans have low vitamin D levels and a compensatory hypersecretion of parathyroid hormone which maximizes urinary retention of calcium and thereby contributes to greater bone mass.

Other studies did not validate this finding between adequate calcium intake and Vitamin

D levels (Meier et al. 1991). However, calcium alone has not been shown to prevent fractures but calcium combined with 17.5-20 ug/d vitamin D results in lower fracture risk in the elderly (Chapuy et al. 1992; Dawson-Hughes et al. 1997). The reduction in fractures from vitamin D supplementation may also be due to improved neuromuscular function, improved balance and fewer falls (Pfeifer et al. 2000). However, calcium is only one of the dietary components that contributes to skeletal health and is not sufficient on its own to ensure adequate bone mass at all life phases (Heaney 1999). Calcium interacts with other nutrients, biocultural variables and other general health aspects such as physical activity and body size. 41

One of the most important and best understood functions of vitamin D is its regulation of calcium absorption in the small intestine. Enzymes in human liver microsomes and mitochondria convert vitamin D to 25(OH)D. The concentration of this metabolite reflects vitamin D nutritional status. The most active form of vitamin D in this regulatory function is 1,25 (OH)2 vitamin D3 (calcitriol) (Stini 2003). The kidney functions as an endocrine gland synthesizing and secreting the hormone 1,25 (OH)2 vitamin D. Production of 1,25 (OH)2 vitamin D is stimulated by low circulating calcium, low phosphate and high parathyroid hormone (PTH). 1,25 (OH)2 vitamin D stimulates the active transport of calcium through intestinal mucosa. Together with calcium, 1,25 (OH)2 vitamin D3 suppresses the parathyroid gland. Together with PTH, 1,25 (OH)2 vitamin D regulates both bone resorption and bone formation, thereby maintaining normal bone and mineral physiology (Vieth 2003) by both helping build adequate bone mass during childhood and slowing the rate of bone loss that occurs with aging.

Magnesium is also an important element in the mineral phase of bone with approximately one half of all body magnesium being found in bone, where it adsorbs to the hydroxyapatite surface (Carpenter et al. 2006; Cohen 1988). Magnesium is important for mineral homeostasis where it regulates PTH secretion and vitamin D activation.

Vitamin D and its metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D enhance intestinal magnesium absorption to a small extent (Hardwick et al. 1991; Krejs et al. 1983). The recommended daily allowance for magnesium for individuals aged 19-30 is 310 mg/day for women and 400 mg/day for men (Food and Nutrition Board 2010). In individuals aged 31-70 RDA increases to 420 mg/day for men and 320 mg/day for females (Food and Nutrition Board 2010). In a 12 month study of magnesium intake in 42

young girls, a significant increase in hip BMC and lumbar spine were noted (Carpenter et al. 2006). Relating to diet, it has been shown that magnesium absorption is lower when protein intake is less than 30g/day (Hunt and Schofield 1969). Magnesium deficiency may be a risk factor for postmenopausal osteoporosis as reductions in serum magnesium and BMC have been described in women with postmenopausal osteoporosis (Reginster et al. 1989). In women with documented osteoporosis, supplementation of 750 mg/day of magnesium for six months followed by 250 mg/day for a following 18 months resulted in an increase in BMD after 12 months, with no successive increase from 12-14 months

(Stending-Lindberg et al. 1993).

Increased Hcy (homocysteine) levels have also been linked with risk of osteoporotic fracture (McLean et al. 2004; van Meurs et al. 2004). Increased levels of

Hcy can result from defects in intracellular Hcy metabolism and can have either a genetic or nutritional basis (Jacques et al. 2002; Selhub et al. 1993). Vitamins B2 (riboflavin), B6

(pyridoxine), B11 (folate), and B12 (cobalamin) act as substrates for the enzymes involved in Hcy metabolism and play a role in Hcy status. An animal study (Herrmann et al. 2009) demonstrated a 40% reduction in bone strength and a 90% removal of spongy bone matrix after a 3-month period of induced hyperhomocysteinemia in adult rats. Other studies have shown that increased concentrations of Hcy stimulates osteoclast activity in vitro (Herrmann et al. 2005; Liu et al. 1997; Raposo et al. 2004). A low cobalamine concentration was also found to be associated with lower hip BMD in men and lower spine BMD in women (Tucker et al. 2005). In a study of individuals over 55 years of age, a significant positive correlation was found between riboflavin and pyridoxine intake and

femoral neck BMD (Yazdanpanah et al. 2007). Folate has also been shown as an independent predictor of BMD in a British study (Baines et al. 2007).

Vitamin C (ascorbic acid) is necessary for the hydroxylation of lysine and proline which are incorporated into the formation of stable collagen helixes which are necessary for stable bone development (Peterkofsky 1991). Animal and human tissue studies demonstrate the stimulation of alkaline phosphatase by ascorbic acid, which is required for the formation of Type I collagen matrix, as well as expression of osteoblastic markers and mineralization (Chan et al. 1990; Kipp et al. 1996; Sugimoto et al. 1986). Tsuchiya and Bates (1994) also found an association between scurvy, a severe deficiency in ascorbic acid, and a decrease in BMD and BMC in guinea pigs; a condition which persisted into adulthood. Ascorbic acid deficiency was also associated with thinner trabeculae, an increase in bone resorption, a decrease in osteoblast differentiation and decreased collagen synthesis. Diets where ascorbic acid is low or nonexistent have also been associated with lower amounts of calcium in the femur and low collagen formation

(Tsunenari et al. 1991). The link between ascorbic acid intake and bone density is one that is complex and may be related to confounding factors such as smoking, estrogen supplements, calcium intake and vitamin E (Ahmadieh and Arabi 2011).

Vitamin E is found in two forms, tocopherol and tocotrienal, both of which are fat-soluble antioxidants. Oxidative stress, occurring when there is an increase in the formation of free radicals, has been shown to increase bone resorption (Garrett et al.

1990). In humans, it has also been demonstrated that a high amount of oxidative stress can lead to reduced BMD (Basu et al. 2001). The relationship between smoking and

vitamin E supplements has also been examined, as smoking itself is a risk factor for age- associated bone loss due to the oxidative stress caused by smoking. A case study of hip fractures among males and females found an inverse relationship between intake of vitamin E and risk of hip fracture; however, this relationship was only found among smokers and previous smokers but not among individuals who never smoked (Zhang et al. 2006). It appears that vitamin E is associated with increased bone mass and a decrease in fracture risk; however, the benefits of vitamin E supplements appears to be modulated by smoking status because of the antioxidant qualities of vitamin E (Ahmadieh and Arabi

2011).

It is also found that vitamin K may play a role in protecting against age-associated bone loss (Shearer 1997). Vitamin K may influence bone metabolism through its effect on urinary calcium (Jie et al. 1993) and may inhibit the production of bone reducing agents such as prostaglandin E2 and interleukin 6 (Reddi et al. 1995). In humans, vitamin

K studies have had mixed results. In healthy young individuals, vitamin K was not shown to be associated with BMD or biochemical markers of bone formation (Rosen et al.

1993). However, in postmenopausal women, it has been demonstrated that vitamin K supplements increase osteocalcin and decrease urinary calcium and hydroxyproline

(Knapen et al. 1989). Studies have also shown low circulating serum vitamin K levels in association with previous hip fracture (Hart et al. 1985; Hodges et al. 1993; Hodges et al.

1991; Kanai et al. 1997). It is also suggested that vitamin K works along with vitamin D as shown in animal models where ovariectomy-induced rats had reduced bone loss when receiving both vitamin D and K, but not when receiving one or the other (Matsunaga et al. 1999; Shiraishi et al. 2002). Studies have shown that low circulating vitamin K levels 45

was associated with decreased BMD, but the protective effects of vitamin K have not been conclusive (Ahmadieh and Arabi 2011).

In addition to adequate amounts of vitamins and nutrients, both overall undernutrition and overnutrition can play a part into bone loss as well. Weight reduction programs that rely upon restriction of energy intake have been shown to increase bone turnover and reduce bone density in obese women (Ricci et al. 2001). Malnutrition associated with alcoholism has also been found to increase the incidence of osteoporosis for men (Santolaria et al. 2000).

3.3.3 The Role of Androgens and Estrogens in Age-Associated Bone Loss

Androgens are a generic term for steroid hormones that control the development and maintenance of male characteristics. Androgens are also the precursor to all estrogens, the female sex hormones. The most well-known androgen is testosterone and other less important androgens are dihydrotestosterone and androtenedione.

Estrogens are a group of compounds that serve as the primary female sex hormones. The three major estrogens in females are estrone, estradiol and estriol. During reproductive years, estradiol is the dominant estrogen whereas during pregnancy, estriol becomes the predominant circulating estrogen. When a female’s body enters the postmenopausal period, estrone become the dominant circulating estrogen. All types of estrogens are synthesized from androgens, or sex steroids. Estrogen receptors are a group of proteins found within the cells; they are activated by estrogen and belong to one of two classes: estrogen receptor (ER) and estrogen G protein-coupled receptor GPR30 (GPER).

ER, once activated, binds to DNA and has the capability of regulating many different

gene activities. There are two variants of ER, α and β, which are both ligand-activated transcription factors and each variant is coded for by a different gene. Both androgens and estrogens play an important role in bone health and risk of osteoporotic fracturing

(Riggs et al. 2002).

Although males have a smaller likelihood of developing osteoporosis, they still have a 15% risk of experiencing an osteoporotic related fracture in their lifetime (Riggs et al. 2002). In males, skeletal androgen action can be mediated directly through activation of the androgen receptor (AR), as well as through ERα following aromatization into estrogens (Riggs et al. 2002). This androgenic dual mode in nonreproductive tissues

(such as bone) has been shown by studies in rodents and humans (Vanderschueren et al.

2004). Studies have shown that both androgens and estrogens play a role in maintaining male trabecular bone mass and structural integrity (Lindberg et al. 2002a; Movérare et al.

2006; Sims et al. 2003). Other studies show that the AR and ERα pathways play a role in cortical bone in males as well (Marcus et al. 2000; Smith et al. 1994; Smith et al. 2008;

Vanderschueren et al. 1997; Venken et al. 2006). AR activation also appears to be the primary mediator of periosteal expansion in males (Vanderschueren et al. 2004).

Callewaert et al. (2009) demonstrate that AR activation is solely responsible for developing and maintaining trabecular bone mass in males but that both AR and ERα activation are necessary for the acquisition of cortical bone in males.

Fuller Albright (1947) first noted a relationship between menopause and osteoporosis with this idea further noted by McLean and Urist (1968) who demonstrated a relationship between bone loss and decreased estrogen secretion is post-menopausal females. This relationship has been noted in numerous studies since then and Seeman

(1999) suggests that estrogen deficiency is the common pathogenesis for both male and females due to the similar pattern of bone loss seen in both sexes.

Riggs and Melton (1986; 1995; 1983) and Riggs et al. (1998; 2002) present the concept that females experience two separate phases to bone loss: the first phase being the phase in the decade following menopause with a second, continuous and gradual phase that is common seen in both males and females. Riggs et al. (2008) suggest the late onset of cortical bone loss has an association with sex-steroid deficiency, while earlier onset trabecular bone loss in sex steroid sufficiency is unexplained; this indicates that current paradigms on the pathogenesis of osteoporosis are incomplete.

Järvinen et al.(2003) present an evolutionary perspective to bone loss among females as a combination of functional factors of the skeleton with reproductive factors of estrogen. With their paradigm, the bones of females accrue a higher mineral density during puberty in preparation for pregnancy, which is driven by estrogen. This density difference persists throughout the fertile period until menopause, where the body

‘unpacks’ the unnecessary bone, driven by estrogen loss.

Riggs et al. (1998) present a comprehensive overview of research finding that estrogen inhibits osteoclastic activity, where the inhibition is lifted at the commencement of the post-menopausal period. Their position posited that this led to an increase in osteoclastic activity at that period, resulting in postmenopausal bone loss. The bone loss that occurs in females after the acute period of estrogen deficiency at the postmenopausal period take about five years to slow down and reach a plateau (Riggs and

Melton 1995). More recent studies have demonstrated the role that estrogen plays in both osteoclastic and osteoblastic activation and activity.

Estrogen plays a large role in maintaining a ratio between osteoblasts and osteoclasts through the induction of osteoclast apoptosis (Krum et al. 2008). It has been demonstrated that lowered estrogen levels lead to lower BMD (Eastell 2003; Wronski et al. 1989), induces apoptosis in osteoclasts (Kameda et al. 1997; Kousteni et al. 2002), and is anti-apoptotic with osteoblasts (Kousteni et al. 2002) which leads to an overall building of bone. Krum et al. (2008) found that estrogen induced apoptosis occurs in the pre-osteoclast stage of osteoclast development and requires the presence of osteoblasts in which the estrogen induction of Fas ligand occurs, rather than osteoclasts. Their research demonstrates that the protective role that estrogen plays result from a paracrine signal that emanates from osteoblasts.

Martin-Millan et al. (2010) further discovered that the protective effects of estrogen were limited to trabecular bone. In their study, they deleted the ERα in mice and found a twofold increase in osteoclast progenitors in the marrow and the number of osteoclasts in trabecular bone as well as a decrease in trabecular bone mass; however cortical bone loss in the experimental group was indeterminate from the control group

(Martin-Millan et al. 2010). Additional mouse models have demonstrated that signaling via ERα protects against ovariectomized (OVX)-induced trabecular bone loss (Börjesson et al. 2011; Lindberg et al. 2002b; Turner 1999b). A more recent study (Ohlsson et al.

2012) noted a difference between peripheral and central ERα activation. The study

(Ohlsson et al. 2012) found that peripheral ERα activation increases bone mass, while central ERα activation decreases bone mass, indicating that the balance between peripheral stimulatory and central inhibitory ERα actions are also important in the regulation of bone mass.

In addition to the sex hormones, pituitary hormones have also been shown to regulate bone mass. Thyroid stimulating hormone (TSH) has been shown to act directly on osteoclasts and osteoblasts (Abe et al. 2003). Follicle-stimulating hormone (FSH) has also been shown to stimulate bone resorption (Sun et al. 2006) and has been theorized to play a role in bone loss during the late peri-menopausal period in conjunction with decreasing estrogen levels (Zaidi 2007). Iqbal et al. (2006) propose that FSH related bone loss occurs independently of the action of low estrogen. The Study of Women’s Health

Across Nations (SWAN), which is a longitudinal study of 2,375 peri-menopausal women, has shown a correlation between high serum FSH levels and bone loss (Sowers et al.

2003). These findings have also been demonstrated in data from the NHANES III study which demonstrates a correlation between serum FSH and femoral neck BMD (Gallagher et al. 2010). Another study found that females with low FSH levels experienced less bone loss (Devleta et al. 2004). Studies have shown that FSH increases osteoclast formation, function and survival (Robinson et al. 2010; Sun et al. 2006; Sun et al. 2010; Wu et al.

2007), while others have documented that it indirectly stimulates osteoclast formation via the release of osteoclastic cytokines (Cannon et al. 2010; Iqbal et al. 2006).

3.3.4 Mechanical Loading and Physical Activity

The observations of Culmann (1866), Roux (1881), von Meyer (1867), and Wolff

(1892) set the stage for the understanding of bone adaptation to the magnitudes and directions of in vivo loading. The relationship of bone to loading is such that if loading on a particular bone increases, the bone responds by modeling itself over time to increase in strength and resist that loading. Under this model, increase in use leads to an increased

bone mass, whereas low gravity or disuse leads to bone loss. Frost (1987; 1996; 2001;

2003b) later proposed the concept of the ‘mechanostat’ whereby bone mass and geometry are regulated by mechanical inputs. When an area of the skeleton is subjected to mechanical strain the sensing of the strain is communicated to effector cells, osteoclasts and osteoblasts, by the secretion of cytokines. The sensing of mechanical loading plays a role in skeletal adaptation during growth and development, maintains bone mass throughout life, and prevents bone loss with increasing age and menopause. Thus, the sensing and transduction of strain signals is essential to maintaining bone health.

The osteocytic lacuna-canalicur network is the primary mechanosensor of strain

(Bonewald 2006). Osteocytes are placed in an ideal location for sensing strain and for directing modeling events; they are connected to each other via gap junctions and are connected to cells of the bone surface and bone marrow (Bergmann et al. 2010).

Osteocytes are sensitive to biomechanical stress through fluid flow in the lacuna- canalicular system (Knothe Tate et al. 1998; Weinbaum et al. 1994). When shear stress is sensed, signals are expressed that repress osteoclasts and initiate osteoblasts (Tan et al.

2007; Vezeridis et al. 2006; You et al. 2008); when loading is absent, osteocytes die through apoptosis (Aguirre et al. 2006). When loading and/or bone damage becomes excessive, an increase in osteocyte apoptosis results in the initiation of the removal of damaged bone (Noble et al. 2003).

Studies have established that mechanical loading influences both modeling and remodeling activities and ultimately bone mass in both cortical and trabecular bone

(Bonnet and Ferrari 2010). Depending upon the skeletal location, loading and the strains

it produces will affect the deposition of bone, increasing it on the periosteal surface and slow the resorption of bone on the endosteal surface (Pearson and Lieberman 2004).

Loading may also signal the direction of remodeling activities in addition to identifying locations in the skeleton. Using computer modeling, Van Oers et al. (2008a) demonstrate that strain-induced osteocyte signals can direct osteoclasts into the dominant loading direction in which the cutting cone can maintain that course over several weeks leading to osteons being oriented in the direction of the dominant loading activity. This explains why osteons tend to be oriented parallel to the direction of loading (Petrtýl et al.

1996). The computer simulation also indicates that the action of unloading leads to abnormal directions of resorption (van Oers et al. 2008a). A further model (van Oers et al. 2008a; van Oers et al. 2008b) indicates similar responses in trabecular bone as well where the BMU travels along the surfaces of trabeculae and does not perforate the bone.

Given that the microstructure of bone is a product of its metabolic activity, a number of studies have explored the relationships between histomorphological features and physical activity. For cortical bone, osteon morphology has been linked with the load history as well as the variability in osteon size, shape and density (number per unit area) which have been proposed as variables that can be used to shed light on questions of intensity of physical activity in the past (Pfeifer and Pinto 2012). A computer simulation by Van Oers (2008b) demonstrates how constraints on the cutting cone are increased with the magnitude of loading leading to a reduction in the overall size of the cutting cone.

This results in smaller osteon area when the remodeling activity of a BMU has completed and smaller osteon size in increased loading environments (van Oers et al. 2008b).

In a comparison of a basal, control, and exercised group of sows (Raab et al.

1991) noted histomorphometric differences with the exercised group only walking on a treadmill for 20 minutes for five days a week for 20 weeks. The exercised group exhibited periosteal modeling with a higher periosteal formation surface and mineral apposition rate (MAR) than the other two groups (Raab et al. 1991). The endosteal surface demonstrated no difference amongst the three groups, but osteonal MAR was also higher in the exercised group. A further study by the same group of researchers

(Tommerup et al. 1993) examined the impact of exercise on non-weight bearing bones on sows that were exercised for 17 weeks. The results that were demonstrated in the femur in the previous study were not apparent in the rib, which is a non-weight bearing bone.

The exercised group of sows showed no benefit from the exercise in osteonal and endocortical MAR or osteonal remodeling frequency.

In addition to the computer simulation mentioned previously, other research indicates variability in osteon size. In general, osteon size varies between individuals and among the bones of the individual, e.g. osteons from the ribs tend to be smaller than ones from midshaft femora. Britz et al. (2009) also demonstrates that femoral osteon size can be attributed to age, weight and sex. In a study by Burr et al. (1996), high strain loads increase remodeling rates only when strains produced by physical activity exceed 2000 microstrain. In vivo measurements of strain levels during various activities in human tibiae found that the highest microstrain was experienced for shear strain for uphill and downhill zig-zag running, which reached nearly 2000 microstrain in all cases (Burr et al.

1996). In comparison, shear strains while walking were ~871 microstrain and while sprinting on a level surface were ~1583 microstrain. 53

In an examination of strain mode, magnitude and strain energy density, Skedros

(1996) reported a correlation between osteon population density (OPD) in areas where compressive strain was dominant in third metacarpals of equines. This study also noted increased porosity in areas where compression was the dominant stran.

In addition to osteon size and density, drifting osteons, osteons that are morphologic variants of secondary osteons have been associated with increased tension

(Keenan et al. 2010). A study examining five regions of adult chimpanzee skeletons found more drifting osteons occurring at regions of tension in the femoral neck while showing no regional variation at the femoral mid-shaft (Keenan et al. 2010). The study also indicated no preference toward drifting toward the endosteum or neutral axis, and suggest that drifting osteons may assist in strengthening cortical bone in areas of high tension (Keenan et al. 2010).

Loading bones has been shown to trigger bone formation and induce bone apposition; however, research on the effects of exercise is less evidence. Forwood (2001) points out that these findings for exercise may be due to the fact that exercise and physical activity are not synonymous with mechanical loading. Exercise can be used to refer to anything from walking to competitive athleticism. Physical activity is generally thought to benefit the skeleton at every decade of life, having a beneficial effect on bone mass. Weight-bearing activity is essential in maintaining bone density, but the optimal amount of exercise differs between the sexes and among individuals and can be dependent upon mitigating factors.

Long-term exercise has been shown to have beneficial effects on the young as well as older adults (Hoshino et al. 1996; Snow‐Harter et al. 1992). In younger individuals, an increase in bone mass is associated with an increase in muscle mass

(Heinonen et al. 2001). An eight-year longitudinal study in children (Gunter et al. 2008) provided evidence that short-term high impact exercise in children increased BMC of the hip. Another longitudinal study found that among boys and girls aged 5-11 that the daily duration of physical activity was an independent factor of femoral neck cross-sectional area (Janz et al. 2007). A longitudinal study of boys and girls aged 8 to 15 demonstrated a positive relationship between estimated amount of physical activity during youth to total body, hip and lumbar spine BMC when measured at the end of the pubertal growth spurt (Baxter-Jones et al. 2008). The most promising results come from studies of high impact exercise, even for short periods of time. Fuchs (2001) reported a 3.5% increase in bone mass when children were submitted to jumping (high impact) exercise for several months. In a longitudinal follow up study of the children, it was reported that there was a large loss of gain three months after the study concluded; however, the results persisted up to 7 years after the study when the exercised group was compared to the non-exercised group (Gunter et al. 2008). Nutritional factors also affect results found with physical activity in children. Chevalley (2008) report that young boys with adequate protein intake coupled with physical activity had the highest BMD.

In middle adults, a controlled study found that women who subjected to 60 minutes of high impact exercise (jumping, running, walking) three times a week for one year resulted in higher BMD of the femur when the acceleration generated by impact was greater than 3.9g (Vainionpää et al. 2006). An additional report of the same groups of 55

women indicated a higher bone circumference at the mid-femur as well (Vainionpää et al.

2007).

In theory, a higher achieved peak bone mass should prevent against bone loss later in life; however, it has been shown that the benefits only persist throughout adult life if the physical activity is continued. Animal models have shown that bone mass levels persist, and may even increase, if the activity is at least moderate (Iwamoto et al. 2000;

Silbermann et al. 1991; Wu et al. 2004). A study of mother-daughter pairs with lifelong exercise estimated via questionnaires found that weight-bearing exercise was correlated with total and peripheral BMD in the daughters but not with the mothers (Ulrich et al.

1996). The study found that with increasing age, other factors such as estrogen use, body weight and calcium nutrition played a larger role in determining bone density (Ulrich et al. 1996). LaFage-Proust et al. (1996) found that a greater expansion of the outer periosteum occurred in adults with physical activity. A ten-year longitudinal study in males and females found that lumbar BMD was related to ground reaction forces from physical activity in males, but not in females (Bakker et al. 2003).

A comparative study of pre- and post-menopausal women subjected to a standardized jumping exercise demonstrated that the effect of exercise was diminished after menopause; however, the mean age of the post-menopausal women was 55 years of age (Bassey et al. 1998). Another study in post-menopausal women showed a small, yet significant, positive effect of exercise in the spine (Maddalozzo et al. 2007).

In elderly individuals, exercise can reduce the risk of fracturing, but vigorous exercise can also cause injury (Kallinen and Markku 1995). One research study on rats

demonstrates that the adaptation of bone in response to mechanical loading decreases with age (Turner et al. 1995). This has also been indicated by Kohrt (2001) who states this is related to decreased muscle mass and strength and/or a decline in hormones and growth factors that may interact with mechanical signals. A research study of females aged 70-79 engaged in controlled trial testing resistance and balance jumping training showed no benefit to BMD or bone geometry in the femoral neck or tibia (Karinkanta et al. 2007). The same study did show that there was an increase in performance among the participants which could, however, lead to a decrease in fracture risk (Karinkanta et al.

2007).

In women, excessive exercise can lead to amenorrhea which can also reduce the amount of circulating estrogens result in a decrease in bone density. In general, however, in addition to the wide range of health benefits, physical activity and muscle strength in older adults also reduce risk of fractures due to trauma from falls. Lack of physical exercise can increase bone loss and complete inactivity can lead to disuse osteoporosis

(Minaire et al. 1974).

Disuse leading to bone loss has been demonstrated in extreme cases such as exposure to hypogravity during spaceflight which has been shown to induce bone loss in weight bearing bones (Collet et al. 1997; Turner 2000; Vico et al. 2000) with similar findings demonstrated with individuals after extended periods of bed rest (Schlecht et al.

2012; Thomsen et al. 2005b; Zerwekh et al. 1998).

In summation, biomechanical loading has been demonstrated to have a direct impact on bone mass and bone strength. The relationship between physical activity itself

and bone mass, however, seems to be a bit uncertain and dependent upon mitigating factors such as age, sex, overall health and nutritional considerations.

3.4 General Skeletal Trends Associated with Age-Associated Bone Loss

As described earlier, osteocytes are responsible for sensing strain and activating the effector cells: osteoclasts and osteoblasts. With age, it has been shown that the density number of osteocytes declines with age from 20-70 years (Mullender et al. 1996). There is also a corresponding decline in lacunar density and lacunar area and an increase in empty lacunae (Mullender et al. 1996; Qiu et al. 2002). When the number of osteocytes is reduced, the inhibitory signal on remodeling is also reduced leading to higher remodeling rates resulting in a net bone loss.

Animal modeling has demonstrated the connection between osteocyte density and osteon dimensions supporting the existence of an inhibitory signal and its impact on osteoblast function (Metz et al. 2003). It is suggested that a threshold signal is perceived sooner in osteons of high osteocyte density, thus shortening the formation period under the assumption that osteoblasts cease matrix production after differentiating into osteocytes or bone lining cells when a threshold inhibitory signal is received (Metz et al.

2003). In normal individuals, it has been shown that there is a natural decrease in the amount of surface covered by osteoblasts with age (Merz and Schenk 1970) indicating that the bone loss is not due solely to an increase in bone resorption, but also a decrease in bone formation. After the age of 40, the amount of bone surface occupied by osteoid seams increases, possibly explained by a delay in mineralization due to the decrease in active osteoblasts.

Osteoclastic resorption usually takes place at sites where the local strains of compression and torsion are greatest. The process of formation takes longer than the process of resorption. During formation, there is a qualitative change in bone in which more highly mineralized primary lamellar bone and primary osteons are replaced by newer less highly mineralized bone tissue. Some evidence also indicates that there is a decrease in mineralization with increasing distance from the Haversian canal (Crofts et al. 1994). During remodeling there is a transient increase in porosity as the resulting resorptive bays fill-in. The resulting Haversian canals formed during cortical bone remodeling lead to increased porosity as a function of the number of Haverisan canals and their size (Atkinson 1964).

Tanizawa et al. (1999) examined bone turnover in three bone envelopes

(trabecular, endosteal and Haversian) of iliac bone. They found the endocortical rates of bone formation as corrected by bone surface (BFR/BS) and the mineralizing surface as corrected for the bone surface (MS/BS) were higher than in the trabecular envelope and that the indicators of bone formation in these envelopes were closely related to the rates in the Haversian envelope. Given that an increased rate of bone turnover contributes to bone loss, the relatively higher rate of remodeling associated with the endorcortical envelope underscores the importance of increased endosteal bone resorption in osteoporosis.

During the fourth decade of life an increase in bone loss takes place, due to endosteal resorption while a small amount of bone apposition continues along the periosteal border (Drusini et al. 2000) resulting in a reduction in cortical thickness (Garn et al. 1969; Ruff and Hayes 1982). With osteoporosis, it is known that medullary 59

expansion causes a decrease in the size of the cortex due to bone loss on the endosteal surface which also leads to a ‘trabecularization’ of the cortex adjacent to the endosteum

(Keshawarz and Recker 1984).

The amount of peak bone mass achieved in young adulthood contributes to the risk of developing osteoporosis since the effect of age-related bone loss will be different in individuals who start with high bone mass compared with those with a lower peak bone mass (Nelson and Villa 2003).

3.4.1 General Age-Associated Trends with Bone Loss in Cortical Bone

Age-associated bone loss affects all of the bone envelopes, but the changes on each differ slightly. Both cortical and trabecular microstructural changes contribute to bone health and determine its fragility (Ostertag et al. 2009). Cortical bone has become an ‘unfashionable subject of study’ (Parfitt 1994), in clinical practice. However, histologic analysis of cortical bone of rib sections (Epker and Frost 1965; Epker et al.

1965; Epker and Frost 1964; Sedlin 1964; Sedlin et al. 1963a; Sedlin et al. 1963b; Sedlin et al. 1963c) laid the foundation for research in age-associated bone loss. The use of cortical bone in histology has certain advantages over trabecular bone, especially for bioarchaeology. With cortical bone, the researcher can orient the section of bone in relationship to the structure of interest. In archaeological contexts, cortical bone also preserves better than trabecular bone and histological samples are easier to prepare.

Cortical bone is denser than trabecular bone, making up approximately 80% of the human skeleton (Polig and Jee 1987) and functions as a major weight-bearing structure. The pattern and distribution of cortical bone throughout the skeleton is a

reflection upon the nature and magnitude of the stresses encountered in each skeletal region. As discussed in Chapter 2, the structural unit of cortical bone is the osteon; at the center of which lies a blood vessel, surrounded by layers of lamellae. These layers contain osteocytes which are connected to each other and the Haversian canal through their canaliculi. This entire system is the Haversian system, a connected network responsible for the exchange of nutrients and metabolic byproducts between the densest sectors of bone and the vascular system.

Through the process of remodeling with age, the original primary lamellar bone is progressively resorbed and replaced by secondary osteons, whereby the system takes on a mosaic-like appearance through time. Remodeling occurs on different rates in different portions of the skeleton.

3.4.2 General Age-Associated Trends with Bone Loss in Trabecular Bone

In clinical practice today, trabecular bone is more commonly examined to evaluate bone health because it is more accessible and, due to an improvement in technology, the components are easier to measure than they were in the past. Along with its accessibility, however, are complexities in its geometric structure. As in cortical bone, remodeling follows the same ARF sequence but the trabecular BMU travels across the plates and struts of trabecular surfaces, thereby digging a trench rather than a tunnel through the bone matrix with a resultant ‘hemi-osteon’ that can vary considerably in orientation to the plane of section.

The structure of trabecular bone consists of a latticework of vertical struts and horizontal plates with the enclosed area being comprised of anywhere between 30-90% 61

open space (Carter and Hayes 1976). The trabeculae develop along lines determined by stresses experienced during growth, and the strength of their latticework structure is dependent upon the density of the components and their connectivity. Trabecular bone is usually the first bony tissue to experience loss of mass because it has greater surface area and, therefore, accessibility for resorption. In early life, trabecular bone is more metabolically active and is the most vulnerable to disruptions in the balance of bone turnover and the first to demonstrate symptoms of osteopenia (Dodds et al. 1989).

With a loss of bone compromising horizontal plates, the vertical struts can buckle and a loss of connectivity ensues (Heaney 1989). With the decline in the weight-bearing capacity of trabeculae, their contribution to the structural integrity of bone decreases, thereby placing increasing stress on cortical bone, to a point where fracture occurs

(Compston et al. 1989). Thomsen et al. (1998) demonstrated that trabecular bone volume gave the strongest correlation with bone strength, and propose that the predictive value of bone volume could not be determined by factors such as connectivity density, trabecular number or trabecular separation.

Parfitt et al. (1983) reported that trabecular bone volume decreased with age as the result of a reduction in plate density, rather than plate thickness and indicates that age-associated bone loss operates as a function of the removal of entire elements of bone.

Where a loss of connectivity is experienced, remaining trabeculae can undergo a thickening process as a compensatory process but the majority of the trabeculae lose thickness with age. Chappard et al. (1988a; 1988b) support these findings by demonstrating the relationship between trabecular mass and spatial configuration. The

authors found a negative correlation between age and trabecular volume and number, in addition to a linear correlation of trabecular volume and thickness as well as trabecular volume and trabecular density; this demonstrates the relationship between spatial configuration of the trabeculae. More specifically, Ding and Hvid (2000) found that the structure of trabeculae in the tibia moved from a plate-like structure to that of more rod- like in the aging skeleton with trabecular thickness declining significantly after 80 years of age. Compston et al. (1989) found that trabecular thinning and erosion were inter- dependent processes and suggest that the activation of new BMUs may not be randomly distributed but may be preferentially located at sites of lower trabecular width.

3.5. The Issue of Intra-skeletal Variability

Age changes with bone remodeling that relate to age-associated bone loss are not the same throughout the skeleton. This means that inter- and intra-skeletal variability must be taken into consideration when selecting a skeletal sampling site, or comparing and evaluating data derived from different sampling sites. As noted above, skeletal elements have differing functional demands based on their placement in the skeleton.

Since different bones are subject to differing strains and stresses, this leads to intra- skeletal variability for bone remodeling variables. Peck and Stout (2007) described the heterogeneity in bone mass from the cortical bone of the six limb bones in males and females of a modern population. Dempster et al. (1993) found that trabecular bone volume in iliac crest samples was larger than in lumbar vertebrae due to the existence of thicker, closely spaced trabeculae in the iliac crest. Thomsen et al. (2002) report similar findings for the iliac crest and vertebral body, noting a weak inter-correlation despite the

demonstration of similar patterns associated with age. Wright et al. (1990) found that the relationship between spinal trabecular bone mineral density and iliac crest mineral bone density was dependent upon other disease processes. In a comparison of iliac crest trabecular bone to the endosteal surface of rib bone, the iliac crest mean wall thickness decreased significantly with age while the mean wall thickness of endosteal BSUs in the rib only decreased slightly with age (Lips et al. 1978). These findings are corroborated by other studies(Verna et al. 1999) and demonstrate that histormorphometric results at one site do not necessarily predict measures at another skeletal site. However, animal model studies have demonstrated that the data obtained from the iliac crest could be used to predict activation frequency and risk of vertebral crush fractures (Mashiba et al. 2005).

3.6 Sex Differences in Age-Associated Bone Loss

Biological differences create varying patterns of bone loss in males and females, although both sexes experience bone loss on all four bone envelopes. Both sexes experience age-associated bone loss but females experience bone loss at much higher levels. If individuals of either sex experience physical inactivity, have a low peak bone mass, low calcium intake and/or other dietary deficiencies, they can succumb to a higher risk of fracture. In both sexes, age-associated bone loss is accompanied by a decrease in mineralization apposition rate, and an increase in activation frequency (Chavassieux and

Meunier 2001). In males, bone loss leading to senile osteoporosis is more gradual and occurs at an older age (~70) whereas in post-menopausal females it is more rapid and precocious leading to an earlier onset of osteoporosis (Ostertag et al. 2009).

Riggs et al. (2008) using quantitative computed tomography (QCT), made a longitudinal, population-based assessment of rates of bone loss over life at the distal radius, distal tibia and lumbar spine. The study was a 3 year study in which QCT values were taken annually. The results indicate that cortical bone loss began during peri- menopause in women and primarily after age 75 in men. In contrast, trabecular bone loss began in young adulthood in both sexes. Substantial trabecular loss began in young adult women and men at all three skeletal sites and continued throughout life with acceleration during peri-menopause in women. Women experienced 37% and men experienced 42% of their total lifetime trabecular bone loss before age 50 compared with 6% and 15% respectively for cortical bone. Post-menopausal women and older men with higher rates of cortical and trabecular bone loss were found to be associated with lower levels of biologically-active sex steroids and higher levels of follicle-stimulating hormone and bone turnover markers.

Men achieve a higher peak bone mass than females in early adulthood, placing females at a greater risk for osteoporosis than males during the period at which bone loss occurs (Stini 1995). The amount of peak bone mass achieved in young adulthood contributes to the likelihood of developing osteoporosis, since the effect of age-related bone loss will be different in individuals who start with high bone mass compared with those with a lower peak bone mass (Nelson and Villa 2003). It has also been found that women who have type I osteoporosis have a period of accelerated bone loss starting with the peri-menopausal period but that rapid period does not continue indefinitely. Stini

(1995) found that approximately 5-10 years after menopause, the rate of bone loss slows to a rate that is comparable to that as men.

A study on the Mediterranean coast of Croatia found that in the trabecular bone of the vertebrae and iliac crest, men and women exhibited differences in peak bone mass and bone loss with age, with a trend in parameters that differed at each skeletal site

(Ostojić et al. 2006). In lumbar vertebrae trabecular bone, bone volume decreased in both males and females with females experiencing a greater loss. Trabecular structural changes in lumbar vertebrae included thinner trabeculae in females, but fewer trabecular and greater separation in men. Ostojić et al. (2006) also report a greater volume of trabecular bone in the iliac crest for both males and females in comparison to trabecular volume in lumbar vertebrae. In the iliac crest, Ostojić et al. (2006) report greater reduction in trabecular thickness, trabecular number, and increase in trabecular spacing in females than males. Overall, their study reported an overall reduction in bone mass of

37% in lumbar vertebrae and 28% with increasing age in individuals aged 41 through 85

(Ostojić et al. 2006). The results of their study further attest to the importance of examining multiple skeletal sites, even when examining the same type of bone tissue, as each skeletal site is subject to differing functional demands.

Other studies indicate that these findings between the sexes do not extend to BMC or BMD (Seeman 1999). During growth, both sexes attain a similar peak vertebral height with vertebral width being larger in men, but trabecular thickness being similar among males and females. Seeman (1999) attributes the differences seen in strength of vertebrae as size dependent, rather than BMD related as seen in the greater periosteal expansion exhibited by males during the growth period.

Increased cortical remodeling results in an increased surface available for bone resorption as cortical bone ‘trabecularizes’ in both sexes. Bone loss in old age accelerates

but it is mainly cortical in both sexes, with a greater relative contribution of trabecular bone loss in men (because women have lost trabeculae) (Stini 1995). Endosteal resorption is also greater in females than males; however, men lose cortical width due to endosteal resorption and due to a greater amount of periosteal expansion during aging

(Seeman 1999; Stini 2003).

In the long bones, it has been noted that bone architectural structure maintains with age but is accompanied with a gradual osteopenia that affects the femur in both sexes with a more marked appearance in females (Drusini et al. 2000). Until the fifth decade, the cortical bone ratio was found to be higher in females than in males (Drusini et al. 2000). In the vertebral column, Ostertag et al. (2009) found that with women, cortical width was associated with vertebral fractures, whereas in men, cortical porosity was more closely associated with fracturing. Males also have wider long bones than women.

