THE PATHOLOGY AND EPIDEMIOLOGY OF SPONTANEOUS DISEASES AND DISORDERS OF THE SPINE IN
THE RHESUS MACAQUES OF CAYO SANTIAGO
Antonietta Maria Cerroni
A thesis submined in conformity with the squirements for the degree of Doctor of Phüosophy Graàuate Department of Anthropology University of Toronto
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cana!! Thesis Title: The Pathology and Epidemiology of Spontaneous Diseases and Disorders of the Spine in the Rhesus Macaques of Cayo Santiago Deeree and Year of Convocation: Doctot of Philosophy, June 2000 -Name: Antonietta Maria Cerroni Graduate Deoartment: Department of Anthropology Universitv: University of Toronto
ABSTRACT
This dissertation is a cross-sectional research project on degenerative spinal disease. bone minerai density (BMD) and metabolic bone disease in a large skeletal population of rhesus monkeys (CPRC Museum collection), derived from the free-ranging colony of Cayo Santiago. Puerto Rico. a colony of historical and contemporary interest to Anthropology. The effects of demographic variables (age, sex, parity, natal group affiliation) on BMD and spontaneous osteopenia, osteoporosis, vertebral osteophytosis (VO), degenerative disc disease (DDD). vertebral osteoarthritis (VOA) and diffuse idiopathic skeletal hyperostosis (DISH) were snidied. The combined frequency of osteopenia and osteoporosis is L2.496 (N = 298). BMD of the Iast lumbar vertebra was measured using DEXA. Females exhibit an initial increase in BMD with age. with peak bone density occurring around 9.5 years. and remahhg constant until 17.2 years. after which there is a steady decline in BMD. Males acquire BMD at a faster rate, and attain a higher peak BMD at an earlier age than do females, at around 7 yean, after which BMD remab relatively constant. The BMD values of the monkeys with vertebral wedge fractures are genedy higher than those of unfktured osteopeniclosteoporotic individuais, thus supporthg the view that BMD alone is not a good predictor of fracture risk. In females, there is a sigair~cantincrease in BMD of the spine with increasing parity, after controhg for age, up to a parity of about 7 offspring. Vertebrae with VO/DDD have abnomially eleviited BMD compared to agelsex- matched controls, even when osteophytes are excluded from the analysis, thus indicating that the trabecular bow was altered by the disease, both quantitatively and qualitatively. VODDD and VOA are present in relatively high frequencies, and there is no significant difference between the sexes in f~quencyor pattern of joint distribution. Thirty-six percent (N = 204) exhibit some degree of VO/DDD, and over 408 (N = 218) exhibit VOA. VO/DDD and VOA increase in severity with advancing age in both sexes.
Degenerative changes in vertebral bodies begin around age 8 to 10 years. and ail monkeys over 15 years exhibit some degree of VOlDDD or VOA. VODDD has a predilection for the thoracic region, and refIects the normal curvatures of the macaque spine. in contmt. VOA is generally distributed throughout the spine. An inverse pattem of VOlDDD and VOA is observed in some joints. These data have implications for our understanding of the human counterparts of these vertebral conditions. DEDICATION
To my husband, James William Zamora This nsearch project was conducted under the auspices of the Department of
Anthropology at the University of Toronto, the Connective Tissue Research Group of the Samuel Lunenfeld Research Institute of Mount Sinai Hospital in Toronto. and the Caribbean Primate Research Center (CPRC)Museum in Puerto Rico. 1 wouid like to thank the Samuel Lunenfeld Research hstitute, especidly Dr. Marc D. Grynpas, for generously providing access to necessary resources and equipment, and the Division of Nuclear Medicine, Mount Sinai Hospital, for the use of its Dual Energy X-ray Absorptiometry (DEXA) machine. 1 am very gratehil to my thesis supervisors, Dr. Jerry Melbye (Dept. of Anthropology. University of Toronto) and Dr. Marc Grynpas (Research Institute. Mount Sinai Hospital and Dept. of Laboratory Medicine and Pathobiology, U. of Toronto), and to Dr. J. Eldon Molto (Core Committee member, Dept. of Anthropology, Lakehead University and adjunci professor, University of Toronto) for their instruction. guidance, support and encouragement. 1 also wish to express my gratitude to the members of my Defence Cornmittee: Dr. Melbye, Dr. Grynpas, Dr. Molto. Dr. Susan Pfeiffer (Dept. of Anthropology, U. of Toronto), Dr. K. P. H. Pntzker (Head, Dept. of Pathology and bboratory Medicine, Mount Sinai Hospital. Toronto, and Dept. of Laboratory Medicine and Pathobiology, U. of Toronto) and Dr. D. J. Simmons (Extemal
Examiner, University of Texas Medical Branch at Galveston) for their participation and for their constructive criticisms. 1 am especiaiiy indebted to Dr. Molto for inspiring an interest in the osteology and pathology of ancient vertebral bones during my undergraduate years. for generously providing opprtunities for coiiaborative research at that the, and for his continued support and mentorship. 1 am also indebted to Dr. Pavicia Stuart-Macadam. whose hiendship, kindness and encouragement during the early stages of the research was very much appreciated. A special th- to Drs. Jean E. Tumquist and Nancy Hong (CPRC Museum) for their enthusiastic support for this research project, for generously providing access to specimens and lab nsources. and for their gracious hospitaiity during the long months of fieldwork in Puerto. Rico. This project was enhanced by interesting discussions with Dr. Tumquist. and her critiques of early drifts of part of this research. Her generosity and thoughtfulness is greatly appreciated, as is the technicd assistance provided by Dr. Hong. Thanks also to Dr. M. Kessler, Director of the CPRC, for his interest in this work. Many thanks to Dr. M. Ichise (Director, Division of Nuclear Medicine, Mount Sinai Hospital) and staff for their expert advice and interest in this project. Finally, 1 gratefuily acknowledge the expert technical assistance of Mr. Richard Cheung, Mr. DougIas Holmyard, Ms. Rita Nespeca (Samuel Lunenfeld Research Institute. Mount Sinai Hospital). Dr. George Tomlinson (Cîinical Epidemiology Unit, Toronto General Hospital) and Mr. Carlos Reyes Rivera (Dept. of Radioiogy, University of Puerto Rico, Medical Sciences Campus). This research was supported by University of Toronto Open Doctoral Feliowships, and by a gant supplement fiom Dr. Marc Grynpas. Support was also provided, in part, by a Comparative Medicine Program Award (RR-03640)from the National Center for Research Resources. NM held by the CPRC, and by the University of Puerto Rico, Medical Sciences Campus. TABLE OF CONTENTS
Page .. ABSTRACT ...... ~...... II DEDICATION ...... iv ACKNOWLEDGMENTS ...... v LiST OF TABLES ...... xiv LIST OF FIGURES ...... xv LIST OF ABBREVLATIONS ...... CHAPTER 1. ~RODUCTION...... *...... 1 1.1. The Cayo Santiago Colony of Rhesus Monkeys (Mocaca mulatta) ...... 3 1.2. Bone MinerPl Density, Osteopenia and Osteopomis: Background and Mq/or Research Questions ...... 9 . . Osteoporosis in Humms ...... 9 Osteoporosis in Animals. Including Non-Human Primates ...... 13 Rhesus Modceys of Cayo Santiago ...... 15 Advantages of a Monkey Model ...... 15 Previous Studies of Bone Mass in Cayo Santiago Rhesus Monkeys ...... 16 Relationship ktween Osteoporosis and Osteoarthrïtis ...... 17 Objectives and Organization of the Study ...... 18 13. Vertebrai Osteophytds (VO) and Degenerative Disc Diseme (DDD): Background and MsJot Research Questions ...... 20 Definition and Description of VO and DDD...... 20 VO and DDD in Animals. including Non-human Primates ...... 22 Objectives and Organization of the Study ...... 24 1.4. Diffuse Idiopathic Skeletai Hypemstosis (DISE): Background and Mqjor Research Questions ...... 25 Definition and Description ...... 25 Organization of the Study...... 26 1.5. Vertebrai -tis (VOA): Backgrod and Mijor Research Questions ...... 27 Definition and Description of VOA ...... 27 Objectives and Organization of the Study ...... 28 CLIAPTER 2. MATERIALS GND METHODS ...... 30 2.1. The Sample...... 30 2.1.1. Bone Density Studies (DEXA), Osteopenia and Osteoporosis ...... 30 2.1.2. VO/DDD, VOA and DISH ...... 33
VOiDDD ...... ,tC,...... o...... 33 VOA ...... ~...... 34 DISH...... 34 2.2. Dual Energy X-ray Absorptiometry (DEXA) ...... *.*...... 34 Description of Technique ...... *...... *.....*...... *.**...... 34 Bone Size ...... 4û 2.3. Examination of Skeletal Material ...... 47 2.3.1. Osteopenia (OPE)and Osteoporosis (OPO) ...... 49 DmC...... 49 Gross Morphology ...... 50 Radiograp hy ...... -50 2.3.2. Vertebral Osteophytosis (VO) / Degenentive Disc Disease (DDD) ...... 52 2.3.3. Vertebrai Osteoarthritis (VOA) ...... 60 2.3.4. Diffuse Idiopathic Skeletai Hyperostosis (DISH) ...... 65 2.4. Data Anaiysis ...... 68 2.4.1. Bone Minerai Density. Osteopenia and Osteoporosis ...... 68 The Smoothing S pline and Broken-line Regression ...... 70 The Generalized Additive Mode1 ...... 71 Conditioning Plots (Co-plots) and Local Estimation Scatter Plot Smoothing (LOESS)...... 73 ûther Statistical Procedures and Graphicd Methods ...... 74 2.4.2. VO/DDD and VOA ...... ,...... 75 Techniques Used to Summarize the Raw Data ...... 75 Graphical Methods ...... 77 PART L BONE MINERAL DENSITY OF THE SPINE ...... 80
CBAPFER 3. THE EFFECTS OF AGE AM) SEX ON BONE MINERAL DENSITY OF TEE SPINE ...... 81 3.1. Resuits: Age, Scx and Bone Minerrl ContenVBone Mimd Density ...... 81 Age. Sex and Bone Mineral Density ...... 8 1 Age. Sex and Bone Minerd Content ...... *..*...**.*....**...... 87 BMD vs .BMC ...... 90 Arca vs .Age ...... 91 BMD by Natal Group ...... 92 3.2. Results: Osteopenia and Osteoporosis ...... 93 Monkeys with Osteopenia / Osteoporosis (without fncture). Diagnosed by Standard Criteria ...... 94 Monkeys with Established Osteoporosis (Vertebral Fnchues) ...... 98 Cornparison of OPWOPO ùidividuals in Tables 3 and 4 ...... 101 3.3. Discussion: Efyects of Age and Sex on Boae Mass ...... *...... **.*,* 128 Bone Minerai Density and Osteoporosis ...... 128 Osteoporosis and Vertebral Fracture Patterns ...... 129 The Causes of Bone FraMty ...... 130 Anatomy of Vertebral Fracture: Architecture and Bone Quality ...... 132 Cayo Santiago Rhesus Monkeys: Studies on Bone Density. Osteopenia I Osteoporosis ...... 133 Cornparison with Previous Studies of Bone Density and Osteopenia I Osteoporosis in Nonhuman Primates ...... 135 Conclusions ...... 139 CBAPTER 4. THE EFFECT OF PARITY, OSTEOPENIA AND OSTEOPOROSIS ON BMD IN FEMALES ...... L41 dl. Results: Parity and Boiw Deasity ...... 141 Parity: Frequencies ...... 14 1 Parity vs .Age ...... 142 BMD vs .Parity ...... 146 BMD vs .Parity. Conditioned on Age (Fig. 37. Co-plots)...... 148 BMD vs. Age. Conditioned on Parîty (Fig .38. Co-plots) ...... 149 4.2. Results: Lon Mty, Osteopenia and Ostcopod ...... 150 4.3. Resuîts: Low Parity. Osteopeaia/Osteopo~b;isand Natal Group Affiiiatiom ...... 151 4.4. Resuits: Puity and Vertebrai OsteopbytosWDegenerative Dise Dlsease ...... 152 45. Discussion: PPrity and BMD ...... 163 Snidies on Human Females ...... 163 Menopause ...... 163 Prepancy and Lactation ...... 164 Iacrease of Bone Density witb Parity in Human Females ...... 169 Non-primate Animai Models ...... 172 Studies on Non-human Primates ...... ,., ...... 174 Macaques as a Mode1 ...... 174 Genetics ...... 174 Age and Parity ...... 175 The Cayo Santiago Female Rhesus Monkeys ...... 177 uicrease of Bone Density with Parity in Cayo Santiago Femaie Monkeys ... 177 Parity. Age and Degenerative Disease in Cayo Santiago Female Monkeys .. 180 Cayo Santiago Femaies: Heritability of Boue Mass ...... 181 Conclusions ...... 183 CHAP'lXR 5. BONE LMINERALDENSITY AND VERTEBRAL OSTEOPHYTOSIS (VO)/ DEGENERATlVE DISC DISEASE (DDD)...... 186 5.1. Results ...... 186 Age by Sex and VOlDDD Group ...... ,...... 188 BMD by Sex and VOlDDD Group ...... 190 Cornparisons Within the Sexes ...... 190 Compûnsons Between Femaies and Males ...... 190 Area by Sex and VO/DDD Group ...... 192 VOlDDD Group 3 Monkeys: Descriptive Statistics and Cornparison of Femes d Md...... 194 5.2 DiSCmjOR.. ..*...... 21 1 Bone Mineral Deasity and Vertebral Osteophytosis I Degemrative Disc Disease ...... 211 The Inverse Relatioaship Between Osteoarthritis of the Peripheral Skeleton and Osteopenid0steoporosis...... 21 2 The Inverse Relationship Behueen OsteopenidOsteoporosis and Vertebral Osteophytosis and Degenerative Disc Disease ...... 214 Cayo Santiago Rhesus Monkeys ...... 215 Conclusions ...... 217
PART II. VERTEBRAL OSTEOPHYTOSIS (VO) / DEGENERATIVE DISC DISEASE @DD) AND DIFFUSE IDIOPATIIlC SKELETAL HYPEROSTOSIS (DISH) ...... 219
C-R 6. VERTEBRAL OSTEOPHYTOSIS (VO)/ DEGENERATLYE DISC DISEASE @DIB) ...... 220 6.1. Results ...... 220 VOIDDD: Distribution Across the Entire Sample. and by Sex ...... 220 Average VODDD by Joint: Overall Patterns in the Sample ...... 223 VOIDDD by Age and Sex ...... 224 VO/DDD by Natal Group...... *...... *.*.. 227 VO/DDD and Parity ...... 227 6.2. Discussion ...... 245 Anatomy and Pathogenesis of Vertebral Degenention ...... 245 Degenerative Spinal Disease in the Rhesus Monkeys of Cayo Santiago: Vertebral Osteophytosis (VO) and Degenerative Disc Disease (DDD) ...... 248 Age/Ser Distribution. Prevalence and Joint Distribution of VOIDDD ...... 248 VODDD: Comparing Femaies and Males ...... 250 Parity and VOlDDD ...... 252 Heritability of VOtDDD ...... 253 Vertebrai Osteophytosis (VO) and Degenerative Disc Disease (DDD) in Non- human Primates and Other Animais ...... 254 VO/DDD in Humans, Past and Resent ...... 259 Conclusions ...... 264 Cm7. DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH).. 266 ...... L...... 7.1. ResuIts ...... L...... 266 7.2. DiSCllSSi011...... 270 DBke Idiopathic Skeletal Hyperostosis (DISH): Description and Differential Diapsis...... ~...... 270 Fractures and DISH ...... ,.*...... *...**..273 DISH in Humans: Epidemiology...... 274 DLTH in Animais: Epidemiology ...... 276 Non-hman Primates ...... 276 Other Animals ...... 278 Conclusions ...... 278
