Variation in Cortical Osteocyte Lacunar Density and Distribution: Implications for Bone Quality Assessment DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Randee L. Hunter, B.S., M.A. Graduate Program in Anthropology The Ohio State University 2015 Dissertation Committee: Clark Spencer Larsen, Advisor Samuel D. Stout Paul W. Sciulli Amanda M. Agnew Copyrighted by Randee Linn Hunter 2015 Abstract The purpose of this study is to investigate variation in cortical bone osteocyte cell populations using their lacunae as a proxy. The osteocytes and their lacunocanalicular network have been identified as the regulator of bone quality and function by exerting extensive influence over metabolic processes, mechanical adaptation, and mineral homeostasis. Recent research has shown that osteocyte malfunction and apoptosis leads to a decrease in bone quality and increase in bone fragility. However, these results are limited to mainly trabecular bone in clinical studies following biopsy or prosthetic replacement in osteoporotic patients and animal studies in which experimental data have been collected. This study is the first to analyze cortical bone variation in osteocyte lacunar density from multiple skeletal sites to establish regional and systemic age and sex related trends. Bone samples were recovered from 30 modern cadaveric individuals (15 males and 15 females) ranging from 49 to 100 years old. Three anatomical sites were utilized for this study: the midshaft femur which is frequently used in anthropological studies of cross-section geometry, age estimation and behavioral interpretations as it is a major load bearing bone; the distal third of the diaphyseal radius as it represents a clinically relevant site for fractures associated with falls especially in older adults; and the midshaft of the 6th rib as this is frequently held as a systemic control. Thin ground histological (80 µm) ii cross-sections were made and imaged under bright field microscopy. Bone size measurements (total subperiosteal area Tt.Ar; cortical area Ct.Ar) were collected using ArcGIS for the larger femora and ImageJ for the radius and rib. ImageJ was then used to optimally manipulate the image in order to identify and count osteocytic lacunae (Ot.Lc.N) automatically over the entirety of the cross-section. Intracortical porosity area (Po.Ar) was collected using a semi-automatic requiring extensive manual verification and used to calculate bone area (B.Ar) for each section (Ct.Ar-Po.Ar). Each variable used for comparisons was normalized for size: osteocyte lacunar density (Ot.Lc.N/B.Ar and Ot.Lc.N/Ct.Ar) and intracortical porosity (%Po.Ar). Independent samples t-tests indicated no significant sex differences between any variables in all elements. Pearson correlations demonstrated an overall decrease in osteocyte lacunar density (Ot.Lc.N/B.Ar) and increase in intracortical porosity (%Po.Ar) with age for each element. Intracortical porosity (%Po.Ar) was significantly negatively correlated with lacunar density (Ot.Lc.N/Ct.Ar) in each element. Lastly, a systemic trend in the decrease in osteocyte lacunar density (Ot.Lc.N/B.Ar) with age was identified between the femur and radius but no relationship between the rib and radius, or rib and femur. All correlations were explored for males and females separately as well as for the pooled sample and demonstrates varying strengths of relationships between sexes. These results indicate that although all elements are experiencing systemically influenced declines in osteocyte lacunar density, there appears to be a differential effect at each anatomical site. It is hypothesized here that the importance of the mechanical environment in osteocyte iii viability (and lacunar existence) may be the underlying cause of this differential intra- individual pattern. This study provides a foundation upon which to build interpretations of osteocyte lacunar density values and distributions in past and present populations. The central role of the osteocyte makes it the “middle man” by which mechanical loading, systemic health, and environmental conditions directly affect change in the skeletal system. Thus, the osteocyte lacunocanalicular network should be utilized in bioarchaeological and clinical studies of bone quality assessment. iv This work is dedicated to my grandmother Carolyn Ravens without whose support none of this was possible. v Acknowledgments I would like to first thank my advisor, Dr. Clark Larsen, for taking a chance on someone with a unique background and facilitating my development as an anthropologist. The support and guidance from the faculty and staff in the Department of Anthropology have been greatly appreciated. Thank you to Dr. Sam Stout for sharing his knowledge of skeletal biology and Dr. Paul Sciulli for his statistical prowess and unique perspective. I would especially like to thank Dr. Jim Gosman who encouraged me to look inside the “black box” of bioarchaeological skeletal research from which this project stems. I sincerely appreciate the support of my family: my Mom, Dad, Step-mom, Grandma and Grandpa for always offering encouraging words and letting me know they are proud. This would not have been possible without The Forsters who have and continue to positively influence my life to no end, and to Kari, Clare, Jenny and Jenny who keep me sane and remind me to embrace life. A special thanks to Dr. Tim Gocha and Victoria Dominguez who shared their extensive knowledge and training in histological techniques and software for analyses; but most importantly, who provided a friendly sounding board for concepts explored here. Lastly, I am deeply grateful to Drs. Amanda Agnew and John Bolte for welcoming me into the IBRC which has challenged me to utilize unique perspectives in vi my research and facilitated my growth as a researcher making this project possible. Thank you to all the members of the IBRC for their help in obtaining samples and feedback throughout this process. A huge thank you to Mandy and John for their encouragement and support in fostering my developing career but especially for their friendship and guidance in life. vii Vita 2002................................................................Francis Howell High School, MO 2006................................................................B.S. Nuclear Medicine Technology, summa cum laude, Saint Louis University 2010................................................................M.A. Anthropology, with Distinction, Department of Anthropology, The Ohio State University 2009-2013 ......................................................Graduate Teaching Associate, Department of Anthropology, The Ohio State University 2011-present ..................................................Graduate Teaching Associate, Division of Anatomy, The Ohio State University Fields of Study Major Field: Anthropology Area of Emphasis: Biological Anthropology Minor: Anatomy viii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Fields of Study ................................................................................................................. viii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiii List of Figures ................................................................................................................... xv Chapter 1: Introduction ....................................................................................................... 1 1.1 Research Context....................................................................................................... 1 1.2 Hypotheses ................................................................................................................ 6 1.3 Significance ............................................................................................................. 10 1.4 Topics Addressed .................................................................................................... 11 Chapter 2: Biological Foundation and Assessment of Cortical Bone Quality ................. 13 2.1 Introduction ............................................................................................................. 13 2.2 Cortical Bone Cells ................................................................................................. 13 2.2.1 Osteoclasts ........................................................................................................ 14 2.2.2 Osteoblastic lineage .......................................................................................... 17 2.3 Cortical Bone Metabolism ...................................................................................... 23 2.3.1 Modeling ........................................................................................................... 23 2.3.2 Remodeling ......................................................................................................