Seeman (1999) concludes that the greater distance of the cortical mineral mass from the neutral axis of a long bone is responsible for the higher bone strength among males.

Seeman (1999) also indicates that males who experience hip fractures tend to have smaller femoral necks, indicating that the overall size differential exists in various locations in the skeleton.

In a study by Burr et al. (1990) an increase was noted in mean Haversian canal size in females over 40 years which resulted in an increase in porosity that was not found in males. The study also found that osteon size decreased with age among males, resulting in a decreased mean wall thickness with age with no change in osteon size found among females. According to these data, osteons are large enough in their sample

population of the Pecos Pueblo to allow significant fatigue inhibiting properties to the bone.

Patterns of trabecular bone loss vary between males and females as well. Mellish et al. (1989) found an age-related decrease in mean trabecular plate thickness with age in both sexes with a significant decrease in trabecular plate density and increase in mean trabecular plate separation with similar but non-significant results in males. More recently, Macho et al. (2005) also showed a statistically significant directional trend in trabecular size in females, which was not observed in males.

Other research indicates that trabecular thickness may decrease in age in men, but suggests that it increases or remains stable in females (Aaron et al. 1987; Macho et al.

2005; Vijayapalan et al. 2003). Macho et al. (2005) found that females tended to have overall thinner trabeculae than males but the increase in thickness with age is more pronounced. These findings suggest the existence of a compensatory mechanism in females between increase in trabecular thickness and trabecular loss with age. From this it follows that the higher incidence of osteoporosis in females may not be from the result of overall bone mass but rather that females experience a high rate in loss of connectivity than men. Ostertag et al. (2009) found that females demonstrated an increase in trabecular separation with age while men demonstrated a decrease in trabecular thickness.

These findings have been reported in other studies as well (Aaron et al. 1987; Parfitt

1993). Males also demonstrate an increase in trabecular separation, also less marked than in females (Chavassieux and Meunier 2001).

The loss in trabeculae can be due to thinning of the structures from age-associated bone loss but it can also be due to fracturing from high localized stresses; each of these mechanisms will result in an interruption of the trabecular structure, additionally removing surfaces whereby osteoblasts could function to replace bone (Keaveny and Yeh

2002).

It has been shown that mean wall thickness for hemiosteons in trabecular bone decreases with increasing age (Birkenhager et al. 1996; Kragstrup et al. 1983; Lips et al.

1978). Mean wall thickness corresponds to the quantity of bone that is replaced or formed at any bone forming center; these findings correspond to a decrease in net bone formation at the level of the BMU and can be used to explain senile osteoporosis.

Males with age-associated bone loss are also characterized by having a decrease in trabecular bone volume and cortical width, but which is less marked than in females.

(Chavassieux and Meunier 2001).

In trabecular bone, the difference lies in a greater loss of connectivity in females, with connectivity explained as the structural integrity of a bone due to the relationship of the struts and plates that is characteristic of the bone tissue (Ostojić et al. 2006; Seeman

1999; Stini 2003).

Seeman (1999) finds that the absolute amount of bone lost in men throughout life is equivalent to that of women. Bone loss is greater among women than men at midlife where a deficiency in estrogen is experienced, leading to acceleration in trabecular bone loss. During this time, many of the trabecular elements disappear, slowing trabecular loss,

while in men it continues, leading to the comparable amounts of bone lost over the lifespan of both males and females.

In males, it has been shown that trabecular bone loss occurs mainly by reduced bone formation and trabecular thinning rather than by increased resorption and loss of connectivity (Seeman 1999). Szulc et al. (2005) describes two opposing activities occurring in the elderly male skeletons: bone loss at the endosteal envelope, which increases bone fragility and periosteal apposition which improves bending strength of bone. In men, bone resorption markers increase with age whereas bone formation markers levels remain stable or increase only minimally suggesting that bone loss is due to increased bone resorption which is not matched by a commensurate increased bone formation. This imbalance between bone resorption and bone formation as the mechanism of bone loss in elderly men is supported by evidence of trabecular thinning and wall width decrease with aging. In contrast, parameters related to bone loss determined mainly by bone resorption decreased with aging. Serum levels of bone formation markers are negatively correlated with the estimates of endosteal bone loss. In contrast, they disclose no association with parameters reflecting periosteal apposition.

Thus, in elderly men, bone formation markers reflect endosteal bone remodeling probably because of the coupling action of resorption and formation. In contrast, they do not reflect the periosteal bone formation, probably because the periosteal surface is smaller and has a slower remodeling rate than the endosteal surface.

Women who have given birth have higher bone densities than those who were childless during their fertile years with a correlation between increased bone density with each childbirths a woman has experienced (Murphy et al. 1994). This correlation is primarily

due to improved efficiency in calcium absorption during pregnancy and breastfeeding, with benefits that are long-term.

3.7 Population-Based Differences in Age-Associated Bone Loss

No population is immune to the effects of age-associated bone loss. In anthropological research, osteopenia and osteoporosis are not commonly recorded due to a) bone loss not being observed in populations due to lower ages of life expectancy of humans in the past and b) the difficulty of diagnosing osteoporotic fractures in the bioarchaeological setting. The use of non-invasive techniques employed by modern clinicians in the medical arena may produce erroneous results in skeletal remains from archaeological contexts if the mineral composition is not an element of the analysis

(Brickley and Agarwal 2003). In archaeological settings, diagenetic changes may not always be discernible from visual gross examination of the bones; bones may appear intact macroscopically but may have actually undergone a considerable amount of change at the tissue level (Bell 1990; Stout and Simmons 1979). Despite limitations, bioarchaeological materials contain great potential to enlighten researchers into the health and lifeways of past populations.

Anthropological research investigations of population-based differences in bone loss include peoples of the past and present (Agarwal and Grynpas 1996; Bennike et al.

1993; Brickley and Howell 1999; Drusini et al. 2000; Martin and Armelagos 1979;

Martin and Armelagos 1985; Mays 2001). Anthropological research has investigated osteoporosis in populations of African descent (Armelagos et al. 1972; Martin and

Armelagos 1979; Martin and Armelagos 1985; Mielke et al. 1972; Mulhern 2000) as well

as Native American (Ericksen 1980; Van Gerven et al. 1969) and European populations

(Bergot and Bocquet 1976; Cho and Stout 2003; Drusini et al. 2000; Mays 2001; Pfeiffer

1998).

Research demonstrates that individuals of African descent suffer from osteoporosis at lower rates than European Americans, with females of light complexions listed as having the highest risk of predisposition (Eskridge et al. 2010; Grossman et al.

2010; Villa 1994). This lower incidence of osteoporosis in people of African descent has been associated with Africans having a higher peak bone mass before bone loss commences (Aufderheide et al. 1998). With the use of DEXA, studies have found higher bone mass among African Americans throughout childhood (Bell et al. 1991; Li et al.

1989; Nelson et al. 1997) whereas volumetric studies with QCT have demonstrated these differences appear during late puberty (Gilsanz et al. 1991). This may reflect the result of the attainment of higher peak bone mass in Americans of African descent where achieving a higher initial bone mass leads to less fatigue damage and less need for repair.

Research comparing native Africans to Europeans living in South Africa indicate higher bone turnover rate among African South Africans; this can be interpreted as evidence of having better bone quality and lower rates of fracture risk (Schnitzler and

Mesquita 2006; Schnitzler et al. 1990; Schnitzler 1993). Other studies report that African

American adults have lower rates of bone turnover than European Americans (Han et al.

1997; Villa 1994; Weinstein and Bell 1988).

Population variation in bone strength may be more dependent on BMD than actual size of the bones as African Americans have greater bone density than age and sex matched European American counterparts (Seeman 1999). The differential expansion of the periosteum seem in males over females is also seen in African Americans over

European Americans and may be a primary reason for variation in peak bone size between the populations (Seeman 1999). This results in wider long bones in early adulthood and places bone mineral mass at a greater distance from the neutral axis of the bone in males than in females and in African Americans than European Americans.

Placement of the bone mineral mass at a greater distance from the neutral axis of the bone confers a mechanical advantage that enhances the strength of long bones especially with resistance to torsional stress (Stini 2003).

Cho et al. (2006) found in a comparison of European Americans and African

Americans that the higher bone mass and slower turnover rates reported for African

Americans may not exist with every age cohort. African American females had higher relative cortical areas and lower remodeling rates than European American males for younger age cohorts but this was reversed in older age cohorts.

Similar findings were reported in Sudanese Nubians where it was found that

Nubian females lost bone at an earlier age than males and modern-day counterparts

(Martin and Armelagos 1979; Martin and Armelagos 1985). The study found higher resorption and slower bone formation rates in females which could be related to

reproductive and nutritional stresses experienced by females. The study also reported significant bone loss at the endosteal surface beginning in the third decade of life for females (Martin and Armelagos 1979; Martin and Armelagos 1985). Mulhern and Van

Gerven (1997) also examined Nubian samples and found that females had a higher amount of fragmentary osteons and lower amounts of intact osteons, interpreted as having higher remodeling rates. The authors interpret the findings as potentially reflecting sexual division of labor that creates differing strains on the femora of males and females.

In the Canary Islands, located 100 km west of Morocco and Saharan Africa, ancient skeletal remains were analyzed for trabecular bone mass (TBM) (González-

Reimers and Arnay-De-La-Rosa 1992). The samples from individuals from the Western

Canary Islands showed no significant differences from a control sample, whereas individuals from two other islands, La Palma and Gran Canaria, demonstrated a high prevalence of osteoporosis. A later study by González‐Reimers et al. (2002) incorporating both histomorphometry and BMD comparisons via DEXA scans showed a decrease in BMD and TBM between younger and older individuals with the same exhibition of greater osteoporosis and osteopenia on Gran Canaria. Trace element analysis of the samples suggests that the inhabitants of La Palma and Gran Canaria subsisted on a mainly vegetarian diet and the authors indicate the possibility of protein malnutrition.

In a comparison of normal, modern British males and females, Rehman et al.

(1994) showed that most parameters in both males and females remained constant in

early adulthood but that bone volume decreased dramatically with age; females losing a larger amount. The authors contribute this to both a decrease in osteoblast activity and an increase in osteoclast activity. The decline in females was first apparent after the age of

40 years.

Most of the studies on prehistoric European populations employed radiography and absorptiometry (Agarwal and Grynpas 1996) and their findings are ambiguous

(Bennike et al. 1993; Kneissel et al. 1994; Mays et al. 2006; S. Mays 1998). In a study of cortical bone among 18th-19th century British Men from the Spitalfields sample, Mays

(2001) found that the patterns of bone loss in cortical bone have remained unchanged for two centuries. In another study, Mays et al. (2006) compared BMD from two medieval populations from Norway and England in order to understand why modern populations in these countries exhibit a disparity in bone mass. Overall, this study found that BMD between the two medieval populations was comparable, suggesting that the differences found in modern groups are of a recent origin.

Pfeiffer (1998) compared variability in osteon size in the rib and femur for 18th century Huguenots from Spitalfields, London with settlers in Ontario. Pfeiffer found smaller osteons in the rib than femur but no correlations to age and sex. When compared to an additional sample from South Africa, Spitalfields specimens had larger femoral osteons but smaller rib osteons. This study again points toward intra-skeletal variability as a compounding factor in studies of bone loss in addition to demonstrating variation between populations.

Cho and Stout (2003) examined the midshaft rib from individuals from the Isola

Sacra necropolis of Imperial Rome and compared them to modern populations of

European American and African American rib samples. The authors found that the

Imperial Romans maintained greater bone mass with respect to absolute and relative cortical area exhibited lower Haversian remodeling rates and may have achieved a greater peak bone mass. The comparison groups of European Americans and African Americans did not differ in terms of cortical bone amounts in their ribs, but African Americans had denser bones due to reduced Haversian remodeling rates and smaller osteon size.

In Native Americans, Burr et al. (1990) investigated population differences between archaic Native Americans from the Pecos Pueblo with European Americans.

Although the Pecos natives were genetically, culturally and nutritionally distinct from

European American populations residing in the United States today, they manifest many skeletal microstructural similarities with modern populations in the same age range. In a comparison of three archaeological populations with a modern autopsy sample, Stout and

Lueck (1995) noted a general increase in cortical bone remodeling activity levels over time with the two intensive foraging populations being more similar and having the lowest remodeling levels. The agricultural sample was found to be intermediate between the foraging populations and the modern sample. Because statistical analysis found that the regression lines for bone remodeling rates against age for the populations were parallel, the observed differences among populations may reflect that earlier populations achieved skeletal maturity at older ages than in modern populations.

Richman et al. (1979) examined three groups of Native Americans: Alaskan

Eskimo skeletons who consumed an excess of protein and fat from meat and fish; Pueblo skeletons who consumed a majority of maize, beans, squash and sunflowers with maize accounting for the majority of the diet; and the Arikara who were known to have a well- balanced diet with maize as a staple food. The study found that Eskimo females had the highest bone remodeling rates which the authors conclude as being attributed to their high protein diet and did not consider nutritional stress due to lactation and pregnancy because of a lack of sexual dimorphism in their study. The authors conclude that the differences were due to the high protein diet associated with increased mineral mobilization from bone, metabolic acidosis and increased bone loss. However, Ericksen

(1980) and Iwaniec (1997) do attribute this difference to demands of lactation and pregnancy. Iwaniec (1997) also established that differences in protein intake did not account for bone remodeling differences between Inuits, with a high protein intake, and

Pueblo agriculturalists, with a diet high in carbohydrates.

In looking at population-level variations, environmental and genetic factors play a part as well. Seasonal variation also exists due to high levels of serum vitamin D in summer and parathyroid hormone (PTH) in winter. Seasonal fluctuations in serum vitamin D levels have been documented in populations where seasonal differences in day length are relatively slight as well as in areas farther from the equator (Fuleihan et al.

2001; Lips et al. 2001; Nakamura et al. 2001; Patel et al. 2001). An example lies in the research by Foldes et al. (1995) who analyzed BMD and histomorphometry in a female skeleton from the Negev Desert of Israel from the 6th century. This individual was found to have suffered osteoporosis and it was hypothesized she may have had a confounding 77

disorder such as hypoparathyroidism which lead to osteoporosis. This interpretation, coupled with comparative ethnographic information stating that females normally stayed indoors or covered the face and body while outdoors and consumed a traditional Bedouin diet that was low in calcium.

3.8 Chapter Summary

A disruption in the activation-resorption-formation sequence of bone remodeling can have drastic consequences, and potentially lead to osteopenia and osteoporosis.

Osteopenia is a condition whereby bone mass and bone strength is decreased, weakening bones, and making them more susceptible to fracturing. Osteoporosis is similar to osteopenia but is characterized by bone mass and bone strength that is reduced to a point where random, non-traumatic fracturing occurs.

Bone loss can occur on all four bony envelopes (periosteal, endosteal, trabecular, and Haversian) and is characterized into several different types. Females tend to succumb to bone loss after reaching menopause, while males are more susceptible to a later onset.

Chronic medical conditions can also lead to osteopenic and osteoporotic states at any decade of life.

In clinical studies, bone loss is typically diagnosed through various radiographic techniques with a comparison of bone mass and/or bone density measures to an age and sex-matched sample, histomorphometric analysis of iliac crest biopsies, and biochemical markers. The diagnosis of bone loss in past populations differs as the researcher typically does not have access to large databases of age and sex-matched samples and must rely upon previous research conducted on similar populations of the past for comparative

measures. Histomorphometric analysis of bone microstructure allows for direct measurement of characteristics related to bone loss on all bone envelopes and can be conducted in both present and past populations.

Research into age-associated bone loss is complex due to the interactions of sex differences and corresponding hormonal differences, levels of physical activities, and nutrition, and underlying genetic factors. Age-associated bone loss displays differing patterns in each bony envelope and throughout regions of the skeleton.

This dissertation will describe the pattern of age-associated bone loss seen in individuals from an ancient Peruvian population. This study describes cortical as well as trabecular bone in identifying variables generally relegated to clinical analyses rather than anthropological inquiries. It also addresses the issue of intra-skeletal variability by using three locations of the skeleton for comparison within each individual.

Chapter 4: The Chiribaya

This study examines individuals of the Chiribaya polity of ancient southern coastal Peru. The Chiribaya lived along the Osmore River Valley, known today as the Ilo or Moquegua Valley, which served as a route from the highlands near Lake Titicaca to the Pacific Ocean. The Chiribaya were heavily influenced by the Tiwanaku culture that developed in the region of Lake Titicaca and expanded into the Andes (Guillén 2004).

Archaeological research in the Osmore drainage was initiated by the Programa

Contisuyo, a branch of the Asociación Contisuyo, a collaborative consortium of archaeologists working out of Moquegua, Peru (Stanish and Rice 1989). The Projecto

Chiribaya was initiated by Dr. Jane Buikstra under the auspices of Programa Contisuyo aimed at more closely defining the cultural and biological patterns of lower Osmore

Valley inhabitants of the Late Intermediate Chiribaya Culture. Initial excavations focused on the Chiribaya sites of Chiribaya Alta, Chiribaya Baja and El Yaral in 1989 by Jane

Buikstra and later incorporated the site of San Geronimo with the work of David Jessup and Elva Torres. Previous studies on the Chiribaya have been multi-disciplinary with the bioarchaeological research focused on the production and redistribution of food, manufactured items, evaluating genetic relationships, and examining social status and resource control (Buikstra 1989). In addition to archaeological interpretations of artifact distributions, the lines of bioarchaeological evidence for Chiribaya lifestyles include:

isotopic evidence of diet (Knudson et al. 2007; Sandness 1992), artificial cranial deformation (Lozada Cerna 1998), skeletal indicators of health (Burgess 1999), and the use of cranial and dental traits for biodistance analysis (Buikstra et al. 2005; Lozada

Cerna 1998; Sutter 2000; Sutter 2009). A primary goal of this project is to illuminate additional factors relating to general health as related to subsistence strategy through the use of bone histomorphometry.

The geographic area provides for excellent preservation of material and biological elements. Human remains are naturally mummified due to the aridity of the area, the high salt content of the soil and methods of funerary practices (Guillén 2004). In addition to human remains, organic artifacts such as textiles and wood are well preserved. The cemeteries were designated areas set aside from agricultural and settlement areas (Guillén

2004). The deceased were placed in rectangular tombs with stones lining the walls; the bodies of the deceased were placed at one end and surrounded by grave goods such as food and pottery (Guillén 2004). Almost all the mummies were preserved naturally

(Aufderheide et al. 1998). Ritualistic items placed with the deceased include coca leaves

(Guillén 2004), commonly chewed by inhabitants of prehistoric coastal southern Peru and was found to be evenly distributed between males and females (Indriati and Buikstra

2001). Mortuary offerings differed between males and females indicating the different activities they engaged in during life.

4.1 General Overview The Osmore River valley is home to several eco-niches that are divided into three sections: a) the Upper Valley which above 2000 meters above sea level (MASL), b) the middle valley which is between 2000 and 1000 MASL and has the most arable land and c) the lower valley which is between 1000 MASL and sea level (Rice 1989). The desert has two seasons: a sunny period from December to March where the mountains received rainfall and a foggy period from June to October when the Andes are dry (Rice 1989).

The desert is crossed by a series of streams and rivers with fluctuating seasonal flows.

The coast is generally fog covered, carrying moisture to support plant life, which is also dependent upon highland runoff for surface water (Moseley 1975). A variety of marine plants supply a food source to invertebrates which themselves serve as a food source for larger marine animals (Moseley 1975). Zaro (2005) states that both long-term and punctuated environmental shifts such as prolonged drought or periodic flooding episode elicit varied agricultural and organizational responses from different sociopolitical levels of society. Isotope analysis of human remains demonstrates seasonal variability in diet

(Knudson et al. 2007). Seasonal variation may be due to poor harvests due to El Niño effects or minimized trading during certain times of the year (Knudson et al. 2007).

Along the lower and coastal Osmore drainage, small communities invested heavily in labor-intensive irrigation and terrace systems, often making efficient use of local resources offered by their micro-environmental contexts.

The overall size and complexity of the Chiribaya is smaller than polities like

Chimu and Chincha in the northern and central coasts (Buikstra et al. 2005; Burgess

1999; Lozada Cerna 1998; Williams et al. 1989). Valleys in southern Peru and northern

Chile are also narrower than ones found in the north and central coasts of Peru and as such, have smaller amounts of arable land useful for cultivation (Rice 1989; Rice et al.

1989; Stanish and Rice 1989).

The rugged topography limited population sizes (Lozada Cerna 1998) as the area would not have been able to accommodate population sizes seen in the Lambayeque and

Viru valleys. The major empires of the highlands such as the Wari, Tiwanaku, and Inka centralized their power in the southern region of the Andes, and the presence of powerful altiplano elites may have also limited expansion of coastal polities (Burgess 1999).

The Chiribaya were first described in terms of their ceramic heritage. Jessup

(1991) organizes the ceramic styles into three phases: 1) the initial or Algorrobal phase characterized by a variety in raw materials and less use of color, 2) the middle or Yaral phase characterized by a standardization in shape and a focus on bowls and jars and 3) the late or San Geronomic phase characterized by a simplification in design and an increase in production. The increase in standardization reflects a unification of settlements and an increase in ceramic production as it became a specialized activity

(Jessup 1991). Jessup (1991) suggests that the pattern demonstrates that Chiribaya settlements were linked through a permanent social hierarchy that was maintained through control over specialized production and distribution of products (Jessup 1991).

Material culture also demonstrates membership into specialist groups and socio- economic statuses.

4.2 Chiribaya Timeframe The cultural periods of Peru were initially described by Lanning (1967) through seriating of ceramic structures and styles, placing Chiribaya in the Late Intermediate

Period (A.D. 1000 – 1476) (Table 1). More recent dendrochronological and radiocarbon dates give the Chiribaya an earliest date of A.D. 900 (Buikstra et al. 2005) which is much earlier than previously thought. Traditionally, it was thought that the Chiribaya had developed out of the remnants of Tiwanaku colonies but it is now thought they may have originated from prior coastal populations when Tiwanaku was at the height of its power

(Buikstra et al. 2005). Chiribaya origins have been examined through the use of strontium isotope, trace element concentration and oxygen isotope data with inconclusive results; however, the data did indicate mobility as adults for certain individuals (Price 2007).

Regardless of their origination, the Chiribaya were an important center of cultural development and demonstrate a presence that outlived Tiwanaku.

Cultural Periods of Peru

Period Dates Some of the Associated Culture(s)

Ceramic

Late Horizon A.D. 1476 - 1534 Inca, Estuquiña

Late Aymara, Estuquiña, Chiribaya, San Intermediate A.D. 1000 - 1476 Miguel

Middle Chiribaya, Tiwanaku, Omo, Chen Chen, Horizon A.D. 600-1000 Tumilaca, Cabuza

Early Tiwanaku, Omo, Chen Chen, Loreto Intermediate A.D. 200- 600 Viejo

Early Horizon 900 B.C.E. - A.D. 200 Late Chiripa, Huaracane, Trapiche

Initial Period 1500 B.C.E. - 900 B.C.E. Early Chiripa, Kotosh, Chiril

Pre-ceramic

Period I-VI 9500 BCE - 1500 BCE Chinchorro, Pre-Chiripa

Table 1. Cultural periods of Peru (from Lanning 1967, Sutter 2000 and Buikstra et al. 2005)

4.3 The Socio-political Environment Chiribaya sociopolitical and economic structure has been interpreted in terms of vertical (Murra 1964) and horizontal (Rostworowski de Diez Canseco 1961) models, representing a continuum of an earlier coastal tradition (Lozada Cerna 1998). In verticality models, populations had access to ecological niches to complement their resource bases and maintain economies (Murra 1964). Resources were distributed along the length of colonial archipelagos through reciprocity and redistribution. In contrast, horizontality (Rostworowski de Diez Canseco 1961) attests to political and economic 85

organization based upon communities of specialists, parcialidades, who were autonomous and endogamous, organized at a higher level into larger political structures referred to as señiorios under the control of a cacique, or chief. The señiorios were based upon loosely organized communities of occupational specialists formed as a response to the coastal environment. The communities of specialists are identified as mercaderes, merchants, pescadores, fishermen, and labradores, farmers (Lozada Cerna 1998) and were first described in an anonymous document found in the library of the Royal Palace in Madrid entitled AVISO (Rostworowski de Díez Canseco 1970). This document details the existence of 600+ merchants who controlled trade routes that extended coastally to

Puerto Viejo, Ecuador and land routes to Cuzco using copper as barter (Rostworowski de

Díez Canseco 1970). The AVISO document also details the existence of over 10,000 fishing specialists, pescadores, who engaged in fishing activities along with the salting and drying of fish for preservation (Rostworowski de Diez Canseco 1975). The labradores were responsible for cultivating lands in addition to defending the lands and guarding the water sources used for irrigation of the cultivated fields (Rostworowski de

Díez Canseco 1990). The labradores were larger in number than the other specialists groups and also had more power in the señorio, as the cacique was generally affiliated with the labradores (Hart 1983; Lozada Cerna 1998).

The caciques were the key to social integration within señiorios as each had responsibilities to reinforce reciprocity such as providing chicha, a drink of great ritual importance in the Andes, and hosting festivities. Within each community or parcialidad, there were lesser lords, who participated in an extensive system of redistribution headed by the cacique. The power of the cacique and the elites was unique in that it crossed

economic boundaries and was founded within the ideology of all inhabitants of the señiorio (Lozada Cerna 1998).

4.4 Chiribaya Subsamples This study examines individuals from three parcialidades: San Geronimo, El

Yaral, and Chiribaya Alta (Figure 8). These parcialidades were joined under a single polity, a señorío. San Geronimo is a community comprised of fishermen, pescadores; El

Yaral is a community of agriculturalists, labradores; Chiribaya Alta is comprised of individuals belonging to a merchant class, mercaderes (Lozada Cerna 1998). The residents of San Geronimo and El Yaral paid a form of tribute to the mercaderes at

Chiribaya Alta. The success of the mercaderes was dependent upon the success of their people, and they returned favors in the form of chicha which was distributed at community taverns (Lozada Cerna 1998).

Figure 8. Map of Chiribaya Sites (Adopted from Knudson et al. 2007)

4.4.1 Chiribaya Alta: Mercaderes Chiribaya Alta lies approximately 5 km from the coast on the valley rim at the foot of the lomas (Jessup 1991) on Pampa del Descando which overlooks the Ilo valley.

Chiribaya Alta is the most complex of the Chiribaya sites and is considered to be the regional center of Chiribaya influence (Rice 1993). Radiocarbon dates for Chiribaya Alta range from 801 A.D. to 1113 A.D., indicating that Chiribaya Alta spanned the occupation of Chiribaya in the Osmore drainage basin (Buikstra 1995; Lozada Cerna 1998)

Chiribaya Alta is interpreted as a ceremonial center and political power as it is characterized by ceremonial buildings, plazas, terraces and fewer residential components

(Lozada Cerna 1998). 9 cemeteries were excavated with 307 interments (Lozada Cerna

1998). Individuals at Chiribaya Alta were members of an elite, merchant class who

organized trade of items with members of other Chiribaya sites, serving as the administrative body (Lozada Cerna 1998). Tombs excavated include a variety of agricultural and marine foods, fine textiles, gold and copper ornamentation, ceramics and camelid crania (Jessup 1991; Lozada Cerna 1998; Sutter 2000). The presence of camelid remains in the burials and woolen textiles suggest animal husbandry and continual access to herds (Jessup 1991). Chiribaya Alta also indicates a high degree in variability in dietary staples and ceramic styles (Lozada Cerna 1998).

Individuals of Chiribaya Alta would have been subjected to lower levels of physical activity than individuals at the other two sites and their primary activities would have included trade, the manufacture and trade of chicha, and ceremonial functions

(Lozada Cerna 1998). Adults of both sexes at Chiribaya Alta have a higher prevalence of spondylolysis than at El Yaral and San Geronimo, as spondylolysis is commonly associated with heavy laborers (Merbs 1996), suggesting they spent their younger years at other sites where heavy labor was necessary.

Isotopic evidence from Chiribaya Alta indicates a diet comprised of both agricultural and marine resources (Knudson et al. 2007; Sandness 1992; Tomczak 2003).

Isotopic signatures examined from individuals at Chiribaya Alta were also found to demonstrate much more variability in dietary resources, supporting the hypothesis that the site was a burial site for elites who originated in different areas (Tomczak 2001;

Tomczak 2003). The isotopic signature of Chiribaya Alta was a mean δ15N value of 17.83

13 ‰ and a mean δ Ccoll value of -13.06‰ (Tomczak 2003). In nitrogen analyses of hair samples, it was found that some individuals from Chiribaya Alta had variable regions as

one moved down the hair shaft, indicating dietary changes within the last 2 years of their lives (Knudson et al. 2007) and suggesting a movement to Chiribaya Alta from another site. The human hair segments from Chiribaya Alta show variability in seasonal consumption of C3 and C4 plants as well as variability in consumption levels among individuals (Knudson et al. 2007). Isotopic evidence also demonstrates that the protein sources in these individuals’ diets were C4 plants and/or terrestrial animals (Knudson et al. 2007). Carbon and nitrogen values were similar and demonstrated a dependence on C4 plants and marine resources (Knudson et al. 2007; Tomczak 2001; Tomczak 2003).

Overall, the Chiribaya Alta diet is slightly less dependent on ocean resources than other parcialidades, but incorporates a small amount of terrestrial meat (Sandness 1992).

There is a negligible amount of highland products in the Chiribaya diet which argues against their trading food products with the altiplano.

4.4.2 San Geronimo: Pescadores San Geronimo is the most extensive Chiribaya site and is located adjacent to the coast (Jessup 1991). Radiocarbon dating indicates dates to around 1100 A.D. (Lozada

Cerna 1998). The maritime subsample of pescadores indicates a sexual division of labor, where females were buried with textiles and ceramics, and males were buried with fishing nets, net weights and harpoons, hooks, and miniature boats (Buikstra 1995). A marine-based diet is indicated with isotopic evidence, reflecting marine resources as their primary resource (Knudson et al. 2007; Sandness 1992; Tomczak 2001; Tomczak 2003), in addition to midden remains consisting of fish bones, shellfish, algae and sea mammal bones (Jessup 1991). The pescadores would have been catching marine fish several kilometers offshore, as well as engaging in other activities (Moseley 1975). Low

prevalence of external auditory exostoses suggest underwater harvesting was not a significant activity at San Geronimo (Burgess 1999), a noted characteristic of cold water deep-sea diving (Standen et al. 1998; Walker et al. 1996). Male pescadores paddled boats into fishing areas, dropped heavy nets in the water and hauled fish-filled nets back into the boat; an activity pattern that would have required a significant amount of body strength (Ramsay 2006). Almost all male burials contained hooks, weights, floats, harpoons, cotton cords and other fishing paraphernalia while some also contained model wooden boats (Jessup 1991).

Thoracic and lumbar osteophytosis is higher at San Geronimo than Chiribaya Alta and El Yaral and is prevalent in younger females suggesting that much of the physically demanding tasks were done by younger females and less strenuous tasks, such as weaving and craft production, were practiced by older females (Burgess 1999).

Isotopic signatures of bone indicate that individuals from San Geronimo had higher amounts of marine-based resources in their diet (Sandness 1992; Tomczak 2001;

Tomczak 2003). This was demonstrated with a mean δ15N value of 20.94 ‰ and a mean

13 δ Ccoll value of -11.98‰ (Tomczak 2003). The carbon isotope ratios fell within the range of C4 plant and marine diets and nitrogen isotope ratios suggested a marine origin for dietary protein (Tomczak 2003).

4.4.3 El Yaral: Labradores El Yaral lies approximately 50 km from the coast at an altitude of 1000 MASL

(Jessup 1991). El Yaral is an extensive residential area containing 99 burials in 2 cemeteries. Ceramics uncovered belonged to Chiribaya and Ilo-Tumilaca/Cabuza

traditions; Chiribaya was the predominant influence but the area was occupied by both

Tumilaca and Chiribaya. Radiocarbon dates place the occupation of El Yaral from 1027

A.D. to 1252 A.D. (Rice 1993). El Yaral is closely associated with irrigation canals and agricultural terraces (Lozada Cerna 1998) and consists of residential terraces and the large mortuary component (Rice 1993). One structure contains large amounts of cantaros, which are clay pots used to create music, in association with maize and may have served as a central storage center for the site (Lozada Cerna 1998). The burial patterns at El Yaral lack the elaborate decor of the sites in the lower valley (Buikstra

1995).

The community at El Yaral built irrigation systems, land terraces, herded llamas, and planted and harvested cultivars (Lozada Cerna 1998; Rice 1993). Excavations of tombs revealed llama bones, textiles of lower quality than those found at Chiribaya Alta and agricultural remains (Buikstra 1995). Agricultural labor would have required intense amounts of digging, hoeing, lifting, pushing, pulling, walking, and carrying and would have subjected the labradores to a considerable amount of biomechanical stress in the upper and lower body (Ramsay 2006).

At El Yaral, isotopic signatures indicate a dependence on maize consumption and less reliance on marine sources than sites in the lower valley (Sandness 1992; Tomczak

2001; Tomczak 2003). This was demonstrated with a mean δ15N value of 11.85 ‰ and a

13 mean δ Ccoll value of -14.00‰ which were the lowest of the Chiribaya sites examined

(Tomczak 2003). At El Yaral, marine dependence is less evident, yet still present, which contrasts with Tiwanaku colonies existing at the same temporal phase (Knudson et al.

2007). Evidence at El Yaral also demonstrates that protein sources were more 92

homogenous over time (Knudson et al. 2007). Isotopic analysis of El Yaral remains indicates a heavy reliance on an agricultural-based diet, but also demonstrates higher than expected values for nitrogen, which implies an inclusion of marine resources (Knudson et al. 2007; Sandness 1992; Tomczak 2001; Tomczak 2003). Isotopic signatures of peoples buried at El Yaral suggest a diet comprised of 1/3 marine-based products, 1/3 maize and

1/3 land plants and animals (Sandness 1992). The presence of marine resources at El

Yaral would have been part of the mercaderes trade goods from San Geronimo that were exchanged with the population at El Yaral and incorporated into their diet.

Males at El Yaral demonstrate significantly higher cases of upper limb arthritis than San Geronimo and Chiribaya Alta (Burgess 1999). A significant difference in cervical ostephytosis in males and females at El Yaral suggests a sexual division of labor where males carried heavy loads on their heads, neck and shoulders while women’s loads were either minor in comparison or carried for a shorter duration of time (Burgess 1999).

Individuals from El Yaral also had a significantly higher number of tuberculosis-like lesions than Sa Geronimo and Chiribaya Alta, but better dental health, as indicated through a lesser number of dental caries (Burgess 1999)

4.4.4 Community Membership Membership into each of the parcialidades was reinforced with occupation- specific cranial deformations (Lozada Cerna 1998). Of the 60% of individuals with cranial deformations, the residents of San Geronimo demonstrate the annular obliqua (an elongated and tubular cranial vault) form of cranial deformation, while residents of El

Yaral demonstrate the tabula oblique (fronto-occipital compression) form of cranial

deformation (Lozada Cerna 1998). The residents of Chiribaya Alta have a mixture of the two deformation types (Lozada Cerna 1998). These findings indicate that cranial deformation was utilized to distinguish themselves from other groups. The deformation style shows a correlation between socio-economic status and membership into a group of specialists.

4.5 Chiribaya: General Health Indicators In examining indicators of general health, the three subsamples demonstrate similar patterns and trends.Burgess (1999) examined the three Chiribaya subsamples for evidence and prevalence of Harris lines, stature variation, cribra orbitalia, porotic hyperostosis, tuberculosis, generalized periosteal reactions, and prevalence of dental caries. Health status of Chiribayans appears to be closely linked with the quality of the diet consumed by individuals; elite status of the Chiribaya community appears to equate with preferential access to goods as well as valuable foods. Overall, the Chiribaya appear to experience relatively high stress and a generally low level of health.

Harris lines, transverse layers in the metaphyseal portions of long bones, reflect episodes of delayed or arrested longitudinal growth of the bone (Larsen 1997). In the three subsamples, no significance in the amount of Harris lines found between the groups or with differences by sex or age (Burgess 1999). Lengths of long bones and estimated stature produced similar means for each of the three subsamples (Burgess 1999).

In all three subsamples, “present” to “high” levels of cribra orbitalia and porotic hyperostosis were found (Burgess 1999). The prevalence was attributed to iron loss through episodic diarrheal disease, thus creating a nutritional iron deficiency (Burgess

1999); however, more recent interpretations of cribra orbitalia and porotic hyperostosis

explain the high incidence of both conditions in archaeological populations as due to a combination of nutritionally adequate diets, poor sanitation, infectious disease, and cultural practices related to pregnancy and breastfeeding (Walker et al. 2009). This interpretation is plausible with the Chiribaya sample given that there is minimal evidence for an elevated parasite load, a possible over-abundance on maize agriculture, an easily contaminated and limited water supply, and a desert environment with a constrained population. The overall prevalence of cribra orbitalia and porotic hyperostosis suggests large segments of the Chiribaya population were affected by systemic disease. Similar trends have been noted in neighboring populations and may indicate a high altitude response (Williams 1990)

Generalized periosteal reactions can be a non-specific response to disease, trauma and inadequate diet (Ortner 2003). Burgess (1999) examined active and healed periosteal lesions in the Chiribaya subsamples and found no difference between groups when the overall number of cases of non-specific skeletal lesions was compared. However, when only moderate and severe lesions were examined, it was noted that individuals at El Yaral had a significantly lower amount moderate and severe lesions than individuals at both

San Geronimo and Chiribaya Alta (Burgess 1999) Chiribaya juveniles also demonstrated the highest ratio of active to inactive lesions, with almost all periosteal lesions being active in juveniles. In adult males and females, active lesions were less frequent than healed lesions, with the exception of San Geronimo females who had 56% of lesions being active (Burgess 1999). The pattern of generalized periosteal lesions demonstrate the stress patterning seen in dietary, parasitic and altitudinal differences between coastal and upland populations.

Burgess (1999) examined Chiribaya subsamples for recognizable patterns of lytic destruction and periosteal remodeling, and evidence of tuberculosis-like lesions was found among all three subsamples. Observations of tuberculosis are also supported by findings of the tuberculosis bacterium in soft tissue analysis of the Chiribaya mummies

(Salo et al. 1994). Individuals at El Yaral have a significantly higher number of affected individuals and those individuals have significantly more elements containing lesions

(Burgess 1999). The San Geronimo sample has a greater number of individuals affected than Chiribaya Alta (Burgess 1999). Chiribaya males also demonstrate a higher frequency of lesions suggesting they were at greater risk for developing the disease

(Burgess 1999). Williams (1990) also reported high levels of tuberculosis-like lesions in

Estuquiña males.

Dental caries result from a disease process through which the dental hard tissues are demineralized by organic acids produced by microbial action (Larsen 1997). Analysis of dental caries in the Chiribaya indicates that the population of El Yaral had better dental health than San Geronimo and Chiribaya Alta (Burgess 1999), corresponding with research by (Elzay et al. 1977) who found significant differences in caries frequency between coastal (Nazca-Ica) and highland (Tiwanaku and Inca) peoples.