CfIAPTER 8. VERTEBRAL OSTEOARTHRITIS (WA)...... 281 8.1. Results ...... 281 VOA: Distribution Across the Entire Sample and by Sex ...... 281 Average VOA by ZAP Joint System: OvedPatterns in the Sarnple ...... 282 VOA by Age and Sex ...... 284 VOA by Natal Group ...... *.....*...... *...... 285 VOA and Parity ...... 286
8.2. Discussion ...... r,...... 301 Degeneration of the Articular Facets of the Spine ...... 301 Vertebral Osteoarthritis (VOA) in the Rhesus Monkeys of Cayo Santiago ...... 304 AgeiSex Distribution. Prevdence. and Joint Distribution of VOA ...... 304 VOA and Parity / VOA by Notai Group ...... 305 VOA in Non-hwnan Primates and Other Animais ...... 306 VOA in Humans. Past and Present ...... *...... 307 Relationship between VOA and VO/DDD in Rhesus Monkeys ...... 310 Conclusions ...... ,...... 313
PART IV. SYNOPSIS. CMPLICATIONS. AND DIRECTIONS FOR FUTURE RESEARCH ...... 315
CaAPTER 9. DECENERATIVE SPINAL DISEASE. BONE MZNERAL DENSITY AND OSTEOPENIA 1OSTEOKDROGIS: SYNOPSIS. IMPLICATIONS AND DIRECTIONS FOR FUTURE RESEARa...... 316
9.1 . VOmDD and VOA. Finai Conclusions...... C.t 3 1 6 9.2. Bone Minerai Density and Osteopenia / Osteoporosis: Final Conclusions...... 320
xii Fracture Patterns in the Vertebrai Column ...... 320 Bone Density ...... 321 9.3. DISH: Fia1 Conclusions ...... 323 9.4. Degenerative Disease and Primate Behaviour ...... 323 9.5. Final Considerations...... 324 Cayo Santiago Rhesus Monkeys: Non-human Primate Models of Disease ...... 324 Directions For Future Research ...... 325 APPENDIX ...... 327 REFERENCES ClTED ...... 330 LIST OF TABLES
Table Page
1 .Descriptive Statistics of the Sample (N = 254) ...... 82 2 .Summary of DMA Results ...... 89 3 .Osteopenic and Osteoporotic Monkeys Identified Using Criteria Endorsed by the W.H.O...... 95 4. Monkeys with Established Osteoprosis: Identified by the Presence of Vertebral Fractures ...... lûû 5 .Descriptive S tatistics on Cayo Santiago Females 2 4 Yean of Age ...... 144 6. Cornparison of Nulliparous and Parous Females in the Same Age Range (8.5 - 9.4 p.)...... 144 7 .Low-Parity Females...... 145 8 .VOlDDD Group Means by Sex in Sample 9+ Years of Age ...... 189 9 .Descriptive Statistics on VOîDDD Group 3 Monkeys ...... 196 10 .Frequencies of VO/DDD and VOA .Calculated Using Average Scores of Individual
xiv LIST OF FIGURES
Figure
1. Photograph of a typical adult female rhesus monkey living on Cayo Santiago...... 8 2 . An example of a typical print-out of a DEXA scan and analysis ...... 46 3. Rhesus monkey skeleton. showing the lateral view of the vertebd column in typical positional behaviour ...... 48 4 . Axial skeleton of the macaque showing the ventral aspect of the vertebral column: AAR joint numbering system ...... 58 5 .Photographs of examples from VODDD scale ...... 59 6. Schematic repnsentation of zygapophyseal (ZAP) joints in a typicai macaque spine, with joint numbering system ...... 63 7. Photographs of examples from VOA scale ...... 64 8. Photognph of a typicd example of DISH. lateral view of part of the lumbar spine .... 67 9. BMD venus age in entire sample ...... 104 10 .BMD versus age in femdes...... 105 11. BMD versus age in males ...... 106 12. Male and female BMD venus age ...... 107 13. Female and male box plots of BMD ...... 108 14. BMC versus age in females ...... 109 15. BMC versus age in males ...... 1 10 16. Male and femaie BMC versus age ...... 111 17. BMD versus BMC in femdes ...... 1 12 18. BMD versus BMC in males ...... 1 13 19. Cornparison of BMD versus BMC in females and males ...... 114 20 .Scan area versus age in femdes ...... 1 15 21 . Scan area versus age in males ...... 1 16 22. Male and fernale scan area versus age ...... ~...... 117 23 .Box plots of BMD in natal groups npresented in the sample among individuals 7+ years of age ...... 118 24 .Osteopeaic females: Radiograph of lefl huwrus and femur fkom two individuals. with control specimens in the center ...... 119 25. Osteopenic males: Radiograph of left humerus aiid femur fiom two individuals. with control specimens in the center ...... 120 26 . Photograph of two lumbar vertebrae (LA and L5) of osteopenic femaie #637...... 121 27 .Lateral radiograph of the vertebral column of osteopenic female ##637...... 123 28 .Photograph of lumbar vertebrae I to 5 (inclusive) of osteoporotic female #852. showing wedge fractures ...... 123 29 . Lateral radiograph of the vertebral column of osteoporotic female #852 ...... 124 30 . Photograph of old female on Cayo Santiago showing dorsal kyphosis of the spine (Dowager's hump) ...... +...... 125 3 1. Probability density estimate of BMD in males over 9 years of age ...... 126 32 . Probability estimate of BMD in females over 1 1 years of age ...... 127 33 . Histogram of parity in females 4 years of age and older ...... 155 34. Parity venus age in females 4 years of age and older: Maximum births per year .... 156 35. Parity versus age in femaies 4 years of age and older: Average parity across the life span ...... 157 36 .Generalized additive moàels: The additive fit for parity and age, with BMD as the response variable ...... 158 37 . BMD by parity. conditioned on age. in females 4 years of age and older ...... 159 38 . BMDby age. conditioned on parity, in femdes 4 years of age and older ...... 160 39 . Parity by age and VODDD in fernales 4 years of age and older ...... 161 40. Box plots of parity by VOlDDD group ...... +...... 162 41 .BMD by age and VO/DDD group in entire simple ...... 197 42 .Scan Area by age and VO/DDD group in entire sample ...... 198 43 .Box plots of age by VO/DDD group in males and females 9 years of age and older ...... 199 44 .Box plots of BMD by VO/DDD group in males and femaies 9 years of age and older ...... 2ûû 45 .Scatter plot of BMD by age and VO/DDD group in females ...... O201 46 .Scatter plot oPBMD by age and VO/DDD group in males ...... CC...... 202 47 .Scatter plots of BMD by age in females and males. with VOIDDD groups graphed separateiy ...... 203 48 .Box plots of scan area by VODDD in males and fernales 9 years of age and older...... 204 49 .Scatter plot of scan area by age and VO/DDD gmup in females ...... +....205 50 . Scatter plot of scan area by age and VO/DDD group in males ...... 206 5 1 . Scatter plots of scan area by age in females and males. with VOlDDD groups graphed separateiy ...... 207 52 .Photograph of typical lumbar vertebrae nom an individual in VOmDD group 3.... 208 53. Photopph of lumbar spine and sacnun of Cat .M46. a male aged 26 years. exhibithg high BMD. DISH and VOIDDD ...... 209 54. Lateral radiograph of spine of Cat .#2065. a male aged 24 years. showing borderline osteopenia, multiple vertebrai wedge hctures. extensive osteophytosis. and DISH ...... t: ...... 210 55 . VO/DDD: 1st and 2nd Principal Components in the entk sample ...... 229 56 . VOIDDD: 1st and 2nd Principal Components in females ...... 230 57 .VO/DDD: 1 st and 2nd Principal Components in mdes ...... 231 58 .Proportion. by joint. of VO/DDD scons in entire sample ...... 232 59 .Proportion. by joint. of VODDD scores in males and females separately ...... 233 60. Average VO/DDD rating in the entire sample at each AAR joint ...... 235 6 1. Average VOlDDD rating by joint in males and fernales separately...... 236 62. Mean VO/DDD I2 standard errors across the entire sample at each AAR joint .... 237 63 . Mean VO/DDD t 2 standard erroa at each AAR joint in males ...... 238 64. Mean VO/DDD I2 standard ermrs at each AAR joint in femaies ...... 239 65 . Average VOlDDD score versus age in enth sample ...... 240 66. Average VO/DDD score versus age in mdes and femdes separately ...... 241 67 . Photograph of lower thoracic vertebrae from a femde aged 14 years. showing typicd VO/DDD; shown with normal vertebrae for cornparison ...... 242 68 .Box plots: VO/DDD average scores by natal grwp...... 243 69. Box plots: Age by natal group of monkeys represented in this study ...... 244 70 .Photogmph of the vertebral column of a monkey with diffuse idiopathic skeletal hyperostosis (DISH) ...... 268 71 . Photograph of the caudal view of a vertebra fiom a spine with DISH. showing ossification of the antecior Iongitudinal ügament ...... 269 72 .VOA: 1st and 2nd Rincipal Components in entire sample ...... 287 73 .VOA: 1st and 2nd Principal Components in females ...... 288 74. VOA: 1st and 2nd Rincipal Components in males ...... +...... 289 75 .Proportion . by joint .of VOA scores in enthe sample ...... 290 76. Proportion. by joint, of VOA scores in males and females separately ...... 291 77 .Average VOA rating in entire sample at each ZAP joint system ...... 292 78 .Average VOA rating. by joint, in males and females separntely...... 293 79 .Mean VOA I2 standard enors at each ZAP joint system in entire sample...... 294 80. Mean VOA I2 standard emaat each ZAP joint system in males ...... 295 8 1. Mean VOA r 2 standard erroa at each ZAP joint system in females...... 296 82 .Average VOA score venus age in entire sample ...... 297 83 .Average VOA score versus age in males and females sepacately ...... 298 84. Photograph of thoracic vertebrae showhg typical VOA in a female aged 8.5 years ...... 299 85. Box plots: VOA average scores by natal group ...... 300 86 .Cornparison of average VOIDDD and VOA rating in entire sample. by joint ...... 312 A 1. Scatter plot of BMD by age. using BMD = BMC/Area ...... 328 A.2. Scatter plot of BMD by age. using BMD = BMC/(AreaAl.S)...... 329 LIST OF ABBREVIATIONS
AAR: Amphiarthodial AU: Antenor Longitudinal Ligament BMC: Bone Mineral Content BMD: Bone Mineral Density DDD: Degenerative Disc Disease DEXA: Dual-Energy X-ny Absorptiomeüy DISH: Diffuse Idiopathic Skeletal Hyperostosis OA: Osteoarthri tis OP: Osteoporosis vo: Vertebral Osteophytosis VOA: Vertebrai Osteoarthritis ZAP: Zygapophyseal INTRODUCTION
This dissertation presents the results of an extensive cross-sectionai research project on degenerative spinal disease and bone mineral density in a large, well-documented skeletal population of free-ranging rhesus monkeys, the Caribbean Primate Research Center Museum collection from Cayo Santiago. Puerto Rico. Lke humans, the rhesus macaques of Cayo Santiago survive to advanced ages despite the physicaî impainnent brought about by degenerative disease. Their highiy sinictured and naturaily fomed social organization, coupled with the lack of predatoa and continuous provisioning, provide an environment that promotes survival, and buffers the effects of aging (DeRousseau. Bito, and Kaufman 1986). The Cayo Santiago monkeys provide a unique mode1 for a wide range of spontaneous diseases and disorders that comrnoniy affect elderiy humans (see below). An ideal research oppomuiity for a cross-sectional snidy of bone mineral density and naWy-occurring degenentive spinal disease is provided by the skeletai collection associated with these rhesus monkeys. The collection is ideal for this study because it is compnsed of large numbea of complete skeletons denved from a well-known. free- ranging colony with accurate identification and long-term observation of individuals. As a result, detailed demognphic and behavioural data are avaiiable for virtualiy dl individuals, thus providing a unique oppomuiity to study, within a population context, the effect of demographic filctors (age, sex, parity, natal group affiiation) on bone density and naturaiiy-occ~gdegenerative conditions sucb as osteopenia, osteoporosis. 1 vertebral osteophytosis, degenerative disc disease, vertebral osteoarthritis and difise idiopaihic skeletal hyperostosis. This population approitch Plso pemits the snidy of the incidence, expression, pathogenesis and interaction of these conditions within r single closed system. The large sample sizes generated by this study will aiso provide the opportunity to test the efficacy of the standard criteria used to diagnose osteopenia 1 osteoporosis and DISH (see below). This colony also has the advantage of uniformity of environment. diet and rean*ng conditions that permits the study of degenerative disease without the usuai confounding variables that are associateci with studies on human subjects. The ultimate goal of this project is to develop a macaque model for BMD, metabolic bone disease and degenerative conditions of the spine - given the ideal characteristics of this panicular colony. To achieve this end, the primary goal is to document the underlying trends in the data for each of the conditions in question. The development of animai models for human conditions is accomplished in seved stages (Pritzker 1994). First of dl. the disease must be identified or induced in a group of animals. Secondly. there must be a sufficient number of animais avdlable for study. under experimentaily controlled conditions. Thirdly. the biological similarities and differences between the model and the human disease must be documented; i.e., one must characterize the functional and structural sirnilarities and differences between the model and the human condition. Finally, when the model is well characterized, it may be used to test hypotheses related to the pathogenesis and etiology of the disease or disorder in question (Pritzker 1994). This thesis accomplishes the htthree stages in the development of a macaque model for the conditions that were studied - by identmng their spontaneous occurrence in a large population ~aredunder ideal conditions, and by dcfining the pathologies in ternis of agelsex and other variables. For each condition, the pssmorphology, incidence, and patterns by age. sex, parity and natal group affiliation are described, and compared to data in the literature on the human counterpart of the condition. Finally, in accordance with stage four. the testing of hypotheses is initiated; the questions are data-âriven as the analyses proceed, and directions for future research are proposed.