Evidence of coca chewing from dental remains and hair analysis has been indicated within individuals from Chiribaya Alta and El Yaral, indicating that coca chewing was a common practice (Indriati and Buikstra 2001). Coca contains alkaloids and has an anesthetic effect (Buck et al. 1968) and has been known to reduce symptoms of altitude sickness in addition to having a religious, medicinal and economic function

(Allen 1988; Weil 1981).

The use of coca has been demonstrated in the Chiribaya previously (Aufderheide

2003; Salo et al. 1994). Chewing of coca leaves is prevalent throughout the Andes today;

Burgess (1999) suggests the combined effects of an added alkaline substances and the abrasive action of the quid may have inhibited caries formation. Burgess also reports that

Chiribayan females reportedly have higher amounts of carious lesions than males.

A further study examining the relationship of coca chewing to carious lesions found a repeated pattern of cervical root caries accompanying root exposure on the buccal surfaces of the posterior dentition, coinciding with the placement of coca quids while chewing (Indriati and Buikstra 2001). In addition to containing alkaloids, coca chewing reduces salivary gland activity and creates a dryness of mouth, and a narcotic effect. Indriati and Buikstra (2001) also found an equivalent number of males and females with carious lesions associated with coca chewing, suggested the practice was not sex-specific.

4.6 Chiribaya: Genetic Relatedness Biodistance analysis of the Chiribaya have been conducted through the use of cranial and dental trait analysis (Lozada Cerna 1998; Sutter 2000; Sutter 2009). Cranial non-metric traits of Chiribaya were found to be significantly different from the Chen

Chen, a neighboring mid-valley Tiwanku site (Lozada Cerna 1998). Chiribaya were also indistinguishable from a formative coastal site that preceded the Chiribaya by several hundred years and predates Tiwanaku influence (Lozada Cerna 1998). This suggests that the Chiribaya arose out of earlier coastal populations and were not descendent of

Tiwanaku (Lozada Cerna 1998). The Chiribaya population itself appears to be genetically homogenous among the various sites. Lozada Cerna (1998) concludes that the Chiribaya

originated on the coast and were not genetically derived from the highlands based on biodistance measurements of non-metric traits.

Through dental trait analysis, Sutter (2000; 2009) hypothesizes a highland origin of the Chiribaya. Using dental trait data for over 900 individuals from 14 mortuary samples examined genetic relatedness and found that South Central Andean populations exhibited very little phenotypic variation (Sutter 2000; Sutter 2009). In regards to their origin, his analysis supports a two-phase diaspora model for explaining the genetic and cultural origins of the Chiribaya. The two phase diaspora model postulates that the

Moquegua Chiribaya represent direct descendants of former middle valley Tiwanaku colonists who dispersed to the coast where they developed a new social identity. The data point to Chiribaya having their cultural and genetic origins among earlier Tiwanaku colonists of the middle Moquegua valley rather than representing an exclusively coastal cultural phenomenon with earlier coastal antecedents. Some individuals interred at

Chiribaya Alta likely had indigenous coastal roots but it also demonstrates that Ilo-

Tumilaca peoples contributed to Chiribaya DNA. He suggests that they began as a polity of economically defined ethnic groups but attracted others to develop a single ethnic identity through economic prosperity and intermarriage.

Regardless of their origins, it appears that minimal amounts of gene flow occurred between the Chiribaya and their neighbors (Lozada Cerna 1998). These data represent genetic homogeneity among the individuals interred at Chiribaya sites with the possibility that some of the individuals interred at Chiribaya Alta having additional Ilo-Tumilaca roots.

4.7 Social Interactions The power of the Chiribaya polity spans the Middle Horizon and Late

Intermediate Period, giving it a time span of 500-600 years. Its power overlaps with the emergence and disappearance of other cultures such as Tiwanaku, Tumilaca and

Estuquiña (Buikstra 1995; Buikstra et al. 2005; Indriati and Buikstra 2001; Lozada Cerna

1998). Tiwanaku state rule in the middle Moquegua Valley occurred circa A.D. 950

(Goldstein 1989; Moseley et al. 1991; Owen and Goldstein 2001). Post-collapse

Tiwanaku popoulations in the Moquegua were given the name Tumilaca and the inhabitants are thought to be the descendants of Tiwanaku populations (Sims 2006). The

Tumilaca is considered the post-Tiwanaku phase but with Tiwanaku cultural manifestations (Augustyniak 2004; Goldstein 1989). It is thought that at least for some point of time, the the Tumilaca culture co-existed with the Chiribaya in the Osmore valley. The Ilo-Tumilaca may have arrived slightly earlier; however, the Chiribaya flourished much longer than the Tumilaca (Late Tiwanaku) (Owen 1993). Chiribaya peoples were probably living on the coast while Tiwanaku colonies flourished within the middle valley. This is demonstrated with the findings of marine consumption with the earliest cemeteries at Chiribaya Alta where it remains in high content among the elites when socio-political inequalities are demonstrated in the archaeological record around

A.D. 1000 (Buikstra et al. 2005). Around the same time, Tiwanaku influence was spreading into the mid-valley of the Osmore (Buikstra et al. 2005). The boundary between Chiribaya and Tiwanaku people had become permeable by A.D. 1000 (Buikstra et al. 2005). Tumilaca people actively sought alliances with coastal groups to establish new power relationships in the absence of Tiwanaku state control (Buikstra et al. 2005).

It is thought that trade existed between the Chiribaya and their neighbors, indicated by

Chiribaya pottery found at Azapa sites (Lozada Cerna 1998). The inclusion of altiplano decorative patterns in pottery indicate that some form of cultural influence occurred, especially at El Yaral (Buikstra 1995), although Sandness (1992) indicates altiplano products played a negligible part in the diet of the Chiribaya which suggests little contact with altiplano centers. It appears that despite the proximity of Chiribaya to Chen Chen and the power of the Tiwanaku culture, that the Chiribaya maintained a very distinct and separate socio-political identity (Buikstra et al. 2005; Lozada Cerna 1998; Sandness

1992). Evidence of trauma in the Chiribaya is relatively low and shows no pattern of interpersonal conflict (Burgess 1999). The relationship of the Chiribaya and Tumilaca is difficult to identify through the archaeological record and it is quite possible their relationship changed throughout the occupation of the Osmore valley (Lozada Cerna

1998).

The Chiribaya culture appears to end abruptly around A.D. 1350. It is largely thought a catastrophic event associated with the effects of El Niño led to its demise and research indicates evidence of a major flooding event around A.D. 1350 (Manners et al.

2007; Reycraft 1999; Sandweiss and Quilter 2008). A flood could have weakened

Chiribaya power and caused the need for restructuring the political dynamic throughout the entire valley (Jessup 1991).

4.9 Chapter Summary The Chiribaya lived along the Osmore River valley in southern Coastal Peru and represent a typical ancient Andean society, although smaller in population size than

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found in ancient polities in the northern and central Peruvian coasts. The Chiribaya were probably limited in population expansion by the rugged topography, narrowness of the river valley and less access to arable lands than their neighbors in the north. Radiocarbon dates give the Chiribaya an earliest date of A.D. 900, after which they succeeded for several hundred years, until reaching their demise around A.D. 1350, probably due to a catastrophic event due to the effects of El Niño.

The contents of this dissertation focus on the human remains of three populations:

Chiribaya Alta, El Yaral, and San Geronimo. These communities, known as parcialidades, served a specific socio-economic function within the larger community, the señorío. San Geronimo consists of a community of fisherman, pescadores; El Yaral is comprised of a community of agriculturalists, labradores; Chiribaya Alta was composed of individuals who belonged to an elite, merchant class who received tribute from the communities of San Geronimo and El Yaral and performed administrative functions of the señorío. Table 2 summarizes the cultural characteristics of each of the Chiribaya sites examined in this study.

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Site Chiribaya Alta San Geronimo El Yaral Radiocarbon 801 A.D. to 1113 1100 A.D. 1027 A.D. to 1252 A.D. Dates A.D Associated Elite, merchant class: Paddling boats, Intense agricultural labor: Physical less physical labor, dropping and digging, hoeing, carrying, Behaviors more administrative hauling fish nets walking tasks, trade and ceremonial functions Diet Both marine and Marine Dependence on maize, agricultural resources: fish, less reliance on marine resources, terrestrial shellfish, sea resources, land plants and meat mammals, algae animals General Health Higher amounts of Thoracic and Cervical osteophytosis, Indicators spondylolysis, cribra lumbar less dental caries, more orbitalia and porotic osteophytosis, tuberculosis-like lesions hyperostotis cribra orbitalia and porotic hyperostosis Cultural Mixture of annular Annular oblique Tabula oblique cranial Affiliation oblique and tabula cranial deformation oblique cranial deformation deformation

Table 2. Summary of Chiribaya Subsample Characteristics

Associated physical behaviors differed between the communities. Individuals at

San Geronimo were involved with the harvesting and processing of ocean fishes and shellfish, a lifestyle demanding of intense upper and lower body strength. Individuals at

El Yaral engaged in heavy labor required of an agricultural lifestyle such as digging, hoeing, carrying, and walking; activities also requiring intense amounts of upper and lower body strength. The activities required of individuals of Chiribaya Alta were much less demanding as they were primarily associated with administrative and ceremonial

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tasks; however, their skeletal health bears witness to individuals’ younger years spent at labor-intensive sites before their relocation to the ceremonial center.

Isotopic analysis, coupled with general health indicators, demonstrate dietary differences at the sites of San Geronimo, Chiribaya Alta and El Yaral. Individuals from

San Geronimo were dependent on marine resources, while the community of El Yaral was primarily dependent on cultivated plants such as maize. Individuals of Chiribaya

Alta demonstrate variability in their dietary habits throughout their lives, suggesting the individuals originated from other Chiribaya sites, where they later relied upon a combination of both marine resources and cultivated products when they resided at

Chiribaya Alta.

Indicators of general health status suggest the Chiribaya people experienced relatively high levels of stress and a generally low-level of health. Health status also appears to be associated with lifestyle, where the community of Chiribaya Alta had preferential access to goods and valuable foods, which were provided as tribute from other sites. Present to high levels of cribra orbitalia and porotic hyperostosis are evident at all three sites, suggesting large segments of the population were impacted by systemic disease. Generalized periosteal reactions were found on the remains of individuals from all three sites, with lower amounts of moderate and severe lesions at San Geronimo.

Tuberculosis-like lesions were present and found to be highest in frequency among individuals at El Yaral and San Geronimo. Dental carious lesions were less prevalent in individuals from El Yaral but present in all subpopulations and evidence of dental carious

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lesions associated with coca chewing have been indicated among individuals from both

El Yaral and Chiribaya Alta.

Biodistance analysis of non-metric cranial and dental traits suggests genetic homogeneity among Chiribaya sites, with altering hypotheses suggesting either coastal or highland origins. Little gene flow occurred with Chiribaya populations and their neighbors and the Chiribaya maintained distinct and separate socio-political entity from neighboring populations. The Chiribaya population represents a unique opportunity to examine bone microstructure in relation to the impacts of age, biological sex and subsistence pattern within a larger community of individuals whose bone growth and development are not further impacted by a large degree of genetic variation.

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Chapter 5: Materials and Methods

The rib, clavicle, and iliac crest of each individual of the Chiribaya population sample were analyzed and a combination of trabecular and cortical bone variables were examined. The contents of this chapter describe the sampling methods for each bone sampled, the variables examined, and the methods used in the analysis.

5.1 Skeletal Sampling Sites

Three skeletal areas were selected for analysis from each individual. These areas were chosen based on areas used in previous research for comparative purposes as well as for examining intra-skeletal variability. Examination of one bone may not represent the entirety of the skeleton as bone mass and other microstructural elements have been shown to differ throughout a single individual (Doyle 2011; Peck and Stout 2007). For this reason, the iliac crest, rib and clavicle were selected for analysis in this study. The selection of these three skeletal sites allowed for the inclusion of both cortical and trabecular bone which represent different biomechanical environments.

5.1.1 Iliac Crest

Studies using iliac crest samples within biological anthropology have been few in number and smaller in scope. In one study, a pathological diagnosis was made using a

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transilial bone core (Weinstein et al. 1981). In another study, total bone mass (TBM) in addition to bone chemistry (González-Reimers and Arnay-De-La-Rosa 1992) was examined and another study examined bone mineral density and bone volume (Foldes et al. 1995).

In clinical research, the iliac crest is one of the major sites of bone biopsy and plays a primary role in diagnosing metabolic bone disease (Melsen and Mosekilde 1981); it has been used for more than four decades to diagnose osteoporosis (Barger-Lux and

Recker 2006). In living individuals, this site is easily accessible and it has proximity to hematopoietic tissue, such that turnover is higher in this region and deviations from normal occur sooner and are of a larger magnitude (Parfitt 1994). As indicated previously in Chapter 3, bone alterations in the iliac crest may also reflect changes in other regions such as the spine or long bones.

In clinical procedures, standardization of the biopsy site ensures comparability of the data obtained between different populations (Anderson 1982); the area used in clinical studies was replicated with sample procurement in this population. The standard clinical site for a bone biopsy lies approximately 2 cm (or 1 inch) behind the anterior superior iliac spine and 2 cm (or 1 inch) below the iliac crest; an area known as

“Bourdier’s triangle” (Recker 1983) (Figure 9).

In clinical examinations, a trephine is generally used to remove a small core of bone from living individuals. Anderson (1982) recommends the use of a 9 mm internal core trephine designed by J. Jowsey at the Mayo Clinic as the most adequate tool to obtain an unadulterated core of iliac crest bone. Other researchers recommend a section of at least 7.5-8.00 mm to conduct proper histologic analysis (Barger-Lux and Recker

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2006; Recker 1983) although trephines commonly used vary in size from 4.0 to 9.0 mm.

Due to the fragility of trabecular bone in archaeological remains, a wedge of bone was removed from each individual rather than a core to ensure the bone within the region of interest was not damaged prior to analysis. The bone samples were provided courtesy of

Jane Buikstra. After procurement of the sample, slides were further prepared using methods described below.

Figure 9. Site of procurement of wedge from iliac crest specimens

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5.1.2 Rib

Ribs have commonly been used in samples for hard tissue histology because they are one of the few bones from metabolically normal people that are available in quantity and can be removed freely at autopsy. There is also a presumed relative homogeneity of its biomechanical functions when compared to bones such as the femur and humerus

(Frost 1963a). The higher remodeling rate of ribs in relation to appendicular bones causes them to reflect the effects of disease, aging and growth earlier than most other bones of the body (Frost 1963a; Frost 1963b; Pirok et al. 1966; Sedlin et al. 1963a; Sedlin et al.

1963b). A cross-section of the midshaft of the 6th rib was removed from each individual

(Figure 10). For the rib, the midpoint of the bone was determined and a section approximately 5-6 cm (2-2.5 inches) in thickness was removed.

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Figure 10. Demonstration of region of rib section removal

(Original drawing by Alexandra Krilevich)

5.1.3 Clavicle

The clavicle is a unique bone in the human body that serves as a strut for the upper limb and is the only bony connection between the upper extremity and the thorax.

Because all loads on the upper extremity are transmitted to the thorax through the clavicle, it represents a more dynamically loaded bone than the rib or iliac crest. The

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clavicle is the first bone in the human body to ossify in the fetus and forms through both intramembranous and endochrondral ossification. Due to its superficial location, the clavicle is also a common site for fracture in the human body (Post 1989). Of all the long bones, the clavicle also has the least amount of bone marrow (Ellis 2002) which should demonstrate differences in bone parameters when compared to the rib. The clavicle has previously been demonstrated to have significant differences with cortical thickness between males and females of varying ages (Anton 1969), supporting the decision to use the clavicle as an additional skeletal site for examining age and sex related effects on bone microstructure.

Similar to the rib, a cross section of the clavicle at midshaft was removed from each individual. For the clavicle, as in the rib, the midpoint of the bone was determined and a sample of approximately 2 to 2 ½ inches was removed (Figure 11). Previous research indicates the midshaft is the most rigid aspect of the clavicle and also experienced the greatest bending and torsional stresses when loaded (Harrington et al.

1993)

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Figure 11. Clavicle demarcating section removed for analysis (Original drawing by Alexandra Keenan-Krilevich)

5.2 Sample Procurement/Preparation

After the removal of bone samples from the rib, clavicle and iliac crest, the cross sections were embedded into small blocks using Buehler Epo-Thin® epoxy resin. This allowed for stabilization of the sections during sectioning, grinding and slide creation.

Some of the elements had been previously embedded by a previous researcher (Ramsay

2006) but did not appear to be vacuum embedded. Therefore, plastic blocks that had previously been created were trimmed down to where the bone was exposed and the bone was embedded again to fill in spaces where air bubbles had formed. The liquid epoxy- resin mixture was placed in a vacuum to remove air bubbles from the liquid before it was dried and solidified.

Rib and clavicle thin sections of bone were further prepared by transversely sectioning five-hundred micrometer (µm) wafers from the embedded samples using an 111

Buehler® Isomet low-speed metallurgical saw (Buehler Instruments, Evanston, IL) then ground and polished to a final thickness of ~70-100 micrometer (µm) wafers using a

Buehler® Tech-met sanding wheel. Rib and clavicle specimens were cleaned using xylenes, dried and then mounted onto standard 25 mm x 75 mm microscopic slides and cover-slipped with the use of Permount® mounting medium.

Iliac crest sections were handled slightly differently due to the fragile nature of the high amount of trabeculae found in that skeletal region. After embedded blocks had hardened, each block was cut using Buehler® Isomet low-speed metallurgical saw and the embedded bone sampled was adhered to 75 mm x 50 mm microscope slide using

Buehler Epo-Thin® epoxy resin. After drying, the block was removed from the slide using the Buehler® Isomet low-speed metallurgical saw, leaving a 500 µm section adhered to the slide. The section was ground and polished while attached to the slide to prevent loss of trabeculae during processing. The slides were then cleaned with xylenes and coverslipped using Permount® mounting medium. Upon drying, 2 cm was measured from the iliac crest and an area of 9 mm to indicate the region of interest that was comparable to the clinical biopsy area was marked with a fine-tipped permanent marker on each iliac crest slide as the region that would be investigated.

5.3 Microscopic Analysis

Each slide was viewed with an Olympus ® BX51 microscope with an attached

SPOT InsightTM QE Color 4.2.1 camera and overlapping photomicrographs were taken of the entire section of bone at a resolution of 100x magnification. The resolution of the photomicrographs was 680 pixels per µm and allowed for precise image analysis. A

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calibration mark was also added to the photomicrographs for use in calibration during data acquisition. Photomicrographs were compiled into a single image using Microsoft®

Image Composite Editor 1.4.4 (ICE). ICE is available as a free download to any user and operates in a similar fashion to the photo stitching element in Adobe® Photoshop software suite but was found to produce more efficient results and required less computer memory to run the software. Variables were then collected using ImageJ® software (U.S.

National Institute of Health; Bethesda, MD) with the use of a PC tablet and stylus. All data were collected in µm and later converted to mm for ease with statistical analyses.

Measuring variables from an image created from compiled photomicrographs has advantages over more traditional methods such as point-count which utilizes an eyepiece reticle (Epker and Frost 1964; Sedlin et al. 1963b). Compiled images allow a researcher to view the thin section multiple times and to save images demonstrating how variables were measured. Photos can also be taken at a great enough resolution to view larger overall features such as areas and perimeters and smaller features such as trabecular width.

5.4 Variables Examined

This study incorporates variables from both cortical and trabecular bone. The variables and descriptions, along with how each measurement was obtained, are described below.

Variables measured from the rib and clavicle specimens include the following:

a) Total Subperiosteal Area (Tt.Ar.): the entire cross-sectional area or the area

within the periosteal border. For the total subperiosteal area and periosteal 113

perimeter, the rib and clavicle sections were traced along the outermost border of

the bone and an area and perimeter measurement were recorded at the same time;

b) Endosteal Area (Es.Ar): the area of the marrow cavity. Endosteal area was

determined by tracing the inner border of the cortex;

c) Periosteal Perimeter (Ps.Pm.): the length of the outermost border of the bone. For

endosteal perimeter and endosteal area, both measurements were recorded at the

same time;

d) Endosteal Perimeter (Es.Pm.): the length of the endocortical surface.

Figure 12 demonstrates an example of these measurements from a compiled image.

From these measurements the following two variables were derived:

e) Cortical Area (Ct.Ar.): the cross-sectional area of cortical bone between the

periosteal and endosteal surfaces and determined by subtracting the endosteal area

from the total area. More specifically, compacted, rather than cortical bone was

used because trabecularized bone is excluded. This modification was made

because trabecularized cortical bone acts biomechanically similar to trabecular

bone;

f) Relative Cortical Area (Rel.Ct.Ar.): the size of the cortical area divided by the

total subperiosteal area;

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Figure 12. Compiled photomicrograph of rib and indication of variables measured

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Variables collected from the iliac crest specimens include the following (Figure 13 gives an example of a compiled photomicrograph of an iliac crest section and Figure 14 demonstrates the variables collected from each individual):

a) Total Area (Tt.Ar.): the entire cross-sectional area of bone that was read; defined

superiorly and inferiorly by the demarcation of the 9 mm section delimited

posteriorly and anteriorly by the extent of cortical bone;

b) Marrow Cavity Area (Es.Ar.): the entire area bounded by the 9mm section and

endosteal boundary of the non-trabecularized cortical bone;

c) Cortical Area (Ct.Ar.): the area of non-trabecularized cortical bone contained

within the 9 mm section of bone;

d) Trabecular Area (Tb.Ar.): the total area of trabecular bone in the sample,

including cortical bone that had become trabecularized;

e) Mean Trabecular Width (Tb.Wi.): the mean diameter of all trabeculae measured

calculated as the sum total width of all trabeculae divided by the number of

trabeculae counted;

f) Total Diameter of Section (Tt.Dm.): a length measurement taken at the point of

greatest anterior-posterior width of the 9 mm region of interest;

g) Cortical Diameter (Tt.Dm): the total diameter of both areas of cortical bone

within the region of interest;

h) Marrow Cavity Diameter (Es.Dm.): the diameter of the marrow cavity taken at the

same location at which total diameter was taken;

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From the measurements that were directly measured from the specimens, the following two variables were derived:

i) Relative Cortical Area (Rel.Ct.Ar.): the size of the cortical area divided by the

total area of the section;

i) Relative Trabecular area (Rel.Tb.Ar.): the size of the trabecular area divided by

the total subperiosteal area;

Figure 13. Example of a compiled image of an iliac crest sample demarcating normal biopsy site (superior border is to the right; anterior is facing the top).

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Figure 14. Variables collected from iliac crest specimens

(right side is superior; anterior surface is toward the top)

5.5 Sample Demographics

Age and sex information was taken from age estimations and sex determinations provided in the dissertation projects of Burgess (1999) and Lozada Cerna (1998). Sex was determined using a combination of several methods utilizing morphological characteristics of the pelvis and cranium including morphology of the sciatic notch, the ventral arc, inferior pubic ramus, the ridge below the pubic symphysis, the pre-auricular sulcus, the mastoid process, supraorbital tori, mental eminence as well as the diameters of the humeral and femoral heads (Buikstra and Ubelaker 1994; Burgess 1999; Lovell 1989;

Phenice 1969; Sutherland and Suchey 1991). Individuals demonstrating ambiguous sex

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characteristics were not included in the demographic sample. Age estimations were obtained primarily through the use of pubic symphyseal morphology (Brooks and Suchey

1990; Buikstra and Ubelaker 1994; Burgess 1999; Katz and Suchey 1986; Suchey 1979), with additional use of cranial suture obliteration ((Buikstra and Ubelaker 1994), fusion of secondary ossification centers, and dental wear (Ubelaker 1978). Age methods resulted in broad age ranges, particularly among older individuals (Burgess 1999). Using the age ranges recorded by Burgess (1999), individuals were placed into one of three adult: young adult (20-34 years), middle adult (35-49 years) or old adult (50+ years), as recommended by Buikstra and Ubelaker (1994). The Suchey-Brooks system of pubic age estimation has been widely used since its publication in 1986 and a variety of studies have examined its usefulness in regional populations (Bednarek et al. 2002; Berg 2008;

Chen et al. 2008; Djuric et al. 2007; Hoppa 2000; Kimmerle et al. 2008; Sakaue 2006;

Schmitt 2004; Sinha and Gupta 1995) with varying results, but with a general finding that age estimation is more accurate among individuals 40 years of age and younger. It should be noted that in using age estimation formulae created from, and tested upon, recent and historical populations with known ages to estimate demographic profiles of past populations, relies upon the assumption that the biological processes related to mortality and fertility that create pubic morphological differences are the same in the past as they are in the present. That being said, it is unknown whether the age estimation methods used in this population are in fact accurate, as it is not possible to test the accuracy of age estimation methods which predict a biological age of individuals from past populations which carry no records of individuals’ chronological age.

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It was found that only 43 individuals in total had all three bone sections of interest

intact and microscopically viable for analysis. These 43 individuals were utilized to

examine intra-skeletal variability. In order to increase the sample size for age, sex and

site comparisons, statistical analysis was conducted separately for each bone element.

The sample demographics of the individuals utilized per bone are detailed below in three

separate tables (Tables 2, 3, 4). Statistical analyses of sex and age, therefore, included

ribs from 62 individuals, clavicles from 54 individuals and iliac crest sections from 62

individuals.

Age 20-34 years Age 35-49 years Age 50+ years Site Rib Samples Totals Site Males Females Males Females Males Females San Geronimo 4 12 8 3 2 7 36 El Yaral 2 1 1 1 1 1 7 Chiribaya Alta 5 5 3 4 1 1 19 Age and Sex Totals 11 18 12 8 4 9 62

Table 3. Demographics of sample used in rib analysis

Age 20-34 years Age 35-49 years Age 50+ years Site Clavicle Samples Totals Site Males Females Males Females Males Females San Geronimo 4 10 7 2 4 7 34 El Yaral 2 1 - 1 1 - 5 Chiribaya Alta 5 3 2 4 - 1 15 Age and Sex Totals 11 14 9 7 5 8 54

Table 4. Demographics of sample used in clavicle analysis

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Iliac Crest Age 20-34 years Age 35-49 years Age 50+ years Site Samples Totals Site Males Females Males Females Males Females San Geronimo 6 12 8 4 3 6 39 El Yaral - 1 1 2 - 2 6 Chiribaya Alta 5 4 3 3 1 1 17 Age and Sex Totals 11 17 12 9 4 9 62

Table 5. Demographics of sample used in iliac crest analysis

5.6 Hypotheses

Four hypotheses were tested in this study:

1. Sex differences exist in histomorphometric areal and perimeter/length

measurements in the Chiribaya sample.

It is expected that similar patterns of sex variation of bone microstructure exist in

the Chiribaya as demonstrated in other populations. In the rib and clavicle, males in

modern populations demonstrate greater periosteal expansion and endosteal resorption

with aging (Seeman 1999; Stini 2003), resulting in larger values for both perimeters and

areal measurements of the cortical envelope. It is expected that males will have larger

values for areal and relative areal measurements in the Chiribaya population. Although

males in other studies have been found to exhibit larger thickness measurements, this

does not translate to larger relative areal measurements. Females in the Chiribaya are

expected to exhibit larger relative cortical area measurements due to the effects of

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estrogens and childbirth; a pattern that is demonstrated in research where females tend toward higher relative cortical bone ratios than males, at least until the fifth decade

(Drusini et al. 2000).

In the iliac crest, Brockstedt et al. (1993) found that modern females have significantly smaller cortical width measurements at the iliac crest in a study of males and females of varying ages. It is expected that Chiribaya males exhibit thicker cortical bone in the iliac crest. It is expected that a similar pattern exists with the Chiribaya sample where Chiribaya males will exhibit larger perimeter and areal measurements than

Chiribaya females.

It is expected that mean trabecular thickness measures will be thinner in females than males, based on previous studies of trabecular thickness in archaeological populations (insert Macho 2005) and modern populations (insert trabecular thickness papers). It is expected that similar findings should be demonstrated among the Chiribaya as found within this archaeological population.

This hypothesis was statistically examined by skeletal sampling site (rib, clavicle, iliac crest) and by each variable examined. Table 6 details the specific hypotheses examining sex differences in the Chiribaya population.

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Statistical Hypotheses Examining Sex Differences:

H1a: Periosteal and endosteal perimeters significantly differ between the sexes.

H1b: Significant differences exist in areal and relative areal bone measurements between the sexes (total area, cortical area, medullary area, trabecular bone area, relative cortical area and relative trabecular area).

H1c: Males have thicker average trabecular width in iliac crest samples than females

H1d: Significant differences exist in total, cortical and medullary thickness/diameter between males and females.

Table 6. Statistical Hypotheses Examining Sex Differences

2. Differences in histomorphometric areal and length/perimeter measurements

exist among individuals from Chiribaya Alta, El Yaral and San Geronimo due

to variation of diet and general subsistence level activities.

Bone remodeling variations have been demonstrated in other studies of archaeological populations varying in subsistence (Burr et al. 1990; González-Reimers and Arnay-De-La-Rosa 1992; Richman et al. 1979; Stout and Lueck 1995). Stout and

Lueck (1995) reported the lowest intra-cortical remodeling rates among foraging groups in a comparison of archaeological populations while Richman et al. (1979) found the highest remodeling rates in Native American fisher-foragers in comparison to

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agriculturalists. Similar patterns are expected with respect to subsistence-level variation in the Chiribaya sample.

It is expected that both perimeters will be larger in San Geronimo individuals

(pescadores) due to periosteal apposition and endosteal expansion due to a net deficit in bone formation through the ARF sequence of bone remodeling. It is expected that San

Geronimo individuals (pescadores) will have smaller areal measurements in comparison to individuals from El Yaral (labradores) and Chiribaya Alta as a result of greater endosteal expansion. It is expected that San Geronimo (pescadores) individuals will have smaller relative areal measurements in comparison to individuals from El Yaral

(labradores) and Chiribaya Alta as a result of higher remodeling rates if there is a decrease in formation in the ARF sequence. It is expected that San Geronimo individuals

(pescadores) will demonstrate smaller thickness/diameter measurements in the iliac crest in comparison to individuals from El Yaral (labradores) and Chiribaya Alta as a result of higher remodeling rates.

With trabecular bone analysis, González-Reimers and Arnay-De-La-Rosa (1992) found that individuals subsisting on a low protein diet had a low protein intake and probably suffered from protein malnutrition. It is expected that San Geronimo individuals will exhibit thinner mean trabecular width measurements in comparison to individuals from El Yaral and Chiribaya Alta due to a disrupted bone balance in trabecular BMUs.

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remodeling due to a high intake of marine-based resources in comparison to individuals from El Yaral who subsisted in a higher amount of carbohydrates due to their agricultural reliance. The individuals from Chiribaya Alta are expected to either be intermediary between San Geronimo and El Yaral as it is thought to be composed of elites drawn from other Chiribaya sites or exhibit larger relative bone areal measurements due to having access to premiere dietary resources.

This hypothesis was statistically examined by skeletal sampling site (rib, clavicle, iliac crest) and by each variable examined. Table 7 details the specific hypotheses examining subsistence-level differences in the Chiribaya population.

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Statistical Hypotheses Examining Subsistence Differences:

H2a: Periosteal and endosteal perimeters differ significantly according to subsistence- level differences at Chiribaya Alta, El Yaral, and San Geronimo.

H2b: Total, cortical, medullary and trabecular areas significantly according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

H2c: There is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya Alta, El Yaral, and San

Geronimo.

H2d: There is a significant difference in trabecular width in iliac crest samples between individuals at Chiribaya Alta, El Yaral and San Geronimo.

H2e: Total, cortical and medullary thickness/diameter of iliac crest sections will significantly differ in individuals according to subsistence-level differences at Chiribaya

Alta, El Yaral, and San Geronimo.

Table 7. Statistical Hypotheses Examining Subsistence Differences

3. Differences in histomorphometric areal and length measurements exist among

different age groups due to the effects of increasing age.

It is expected the periosteal and endosteal perimeters of the rib and clavicle will both demonstrate differences with relation to age. Past research on modern populations indicates a general trend with an increase in the periosteal perimeter due to apposition with age, with an accompanying increase in the endosteal perimeter due to resorption 126

(Epker and Frost 1965; Epker et al. 1965). It was discussed previously that males have been found to experience a greater amount of periosteal expansion during aging (Seeman

1999; Stini 2003), but it is expected that within both sexes, that larger perimeters will be noted in individuals from the older age categories.

In relation to the expansion of the periosteal and endosteal perimeters, it is expected that the medullary and total areas will be larger with relationship to increasing age due to expansion of the cortex, while cortical area and trabecular area will be reduced. Research on modern populations indicates a general decrease in bone mass with age (Epker et al. 1965; Sedlin et al. 1963b) in both the cortical and trabecular envelopes

(Mellish et al. 1987; Ostertag et al. 2009). It is expected the same pattern will be found in this archaeological population with respect to increasing age.

Research with clinical samples has demonstrated that the majority of trabeculae decrease in thickness with increasing age (Dalle Carbonare et al. 2005; Mellish et al.

1989; Parfitt et al. 1983) which should be evident in a comparison of mean trabecular width of all trabeculae measured. It is also suggesting that the structure of trabeculae move from a plate-like to a rod-like structure with increasing age (Ding and Hvid 2000), which when viewed two-dimensionally, will be demonstrated as thinner trabeculae overall because plates when viewed from the side will appear as struts.

Previous research on modern samples indicates a relationship between decreased cortical thickness and a reduction in cortical bone mass in the iliac crest with increasing age (Christiansen et al. 1993; Vedi et al. 2011). It is expected that cortical width values will be negatively correlated with increasing age cohort in this sample, whereas

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medullary thickness will either remain the same or increase due to remodeling at the endocortical envelope.

This hypothesis was statistically examined by skeletal sampling site (rib, clavicle, iliac crest) and by each variable examined. Table 8 details the specific hypotheses examining age differences in the Chiribaya population.

Statistical Hypotheses Examining Age Differences:

H3a: Periosteal and endosteal perimeters increase significantly with age.

H3b: Total, cortical, medullary and trabecular bone areas will exhibit significant relationships to increasing age.

H3c: There is a significant difference in relative cortical area and relative trabecular area between young, middle and older adults.

H3d: A significant difference exists in trabecular width in iliac crest samples among age groups.

H3e: Significant differences in the total, cortical and medullary thickness/diameter of iliac crest sections exist among age groups.

Table 8. Statistical Hypotheses Examining Age Differences

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4. Variability in relative area measurements due to differential loading throughout

the skeleton exist.

Previous research in both modern and archaeological samples indicates that intra- skeletal variability is intricately linked to the specific mechanical loading environment of each bone (Doyle 2011; Peck and Stout 2007). It is expected that due to each region of the skeleton being subjected to differential loading, the bone microarchitecture will reflect the function of each. In this study, it is expected that the clavicle will have a higher relative cortical area than the iliac crest and rib. This is expected because throughout life, the clavicle under greater amounts of loading, and dynamic loading rather than static loading as in the rib and iliac crest. The clavicle also has higher amounts of cortical bone with a smaller marrow cavity with less bone marrow than the rib. Thus, a loss in bone mass should appear first in the iliac crest and rib.

This hypothesis was statistically examined by skeletal sampling site (rib, clavicle, iliac crest) for relative cortical area. Table 9 offers the statistical hypothesis that was tested to examine this research question in the Chiribaya population.

Statistical Hypothesis Examining Intra-skeletal Variability:

H4: Relative cortical area differs significantly among the rib, clavicle and iliac crest.

Table 9. Statistical Hypothesis Examining Intra-skeletal Variability

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5.7 Analyses Employed

Statistical analysis was performed using SAS® 9.2 statistical software and

ImageJ® was employed for image analysis. The SNR plugin (Sage 2010) was added to determine the root mean square error (RMSE) of images, which yielded RMSE values of

0.0 for compiled photomicrographs used in analysis.

Intra-observer error was initially conducted for each variable of each bone sample using a one-way ANOVA test with a significance level of 0.05. RMSE values are exhibited for each variable for intra-observer comparison in Chapter 6.

Normality was examined for datasets by bone and variable. Amounts of skewness

(g1) and kurtosis (g2) were obtained using the univariate procedure in SAS® (2011). G1 and g2 values were further applied to the K2 omnibus test of normality which detects deviations in normality due to skewness or kurtosis (D'Agostino and Pearson 1973;

D'Agostino et al. 1990). Variables with distributions that had high degrees of skewness and/or kurtosis were square root transformed or log(square root) transformed.

Sex comparisons and site comparisons of Chiribaya Alta and San Geronimo were conducted using a series of t-tests. The elements available from the El Yaral sample were too few in number to obtain a normal distribution even after applying square root and log transformations; therefore, the data from the El Yaral sample are described qualitatively in the results section. Age group comparisons between young, middle and old adults were conducted using a series of one-way and two-way ANOVA tests. Intra-skeletal variability was examined using 43 individuals who had all three skeletal sites present. A randomized

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block ANOVA was applied to test significance of relative cortical area (Rel.Ct.Ar.) between the three skeletal sites of each individual.

5.8 Chapter Summary This chapter describes the skeletal samples chosen (rib, clavicle, iliac crest), methods of procurement and preparation, methods of microscopic analysis, variables examined, sample demographics, hypotheses, and statistical analyses employed in this study.

The rib was selected because it is commonly used in hard tissue histology and is thought to reflect the effects of age, disease, and growth earlier than appendicular bones.

The clavicle was selected because it is thought to be more dynamically loaded than the rib as it is a major strut for the upper limb and has also been shown to exhibit age and sex differences. The iliac crest was selected as a third area of the skeleton because it is comprised of both cortical and trabecular bone, and is a primary location for bone biopsy studies in clinical research.

Rib and clavicle sections were removed at the midshaft where 5-6 cm of each rib and clavicle was removed. Thin sections were produced from the mid-region of the section and prepared using standard histological methods to a thickness of ~70-100 micrometers (µm). Iliac crest sections were removed at the site of Bourdier’s triangle; 2 cm below the iliac crest and 2 cm behind the anterior superior iliac spine. The region of interest of iliac crest sections corresponds to the thickness of trephines used to remove bone samples in clinical biopsies.

Thin sections were imaged using an Olympus ® BX51 microscope with an attached SPOT InsightTM QE Color 4.2.1 camera and overlapping photomicrographs were

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taken of the entire section of bone at a resolution of 100x magnification. Variables were then collected using ImageJ® software with the use of a PC tablet and stylus.

Variables measured in the rib and clavicle include total subperiosteal area

(Tt.Ar.), endosteal area (Es.Ar.), periosteal perimeter (Ps.Pm.), endosteal perimeter

(Es.Pm.), cortical area (Ct.Ar.), and relative cortical area (Rel.Ct.Ar.). Variables measured in the iliac crest include total area (Tt.Ar.), marrow cavity area (Es.Ar.), cortical area (Ct.Ar.), trabecular area (Tb.Ar.), mean trabecular width (Tb.Wi.), total diameter of section (Tt.Dm.), cortical diameter (Ct.Dm.), marrow cavity diameter

(Es.Dm.), relative cortical area (Rel.Ct.Ar.), and relative trabecular area (Rel.Tb.Ar.).

Age and sex estimates of the Chiribaya sample were taken from the dissertation projects of Burgess (1999) and Lozada Cerna (1998). Individuals were characterized by sex, age group (young, middle, old), subsistence pattern as related to geographic site

(Chiribaya Alta, El Yaral, San Geronimo). A total of 62 ribs, 54 clavicles and 62 iliac crest sections were used to test the following three hypotheses: a) sex differences exist in histomorphometric areal and perimeter/length measurements in the Chiribaya sample, b) differences in histomorphometric areal and perimeter/length measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo due to variation in diet and general subsistence level activities, and c) differences in histomorphometric areal and length measurements exist among differing age groups due to the effects of increasing age.

Measurement of relative cortical area (Rel.Ct.Ar.) was recorded on 43 individuals with all three skeletal sampling sites intact to test the fourth hypothesis: variability in relative area measurements due to differential loading throughout the skeleton exist.

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Statistical analysis was performed using SAS®9.2 statistical software.

Intraobserver error was conducted using a one-way ANOVA test with a significance value of 0.05. Normality was examined using the K2 omnibus test of normality and variables with high degrees of skewness and kurtosis were transformed using either the square root transformation or log(square root) transformation. T-tests were used to examine sex and subsistence-level differences of the Chiribaya sample. One-way and two-way ANOVA tests were employed to examine differences between age categories, and a randomized block ANOVA was used to examine intra-skeletal variability in relative cortical area.

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Chapter 6: Results

The contents of this chapter represent the statistical and non-statistical examination of data measured within the rib, clavicle, and iliac crest data within the

Chiribaya sample. This chapter includes a description of normality measures employed, measures of intra-observer error, and statistical tests applied for comparative analyses.

Variables were categorized into datasets and normality was determined for each dataset before statistical analyses were conducted. Descriptive statistics are also listed for variables that could not be tested significantly.

6.1 Intra-Observer Error

Intra-observer error rates were conducted using a one-way ANOVA procedure using the initial measurements taken for 30 individuals and comparing them to measurements that were re-taken on the same individuals three months later. The root mean square error in mm2 (RMSE) for each variable is indicated in Table 10.

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RMSE RMSE Variable (mm2) Variable (mm2) Rib Total Area 0.26073 Clavicle Relative Cortical Area 0.00294 Rib Periosteal Perimeter 0.15969 Iliac Crest Cortical Diameter 0.07449 Rib Medullary Area 0.66365 Iliac Crest Medullary Diameter 0.04852 Rib Endosteal Perimeter 0.42935 Iliac Crest Total Diameter 0.04205 Rib Cortical Area 0.7451 Iliac Crest Total Area 0.58284 Rib Relative Cortical Area 0.00981 Iliac Crest Medullary Area 0.48336 Clavicle Total Area 0.24075 Iliac Crest Cortical Area 0.58879 Clavicle Periosteal Perimeter 0.11465 Iliac Crest Relative Cortical Area 0.00851 Iliac Crest Average Trabecular Clavicle Medullary Area 0.23691 Width 0.01765 Clavicle Endosteal Perimeter 0.27171 Iliac Crest Total Trabecular Area 0.3049 Iliac Crest Relative Trabecular Clavicle Cortical Area 0.32036 Area 0.00695

Table 10. Intra-observer Error Rates for Each Variable

6.2 Normality Tests

Datasets from each bone used in sex, site, age, and intra-skeletal variability analysis was checked for normality by obtaining G1 and G2 values through the univariate procedure in SAS®. G1 and G2 values were then applied to the K2 omnibus test of normality, where the critical value for K2 is 5.991 (D'Agostino and Pearson 1973;

D'Agostino et al. 1990). The values for G1, G2, the z-scores for each and the K2 value for each variable are presented in Table 11-Table 19. Variables that were transformed are indicated with a “*” with the adjusted normality measures indicated on the line below the original results. 135

6.2.1 Normality Measures by Bone for Examination of Sex

Rib Normality by Sex n g1 g2 zg1 zg2 K2 (Crit. Value=5.991) Male Tt.Ar. 27 1.03786 2.7593 2.2346 2.23359 9.9823122 *Sqrt Trans. Male Tt.Ar. 27 0.42271 1.1535 0.9838 1.328 2.7314599 Female Tt.Ar. 35 -0.2232 0.0992 -0.5902 0.36369 0.4805875 *Sqrt Trans. Female Tt.Ar. 35 -0.7124 1.1958 -1.7879 1.45845 5.3238091 Male Es.Ar. 27 1.04678 2.7459 2.2507 2.22765 10.028105 *Sqrt Trans. Male Es.Ar. 27 0.29054 1.2517 0.6829 1.39839 2.4218851 Female Es.Ar. 35 0.04555 -0.4508 0.1212 0.78181 0.6257737 *Sqrt Trans. Female Es.Ar. 35 -0.4106 0.0217 -1.0701 0.25946 1.2124169 Male Ct.Ar. 27 0.63123 -0.0517 1.4379 0.29706 2.1557372 Female Ct.Ar. 35 -0.3089 -0.2622 -0.8122 0.5677 0.9818625 Male Rel.Ct.Ar. 27 0.22689 -0.1315 0.5352 0.39169 0.4398688 Female Rel.Ct.Ar. 35 0.10167 0.5031 0.2703 0.83743 0.7743351 Male Ps.Pm. 27 -0.0999 -0.4022 -0.2367 0.68446 0.5245232 Female Ps.Pm. 35 -0.1905 0.1076 -0.5047 0.37464 0.3950515 Male Es.Pm. 27 0.11434 0.0114 0.2709 0.24769 0.1347139 Female Es.Pm. 35 -0.255 -0.279 -0.6732 0.58771 0.7985977 Table 11. Rib Normality Measures of Males and Females *Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

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Clavicle Normality by Sex n g1 g2 zg1 zg2 K2 (Crit. Value=5.991) Male Tt.Ar. 25 1.1107 0.8354 2.2967 1.0580 6.3943 *Sqrt Trans. Male Tt.Ar. 25 0.8192 0.2819 1.7659 0.5499 3.4209 Female Tt.Ar. 29 0.6668 0.4873 1.5577 0.7828 3.0393 *Sqrt Trans. Female Tt.Ar. 29 0.33753 0.0233 0.8159 0.2625 0.7345 Male Es.Ar. 25 1.4325 2.7651 2.8163 2.1951 12.7499 *Sqrt Trans. Male Es.Ar. 25 0.6362 1.1214 1.4027 1.2773 3.5988 Female Es.Ar. 29 0.7974 1.1481 1.8303 1.3501 5.1728 *Sqrt Trans. Female Es.Ar. 29 -0.1257 0.0379 -0.3072 0.2809 0.1733 Male Ct.Ar. 25 0.8652 0.4721 1.8536 0.7392 3.9822 Female Ct.Ar. 29 0.7731 1.0303 1.7807 1.2603 4.7592 Male Rel.Ct.Ar. 25 -0.1852 0.0724 -0.4230 0.3196 0.2811 Female Rel.Ct.Ar. 29 -0.2021 -0.0155 -0.4925 0.2526 0.3063 Male Ps.Pm. 25 0.6125 -0.3005 1.3541 0.5692 2.1575 Female Ps.Pm. 29 0.4174 0.5139 1.0022 0.8090 1.6589 Male Es.Pm. 25 -0.0846 -0.3321 -0.1937 0.6015 0.3994 Female Es.Pm. 29 0.1203 0.8491 0.2942 1.1133 1.3261

Table 12. Clavicle Normality Measures of Males and Females *Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

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Iliac Crest Normality by Sex n g1 g2 zg1 zg2 K2 (Crit. Value=5.991) Male Tt.Ar. 27 -0.0044 1.3324 -0.0105 1.45429 2.1150833 Female Tt.Ar. 34 -0.133 0.4071 -0.3486 0.729 0.6529628 Male Es.Ar. 27 0.09304 0.0351 0.2205 0.27677 0.1252208 Female Es.Ar. 34 0.18099 -0.088 0.4736 0.34802 0.3454202 Male Ct.Ar. 27 -0.4777 -0.7546 -1.1062 1.01233 2.2485421 Female Ct.Ar. 34 -0.0383 -0.1512 -0.1006 0.42907 0.1942281 Male Tb.Ar. 27 1.21807 1.7575 2.5489 1.72393 9.4689836 *Sqrt Trans. Male Tb.Ar. 27 0.66581 0.3903 1.5104 0.67242 2.7333103 Female Tb.Ar. 34 0.37861 -0.0569 0.9773 0.30711 1.0493997 *Sqrt Trans. Female Tb.Ar. 34 -0.5284 0.8688 -1.3426 1.1799 3.1946809 Male Rel.Ct.Ar. 27 0.55657 -0.3323 1.2786 0.61266 2.0100605 Female Rel.Ct.Ar. 34 0.16974 -0.7528 0.4444 1.07573 1.3546889 Male Rel.Tb.Ar. 27 0.68489 1.0412 1.55 1.24421 3.9504887 Female Rel.Tb.Ar. 34 0.3607 -0.2346 0.9325 0.53158 1.1522128 Male Tb.Wi. 27 1.46878 2.9348 2.9496 2.3091 14.031904 *Log(sqrt) Trans. Male Tb.Wi. 27 0.72782 1.3007 1.6382 1.43252 4.7357023 Female Tb.Wi. 34 1.26392 3.2031 2.8604 2.55568 14.713554 *Log(sqrt) Trans. Female Tb.Wi. 34 0.00901 1.2457 0.0237 1.48455 2.2044367 Male Tt.Dm. 27 -0.0601 3.6415 -0.1426 2.5824 6.689111 Female Tt.Dm. 34 0.22673 0.7295 0.5919 1.05414 1.4615831 Male Ct.Dm. 27 -0.2987 -0.6946 -0.7018 0.96014 1.4143863 Female Ct.Dm. 34 0.39113 -0.3137 1.0084 0.62435 1.4067683 Male Es.Dm. 27 0.37115 1.5145 0.8675 1.57464 3.2320248 Female Es.Dm. 34 0.58913 0.158 1.4857 0.43756 2.398743 Table 13. Iliac Crest Normality Measures of Males and Females

*Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

(Total diameter for males could not be normalized using transformations)

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6.2.2 Normality Measures by Bone for Examination of Subsistence by Site Affiliation

Rib Normality by Site n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Chiribaya Alta Total Area 36 -0.051831 -0.189373 -0.139661 0.4811534 0.2510138 El Yaral Total Area 7 -0.114295 -1.107885 unable to test San Geronimo Total Area 19 1.256664 3.763159 2.300318 2.4332289 11.212066 Chiribaya Alta Per. Perimeter 36 0.309823 -0.370799 0.8247223 0.6981342 1.1675581 El Yaral Per. Perimeter 7 -0.882354 0.445853 unable to test San Geronimo Per. Perimeter 19 0.145369 1.093966 0.2943888 1.165015 1.4439247 Chiribaya Alta Medullary Area 36 0.047029 -0.733248 0.136729 1.0740015 1.1695395 El Yaral Medullary Area 7 -0.540679 -0.15247 unable to test San Geronimo Medullary Area 19 1.055393 2.338589 1.9828132 1.8678761 7.4205091 Chiribaya Alta End. Perimeter 36 0.544466 0.303845 1.4146919 0.62060789 2.3865073 El Yaral End. Perimeter 7 -1.651384 3.161139 unable to test San Geronimo End. Perimeter 19 -0.195345 0.73572 -0.395054 0.9035805 0.972525 Chiribaya Alta Cortical Area 36 0.223773 -0.480213 0.5991981 0.8189768 1.0297614 El Yaral Cortical Area 7 0.275242 -1.365317 unable to test San Geronimo Cortical Area 19 0.3927 1.528983 0.7869359 1.4416362 2.6975832 Chiribaya Alta. Rel. Cortical Area 36 0.194614 0.449775 0.5219522 0.7860557 0.8903177 El Yaral Rel. Cortical Area. 7 -0.526714 -0.34877 unable to test San Geronimo Rel. Cortical Area 19 -0.071237 -1.112409 -0.144451 1.1775823 1.4075661 Table 14. Rib Normality Measures by Site

*Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed (El Yaral sample was too small to normalize for statistical analysis)

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Clavicle Normality by Site n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Chiribaya Alta Total Area 34 0.11867 -0.5072 0.31129 0.8355351 0.7950203 El Yaral Total Area 5 -0.3046 -2.9472 unable to test San Geronimo Total Area 15 0.57405 -0.907 1.026722 0.9626443 1.9808427 Chiribaya Alta Per. Perimeter 34 0.02202 -0.2023 0.05785 0.492438 0.2458418 El Yaral Per. Perimeter 5 -0.145 -2.6399 unable to test San Geronimo Per. Perimeter 15 0.3018 -1.4304 0.547293 1.2934689 1.9725918 Chiribaya Alta Medullary Area 34 0.49504 0.12066 1.262703 0.3903029 1.746754 El Yaral Medullary Area 5 -0.0679 -2.5441 unable to test San Geronimo Medullary Area 15 1.36436 1.50087 2.267269 1.3341201 6.9203848 Chiribaya Alta End. Perimeter 34 -0.0936 -0.0738 -0.24566 0.329382 0.1688412 El Yaral End. Perimeter 5 -0.3084 -2.6844 unable to test San Geronimo End. Perimeter 15 0.29878 -0.5745 0.541876 0.7209206 0.8133562 Chiribaya Alta Cortical Area 34 0.28008 -0.5585 0.728714 0.8881461 1.3198282 El Yaral Cortical Area 5 0.79238 -0.572 unable to test San Geronimo Cortical Area 15 0.42075 -0.9217 0.759136 0.972662 1.5223592 Chiribaya Alta. Rel. Cortical Area 34 -0.0134 -0.3169 -0.035341 0.6280276 0.3956676 El Yaral Rel. Cortical Area. 5 0.0114 -2.0102 unable to test San Geronimo Rel. Cortical Area 15 -0.7203 0.21823 -1.274992 0.4273927 1.8082685 Table 15. Clavicle Normality Measures by Site

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Iliac Crest Normality by Site n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Chiribaya Alta Total Area 39 -0.1327 0.19069 -0.369674 0.4892755 0.3760496 El Yaral Total Area 5 -0.9821 1.5919 unable to test San Geronimo Total Area 17 0.79695 1.90484 1.475095 -0.108842 2.1877513 Chiribaya Alta Cortical Area 39 -0.1999 -0.4143 -0.555426 0.7615258 0.8884195 El Yaral Cortical Area 5 -0.1375 -2.5334 unable to test San Geronimo Cortical Area 17 0.04935 -0.0436 0.095206 0.2740923 0.0841908 Chiribaya Alta Medullary Area 39 0.21591 -0.3762 0.599213 0.7175394 0.8739183 El Yaral Medullary Area 5 0.85223 2.05725 unable to test San Geronimo Medullary Area 17 0.98658 1.55372 1.790794 -0.108842 3.2187908 Chiribya Alta Trab. Area 39 1.29367 2.74203 3.067419 -0.107443 9.4206017 El Yaral Trab. Area 5 0.66692 1.69142 unable to test San Geronimo Trab. Area 17 0.42347 -0.7239 0.806984 0.865681 1.4006264 Chiribaya Alta. Rel. Cortical Area 39 0.24082 -0.3869 0.667308 0.7300072 0.9782109 El Yaral Rel. Cortical Area. 5 -1.7844 3.45943 unable to test San Geronimo Rel. Cortical Area 17 1.0056 1.17419 1.821499 1.1810395 4.7127123 Chiribaya Alta Rel. Trab. Area 39 0.06387 0.35517 0.178302 0.6928529 0.5118368 El Yaral Rel. Trab. Area 5 1.0505 0.68525 unable to test San Geronimo Rel. Trab. Area 17 0.13773 1.02369 0.265419 1.0810398 1.2390944 Chiribaya Alta Mean Trab. Width 39 1.18374 2.56698 2.864571 -0.107443 8.2173104 El Yaral Mean Trab. Width 5 0.51031 -0.7119 unable to test San Geronimo Mean Trab. Width 17 -0.3775 -0.1042 -0.721202 0.3339275 0.6316398 Chiribaya Alta Total Diameter 39 0.25079 1.16901 0.694488 1.4814058 2.676877 El Yaral Total Diameter 5 -0.4124 -0.5078 unable to test San Geronimo Total Diameter 17 0.33013 -0.0192 0.632177 -0.108842 0.4114947 Chiribaya Alta Cortical Diameter 39 0.13395 -0.524 0.373229 0.883365 0.9196334 El Yaral Cortical Diameter 5 0.9565 1.38526 unable to test San Geronimo Cortical Diameter 17 0.134 0.03782 0.258257 -0.108842 0.0785431 Chiribaya Alta Medullary Diameter 39 0.66608 0.73718 1.760707 1.103119 4.3129778 El Yaral Medullary Diameter 5 1.72883 3.23655 unable to test San Geronimo Medullary Diameter 17 0.47464 -0.0773 0.901692 -0.108842 0.8248946 Table 16. Iliac Crest Normality Measures by Site

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6.2.3 Normality Measures by Bone for Examination of Age

Rib Normality by Age n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Young Adult Tt.Ar. 29 1.00748 3.51638 2.24 2.57938 11.673211 *Sqrt Trans. Young Adult Tt.Ar. 29 0.08457 2.2434 0.20692 2.02611 4.1479548 Middle Adult Tt.Ar. 20 0.20221 -0.7839 0.41827 0.95471 1.0864146 *Sqrt Trans. Middle Adult Tt.Ar. 20 -0.0258 -0.7585 -0.0536 0.9349 0.8769169 Older Adult Tt.ar. 13 -0.0534 -0.8261 -0.0912 0.86504 0.7566096 *Sqrt Trans.Older Adult Tt.Ar. 13 -0.2973 -0.5754 -0.5056 0.68814 0.7291522 Young Adult Ps.Pm. 29 0.51183 0.45378 1.21771 0.7492 2.0441218 Middle Adult Ps.Pm. 20 -0.2004 -0.7342 -0.4146 0.91575 1.0104793 Older Adult Ps.Pm. 13 0.03892 1.39524 0.06645 1.21943 1.4914147 Young Adult Es.Ar. 29 1.06249 2.25451 2.34226 2.03182 9.6144733 *Sqrt Trans. Young Adult. Es.Ar. 29 0.26759 0.61719 0.64987 0.90777 1.2463798 Middle Adult Es.Ar. 20 0.555 -0.1464 1.12349 0.38788 1.4126812 *Sqrt Trans. Middle Adult Es.Ar. 20 0.27369 -0.4791 0.5645 0.70292 0.8127542 Older Adult Es.Ar. 13 -0.5885 -0.4868 -0.9887 0.62202 1.3644809 *Sqrt Trans. Older Adult Es.Ar. 13 -0.9201 0.46014 -1.5125 0.60175 2.6496287 Young Adult Es.Pm. 29 0.68919 0.75944 1.60547 1.03636 3.6515971 Middle Adult Es.Pm. 20 -0.0531 -0.7852 -0.1102 0.95571 0.9255185 Older Adult Es.Pm. 13 0.30765 4.11092 0.52297 2.36515 5.8674468 Young Adult Ct.Ar. 29 0.20225 0.84326 0.49289 1.10841 1.4715239 Middle Adult Ct.Ar. 20 0.18726 -0.7915 0.38752 0.9606 1.0729199 Older Adult Ct.Ar. 13 0.64681 -0.7622 1.08311 0.82128 1.8476163 Young Adult Rel.Ct.Ar. 29 -0.1115 -0.07 -0.2726 0.32095 0.1772993 Middle Adult Rel.Ct.Ar. 20 -0.1393 -0.0139 -0.2886 0.24828 0.1449101 Older Adult Rel.Ct.Ar. 13 0.41938 0.66839 0.71016 0.75544 1.0750135

Table 17. Rib Normality Measures for Each Age Category *Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

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Clavicle Normality by Age n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Young Adult Tt.Ar. 25 1.00726 1.09568 2.1151 1.25855 6.0575705 *Sqrt Trans. Young Adult Tt.Ar. 25 0.55249 0.12048 1.22919 0.37473 1.6513366 Middle Adult Tt.Ar. 16 1.28672 1.1518 2.20687 1.14598 6.1835513 *Sqrt Trans. Middle Adult Tt.Ar. 16 1.05306 0.36721 1.85423 0.56598 3.7584932 Older Adult Tt.Ar. 13 -0.1374 1.47687 -0.2344 1.26567 1.6568354 *Sqrt Trans. Older Adult Tt.Ar. 13 -0.5103 1.80121 -1.0368 1.61785 3.6923151 Young Adult Es.Ar. 25 1.06333 1.7167 2.21441 1.66568 7.67811 *Log(sqrt) Trans. Young Adult Es.Ar. 25 0.41585 0.27565 0.77206 -0.1081 0.6077624 Middle Adult Es.Ar. 16 2.03482 5.52364 3.17763 2.85349 18.239768 *Log(sqrt)Trans. Middle Adult Es.Ar. 16 0.41585 0.27565 0.77206 0.48655 0.8328069 Older Adult Es.Ar. 13 0.81992 1.48055 1.35779 1.26773 3.4507318 *Log(sqrt) Trans. Older Adult Es.Ar. 13 -0.6441 0.8507 -1.0787 0.88164 1.9408987 Young Adult Ct.Ar. 25 0.94569 0.34241 2.00352 0.61203 4.388656 Middle Adult Ct.Ar. 16 0.8378 0.93814 1.50741 1.00352 3.2793288 Older Adult Ct.Ar. 13 -0.6043 0.33404 -1.0144 0.50323 1.2823102 Young Adult Rel.Ct.Ar. 25 0.06285 -0.6976 0.14403 0.943 0.9099891 Middle Adult Rel.Ct. 16 -0.6482 0.77247 -1.1852 0.88575 2.182992 Older Adult Rel.Ct.Ar. 13 -0.5737 -0.4948 -0.9646 0.62808 1.32501 Young Adult Ps.Pm. 25 0.43587 -0.2202 0.98032 0.48465 1.19592 Middle Adult Ps.Pm. 16 0.98776 -0.1532 1.75121 0.37552 3.2077636 Older Adult Ps.Pm. 13 -0.0977 2.19151 -0.1668 1.63092 2.6877449 Young Adult Es.Pm. 25 -0.0983 -0.6633 -0.2252 0.91328 0.8847871 Middle Adult Es.Pm. 16 0.65849 -0.1448 1.20316 0.36774 1.58282 Older Adult Es.Pm. 13 0.01322 0.11548 0.02258 0.32153 0.1038933 Table 18. Clavicle Normality Measures for Each Age Category *Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

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Iliac Crest Normality by Age n g1 g2 zg1 zg2 K2 (Crit. value=5.991) Young Adult Tt.Ar. 28 0.0093 0.15936 0.02243 0.42628 0.1822197 *Log(sqrt) Trans. Young Adult Tt.Ar. 28 -0.9302 1.80737 -2.0649 1.76991 7.396228 Middle Adult Total Area 20 1.44997 2.9872 2.63091 2.17386 11.647346 *Log(sqrt) Trans. Middle Adult Tt.Ar. 20 0.83293 1.07842 1.63953 1.17121 4.0597983 Older Adult Tt.Ar. 13 0.1018 -0.6309 0.17375 0.72858 0.5610141 *Log(sqrt) Trans. Older Adult Tt.Ar. 13 -0.7816 0.10644 -1.2977 0.31369 1.7824897 Young Adult Ct.Ar. 28 -0.19 -0.591 -0.4561 0.87507 0.9737628 Middle Adult Ct.Ar. 20 0.09756 -1.0458 0.20233 1.14836 1.3596627 Older Adult Ct.Ar. 13 -0.6649 -0.1396 -1.1123 0.3423 1.3543028 Young Adult Es.Ar. 28 0.1545 -0.0361 0.37147 0.27846 0.2155267 Middle Adult Es.Ar. 20 0.79495 1.09095 1.57146 1.17991 3.8616939 Older Adult Es.Ar. 13 0.45381 -0.9586 0.76736 0.95297 1.4966919 Young Adult Tt.Tr.Ar. 28 0.10093 -0.757 0.24305 1.0245 1.1086801 *Log(sqrt) Trans. Young Adult Tt.Tr.Ar. 28 -1.0963 1.6832 -2.3717 1.69581 8.5009451 Middle Adult Tt.Tr. Ar. 20 1.66294 2.584 2.92572 2.00385 12.575219 *Log(sqrt) Trans. Middle Adult Tt.Tr.Ar. 20 0.95485 0.14368 1.85247 0.38514 3.5799736 Older Adult Tt. Tr. Ar. 13 0.59303 -0.4179 0.99606 0.56924 1.3161693 *Log(sqrt) Trans. Older Adult Tt.Tr.Ar. 13 -1.6372 3.83768 -2.5189 2.27684 11.528845 Young Adult Rel.Ct.Ar. 28 -0.0158 -0.5084 -0.038 0.79641 0.6357097 Middle Adult Rel.Ct.Ar. 20 0.97567 0.99887 1.88799 1.11504 4.8077999 Older Adult Rel.Ct.Ar. 13 0.26319 -1.2082 0.44793 1.10943 1.4314778 Young Adult Rel.Tr. Ar. 28 0.23319 -0.4439 0.55875 0.73278 0.8491721 Middle Adult Rel.Tb.Ar. 20 1.17974 0.875 2.22297 1.02424 5.9906513 Older Adult Rel.Tb.Ar. 13 0.05052 0.83891 0.08626 0.87369 0.7707833 Young Adult Tr. Wi. 28 1.71952 4.47559 3.35296 2.87664 19.517387 *Log(sqrt) Trans. Young Adult Tr.Wi. 28 0.72697 1.13339 1.66097 1.32634 4.5180154 Middle Adult Tr. Wi. 20 1.33714 2.12942 2.46538 1.78992 9.2818815 *Log(sqrt) Trans. Middle Adult Tr.Wi. 20 0.65208 0.87157 1.30837 1.02166 2.7556125 Older Adult Tr. Wi. 13 -0.0096 1.20851 -0.0164 1.1096 1.2314843 *Log(sqrt) Trans. Older Adult Tr.Wi. 13 -1.1447 2.95426 -1.8472 2.27684 8.5960664 Young Adult Tt.Dm. 28 0.3538 2.04926 0.84119 1.90601 4.3404664 Middle Adult Tt.Dm. 20 0.03587 -0.4239 0.07445 0.65386 0.4330747 Older Adult Tt.Dm. 13 0.46865 0.30876 0.79195 0.48294 0.8604162 Young Adult Es.Dm. 28 0.08911 0.36247 0.23146 0.64956 0.4679979 Middle Adult Es.Dm. 20 -0.041 -0.5893 -0.0851 0.79759 0.6433924 Older Adult Es.Dm. 13 0.90833 0.46674 1.49451 0.60678 2.6017403 Young Adult Ct.Dm. 28 0.30423 -0.7509 0.72591 1.01918 1.5656735 Middle Adult Ct.Dm. 20 -0.0287 -0.0198 -0.0595 0.2547 0.0684095 Older Adult Ct.Dm. 13 0.69658 -0.1225 1.16298 0.32757 1.4598159 Table 19. Iliac Crest Test Normality Measures for Each Age Category *Indicates the variable was square root transformed; normality values of transformed variables are listed below the variables that were transformed

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6.2.4 Normality Measures for Examination of Intra-Skeletal Variability

n g1 g2 zg1 zg2 K2 (Crit. Value=5.991) Rib 43 0.2198 -0.0622 0.63681 0.31677 0.5058772 Clavicle 43 -0.0096 -0.2764 -0.0282 0.60986 0.3727186 Iliac Crest 43 0.48838 -0.4755 1.37901 0.85095 2.6257741 Table 20. Intra-skeletal Variability Normality Measures

6.3 Examination of Sex

In order to test Hypothesis 1, Sex differences exist in the histomorphometric areal and perimeter/length measurements in the Chiribaya sample, a series of t-tests were applied on all variables measured in the rib, clavicle and iliac crest to examine sex-based differences. When the variances were equal for male and female datasets, the pooled results were used. When variances were significantly different in the male and female datasets, the Sattherthwaite results were used. The following section details the statistical results for each t-test conducted. Histograms that accompany each comparison are included in Appendix A.

6.3.1 Sex Comparisons Using Rib Variables

A series of t-tests were conducted for each rib variable. The output values for the t-tests of each rib variable are summarized in Table 21. T-test results for male/female comparisons of all rib data are summarized in Table 22, with significant values highlighted in grey.

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Variable/Sex N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Male 27 8.7631 1.3264 0.2553 6.1841 12.4702 Tt.Ar. Female 35 8.1728 1.0935 0.1848 4.86 9.9255 Ct.Ar. Male 27 36.28 11.322 2.1789 21.257 65.2183 Ct.Ar. Female 35 34.9081 9.0366 1.5275 13.4944 52.8785 Es.Ar. Male 27 6.399 1.143 0.22 3.8324 9.502 Es.Ar. Female 35 5.6637 0.9995 0.169 3.182 7.4461 Rel.Ct.Ar. Male 27 0.4701 0.074 0.0142 0.3146 0.6241 Rel.Ct.Ar. Female 35 0.5235 0.0832 0.0141 0.3347 0.7421 Ps.Pm. Male 27 37.856 6.413 1.2342 24.4866 49.6315 Ps.Pm. Female 35 34.8682 4.441 0.7512 24.6906 44.0916 Es.Pm. Male 27 34.6631 7.3242 1.4095 18.7555 49.8589 Es.Pm. Female 35 31.1456 4.7095 0.796 21.5883 40.3567 Table 21. T-test Output of Rib Variables Analyzed for Sex Comparisons

Rib Sex T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.2886 Pooled 60 1.92 0.0596 Ct.Ar. 0.216 Pooled 60 0.53 0.5975 Es.Ar. 0.4583 Pooled 60 2.7 0.0091 Rel.Ct.Ar. 0.5408 Pooled 60 -2.63 0.0109 Ps.Pm. 0.0454 Satterthwaite 60 2.07 0.0445 Es.Pm. 0.0163 Satterthwaite 41.966 2.17 0.0355

Table 22. Summary of T-Test Results for Rib Analysis by Sex

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In the rib, males demonstrate larger sized ribs than females (Tt.Ar.) on average, although the difference is not significant (p=0.0596). The size of the area containing cortical bone (Ct.Ar.) is slightly larger in males on average with this difference not showing significance (p=0.5975). Significant differences were found with areal measurements of Es.Ar. (p=0.0091), indicating males have significantly larger medullary cavities than females. Rel.Ct.Ar. was significantly different between the sexes

(p=0.0109), with females demonstrating larger cortical bone areas when bone size is taken into account. In regards to perimeter measurements, both periosteal perimeter

(Ps.Pm.) and endosteal perimeter (Es.Pm.) were significantly different between the sexes

(p=0.0445 and p=0.0355, respectively). Consistent with the area results, males demonstrated larger overall perimeters at both the periosteal and endosteal surfaces than females. The findings in rib data analysis support Hypothesis 1 and the statistical hypotheses: H1a: periosteal and endosteal perimeters significantly differ between the sexes and H1b: significant differences exist in areal and relative areal bone measurements between the sexes.

6.3.2 Sex Comparisons Using Clavicle Variables

T-test output values for clavicle sex comparisons are listed in Table 23. Table 24 details t-test results with significance at the 0.05 level indicated in grey. All variables examined for sex differences in the clavicle demonstrated significance at the 0.05 level.

This finding indicates that sex differences at the microstructural level are more apparent in the clavicle than the rib in Chiribaya individuals.

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Variable/Sex N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Male 25 9.5157 1.3512 0.2702 7.5146 12.7312 Tt.Ar. Female 29 8.0523 1.0017 0.186 6.4622 10.3942 Ct.Ar. Male 25 58.7816 16.2439 3.2488 35.1358 100.3 Ct.Ar. Female 29 47.141 9.6076 1.7841 29.673 74.5733 Es.Ar. Male 25 5.6313 1.3723 0.2745 3.2581 9.065 Es.Ar. Female 29 4.1283 1.2972 0.2409 1.6189 7.0238 Rel.Ct.Ar. Male 25 0.6471 0.1036 0.0207 0.4265 0.8707 Rel.Ct.Ar. Female 29 0.733 0.1154 0.0214 0.4565 0.9372 Ps.Pm. Male 25 35.8673 5.08 1.016 28.424 47.326 Ps.Pm. Female 29 30.1276 3.7134 0.6896 24.108 39.872 Es.Pm. Male 25 24.7971 5.4875 1.0975 14.213 35.6966 Es.Pm. Female 29 17.9504 5.8813 1.0921 6.3363 33.328

Table 23. Descriptive Statistics of Clavicle Dataset Analyzed for Sex Comparison (Tt.Ar. and Es.Ar. were square root transformed)

Clavicle Sex T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.1291 Pooled 52 4.56 <0.0001 Ct.Ar. 0.0085 Satterthwaite 37.718 3.14 0.0033 Es.Ar. 0.769 Pooled 52 4.13 0.0001 Rel.Ct.Ar. 0.5948 Pooled 52 -2.86 0.0061 Ps.Pm. 0.1123 Pooled 52 4.78 <0.0001 Es.Pm. 0.7353 Pooled 52 4.4 <0.0001 Table 24. Summary of T-Test Results for Clavicle Analysis by Sex (Significant values are indicated in grey)

Chiribaya males exhibit larger clavicles than females (Tt.Ar.: p= <0.0001).

Chiribaya males also have significantly larger cortical areas (Ct.Ar.: p=0.0033) and larger

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medullary cavities (Es.Ar.: p=0.0001) than Chiribaya females; however, when size is taken into account, the relative amount of cortical bone is actually higher in Chiribaya females than males (Rel.Ct.Ar.: p=0.0061). Similar to findings in Chiribaya ribs, male perimeters are significantly larger at both the periosteal and endosteal surfaces (Ps.Pm.: p<0.0001; Es.Pm.: p<0.0001). The findings in clavicle data analysis support Hypothesis 1 and the statistical hypotheses: H1a: periosteal and endosteal perimeters significantly differ between the sexes and H1b: significant differences exist in areal and relative areal bone measurements between the sexes.

6.3.3 Sex Comparisons Using Iliac Crest Variables

Output values for t-test comparisons of iliac crest data for sex are listed in Table

25. Results of t-tests run for sex comparisons of iliac crest variables are summarized in

Table 26 with significant values shaded in grey. Thickness of iliac crest section, measured as Tt.Dm. could not be normalized and was not included in the statistical analysis. As such, sex differences reflected in iliac crest Tt.Dm. crest are described at the end of this section.

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Variable/Sex N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Male 27 60.5575 15.4201 2.9676 20.5933 97.3606 Tt.Ar. Female 34 50.8511 13.7427 2.3569 18.259 82.2479 Ct.Ar. Male 27 17.3385 4.8233 0.9282 7.318 24.571 Ct.Ar. Female 34 17.2775 5.4011 0.9263 4.532 28.181

Es.Ar. Male 27 43.219 14.1075 2.715 11.982 74.9321 Es.Ar. Female 34 33.5736 11.5524 1.9812 10.071 57.67 Rel.Ct.Ar. Male 27 0.2972 0.0913 0.0176 0.1592 0.4915 Rel.Ct.Ar. Female 34 0.3485 0.0983 0.0169 0.1592 0.5565 Tt.Tb.Ar. Male 27 3.2123 0.736 0.1416 1.9109 5.1183

Tt.Tb.Ar. Female 34 2.8523 0.7582 0.13 0.7221 4.3078 Rel.Tb.Ar. Male 27 0.175 0.0501 0.00964 0.0963 0.3158 Rel.Tb.Ar. Female 34 0.1669 0.0664 0.0114 0.0286 0.307 Tb.Wi. Male 27 -0.8445 0.1011 0.0195 -1.0187 -0.5821 Tb.Wi. Female 34 -0.8479 0.143 0.0245 -1.2309 -0.4738 Ct.Dm. Male 27 1.955 0.6405 0.1233 0.682 2.993 Ct.Dm. Female 34 2.1202 0.7839 0.1344 0.497 3.85 Es.Dm. Male 27 5.3975 1.6729 0.322 1.497 9.949 Es.Dm. Female 34 4.3381 1.3998 0.2401 1.975 7.702

Table 25. Output Values for Iliac Crest Analysis by Sex (Tb.Ar. was square root transformed; Tb.Wi. was log(square root) transformed)

Iliac Crest Sex T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.5269 Pooled 59 2.6 0.0119 Ct.Ar. 0.5573 Pooled 59 0.05 0.9635 Es.Ar. 0.2765 Pooled 59 2.94 0.0047 Rel.Ct.Ar. 0.7036 Pooled 59 -2.09 0.0408 Tt.Tb.Ar. 0.8854 Pooled 59 1.87 0.067 Rel.Tb.Ar. 0.1417 Pooled 59 0.52 0.6033 Tb.Wi. 0.0725 Pooled 59 0.1 0.919 Ct.Dm. 0.2918 Pooled 59 -0.88 0.3798 Es.Dm. 0.331 Pooled 59 2.69 0.0092

Table 26. Summary of T-Test Results for Iliac Crest Analysis by Sex

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For the iliac crest, males were significantly larger than females with regards to overall size of section (Tt.Ar.: p=0.0119); because the length of the section was standardized to reflect the size of a core taken in biopsy, the differences demonstrate variations due to thickness, rather than length. Endosteal area (Es.Ar.) was also significantly different between the sexes (p=0.0047) with males exhibiting larger overall medullary areas. Cortical area was not significantly different between the sexes; however, cortical area with size taken into consideration (Rel.Ct.Ar.) was significantly larger in females than males (p=0.0408), which is similar to findings in the rib and clavicle.

Amount of trabecular bone and relative trabecular bone was not significantly different between males and females, although on average, Chiribaya males exhibit higher amounts of trabecular bone than Chiribaya females. Average trabecular width (Tb.Wi.) was not significantly different between the sexes. In examining thickness of the sections, males demonstrate larger, on average, Tt.Dm. although this could not be normalized to test statistically (descriptive statistics for Tt.Dm. are given in Table 27). The thickness of cortical bone in the iliac crest (Ct.Dm.) was not significantly different between Chiribaya males and females (p=0.3798) but cortical thicknesses are slightly larger in males.

Es.Dm. was significant when statistically examined (p=0.0092) with males demonstrating larger medullary areas than females.

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Table 27. Descriptive Statistics of Tt.Dm. of Iliac Crest by Sex

In the iliac crest, the statistically significant findings of total area (Tt.Ar.), medullary area (Es.Ar.), relative cortical area (Rel.Ct.Ar.) and medullary thickness

(Es.Dm.) support Hypothesis 1: Sex differences exist in the histomorphometric areal and perimeter/length measurements in the Chiribaya sample. More specifically, iliac crest data analysis supports the statistical hypotheses: H1b: significant differences exist in areal and relative areal bone measurements between the sexes, with regards to total area

(Tt.Ar.), medullary area (Es.Ar.) and relative cortical area (Rel.Ct.Ar.) and H1d: significant differences exist in total, cortical and medullary thickness/diameter between males and females, with regards to medullary thickness (Es.Dm.) only. Hypothesis 1c: males have thicker average trabecular width in iliac crest samples than females, was not supported by statistical findings.