1.1. The Cayo Santiago Colony of Rhesus Monkeys (Macaca mulatta)
The rhesus monkeys of Cayo Santiago are the descendants of an original stock of 409 rhesus macaques fmm Lucknow, India, released ont0 the island in 1938 by Clarence Ray Carpenter, a primatologist, for the purpose of observing primate behaviour in a free- ranging state (Carpenter 1973; Kessler 1989; Kessler and Berard 1989; Rawlins and Kessler 1988). The colony is managed by the Caribbean Primate Resevch Center (CPRC). The CPRC consists of a number of facilities: the "fiee-ranging", semi-naturai colony of rhesus monkeys on Cayo Santiago, an 18 hectare island located L km off the southeastern Coast of Puerto Rico; the Sabana Seca Field Station West of San Juan,
Puerto Rico; and the CPRC Museum located at the University of Puerto Rico. Medical Sciences Campus (Kessler and Berard 1989). Figure 1 shows a photograph of a typical adult fernale rhesus monkey living on Cayo Santiago today. The monkeys on Cayo
Santiago, those living at the CPRC's Sabaaa Seca Field Station, and the associated skeletal collection at the CPRC Museum, are aii derived from the same gene pl; Le., they are al1 descended fkom the members of the original colony estabiished in 1938. Macaques iîve in multi-male. multi-femaie social groups with a strong dominance hiemhy in both males and fernales. in which kinship plays an important role (Caldecott 1986; Fa 1989; Napier and Napier 1967). On Cayo Santiago, the monkeys in the original colony organized themselves spontaneously into two social gmups, designated A and B. Over time, these original groups Fissioned into multiple social groups, dividing dong matdineal Lines (Sade et al. 1976). Maternai kinship lineages are the key subunit of social organization in rhesus macaques. Femdes remain in their natal groups for life, wtde males transfer to another socid group at puberty (Sade 1967; Sade 1972). The colony has been dosed since its establishment. and matrilineages are known for the last 12 generations. The genus Macaca is exceptional for its wide geographic distribution; one of the most outstanding features of the genus is that many species are adapted for a predominantly terresvia1 lifestyle, ascending trees primuily to sleep (Manin 1990). Rhesus macaques are medium-sized monkeys. with a quadnipedai pattern of locomotion on the ground as well as in trees. The tail is about half the body length and limbs are of almost equal proportions (Napier and Napier 1967). As they age, the positionai behaviour of the macaques on Cayo Santiago includes a greater proportion of time spent on the ground, walking quadrupedaily, and feeding from standing or sitting positions. Limb movement is primariiy in an anterior-postenor plane. and acrobatie or suspensory activity is infrequent in adults (DeRousseau 1988). There is moderate sexual dimorphism in rhesus monkeys; the adult body length of Cayo Santiago males is 58.8 I
2.2 cm (SD), and that of females is 53.6 t 19cm (SD) (Cheverud 1981; Tuniquist and Kessler 1989). The average body weight of Cayo Santiago males aged 6 to 9 yean is 10.5 kg, while that of females in the same age range is 8.8 kg; the average weight for males aged 10 to 14 yem is 11.9 kg, and that of females is 9.6 kg. in 15 to 19 year old males, the average body weight is 10.9 kg, and the average weight for females is 9.7 kg. The average body weight of males 20+ years of age decreases to 9.4 kg. and that of females aged 20+ yean also declines, to 8.6 kg (Tunquist and Kessler 1989). Rhesus monkeys have a seasonal reproductive cycle. In the monkeys of Cayo
Santiago, mating occurs from mid-luly to early December, and the birth season mns from mid-December to mid-lune. Femde rhesus monkeys reach sexual maturity at 3 yeys of age and usually produce their fitoffspring sometime in their fourth year (Koford 1965). Appmximately 80% of adult females 4 years of age and older give birth each year, 97% of which are live births. The female rhesus monkeys in this sarnple continued to nproduce until the end of their lives. The length of gestation in rhesus monkeys is 165 days. The average interbirth interval for females delivering and weaning offspring in consecutive years is 372 f 33 days. The average interbirth intervd is 336 t 29 days for femdes who have stiilbirths or spontaneous abortions. or whose infants die prior to
weaning. Old females tend to skip a. year occasionally, so their births may be spaced further apart (Raw lins, Kessler, and Tumquist 1984).
Regular provisioning of the colony on Cayo Santiago began in 1956. as did the cumnt system of individual identification of animals via unique tattoos. Longitudinal
data on the colony are available from 1956 to the present; life history information on over 4100 monkeys is available, derived from the daily census initiated by Stuart
Altmann in 1956. Prior to 1971, there was no systematic retrieval of the skeletons of rnonkeys that died on the island, so the early CPRC Museum collection consists of only isolated bones found on the island, as well as about 20 skeletons of animals found immediately after death and macerated (Tumquist and Hong 1989). Until recentiy, these bodies were skeletalized by a nadwater maceration technique, following evisceration. The present systematic skeletd coilection kgan in 1972. and thus, for al1 individuals in the sample, life history information is available in coajunction with the skeletai remaïns, both of which are curated at the CPRC Museum. This museurn presently curates over 2000 rhesus monkey skeletons (Tuniquist and Hong 1989). The identity (tattoo number and catalogue number), date of biah, sex, maternity, natal group affiliation and age at death are among the life history data contained in the associated database (Kessler and
Berard 1989). This is the only collection currently known to this author which attempts to integrate genealogical information, behavioural and dietary data. and medical history of a non-human primate with its osteological remains. This collection is also unique in the extent of the postcranial materid that is available: most museum collections consist primarily of crania and/or partial postcranial bones. Thus, the unique nature of this skeletd collection provides an opportunity for the smdy of certain diseases and disorders from an epidemiological perspective. Within the protective environment of the CPRC. rhesus monkeys develop the naturallysccumng degenerative conditions that arc commonly found in elderly humans. in this population. the development of spinal disease and the loss of passive joint mobility are congruent with the rate of ontogeny, showing r th-fold increase relative to human developrnent.
The growth pend and longevity are about one-third those of humans. This consistent pattern is valuable for developing a nonhuman primate mode1 for human conditions (DeRousseau 198Sa; DeRousseau 198%). The Cayo Santiago monkeys have thus provided unique models of a wide range of spontaneous diseases and disorders, including a fom of degenerative arthritis known as Calcium Pyrophosphate Dihydrate Crystai Deposition Disease, or CPPD (Pritzker et al. 1989), diabetes (Howard, Kessler, and Schwartz 1989), ocular disorders (Dawson et al. 1989). obesity (Schwartz 1989). and age- dependent loss of joint mobility (Tumquist 1986). Congenital anomalies (Rawlins and Kessler 1983), and healed fractures (Buikstra 1975) have also been ~ported. These rhesus monkeys have also provided a unique primate mode1 of spontaneous osteoarthritis of tbe appendicuiar joints (Chateauvert et ai. 1990; DeRousseau 198%: DeRousseau 1988; Riaker et al. 1989; Rawhs and Kesslec 1986a), and of osteoporosis (DeRousseau
198%; Grynpas 1992; Grynpas et al. 1989). In manner of expression, progression, gross morphology and histology, the peripheral joint OA observed in these monkeys closely nsembies its human counterpnrt (Pritzker et al. 1989). The rhesus monkeys of Cayo Santiago also develop spinai osteophytosis. vertebral osteoarthritis and degenerative disc disease spontaneously with advancing age. similar to humans (Cerroni 1992; DeRousseau 1988; DeRousseau, Bito, and Kauhan 1986).
9 If Bone Minerai Density, Osteopenia and Osteoporosis: Background and Mqjor Research Questions
Osteoporosis is a rnultifactorial, age-related metabolic bone disease characterized by low bone mineral density and the deterioration of the microarchitecture of cancellous bone, leading to enhanced bone fragility and a consequent increase in risk of fracture
(Kanis L 994a; Wasnich 1996). A normal mineraVcollagen ratio in osteoporosis distinguishes it from osteomalacia a disease characterized by a relative deficiency of minera1 in relation to collagen (Wasnich 1996). There are two main types of osteoporosis: 1) Primarv Osteo~orosis(includes involutional and juvenile osteoporosis) and 2) Secondary Osteomrosis (includes osteoporosis caused by endocrine and chronic diseases. dietary deficiencies- of calcium, vitamin C or D, inbom enors of metabolism. and the use of certain drugs). Involutional osteoporosis is a term that refea to the graduai progressive bone loss, often accompanied by fractures, that is observed in postmenopausal women, and in both men and women with advancing age. Involutional osteoporosis represents two syndromes - postmenopausal osteoporosis (type i) and senile osteoporosis (type [D (Genant 1996; Riggs 1991). Senile osteoporosis occurs in elderly men and women. usually 75 years of age and older, and is characterized by fractures of the hip, pelvis and proximal humerus. Postmenopausal osteoporosis occurs in a subset of postmenopausal women between the ages of 50 and 65 years, and is characterized by accelerated tnbecular boue resorption (related to estrogen deficiency) in the spine and wrist, and consequent hgîlity fracnues in these matornical regions (Genant 1996). Osteoporosis has been cdied the 'silent epidemic'; it affects one in every four posmienopausai women in North Amenca, causing considerable morbidity and moriaüty, as well as high costs in terms of health care (Bassey 1995; bis1994b; Melton III 1988; Orwoll 1996; Wasnich 1996). Although not as commoa as in wornen, osteoporosis in men is also a si-cant health problem; about one-third of dl osteoporotic hip fractures world-wide occur in men, and the vertebrai fracture rate is about hdf that of women (Cooper and Melton 1992). Osteoporosis is the most prevaient metabolic bone disease in the United States and other developed countries (Wasnich 1996). It is a major public health problem; in the United States, more than 1.5 million fractures rire attnbuted to osteopomsis each year, and the costs associated with them is estimated to be close to $10 billion per year (Riggs 199 1). As the proportion of the population over 65 years of age continues to increase, osteoporosis wül become an even mon significant health problem in the near future (Melton III 1988; Wasnich 1996). The main sites of these fractures are the vertebral column. the proximal femur, and the distd radius (Colles' fracture). It is estimated thût one third of women over 65 years of age will, at some time duhg their later years, incur vertebral fractures (Riggs 199 1). A disproportionate loss of trabecular bone fmm the axial skeleton is a distinguishing chancieristic of spinal osteoporosis (Riggs et al. 198 1). Age is known to be the rnost sipififant predictor of bone mass in any given individual, fernale or male. and decreased bone density is a significant risk fxtor for osteoporosis. In humans, fracnires occur in the femur and spine when bone density fdls below a critical threshold level, which is about 1 @cm2 for both sites (Riggs, Peck, and Bell 199 1). In human fernales, peak bone minerai density is attained by the late 20dearly 30s, after which it declines steadily with age. Men attain peak bone density somewhat later than wornen, and their peak bone mass is higher (Riggs, Peck, and BeU 1991). Mer bone density peaks, losses in bone mass begin. and continue ihroughout Me; this loss is attributed to an imbalance in skeletal remodelling (boae turnover), in which the rate of 11 bone formation is less than that of bone ~sorption(Baron 1996; Candis 1996; Riggs and Melton lii 1988; Riggs, Peck. and Bell 199 1). Studies have show that osteoporosis is a disorder of complex etiology; there is an interaction of nuuitiond, endocrinological and local factors that are subject to various environmental and genetic influences (Stini 1990). Most risk factors for osteoporosis fidi into six major categories (Riggs. Peck, and Bell 199 1; Wasnich 1996)-as follows:
Age, or Aee-related (each decade after the age of 30 years is associated with 1.4 to 1.8-fold increased risk) Gender (fernales are more predisposed) -Genetic (pe tite/thin build. positive family his tory) Environmental (inadequate nutrition, calcium deficiency, limited physicd activity, medications such as conicosteroids, premature/surgically-induced menopause. cigarette smoking. alcohol abuse)
Endoeenous Hormones and Chronic Diseases (estrogen/androgen deficiency. hyperthyroiâism. cirrhosis) Phvsical Charactenstics of Bone (density, size, geometry, microarchitecture, composition).
Women over 65 years of age are at greater risk for incming osteoporotic fractures cornpared to men; the Iifetime risk for hip fracture in women is 15%. compared to 5% in men (Riggs 1991). Osteoporosis is diagnosed when a value for bone mineral density or bone minerai content is 2.5 standard deviations or more below the mean of the young adult ceference range (Kanis 1994a). Thus, many studies have sought to identify the reproductive and Mestyle correlates of boue mass in women. hoping to identify the greatest risk factors for developing osteoporosis later in life. Two reproductive correlates, pregnancy and lactation. are associated with profound changes in hormone levels and calcium metnbolisrn, imposing stress on the calcium homeostasis of the mother, particularly on the skeleton, which is the main repository of calcium in the body. Many stuâies have assessed and debated, the impact of pregnancy and lactation on the human maternai skeleton (Atkinson and West 1970; Chtistiansen, R~dbro,and Heinild
1976; Dequeker et al. 1987b; Drinkwater and Chesnut III 199 1; Dunne et ai. 1993; Fox et al. 1993: Goldsmith and Johnston 1975; Hayslip et al. 1989; Kent et ai. 1990; Khastgir and Studd 1994; Koetting and Wardlaw 1988: Lamke, Brundin. and Moberg 1977;
Nilsson 1969: Nordin and Roper 1995; Sowea et al. 1991; Sowen et al. 1993; Valenzuela et al. 1987; Waiker 1972; Walker, Richardson, and Walker 1972; Wacdlaw and Pike 1986). Some retrospective and prospective studies on human femdes report increased bone density with parity (Aloia et ai. 1983; Fox et al. 1993: Goldsmith and Johnston 1975; Murphy et al. 1994: Nilsson 1969; Sowers et ai. 199 1; Valenzuela et al. L98?), and with lactation (Melton III et al. 1993), but some prospective studies show constant bone mass during pregnancy (Christiansen, Rdbro, and Heinild 1976), while others reveal signifiant bone loss (Drinkwater and Chesnut III 1991; Hayslip et al. 1989;
Lissner, Bengtsson, and Haasson 1991; Yamamoto et ai. 1994). One report cites nulliparity as one of the factors associated with decreased bone density among the women in their study, and hence. with increased nsk for osteoporosis (Stevenson et al. 1989). Thus, the research to date bas produced conflicting and inconclusive results. Longinidinal snidies are notoriously difficult to conduct on human subjects; thus, such snidies are usuaily ümited by smaii sample size. Osteomrosis h Animals. Indudina Non-Humsn Primates
Some studies on bone mus in animais, hcluding non-human primates, bave also ken conducted (Draper 1994; Geusens 1992; Hiyaoka et ai. 1996; Kimmel 1994; Miller, Bowman, and Jee 1995). Many animal models of osteopenia have been developed, including the rat, mouse. beagle dog, sheep, kmt. minipig and monkey (Draper 1994; Geusens 1992; Miller, Bowman. and Jee 1995). Age-related osteopenia has been noted in rhesus macaques (Bowden et ai. 1979: Williams and Bowden 1984). and other studies also report age-related bone loss in various species of macaques and baboons (Jayo et al. 1994; Kammerer, Sparks, and Rogers 1995: Pope et al. 1989; Przybeck 1985). Some evidence for age-related osteopenia of the spine was noted in gonilas (Ohman. Mensforth. and Latimer 1997). and age-nlated bone loss, especially from femoral corticai sites. was observed in femde chimpanzees from Gombe (Sumner, Morbeck. and Lobick 1989). Another study also noted bone loss with age in the long bones of both a femaie and a male chimpanzee from Gombe (Zihirnan. Morbeck, and Goodail 1990). Many studies have examined the effect of ovariectomy, i.e., experimentnlly induced hormone deficiency, on bone mas in macaques and baboons (Bowles et al. 1985; Jayo et al. 1994; Jayo et ai. 1990; Jerome et al. 1994; Jerome et al. 1986; Jerome, Lees, and Weaver 1995; Jerome et ai. 1992; Longcope et al. 1989: Lundon, Dumiuiu, and Grynpas 1994; Lundon, Dumitrïu, and Grynpas 1997; Lundon and Grynpas 1993; Mann, Gould, and Collins 1990; Miller et al. 1986). and some investigators have conducted cross-sectional and longitudinal studies of bone density by age and sex in macaques and baboons (Champ et al. 19%; Jayo et al. 1991b; Kammerer et ai. 1994; Kammerer, Sparks, and Rogea 1995;
Pope et al. 1989). The effect of ovariectomy on the quaiity and quantity of cortical and cancellous bone was studied in cynomolgus macaques (Lundon, Dumitriu, and Grynpas 14
1994; Lundon and Grynpas 1993). Cross-sectional and longitudinal studies of bone mass in female cynomolgus monkeys have been conducted (Jayo et al. 1994; Jayo et al. 199 lb), and the effects of age. sex and heredity on bone mass have been studied in pedigreed baboons (Kamrnerer et al. 1994; Karnrnerer, Sparks, and Rogers 1995). The effects of age and sex on bone density have been studied in rhesus monkeys at the Yerkes Primate Center (Pope et al. 1989). and the effect of age on bone minerd content has been studied in female rhesus monkeys at the Wisconsin Regional Primate Research Center (Champ et al. 1996). Bone mass and cortical remodelling of the rib was also studied in rhesus monkeys (Pnybeck 1985). However. studies which examine the effects of reproductive parameters such as parity and lactation on bone mass in non-human primates an rare (Bowden et al. 1979;
DeRousseau 1985b; Grynpas 1992; Grynpas et al. 1989; Grynpas et ai. 1993b; Hiyaoka et al. 1996; Sumner, Morbeck, and Lobick 1989). A number of non-primate models, such as the rat model. have been developed over the years which demonstrate bone loss from ovariectomy or from pregnancy and lactation (Currey and Hughes 1973; Ellinger et al. 1952; Gare1 1987; Kalu 1991; Peng et al. 1987; Rasmussen 1977). Ovariectomy in beagle dogs also produces a model of hormonedeficiency bone loss (Martin et al. 1987). Although these models have beea informative, an animal model which is physiologicaily and genetically closer to humans would be more desinble, especially one that is naturd and spontaneous rather than derived experimentdy. The ultimate aim of the present study is to provide such a model. -Rhesus Monkevs of Cavo Santiano
There is a need to explore the relationship between the natunl phenornena of normal aging (i.e., naturd menopause. natumi age-related hormone deficiency) and bone density and spontaneous osteopenia/osteoporosis in a large population. This cross- sectionai study investigated the relationship between age, natal group affiliation and bone mineral content (BMC) I bone minera1 density (BMD)of the lumbar spine in males and femaies of P large, well-documented population of rhesus monkeys (Mraca mulatta) from the CPRC Museum collection from Cayo Santiago. Puerto Rico. This cross- sectionai study was also designed to examine the relationship between parity and bone minerd content (BMC)1 bone mineral density (BMD) of the lurnbar spine in the fernale rhesus monkeys from this collection. The relationship between age and parity in this popdation was also examined; the result is a profile of the fertility of the Cayo Santiago femaies in the present sample. The relationship between parity and osteopenia/osteoporosis was also investigated, as was any possible associations with the spinal diseases of vertebral osteophytosis (VO)and degenentive disc disease (DDD).
Advanta~esof a Monkev Mode1
One dvantage of developing a monkey mode1 for the study of parity and bom density is the lack of confounding factors usually found among humans - variables for which adjustments ;ire required in studies that use human subjects. These factors include iifestyle variables such as physical activity, cigarette smoking, alcohol intake, and the use of oral contraceptives, estrogen replacement thenpy, and calcium supplements. Another advantage is that the diet of the colony is constant; the monkeys are provisioned with a commercial monkey chow, which is avdable on demand and nadily accessible to al1 members of the population. Therefore. gJ females in the colony have essentially the same diet - unlike the great variability in diet observed arnong human subjects. Although this commercial preparation is high in protein and contains calcium and vitamin D supplements as per govemment standards. a study has demonsirated that increasing the protein content in the diet of these monkeys does not change the mineral content of their bones; variation in the commercial diet has no significant effect on the calcium content in their bones (Grynpas et al. 1993a). Lactation history is also not as variable as in humans; dl female monkeys who give binh lactate, and nurse their infants for a similar length of time. With little variability, female rhesus monkeys mach sexud maturity at 3 years of age and usually produce their fitoffspring in their fourth year of life (Koford
1965). Age adjustment with respect to age at menarche, which is often done in studies on humans, is therefore not necessary. It is also not necessary to adjust age with respect to years since menopause, ais0 a requirement in studies on human subjects, since the rhesus monkeys in this sarnple have continued to reproduce until the end of their Iives (Rawlins, Kessler, and Tumquist 1984).
Previous Studies of Bone Mas in Cavo Santiago Rhesus Monkevs
There are a few previous studies of bone density and bone mineralization conducted on the rhesus macaques of Cayo Santiago; these snidies demonstrate the potential utility of this colony for mode1 development for bone density and osteoporosis (Aguil6 and
Cabrera 1989; DeRousseau 198%; Grynpas et al. 1993a; Grynpas et al. L989; Grynpas et ai. 1993b). A single photon absorptiometry (SPA) study of raàii and femora hm individuals in this colony noted an age-celated deccease in bone density (AguilO and Cabrera 1989). Other previous studies on Cayo Santiago macaques have noted age- related osteopenia among fernales, and demonstrated that osteoporosis is associated with age, that fernales are predilected, and that high par@is a pmtective factor (Grynpas 1992; Grynpas et al. 1989). These earlier stwlies of vertebrai bone density in this population have found that pdty has a significant impact on the vertebnte of femde monkeys: they report a weak positive correlation between parity and vertebral bone density, and between bone density and weight in these lemde rhesus macaques (DeRousseau 1985b; Grynpas 1992; Grynpas et al. 1993b). Other previous studies on this coloay include an investigation of the effects of age and osteoarthritis on bone minerai (Grynpas et al. 1993b). a study of the effects of diet, age and sex on the minerai content of bones (Grynpas et al. 1993a). and a study of bone minerd and osteoporosis in aged rhesus monkeys (Grynpas et al. 1989). Some of these previous studies were limited by poor sex ratios. the technology available at the time, or by small sample size.
Reiationshi~between Osteonorosis and Osteoarthritis
An inverse relationship between osteoarthritis and osteoporosis has been inferred from clinicd studies on human subjects, and an inverse relationship between osteoporosis and osteophytosis of the vertebral column has aiso been reported (Belmonte-Serrano et al. 1993; Dequeker 1985; Dequeker et al. 1996; Hart et al. 1994; Liu et al. 1997; Marcelli et al. 1995; Peel et al. 1995; Versmeten et al. 1991; von der Recke et ai. 1996; Yu et al. 1995). Patients with osteoprosis tend to have lower frequencies and milder expressions of vertebral osteophytosis and disc degeneration (Dequeker 1985). Some studies have also shown that vertebrai osteophytosis (VO), degenerative disc discase (DDD)and vertebral osteoanhntis (VOA) introduce bias into a DEXA study (Dequeker et al. 1996; Drinka et ai. 1992; Hamerman and Stanley 1996; Masud et al. 1993; Omoll. Oviatt, and Mann 1990: von der Recke et al. 1996). The present study has found that these conditions interfere with DEXA anaiyses by causing changes in the density and bone quality of the centmm (se<:chapter 2). Thus, for certain parts of the study, osteophytic bones were removed in order to avoid bias, but they were later re-incorporated into the smple to conduct other anaiyses (see below). The associations between BMD, osteopenia, osteoporosis and VO/DDD were investigated during the course of this DEXA study.
Ob-iectivesand Oreankation of the Studv
The first piut of this project involves a detailed investigation of bone mineral density, osteopenia and osteoporosis of the spine that was conducted on a large sample of skeletons derived from this rhesus monkey colony. This study utilized the most advanced bone densitometry available (dual energy X-ray absorptiometry - DEXA) to study the intricate relationship between parity and bone minerai density in greater detail, to establish a natuai non-human primate model for the effect of pari& on bone density and nanirally-occumng osteopenia and osteoporosis. and a nawal primate model for the effects of age and sex on BMD. The relationship between bone density and VO/DDD was also explored. The main goal of this research is to assess the role of demographic variables in the spontaneous developrnent of osteopenia/osteoporosis of the vertebrai column in both females and males. More specifically, the objectives of the fust part of this study were as follows: 1. To describe changes in bone mass across age in both males and fernales, using dual- energy X-ray absorptiometry (DEXA);
2. To detennine the age at which male and fernale rhesus monkeys reach peak bone mineral density;
3. To determine if. and when, an age-related decrease in bone density OCCLUS; 4. To investigate the relationship between parity and bone density, and between parity and osteopenia/osteoporosis; 5. To explore the relationship between parity and vertebrai osteophytosis (VO) / degenerative disc disease (DDD): 6. To determine whether there is a relationship between natal group affiliation and bone minerai density in this colony;
7. To look for cases of osteopenia (using DEXA andysis), and to determine the frequency of osteopenia in this skeletd collection; 8. To search for, and describe, cases of osteoporosis. as determined by vertebrai compression or wedge fractures and DEXA. and to detennine their frequency: 9. To study disease interaction - the relationship between bone density and vertebrai osteophytosis (VO) / degenerative disc disease (DDD),and the interaction between
the latter and osteopenia/osteoporosis.
It is important to note that body weight was not included as a variable in ihis study because consistent records arr not available for this skeletal collection. Data on body weight is iimited, and consists of death weights for a few Cayo Santiago animals in the skeletal collection and iive weights for others. Colony means and standard deviations for each agdsex category are available, but did not yield usehil data for the purposes of this study .
The results of the fitpart of this project are presented in chapters 3,4 and 5. The effects of age and sex on bone density of the spine are presented in chapter 3, and the findings on osteopenia and osteopomsis are also discussed. The effect of parity, osteopenia and osteoporosis on BMD is discussed in chapter 4, as is the relationship between par@ and VOIDDD. Chapter 5 presents the results obtained from the study of the association between BMD and VO/DDD, and between VO/DDD and osteopenia/osteoporosis. in chapter 5, the osteophytic individuais were re-incorporated into the sample in order to study the effect of VOlDDD on bone density and to examine the relationship between VOIDDD and osteopenia/osteoporosis.
1.3. Vertebml Osteophytosis (VO)and Degenerative Disc Disease (DDD): Background and Mqjor Research Questions
Dcfinitioa and Desrriinlon of VO and DDD
Severai distinct degenerative processes affect the articulations of the vertebral column. and classification is based on the type of joint that is involved. There are two distinct intervertebrai anicular systems: the zygapophyseal joints (articular facets) and the amphiarthrodial joints (vertebral bodies and intervertebrai discs) (Hough Jr. and Sokoloff 1989). The zygapophyseal joints of the spine are synovial in nature, and thus, osteophytosis and remodeiüng of the joint surfaces are interpreted as the diagnostic feanires of osteoarthritis, Le., vertebral osteoaithritis (VOA); VOA is discussed in section 1.5. The joints between the vertebrai bodies of the qine are fibrocartiIapinous, and are thus subject to degenerative disc disease (DDD) and vertebral osteophytosis (VO). Each disease is associated with characteristic radiographie and pathologie abnormalities. and accompanied by significant clinical manifestations (Resnick 1985). Disc degeneration refea to the stmctunl alteration of the intervertebral disc, changes which include reduced water content? modified proteoglycan content, reduced disc height, and fisswing or clefts at the insertion of the annulus fibrosus into the marginal ring (François, Eulderink, and Bywatea 1995). Disc degeneration is often accompanied, or followed, by osseous changes in the vertebrai body, mainly consisting of osteophyte formation (bone spm) and sclerosis. When bone changes are present, the term 'discanhrosis' is often used; however, some investigaton consider disc degeneration and discarthrosis to k one continuous process, and employ the term 'degenerative disc disease' (François, Euldennk. and Bywaten 1995). This practice is followed in the present study; the acronym 'VOIDDD' is used to denote 'vertebral osteophytosis' and 'degenerative disc disease'. The prolapse of the intervertebral disc superiorly and inferiorly into the vertebrai bodies results in Schmorl's nodes (Schmorl and Junghanns,
1971). Bony changes of the vertebrai body may also include disruptïon of the endplate, which may eventually lead to a defect cailed a 'Schmorl's node', in which there is extrusion of the disc into the centnim (intravertebnl disc hernia), forrning a cartilaginous node within the venebral body that is continuous with the intervertebrai disc. with subsequent formation of an osseous casing surroundhg the prolapsed disc tissue (Schmorl and Junghanns, LW 1). Changes in the subchondrai bone hclude sclerosis, pitting and ebumation. In skeletal remains, VO/DDD is manily characterized by the marginal development of osteophytes, and the presence of sclerosis, pitting and ebumation in severe manifestations of the disease. Osteophytes may develop on both the superior/craaid and uiferiorlcaudai rims of the vertebral bodies in al1 sections of the spine, developing a few millimeten fiom the discovertebral junction and extending in a ventral plane. Such osteophytes are outgrowths of the vertebral centmm ûad consist of both cortical and trabecular bone. Advanced States of the disease may exhibit fusion of vertebral bodies at end-stage (Dihlmann 1985; Moskowitz and Goldberg 1988). Genedized pitting or remodelling of ail, or part, of the articular surface of the centrum is often associated with VOIDDD (Ortner and Putschar 1985). The diagnosis of VO/DDD in skeletal materiai may be complicated by the pnsence of other spinal diseases such as difise idiopathic skeletal hyperostosis (DISH) (Hough Jr. and Sokoloff 1989), described in the next section.