6.3.4 Summation of Sex-Related Differences In the rib, clavicle and iliac crest, sex differences between Chiribaya males and females indicate males having larger sized bones than females; however, when size is taken into consideration, females have a higher ratio of bone mass than males at all three skeletal sites. Males demonstrate larger perimeters in the rib and clavicle indicating

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larger overall size and larger medullary cavities than females. In the iliac crest, significant differences were noted with variables in cortical bone but not trabecular bone, suggesting that in this sample, the trabecular bone variables measured are not as sensitive to the effects of sex as cortical bone. Overall, all three skeletal sampling sites support

Hypothesis 1: Sex differences exist in the histomorphometric areal and perimeter/length measurements in the Chiribaya sample.

6.4 Examination of Subsistence as Indicated by Cultural Site Affiliation

To examine differences between sites, a series of t-tests was employed to test

Hypothesis 2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo due to variation of diet and subsistence level activities. The El Yaral sample was too small to be normalized for statistical analysis and was not included in initial statistical tests. A series of t-tests was conducted to examine differences in means between individuals at Chiribaya Alta (mercaderes) and San Geronimo (pescadores). After these results were obtained and examined, individuals from El Yaral (labradores) were combined with the San Geronimo dataset, and a second series of t-tests was conducted to examine differences between elites at Chiribaya Alta and laborers (pescadores and labradores) at San Geronimo and El Yaral. Descriptive statistics for data from all three sites are given toward the end of this section. This section contains results of t-tests conducted. Histograms for t-tests are included in Appendix A.

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6.4.1 Examination of Subsistence Differences Demonstrated in Rib Data

An initial series of t-tests was conducted to examine differences between data

from San Geronimo individuals and Chiribaya individuals. The t-test output values the

rib are given are given in Table 28. Results of significance are given in Table 29 with

significant values shaded in grey.

Variable/Site N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Chiribaya Alta 36 8.1155 1.1241 0.1874 4.8083 9.9442 Tt.Ar. San Geronimo 19 8.9936 1.2976 0.2977 6.1436 12.4501 Ct.Ar. Chiribaya Alta 36 34.0363 9.4483 1.5747 13.4944 52.2794 Ct.Ar. San Geronimo 19 39.341 10.2791 2.3582 19.2528 65.2183 Es.Ar. Chiribaya Alta 36 5.6565 1.0432 0.1739 3.1025 7.3695 Es.Ar. San Geronimo 19 6.4554 1.2439 0.2854 3.7666 9.4756 Rel.Ct.Ar. Chiribaya Alta 36 0.5139 0.0865 0.0144 0.3146 0.7421 Rel.Ct.Ar. San Geronimo 19 0.4865 0.085 0.0195 0.3347 0.6241 Ps.Pm. Chiribaya Alta 36 35.6409 5.4521 0.9087 25.3049 47.509 Ps.Pm. San Geronimo 19 37.6884 5.6799 1.3031 24.4866 49.6315 Es.Pm. Chiribaya Alta 36 33.0135 6.7201 1.12 21.6158 49.8589 Es.Pm. San Geronimo 19 32.7367 5.7989 1.3304 18.7555 42.1424

Table 28. Output Values of Rib Dataset Analyzed for Site Comparisons (Tt.Ar. and Es.Ar. were square root transformed)

Rib Site T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.4554 Pooled 53 -2.61 0.0117 Ct.Ar. 0.6495 Pooled 53 -1.92 0.0601 Es.Ar. 0.364 Pooled 53 -2.53 0.0146 Rel.Ct.Ar. 0.9677 Pooled 53 1.12 0.2669 Ps.Pm. 0.8084 Pooled 53 -1.31 0.1973 Es.Pm. 0.5121 Pooled 53 0.15 0.8798

Table 29. Summary of T-Test Results for Rib Analysis by Site

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Significant differences were noted in Tt.Ar. (p=0.0117) and Es.Ar. (p=0.0146) at the 0.05 level. Individuals at San Geronimo had, on average, significantly larger ribs and larger medullary areas than individuals at Chiribaya Alta. Ct.Ar. was larger, on average, in San Geronimo individuals as well but the difference was not significant. Relative cortical area (Rel.Ct.Ar.) was not significant (p=0.2669). Periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.) were not significantly different between San Geronimo and Chiribaya Alta ribs. On average, periosteal perimeter (Ps.Pm.) was larger in San

Geronimo ribs and endosteal perimeter (Es.Pm.) was larger in Chiribaya Alta ribs. The results indicate that San Geronimo ribs are significantly larger than Chiribaya Alta ribs but when adjusted for size, the ratio of cortical bone to total bone size is not significantly different. The analysis of these data with regards to total area (Tt.Ar.) and endosteal area

(Es.Ar.) support H2b: total, cortical, medullary and trabecular areas significantly differ according to subsistence-level differences at Chiribaya Alta, El Yaral, and San

Geronimo. The findings do not support H2a: periosteal and endosteal perimeters differ significantly according to subsistence-level differences at Chiribaya Alta, El Yaral, San

Geronimo or H2c: There is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

It was not possible to statistically analyze the El Yaral (labradores) data; therefore, the mean, standard deviations, minimum and maximum for each variable of El

Yaral ribs are given in Table 30 and can be compared with the results of San Geronimo and Chiribaya Alta data from Table 28. Both perimeter measurements, periosteal

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(Ps.Pm.) and endosteal (Es.Pm.), are smaller in the El Yaral sample on average than both

Chiribaya Alta and San Geronimo. Mean Tt.Ar. of El Yaral ribs falls in between that of

Chiribaya Alta and San Geronimo, with San Geronimo being the largest in average size.

Mean Es.Ar. for El Yaral also falls in between means of Chiribaya Alta and San

Geronimo, with Chiribaya individuals demonstrating the highest endosteal areas. Mean

Ct.Ar. and mean Rel.Ct.Ar. was smallest on average in the El Yaral sample than

Chiribaya Alta and San Geronimo.

Variable n Mean Std Dev Min. Max. TtAr 7 69.174715 18.890838 40.911705 94.338251 PsPm 7 34.763737 5.597979 24.69055 40.205044 EsAr 7 36.523416 8.8410938 21.628407 46.333128 EsPm 7 30.788125 4.5167165 21.588285 34.884574 CtAr 7 32.651299 10.781405 19.283298 48.13902 RelCtAr 7 0.4672949 0.0431737 0.3953364 0.5177766

Table 30. Descriptive Statistics for Rib Data from El Yaral Sample

A second set of t-tests was conducted with San Geronimo and El Yaral data pooled into one group and compared to the means of Chiribaya Alta data. Output values for this series of t-tests are listed in Table 31. T-test results are given in Table 32.

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Table 31. T-Test Output Values for Combined Site Rib Comparisons

Table 32. T-Test Results for Combined Site Rib Comparisons

T-tests conducted on a combined sample of San Geronimo (pescadores) and El

Yaral (labradores) rib data compared to Chiribaya Alta rib data resulted in similar results as the initial t-tests conducted. Significance was noted in total area (Tt.Ar.)(p=0.0314) and endosteal area (Es.Ar.) (p=0.0314). The combined data indicate that the elites buried at Chiribaya Alta demonstrate significantly higher total areas (Ct.Ar.) and larger mean 157

endosteal areas (Es.Ar.) than the laborers at San Geronimo and El Yaral but overall size measurements (perimeters) and relative cortical bone amounts were not significantly different. These findings of the combined laborer dataset (pescadores and labradores) compared to Chiribaya Alta (mercaderes) are consistent with the findings between

Chiribaya Alta (mercaderes) and San Geronimo (pescadores), where H2b is supported, but H2a and H2c are not supported. Box and whisker plots were created for each rib variable and grouped by site affiliation. These variables can be visualized in Figure 15 through Figure 20.

Figure 15. Boxplot of Rib Total Area (Tt.Ar). by Site

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Figure 16. Boxplot of Rib Cortical Area (Ct.Ar.) by Site

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Figure 17. Boxplot of Rib Endosteal Area (Es.Ar.) by Site

Figure 18. Boxplot of Rib Relative Cortical Area (Rel.Ct.Ar.) by Site

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Figure 19. Boxplot of Rib Periosteal Perimeter (Ps.Pm.) by Site

Figure 20. Boxplot of Rib Endosteal Perimeter (Es.Pm). by Site

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6.4.2 Examination of Subsistence Differences Demonstrated in Clavicle Data

An initial series of t-tests was conducted on clavicle data to examine significant differences in clavicle data between Chiribaya Alta individuals (mercaderes) and San

Geronimo individuals (pescadores) to test the general hypothesis: H2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo. The El Yaral (labradores) sample size was too small to be normalized and incorporated in initial site comparisons and was not included in the original series of t-tests. Output values for the initial set of t-tests conducted are given in Table 33. T-test results are given in Table 34 with significant values shaded in grey.

Variable/ Site N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Chiribaya Alta 34 70.5235 17.433 2.9897 41.76 108 Tt.Ar. San Geronimo 15 96.795 34.2917 8.8541 57.9063 162.1 Ct.Ar. Chiribaya Alta 34 48.662 9.9036 1.6985 29.673 71.45 Ct.Ar. San Geronimo 15 61.9582 19.9064 5.1398 36.4898 100.3 Es.Ar. Chiribaya Alta 34 4.4684 1.3972 0.2396 1.6189 7.1421 Es.Ar.San Geronimo 15 5.6991 1.5891 0.4103 3.4697 9.065 Rel.Ct.Ar. Chiribaya Alta 34 0.7084 0.1233 0.0211 0.4565 0.9372 Rel.Ct.Ar. San Geronimo 15 0.6543 0.104 0.0269 0.4265 0.8075 Ps.Pm. Chiribaya Alta 34 31.3651 4.0721 0.6984 24.108 39.872 Ps.Pm. San Geronimo 15 36.2278 6.488 1.6752 28.2496 47.326 Es.Pm. Chiribaya Alta 34 19.7162 6.5256 1.1191 6.3363 33.328 Es.Pm. San Geronimo 15 24.4642 6.114 1.5786 14.1309 35.6966

Table 33. Output Values of Clavicle Dataset Analyzed for Site Comparison (Es.Ar. was square root transformed)

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Clavicle Site T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.0014 Satterthwaite 17.279 -2.81 0.0119 Ct.Ar. 0.001 Satterthwaite 17.138 -2.46 0.025 Es.Ar. 0.5064 Pooled 31.574 1.58 0.1234 Rel.Ct.Ar. 0.5259 Pooled 47 -2.73 0.009 Ps.Pm. 0.0277 Satterthwaite 19.045 -2.68 0.0148 Es.Pm. 0.8245 Pooled 47 -2.39 0.0208

Table 34. Summary of T-Test Results for Clavicle Analysis by Site

In Chiribaya clavicles, significant differences were noted in all variables except endosteal area (Es.Ar.) Mean total area (Tt.Ar.) (p=0.0119) and mean cortical area

(Ct.Ar.) (p=0.025) were significantly larger in San Geronimo (pescadores) clavicles than

Chiribaya Alta (mercaderes) clavicles. Both perimeter measurements were significantly larger in San Geronimo individuals than Chiribaya Alta individuals (periosteal perimeter

(Ps.Pm.): p=0.0148; endosteal perimeter (Es.Pm.): p=0.0208). Mean relative cortical area

(Rel.Ct.Ar.) was significantly larger in Chiribaya Alta remains than San Geronimo remains, demonstrating that bone mass was larger in Chiribaya Alta clavicles when given as a ratio to clavicle size.

The mean, standard deviation, minimum and maximum for each variable for El

Yaral (labradores) data are given in Table 35. Mean total area (Tt.Ar.) for El Yaral clavicles is larger than Chiribaya Alta (mercaderes) but smaller than San Geronimo

(pescadores), similar to the pattern seen in Chiribaya rib data. Mean cortical area

(Ct.Ar.) and mean relative cortical area (Rel.Ct.Ar.) for El Yaral individuals lies between the means for Chribaya Alta and San Geronimo. Mean endosteal area (Es.Ar.) is larger among El Yaral clavicles than San Geronimo and Chiribaya Alta.

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With regards to both perimeter measurements, mean periosteal perimeter (Ps.Pm.) and mean endosteal perimeter (Es.Pm.) for El Yaral data falls in between means for San

Geronimo and Chiribaya Alta, with San Geronimo being the largest on average of the three sites.

Variable n Mean Std Dev Min. Max. TtAr 5 73.24914 15.49102 55.78283 88.90613 PsPm 5 32.11007 3.95714 27.46083 36.32789 EsAr 5 22.69921 11.87699 9.69287 36.56597 EsPm 5 20.63495 6.24689 13.46135 27.52097 CtAr 5 50.54992 4.75661 46.08996 57.53519 RelCtAr 5 0.70713 0.10558 0.57549 0.82624

Table 35. Descriptive Statistics for Clavicle Data for El Yaral Sample

A second series of t-tests was conducted on clavicle data with San Geronimo

(pescadores) and El Yaral (labradores) data pooled into one group and compared to the means of Chiribaya Alta (mercaderes) data. Output values for this series are given in

Table 36. T-test results are given in Table 37.

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Table 36. T-Test Output Values for Combined Site Data Set of Clavicle

Table 37. T-Test Results for Combined Site Data Set of Clavicle

When the two groups of laborers (San Geronimo and El Yaral) are combined and compared to elites buried at Chiribaya Alta, there are fewer significant differences seen in the clavicle. Statistical significance was found in total area (Tt.Ar.), periosteal perimeter

(Ps.Pm.) and endosteal perimeter (Es.Pm.) Mean total area (Tt.Ar.) was larger in

Chiribaya Alta clavicles than the pooled data set of San Geronimo and El Yaral clavicle data. Both periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.) were larger on 165

average in Chiribaya Alta individuals than the pooled San Geronimo and El Yaral clavicle data set. These data indicate that when the pescadores and labradores are combined, they are more similar to elites, providing evidence for the suggestion that

Chiribaya Alta was comprised of individuals who originated from other sites.

Analysis of clavicle variables for subsistence-related site differences support statistical hypotheses H2a: periosteal and endosteal perimeters differ significantly according to subsistence-level differences at Chiribaya Alta, El Yaral and San Geronimo,

H2b: total, cortical, medullary and trabecular areas significantly differ according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo, and H2c: there is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya Alta, El Yaral, and San

Geronimo. A series of box and whisker plots was created to visualize data patterns among the three sites. Each clavicle variable is represented in Figure 21 through Figure

26.

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Figure 21. Boxplot of Clavicle Total Area (Tt.Ar.) by Site

Figure 22. Boxplot of Clavicle Cortical Area (Ct.Ar.) by Site 167

Figure 23. Boxplot of Clavicle Endosteal Area (Es.Ar.) by Site

Figure 24. Boxplot of Clavicle Relative Cortical Area (Rel.Ct.Ar.) by Site

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Figure 25. Boxplot of Clavicle Periosteal Perimeter (Ps.Pm.) by Site

Figure 26. Boxplot of Clavicle Endosteal Perimeter (Es.Pm.) by Site

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6.4.3 Examination of Subsistence Differences Demonstrated in Iliac Crest Data

An initial series of t-tests was conducted on iliac crest data to examine significant differences between Chiribaya Alta individuals (mercaderes) and San Geronimo individuals (pescadores) to test the general hypothesis: H2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo. The El Yaral (labradores) sample size was too small to be normalized and incorporated in initial site comparisons and was not included in the original series of t-tests, which compared Chiribaya Alta (mercaderes) iliac crest data to San Geronimo (pescadores) iliac crest data. Output values for t-tests of iliac crest variables are indicated in Table 38. T-tests results with significance are listed in Table 39 with significant values shaded in grey.

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Variable/ Site N Mean St.Dev. Std. Err. Min. Max Tt.Ar. Chiribaya Alta 39 53.5869 15.927 2.5504 18.259 86.791 Tt.Ar. San Geronimo 17 60.6191 13.89 3.3688 38.8218 97.3606 Ct.Ar. Chiribaya Alta 39 17.1793 5.0789 0.8133 4.532 26.536 Ct.Ar. San Geronimo 17 16.7069 5.5063 1.3355 7.318 28.181 Es.Ar. Chiribaya Alta 39 36.4076 13.8766 2.222 10.071 66.122 Es.Ar. San Geronimo 17 43.9122 11.7085 2.8397 29.3794 74.9321 Rel.Ct.Ar. Chiribaya Alta 39 0.3345 0.0954 0.0153 0.1592 0.5565 Rel.Ct.Ar. San Geronimo 17 0.2763 0.0802 0.0194 0.1592 0.4605 Tt.Tb.Ar. Chiribaya Alta 39 2.9037 0.8155 0.1306 0.7221 5.1183 Tt.Tb.Ar. San Geronimo 17 3.3674 0.553 0.1341 2.4941 4.3078 Rel.Tb.Ar. Chiribaya Alta 39 0.1642 0.0639 0.0102 0.0286 0.3158 Rel.Tb.Ar. San Geronimo 17 0.1919 0.0445 0.0108 0.0963 0.2861 Tb.Wi. Chiribaya Alta 39 0.439 0.0614 0.00984 0.292 0.6226 Tb.Wi. San Geronimo 17 0.4147 0.0311 0.00754 0.3426 0.4593 Tt.Dm. Chiribaya Alta 39 6.8022 1.9862 0.318 2.179 12.399 Tt.Dm. San Geronimo 17 7.2241 1.0175 0.2468 5.5157 9.479 Ct.Dm. Chiribaya Alta 39 2.0134 0.7435 0.1191 0.497 3.52 Ct.Dm. San Geronimo 17 2.0106 0.6246 0.1515 0.82 3.236 Es.Dm. Chiribaya Alta 39 4.7889 1.7732 0.2839 1.497 9.949 Es.Dm. San Geronimo 17 5.2135 1.0471 0.254 3.6803 7.5483

Table 38. Output Values for Site Comparisons of Iliac Crest Data (Tb.Ar. and Tb.Wi. were square root transformed)

Iliac Crest Site T-Test Eq. of Var. Method DF t-value Pr>|t| Tt.Ar. 0.5667 Pooled 54 -1.58 0.1208 Ct.Ar. 0.6591 Pooled 54 0.31 0.7562 Es.Ar. 0.4708 Pooled 54 -1.95 0.0569 Rel.Ct.Ar. 0.4584 Pooled 54 2.19 0.0326 Tt.Tb.Ar. 0.0967 Pooled 54 -2.13 0.0374 Rel.Tb.Ar. 0.1216 Pooled 54 -1.63 0.1099 Tb.Wi. 0.005 Satterthwaite 52.636 1.96 0.0552 Tt.Dm. 0.0058 Satterthwaite 52.41 -1.05 0.2994 Ct.Dm. 0.4588 Pooled 54 0.01 0.9895 Es.Dm. 0.0266 Satterthwaite 48.857 -1.11 0.2704

Table 39. T-Test Results of Iliac Crest Data for Site Comparisons

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Significant differences in iliac crest data in initial site comparisons was only found in relative cortical area (Rel.Ct.Ar.) (p=0.0326) and total trabecular area

(Tt.Tb.Ar.) (p=0.0374). Mean relative cortical area (Rel.Ct.Ar.) was larger in Chiribaya

Alta iliac crest sections than San Geronimo sections. Mean total trabecular area

(Tt.Tb.Ar.) was larger in San Geronimo iliac crest sections than Chiribaya Alta sections.

As with rib and clavicle site comparisons, El Yaral (labradores) data could not be normalized and included in statistical analyses for the iliac crest. Table 40 lists the descriptive statistics for iliac crest variables for El Yaral. Mean total area (Tt.Ar.) for El

Yaral was smaller than San Geronimo and Chiribaya Alta, with San Geronimo individuals demonstrating the highest mean total area (Tt.Ar.) Mean cortical area (Ct.Ar.) is largest for El Yaral; when standardized for total area, relative cortical area (Rel.Ct.Ar.) is also largest in El Yaral iliac crest. Mean endosteal area (Es.Ar.) is also the smallest in

El Yaral iliac crests in comparison to San Geronimo and Chiribaya Alta.

Trabecular bone, however, shows a different pattern. Tt.Tb.Ar. is also largest in

El Yaral iliac crest sections, with Chiribaya Alta exhibiting the smallest mean values.

However, when standardized for total area, Rel.Tb.Ar. exhibits the opposite trend, with

El Yaral iliac crest sections demonstrating the lowest mean Rel.Tb.Ar. of the three sites, and San Geronimo having the highest mean Rel.Tb.Ar. Mean Tb.Wi. is smallest in El

Yaral and largest in Chiribaya Alta iliac crest.

In looking at overall size and thickness of iliac crest sections, El Yaral demonstrates the highest meant for total diameter (Tt.Dm.) with Chiribaya Alta and San

Geronimo being smaller in overall thickness. Cortical diameter (Ct.Dm.) is also largest in

El Yaral with Chiribaya Alta and San Geronimo demonstrating similar mean Ct.Dm.

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Mean medullary diameter (Es.Dm.) is smallest among El Yaral iliac crest sections with

San Geronimo demonstrating the highest mean value. These findings support H2b: total, cortical, medullary, and trabecular areas differ significantly according to subsistence- level differences at Chiribaya Alta, El Yaral, and San Geronimo, but only in terms of total trabecular area (Tt.Tb.Ar.) and H2c only with regards to relative cortical area

(Rel.Ct.Ar.): there is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo. These findings do not support H2d: there is a significant difference in trabecular width in iliac crest samples between individuals at Chiribaya Alta, El Yaral, and San Geronimo and H2e: total, cortical and medullary thickness/diameter of iliac crest sections will significantly differ in individuals according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

Variable n Mean Std Dev Min. Max. TtAr 5 48.7157 8.59392 35.2334 58.323 EsAr 5 28.4026 8.8855 17.717 42.279 CtAr 5 20.3131 3.54668 16.044 24.0043 RelCtAr 5 0.42627 0.08824 0.27509 0.49715 TtTbAr 5 7.30299 3.33908 3.16069 12.3876 RelTbAr 5 0.14679 0.05658 0.08971 0.2345 TbWi 5 0.19694 0.05681 0.13344 0.27713 TtDm 5 5.99999 1.4699 3.92774 7.691 EsDm 5 3.56652 1.30076 2.4901 5.781 CtDm 5 2.43347 0.90718 1.43764 3.85

Table 40. Descriptive Statistics for Iliac Crest Variables from El Yaral Sample

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A second series of t-tests was conducted, pooling San Geronimo and El Yaral iliac crest data together to compare with the mean of Chiribaya Alta iliac crest data. The output values for the second series of t-tests is given in Table 41 with t-test results listed in Table 42.

Table 41. Output Values for T-Test Results of Grouped Site Data Set for Iliac Crest

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Table 42. T-Test Results for Combined Site Data Set of Iliac Crest Data

When San Geronimo and El Yaral iliac crest data was pooled together, none of the comparisons to Chiribaya Alta data was statistically significant. In the first series of t- tests, relative cortical area (Rel.Ct.Ar.) and total trabecular area (Tt.Tb.Ar.) demonstrated significance between Chiribaya Alta and San Geronimo. When the two sites of laborers are combined, pescadores and labradores, the iliac crest data is more similar to Chiribaya

Alta (mercaderes) data supporting the suggestion that Chiribaya Alta was comprised of a class of elite individuals who originated from other sites. The second series of statistical tests do not support the statistical hypotheses regarding the assumptions of subsistence- related differences in iliac crest data: H2b-H2e.

A series of box and whisker plots was created to demonstrate the variation exhibited in each iliac crest variable by Chiribaya site. Box and whisker plots of iliac crest site-specific data are presented in Figure 27 through Figure 35.

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Figure 27. Boxplot of Iliac Crest Total Area (Tt.Ar.) by Site

Figure 28. Boxplot of Iliac Crest Cortical Area (Ct.Ar.) by Site

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Figure 29. Boxplot of Iliac Crest Marrow Cavity Area (Es.Ar.) by Site

Figure 30. Boxplot of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) by Site 177

Figure 31. Boxplot of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) by Site

Figure 32. Boxplot of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) by Site

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Figure 33. Boxplot of Iliac Crest Total Diameter (Tt.Dm.) by Site

Figure 34. Boxplot of Iliac Crest Cortical Diameter (Ct.Dm.) by Site

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Figure 35. Boxplot of Iliac Crest Marrow Cavity Diameter (Es.Dm.) by Site

6.4.4 Summary of Findings Regarding Subsistence-Level Differences The three Chiribaya sites examined demonstrate a different pattern in each of the three skeletal sites examined. San Geronimo individuals demonstrate larger ribs than the other two sites, although Chiribaya Alta ribs have a larger mean bone mass. In the clavicle, bone mass is higher in individuals from El Yaral, while Chiribaya Alta individuals have higher means for total size. In the iliac crest, bone mass is also higher in

El Yaral than San Geronimo and Chiribaya Alta. San Geronimo iliac crest sections demonstrate higher means of total trabecular bone and trabecular bone mass than

Chiribaya Alta and San Geronimo. These data partially support Hypothesis 2:

Differences in histomorphometric areal and length/perimeter measurements exist among

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individuals from Chiribaya Alta, El Yaral, and San Geronimo due to variation of diet and general subsistence level activities. The findings indicate that subsistence patterns have an impact on bone mass and variables impacting bone strength; however, one must take the skeletal site examined into consideration as each bone exhibits the effects of subsistence uniquely.

When El Yaral (labradores) and San Geronimo (pescadores) data are pooled together, they are more similar to Chiribaya Alta (mercaderes) data. This supports the suggestion that individuals from Chiribaya Alta may have originally been drawn from other sites before claiming residence at the administrative locale.

6.5 Examination of Age

A series of one-way and two-way ANOVA tests were used to examine age differences in rib, clavicle, and iliac crest data to test the general hypothesis H3: differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age. After examining sex and site variability of each bone, only those variables demonstrating no significant differences in due to sex or subsistence were incorporated into age comparisons between young, middle, and older adult males and females. Box and whisker plots for each ANOVA test can be found in

Appendix B.

6.5.1 Examination of Age in Rib Variables

Comparisons of male and female data demonstrated statistical significance in endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.)

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and endosteal perimeter (Es.Pm.). Subsistence differences indicated by site demonstrated significant differences in total area (Tt.Ar.) and endosteal area (Es.Ar.). Sex and site comparisons drew significance from these variables, leaving cortical area (Ct.Ar.) as a remaining rib variable not influenced by sex or site, and the only variable chosen to be examined for comparison between age groups. Table 43 lists results of a one-way

ANOVA run to examine the interaction of age in cortical area (Ct.Ar.). The one-way

ANOVA was not significant (p=0.2585)

Source DF Sum of Mean F Value Pr > F Model 2 275.115504 137.55775 1.38 0.2585 Error 59 5862.89818 99.371156 Corrected 61 6138.01368

R-Square Coeff Var Root MSE CtAr Mean 0.044822 28.07592 9.968508 35.50554 Table 43. One-Way ANOVA Table for Rib Cortical Area (Ct.Ar.)

A two-way ANOVA was also conducted to examine the interaction of age and sex for rib cortical area (Ct.Ar.) The two-way ANOVA results were also insignificant at the 0.05 level (p=0.2759) and no significance noted with the interaction of sex with age

(Table 44).

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Sum of Mean Source DF F Value Pr > F Squares Square Model 3 317.173 105.724 1.05 0.3759 Error 58 5820.84 100.359 Corrected 61 6138.01 Total

R-Square Coeff Var Root MSE CtAr Mean

0.05167 28.2152 10.018 35.50554

Mean Source DF Anova SS F Value Pr > F Square sex 1 28.6896476 28.689648 0.29 0.5896 age 2 275.115504 137.55775 1.41 0.2523 sex*age 2 377.258009 188.629 1.94 0.1539

Table 44. Two-Way ANOVA Table of Rib Cortical Area (Ct.Ar.)

The findings in the rib do not support general hypothesis H3 or the statistical hypotheses relegated toward rib variables H3a:periosteal and endosteal perimeters increase significantly with age, H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a significant difference in relative cortical area and relative trabecular area between young, middle, and older adults.

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6.5.1.1. Descriptive Statistics of Rib Data

Descriptive statistics for Chiribaya rib data categorized by age are listed in Table

40. The mean, standard deviation, minimum and maximum for each variable are listed for age categories of young, middle and old adults. When observed together, male and female Chiribaya ribs demonstrate a general decrease in relative cortical area (Rel.Ct.Ar.) with increasing age category; however, perimeter measurements of the outer cortex

(Ps.Pm.) is similar among the three age categories. Mean total area (Tt.Ar.) and mean cortical area (Ct.Ar.) are larger in young adults and smallest in older adults Mean endosteal area (Es.Ar.) also increases with increasing age category, representing endosteal resorption with increasing age.

Age n Variable Mean Std Dev Min. Max. Young Adult 29 TtAr 74.2784 24.3215 23.1196 155.006 PsPm 36.3743 5.55194 25.3049 49.6315 EsAr 36.5814 16.7929 9.62521 89.7873 EsPm 32.3817 6.32205 21.6158 49.8589 CtAr 37.6969 10.523 13.4944 65.2183 RelCtAr 0.52243 0.09592 0.31456 0.74214 Middle Adult 20 TtAr 70.3404 17.8555 40.9117 100.526 PsPm 35.9995 5.7942 24.6906 44.3979 EsAr 36.2455 10.1527 21.6284 58.2435 EsPm 32.8567 6.10448 21.5883 43.6657 CtAr 34.0949 9.91611 19.2528 52.2794 RelCtAr 0.48355 0.06452 0.33474 0.59217 Old Adult 13 TtAr 69.67 18.0647 37.7435 98.0161 PsPm 35.9732 5.5798 24.4866 47.509 EsAr 36.8827 11.1563 14.1873 51.2547 EsPm 33.061 6.48828 18.7555 48.748 CtAr 32.7873 8.63186 22.1421 48.139 RelCtAr 0.47651 0.06905 0.3607 0.62411

Table 45. Descriptive Statistics of Female Rib Data Categorized by Age and Sex

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When observed separately, male and female Chiribaya ribs demonstrate a different pattern with increasing age category. Descriptive statistics for male Chiribaya ribs are given in Table 45. The mean, standard deviation, maximum and minimum for each age category are listed. Perimeter measurements in males demonstrate larger means for periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.) in young and middle adult males than older adult males. Because males generally exhibit an increase in periosteal perimeter with age due to periosteal apposition and an increase in endosteal perimeter with age due to endosteal resorption, these data indicate that larger males are absent from the old adult age category, unless periosteal resorption has occurred. This trend is also visible with bone mass (Rel.Ct.Ar.) where middle adult males exhibit the highest mean value of the three age categories. These data also demonstrate a decline in total area (Tt.Ar.), cortical area (Ct.Ar.) and endosteal area (Es.Ar.) with increasing age category in males.

One must consider the difference in sample size for each age category of males.

The sample size for older adult males (n=4) is much smaller than young adult males

(n=11) and middle adult males (n=12); the small sample size may be reflected in the pattern of age effects in male ribs.

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Sex Age n Variable Mean Std Dev Min Max Male Younger Adult 11 TtAr 91.01385 25.01226 60.1125 155.0056 PsPm 39.93975 5.583718 32.34598 49.63148 EsAr 49.75914 16.73002 30.56666 89.78731 EsPm 36.58643 6.650925 27.32367 49.85889 CtAr 41.25472 11.54051 23.61535 65.21832 RelCtAr 0.456977 0.080647 0.314564 0.590219 Middle Adult 12 TtAr 71.30184 19.81435 44.97808 100.5262 PsPm 36.57524 6.075703 27.74 44.39791 EsAr 36.60549 10.72983 22.62581 58.24348 EsPm 33.29842 6.246218 24.80565 43.66574 CtAr 34.69635 10.66618 21.25701 52.27943 RelCtAr 0.48572 0.053087 0.420614 0.575095 Older Adult 4 TtAr 62.20993 19.32697 37.74355 80.11977 PsPm 35.96774 9.482355 24.48658 47.50896 EsAr 34.85919 14.97765 14.18725 47.9267 EsPm 33.4679 12.30811 18.7555 48.74802 CtAr 27.35074 6.540756 22.14215 36.66318 RelCtAr 0.45944 0.116875 0.360705 0.624115

Table 46. Descriptive Statistics of Male Rib Data by Age Category

Table 46 lists descriptive statistics for rib variables for Chiribaya females of each age category. In females, mean total area (Tt.Ar.) and mean endosteal area (Es.Ar.) exhibit larger areas with older age category. Cortical area (Ct.Ar.) is relatively similar between the three age categories while relative cortical area (Rel.Ct.Ar.) is larger in young adult females but has similar means for middle and older adult females. In females, both perimeter measurements, periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.) demonstrate an increase with increasing age category.

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Sex Age n Variable Mean Std Dev Min Max Female Younger Adult 18 TtAr 64.05111 17.72871 23.11962 95.00242 PsPm 34.19544 4.379109 25.30494 42.52135 EsAr 28.5284 10.89963 9.625212 47.58204 EsPm 29.81221 4.623771 21.61577 37.89878 CtAr 35.52272 9.5313 13.49441 52.87849 RelCtAr 0.562426 0.082839 0.378062 0.742138 Middle Adult 8 TtAr 68.89833 15.63473 40.91171 95.45717 PsPm 35.13592 5.630055 24.69055 44.09164 EsAr 35.70561 9.915097 21.62841 54.94501 EsPm 32.19423 6.245111 21.58829 40.35667 CtAr 33.19272 9.304084 19.25278 42.2661 RelCtAr 0.480301 0.082769 0.33474 0.592174 Older Adult 9 TtAr 72.98564 17.58494 51.35582 98.01613 PsPm 35.97568 3.603194 31.34975 41.79635 EsAr 37.78208 9.980678 24.84961 51.25473 EsPm 32.88019 2.493702 27.97774 36.70746 CtAr 35.20355 8.624048 26.04147 48.13902 RelCtAr 0.4841 0.042644 0.412074 0.55463

Table 47. Descriptive Statistics of Female Rib Data by Age Category

In order to visualize the patterns of each variable more efficiently, a series of boxplots was created for each variable measured in the rib with age and sex identified as class variables to separate the range of values. Each of these is demonstrated below in

Figure 36 through Figure 41.

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Figure 36. Boxplot of Rib Total Area (Tt.Ar.) by Age and Sex

Figure 37. Boxplot of Rib Cortical Area (Ct.Ar.) by Sex and Age Category

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Figure 38. Boxplot of Rib Endosteal Area (Es.Ar.) by Age and Sex

Figure 39. Boxplot of Rib Relative Cortical Area (Rel.Ct.Ar.) by Age and Sex

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Figure 40. Boxplot of Rib Periosteal Perimeter (Ps.Pm.) by Age and Sex

Figure 41. Boxplot of Rib Endosteal Perimeter (Es.Pm.) of Age and Sex

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6.5.2 Examination of Age in Clavicle Data

Statistical examination of clavicle data in age and subsistence comparisons demonstrated significance in every variable; therefore, ANOVA tests were not conducted on clavicle data for age differences. Therefore, H3: differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age, and the statistical hypotheses H3a:periosteal ad endosteal perimeters increase significantly with age, H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a significant difference in relative cortical area and relative trabecular area between young, middle, and older adults were untested.

6.5.2.2 Descriptive Statistics of Clavicle Data

The mean, standard deviation, minimum and maximum for each age category of

Chiribaya data is listed in Table 48. When male and female data is considered together, the clavicle demonstrates trends with increasing age category. Mean total area (Tt.Ar.) is similar among young, middle and older adults, yet smaller on average in older adults.

Mean cortical area (Ct.Ar.) steadily decreases by age category on average, while mean relative cortical area (Rel.Ct.Ar.) is largest among young adults and similar among middle and older adults. Both perimeter measurements, periosteal (Ps.Pm.) and endosteal

(Es.Pm.) are similar between young, middle and older adults.

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Table 48. Descriptive Statistics of Clavicle Data by Age Category

When Chiribaya male clavicles are examined separately from female clavicles, the clavicle data demonstrates a differing pattern from sex-pooled data (Table 49). Mean total area (Tt.Ar.), mean cortical area (Ct.Ar.), and mean endosteal area (Es.Ar.) decrease with increasing age in male clavicles. Mean relative cortical area (Rel.Ct.Ar.) is similar with all three age categories but slightly decrease with increasing age. With perimeter measurements, mean periosteal perimeter (Ps.Pm.) decreases with increasing age category, while endosteal perimeter (Es.Pm.) is similar across all three age categories.

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Sex Age n Variable Mean Std Dev Minimum Maximum Male Young 11 TtAr 99.704102 29.904154 63.746 162.083833 Adult PsPm 37.315789 5.0657553 31.072 47.326019 EsAr 34.30267 15.96034 10.615 73.8613123 EsPm 25.118388 5.1222876 14.213 33.831154 CtAr 65.401432 19.416355 38.491 100.254745 RelCtAr 0.6599884 0.0998295 0.5048662 0.8706529 Middle 9 TtAr 88.48867 30.206753 56.469 143.285108 Adult PsPm 35.12738 6.155326 28.424 43.904892 EsAr 33.561388 21.289456 11.236 82.1733594 EsPm 24.806013 7.0364884 14.887 35.696648 CtAr 54.927282 13.275516 35.135807 79.9089196 RelCtAr 0.6454469 0.1145607 0.4265045 0.8010236 Older 5 TtAr 82.875042 8.6602683 75.26 97.002 Adult PsPm 34.012454 1.8990314 32.43027 37.213 EsAr 31.719179 11.775103 22.6 51.009 EsPm 24.074011 3.8883722 19.757 29.823 CtAr 51.155862 7.8483044 40.408 58.924 RelCtAr 0.6219016 0.1091824 0.4741449 0.7180005

Table 49. Descriptive Statistics of Clavicle Variables for Males by Age Category

Descriptive statistics of female clavicle data is listed in Table 50. Female

Chiribaya clavicle data represents a different pattern from Chiribaya males. Mean total area (Tt.Ar.) is largest in middle adults, smallest in young adults and in between those two in older females. Mean endosteal area (Es.Ar.) demonstrates a similar pattern where the largest mean is found in middle adults, smallest in young adults and in between those two in older adults. Mean cortical area (Ct.Ar.) in female clavicles indicates a decrease with increasing age category. Relative cortical area (Rel.Ct.Ar.) is largest in young adults with mean Rel.Ct.Ar. being similar in middle and older adults. With length measurements, periosteal perimeter (Ps.Pm.) has a similar mean among all three age

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categories, while mean endosteal perimeter (Es.Pm.) is slightly larger between young and middle adults and similar among middle and older adults.