---œVO and DDD in Animals, including Non-human Primates
The occunence of degenerative disc disease. vertebrai osteophytosis, and other skeletal anornidies have been reported in rhesus monkeys from other primate research centen in addition to the CPRC, and in monkeys from zoos (Davis and Leathea 1985; DeRousseau 1985a; DeRousseau 198%; Sokoloff, Snell, and Stewart 1968). A ment review of muscdoskele ta1 diseases, including bone diseases, degenerative spinal disorders and osteoarthntis is provided by Fritzker and Kessler (1998), and Pritzker (1994) discusses the processes, problems and prospects associated with the development of mimai models for osteoarthritis. VO/DDD and other diseases of the spine occur spontaneously in many species of monkeys and apes, as well as in other mammals (Bauza, htimer, and Scoles 1991; Clauser 1981; DeRousseau 1988; Fox 1939; Jurmain 1989b; Love11 1987; Lovell 1990; Lovell 199 1; Rothschiid and Woods L992; Schultz
1956; Sokolo& Snell, and Stewart 1968). One study found that gorillas, chimpanzees 23 and ormgutans exhibit relatively higher frequencies of these diseases than do monkeys (Clauser 1981). The occurrence of VO/DDD in quadrupeda1 primates challenges the popular assertion that its ubiquitous presence in human populations is primarily due to the unique biomechanical stresses of habitual bipedal locomotion. The rhesus monkeys of Cayo Santiago provide a unique non-human primate model of spontaneous OA (Chateauvert et al. 1990; DeRousseau 198%; DeRousseau 1985b; DeRousseau 1988; Pritzker et al. 1989; Rawlins and Kessler 1986a). These monkeys also develop degenentive spinal diseases spontaneously with advancing age (DeRousseau
1988; DeRousseau, Bito. and Kaufman 1986). According to DeRousseau et ai (1986). spinal degeneration in the üve population of rhesus monkeys on Cayo Santiago is most prominent in the thoracic region of the spine. and well advanced by the time the animal is
25 years of age; moderate to severe VODDD is comrnon in individuais over 20 years of age. Studies have shown that OA of the appendicular joints in this population is more fi-equent in older animals, especidly in females with high parity (Chateauvert et al. 1990; Ritzker et al. 1989). These monkeys develop VOlDDD of the spine and OA of major appendicular joints in high frequencies. in both males and females. DeRousseau has shown that, in this population of rhesus monkeys, the development of spinal VO/DDD and the loss of passive joint mobility are congruent with the rate of ontogeny, showing a three-fold increase relative to human development. Also, the growth period and longevity are about one-third those of humans. This consistent pattern is valuable for developing a non-human primate model for human conditions (DeRousseau 1985a; DeRousseau 198%). Indeed. the spinal VO/DDD, OA and DISH exhibited by these monkeys closely resemble their buman counterparts in pathology and gross morphology (see below). DeRousseau (L985a) aiso noted that Cayo Santiago monkeys develop spinal and hip diseases earlier in ontogeny and at a faster rate than caged rhesus monkeys at a facüity in 24
Wisconsin; she reasoned that differences in locomotorlpositional behaviour are r~sponsiblefor this variation.
Obiectives and Omanization of the Shidv
The second part of this project involved a detailed investigation of natunlly accurcing VO/DDD in a large sample of complete spines from skeletons derived lrom this rhesus monkey colony. This part of the study used gross morphology and radiography to study the pattem of spontaneous joint degenention of the vertebnl body in both maies and lemales. The role of demognphic variables (age, sex, parity and natal group affiliation) in the spontaneous development and expression of VOlDDD was assessed, in addition to population frcquencies. The uitimate goal of this project is to establish a natucal non-human primate mode1 br VOiDDD. The results of the second part of this project are presented in chapter 6. The objectives of this part of the study were as follows:
1. To describe the pattern of VOfDDD in the rhesus monkey spine, by intervertebral joint, and to describe colony trends;
2. To describe age-related trends in the developrnent and expression of VOlDDD in both males and females, using the techniques of gross morphoiogical analysis aad
radiography ; 3. To determine whether or not there are differences between males and females with
regard to the development, expression and severity of VODDD; 25
4. To mer investigate the relationship between parity and VODDD. using the methods of this part of the study (Le.. complete spines, gross morphoiogy); 5. To determine whether thcn is a relationship between aatd group affiliation and VO/DDD in this colony; 6. To determine the fiequency of VOIDDD in this skeletal collection. 7. To investigate any relationship between VO/DDD and osteo;irthntis of the facet joints (VOA).
1.4. Diffuse Idiopathic Skeletal Hyperostosis (DISH): Background and Major Research Questions
Definition and ~rintioa
Diffuse [diopaihic Skeletal Hyperostosis (DISH). dso known as 'ankylosing hyperostosis of Forestier', is a striking, asymptomatic disorder of middle- and old- age in humans, that has a higher prevdence in males (Resnick 1988). Forestier and Rotes-
Querol (1950) coined the term "senile ankylosing hyperostosis of the spine" in 1950 to denote this distinctive disorder. After evaluating over 200 patients with this disorder,
they noted that 65% were males. DISH is marked by flowing calcification and ossification of the anterior longitudinal ligament (W)dong the anterohteral aspect of vertebrai bodies, thus forming ankyiotic bridges across contipous vertebral centra, especiaiiy in the thoracic and lumbar regions. The result of this process is a bumpy spinal contour, particularly overlying intervertebral discs. where the new bone is the most dense (Forestier and Rotes-Querol 1950; Resnick et al. 1978). The ankylotic bridging is accomplished by a type of osteophyte cailed an enthesophyte. This disorder is aiso characterized by the absence of disc degeneration. and by occasional excessive osteophytosis (bony spurs) in peripheral joints. The cause of DISH is unknown and no consensus currently exists on the 'bounds' of the DISH syndrome; thus, it is often conhsed with VO/DDD (Hough Jr. and Sokoloff 1989: Moskowitz and Goldberg 1988).
It has ken noted that DISH tends to modi@ the pathogenesis and expression of other spinal diseases such as rheumatoid arthntis, degenerative disc discase and osteoporosis (Doyle and Littlejohn 1986). In the study of both human and nonhuman primate spinal disease, it is important to distinguish the various disordea. However, it is also cntical to appreciate that some of the processes are related. that they freqwntly occw simultaneously at the sarne vertebral level. and that the presence of one process may affect the pathologie and radiologie expression of another (Resnick and Niwayma 1988).
Onanization of the Study
The second part of this project also involved a detailed investigation of nanunlly ccchng DISH in this rhesus monkey colony. This part of the s~dyalso used gross morpbology and ndiography to snidy the pattern of spontaneous DISH in the sane sample of rhesus monkey skeletons. The individuals diagnosed with DISH are described in chapter 7. The relationship between the osteophytes of VO/DDD and the enthesophytes of DISH is aiso discussed. and the coexistence of any other disorder is noted. 1.5. Vertebrai OsteoarthFitis (VOA): Background and Mq(ot Research Questions
Definition and Descri~tionof VOA
Osteoarthritis may affect any of the synovium-lined joints of the vertebd column, including the zygapophysed, costovertebrai. transitional lumbosacnl, median atlantoaxial, and sacroiliac articulations (Resnick 1985). The primary, degenerative fom of OA develops on the basis of age-related changes and degeneration of articula cartilage (Ortner and Putschar 1985). Degenerative change of the zygapophysed joints involves marginal lipping, pitting, and ebumation. genedy progressing in this order. in advanced cases, there is enlargement and remodelling of the articular surface, which may lead to fusion of the joint in severe conditions (Resnick 1985). François (1995) uses the terni 'zygarthrosis' (short for 'zygapophyseal osteoanhrosis') to denote 'osteouthrosis' of the zygapophyseai joint. The tem 'os teoarthrosis' and 'os teoanhri tis' are synon ymous, and the term 'vertebrai osteoarthritis' or VOA is employed in the present study, to refer to OA of the zygapophysed joint. The transition from normal to pathological remodelling is a problematic stage of joint degeneration. Ii is difficuît to diagnose since the limits of normal variation are not known, and there is no clear minimal definition of the pathology. In OA of the large appendicular joints, the preliminary stages of pathology tend to be indistinguishable from
normal variation, and the entire process is age-progressive and preceded by long-term
chemicai alterations of articular canilage (Chateauvert et ai. 1989; Octner and Putschar 1985; Petzker. Chateauvert, and Grynpas 1987). Thus, only defiaite expressions of OA shouid be considend in studies of macerated bone. The etiology of OA is widely acknowledged to be multifactoriai in nahue (Hough Jr. and Sokoloff 1989; Moskowitz and Goldberg 1988). and to have a genetic component. A genetic defect responsible for the development of human "primary generalized osteoarthritis" was identified in a fiunily
with a long history of the disease, which was associated with a mild chondrodysplasia (Na-Kokko et ai. 1990). The present research may provide data in support of a genetic component to the etiology of VOA, in the fom of indirect evidence from natal group affiliation. Revious studies have shown that OA of the major peripheral joints in the rhesus macaques of Cayo Santiago is very similar in distribution and clinical course to the human disease (Chateauvert et al. 1989; Prinker, Chateauvert, and Grynpas 1987: Ritzker et al. L989). These studies have demonstrated that aging. and other variables
such as high pacity in fernales. obesity in younger individuais. and level of activity. are important in the development of OA (Chateauvert et al. 1990, Chateauvert et al. 1989;
DeRousseau 1984). These studies also show that there is an initial physical change in the peripheral joints that is impossible to quantify, hence the difficulty in delineating the boundary between pathology and normal variation.
Objectives aOrgmizstion of the Study
The third part of this project involved a detded investigation of naturaily occucring vertebnl osteoarthritis (VOA) in this rhesus monkey colony. This part of the study used
gross morphology and radiography to study the pattern of spontaneous VOA in the macerated spines of a large sample of rhesus male and fernale monkeys. The role of demographic variables (age, sex, parity and natai group affiliation) in the spontaneous
development and expression of VOA was assessed As befoce. the uitimate goal of this project is to establish a natural non-human primate mode1 for vertebral osteo~tis.The results of the thud part of this project are presented in chapter 8. The objectives of this part of the study were as follows:
To descnbe the pattern of VOA in the rhesus monkey spine, by zygapophyseal joint, and to describe colony trends; To describe age-related trends in the development and expression of VOA in both males and females. using the techniques of gross morphological analysis and
radiography ;
To determine whether or not there are differences between males and females with regard to the development, expression and seventy of VOA; To investigate the relationship between par@ and VOA; To determine whether there is a relationship between natal group affiliation and VOA in this colony; To determine the kquency of VOA in this skeletal collection; To study disease interaction; Le., the interaction between VOA and VO/DDD. CHAPTER 2
MATERIALS AND METHODS
2.1. The Sample
The individuds included in this study were those with complete, or virtually complete. spines and complete demopphic information - age at death. sex. parity and natal group affiliation.
--2.1.1. Bone Densitv Studks (DEXA). Osteownia and Osteowrosis
The ~amplein the DEXA study which examined agdsex effects on BMD (chapter 3) consists of the 1st lumbar vertebrn from the water rnacerated spines of 254 free- ranging rhesus monkeys ftom Cayo Santiago. aged 1.0 to 20+ years: 13 1 fernales aged 1- 22.2 years, and 123 males aged 1- 18.5 years. The last lumbar vertebra, typically the seventh, was chosen for DEXA scanning. Due to numerical vaciability in the primate vertebnl column, the sample contains a few sixth and eighth lumbar vertebrae from individuais in which these were the 1st bones in the lumbar spine. Taking the last lumbar vertebra rather than the vertebra of a specifc number ensured consisiency. Also, the choice of the third, fourth or fifth lumbar vertebra would have presented a problem in terms of sample size; a previous investigator conducted destructive sampling on this section of the spine in the same group of animais. Thus. the last Iumbar vertebra was also 30 chosen in order to maximue sample size for DEXA. Of the original sample of 298 specimens, most older individuals, females older than 22.2 yem and males older than 18.5 years, had to be excluded from this part of the DW(A andysis (chapter 3) due to the presence of vertebrai osteophytosis (VO) andor degenerative disc disease (DDD) which resdted in inaccurate readings (see below). These diseases introduce bis into a DEXA sample when the goal of the study is to produce normative data on bone demity andor diagnose osteopenia~osteoporosis. Since only the centrum was analyzed by DEXA, OA of the zygapophyseal joints was not a concem in this study. Of a totai of 298 individuals scanned 43 were excluded fiom the anaiysis because of venebd osteophytosis and degenerative disc disease, and another was eliminated for other reasons (post-mortem damage), leaving a sample size of 254 for the DEXA study on the effects of age and sex on BMD. h order to determine the overall incidence of established osteoporosis in this collection, these 44 individuals were included in r sepamte survey (N = 298) which examined complete spines macroscopically for the presence of vertebrai compression or wedge fnctures. The sample in the DEXA study of par@ and BMD (chapter 4) consists of the last lumbar vertebra from the water macented spines of 119 sexually mature femaie rhesus monkeys from Cayo Santiago, aged 4.0 to 22.2 yems. Of an original sample of 150 lemde spccimens, most older individuais, femdes older than 22.2 years of age, were also excluded from the DMA analysis due to the presence of VO/DDD. Of a totai of 150
females scanned 18 were excluded from the analysis because of VOIDDD, one was eliminated due to post-mortem damage, and 12 immature individuals 1.O to 3.9 years of age were removed, leaving a sample size of 119 females aged 4.0 to 22.2 years for this section of the DEXA study. In order to determine the overall incidence of osteoporosis in these females, and to study the interaction between parity and VO/DDD, the 18 individuds with VO/DDD were included in a separate survey (N = 137) which examined complete spines for the presence of vertebral fractures. and investigated the effect of VOlDDD on parity (chapter 4). At the end of the DEXA studies on the effects of age, sex and parity on BMD,these osteophytic individuds were re-incorporated into the sample in order to study the effect of VO/DDD on bone density, and to examine the relationship between VO/DDD and osteopenia/osteoporosis (chapter 5). The presence of vertebral bones with VOmDD in ihis sample has afforded the opportunity to study the relationship between this disease and osteopenia/osteopomsis. These osteophytic individuals were used to study the so- called inverse relationship between osteoporosis and osteophytosis of the vertebrai column (chapter 5) (Dequeker 1985; Dequeker et ai. 1996; Dequeker, Mokassa, and
Aerssens 1995). The entire sample (N = 298) was divided into theVOlDDD Groupings according to the degree of osteophytosis and remodelling of the cenuum: Group 1 = Normal, Group 2 = Mild VOmDD. and Group 3 = Moderate to Severe VOAIDD. The protocol for assessing VO/DDD is described in section 2.3. VOfDDD group membership was assessed on the lurnbar vertebrae that were used in the DEXA study. Note that, in the fmt two sections of the DEXA study (chûpters 3 and 4), only Group 3 individuals were removed from the DEXA sample before final analyses were conducted. In chapter
5, the entire siunpie was re-constituted and divided into the ahe-mentioned groups to study disease interaction. -2.1.2. VO/DDD, VOA and DISH
For these parts of the study, a total of 228 vertebral columns were examuied initially. from individuals ranging in age From 7 to 29 years. However, only individuals with complete. or virtually complete spines, and with the nomal complement of vertebrae for rhesus monkeys (7 cervicd, 12 thoracic and 7 lumbu) were actualiy used in the study of VODDD, VOA and DISH. The sarnple sizes Vary. depending on the completeness of the data on disease for each spine (see below). However, al1 individuals have cornplete demographic data associated with their skeletons. By age 7 years, most monkeys exhibit complete closure of the vertebrd endplates, which is why only individuals 7+ years of age were examined in these sections of the thesis.
The simple consisis of 204 vertebrai columns. There are 102 males nnging in age from 7.0 to 29.0 yeus, and 102 females ranging in age from 7.0 to 23.0 years. Each spine has 26 amphiarhrodial (AAR) joints. The total number of AAR joints examined is 5,304
(204 spines x 26 AAR joints per spine). Each AAR joint score is based on the raw scores for two articular surfaces, for a total of 10,608 vertebral body articular surfaces examined
(26 AAR joints x 2 artïcular surfiu1es x 204 spines). Divided by sex, there are a total of 2,652 AAR joints and 5,304 articulnr surfaces for males and females separately. VOA
For the study of VOA, the sample consists of 2 18 vertebrai columns. There are 103 mdes ranging in age fiom 7.0 to 29.0 yean, and 115 fernales ranging in age from 7.0 to 23.0 years. Each spine has 26 zygapophysed (ZAP) joint systems. The totai number of ZAP joint systems examined is 5,668 (218 spines x 26 ZAP joint systems per spine). Each surnmary ZAP joint score is bosed on the raw scores of four articular fxets, for a total of 22,672 articular fûcets examined (26 ZAP joint systerns per spine x 4 articular fxets per ZAP joint system x 218 spines). For the femaies, the total number of ZAP joint systems is 2.990 and the totai number of articular facets is 1 L ,960. For the moles. there are 2,678 ZAP joint systems with a total of 10,7 12 articular facets.
The totai number of spines examined for the presence of DISH is 228. There are 120 femaies ranging in age from 7.0 to 24 years, and 108 mdes between the ages of 7.0 and 29.0 years.
Most methods useâ for measu~gbone mass are based upon differentiol absorption of ionizing radiation by bone and soft tissue. DJXA uses an x-ray tube as the soum of photons, and measures the quantity of hydroxyapatite in bone, the results being expressed as gnuns of mineral per unit area scanned (Johnston and Melton DI 1995). During a scan, an X-ray beam passes through the patient and up into the detector in the scan ami. The beam passes through sob tissue easily, but bone attenuates it. As the bem moves across the patient during r scan, it passes through large areas of soft tissue that do wt absorb photons. As tbe beam moves from tissue into bone, the denser bone materiai attenuates the photons, and the nurnber of photons reaching the detector decreases sharply. The detector counts the number of photons that pass through the patient and calculates the density at each sûmple point. The scanner continuously samples the bone minerd content
(BMC)as it moves in a rectilinear pattern over the lumbar spine or other area of interest. As the detector moves across the bone, the program plots the difTerent density patterns of the cortical and uabecular bone, drawing a profile of the densities of the samples taken during each üne of the scan. The result is a series of transverse profiles, which provide the database for quantitative analysis of BMC. The program calculates bone density by subtracting soft tissue beam attenuation from the totd attenuation during anaiysis. The caiculated BMC is calibrated against a set of bone simulating standards of known bone mineral content. The quantities measured by DEXA are: projectional area (in cm2), bone mineral content (BMC,in giams) and bone minerai density (BMD, in @m2); BMD is the BMC normalized for bone size. Thus, BMD (glcm2) = BMC (g) / projectional Ana (cm2), giving a measurement of areal bone density for the selected region of interest. The
DEXA system by Lunar has had a primary caiibration for bone minera1 content and bone mineral density against standards which have been calibrated agaînst ashed bone sections at the University of Wisconsin (DEXA Operator's Manual, Lunar). Instruments iike DEXA emphasue areal density, which is the bone mineral content per unit ana of the projected silhouette of the bone or region of the skeleton king measured (Heaney and Matkovic 1995). DWCA was designed to measure BMC vivo, and BMD was devised as a means of nonnaiizing for bone size vivo, where a volume memurernent is not possible; thus areai density is emphasized. The expression of bone density in this manner provides a degree of standardization for differences in bone size between individuals (see below), and permits cornparison of an individual with a reference population. Knowing the depth of the bone in the path of the scan is not essentiai to the calculation of bone density, as the process of attenuation of the X-ray beam accounts for depth: the thicker the bone, the greater the attenuation of the bem. The main clinical application of DEXA is in the measurernent of spine, hip and total body mineral content. This method allows measurements of body areas, such as the spine and hips, where single photon scanning would be impossible: with DEXA, the entire body rnay be scanned to determine total body minerai content and its distribution. DEXA scanners cm perform scans of the lumbar spine and hip, fore- scans without a water bath, lateral scms of the spine that isolate the vertebral body. and total body scans for bone mass and body composition studies (Fogelman and Ryan 1992; Mazess 1981; Mazess 1983). Because bone minerai content is associated with bone strength and risk of fncture, it has ken considered useful in the evduation of bone disease (Mazess 198 1;
Riggs, Peck, and Bell 1991). With the DEXA method, bone loss may be assessed with high precision, thus permitting a quantitative diagnosis of osteopenia and osteoporosis (Fogelman and Ryan 1992). The bone densitometry hardware used in this study is a Lunar DPX-L Bone Densitometer which emits ionized radiation (X-rays) when energized. The softwam is the Lunar DPX-L X-ray Bone Densitometer SmaU Animal software version l.Oc, 1992 (Lunar Corporation). This software was designed for use on small animais or single bones - ksh or macerated. The DEXA hardware is aiso designed to work with this software, providing the proper attenuation and caiibration for small animal bones. Macerated bones must be encwd in a lucite (plastic) container. In this study, single bones were placed in a lucite box with sides approximately 1.5 cm thick, and scanned with the software's 'appendicular' scan modes. Each vertebn was clamped, by its spinous process. to a special lucite device installed in the box: the vertebn was suspended in a horizontai position in the center of the box, with the cranial aspect of its centrum inferior and the caudal aspect superior. Careful positioning is important in order to prevent excessive shadows around the scan image. which make edge-detection more dificult when drawing the region of interest (Roi) for analysis (see below). The cranio-caudal projection was chosen in order to focus on the trabecular bone of the vertebrai bodies without interference from the posterior elements. Another advantage of this projection is that vertebrai osteophytes cm be detected and excluded from the anaiysis. In clinical settings. the lateral projection of the spine is now favoured for the sarne reasons; a greater percentage of vertebrai tnbecular bone is evaiuated with DEXA by including only the vertebrai bodies without the posterior elements. and any anomalies that rnight bias the
anaiysis cm be detected more easily (Jergas and Genant 1997; Kanis 1994b; Mazess et ai. 1990; Shore and Poznanski 1996).
The bones were scanned individuaily, and ail scans were done with the following settings: high resolution mode, 76 kVp (voltage), 150 pA (current), fine collimation, 0.15 x 0.3 sample size. L/64 sample interval, 60 mm x 60 mm scan widih and length. The
'mode' indicates the speed of the scan; 'high resolution' provides the highest image
resolution for scanning small areas. Subjects receive 1.92 rnrem of radiation, and the
scan of an area 60 mm by 60 mm lasu approximately 15 minutes. The voltage applied to the X-ray tube, measured in kilovolts, is autornaticaily set by the program when a scan
mode is selected. The program ais0 automatically sets the current, measured in microamps, that flows through the X-ray tube to produce X-rays. The X-ray cumnt for dl appendicular scan modes is 150.0 M. 'Collimation' refers to the size of the X-ray beam at the source; the program automaticdly sets the collimation when a scan mode is chosen. The source collimation for dl appendicular scan modes is 'fine' (0.84 mm). 'Sample size' refea to the size of each sample point during the scan. The sample size for the high resolution mode is 0.15 mm x 0.3 mm. which is set automaticdly. 'Sample interval' controls the sarnpling time for each sample point and is measured in seconds. The sûmple interval for dl appendicular modes is 1/64; the program automaticdly sets the sample interval when a scan mode is selected. The 'Scan Width' sets the limit for transverse movement of the detector during the scan; 'Scan Length' sets the lirnit for longitudinal movement of the scan ami. Both are measured in millimeters and set manually in an appendicular scan. Proper procedure requires that a qudity assurance (QA) test be conducted on the system belore beginning each scanning session. The computer program prompts the user to initiate the test. and it does not proceed to scan until there is a successful QA result.
Bone mineral content (BMC) and bone mineral density (BMD)values are highly replicable. DEXA of the spine and hip has a reported precision of L to 2%; it is a precise method of measuring bone mineral content and bone mined density in the lumbar spine
(Hansen et ai. 1990; Johnston. Slernenda, and Melton Ei 1996). Jayo et al. ( 199 1) tested the DEXA method on live female cynomolgus macaques, and found that sequential in vivo DEXA scans of lumbar vertebrae are significantly correlated. These researchers also revealed that in vivo BMC of lumbar vertebrae of macaques is significantly correlated with acnial ash weight. This study showed that in vivo DMA is a precise and effective method of measunng lumbar BMC in macaques (layo et al. 199 1a). In the pnsent study, vimiaily identical results wen obtained in a test of the system whereby the same specimen was scanned five times on different occasions; the coefficient of variation (CV) obtained for BMC was 1.896, and that for BMD was 1.65%. AU subjects were macented lumbar vertebrae: Bone size bas not affected precision (see below), nor has operator technique; al1 scanning and analysis using the DEXA system was conducted by the author, employing the sme procedures and settings as described above. After the scans were completed, they were andyzed using the sarne software. The prograrn produces an image of the scanned bone upon which one may indicate the areas of the bone that are to be analyzed. This is occornplished by applying a 'region of interest' (ROI)on the image. which the prograrn then analyzes for bone minerai content (BMC) and bone minerai density (BMD). For this research. an ROI was drawn around the centrum of each vertebra, following the outline of the endplate. The endplate appears as a bright white margin on the scan image: the placement of the ROI around this margin not oniy ensures consistency but aiso eliminates osteophytes, should they be present, from the scan andysis. in this study, the projectional area is that of the centmm only. imaged in a caudaücraniai plane, which gives direct access to the trabecular bone of the vertebrd body. Figure 2 shows an example of r typicd pnnt-out of a DEXA scan and andysis, showing the vertebrai body with the ROI drawn around the contours of the endplate, and the calculated BMC,projectional area and BMD.
The Iast lumbar vertebn from eafh individual in the totai sample (N = 298) was scanned and analyzed by DEXA. However, as descnbed earlier, bones that exhibited moderate to seven vertebrai osteophytosis or degenemtive disc disease - the presence of large osteophytes and excessive remodelling of the centmm - were excluded (N = 43) hmchapten 3 and 4 of this investigation due to the abnomaiiy high BMC and BMD values confened by these conditions. It appears that remodelling of the centrum caused by vertebral osteophytosis and degenerative dix disease changes the bone qudity and density of the centnun. producing an abnormally elevated BMC and BMD compared to age- and sex- matched controis. These osteophytic individuals were identified macroscopicaiiy by an osteophytosis score of 2+ or higher for the last lumbar vertebra (referring to mildlmodente to severe osteophytosis and remodelling). and by a BMC equal to. or higher thi~,3.3 grams. Other studies have also confied that this disease introduces bias into a DEXA sample (Dequeker et al. 1996; Drinka et ai. 1992:
Hamerman and Stanley 1996; Mnsud et ai. 1993; Onuoll. Oviatt. and Mann 1990; von der Recke et al. 1996). In the fitpu& of the DEXA study (chaptea 3 and 4). vertebrae with very small osteophytes but no remodelling of the centrum were retained in the sample, since the ROI could be placed in a position that excluded these osteophytes from the DEXA analysis. Vertebral osteoarthritis of the zygapophysed joints was not a concem in this study because only the centrum was scanned and analyzed by DEXA.
--Bone Size
As mentioned earlier. a degree of standardization for differences in bone size between individuals is provided by the DEXA system. The system assesses the BMC of an entire bone. or of r panicular region of interest (ROI) that is selected on the image of the scanned bone. in this study, the bone minerai content of the vertebral body was assessed. The ROI was placed on the scan image of the vertebrai body, as described above. The 'area' that is used in the cdculation of BMD is based on the projectional area of the ROI. This projectional area provides some degree of normalization when BMC is divided by this value. The system, howevet, does not give any information on the depth of the bone in the path of the scan. Thus, the result from this procedure is not a "me" bone density value (i.e.. not a volumetric BMD), but an "areai BMD". However, as noted eariier. it is not essential to know the depth of the bone in the scan path, since the program accounts for depth via attenuation of the X-ray beam, as described above. This attenuation, combincd with projectional ma,provides the normalization for bone size. Although nomaiization for bone size is provided by the system, an allometric analysis was conducted on this sarnple, as an exercise, in order to test whether or not centrum size has affected the bone density rneasurements. A geometric correction was appiied to the data. Areai bone density was compared to an estirnated volumetnc BMD, which was calculated by raising area to the exponent of 1.5 (which provides the third dimension to the area measurement), then dividing BMC by this new measurement. The rationale for this type of geomevic correction is as follows: since BMC is measured in gram and area in square centimeten, an increase in centrum size leads to a mon npid increase in volume compared to area by a power of 1.5; in essence, one is dividing a cube by a square. which yields the exponent of 1.5. This geomeuic correction is one of several geometric or mathematicai procedures that have been attempted in previous studies. The main disadvantage of such normalization procedures is that data are forced to fit pndefined relationships; each method has its own inherent assumptions. The geometric method described above is based on the fact that 'ka'is a known value, and if the third dimension is proportionai to the other two, then the following equation applies: Volume = k (ka) where k is the constant of proportionality. and E is the exponent to which 'Areabis raised. if the volume-area relationship is the same for al1 animais. then it does not matter if k is omined from the calculations; al1 the volume calculations wili be off by the sarne factor -of k. However, if k differs for different groups of animals (eg. for males and femaies, or for young and old individuals), then omission of k wil1 mean that the densitv will k off by a different factor for different animais. For this sample, the value of k is unknown, and the exponent of 1.5 is based on geometric relationships without reference to actual bone
morphology. It should aiso be emphasized that this geometric rnethod rnay acniaiiy result in an over-correction of BMD due to the fûct that the vertebrai body is not a perfect cube.
nor a perfect cylinder; it is 'pincheb slightly around the middle. This irregular shape may result in an inflated estimate of volume by this method, and an over-correction when
cdculating BMD. if the cross-sectional mais not the same at al1 points in the bone (Le.,
the volume is not simply 'area x height'), then multiplying by height. or an estimate of height will overestimate volume. Also noteworthy is the fact that the bone density of a
typicd vertebral bone is not uniform throughout its trabecular component. When viewed in frontal cross-section, the vertebra reveds a 3-zond arrangement of tnbecular structure. with a relatively open structure in the center. and a denser arrangement of trabeculae in
the superior and infenor zones. This anatomv also sunnests that an exDonent is not suitable. since it assumes uniformitv. The best rnethod of assessing the volume of a bone
is by water displacement, but dry bones either float or absorb water, rendenng this technique futile. According to Mazess (1983). one of the inventors of DEXA, proper nomdization
must both reduce vuiance and provide discrimination of abnormality. He noted that dividing bone mass by bone size to give an estimate of 'me' density reduces variation, but does not enhance discrimination of abnormality because it aiso reduces the differences
between normal and abnomal subjects. Mazess (1983) concluded that the use of normalization procedures based on bone size does not provide the nonnalization potential of multiple regressions based on bodv size (Mazess 1983). Since body size estimates were not available for the animais in this study, this kind of nomdization using multiple
regcession was not feasible. However, despite the limitations of geomevic corrections, the normalization procedure descnbed above was tested on the DEXA data, and the results compared to the original areal density measurements. The two methods of caiculating BMD yielded similar results, and the overall trends and relationshios between the variables wen the same. The results are presented in the Appendix, in graphical format; the first pph (Fig. AL) shows original. mal BMD plotted against age, and the second shows revised BMD versus age (Fig. A2). The pattern and disuibution of the data are similx in the two graphs, and the fitted curves are also similv in shape. It is noteworthy that, contrary to expectations. the geometnc correction did reduce the variability in the sarnple: in fact. there is more scatter in the data in both young and mature age groups. It is thus possible that k. the constant of proportionality, differs between young and old individuds in this sample. and rnay even differ by sex. Recall that k is not known for this smple. and had to be omitted from the caiculations, thereby making the assumption that the volume-area relationship is the same for al1 individuals; this rnay not be a valid assumption. It is concluded that this assumption must be tested, and that rdsing dl the 'ha'values to the power of 1.5 is too simplistic. Therefore, this exercise was not puaued hrther since this geometric correction procedure requires validation, and this was beyonci the scope of this thesis.