Sex Age n Variable Mean Std Dev Minimum Maximum Female Young 14 TtAr 64.342014 21.25099 41.76 108.039 Adult PsPm 29.59964 4.8600313 24.108 39.872 EsAr 15.611684 12.327222 2.6208936 49.334 EsPm 16.181195 7.1306576 6.336297 33.328 CtAr 48.730331 11.533815 34.83 74.5732703 RelCtAr 0.7832014 0.1134213 0.5433686 0.9372391 Middle 7 TtAr 69.224662 13.111685 57.906301 91.346039 Adult PsPm 30.931523 2.7374015 28.24959 35.488717 EsAr 22.349969 8.4961015 11.523401 37.9424965 EsPm 19.704918 3.4450866 13.461346 24.739774 CtAr 46.874693 7.6278127 36.48977 59.118 RelCtAr 0.6830269 0.0774021 0.584629 0.8018837 Older 8 TtAr 65.386519 9.2992744 45.605 75.611 Adult PsPm 30.347993 1.9001006 26.025 32.133 EsAr 20.793798 8.975175 7.951 35.334 EsPm 19.51121 4.6514818 12.79 27.845 CtAr 44.592721 7.7300029 29.673 52.414 RelCtAr 0.6889571 0.1202445 0.4564585 0.8256551

Table 50. Descriptive Statistics of Female Clavicles by Age Category In order to visualize the patterns of each variable more efficiently, a series of boxplots was created for each variable measured in clavicles with age and sex identified as class variables to separate out the range of values. Each of these is demonstrated below in Figure 42 through Figure 47.

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Figure 42. Boxplot of Clavicle Total Area (Tt.Ar.) by Age and Sex

Figure 43. Boxplot of Clavicle Cortical Area (Ct.Ar.) by Age and Sex

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Figure 44. Boxplot of Clavicle Endosteal Area (Es.Ar.) by Age and Sex

Figure 45. Boxplot of Clavicle Relative Cortical Area (Rel.Ct.Ar.) by Age and Sex

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Figure 46. Boxplot of Clavicle Periosteal Perimeter (Ps.Pm.) by Age and Sex

Figure 47. Boxplot of Clavicle Endosteal Perimeter (Es.Pm.) by Age and Sex

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6.5.3 Examination of Age in Iliac Crest Data

Iliac crest sections were also examined for differences between age categories.

Variables demonstrating no significance for sex differences and/or subsistence related differences were examined for age significance; therefore, one-way and two-way

ANOVA tests to examine age differences were only conducted on cortical area (Ct.Ar.), relative trabecular area (Rel.Tb.Ar.), trabecular width (Tb.Wi.), and cortical thickness

(Ct.Dm.). Box and whisker plots for ANOVA tests conducted on iliac crest variables are included in Appendix B.

Cortical area (Ct.Ar.) did not demonstrate significance with age category using a one-way ANOVA at the 0.05 level t results at the 0.05 level (p=0.3016)(Table 51), A two-way ANOVA examining the interaction of sex as well as age was also not statistically significant (p=0.3852)(Table 52).

Source DF Sum of Mean F Value Pr > F Model 2 63.47253 31.736265 1.22 0.3016 Error 58 1504.11817 25.933072 Correcte 60 1567.5907

R-Square Coeff Root MSE CtAr Mean 0.04049 29.4285 5.092452 17.3045

Table 51. One-Way ANOVA Table for Iliac Crest Cortical Area (Ct.Ar.) for Age

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Sum of Mean Source DF F Value Pr > F Squares Square Model 5 139.363 27.8727 1.07 0.3852 Error 55 1428.23 25.9678 Corrected 60 1567.59 Total

CtAr R-Square Coeff Var Root MSE Mean 0.088903 29.44817 5.09586 17.3045

Type I Mean Source DF F Value Pr > F SS Square sex 1 0.05602 0.05602 0 0.9631 age 2 63.5376 31.7688 1.22 0.3021 sex*age 2 75.7697 37.8848 1.46 0.2413 Type III Mean Source DF F Value Pr > F SS Square sex 1 0.70705 0.70705 0.03 0.8695 age 2 44.8624 22.4312 0.86 0.4272 sex*age 2 75.7697 37.8848 1.46 0.2413

Table 52. Two-way ANOVA Table for Iliac Crest Cortical Area (Ct.Ar.)

Relative trabecular area (Rel.Tb.Ar.) was also examined using one-way and two- way ANOVA tests. The one-way ANOVA was not statistically significant

(p=0.648)(Table 53). A two-way ANOVA of relative trabecular area (Rel.Tb.Ar.) examining the interaction of age and sex was also not statistically significant (p=0.8024)(

(Table 54).

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Source DF Sum of Mean F Value Pr > F Model 2 0.00314417 0.0015721 0.44 0.648 Error 58 0.20855687 0.0035958 Correcte 60 0.21170104

R-Square Coeff Root MSE RelTbAr 0.01485 35.1728 0.059965 0.170487

Table 53. One-Way ANOVA Table of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.)

Sum of Mean Source DF F Value Pr > F Squares Square Model 5 0.01227 0.00245 0.68 0.6429 Error 55 0.19943 0.00363 Correcte 60 0.2117 d Total

RelTbAr R-Square Coeff Var Root MSE Mean 0.05796 35.3206 0.06022 0.17049

Type I Mean Source DF F Value Pr > F SS Square sex 1 0.00098 0.00098 0.27 0.6061 age 2 0.00266 0.00133 0.37 0.6949 sex*age 2 0.00864 0.00432 1.19 0.3117 Type III Mean Source DF F Value Pr > F SS Square sex 1 0.00225 0.00225 0.62 0.4342 age 2 0.00067 0.00034 0.09 0.9114 sex*age 2 0.00864 0.00432 1.19 0.3117 Table 54. Two-Way ANOVA Table of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.)

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Cortical thickness of iliac crest sections (Ct.Dm.) was examined using a one-way

ANOVA for age and a two-way ANOVA for age and sex interaction. The one-way

ANOVA was not significant at the 0.05 level (p=0.1269)(Table 55). The two-way

ANOVA was also not significant at the 0.05 level (p=0.3366)(Table 56).

Source DF Sum of Mean F Value Pr > F Model 2 2.15429239 1.0771462 2.14 0.1269 Error 58 29.2028154 0.5034968 Correcte 60 31.3571077

R-Square Coeff Root MSE CtDm 0.0687 34.663 0.709575 2.047066

Table 55. One-Way ANOVA Table of Iliac Crest Cortical Thickness (Ct.Dm.)

Sum of Mean Source DF F Value Pr > F Squares Square Model 5 3.00987 0.60197 1.17 0.3366 Error 55 28.346 0.51538 Correcte 60 31.3559 d Total

CtDm R-Square Coeff Var Root MSE Mean 0.09599 35.0703 0.7179 2.04704

Type I Mean Source DF F Value Pr > F SS Square sex 1 0.41065 0.41065 0.8 0.3759 age 2 2.1137 1.05685 2.05 0.1384 sex*age 2 0.48552 0.24276 0.47 0.6269 Type III Mean Source DF F Value Pr > F SS Square sex 1 0.1705 0.1705 0.33 0.5675 age 2 1.64347 0.82173 1.59 0.2123 sex*age 2 0.48552 0.24276 0.47 0.6269 Table 56. Two-Way ANOVA Table of Iliac Crest Cortical Thickness (Ct.Dm.)

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Mean trabecular thickness (Tb.Wi.) was examined with a one-way ANOVA for age effects and a two-way ANOVA for age and sex interaction. The one-way ANOVA of

Tb.Wi. was not significant at the 0.05 level (p=0.7877)(Table 57). The two-way

ANOVA was not significant at the 0.05 level (p=0.9247)(Table 58).

Source DF Sum of Mean F Value Pr > F Model 2 0.00770495 0.0038525 0.24 0.7877 Error 58 0.93266695 0.0160805 Correcte 60 0.94037191

R-Square Coeff Root MSE lsTrWi Me 0.00819 -14.9823 0.126809 -0.846393 Table 57. One-Way ANOVA Table of Iliac Crest Mean Trabecular Thickness (Tb.Wi.)

Sum of Mean Source DF F Value Pr > F Squares Square Model 5 0.04918 0.00984 0.61 0.6948 Error 55 0.89122 0.0162 Correcte 60 0.9404 d Total

lsTrWi R-Square Coeff Var Root MSE Mean 0.05229 -15.04 0.1273 -0.8464

Type I Mean Source DF F Value Pr > F SS Square sex 1 0.00017 0.00017 0.01 0.9197 age 2 0.00754 0.00377 0.23 0.7932 sex*age 2 0.04147 0.02073 1.28 0.2863 Type III Mean Source DF F Value Pr > F SS Square sex 1 4.6E-07 4.6E-07 0 0.9957 age 2 0.00765 0.00382 0.24 0.7906 sex*age 2 0.04147 0.02073 1.28 0.2863 Table 58. Two-Way ANOVA Table of Iliac Crest Average Mean Trabecular Thickness (Tb.Wi.)

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6.5.3.1 Descriptive Statistics of Iliac Crest Data Descriptive statistics of iliac crest data are given in Table 59-Table 61 which list the mean, standard deviation, minimum and maximum values for all Chiribaya iliac crest data. Table 59 lists the mean, standard deviation, minimum and maximum values for all

Chiribaya sections measured.

Age n Variable Mean Std Dev Min. Max. Young 28 TtAr 53.286807 13.325652 22.709 82.2479 Adult EsAR 35.156923 11.368922 10.071 57.67 CtAr 18.129885 5.4804483 7.318046 28.181 RelCtAr 0.3492465 0.1004276 0.159185 0.556519 TtTbAr 9.2220804 3.736711 1.955691 15.55364 RelTbAr 0.172852 0.0596867 0.066639 0.307042 TbWi 0.1926037 0.0556875 0.1174 0.387685 TtDm 6.6509107 1.4314747 3.418 10.779 EsDm 4.4070035 1.1942035 1.975 7.259 CtDm 2.2439072 0.6693385 1.249226 3.52 Middle 20 TtAr 60.875197 12.443985 46.72249 97.36057 Adult EsAR 43.525574 12.062652 25.417 74.93207 CtAr 17.349623 4.3503244 10.761 24.571 RelCtAr 0.2921633 0.0855504 0.159222 0.491538 TtTbAr 10.968774 5.2194261 6.109 26.19699 RelTbAr 0.1759778 0.056808 0.114397 0.315771 TbWi 0.1909193 0.0459385 0.1292 0.312167 TtDm 7.3541547 1.1598314 5.380552 9.542 EsDm 5.4200634 1.3727393 2.811036 7.702 CtDm 1.9340913 0.5408649 0.819973 2.951 Older 13 TtAr 50.3428 20.499303 18.259 86.791 Adult EsAR 34.88547 17.707567 11.982 66.122 CtAr 15.45733 5.2724427 4.532 22.35922 RelCtAr 0.3270129 0.1023386 0.17219 0.497152 TtTbAr 8.5139395 5.4507823 0.521437 19.147 RelTbAr 0.1569423 0.0652267 0.028558 0.288996 TbWi 0.1824005 0.0468654 0.085286 0.276899 TtDm 6.5221958 2.7796384 2.179 12.399 EsDm 4.7254325 2.3790676 1.497 9.949 CtDm 1.7967632 0.9809314 0.497 3.85

Table 59. Descriptive Statistics of Iliac Crest Data by Age Category

Areal measurements of iliac crest data demonstrate the largest values for total area

(Tt.Ar.) in middle adults; a pattern that is duplicated in total thickness (Tt.Dm.),

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medullary area (Es.Ar.), and medullary thickness, where the largest means are in the middle adult category. Mean cortical area (Ct.Ar.) is highest in younger adults and decreasing with increasing age category; this pattern of decrease with increasing age is also demonstrated in cortical thickness (Ct.Dm.). The standardized ratio of cortical bone to bone size, relative cortical area (Rel.Ct.Ar.), has the highest mean in younger adults and the smallest mean among middle adults, indicating a trend in lower mean bone ratios in older individuals.

In measurements of trabecular bone in the iliac crest, mean total trabecular area

(Tt.Tb.Ar.) is highest in middle adults and lowest in older adults. When adjusted for size, relative trabecular area (Rel.Tb.Ar.) is similar among young and middle adults and demonstrates a slightly larger mean in older adults. Mean trabecular thickness (Tb.Wi.) demonstrates no pattern between age categories

When males and females are considered separately, a different pattern in each sex is apparent. Table 60 lists descriptive statistics for male iliac crest sections by increasing age category. Mean total area (Tt.Ar.) in males exhibits a higher value in middle adults than younger and older adults; however, mean total thickness (Tt.Dm.) demonstrates a mean decrease with increasing age category in males. Cortical area (Ct.Ar.) exhibit higher values in middle adults than younger and older adults, but thickness of cortical bone (Ct.Dm.) demonstrates smaller means with increasing age categories. In males, relative cortical area (Rel.Ct.Ar.), demonstrates a slight reduction in mean values with increasing age. Mean medullary area (Es.Ar.) demonstrates a larger mean with increasing age; indicating that resorption at the inner cortex may also be apparent in iliac crest sections as well as male ribs and clavicles; this is not supported by measurements of

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medullary thickness at the iliac crest (Es.Dm.), which decreases with increasing age category.

In trabecular bone, mean total trabecular area (Tt.Tb.Ar.) of males is highest in middle adults, similar to total area (Tt.Ar.) and cortical area (Ct.Ar.) The same pattern is evidence in relative trabecular area (Rel.Tb.Ar.) where middle adult males exhibit larger means than older and younger males. Mean trabecular width (Tb.Wi.) is larger in middle adult males than young adults and middle adults.

With examination of iliac crest data for significant sex differences, Hypothesis 3 was not supported. The accompanying statistical hypotheses were also not supported:

H3a:periosteal and endosteal perimeters increase significantly with age, H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a significant difference in relative cortical area and relative trabecular area between young, middle, and older adults, H3d: A significant difference exists in trabecular width in iliac crest samples among age groups, H3e:

Significant differences in the total, cortical and medullary thickness/diameter of iliac crest sections exist among age groups.

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Sex Age n Variable Mean Std Dev Min. Max. Male Young 11 TtAr 56.200347 10.698964 41.995 70.16108 Adult EsAr 39.485979 10.799148 25.447 54.30507 CtAr 16.714369 5.3046646 7.318046 23.679 RelCtAr 0.3024004 0.1042197 0.159185 0.482006 TtTbAr 9.1772645 3.5492088 4.872 15.55364 RelTbAr 0.1606771 0.0424603 0.096315 0.223288 TbWi 0.1800021 0.0317499 0.130357 0.2514 TtDm 6.9270625 0.8450806 5.653 8.021732 EsDm 4.8994868 0.8801683 3.494 6.117331 CtDm 2.0275757 0.5365033 1.249226 2.582 Middle 12 TtAr 63.755229 13.977752 47.08785 97.36057 Adult EsAr 45.258099 13.329434 25.417 74.93207 CtAr 18.49713 4.0715744 11.70472 24.571 RelCtAr 0.2985322 0.0818871 0.184174 0.491538 TtTbAr 12.108086 5.714608 6.672 26.19699 RelTbAr 0.1859017 0.0584206 0.114397 0.315771 TbWi 0.202779 0.0494481 0.133438 0.312167 TtDm 7.4479418 1.1304606 5.515661 9.542 EsDm 5.5005531 1.306961 3.431 7.548311 CtDm 1.9473887 0.5914324 0.819973 2.951 Older 4 TtAr 62.946509 29.063382 20.59335 86.791 Adult EsAr 47.3675 24.371483 11.982 66.122 CtAr 15.579009 6.0097084 8.611345 20.669 RelCtAr 0.2786426 0.1040719 0.17219 0.418162 TtTbAr 11.611995 6.7855946 3.651374 19.147 RelTbAr 0.1815002 0.0438215 0.12159 0.22061 TbWi 0.1692976 0.0308719 0.145615 0.211 TtDm 8.23575 4.3199293 2.179 12.399 EsDm 6.45775 3.5998086 1.497 9.949 CtDm 1.778 1.1187353 0.682 2.993 Table 60. Descriptive Statistics of Male Iliac Crest by Age Category

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Table 61 lists descriptive statistics for female iliac crest sections by increasing age category. In females, mean total area (Tt.Ar.) and mean total thickness (Tt.Dm.) are higher in middle adults than younger and older adults. In female iliac crest sections, mean cortical area (Ct.Ar.) and mean cortical thickness (Ct.Dm.) demonstrate a slight decrease with increasing age category. When adjusted for size, mean relative cortical area

(Rel.Ct.Ar.) indicate middle adults have the lowest bone mass than younger and older adults. Mean medullary area (Es.Ar.) and mean medullary thickness (Es.Dm.) are higher in middle adults than young adults, with older adults having the smallest mean value.

In trabecular bone, mean total trabecular bone area (Tt.Tb.Ar.) is similar for young adults and middle adults but there is a reduction in mean Tt.Tb.Ar. in older adults.

When adjusted for size, relative trabecular area (Rel.Tb.Ar.) demonstrate a pattern of decrease with increasing age category. Mean trabecular width (Tb.Wi.) is highest in young adults; however, older adults demonstrate a higher mean Tb.Wi. than middle adults. This may be evidence of a compensatory mechanism in trabecular thickening with increasing age.

In order to visualize the pattern of each variable more effectively, a series of boxplots was created for each iliac crest variable by age and sex. Each of these is demonstrated in Figure 48-Figure 57.

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Sex Age n Variable Mean Std Dev Min. Max. Female Young 17 TtAr 51.4016 14.7817 22.709 82.2479 Adult EsAr 32.3558 11.1357 10.071 57.67 CtAr 19.0458 5.55235 8.01413 28.181 RelCtAr 0.37956 0.08801 0.20643 0.55652 TtTbAr 9.25108 3.96071 1.95569 15.5229 RelTbAr 0.18073 0.06868 0.06664 0.30704 TbWi 0.20076 0.06651 0.1174 0.38768 TtDm 6.47222 1.71032 3.418 10.779 EsDm 4.08834 1.28365 1.975 7.259 CtDm 2.38389 0.72328 1.443 3.52 Middle 8 TtAr 56.5551 8.81729 46.7225 67.585 Adult EsAr 40.9268 10.1431 26.0798 56.824 CtAr 15.6284 4.43562 10.761 24.0043 RelCtAr 0.28261 0.09568 0.15922 0.47928 TtTbAr 9.25981 4.13073 6.109 18.5574 RelTbAr 0.16109 0.05454 0.11612 0.28615 TbWi 0.17313 0.03582 0.1292 0.24289 TtDm 7.21347 1.26704 5.38055 9.164 EsDm 5.29933 1.55011 2.81104 7.702 CtDm 1.91415 0.49355 1.22172 2.56952 Older 9 TtAr 44.7412 14.1014 18.259 66.796 Adult EsAr 29.3379 11.6222 13.727 51.756 CtAr 15.4033 5.30505 4.532 22.3592 RelCtAr 0.34851 0.09979 0.22516 0.49715 TtTbAr 7.13703 4.5131 0.52144 15.3479 RelTbAr 0.14603 0.07229 0.02856 0.289 TbWi 0.18822 0.05304 0.08529 0.2769 TtDm 5.76062 1.57187 3.287 7.898 EsDm 3.95551 1.2096 2.4901 6.326 CtDm 1.8051 0.98679 0.497 3.85 Table 61. Descriptive Statistics of Female Iliac Crest by Age Category

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Figure 48. Distribution of Iliac Crest Total Area (Tt.Ar.) by Sex and Age

Figure 49. Distribution of Iliac Crest Cortical Area (Ct.Ar.) by Sex and Age

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Figure 50. Distribution of Iliac Crest Area Containing Trabecular Bone (Es.Ar.) by Sex and Age

Figure 51. Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.) by Sex and Age 210

Figure 52. Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.) by Sex and Age

Figure 53. Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.) by Sex and Age

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Figure 54. Distribution of Iliac Crest Mean Trabecular Width (Tb.Wi.) by Sex and Age

Figure 55. Distribution of Iliac Crest Total Diameter/Thickness (Tt.Dm.) by Sex and Age

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Figure 56. Distribution of Iliac Crest Marrow Cavity Diameter/Thickness (Es.Dm.) by Sex and Age

Figure 57. Distribution of Iliac Crest Cortical Diameter/Thickness (Ct.Dm.) by Sex and Age

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6.6 Examination of Intra-skeletal Variability To examine intra-skeletal variability, relative cortical area (Rel.Ct.Ar.) from rib, clavicle, and iliac crest sections of 43 individuals with all three bones present was used.

Rel.Ct.Ar. was selected for comparison as this variable is standardized and has no units.

A randomized block ANOVA was conducted to examine intra-skeletal variability in the

Chiribaya sample. ANOVA results are in Table 62 and garnered a significant results

(p<0.0001). The “blocks” in the model represent the three skeletal sites selected: rib, clavicle and iliac crest.

Sum of Mean Source DF F Value Pr > F Squares Square Model 44 3.84232 0.08733 13.85 <.0001 Error 84 0.52951 0.0063 Corrected 128 4.37184 Total

lsTrWi R-Square Coeff Var Root MSE Mean 0.878881 15.7597 0.0794 0.50379

Type I Mean Source DF F Value Pr > F SS Square ind 42 0.8867 0.02111 3.35 <.0001 block 2 2.95563 1.47781 234.43 <.0001

Table 62. Randomized Block ANOVA Table Testing Intra-skeletal Variability

The results of the ANOVA indicate that intra-skeletal variability is a factor that should be considered in histological analyses and that each skeletal element should be considered differently with examination of bone remodeling and bone loss.

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6.7 Chapter Summary The contents of this chapter represent the statistical and non-statistical examination of data measured in the rib, clavicle, and iliac crest within the Chiribaya sample. Intra-observer or each variable was tested with 30 repeated measurements of each variable using a one-way ANOVA. Normality of variables grouped by sex, subsistence by site affilitation, and age category was examined using the K2 omnibus test of normality with a critical value of 5.991. Variables that were skewed and/or kurtotic were transformed using either a square root or log(square root) transformation.

A series of t-tests were conducted to test Hypothesis 1: sex differences exist in the histomorphometric areal and perimeter/length measurements in the Chiribaya sample. In rib data, statistical significance was found with endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.), and endosteal perimeter (Es.Pm.). In clavicle comparisons, all variables (Tt.Ar., Ct.Ar., Es.Ar., Rel.Ct.Ar., Ps.Pm., Es.Pm.) were significant at the 0.05 level. In the iliac crest, total area (Tt.Ar.), medullary area

(Es.Ar.), relative cortical area (Rel.Ct.Ar.), and medullary thickness (Es.Dm.) were statistically significant. These findings in the rib, clavicle, and iliac crest support H1 and the statistical hypotheses: H1a: periosteal and endosteal perimeters significantly differ between the sexes, H1b: significant differences exist in areal and relative areal bone measurements between the sexes, and H1d: significant differences exist in total, cortical and medullary thickness/diameter between males and females. H1c was not supported by statistical testing: males have thicker on average trabecular width in iliac crest samples than females. Table 58 summarizes the statistical findings for each variable for each skeletal sampling site.

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Sex Examination: Summary of Statistical Significance (Pr>|t|) Variable Rib Clavicle Iliac Crest Total Area (Tt.Ar.) 0.0596 <0.0001 0.0119 Cortical Area (Ct.Ar.) 0.5975 0.0033 0.9635 Endosteal Area (Es.Ar.) 0.0091 0.0001 0.0047 Periosteal Perimeter (Ps.Pm.) 0.0445 <0.0001 Endosteal Perimeter (Es.Pm.) 0.0355 <0.0001 Relative Cortical Area (Rel.Ct.Ar.) 0.0109 0.0061 0.0408 Relative Trabecular Area (Rel.Tb.Ar.) 0.6033 Total Trabecular Area (Tt.Tb.Ar.) 0.067 Trabecular Thickness (Tb.Wi.) 0.919 Cortical Thickness (Ct.Dm.) 0.3798 Medullary Thickness (Es.Dm.) 0.0092 Figure 58. Summary of Statistical Significance Found in Sex Examinations

To examine differences between subsistence indicated by cultural site affiliation, a series of t-tests was employed to test Hypothesis 2: differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta,

El Yaral, and San Geronimo due to variation of diet and subsistence level activities. The sample from El Yaral (labradores) was too small in size to normalize (n=7) and was not included in initial t-tests. Differences in means were initially compared between individuals at Chiribaya Alta (mercaderes) and San Geronimo (pescadores). After initial t-tests were conducted, another series of t-tests were conducted to compare Chiribaya

Alta data to a combined sample of San Geronimo and El Yaral data. In the ribs, total area

(Tt.Ar.) and endosteal area (Es.Ar.) were significant in both series of t-tests. In the clavicle, initial t-tests comparing the means of Chiribaya Alta and San Geronimo noted significance in total area (Tt.Ar.), cortical area (Ct.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.), and endosteal perimeter (Es.Pm.). The secondary round of t-tests comparing Chiribaya Alta to a pooled dataset of San Geronimo and El Yaral only

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found significance in total area (Tt.Ar.), periosteal perimeter (Ps.Pm.) and endosteal perimeter (Ps.Pm.). Within iliac crest data, initial t-tests found significance in relative cortical area (Rel.Ct.Ar.) and total trabecular area (Tt.Tb.Ar.). A secondary round of t- tests found no significant differences in iliac crest data between Chiribaya Alta indivdiuals and a pooled sample of San Geronimo and El Yaral individuals. Statistical findings for subsistence/site differences are condensed in Table 59.

Site Affiliation/Subsistence Examination: Summary of Statistical Significance (Pr>|t|) Chiribaya Alta Tested Against San Geronimo Variable Rib Clavicle Iliac Crest Total Area (Tt.Ar.) 0.0117 0.0119 0.1208 Cortical Area (Ct.Ar.) 0.0601 0.025 0.7562 Endosteal Area (Es.Ar.) 0.0146 0.1234 0.0569 Periosteal Perimeter (Ps.Pm.) 0.1973 0.0148 Endosteal Perimeter (Es.Pm.) 0.8798 0.0208 Relative Cortical Area (Rel.Ct.Ar.) 0.2669 0.009 0.0326 Relative Trabecular Area (Rel.Tb.Ar.) 0.1099 Total Trabecular Area (Tt.Tb.Ar.) 0.0374 Trabecular Thickness (Tb.Wi.) 0.0552 Total Thickness (Tt.Dm.) 0.2994 Cortical Thickness (Ct.Dm.) 0.9895 Medullary Thickness (Es.Dm.) 0.2704 Chiribaya Alta Tested Against San Geronimo & El Yaral Variable Rib Clavicle Iliac Crest Total Area (Tt.Ar.) 0.0314 0.0187 0.289 Cortical Area (Ct.Ar.) 0.1768 0.0621 0.8014 Endosteal Area (Es.Ar.) 0.0314 0.0651 0.2735 Periosteal Perimeter (Ps.Pm.) 0.3818 0.0124 Endosteal Perimeter (Es.Pm.) 0.619 0.038 Relative Cortical Area (Rel.Ct.Ar.) 0.1291 0.2834 0.3614 Relative Trabecular Area (Rel.Tb.Ar.) 0.2724 Total Trabecular Area (Tt.Tb.Ar.) 0.1432 Trabecular Thickness (Tb.Wi.) 0.1617 Total Thickness (Tt.Dm.) 0.7593 Cortical Thickness (Ct.Dm.) 0.6321 Medullary Thickness (Es.Dm.) 0.9075 Figure 59. Site Affiliation/Subsistence Examination: Summary of Statistical Significance 217

Statistical analysis of rib, clavicle, and iliac crest data partially support

Hypothesis 2: differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo due to variation of diet and general subsistence level activities. These data indicate that differences due to subsistence patterns have an impact on bone mass and variables that impact bone strength; however, the skeletal sampling site must be taken into consideration as variables are impacted to varying degrees in different areas of the skeleton. When the two sites of non-elites were pooled together and compared to the site of elites, the findings of statistical significance were fewer in number, suggesting there are more similarities within the Chiribaya subsamples when they are considered together.

Examination of age was only conducted on variables not demonstrating significant differences due to sex and/or subsistence. In the rib, cortical area (Ct.Ar.) was examined for statistical significance using both one-way and two-way ANOVA tests, both of which were not significant. In the clavicle, all variables noted statistical significance in sex and subsistence and as such, were not statistically examined for age differences. With iliac crest data, cortical area (Ct.Ar.), relative trabecular area

(Rel.Tb.Ar.), mean trabecular thickness (Tb.Wi.), and cortical thickness (Ct.Dm.) were examined using one-way and two-way ANOVA tests. None of these comparisons were significant at the 0.05 level. Descriptive statistics were listed and boxplots were created for a visual comparison of the means and medians for all variables according to age and sex category by skeletal sampling site.

Intra-skeletal variability was examined using 43 individuals with all three skeletal sampling areas intact. A random block ANOVA was used to compare relative cortical

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area (Rel.Ct.Ar.) measurements for these three sites within each individual, as this measurement is a standardized ratio and not dependent upon size differences between different areas of the skeleton. The random block ANOVA was significant at the 0.05 level and demonstrated the importance in examining more than one skeletal sampling site within a single individual.

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Chapter 7: Discussion

This chapter discusses the results achieved in statistical analysis and non- statistical comparisons of the Chiribaya data set. This chapter is grouped into separate discussions related to hypotheses tested to examine the effects of sex, site and subsistence, age, and intra-skeletal variability. Within each section, the results obtained on the rib, clavicle, and iliac crest are further discussed. This chapter places the results of

Chiribaya histomorphometric observations within findings reported by other authors on both modern and archaeological populations. Limitations of the study are addressed and potential for future research is discussed.

7.1 Examination and Discussion of Sex Differences

To test Hypothesis 1: Sex differences exist in the histomorphometric areal and perimeter/length measurements in the Chiribaya sample, a series of t-tests were conducted on each variable in the rib, clavicle, and iliac crest examining differences between males and females. This hypothesis was supported in all skeletal sampling sites, with different variables demonstrating significance at each site; as such, the rib, clavicle, and iliac crest are discussed separately and statistical hypothesis are re-examined for each bone.

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7.1.1. Examination of Sex Differences in the Rib

In the rib, statistical significance was found in t-tests conducted on sex differences in endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter

(Ps.Pm.) and endosteal perimeter (Es.Pm.). Relative cortical area was significantly larger in females, a finding supported by previous authors indicating that the ratio of cortical bone to total area is generally higher in females than males, at least until the fifth decade

(Drusini et al. 2000). Endosteal area was significantly larger in males, supporting previous studies demonstrating an increase in endosteal resorption in male ribs (Seeman

1999; Stini 2003). Total area and cortical area were not significantly different in male and female ribs; however, males were found to have higher mean values than females, although the difference was not significant. Both perimeter measurements, periosteal and endosteal, were significantly larger in males. Larger perimeters were expected to be seen in male ribs due to findings in previous research indicating trends in bone resorption at the endosteal surface and expansion at the periosteum with age. Endosteal resorption increases bone fragility, which is compensated for at the periosteal surface, which increases bending strength (Szulc et al. 2005).

The findings in rib data analysis support Hypothesis 1 and the statistical hypotheses directed toward rib variables H1a: periosteal and endosteal perimeters significantly differ between the sexes and H1b: significant differences exist in areal and relative areal bone measurements between the sexes.

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7.1.2 Examination of Sex Differences in the Clavicle

T-tests conducted on clavicle variables were significant in all comparisons. This finding demonstrates that the clavicle, in the Chiribaya population, appears to be more sensitive to sex differences than the rib. Both perimeters, endosteal and periosteal, were found to be significantly larger in males than females, a general finding that was expected in both the rib and clavicle due to previous observations of increased endosteal resorption and periosteal expansion in males (Seeman 1999; Stini 1995). All areal measurements were larger in males than females, with the exception of relative cortical area (Rel.Ct.Ar.) which was found to be higher in females than males. This was expected due to evidence from past studies demonstrating larger cortical bone ratios in females due to the impact of pregnancy and childbirth (Drusini et al. 2000). The findings in the clavicle support

Hypothesis 1 in addition to the statistical hypotheses address clavicle variables H1a: periosteal and endosteal perimeters significantly differ between the sexes and H1b: significant differences exist in areal and relative areal bone measurements between the sexes.

7.1.3 Examination of Sex Differences in the Iliac Crest

Iliac crest t-tests were significant for total area (Tt.Ar.), endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), and medullary thickness (Es.Dm.). Males were larger in these variables with the exception of relative cortical area (Rel.Ct.Ar.); a finding that was also noted in the rib and clavicle. Other studies have reported smaller cortical thicknesses in female iliac crest sections (Brockstedt et al. 1993),whereas other have found no six differences in section or cortical thickness (Vedi et al. 2011). Total thickness was not statistically examined due to a high degree of kurtosis. These findings support 222

Hypothesis 1 and the statistical hypotheses H1b: significant differences exist in areal and relative areal bone measurements between the sexes, with regards to total area

(Tt.Ar.), medullary area (Es.Ar.) and relative cortical area (Rel.Ct.Ar.), as well as H1d: significant differences exist in total, cortical and medullary thickness/diameter between males and females, but only with regards to medullary thickness. H1c: males have thicker average trabecular width in iliac crest samples than females, was not supported in the

Chiribaya sample.

7.1.4 Summary of Sex Examination in Rib, Clavicle, and Iliac Crest

Sex comparisons were conducted to test Hypothesis 1: Sex differences exist in histomorphometric areal and perimeter/length measurements in the Chiribaya sample.

This general hypothesis was supported by results at each skeletal sampling site, but with different variables in each area of the skeleton. This general hypothesis was further examined by the following statistical hypothesis: H1a: Periosteal and endosteal perimeters significantly differ between the sexes, H1b: significant differences exist in areal and relative areal bone measurements between the sexes (total area, cortical area, medullary area, trabecular bone area, relative cortical area, and relative trabecular area, H1c: Males have thicker average trabecular width in iliac crest samples than females and H1d: Significant differences exist in total, cortical, and medullary thickness/diameter between males and females.

In both the rib and clavicle, the periosteal and endosteal perimeters (Ps.Pm. and

Es.Pm.) were found to be statistically significant between the sexes, with males being significantly larger. It was expected, and found, that in both the rib and clavicle would

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have larger perimeter measurements based on males experiencing greater periosteal expansion and endosteal resorption throughout life and a general tendency toward larger bones.

With regards to areal measurements, rib endosteal area (Es.Ar.) and relative cortical area (Rel.Ct.Ar.) was statistically significant with endosteal area significantly larger in males and relative cortical area significantly larger in females. In the clavicle, all areal and relative areal measurements were significant between the sexes, with males found to be larger in all with the exception of relative cortical area (Rel.Ct.Ar.). In the iliac crest, significant sex differences were found in total area (Tt.Ar.), endosteal area

(Es.Ar.), relative cortical area (Rel.Ct.Ar.), but not in cortical area (Ct.Ar.), total trabecular area (Tt.Tb.Ar.) or relative trabecular area (Rel.Tb.Ar.). It was expected that males would have larger values for areal measurements because males achieve a higher peak bone mass than females in early adulthood (Stini 1995) which generally persists throughout life. Based on previous research (Drusini et al. 2000), it was assumed, and found, that Chiribaya females would have a higher cortical bone ratio than Chiribaya males.

It was expected that females would have, on average, thinner trabeculae than males, based on research presented (Macho et al. 2005). The findings in this study found no difference in mean trabecular width (Tb.Wi.) between males and females. Because this study grouped all adults over the age of 50 into one category, it was not possible to determine whether any of the females had lived to an age where a proposed compensatory thickening mechanism is thought to occur in females (Macho et al. 2005).

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It was expected that Chribaya females would have smaller cortical thickness

(Ct.Dm.) measurements at the iliac crest based on previous studies (Brockstedt et al.

1993)but this was not found in Chiribaya females. The Chiribaya population exhibits no statistical significance with regards to cortical thickness (Ct.Dm.) at the iliac crest.

However, the amount of medullary thickness (Es.Dm.) was found to be significantly larger in males and females. Total thickness (Tt.Dm.) was not tested significantly as the dataset was kurtotic. Observations of thicknesses at the iliac crest suggest that endosteal resorption, with a possible compensatory action of periosteal apposition, occurs at the iliac crest as well as in the rib and clavicle.

The variables measured within trabecular bone (Tt.Tb.Ar. and Rel.Tb.Ar.) did not demonstrate significance between males and females. This may be the result of having a smaller number of older individuals in the study sample as these differences are generally not apparent until the post-menopausal period in women, and because bone loss generally occurs at a much later age in males (Riggs et al. 1998; Riggs and Melton 1986; Riggs and

Melton 1983).

The Chiribaya population was found to have sex differences in all three skeletal sites examined: rib, clavicle, and iliac crest; however, the differences noted varied by bone. The clavicle was significant in all histomorphometric variables examined, while the rib was only statistically significant in perimeter measurements, relative cortical area

(Rel.Ct.Ar.) and endosteal area (Es.Ar.). The iliac crest demonstrated statistical significance in total area (Tt.Ar.), medullary area (Es.Ar.), relative cortical area

(Rel.Ct.Ar.) and endosteal diameter (Es.Dm.). Histomorphometric analysis of trabecular

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bone in the iliac crest did not demonstrate any statistical significance for sex differences.

Sex comparisons of the three skeletal sites stress the importance in examining more than one site within skeleton comparison studies involve similar sampling sites.

7.2 Examination and Discussion of Subsistence by Site Affiliation

In order to test Hypothesis 2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral, and San Geronimo due to variation of diet and general subsistence level activities, a series of statistical were conducted on variables from the three Chiribaya subsamples.

However, the sample size for El Yaral was too small to be normalized for skewness and kurtosis to be statistically analyzed in a site-to-site comparison. Therefore, an initial series of t-tests was conducted comparing the means of Chiribaya Alta and San Geronimo data only. In order to incorporate El Yaral data, El Yaral data was merged with San

Geronimo data, effectively creating a merged data set of laborers (pescadores and labradores) with which to compare with elites of Chiribaya Alta. A second series of t- tests was conducted using a pooled population of El Yaral and San Geronimo data which was compared to the means of Chiribaya Alta. This was conducted to determine whether significance could be found between Chiribaya Alta, which is thought to be comprised of a class of elite individuals, and individuals who were considered laborers: pescadores of

San Geronimo and labradores of El Yaral. The pooled data set of pescadores and labradores resulted in fewer findings of significance, indicating that when non-elite

Chiribaya sites are pooled together, they become more similar to the elite site. This lends evidence toward the interpretation that Chiribaya Alta is comprised of an elite class of

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individuals who originated from other sites, by demonstrating the overall homogeneity of the Chiribaya population.

7.2.1 Examination of Site and Subsistence Level Differences in Rib Data

Initial t-tests of rib data comparing San Geronimo to Chiribaya Alta found significance in total area (Tt.Ar.) and endosteal area (Es.Ar.). Cortical area (Ct.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.) and endosteal perimeter

(Es.Pm.) were not statistically significant. Total area was found to be significantly larger in San Geronimo ribs than Chiribaya Alta ribs, and with visual comparisons to El Yaral means and standard deviations, San Geronimo ribs are larger in general than both of the other two sites. Cortical area and relative cortical area was not found to be statistically significant; however, when plotted, rib cortical area is largest among San Geronimo individuals than Chiribaya Alta and El Yaral. When cortical bone is examined as a ratio to overall rib size, this amount is actually highest among Chiribaya Alta individuals, although this difference was not statistically significant.