Furthemore. cornparisons of the results drawn from calculating bone minerai density by the two different methods indicate that they lead to essentidiy the same substantive conclusions, and thus, for the purposes of this thesis, there is little or nothing to be gained from the alternative method. It is also noteworthy that the sme individuals (males and females of different ages) appear in an osteopenic outiier group neat the bottom of both graphs (dark circles), in nearly the same positions. Thus. it appears that centrum size has not affected the results in this study, so adjustments for bow size are not necessary, and may in fact be counter-productive, as noted above. This test reveals that the projected areai measurements assessed by DEXA appear to provide an effective normalization for this sample. The fact that the data consist of bone density measurements of the sme sinde bone from each spine may have helped to reduce the effects of bone size. BMD reported as areal bone density is very cornmon in the litenture. Therefore. BMD assessed as BMClprojectional ana was used in this study so that the results may be comparable to other cross-sectionai and longitudinal studies of bone density in humans and other primates. Similar tests in the Iiterature confirm that adjustment for bone size is not necessary for vertebnl bones. Nilas et al ( 1986) reviewed adjustments of vertebrai. radius and total body bone mineral in 161 women. and found that adjustment of lumbar spine BMC for bone size reduced the SD only slightly, from 16 to 13%; adjustment for bone size in the distai radius only decreased the SD from 14 to 12% (Nilas. Gotfredsen, and Christiansen 1986). Another study reviewed different methods of adjusting spinal BMD for bone width and ma. and although the SD was reduced somewhat, the authoa report that such adjustments did not enhance diagnostic sensitivity; the signifcance of the difference between nonniil and osteoporotic women was not greatly increased (Krolner 1982).
Riggs et al. ( 198 l), as reported in Mazess (1983). found that even without normalization, bone density measurements of the lumbar spine discriminated osteoporosis with severd tirnes the sensitivity of compact bone measurements (Mazess 1983). A recent cross- sectional DMA study by Champ et al (1996) of bone density in live female rhesus monkeys from the Wisconsin Regionai Primate Research Center supports the methods
used in this thesis and corroborates the results on bone density presented hecein. Champ
and colleagues used the same DEXA technology that was used in this thesis, and while they used pediatric software For their malyses, the bone density scans for this thesis were analyzed with software designed for small animais and macerated bones. Since body weight measwements were available to Champ et al (L996). they were able to obtain a body-weight and bone-area adjusted BMC for each animal. Their study of the effect of age on bone-area and body-weight adjusted BMC of the lumbar spine (Ll-4) produced similar results to those reported in this thesis, thus confirming that adjustments for bone size are not necessary for the data in ibis thesis. in fact, in sorne ways, the analyses executed in this thesis yielded superior results due to the more direct manner of scanning which targeted trabecular bone. and to the caceful elimination of specimens with vertebnl osteophytosis and degenerative disc disease. Cornparisons between the study by Champ et d (1996) and the results presented herein are discussed in more detil in Chapter 3. Manual Analysis* DEXQ Cal ibration 8 CEilTER hbr Of WîC fiREfi BMD x,y üertica gr crit g/d 1 222, (IL 25 1.717 2.712 B.633
+Data alte4 exclirsion RûI *ta Cor onlq
Fig. 2. An exarnple of a typical print-out of a DEXA scan and analysis, showing the veitebral body with the region of interest (ROI) drawn around the contours of the endplate (bluck arrow), and the calculated bone mineral content (BMC) in grams, area in cm2, and bone minera1 density (BMD) in glcm2. 2.3. Examination of Skeletal Material
The vertebnl column of each individual in the sample was articulated by the author. and examined for the presence of Vertebrai Osteophytosis (VO) / Degenerative Disc Disease (DDD),Vertebrd Osteoarthntis (VOA). Diffuse Idiopathic Skeletal Hyperostosis (DISH), Osteopenia (OPE) and Osteoporosis (OPO) by three methods: gross morphology, radiography and dual energy X-ray absorptiometry (DEXA). The presence of VOIDDD, VOA and DISH was assessed by gross morphology and radiography, and the diagnosis of OPE and OP0 was determined by gros morphology. radiography and
DEXA. Figure 3 is a àiagnm of a rhesus monkey skeleton, showing a laterd view of the spine in typical positiond behaviour; the themain regions of the spine are labelled. A typical rhesus macaque spine hiis 7 cervical. 12 thoracic and 7 lumbar vertebrae. in
macaques. numencal vaiability may occur at the thoraco-lurnbar and lumbar-sacral transitional regions of the spine: the total number of thoracic and lumbar vertebrae mges from 18 to 20, and the average number is 19 (Schultz 1969. p. 75). Gross morphological data on VODDD and VOA was collected separately on amphiarthrodial and zygapophyseal joints respectively, according to the ordinal scaiing systems described in sections 2.3.2 and 2.3.3, which were adapted from Stewart (1979).
DeRousseau ( 1988) and Jurmain (1990) (DeRousseau 1988; Jumain 1990; Stewart
1979). The reader should note that these data were collected pnor to the publication of new ncording standards for data collection of pathology in skeletal collections (Buikstm, Ubelaker, and Afiandüian 1994); however, the methods used in this study are in general agreement with the new ncording standards. A discussion of intmobserver reliability for the VO/DDD and VOA ordinal scaling systems is included in section 2.32 Fig. 3. Rhesus monlcey (Macaca mulatta) skeleton, showing the lateral view of the vertebral column in typical positionai behaviour. The regions of the spine are labclied. Adapted hmCarolina Biologicai Supply Co. Bioreview Sheet 5850. -2.3.1. Ostemenia [OPE) and Osteo~~mis(OP01
DEXA
Bone mineral measurements have been used to diagnose osteopenia and osteoporosis. and to follow their progression. Four generd diagnostic categories have been established for adult women, and the following cnteria are accepted by the National Osteoporosis Foundation of the United States, the European Foundation for Osteoporosis and Bone Disease, and the World Health Organization (W.H.O.)(Kanis 1994b; Wasnich 1996):
1. Normal: a vaiue for BMD or BMC not more than 1 SD (Standard Deviation) below
the average vaiue of young adults;
2. Osteo~enia(Iow bone massl: a value for BMD or BMC more than I SD below the
young adult average. but not more than 2.5 SD below;
3 Osteowrosis: a value for BMD or BMC more than 2.5 SD below the young adult
average value;
4. Severe Osteo~orosis(established osteomrosis): a value for BMD or BMC more than 2.5 SD below the young adult average value. and the presence of one or mon fragility fractures*
These criteria were applied to the DEXA data. According to Genant (1988, 1996). osteopenia, meaning 'poverty of bone', is now acceptable as a non-specific. gross descriptive tem for generalized or regional rarefaction of the skeleton; it is broadly used to describe diminished bone density when the histopathologic nature of the bone loss is uncertain (Genant 1996; Genant, Vogler, and BIock 1988). The term osteoporosis refers to generaiized loss of bone density (osteopenia), accompanied by relatively atraumatic fractures of the spine, wnst, hip or nbs (Genant 1996; Genant, Vogler, and Block 1988). This terminology is employed in this study.
Gross Momholopy
in addition to DEXA, the diagnosis of osteoporosis in the vertebrai column was determined by gross morphology and radiography. Gross morphological analysis involves the detection of vertebd deformities such as vertebral compression fnctures and anterior wedging of vertebral bodies (Mensforth and Latimer 1989: Riggs and Melton üI 1988). Then are many types of vertebrai fncture. in wedge fractures. the postenor height of the centrum is relatively preserved, but there is collapse of its anterior aspect. Compression or cnish fnctures refer to the compression of the entire centrurn, including the posterior aspect. A concave or biconcave deformity of the vertebnl body indicates collapse of the supior or inferior endplates (or both), with relative preservation of posterior and anterior heights of the cenuum. 'Angling of the endplates' is another morphological defomiity (Riggs and Melion iü 1988). Vertebral compression fractures and antenor wedging of the centra were scored as follows: O = Normal; 1 = Anterior Wedging; and 2 = Compression Fracture.
The primary diagnostic tools for osteopenialosteoporosis were DEXA and gmss morphology. Lateral radiographs were used to confi the presence of compression fractures or wedged morphology of the centrum. The radiographie assessrnent of osteoporosis and osteopenia is characterized by a decrease in radiodensity of bone, which is visible as accentuation of the vertebral endplates, and disappearance of trabeculae (Riggs, Peck, and Be11 1991). For the diagnosis of osteoporosis (Riggs and Melton iII 1988), the radiologie cnteria include:
Generalized osteopenia (most prominent in the spine); Thinning and accentuation (opaqueness) of the vertebral endplates, which are composed of cortical bone; Marked thinning and dissolution of transverse (horizontal) trabeculae with relative preservation of the primary (vertical) trabeculae or those aligned with the ais of stress; Striated appearance of the primary trabeculae caused by their reinforcement; Changes in vertebral body shape (wedge-shaped. biconcave, compression); Spontaneous fractures of the spine. wrist. hip or ribs.
A stepwedge penetrometer produced a gray-scale (sensitomeuic strips) on the radiograph, which helped to determine the relative density of bone, and also served as a measure of quality control. Before the adveni of DEXA. the plain raâiograph was the clinicai standard for the diagnosis of osteopomsis.
For spines suspected to have fractures. a grading scde devised by Riggs and Melton (Riggs and Melton Di 1988) was used on radiographs of these specirnens to assess the presence and degree of vertebrai compression or anterior wedging. Relative anterior compression was caicdated as a percentage and graded as follows: Relative Anterior Compression: Postenor Heieht - Antenor Heinht x 100 Postenor Height
Grade O Nomal: Less than 15% diffennce between anterior and posterior heights;
Grade H Minimai Vertebd Compression: 15 - 20% (inclusive) reduction of anterior height relative to posterior height. Not considered a me osteoporotic Fracture;
Grade 1 Mild Compression Fncture: Anterior wedging, greater than 20% and less than, or equd to. 25%;
Grade 2 Moderate Vertebrai Fracture: Furlher mterior wedging (greater than 25%). or postenor or mid-height deformities;
Grade 3 Severe Vertebrai Fracture: Marked deformity with loss of volume or projected area of greater than 40% relative to adjacent unfmctured vertebrae.
-2.3.2. Vertebral Osteo~hvtosis(Vol! Deponerative Dise Disease (DDD)
VOfDDD of the amphiarthrodial joints (vertebral centra) wûs scored according to the following ordinal scaling system. The cranial and caudal rims and surfaces of erh vertebn in the spine were assigned a score besed on the presence and degree of osteophytosis and bone remodebg: Normal. Body rim is smooth.
Body rim is sharp or somewhat irregular, but essentiallv normal. This morphology is the precunor to later traction spur (osteophyte) development. There may be a continuous "wnnkling" of bone adjacent to the disco-vertebral margin.
There is obvious, but slight marginal bone deposition. more developed than W. with tiny traction spurs beginning to develop. These spurs ;ire discontinuous; Le.. there is no connection between them. This is the first stage of actual ~atholonv.
As above. but the traction spun are somewhat larger, and they are continuous around the margin; Le.. bony connections exist between them. These marginal osteophytes originate From an area about 1 to 2 mm away from the disco-vertebnl margin.
Definite osteophyte development; conditions as above, but more developed. The traction spurs are larger, and continuous around the rim of the centrum (ring of osteophytes). The spurs are oriented in the donal-venual plane.
As above; traction spurs are continuous around the margin and oriented in the dorsal-ventral plane. Development of osteophytes is still moderate, but mater than code 2. ûccasionaily. small ~itsoccur at the base of the traction spur, or in the adjacent pan of the centrum.