Analysis of rib data for subsistence-related site differences, the significant findings of total area (Tt.Ar.) and endosteal area (Es.Ar.) support Hypothesis 2, as well as the statistical hypotheses H2b: total, cortical, medullary and trabecular areas significantly differ according to subsistence-level differences at Chiribaya Alta, El

Yaral, and San Geronimo. The findings do not support H2a: periosteal and endosteal perimeters differ significantly according to subsistence-level differences at Chiribaya

Alta, El Yaral, San Geronimo or H2c: There is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya

Alta, El Yaral, and San Geronimo. 227

7.2.2 Examination of Site and Subsistence Level Differences in Clavicle Data

Initial t-tests conducted on clavicle data between Chiribaya Alta and San

Geronimo data demonstrate statistical significance with endosteal area (Es.Ar.), total area

(Tt.Ar.), cortical area (Ct.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter

(Ps.Pm.), and endosteal perimeter (Es.Pm.)

A second series of t-tests conducted on a pooled dataset of San Geronimo and El

Yaral data compared to the means of Chiribaya Alta generated significance in total area

(Tt.Ar.), periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Pm.); all three of these variables were larger in Chiribaya Alta individuals, who are thought to be elite individuals.

When examining means and distributions of the three sites, relative cortical area

(Rel.Ct.Ar.) appears to be larger in El Yaral individuals (labradores), although this was not tested significantly. Examination of a larger dataset of the El Yaral community would confirm whether this trend of larger clavicles at Chiribaya Alta, while larger relative ratios of cortical bone amounts were apparent at El Yaral. The clavicle sample size for El

Yaral was small (n=5) in comparison to Chiribaya Alta (n=15) and San Geronimo

(n=34); therefore, means and standard deviations, as well as statistical significance, may be reflective of small sample sizes.

Analysis of clavicle variables for subsistence-related site differences support

Hypothesis 2 as well as the statistical hypotheses addressing clavicle variables H2a: periosteal and endosteal perimeters differ significantly according to subsistence-level differences at Chiribaya Alta, El Yaral and San Geronimo, H2b: total, cortical, medullary and trabecular areas significantly differ according to subsistence-level

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differences at Chiribaya Alta, El Yaral, and San Geronimo, and H2c: there is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

7.2.3 Examination of Site and Subsistence Level Differences in Iliac Crest Data

Initial t-tests comparing Chiribaya Alta data to San Geronimo data found significance in relative cortical area (Rel.Ct.Ar.) and total trabecular area (Tt.Tb.Ar.).

Relative cortical area was significantly larger in Chiribaya Alta remains while total trabecular area was significantly larger in San Geronimo remains. This indicates that

Chiribaya Alta individuals had significantly higher ratios of bone mass in the iliac crest, then the pescadores at San Geronimo. The lack of significance in other parameters measured in the iliac crest suggests this region is not as sensitive to effects of subsistence related activity. Secondary t-tests conducted, testing Chiribaya Alta data against a pooled group of San Geronimo and El Yaral data found no significance between the two groups.

Non-statistical comparison of data suggests differing trends, but lack statistical significance because the El Yaral data sample was limited in size. Visual comparisons suggests that individuals from El Yaral express larger cortical areas, relative cortical areas and cortical thickness, while San Geronimo individuals express larger total sectional areas, endosteal areas, total trabecular areas, and thicknesses at the iliac crest. In visually examining the spread of data from all three sites, it suggests that in iliac crest data, San Geronimo individuals have thicker iliac crests, but that individuals from El

Yaral have denser bones, although this assumption requires further statistical examination. This may also be reflective of unequal sample sizes in iliac crest specimens from each site (San Geronimo n=39; El Yaral n=5; Chiribaya Alta n=15). 229

These findings partially support H2b: total, cortical, medullary, and trabecular areas differ significantly according to subsistence-level differences at Chiribaya Alta, El

Yaral, and San Geronimo, but only in terms of total trabecular area (Tt.Tb.Ar.) when laborer sites are not pooled and H2c only with regards to relative cortical area

Yaral, and San Geronimo and H2e: total, cortical and medullary thickness/diameter of iliac crest sections will significantly differ in individuals according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

7.2.5 Summary of Site and Subsistence Level Differences in the Chiribaya Population Subsistence differences as indicated by site specialization were examined using t- tests to test Hypothesis 2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral and San

Chiribaya Alta, El Yaral, and San Geronimo, H2b: Total, cortical, medullary and 230

trabecular areas significantly according to subsistence-level differences at Chiribaya

Alta, El Yaral, and San Geronimo, H2c: There is a significant difference in relative cortical area and relative trabecular area according to subsistence-level differences at

Chiribaya Alta, El Yaral, and San Geronimo, and H2d: There is a significant difference in trabecular width in iliac crest samples between individuals at Chiribaya Alta, El Yaral and San Geronimo, and H2e: Total, cortical and medullary thickness/diameter of iliac crest sections will significantly differ in individuals according to subsistence-level differences at Chiribaya Alta, El Yaral, and San Geronimo.

It was expected that statistically significant differences would be found between

Chiribaya sites based on subsistence-level differences regarding diet and variation in physical activities. It was expected that individuals at Chiribaya Alta would exhibit parameters suggesting better “bone health” as indicated by larger relative areal measures.

This expectation was supported by statistically significant comparisons of higher relative cortical area (Rel.Ct.Ar.) in the clavicle and iliac crest of Chiribaya Alta individuals, but not in their ribs.

In all three skeletal sampling areas examined, San Geronimo individuals appear to be larger in overall size, yet lower in bone mass ratios when size is accounted for. The differences may be indicative of diet coupled with differences in physical exertion and types of physical activity each community engaged in, although the differences noted in statistical analysis were not as dramatic as originally hypothesized. Previous studies have reported histomorphometric variation in populations with differing subsistence bases

(Burr et al. 1990; González-Reimers and Arnay-De-La-Rosa 1992; Richman et al. 1979;

Stout and Lueck 1995). The minimal amount of statistical differences noted among the

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Chiribaya sample may be due to variation in sample sizes between the subsamples or may indicate another line of evidence suggesting Chiribaya Alta individuals originated at other Chiribaya sites, before claiming the ceremonial center as their home.

The larger sized bones seen in San Geronimo remains may be indicative of a disruption of the bone balance due to a dietary imbalance, as the larger periosteal perimeters are accompanied with larger endosteal perimeters. Isotope studies demonstrate a reliance on marine resources at San Geronimo (Knudson et al. 2007), which may suggest having lower amounts of dietary protein than the other two sites, as protein- calorie malnutrition can be indicated by amplified periosteal expansion and endosteal resorption (Garn et al. 1969). However, the suggestion of evidence of low protein-intake is not supported by findings in trabecular bone areal measurements in the iliac crest.

Previous studies found lower amounts of trabecular bone in populations with low protein intake (González-Reimers and Arnay-De-La-Rosa 1992; González‐Reimers et al. 2002); however, in the Chribaya sample, San Geronimo individuals demonstrate statistically significantly higher total trabecular areas in comparison to Chiribaya Alta and statistically insignificant differences in relative trabecular areas. The differences noted may be solely indicative of a size differential between individuals at the two sites, rather than indicate a nutritional inadequacy.

The iliac crest demonstrated less statistical significance in subsample comparisons than the rib and clavicle. It was initially expected that individuals from San Geronimo would exhibit thinner mean trabecular width measurements in comparison to El Yaral and Chiribaya Alta due to a disrupted balance in trabecular BMUs, but significance was not found in mean trabecular width in any of the site comparisons. Mean trabecular width

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does not appear to be responsive to subsistence/site-specific variation and may also be a reflection of the smaller sample size of older individuals in the sample population, as differences in trabecular thickness are generally not detected until older age (Macho et al.

2005). Total thickness at the iliac crest was similar at all three sites and none of the statistical comparisons conducted on iliac crest thickness resulted in significant differences.

In summation, Chiribaya Alta individuals demonstrate larger relative cortical areal measurements in the clavicle and iliac crest, indicating a higher amount of bone mass in the individuals of the elite, administrative community. San Geronimo individuals appear to have larger, thicker bones than individuals at Chiribaya Alta, but this difference does not translate to larger ratios of cortical or trabecular areas when size is taken into consideration. The findings suggest slight alterations in bone strength at all three sampling areas of the skeleton with regards to subsistence, but the differences found are unique to each skeletal sampling site and appear to be dependent upon the unique biomechanical environment of each area of the skeleton.

7.3 Examination and Discussion of Age Differences To test the general Hypothesis 3: Differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age, only variables that did not demonstrate significant differences with respect to sex and site were incorporated into statistical analyses for age comparisons. The additional statistical hypotheses that were examined were: H3a: Periosteal and endosteal perimeters increase significantly with age, H3b: Total, cortical, medullary and trabecular bone areas

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will exhibit significant relationships to increasing age, H3c: There is a significant difference in relative cortical area and relative trabecular area between young, middle and older adults, H3d: A significant difference exists in trabecular width in iliac crest samples among age groups and H3e: Significant differences in the total, cortical and medullary thickness/diameter of iliac crest sections exist among age groups. One-way

ANOVA tests were first conducted to examine the means of each variable on three age categories: young adult, middle adults and older adult. None of the initial tests were significant. Two-way ANOVA tests were conducted on the same variables to examine differences related to age, sex, and the interaction of age and sex on each variable. Again, none of these observations was statistically significant.

7.3.1 Examination of Age Differences in the Rib

In the rib, a one-way ANOVA test was only conducted on cortical area. Other variables demonstrated significance in sex and site differences, creating a difficulty in determining whether any variation demonstrated was actually attributed to sex. The one- way ANOVA test of cortical area (Ct.Ar.) was not significant for age effects. A two-way

ANOVA was also conducted on cortical area, demonstrating no significance with relation to age, sex and the interaction of age and sex. No significant differences were noted in the rib with increasing age or with increasing age when sex is considered.

In examining the spread of rib data visually and non-statistically, means for total area (Tt.Ar.), cortical area (Ct.Ar.), and relative cortical area (Rel.Ct.Ar.) suggest a general increase with increasing age category. Periosteal perimeter measurements are similar with all age categories, but endosteal perimeter demonstrates an increase in size

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with increasing age, thus suggesting an increase in the endosteal resorption with increasing age category

When males and females are considered separately, the two sexes suggest a different pattern in skeletal microstructure with increasing age. In males, perimeter measurements of the periosteal and endosteal surfaces are larger in younger age categories than among older males. General observations in other studies indicate periosteal expansion with increasing age with increasing endosteal perimeters due to resorption at the endosteal cortex, the findings of this study seem to demonstrate a lacking of larger males in the older age category (Drusini et al. 2000; Garn et al. 1969)

Visual examination of the means and distribution of female rib data suggest an increase in both periosteal and endosteal perimeter measurements with increasing age. In males, rib areal measurements (Tt.Ar., Ct.Ar., and Es.Ar.) are smaller on average with increasing age category. The pattern is reversed in females, where rib areal measurements of total area and endosteal area exhibit an increase with increasing age category, while cortical area is similar among all three age categories. Mean relative cortical area

(Rel.Ct.Ar.) in males is larger in middle adults, whereas in females, it is larger in young adult females and has similar means among young adult females and older adult females.

Although trends in bone microstructure appear visually by age category, the findings of non-statistical significance do not support Hypothesis 3 or the statistical hypotheses relegated toward rib variables H3a:periosteal and endosteal perimeters increase significantly with age, H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a

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significant difference in relative cortical area and relative trabecular area between young, middle, and older adults.

7.3.2 Examination of Age Differences in the Clavicle

In the clavicle, all variables achieved significant differences with sex comparisons and all variables, with the exception of endosteal area (Es.Ar.), was found to be significantly different in site comparisons. For this reason, ANOVA tests were not conducted on clavicle data for age because it was not possible to factor out whether significance was due to sex or site differences.

When the distribution of clavicle data is examined visually and non-statistically, the general pattern of both sexes compiled indicate a general decrease in total area and cortical area with age. Relative cortical area (Rel.Ct.Ar.) is similar among middle and older adults. Periosteal (Ps.Pm.) and endosteal perimeter (Es.Pm.) measurements in the clavicle were similar between all three age categories.

When each sex is examined separately, Chiribaya clavicles suggest a sex-specific pattern with increasing age. In males, total area, cortical area and endosteal area decrease with increasing age. In females, mean clavicle total area and endosteal area is largest in middle adults, while cortical area demonstrates a linear pattern of decrease with increasing age. Visual examination of male clavicle data suggests a decrease in periosteal perimeter (Ps.Pm.) with increasing age, while females have similar means among all three age categories. Male clavicle endosteal perimeter (Es.Pm.) is similar among all three age categories, whereas in females, clavicle endosteal perimeter (Es.Pm.) is similar among middle and older adults but larger in young adults. 236

The variation visually identified in clavicle data was not statistically examined, therefore, Hypothesis 3 and the statistical hypotheses H3a:periosteal ad endosteal perimeters increase significantly with age, H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a significant difference in relative cortical area and relative trabecular area between young, middle, and older adults could not be supported by clavicle data.

7.3.3 Examination of Age Differences in the Iliac Crest

In the iliac crest, one-way and two-way ANOVAs conducted on variables not demonstrating statistical significance in sex and site effects demonstrated no statistical significance. One-way and two-way ANOVA tests were conducted on iliac crest cortical area (Ct.Ar.), relative trabecular area (Rel.Tb.Ar.), mean trabecular width (Tb.Wi.) and cortical thickness (Ct.Dm.).

Visual, non-statistical consideration of iliac crest data suggests sex-specific aging patterns. When the population is considered as a whole, middle adults have higher means for total area (Tt.Ar.) and section thickness (Tt.Dm.). Medullary area (Es.Ar.) and medullary thickness (Es.Dm.) appears to be larger in middle adults. Due to noted trends in the literature with decreases in section thickness and area with increasing age

(Brockstedt et al. 1993; Melsen et al. 1978), this finding suggests that individuals with larger thicknesses were not present in the older age category. Mean values for cortical area (Ct.Ar.) and relative cortical area (Rel.Ct.Ar.) are larger in young adults and suggest decreasing amounts of cortical bone with increase of age category.

Mean values of trabecular measurements in the iliac crest suggest higher total trabecular area (Tt.Tb.Ar.) in middle adults and lowest in older adults. Relative trabecular 237

area (Rel.Tb.Ar.) was similar among young and middle adults, but was larger in the older adult category. Mean trabecular thickness (Tb.Wi.) was not significant when tested statistically, and did not demonstrate a pattern of increase or decrease in size with age category.

In visual examination of male iliac crest data, total area (Tt.Ar.) exhibited a higher mean among middle adults, whereas total thickness (Tt.Dm.) demonstrates a decrease in mean between increasing age category. In males, cortical area (Ct.Ar.) was highest in middle adults but cortical thickness (Ct.Dm.) and relative cortical area (Rel.Ct.Ar.) decreased with increasing age. Endosteal area (Es.Ar.) was larger with increasing age category, indicating that resorption at the inner cortex may be a factor at the iliac crest as well as in the rib and clavicle. Total trabecular bone area (Tt.Tb.Ar.) and relative trabecular area (Rel.Tb.Ar.) in males was highest in middle adults, similar to total and cortical area.

In females, total area (Tt.Ar.) and total thickness (Tt.Dm.) are higher in middle adults; the same pattern seen in males. Cortical area (Ct.Ar.) and cortical thickness

(Ct.Dm.) gradually decrease in means with each increasing age category, whereas endosteal area (Es.Ar.) increases in means from young to middle adults but is much lower on average in older adults. Female iliac crest relative cortical area (Rel.Ct.Ar.) is higher in young adults, but older adults show a higher mean than middle adults. Total trabecular area (Tt.Tb.Ar.) and relative trabecular area (Rel.Tb.Ar.) in females gradually decreases by increasing age category. Mean trabecular width (Tb.Wi.) is similar across all female age categories.

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7.3.5 Summary of Age Examination in the Chiribaya Population

Hypothesis 3: Differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age and the accompanying statistical hypotheses generated to examine age-associated variability in bone parameters H3a:periosteal and endosteal perimeters increase significantly with age,

H3b: total, cortical, medullary, and trabecular bone areas will exhibit significant relationships to increasing age, and H3c: there is a significant difference in relative cortical area and relative trabecular area between young, middle, and older adults, H3d:

A significant difference exists in trabecular width in iliac crest samples among age groups, H3e: Significant differences in the total, cortical and medullary thickness/diameter of iliac crest sections exist among age groups were not supported in the Chiribaya dataset in the rib, clavicle, or iliac crest.

Variables examined statistically demonstrated no significance in the rib (Ct.Ar.) and the iliac crest (Ct.Ar., Rel.Tb.Ar.,Tb.Wi., Ct.Dm.). Clavicle data was not statistically tested due to the compounding factors of sex and site demonstrating significance in all variables. Descriptive statistics in some variables suggest a trend according to age category with males and females suggesting a differing pattern of bone parameters in each age category. Research demonstrates that in males, bone loss leading to osteoporosis is more gradual and occurs at an older age (~70 years), whereas in females, the process is more rapid and precocious (Ostertag et al. 2009). Other studies indicate that bone loss occurs after age 75 in males and during the peri-menopausal period in females (Riggs et al. 2008; Stini 2003; Stini 1995). The small number of older individuals in the sample utilized in this study may not be allowing for an accurate representation of age categories 239

for the Chiribaya population as a whole. The older age category of this study was limited to 50+ and the exact ages of individuals in the sample are not known; it is not known whether the individuals in this sample had lived long enough to exhibit the full pattern of bone loss demonstrated in other populations.

With the Chiribaya sample, it was expected that the periosteal (Ps.Pm.) and endosteal perimeters (Es.Pm.) of ribs and clavicles would have larger means with increasing age category. This finding was not substantiated statistically and the trend in means per age grouping suggests an increase in perimeters from younger to middle adults, but suggests a mean decrease in perimeter lengths from middle adults to older adults. This finding may be the result of having a small number of older individuals in the data set and/or a lack of larger individuals in the older age category. Female clavicle perimeters are similar across all age groupings and male clavicle perimeters demonstrate smaller mean periosteal perimeters (Ps.Pm.) with successive age categories, while endosteal perimeters (Es.Pm.) in males is similar across all three age categories. These data suggest that younger adults of both sexes have larger sized ribs than older adult of both sexes, which suggests a lacking of older adults in the Chiribaya sample, because perimeter measurements increase with age, rather than decrease (Drusini et al. 2000).

It was expected that with the expansion of the periosteal and endosteal perimeters, an increase in medullary (Es.Ar.) and total areas (Tt.Ar.) would be larger with increasing age category, reflecting the expansion of the cortexes. This expectation was not seen across all age categories in the rib and clavicle. Rib data for both sexes suggest a general pattern in increasing total area (Tt.Ar.) with increasing age category. Rib areal measurements in females do suggest a slight increase in areal measurements with 240

increasing age. In clavicles, male total area (Tt.Ar.) and endosteal area (Es.Ar.) have smaller means with increasing age categories. In female clavicles, total area (Tt.Ar.) and endosteal area (Es.Ar.) is larger in middle adults, with younger adults and older adults having smaller means. Areal measurement data supplements observations of perimeters, in that the older age sample of males and females may be lacking in larger-sized individuals.

It was expected that cortical area (Ct.Ar.) in each skeletal sampling site would have smaller means per increasing age category due to an increase in cortical bone loss beginning during the fourth decade of life, resulting in a reduction in cortical thickness

(Garn 1972; Garn et al. 1969; Ruff and Hayes 1982). One-way and two-way ANOVA tests in the rib found no significance in cortical area (Ct.Ar.) across sexes and ages. Male ribs and clavicles have smaller means per increasing age category, but female means are similar in number for each age group in both the rib and clavicle. One-way and two-way

ANOVA tests of cortical area (Ct.Ar.) and cortical thickness (Ct.Dm.) in the iliac crest between sexes and age categories and sexes were not significant. The ratio of cortical bone adjusted for bone size, relative cortical area (Rel.Ct.Ar.), has similar means in male ribs and clavicles across all age categories. Female ribs and clavicles have larger mean relative cortical area (Rel.Ct.Ar.) in younger adults, but middle and older adults have similar means in both skeletal locations. These data suggest that cortical bone loss was not significantly noted in the Chiribaya sample. These findings suggest that cortical bone loss was not experienced in the Chiribaya until an older age, possibly due to the positive impact of strenuous physical activity prior to the fourth decade of life.

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It was expected that a reduction in amounts of trabecular bone (Tt.Tb.Ar.) and relative trabecular bone areas (Rel.Tb.Ar.) would be evident in older age categories because trabecular bone is thought to experience loss of mass first because it has greater surface area, and more accessible areas for resorption (Dodds et al. 1989; Parfitt 1984).

Trabecular bone is more metabolically active and more vulnerable to disruptions in the balance of bone turnover and the first to demonstrate symptoms of osteopenia (Dodds et al. 1989). One-way and two-way ANOVA tests of relative trabecular area (Rel.Tb.Ar.) were not significant between age categories between males and females. Means of relative trabecular area (Rel.Tb.Ar.) are similar across the three age categories in males and females. Total trabecular bone areal measurements (Tt.Tb.Ar.) were also similar in both sexes across age categories. The lack of significant findings in the Chiribaya data sample suggest that trabecular bone loss, in addition to cortical bone loss, was not apparent with increasing age grouping. Although trabecular bone is thought to be more sensitive to bone turnover than cortical bone, it appears that both bone tissues in this

Chiribaya sample do not exhibit the characteristic decrease in bone mass with age.

One-way and two-way ANOVA tests were not significant for mean trabecular width (Tb.Wi.) measurements in males and females of the three age categories. A significant decrease in mean trabecular width was expected to be visualized with increasing age category (Mellish et al. 1989; Parfitt 1984). This finding, coupled with the lack of significance in mean trabecular width (Tb.Wi.) in sex and subsistence examination, suggests that thickness of trabeculae is a characteristic of bone microstructure not heavily influenced by sex differences, dietary differences, and activities related to subsistence strategy. The lack of findings in mean trabecular width 242

(Tb.Wi.) may also be due to a small number of older individuals, and the possibility that the older adult category did not contain individuals who had lived to an age where trabecular thinning occurs.

7.4 Discussion of Intra-skeletal Variability Results

To test Hypothesis 4: Variability in relative area measurements due to differential loading throughout the skeleton exist, and the statistical hypothesis H4:

Relative cortical area differs significantly among the rib, clavicle and iliac crest, relative cortical area was used to examine intra-skeletal variability within the Chiribaya sample.

Relative cortical area is common to all three skeletal samples used in the project and is a standardized ratio of areal cortical bone to areal total size and is not dependent upon bone size. Forty-three individuals with intact rib, clavicle and iliac crest were included in statistical analysis for intra-skeletal variability.

Application of a randomized block ANOVA detected a significant difference between relative cortical area of the three bones within each individual (p<0.0001). The findings within this study are consistent with other studies in intra-skeletal variability

(Dempster et al. 1993; Lips et al. 1978; Peck and Stout 2007; Thomsen et al. 2002; Verna et al. 1999; Wright et al. 1990). The significance detected with these results provides further evidence toward the importance in considering the unique biomechanical environment of skeletal sampling sites selected for histomorphometric analysis.

Based on previous studies (Doyle 2011; Peck and Stout 2007), it was thought that the rib, clavicle and iliac crest would exhibit significant differences in bone mass based on differences in their mechanical environments. In this particular study, which included males and females of varying ages, mean relative cortical area for the clavicle (0.69) was 243

higher than rib mean relative cortical area (0.50) and iliac crest mean relative cortical area (0.32). Results of the random block ANOVA support Hypothesis 4 such that the results demonstrate no pattern in bone mass with regards to age or sex.

7.5 General Chiribaya Histomorphometrics in Comparison to Other Populations

The Chiribaya population as a whole offers insight into bone microstructural variation in ancient South American populations. The contents of this section discuss other studies with comparable variables and published data and/or descriptive statistics.

7.5.1 Chiribaya Rib and Clavicle Histomorphometry

Published studies including raw data are limited, but several published studies of rib and clavicle histomorphology include descriptive statistics of rib and clavicle areal measurements (Cho et al. 2002; Epker et al. 1965; Harrington et al. 1993). A seminal study of rib histomorphometry of 140 normal, modern individuals (Epker et al. 1965) lists the means by age decade for ribs. When adjusted to reflect means of age categories utilized in this study of Chiribaya ribs, the means of total area (Tt.Ar.) are: 54.78 mm2 for young adults, 54.39 mm2 for middle adults, and 58.68 mm2 for older adults. In comparison to the results of this study, mean total area (Tt.Ar.) for Chiribaya ribs in males 76.73 mm2; mean total area for Chiribaya females is 66.74 mm2. The means found in this study are much larger for each age category than reported in the modern study. By age category, Chiribaya female ribs have means of 64 mm2 (young adult), 68.89 mm2

(middle adult) and 72.98 mm2 (older adults). By age category, Chiribaya males have means of 91 mm2 (young adult), 71 mm2 (middle adult) and 62.21 mm2 (older adult). In metabolically normal humans, the rib should increase in total area throughout life, 244

corresponding to an increase in endosteal perimeter; this demonstrates a balance between resorption and formation at the two bone surfaces. The pattern of increased periosteal extension is not visible in Chiribaya male ribs through total area or periosteal perimeter measurements and may be reflective of bias in sample size, such that larger and smaller individuals are not present for all age categories in this sample. Similar findings with total area (Tt.Ar.) are evidence at the clavicle midshaft, a clinical study reports a mean total area (Tt.Ar.) of 67 mm2 in modern individuals (Harrington et al. 1993). At midshaft,

Chiribaya male clavicles average 90.44 mm2 and Chiribaya females average 64.8 mm2.

Both Chiribaya males and females have higher means than data from the modern sample.

The Chiribaya subsample represents larger sized ribs and clavicle than published data on modern populations.

Chiribaya ribs also have higher means for cortical area (Ct.Ar.) in comparison to modern data, corresponding to a propensity for larger sized ribs. Rib cortical area (Ct.Ar.) has the following reported values in normal, modern samples by age-decade (Epker et al.

1965): 25.68 mm2 for ages 20-29; 21.37 mm2for ages 30-39; 20.26 mm2for ages 40-49;

20.38 mm2for ages 50-59; and 19.75 mm2 for ages 60-69. Chiribaya males average 36.28 mm2 for rib cortical area (Ct.Ar.); Chiribaya females average 34.91 mm2 for rib cortical area (Ct.Ar.). By age category, Chiribaya males exhibit mean rib cortical areas of 41.25 mm2 (young adult), 34.69 mm2 (middle adult), 27.35 mm2 (older adult). Chiribaya females exhibit mean rib cortical areas of 35.53 mm2 (young adult), 33.19 mm2 (middle adult), and 35.20 mm2 (older adult). Because total area (Tt.Ar.) and cortical area (Ct.Ar.) are areal measures not standardized for size, the larger means of total area and cortical

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area in the Chiribaya are largely reflective of individuals with larger body sizes, and larger sized ribs, than more recent populations.

Relative cortical area (Rel.Ct.Ar.) of Chiribaya remains is much larger than reported findings in clinical studies and recent populations. A modern study reports relative cortical area (Rel.Ct.Ar.) of the rib in African Americans (0.35) and European

Americans (0.343) (Cho et al. 2002). Chiribaya males were found to have a mean relative cortical area ratio of 0.47 and females had a mean of 0.52. By age category, mean relative cortical area ratios for Chiribaya males are 0.45 (young adult), 0.48 (middle adult), and

0.46 (older adult). Chiribaya female mean relative cortical area ratios were 0.56 (young adult), 0.48 (middle adult), and 0.48(older adult). Relative cortical area is larger in all age and sex categories than modern reported data. Because relative cortical area (Rel.Ct.Ar.) is standardized for body size, and not reflective of Chiribaya having larger sized ribs, this suggests that Chiribaya individuals have higher amounts of bone mass than more recent individuals, possibly due to the bone modeling demands brought about by physically intensive subsistence strategies.

It appears that despite the hardships associated with a high altitude environment, the prevalence of generalized periosteal reactions, cribra orbitalia and porotic hyperostosis, and other indicators of poor health, and Chiribaya had larger sized ribs and larger areas of cortical bone; however, when accounting for size, the ratio of cortical bone to total size is higher than reported samples, indicating that the Chiribaya had higher amounts of bone mass than values reported in other studies.

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7.5.2 Chiribaya Iliac Crest Histomorphometry

The Chiribaya demonstrate differences to modern, clinical samples in both trabecular and cortical bone parameters. Reported thicknesses of trephines used to remove iliac crest sections vary from 5mm to 10mm and data are not comparable.

Therefore, relative areal measurements, which are standardized ratios of amount of bone

(trabecular and cortical) and take size into consideration, are discussed in addition to diameters measured in iliac crest sections. In Chiribaya males and females, cortical thickness is much higher than reported data on modern, normal populations. The

Chiribaya sample was found to have lower amounts of relative cancellous bone than studies using modern individuals.

A study examining modern female patients with primary hyperparathyroidism

(PHP) reports data for section thickness (Tt.Dm.) and cortical width (Ct.Dm.) of the iliac crest in both PHP patients and a control sample of normal females (Christiansen et al.

1993). Published data for section thickness in modern females under 50 years of age with

PHP is 7.2 mm and a mean of 7.5 for the control group. Females over 50 years had larger section thicknesses with a mean of normal and 7.9 mm in patients with PHP and 8.6 mm for the control. Another study, using a modern population from Denmark, reports mean total thickness of 7.6 mm for females aged 19-80 years (Brockstedt et al. 1993). In

Chiribaya females, the young adult age category has a mean of total section thickness of

6.47 mm, whereas middle adults have a mean of 7.21 and older adults have a mean of

5.76. The Chiribaya sample does not represent the same increase in total section thickness with age as is represented in the modern female study. Chiribaya females also demonstrate smaller thickness of iliac crest sections and smaller thickness in cortical

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width in all age categories with comparison to modern data. The average total section thickness of males in a modern study is 8.00 mm aged 21-90 (Brockstedt et al. 1993).

Chiribaya males have a mean iliac crest section thickness of 7.35 mm. The mean of young male Chiribaya total section thickness is 6.93 mm, middle adults is 7.48 mm and older adults in 8.23 mm. Age and sex cohorts indicate an increase in total thickness with age, suggesting expansion of the outer cortex with age. The lower mean in Chiribaya males, when compared to modern males, may be reflective of having fewer older males available for measurement in the Chiribaya sample.

Cortical thickness at the iliac crest was also reported in the (Christiansen et al.

1993). Cortical thickness in the iliac crest was reported as 1.09 mm in females with PHP, and 1.19 mm in a control group, for females under 50 years. Females over 50 years had a mean of 0.90 mm among PHP patients and 0.91 in the control. Melsen et al. (1978) also report data on cortical thickness at the iliac crest with data grouped by twenty year age categories. Cortical thickness on normal, modern females was reported as: 1.60 mm in females aged 10-29; 1.05 mm in females aged 30-49; 0.90 mm in females aged 50-69; and 0.90 mm in females aged 70+. Another study of modern humans from Denmark, reports an overall mean cortical thickness of 1.1 mm in females aged 19-80 years

(Brockstedt et al. 1993). A modern population from the UK noted a mean of 1.162 mm in females with a mean age of 47 years (Vedi et al. 2011). Chiribaya cortical thickness was taken as a total measure of both cortexes; this number is halved here to be comparable to the literature. In young adult females of the Chiribaya sample, the mean is 1.19 mm; in middle adults, the mean is 0.96 mm and in older adults, the mean is 0.90 mm. Despite having thinner total sections than modern samples, Chiribaya females have comparable

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thickness of cortical bone in the iliac crest with modern females. The trend of decrease in cortical thickness with increasing age category is a reflection of marrow cavity expansion.

Similar comparisons may be made in Chiribaya males when compared to modern data. Reported data on normal, modern male iliac crest sections (Melsen et al. 1978) report cortical thicknesses of: 1.27 mm in males aged 10-29; 1.08 in males aged 30-49;

0.86 in males aged 50-69; and 0.95 in males aged 70+. A Danish study of modern males found a mean cortical thickness of 0.9 mm in modern males ranging in age from 21-90 years (Brockstedt et al. 1993). A modern sample of UK males found a mean of 0.935 mm in cortical thickness (Vedi et al. 2011). Male Chiribaya cortical thicknesses found in this study are 1.01 mm in young adult males, 0.975 mm in middle adult males, and 0.894 in older adult males. Thickness of cortical bone in Chiribaya males is comparable, and slightly larger, than modern studies.

Diameter, or thickness, measurements at the iliac crest (Tt.Dm., Ct.Dm., Es.Dm.) are also reflective of bone size and body size, as they are not standardized measures. The smaller thicknesses at the iliac crest in the Chiribaya, when compared to data of other samples, does not correlated with the larger sized ribs and clavicles noted in the

Chiribaya sample. This may suggest that the intense physical labor required of the

Chiribaya population did not place the same amount of modeling demands on the iliac crest than on the rib and clavicle.

Width of the area containing trabecular bone (recorded as Es.Dm.) was recorded as 4.89 mm in young males, 5.5 mm in middle adult males, and 6.45 in older adult males for the Chiribaya sample. Chiribaya females had means of 4.1 mm in young adults, 5.3

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mm in middle adults, and 3.96 mm in older adults. Data reported by Brockstedt et al.

(1993) indicate a mean of 5.4 mm in normal adult females aged 19-80 years and a mean of 6.3 mm in adult males aged 21-90 years. In males, this pattern reflects expansion of the marrow cavity. In females, the increase in width from young adults to middle adults and the subsequent decrease from young adults to older adults, does not mirror the pattern of cortical thickness seen in each age category (Brockstedt et al. 1993).

Relative trabecular bone (all trabecular bone measured within the section given as a ratio to total bone size) is smaller in Chiribaya females and males in comparison to modern data. Published data on normal, modern males lists the following means for relative trabecular bone area in males by age-decade cohorts (Melsen et al. 1978):

24.49% in males aged 10-19; 24.55% in males aged 20-29; 22.92% in males 30-39;

17.37% in males aged 40-49; 20.41% in males aged 50-59; 13.74% males aged 60-69;

16.24% in males aged 70-79; and 15.35% in males over the age of 80. Relative trabecular volume in Chiribaya males was found to be 16% in young adults, 18.5% in middle adults, and 18.2% in older adults. Relative trabecular volume is much lower in Chiribaya males at all age ranges and be reflective of having denser cortical bone at the iliac crest, and less need for trabecular strength.

In females, reported data of relative trabecular area of normal modern individuals per age cohort are (Melsen et al. 1978): 31.78% in females aged 10-19; 26.76% in females aged 20-29; 26.49% in females aged 30-39; 22.54% in females aged 40-49;

20.1% in females aged 50-59; 15.49% in females aged 60-69; 16.97% in females aged

70-79; and 17.65% in females over 80 years of age. Means for Chiribaya female relative trabecular volume in the iliac crest were 16% in young adults, 18.6% in middle adults,

250

and 18.15% in older adults. The ratio of trabecular bone in Chiribaya females is much lower than reported data on modern human populations, and as in males, may be reflective of having denser cortical bone at the iliac crest, and less need for trabecular strength.

7.6 Anthropological Considerations

The hypotheses tested in this study benefit physical anthropology for a variety of reasons. This study describes bone microstructure of an ancient population from a geographic region that is not well-represented in bone histology literature. It is known that bone mass and other bone parameters differ greatly between populations with variation generated from factors such as genetics, ecology as represented through subsistence adaptation, and activity levels and patterns. The results of this study present an overview of both cortical and trabecular bone parameters in an Andean population that can be used as a means of comparison for future studies in South American populations.

In regards to the focal population of this study, the results presented in this study complement other research conducted on the Chiribaya population such as skeletal pathology (Burgess 1999; Martinson 2002), agriculture and risk management (Zaro and

Alvarez 2005), archaeology (Jessup 1991; Owen 1993; Owen 2005), textile analysis

(Wallert and Boytner 1996), coca chewing (Indriati and Buikstra 2001), isotope analysis

(Knudson et al. 2007; Tomczak 2001), and comprehensive bioarchaeological studies

(Buikstra 1989; Buikstra 1995; Buikstra et al. 2005). Analysis of bone microstructure adds another component, offering a more complete view of Chiribaya lifeways.

With the inclusion of both trabecular and cortical bone data, the results of this study offer a comprehensive overview of bone microstructure in a past population. This 251

allows anthropological studies to be comparable to modern, clinical studies on bone microarchitecture, which allows for a more complete evolutionary picture of human bone health as a whole. Anthropological research regarding microstructural bone parameters differs from clinical studies in bone microstructure in that anthropology has an overbridging goal of examining patterns in the human evolutionary past.

7.7 Limitations

There are several limitations to the study that should be considered as they can potentially affect the interpretation of the results.

1. The sample chosen for this study is an ancient population with no documentation

of sample demographics. Age and sex analyses were conducted by previous

researchers and are estimated based on morphological characteristics. The age

categories utilized are wide in range (20-34 years, young adult; 35-49 years,

middle adult; 50+ years, older adult). Many differences exhibited in bone mass

and bone structure do not present themselves significantly until the post-

menopausal period in women, and in older ages in men. Having larger data

samples of older individuals or the use of aging methods such as seriation could

be beneficial to studies investigating age-associated bone loss such as this study.

2. Data collection for the study was collected two-dimensionally; however,

Haversian systems are oriented in three-dimensions. Advancements in technology

have led to improvements in three-dimensional imaging that present a better

representation of the microstructural properties of the bone specimen (Chappard

252

et al. 2008; Cooper et al. 2004; Cooper et al. 2003; Kuhn et al. 2007; Nägele et al.

2004; Rühli et al. 2007)

3. The study is not longitudinal in that the information is not taken from individuals

at various age stages in their life so it is not completely representative of growth

differences that occur in a single individual throughout life.

7.8 Directions for Future Research

Within the Chiribaya sample, the addition of additional variables in both cortical and trabecular bone will offer a more comprehensive view of Chiribaya bone structure alnd allow the Chiribaya data to be comparable to other studies in past populations.

Osteon population density (OPD), osteon area, and osteon circularity are variables that were not included in this study. Previous studies have indicated that OPD is higher in areas where compressive strain is dominant (Skedros et al. 1996). Analyzing OPD will also allow for the examination of activation frequency, which is an indicator of the overall intensity of bone remodeling (Parfitt 2003), and can indicate whether the demand for remodeling is high or low. Osteon morphology, in terms of size and shape have also been proposed as variables that can indicate trends in physical activity in past populations

(Pfeifer and Pinto 2012). Assessment of these variables in the Chiribaya dataset will shed more light on the effects of subsistence-level behavioral differences and may indicate behavioral differences between the three Chiribaya subpopulations.

Variables collected and analyzed from trabecular bone of the iliac crest were limited to trabecular area, relative trabecular area, and average trabecular width. Previous studies have indicated female indicators of osteoporosis in the trabecular cortex may not be resultant of trabecular thinning, but rather from an increase in the spacing between 253

trabeculae (Aaron et al. 1987; Ostertag et al. 2009). Collecting data on trabecular spacing would verify whether this trend is visible in the Chiribaya population as well.

The thin sections used in this study were created in a manner such that the areas above and below the site of clinical biopsy could be examined in a future study. A clinical study using non-pathological modern humans of both sexes and varying ages found no differences in parameters of bone mass and bone remodeling in areas adjacent to the site of clinical biopsy (Melsen et al. 1978). An examination of the areas adjacent to the standard clinical biopsy site would support previous findings and demonstrate homogeneity in this region in a human population from the past.

There are very few studies regarding microstructural bone parameters in South

American populations (Robling 1998); examining histomorphometrics in neighboring populations will offer a more complete analysis of microstructural bone dynamics in this geographic region. Data from neighboring populations will also demonstrate whether histological indicators of bone biology of the Chiribaya are unique to this population or common to other Andean populations.

7.9 Chapter Summary

This chapter discusses the hypotheses tests and the test results used in the examination of each of the hypotheses. Sex comparisons were conducted to test

Hypothesis 1: Sex differences exist in histomorphometric areal and perimeter/length measurements in the Chiribaya sample. This general hypothesis was supported by results at each skeletal sampling site, but with different variables in each area of the skeleton. The clavicle was significant in all histomorphometric variables examined, while

254

the rib was only statistically significant in perimeter measurements, relative cortical area

(Rel.Ct.Ar.) and endosteal area (Es.Ar.). The iliac crest demonstrated statistical significance in total area (Tt.Ar.), medullary area (Es.Ar.), relative cortical area

(Rel.Ct.Ar.) and endosteal diameter (Es.Dm.). Histomorphometric analysis of trabecular bone in the iliac crest did not demonstrate any statistical significance for sex differences.

Sex comparisons of the three skeletal sites stress the importance in examining more than one site within skeleton comparison studies involve similar sampling sites.

Subsistence differences as indicated by site specialization were examined using t- tests to test Hypothesis 2: Differences in histomorphometric areal and length/perimeter measurements exist among individuals from Chiribaya Alta, El Yaral and San

Geronimo due to variation of diet and general subsistence level activities. This general hypothesis was supported by results at each skeletal sampling site, but with different variables in each area of the skeleton. There was less support for this general hypothesis when communities of heavy laborers (El Yaral and San Geronimo) were pooled together and compared with a site of administrative elites (Chiribaya Alta). Chiribaya Alta individuals demonstrate larger relative cortical areal measurements in the clavicle and iliac crest, indicating a higher amount of bone mass in the individuals of the elite, administrative community. San Geronimo individuals appear to have larger, thicker bones than individuals at Chiribaya Alta, but this difference does not translate to larger ratios of cortical or trabecular areas when size is taken into consideration. The findings suggest slight alterations in bone strength at all three sampling areas of the skeleton with regards to subsistence, but the differences found are unique to each skeletal sampling site

255

and appear to be dependent upon the unique biomechanical environment of each area of the skeleton.

Clavicle data was not statistically tested due to the compounding factors of sex and site demonstrating significance in all variables. Descriptive statistics in some variables suggest a trend according to age category with males and females suggesting a differing pattern of bone parameters in each age category. The small number of older individuals in the sample utilized in this study may not be allowing for an accurate representation of age categories for the Chiribaya population as a whole. The older age category of this study was limited to 50+ and the exact ages of individuals in the sample are not known; it is not known whether the individuals in this sample had lived long enough to exhibit the full pattern of bone loss demonstrated in other populations.

These data suggest that cortical bone loss was not significantly noted in the

Chiribaya sample. These findings suggest that cortical bone loss was not experienced in the Chiribaya until an older age, possibly due to the positive impact of strenuous physical activity prior to the fourth decade of life. The lack of significant findings in the Chiribaya data sample suggest that trabecular bone loss, in addition to cortical bone loss, was not

256

apparent with increasing age grouping. Although trabecular bone is thought to be more sensitive to bone turnover than cortical bone, it appears that both bone tissues in this

Chiribaya sample do not exhibit the characteristic decrease in bone mass with age.

Statistical testing generated support for Hypothesis 4: Variability in relative area measurements due to differential loading throughout the skeleton exist, where relative cortical area was used to examine intra-skeletal variability within forty-three individuals within the Chiribaya sample. Relative cortical area is common to all three skeletal samples used in the project and is a standardized ratio of areal cortical bone to areal total size and is not dependent upon bone size. Application of a randomized block ANOVA detected a significant difference between relative cortical area (Rel.Ct.Ar.) of the three bones within each individual.

This chapter also addresses the limitations of the study. The sample examined in this analysis is an ancient population with no documentation of sample demographics.

The demographic characteristics were interpreted in previous dissertations. Data collection was conducted two-dimensionally, but Haversian systems are oriented in three- dimensions. The study is also not longitudinal and the information is not taken from individuals at various age stages in their lives so it is not completely representative of growth differences that occur in a single individual throughout life.

Directions for future research include examining more variables within the

Chiribaya sample to obtain a more conclusive view of Chiribaya bone health and allow for comparisons with other archaeological populations, examining the areas of the iliac crest above and below the site of trephining, and examining the histology of neighboring

Andean populations. Osteon population density (OPD), osteon area, and osteon 257

circularity are variables that were not included in this study and can shed light on differences in physical activity. Analyzing OPD will also allow for the examination of activation frequency, which will allow for the examination of intensity of bone remodeling. Variables collected and analyzed from trabecular bone of the iliac crest were limited to trabecular area, relative trabecular area, and average trabecular width.

Collecting data on trabecular spacing would verify whether this trend is visible in the

Chiribaya population as well. Data from neighboring populations will also demonstrate whether histological indicators of bone biology of the Chiribaya are unique to this population or common to other Andean populations.

258

Chapter 8: Research Summary and Conclusions

The purpose of this study was to examine histomorphometric variables in an ancient population to examine skeletal variation due to sex, age, and subsistence. This study also examined intra-skeletal variability within individuals. Skeletal samples were taken from: a) the rib, a commonly utilized site in anthropological histological analyses, b) the iliac crest, an area commonly examined in clinical bone studies and c) the clavicle, a third skeletal site representing a different biomechanical environment within the skeleton.

The findings of this study are important for skeletal biologists, bioarchaeologiss and clinicians in the medical field. This study presents a comprehensive analysis of histomorphometric variables for both cortical and trabecular bone that should be considered in future studies by skeletal biologists examining bone remodeling imbalances. The results of this study also confirm the need in examining multiple skeletal sites in bone histomorphometry. With regards to bioarchaeological inquiries, the results of this study demonstrate the need in adding microscopic analysis to gross macroscopic analysis of past populations, as many of the factors impacting bone remodeling may not be manifest in visual and metric observations of human skeletons.

Furthermore, the investigation of bone loss in past populations provides a perspective on

259

bone remodeling imbalances that can aid in the interpretation of the prevalence of osteopenia and osteoporosis in the western world by clinicians.

This project examined four hypotheses:

1. Variability in relative area measurements due to differential loading throughout the

skeleton exist.

2. Differences in histomorphometric areal and length measurements exist among

different age groups due to the effects of increasing age.

3. Differences in histomorphometric areal and length/perimeter measurements exist

among individuals from Chiribaya Alta, El Yaral and San Geronimo due to variation

of diet and general subsistence level activities.

4. Sex differences exist in histomorphometric areal and perimeter/length measurements

in the Chiribaya sample

The variables used in this study include total area (Tt.Ar.), cortical area (Ct.Ar.), endosteal area (Es.Ar.), relative cortical area (Rel.Ct.Ar.), periosteal perimeter (Ps.Pm.) and endosteal perimeter (Es.Ar.) in the ribs and clavicles. In the iliac crest, total area of the section (Tt.Ar.), marrow cavity area (Es.Ar.), cortical area (Ct.Ar.), relative cortical area (Rel.Ct.Ar.), trabecular area (Tb.Ar.), relative trabecular area (Rel.Tb.Ar.), mean trabecular width (Tb.Wi.), total section diameter (Tt.Dm.), total cortical diameter

(Ct.Dm.) and marrow cavity diameter (Es.Dm.).

This study incorporated variables measured on 62 ribs, 54 clavicles and 62 iliac crests of males and females of varying ages. Intra-skeletal variability was examined in

43 individuals for whom all three skeletal sampling sites were represented and intact. Rib and clavicle sections were created from wedges taken from the midshaft of each bone. 260

Iliac crest sections were taken from a wedge removed from the site of clinical bone biopsies. Histomorphometric data was collected using compiled photomicrographs and

ImageJ® software on a PC Tablet. Data collection included areal and perimeter/length measurements on cortical bone in the rib and clavicle, and a combination of cortical and trabecular bone in iliac crest specimens.

Sex comparisons were conducted to test Hypothesis 1: Sex differences exist in histomorphometric areal and perimeter/length measurements in the Chiribaya sample.

This general hypothesis was supported by results at each skeletal sampling site, but with different variables in each area of the skeleton. The clavicle was significant in all histomorphometric variables examined, while the rib was only statistically significant in perimeter measurements, relative cortical area (Rel.Ct.Ar.) and endosteal area (Es.Ar.).

The iliac crest demonstrated statistical significance in total area (Tt.Ar.), medullary area

(Es.Ar.), relative cortical area (Rel.Ct.Ar.) and endosteal diameter (Es.Dm.). Sex comparisons of the three skeletal sites stress the importance in examining more than one site within skeleton comparison studies involve similar sampling sites.

261

individuals demonstrate larger relative cortical areal measurements in the clavicle and iliac crest, indicating a higher amount of bone mass in the individuals of the elite, administrative community. The findings suggest slight alterations in bone strength at all three sampling areas of the skeleton with regards to subsistence, but the differences found are unique to each skeletal sampling site and appear to be dependent upon the unique biomechanical environment of each area of the skeleton.

Hypothesis 3 was not supported by statistical examination: Differences in histomorphometric areal and length measurements exist among different age groups due to the effects of increasing age and the accompanying statistical hypotheses generated to examine age-associated variability in bone parameters were not supported with the findings in this study. Variables examined statistically demonstrated no significance in the rib and iliac crest. The small number of older individuals in the sample utilized in this study may not be allowing for an accurate representation of age categories for the Chiribaya population as a whole. The older age category of this study was limited to 50+ and the exact ages of individuals in the sample are not known; it is not known whether the individuals in this sample had lived long enough to exhibit the full pattern of bone loss demonstrated in other populations. This study suggests that cortical and trabecular bone loss was not significantly noted in the Chiribaya sample, possibly due to the positive impact of strenuous physical activity prior to the fourth decade of life. The lack of significant findings in the Chiribaya data sample suggest that trabecular bone loss, in addition to cortical bone loss, was not apparent with increasing age grouping.

262

Testing of Hypothesis 4: Variability in relative area measurements due to differential loading throughout the skeleton exist, was supported statistically with a randomized block ANOVA. Relative cortical area was used to examine intra-skeletal variability within forty-three individuals within the Chiribaya sample. Relative cortical area is common to all three skeletal samples used in the project and is a standardized ratio of areal cortical bone to areal total size and is not dependent upon bone size.

The contents of this study demonstrate the usefulness of cortical and trabecular histomorphometry in past populations to examine sex and subsistence level variations.

The results indicate a general pattern in skeletal microstructural changes in the rib, clavicle, and iliac crest of the Chiribaya with increasing age, but evidence for age- associated bone loss in this ancient population was not comparable to the high rates reported in modern clinical studies. This study also presents a pattern of bone histomorphology in an ancient Andean archaeological population which can be used for comparison in future studies from neighboring populations.

263

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Appendix A: Histograms from T-Test Analyses

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A.1 Sex Comparisons of Rib Data

Figure 60. Sex Distribution of Rib Total Area (Tt.Ar.)

Figure 61. Sex Distribution of Rib Cortical Area (Ct.Ar.)

311

Figure 62. Sex Distribution of Rib Relative Cortical Area (Rel.Ct.Ar.)

Figure 63. Sex Distribution of Rib Endosteal Area (Es.Ar.)

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Figure 64. Sex Distribution of Rib Periosteal Perimeter (Ps.Pm.)

Figure 65. Sex Distribution of Rib Endosteal Perimeter (Es.Pm.)

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A.2 Sex Comparisons of Clavicle Data

Figure 66. Sex Distribution of Clavicle Total Area (Tt.Ar.)

Figure 67. Sex Distribution of Clavicle Cortical Area (Ct.Ar.)

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Figure 68. Sex Distribution of Clavicle Endosteal Area (Es.Ar.)

Figure 69. Sex Distribution of Clavicle Relative Cortical Area (Rel.Ct.Ar.)

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Figure 70. Sex Distribution of Clavicle Periosteal Perimeter (Ps.Pm.)

Figure 71. Sex Distribution of Clavicle Endosteal Perimeter (Es.Pm.)

316

A.3 Sex Comparisons of Iliac Crest Data

Figure 72. Sex Distribution of Iliac Crest Total Area (Tt.Ar.)

Figure 73. Sex Distribution of Iliac Crest Cortical Area (Ct.Ar.) 317

Figure 74. Sex Distribution of Iliac Crest Marrow Cavity Area (Es.Ar.)

Figure 75. Sex Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.)

318

Figure 76. Sex Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.)

Figure 77. Sex Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.)

319

Figure 78. Sex Distribution of Iliac Crest Mean Trabecular Width (Tb.Wi.)

Figure 79.Sex Distribution of Iliac Crest Cortical Diameter (Ct.Dm.) 320

Figure 80. Sex Distribution of Iliac Crest Marrow Cavity Diameter (Es.Dm.)

321

A.4 Site Comparisons of Rib Data

Figure 81.Site Distribution of Rib Total Area (Tt.Ar.)

Figure 82. Site Distribution of Rib Cortical Area (Ct.Ar.)

322

Figure 83. Site Distribution of Rib Endosteal Area (Es.Ar.)

Figure 84. Site Distribution of Rib Relative Cortical Area (Rel.Ct.Ar.)

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Figure 85. Site Distribution of Rib Periosteal Perimeter (Ps.Pm.)

Figure 86.Site Distribution of Rib Endosteal Perimeter (Es.Pm.)

324

A.5 Site Comparisons of Clavicle Data

Figure 87. Site Distribution of Clavicle Total Area (Tt.Ar.)

Figure 88. Site Distribution of Clavicle Cortical Area (Ct.Ar.)

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Figure 89. Site Distribution of Clavicle Endosteal Area (Es.Ar.) (Square Root Transformed)

Figure 90. Site Distribution of Clavicle Relative Cortical Area (Rel.Ct.Ar.) 326

Figure 91. Site Distribution of Clavicle Periosteal Perimeter (Ps.Pm.)

Figure 92. Site Distribution of Clavicle Endosteal Perimeter (Es.Pm.)

327

A.6 Site Comparisons of Iliac Crest Data

Figure 93. Site Distribution of Iliac Crest Total Area (Tt.Ar.)

Figure 94. Site Distribution of Iliac Crest Cortical Area (Ct.Ar.) 328

Figure 95. Site Distribution of Iliac Crest Endosteal Area (Es.Ar.)

Figure 96. Site Distribution of Iliac Crest Total Trabecular Area (Tt.Tb.Ar.)

329

Figure 97. Site Distribution of Iliac Crest Relative Cortical Area (Rel.Ct.Ar.)

Figure 98. Site Distribution of Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.)

330

Figure 99. Site Distribution of Iliac Crest Mean Trabecular Width (Tb.Wi.)

Figure 100.Site Distribution of Iliac Crest Total Diameter (Tt.Dm.)

331

Figure 101. Site Distribution of Iliac Crest Cortical Diameter/Thickness (Ct.Dm.)

Figure 102. Site Distribution of Iliac Crest Marrow Cavity Diameter (Es.Dm.) 332

Appendix B: Box and Whisker Plots for ANOVA Tests

333

B.1. ANOVA Plots for Rib Data

Figure 103. One-Way ANOVA Box and Whisker Plot of Age and Rib Cortical Area (Ct.Ar.)

Figure 104. One-Way ANOVA Box and Whisker Plot of Age and Rib Relative Cortical Area (Rel.Ct.Ar.)

334

Figure 105. Two-Way ANOVA Interaction Plot for Age, Sex and Rib Cortical Area (Ct.Ar.)

Figure 106. Two-Way ANOVA Interaction Plot for Age, Sex and Rib Relative Cortical Area (Rel.Ct.Ar.)

335

B.2. ANOVA Plots for Iliac Crest Data

Figure 107. One-Way ANOVA Box and Whisker Plot for Age and Iliac Crest Cortical Area (Ct.Ar.)

Figure 108. One-Way ANOVA Box and Whisker Plot for Age and Iliac Crest Relative Trabecular Area ( Rel.Tb.Ar.)

336

Figure 109. One-Way ANOVA Box and Whisker Plot of Age and Iliac Crest Cortical Thickness/Diameter (Ct.Dm.)

Figure 110. One-Way ANOVA Box and Whisker Plot of Age and Iliac Crest Mean Trabecular Width (Tb.Wi.)

337

Figure 111. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Mean Trabecular Width (Tb.Wi.)

Figure 112. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Cortical Diameter (Ct.Dm.)

338

Figure 113. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Cortical Area (Ct.Ar.)

Figure 114. Two-Way ANOVA Interaction Plot for Age, Sex and Iliac Crest Relative Trabecular Area (Rel.Tb.Ar.)

339

Appendix C: Raw Data

340

Table 63. Raw Data for Rib

Individual Total Area Periosteal Medullary Endosteal Cortical Relative (mm2) Perimeter Area Perimeter Area Cortical (mm) (mm2) (mm) (mm2) Area CHA1 T4 48.74138 27.74000 26.34209 25.23088 22.39930 0.45955 CHA1 T7 74.96803 37.47500 47.92670 34.66231 27.04133 0.36070 CHA1 T14 54.31736 29.92800 29.90784 25.50885 24.40952 0.44939 CHA1 T16 52.47400 31.64800 26.43253 27.97774 26.04147 0.49627 CHA1 T17 44.97808 27.82000 23.72107 24.80565 21.25701 0.47261 CHA1 T19 37.91361 23.69300 16.79514 21.22954 21.11848 0.55702 CHA1 T20 56.53383 30.41500 23.93739 25.61139 32.59643 0.57658 CHA1 T34 39.04094 24.37200 13.70753 17.28162 25.33341 0.64889 CHA T101 71.12000 38.21600 39.20207 34.39123 31.91794 0.44879 CHA1 71.37443 34.62560 29.10833 33.83501 42.26610 0.59217 T1804A CHA2 T211 23.11962 25.30494 9.62521 22.95449 13.49441 0.58368 CHA2 T240 78.33498 37.92329 32.56368 34.89414 45.77130 0.58430 CHA3 T302 59.04689 32.76478 32.48302 33.38350 26.56387 0.44988 CHA3 T304 97.05100 39.89000 54.30950 38.56425 42.74151 0.44040 CHA3 T308 74.07000 39.69200 41.56304 40.35667 32.50696 0.43887 CHA3 T312 60.54435 30.94586 27.35444 26.21261 33.18991 0.54819 CHA3 T317 80.11977 47.50896 43.45660 48.74802 36.66318 0.45760 CHA3 T318 77.09400 46.99400 43.62482 49.85889 33.46918 0.43413 CHA3 T320 48.69400 28.91100 16.73258 21.61577 31.96142 0.65637 CHA3 T324 63.87987 35.65688 28.45016 32.12760 35.42971 0.55463 CHA3 T325 73.10718 36.09432 29.94789 31.01479 43.15929 0.59036 CHA3 T328 54.04683 35.30230 30.86810 33.43851 23.17872 0.42886 CHA4 T401 98.01613 41.79635 51.25473 36.70746 46.76140 0.47708 CHA4 T402 38.99708 29.13811 13.61747 25.50130 25.37961 0.65081 CHA4 T404 98.88800 44.09900 46.60857 43.66574 52.27943 0.52867 CHA4 T407 94.36791 42.02473 43.03433 36.59755 51.33358 0.54397 CHA4 T409 59.12699 32.22764 15.24662 25.37409 43.88037 0.74214 CHA4 T411 76.38801 37.17942 35.95281 33.56447 40.43520 0.52934 CHA4 T437 65.05100 34.78700 35.01330 31.47920 30.03771 0.46176 CHA6 T609 90.34200 36.46000 38.43762 32.41662 51.90438 0.57453 CHA6 T610 51.82001 36.24385 22.62581 35.05618 29.19420 0.56338 CHA7 T728 75.07322 36.63620 51.45786 40.33853 23.61535 0.31456 (Continued)

341

Table 63. Continued

Individual Total Area Periosteal Medullary Endosteal Cortical Relative (mm2) Perimeter Area Perimeter Area Cortical (mm) (mm2) (mm) (mm2) Area CHA7 T736 88.37800 42.52135 47.58204 37.89878 40.79596 0.46161 CHA7 T746 51.35582 31.34975 24.84961 31.56624 26.50622 0.51613 CHA7 T760 56.61271 31.74342 25.66063 31.52270 30.95208 0.54673 CHA7 T763 54.99592 33.57240 23.12917 27.79237 31.86674 0.57944 CHA9 T901 81.25521 36.24765 47.77207 34.96994 33.48314 0.41207 CHA9 T902 70.98242 42.18962 30.16081 38.84143 40.82161 0.57509 M8 T116 64.97467 32.57369 36.32253 29.51271 28.65214 0.44097 M8 T131 40.91171 24.69055 21.62841 21.58829 19.28330 0.47134 M8 T137 84.17363 39.38574 40.59049 34.88457 43.58314 0.51778 M8 T225 56.00836 34.40041 33.86622 31.70578 22.14215 0.39534 M8 T229 83.70389 40.20504 46.33313 34.29169 37.37076 0.44646 M8 T234 94.33825 39.74476 46.19923 33.23774 48.13902 0.51028 M8 T246 60.11250 32.34598 30.72391 30.29611 29.38859 0.48889 SG R8 75.50725 39.23003 46.96080 35.16935 28.54645 0.37806 SG R9 37.74355 24.48658 14.18725 18.75550 23.55630 0.62411 SG R15 95.12866 38.77243 32.65923 27.23257 62.46943 0.65668 SG R30 92.86957 39.36113 58.50808 34.65354 34.36149 0.37000 SG R47 85.38458 36.35701 43.39534 31.56030 41.98925 0.49177 SG R48 68.52358 33.84221 29.93649 29.45982 38.58709 0.56312 SG R63 75.30584 39.26345 42.64556 34.56356 32.66028 0.43370 SG R66 100.52625 44.39791 58.24348 41.03505 42.28277 0.42061 SG R69 41.47203 28.57199 25.03299 26.02025 16.43904 0.39639 SG R80 57.51562 33.13673 38.26284 31.74923 19.25278 0.33474 SG R86 56.49085 32.37009 35.45031 30.09517 21.04054 0.37246 SG R92 155.00563 49.63148 89.78731 42.14239 65.21832 0.42075 SG R110 68.78606 34.11130 30.58134 29.64415 38.20472 0.55541 SG R112 79.03087 39.39789 40.42308 35.98366 38.60779 0.48852 SG R121 72.96291 36.19092 29.81230 29.42618 43.15061 0.59140 SG R124 74.59264 35.22195 30.56666 27.32367 44.02599 0.59022 SG R126 63.99630 34.58164 27.17703 30.28879 36.81927 0.57533 SG R128 85.66915 37.83566 44.64046 33.28423 41.02870 0.47892 SG R129 85.26831 35.77052 42.31463 30.56454 42.95368 0.50375 SG R130 110.03961 46.82100 61.28697 42.00047 48.75264 0.44305 SG R132 95.00242 37.91861 42.12392 33.37589 52.87849 0.55660

(Continued)

342

Table 63. Continued

Individual Total Area Periosteal Medullar Endosteal Cortical Relative (mm2) Perimeter y Area Perimeter Area Cortical (mm) (mm2) (mm) (mm2) Area SG R141 66.94613 33.82924 34.24796 27.24547 32.69817 0.48843 SG R146 95.45717 44.09164 54.94501 39.75487 40.51216 0.42440

343

Table 64. Raw Data for Clavicle

Individual Total Area Periosteal Medullary Endosteal Cortical Relative (mm2) Perimeter Area Perimeter Area Cortical (mm) (mm2) (mm) (mm2) Area CHA1 T17 80.14200 32.85500 22.60000 19.75700 57.54200 0.71800 CHA1 T19 41.70100 23.95600 12.76540 12.40679 28.93560 0.69388 CHA3 T317 97.00200 37.21300 51.00900 29.82300 45.99300 0.47414 CHA1 T4 60.75000 28.58300 14.09600 17.54800 46.65400 0.76797 CHA1 T7 75.26000 33.44100 34.85200 25.87200 40.40800 0.53691 CHA1 T14 64.55900 30.58300 19.11800 18.71700 45.44100 0.70387 CHA1 T16 65.00700 30.58200 35.33400 27.84500 29.67300 0.45646 CHA1 T20 65.88551 31.03056 10.88700 15.35100 54.99851 0.83476 CHA1 T34 33.84830 21.77282 6.31244 9.94814 27.53586 0.81351 CHA1 T38 84.71600 34.12300 25.79200 22.04600 58.92400 0.69555 CHA1 T101 75.61100 32.13300 23.19700 20.09500 52.41400 0.69321 CHA1 81.94700 33.62600 22.82900 21.08700 59.11800 0.72142 T1804A CHA2 T211 49.85300 26.43300 15.02300 16.58000 34.83000 0.69865 CHA2 T240 108.03900 39.87200 49.33400 33.32800 58.70500 0.54337 CHA3T392 55.68872 27.70768 14.02832 16.26934 41.66040 0.74809 CHA3 T302 64.75300 30.02100 13.57400 20.39600 51.17900 0.79037 CHA3 T304 82.06600 33.43600 10.61500 14.21300 71.45100 0.87065 CHA3 T308 71.80400 31.50500 27.32100 21.05100 44.48300 0.61951 CHA3 T318 63.74600 31.07200 20.83600 20.12200 42.91000 0.67314 CHA3 T320 43.90320 25.08974 3.55715 8.50410 40.34605 0.91898 CHA3 T324 45.60500 26.02500 7.95100 12.79000 37.65400 0.82566 CHA3 T328 64.05700 29.88400 21.22100 18.89400 42.83600 0.66872 CHA4 T401 61.26178 30.62268 13.01200 14.13100 48.24978 0.78760 CHA4 T402 41.76000 24.10800 2.62089 6.33630 39.13911 0.93724 CHA4 T404 87.16800 35.52200 29.15800 23.51700 58.01000 0.66550 CHA4 T407 96.32600 36.29800 32.72000 28.55000 63.60600 0.66032 CHA4 T409 41.90088 24.34261 2.92755 7.35016 38.97333 0.93013 CHA4 T437 56.46900 28.42400 11.23600 14.88700 45.23300 0.80102 CHA6 T609 88.56300 34.60400 23.26600 21.52600 65.29700 0.73729 CHA6 T610 64.79881 29.85351 29.66300 22.89600 35.13581 0.54223 CHA7 T728 84.31007 35.52869 30.79793 26.19891 53.51214 0.63471 (Continued)

344

Table 64. Continued

Individual Total Area Periosteal Medullar Endosteal Cortical Relative (mm2) Perimeter y Area Perimeter Area Cortical (mm) (mm2) (mm) (mm2) Area CHA7 81.02096 33.52558 23.74307 19.28231 57.27789 0.70695 T736 CHA7 73.02051 32.02855 28.63910 21.57570 44.38141 0.60779 T746 CHA7 61.69154 29.38054 18.30714 20.94939 43.38440 0.70325 T760 CHA7 43.32909 24.17967 5.70235 9.74483 37.62674 0.86839 T763 CHA9 71.00532 30.91872 21.12857 18.51779 49.87675 0.70244 T901 CHA9 100.46998 39.57022 41.22492 30.86769 59.24506 0.58968 T902 M8 T131 58.16482 28.76556 11.52340 13.46135 46.64142 0.80188 M8 T137 55.78283 27.46083 9.69287 14.64462 46.08996 0.82624 M8 T225 77.25521 32.43027 24.34290 22.87205 52.91231 0.68490 M8 T229 88.90613 35.56582 31.37094 24.67573 57.53519 0.64715 M8 T246 86.13670 36.32789 36.56597 27.52097 49.57073 0.57549 SG R141 59.46254 28.24959 19.09897 19.69197 40.36357 0.67881 SG R30 76.24000 32.18800 37.74900 26.88700 38.49100 0.50487 SG R47 66.82854 30.45299 23.51471 20.73919 43.31383 0.64813 SG R48 63.94193 29.82167 16.31838 17.94600 47.62355 0.74479 SG R63 122.57213 43.40780 42.66321 30.57479 79.90892 0.65193 SG R66 143.28511 43.90489 82.17336 35.69665 61.11175 0.42650 SG R80 57.90630 29.06413 21.41653 19.95734 36.48977 0.63015 SG R92 162.08383 47.32602 73.86131 33.83115 88.22252 0.54430 SG R110 62.53047 29.07112 12.03902 14.13091 50.49145 0.80747 SG R112 78.09623 33.18667 25.68322 27.21895 52.41302 0.67113 SG R121 99.00695 36.24191 24.43368 22.02023 74.57327 0.75321 SG R124 143.50640 43.81053 43.25166 29.30672 100.25475 0.69861 SG R129 116.88362 41.14867 34.25586 26.37922 82.62776 0.70692 SG R130 104.30337 39.46606 34.75971 25.64157 69.54367 0.66674 SG R132 82.02777 33.77540 19.07485 19.42089 62.95292 0.76746 SG R146 91.34604 35.48872 37.94250 24.73977 53.40354 0.58463

345

Table 65. Raw Data for Iliac Crest

Individual Total Area Medullary Total Relative Total (mm2) Area Cortical Area Cortical Area Trabecular Area (mm2) (mm2) (mm2) CHA1 T7 20.59334 11.98200 8.61134 0.41816 3.65137 CHA1 T12 49.98800 25.41700 24.57100 0.49154 9.48110 CHA1 T14 52.26000 34.41100 17.84900 0.34154 9.42183 CHA1 T16 18.25900 13.72700 4.53200 0.24821 0.52144 CHA1 T19 75.02500 61.05500 13.97000 0.18620 13.38635 CHA1 T20 74.79200 48.25600 26.53600 0.35480 15.33559 CHA1 T24 48.98900 31.16800 17.82100 0.36378 6.69400 CHA1 T38 86.79100 66.12200 20.66900 0.23815 19.14700 CHA1 T101 44.12600 33.02300 11.10300 0.25162 7.74745 CHA1 59.97900 43.11100 16.86800 0.28123 8.61370 T1804A CHA2 T211 22.70900 10.07100 12.63800 0.55652 1.95569 CHA2 T223 39.34500 21.30600 18.03900 0.45848 5.38500 CHA2 T240 43.37572 25.09609 18.27962 0.42143 6.63141 CHA3 T302 40.25900 26.59102 13.66798 0.33950 5.14778 CHA3 T304 54.94300 32.23200 22.71100 0.41336 9.47718 CHA3 T308 49.45475 37.84700 11.60775 0.23471 6.10900 CHA3 T312 64.34400 41.83500 22.50900 0.34982 6.95700 CHA3 T317 72.65069 60.14100 12.50969 0.17219 8.83361 CHA3 T318 41.99500 30.95200 11.04300 0.26296 5.39700 CHA3 T320 48.62000 31.29332 17.32668 0.35637 3.24000 CHA3 T325 82.24790 57.67000 24.57790 0.29883 8.72200 CHA3 T328 52.66300 39.23800 13.42500 0.25492 6.94427 CHA4 T402 32.04000 15.78200 16.25800 0.50743 4.98176 CHA3 T404 62.42500 40.24200 22.18300 0.35535 7.15651 CHA4 T407 82.96200 60.97800 21.98400 0.26499 26.19699 CHA4 T409 60.06700 35.69100 24.37600 0.40581 13.17112 CHA4 T411 67.58500 56.82400 10.76100 0.15922 7.84800 CHA4 T412 62.16200 40.55800 21.60400 0.34754 9.81300 CHA4 T437 66.20900 50.53400 15.67500 0.23675 14.88873 CHA5 T517 49.65300 31.55400 18.09900 0.36451 10.60700 CHA6 T609 49.05899 28.43245 20.62654 0.42044 8.25817 CHA6 T610 61.65171 47.72886 13.92284 0.22583 15.87179 CHA7 T713 48.14100 31.31400 16.82700 0.34954 7.66900 (Continued)

346

Table 65. Continued

Individual Total Medullar Total Cortical Relative Total Area y Area Area (mm2) Cortical Area Trabecular (mm2) (mm2) Area (mm2) CHA7 T728 68.27700 51.66000 16.61700 0.24338 10.64280 CHA7 T746 53.10748 37.16279 15.94469 0.30023 15.34786 CHA7 T760 36.49582 22.94217 13.55365 0.37138 9.70593 CHA7 T763 50.55616 33.63347 16.92269 0.33473 15.52288 CHA9 T901 58.43350 36.07428 22.35922 0.38264 6.62102 CHA9 T902 63.55262 51.84790 11.70472 0.18417 9.51450 CHA9 T904 49.12600 25.44700 23.67900 0.48201 4.87200 M8 T116 58.32300 42.27900 16.04400 0.27509 6.67200 M8 T131 50.08406 26.07977 24.00429 0.47928 6.39262 M8 T137 52.82681 29.25318 23.57362 0.44624 12.38762 M8 T225 35.23339 17.71703 17.51636 0.49715 3.16069 M8 T234 47.11100 26.68400 20.42700 0.43359 7.90200 SG R8 38.82177 30.80764 8.01413 0.20643 10.25498 SG R9 71.75100 51.22500 20.52600 0.28607 14.81600 SG R30 64.58624 54.30507 10.28117 0.15919 6.22064 SG R48 65.62295 49.46313 16.15982 0.24625 11.66226 SG R63 47.08785 32.16935 14.91850 0.31682 9.06617 SG R66 97.36057 74.93207 22.42850 0.23037 18.34541 SG R80 46.72249 35.59667 11.12581 0.23813 7.22649 3619 SG R80 66.79600 51.75600 15.04000 0.22516 12.40000 3617 SG R92 69.65735 49.47862 20.17873 0.28969 15.55364 SG R110 51.13227 34.53677 16.59551 0.32456 7.63873 SG R112 64.45122 52.52417 11.92705 0.18506 11.60636 SG R121 52.29235 29.37943 22.91292 0.43817 9.68535 SG R124 58.94215 43.95846 14.98370 0.25421 12.66843 SG R126 61.19000 33.00900 28.18100 0.46055 13.52700 SG R128 61.08100 42.00000 19.08100 0.31239 8.86900 SG R129 42.46800 35.14995 7.31805 0.17232 7.55369 SG R130 70.16108 51.56221 18.59887 0.26509 13.61236 SG R141 64.85194 47.17872 17.67322 0.27252 18.55737 (Continued)

347

Table 65. Continued

Individual Relative Average Total Medullary Cortical Trabecular Trabecular Diameter Diameter Diameter (mm) Area Width (mm) (mm) (mm) CHA1 T7 0.17731 0.14620 2.179 1.497 0.682 CHA1 T12 0.18967 0.19800 6.382 3.431 2.951 CHA1 T14 0.18029 0.18400 5.658 4.111 1.547 CHA1 T16 0.02856 0.19500 3.287 2.790 0.497 CHA1 T19 0.17843 0.18000 10.891 8.803 2.088 CHA1 T20 0.20504 0.14957 8.662 5.165 3.497 CHA1 T24 0.13664 0.17133 5.653 3.606 2.047 CHA1 T38 0.22061 0.14562 12.399 9.949 2.450 CHA1 T101 0.17558 0.08529 6.127 4.937 1.190 CHA1 0.14361 0.19343 9.164 6.940 2.224 T1804A CHA2 T211 0.08612 0.12880 3.418 1.975 1.443 CHA2 T223 0.13687 0.17740 5.715 4.180 1.535 CHA2 T240 0.15288 0.15300 5.970 3.077 2.893 CHA3 T302 0.12787 0.18000 4.444 3.231 1.213 CHA3 T304 0.17249 0.25140 7.063 4.709 2.354 CHA3 T308 0.12353 0.12920 6.800 5.013 1.787 CHA3 T312 0.10812 0.18000 7.325 4.167 3.158 CHA3 T317 0.12159 0.17438 8.886 7.899 0.987 CHA3 T318 0.12852 0.18867 5.671 4.406 1.265 CHA3 T320 0.06664 0.18100 6.304 4.519 1.785 CHA3 T325 0.10605 0.20925 10.779 7.259 3.520 CHA3 T328 0.13186 0.16658 5.907 4.380 1.527 CHA4 T402 0.15549 0.26600 3.998 2.096 1.902 CHA3 T404 0.11464 0.16390 7.926 5.071 2.855 CHA4 T407 0.31577 0.19096 9.542 7.349 2.193 CHA4 T409 0.21927 0.20269 7.331 5.793 1.538 CHA4 T411 0.11612 0.14850 8.934 7.702 1.232 CHA4 T412 0.15786 0.31217 7.527 5.290 2.237 CHA4 T437 0.22487 0.28878 7.777 6.392 1.385 CHA5 T517 0.21362 0.24300 6.266 3.823 2.443 CHA6 T609 0.16833 0.19800 7.681 5.224 2.457 CHA6 T610 0.25744 0.18700 7.968 6.238 1.730 (Continued)

348

Table 65. Continued

Individual Relative Average Total Medullary Cortical Trabecular Trabecular Diameter Diameter Diameter Area Width (mm) (mm) (mm) (mm) CHA7 T713 0.15930 0.24289 6.595 4.218 2.377 CHA7 T728 0.15588 0.18012 7.390 6.117 1.272 CHA7 T746 0.28900 0.27690 6.236 3.818 2.418 CHA7 T760 0.26595 0.22725 4.844 2.873 1.972 CHA9 T901 0.11331 0.22276 7.161 4.628 2.534 CHA9 T902 0.14971 0.20193 7.322 5.547 1.774 CHA9 T904 0.09917 0.13036 6.076 3.494 2.582 M8 T116 0.11440 0.13344 7.691 5.781 1.910 M8 T131 0.12764 0.19005 5.381 2.811 2.570 M8 T137 0.23450 0.27713 5.951 3.550 2.400 M8 T225 0.08971 0.22631 3.928 2.490 1.438 M8 T234 0.16773 0.15776 7.050 3.200 3.850 SG R8 0.26416 0.14800 5.968 4.072 1.896 SG R9 0.20649 0.21100 9.479 6.486 2.993 SG R30 0.09632 0.14476 6.672 5.423 1.249 SG R48 0.17772 0.16418 6.473 4.551 1.922 SG R63 0.19254 0.18776 5.516 3.680 1.835 SG R66 0.18843 0.18941 8.368 7.548 0.820 SG R80 0.15467 0.14488 7.114 5.892 1.222 3619 SG R80 0.18564 0.17260 7.898 6.326 1.572 3617 SG R92 0.22329 0.16247 8.022 5.451 2.571 SG R110 0.14939 0.11740 6.075 4.348 1.728 SG R112 0.18008 0.21363 8.423 6.591 1.833 SG R121 0.18522 0.16495 6.307 3.786 2.521 SG R124 0.21493 0.18888 7.085 4.945 2.141 SG R126 0.22107 0.21057 8.002 4.766 3.236 SG R128 0.14520 0.19600 7.699 5.568 2.131 SG R129 0.17787 0.16774 6.897 4.426 2.471 SG R130 0.19402 0.19630 7.988 6.094 1.894 SG R141 0.28615 0.17191 7.248 5.267 1.980

349