Same as above (2+), but with the start of subwriosteal bone dewsitioq on the vertebrai body's venuo-lateral aspect, with or without associated pitting on its ar Pronounced osteo~hvtosis; Iarger osteophytes. continuous around the body rim. Larger traction spurs (curved soicules) are just beginning to curw over the intervertebral disc space, usuaiîy extending beyond the continuous ring of osteophytes. At this stage. they are just beginning to assume the claw-type morphology of the next stage of degeneration, but they are stiii traction spurs. There is further develo~mentof submriosteai ossification (new bone formation) on the vertebrai body's ventro-lateral aspect. The larger spurs in this category are continuous with this subperiosteal bone deposition. Centnun sizelshaw chanee is mild or modente in this category, accomplished by subperiosteal bone deposition. 3+ As above, but with the adaition of some degree of remodelline of these curved osteo~hvtesor remodelling of the recentiy deposited ventro-lated portion of the joint surface (the extended new articular surface created by subperiosteal ossification on the vertebrai body's ventro-laterai aspect). The body's original yticular surface exhibits no change. Centmm sizelshape change is still mild or moderate. 4- As above, but more severe development, with a severe change in centrum size or sha-K. At this stage, the centrum develops a "splayed-out" morphology, with some pitting of its original articula surface. Occasionally, the large spun from the cranial and caudal rims of the same vertebrd body approximate one another. creating considerable apposition on the vend surface, giving the centrum a 'pinched' appearance. 4 Severe osteo~hvtosis, with comr>letelv remodelled joint surface, and severe subperiosted ossification on the corticai bone of the ventro-lateral aspect of the centnun. Claw-like osteophytes are noticeably curved in a cmnial or caudal direction, and thus project over the intervertebrai disc space. When spua take on a definite orientation. they tend to be positioned ventro-laterally, adjacent to the body rim. As above, there is a severe change in the sizelshape of the cenmrn. 4+ As above, plus ebumation on the articular surface of the vertebrai body, or on osteophytic outgrowths. Although rmly seen in amphi;Lfthtodialjoints, eburnation may occur afier the intervertebral disc has degenerated completely, allowing bone- on-bone contact between the artïcular surfaces of two contiguous vertebrai bodies, or their osteophytes. 5 This cateogory is distinguished by the presence of huee claw osteo~hvtesthat grow hmthe coatiguous body rims of two vertebrae, corne into close approximation but do not fuse tonether (Le., there is no fusion of osteophytes or of ;irticular body surfaces). Or, there may be one very large osteophyte curving over intervertebnl space to meet a much srnalier one on the next vertebra. There is remodellin~of the orininal ioint surface of the centrum, but no fusion. 5+ Ankylosis (segmental immobility); amphiarthrodial joint fusion. This ordinal scding system yields detailed information on the pattern of degenerative change in the vertebral body. However, the stages of degeneration may be summarized in order to highlight the main &atm observed at each successive stage. Osteophytosis is present at al1 stages except 'O', and the degree of osteophytosis increases from stage 1 to 5; the following list highlights the additional features that are present at each successive stage: Surnmary of menerative Changes (V0) O Normal; 1 initial evidence of pathology; 2 Osteophytes present, but small. There is no red involvement of the vertebral body; - 3 De finite body involvement (subperiosteal ossification on the vertebral body's ventro- lateral aspect), but no involvement of the original articuiar surface; 4 Original acticular surface involvement; 5 Severe changes ending in ankylosis. The more detaüed version of this ordinal scaie was used. Figure 4 illustrates the joint numbering system for amphiarthrodial (AAR) joints used in this study; the illustration is adapted fiom Schultz (1961). Each AAR joint is comprised of the caudal aspect of the centru.cn of one vertebra and the craaial aspect of the centmm of the next vertebca, and the joints are numbered from 1 to 26. Figw 5 illustrates a few examples of the scoring system outlined above. The additional codes utilized in this study are described below. Other Codes: 5- This category is reserved for the presence of hune claw osteo~hvteschat grow from the contiguous body rims of two vertebrae (as in code #5 above), exceDt that there is no remodelling of the original joint surface. This code was used to distinguish the enthesophytes of DISH from the osteophytes of VO/DDD when they occurred in the same vertebral column. It also helped to detect the pnsence of incipient or probable DISH (see below), and aided in the study of disease interaction. X Not able to observe due to postmortem damage. / Missing vertebra, or not applicable. Intra-observer Reliability A recent study illustrated the importance of inter-observer variation in coding VOA and osteoanhntis of the priphenl joints (Waldron and Rogers 199 1). Certain mesures were taken to ensure inin-observer reliability for the ordinal scaling systems used in this study. The data on VO/DDD and VOA were collected by the author at the CPRC museum in Puerto Rico over a pcriod of five months. in order to ensure consistency during data collection, photographs representing each stage of degeneration were used in conjunction with the descriptions of each code in the ordinal scaling systems. Also, at the start of each major session of data collection, a few spines that were examined at an earlier tirne were reviewed, dong with the photographs and descriptions of the codes. In a test of the VOlDDD scaiing system, tea complete vertebral columns were xored thee separate times during the course of data collection. Eighty-sevea percent of the vertebral symphyses were scored within the same code level during these trials. Although more replications of these tests would have been desuable, they were nonetheless not possible during fieldwork. However. the tests that were conducted indicate a relatively high degree of intra-observer precision. Also, the results obtained from these data (chapters 6 and 8) attest to the intra-observer reliability. and efficacy of the ordinal scaiing systems. AAR JOINT NO. Fig. 4. Axial skeleton of the macaque showing the ventral aspect of the vertebral column. The amphiarthtodial (AAR) joints are indicated, dong with the joint numbering system used in this study; C = cervical, T = thoracic, L = Iumôar, and S = sacnun. Each AAR joint is comprised of the caudal aspect of the centnun of one vertebra and the cranial aspect of the centrum of the next vertebra, and numbered hm1 to 26. Figure is adapted fiom AB. SchulQ 1961, pl Fig. 5. Phobgraphs of exampies fram VOIDDO scaîe, shawing Grades O. 3+, &,4+ and 5. The pidum of 4+ depids ebumation of aie articulai, surhce of a lumber verteka; abugh raie, this condition was kund in a fW spedmens Vertebrai OsteoaRhntis (VOA) of the zygapophyseal joints was scored in al1 four yticular facets of each vertebn in the spine. The articular face& were identified as follows: LEFr SIDE WGHT SIDE Cranial Articular Facets A B Caudal Articular Facets C D OA of the zygapophyseal joints was scored according to the presence and degree of marginal lipping and surface change in each articular facet. Each anicular facei was scored according to the following ordinal scaling system, which was designed to record as much detnil as possible about joint degeneration: 0 Normal; smooth margin and anicular surface. O+ Facet has a shm rnarain and may have a 'sheared-off shape. There is no lipping; Le.. there is no deposition on the donal laminae adjacent to the sbeared facet's margin. The sheared facet may have a jagged edge to it. and its margin is sometimes serrated. Very shqspicules of bone may be present on the facet rim. This morphology marks a precursor stage to later deveiopment of OA. There is no anicular surface change or marginal deposition of bone. This is the stage of 1 This is the fust.There is slipht but obvious li ~inqof the facet rim. If the articular facet is on the cranial aspect of the vectebra (cranial zygapophyxal joint). the= may be slight deposition onto the caudal edges of the f'et, ont0 the laminae. If the facet is on the caudal aspect (caudal zygapophyseal joint). and it is sheand. there may be slight deposition ont0 the laminae donai to the facet. giving it a "Lipped-back appearance. The joint articular surface is not affected. 2 As above, but more develomd (but stili moderate). The joint articular surface is not affected. This stage is an exaggeration of stage 1. 3 Funher development of conditions described in stage 1, with pronounced liming and ~ittinq,of up tu onequarter or less, of the articular surface. This is the beginning of acticulûr surface change. 3+ As above. but with pronounced enlargement of the ioint surface ma, caused by excessive lipping of joint margins. 4 Vew severe Iiopine with ioint enlargement; the joint surfxe is pitted on >LI4 of the articular surface. The lumbar facet joints. which are concave/convex, tend to have curled-in margins by this stage. These facets have changed shape from circular, to long and nurow, with enlarged surface area in the cranial-caudal plane. The convex facets are severely lipped-back. 4+ As above (code 4). with ebumation. 5- Very severe lipping with joint enlargement. The concavefconvex fwets of the lumbar vertebrae have verv curled-in mareins resulting in the lockine-toeether of the facet joints of two contiguous vertebrae to the extent that they cmnot be disarticulated. This is partial segmental immobility; there is restriction of the range of motion at this vertebral level. 5 Ankyiosis (segmental immobility); joint hision. Other Codes: 4* This code was reserved for instances where the joint was completely remodelled, resulting in a reduction in size and a change in shape (peg-like appearance). This morphology was rare. Eburnation was often present in these remodelled facets, in w hich case the facet was scored 4+. Figure 6 shows a schematic representation of the zygapophysed (ZAP) joints in a typical macaque spine, as viewed from the dorsal aspect. The articula- facets of each vertebra are labelled from A to D; each intervertebrai joint is composed of the caudal &cular facets (C and D) of one vertebm and the cranid articular facets (A and B) of the next, as shown in the drawing. Facet C articulates with A. and lacet D articulates with B, forming two separate intervertebrai joints (CIA and Dm)which are summarized as one unit in this study (see befow), and numbered from 1 to 26. Figure 7 illustrates a few examples of the VOA scoring system. Lateral radiographs were also used to diagnose VOA and VO. They revealed areas of sclerosis (Le., increased density) in both joint systems of the spine if present, and aided in locating the point of origin of osteophytes in the cenuum, that is, the initiai site of ossification or calcifïcation. Lateral radiographs were thus used to differentiate the osteophytes of VO from the enthesophytes of DISH, which was especidly useN in individuals with multiple spinal diseases. JOll JO. Fig. 6. Schcmatic nprrsentation of the zygapophyscal (ZAP) joints in a typicai macaque spine, as vicwed hmthe dorsal as* The rectangles rcprcsent individuai vcrtebrac, hm Cl to SI, as indicatcd on the Ieft of the figure; C = cervical, T = thomCc,L = lumbar, and S = sacnini. The aiticular fa- ofeach vcrtebra an labelleci A, B, C, D as shown. Each intervertcbraljoint is comp 1. Presence of continuous calcification and ossification along the antecolaterd aspect of at least four contiguous vectebnl bodies; 2. Essentiaily normal disc space height, as inferred from spaces between vertebrae connected by ossified W; 3. In radiographs. a ndiolucent line between the deposited bone and the anterior vertebrai surface; 4. Absence of zygapophyseal joint ankylosis; 5. Absence of sacroiliac (SI) joint fùsion (intra-anicular joint fusion) and absence of SI joint erosions; 6. Proliferative enthesopathy and new bone formation. A definite diagnosis is made when al1 these criteria are met. The existence of DISH is terrned probable when criteria 2 to 6 are met, and there is continuous calcification and ossification along the anterolaterai aspect of at least two contiguous vertebd bodies (Doyle and Littlejohn 1986; Resnick 1988; Resnick et ai. 1978; Resnick. Shaul. and Robins 1975; Utsinger 1984). Extra-spinal manifestations of DISH are distinctive, and involve hyperostosis (bone spurs) at the attachment sites of tendons, ligaments and capsules; sites include the pelvis (para-articuiar osteophytes dong the inferior aspect of the SI joint), the pateiia, dista1 tiiia and fibula, the foot (talar and calcaneal spurs), and elbow (olecranon spurs) (Resnick. Shaul, and Robins 1975). An example of DISH from this sample of monkeys from Cayo Santiago is shown in Figure 8. The enthesophytes of DISH were scored according to the following ordinal scale: O Normal 1 Present: small enthesophyte 2 Present; large enthesophyte, bridging at lest one-half of the intervertebral space 3 Present, vertebra connected to its neighbour by ossified ALL (intervertebrai-space bridging complete) Fig. 8. Photograph of a typical example of DlSH, lateral aspect of part of aie lumbar spine. Note cantinuous ossification of the anbrior bngitudinal ligament. 2.4. Data Analysis This study employed a variety of statistical and graphical techniques to anaiyze and present the different kinds of data that were generated. Broken-line ngression and smoothing spiines (nonlinear regression), scatter plots. box-plots and histograms were used in various parts of the study. as were Generaiized Additive Models (GAMs) and statistics such as t-tests, ANOVA. Mann-Whitney Rank Sum Test. and the generalized F- test. For such tests, alpha was set at 0.05. Two-tailed statistical tests were employed throughout this thesis. Al1 statistical testing is based on two-tailed designs given the lack of consensus in the literature with regard to some aspects of the diseases/disordea under investigation. Conditioning plots (CO-plots)and Local Estimation Scatter Plot Smoothing (LOESS) were aiso used in one part of the study. as described below. Principal Components Analysis (PCA). scatter plots and line pphs were used to andyze and present the data on VOIDDD and VOA. The following sections describe these statistical and gnphical methods in more detail. --2.4.1. Bone Mineral Densitv, Ostmwnia and Osteomrasis Regression is commoniy used for two different purposes: 1) to estimate an underlying trend. and 2) to develop a mode1 for prediction (Olson 1988). The main empbasis in this study was to estimate undedying trends. The andyses of the DEXA data followed this logic: 1. Fitting a type of regression line for the mean of the data was the main goal. A general nonlinear regression curve was fitted - a smoothing spline (S.S.). Broken-line regression (b.1.) was dso fitted, making the restrictive assumptions (compared to the S.S.) that there is a penod of growth followed by a plateau. The S.S. and b.1. were compared. and it was found that the b.1. is no worse than the S.S., and is sometuaes better. Linear regression was not considered through dl the data, as it did not make sense biologically, and was not suggested by the data (see below). Furthemore, any test of a iinear regression against S.S. or b.1. showed the linear regression to be lar inferior. Most scatter plots in Part I of this study exhibited a curvilinear relationship between the dependent and independent variables. To iavestigate these relationships, both smoothing splines and broken-line regression were used; regression lines for the mean of the data were fitted. Nonlinear regression is used for mode1 building when the relationship between the dependent and independent variables is curvilinear. so that the straight-line description is inadequate. Often. linear regession is an inadequate model when applied to biological data because it implies that there are no "floor" or "ceiling" constraints; i.e., that values increase and decrease without bound. Thus. Linear regression was aot applicable to the data in this study. Broken-line regression and smoothing splines were used for different purposes. Broken-üne regression was usehil For the detemination of the age of peak bone mas in males and fernales. This particular model was applied because of the expected increase in BMC and BMD with maturation. and anticipated stabilization or decrease of these variables with advancing age. The smoothing spline was used to determine if, and at what age, males and $males experience a decline in bone mineral density. Although the broken-line regression miiy show a descending slope, it does not reveal the -ge iit which BMD bains to decline. The smoothing spline, however, is able to show both a plateau fouowed by a negative slope, if one exists. and the approximate age at which a decline is observed. Broken-line regression is only able to show either a plateau a descending slope. Therefore, both tbe smoothing spline and broken-line regression were employed, with both often show on the same pphs. The Smoothing S~lineand Broken-line Remession A 'smoother' is a tool for surnmiuizing the trend of a response variable as a function of one or more predictor variables. It is a tool for nonpmetric regression; it does not assume a ngid form for the dependence of y on x. The estimate produced by a smoother is called a 'smooth'. The single predictor case is the most cornmon, and is called 'scatter plot smoothing' (Hastie and Tibshirani 1990). For an independent variable Xi and a dependent variable Yi, a smoothing spline is a curve f(x) that minimizes the residuai sum of squares E (€(xi) subject to f(x) being smooth. One advantage of smoothing splines is that there is no need to mûke assumptions about the exact functiond form (e.g., quaciratic, logarithmic) of the relationship between x and y. A second advantage is that fitting of f(x) is local, so that vaiues of f(x) in the region of depend mainly on values of y near to xg. The fitted values f(x) act as a kind of locaî average. This local fitting means that the shape of the curve at one end of the plot is not affected by the number or scatter of points at the other end. Statistical uncertainty about the shape of the smoothing spline fit was show by plotting a 95% confidence band around the curve. This confidence band indicates that there is a 95% certainty that the true function lies in this region (Hastie and Tibshirani 1990). Smoothers have two main uses. The first use is as a descriptive tool, as outlined above; a scatter plot smoother illustrates and summackes the trend in a plot. A smwther mq also be used to estimate the dependence of the rnean of the response variable (y) on the padictors, thus serving as a building block for the estimation of additive models, as described in the next section (Hastie and Tibshirani 1990). A regression model intemediate between the smoothing spline and simple linear regression is broken-line regression. another type of non-linear regression. In this case, two separate linear regressions are forced to meet, forming a "hockey-stick-shaped curve. The x-value where they meet is chosen to rninimize the overail residual sum of squares, and is known as the break-point of the broken-line regression (Hastie and Tibshirani 1990). For a regression of BMD on age, the slope OF the initial, increasing line segment was used to estimate the gain of bone density per year for both females and males (chapter 3). The same procedure was used to estimate the gain of bone minerai content per year in both sexes. The break-point of the broken-line regression (the age at which a slope change occurs) was used to assess the age at which males and fernales main peak BMD and BMC (chapter 3). Generalized Additive Mode1 Statistical tests of hypotheses regarding the shape of curves and differences between males and femaies were made in the context of the generalized additive model (GAM) (Hastie and Tibshirani 1990). The model is nonpanmetric in the sense that a parametric form is not imposed on the hnctions; instead, the hnctions are estimated in an itentive manner using scatter plot smoothers. Generaiized additive models are additive in the predictor effects. The tenn 'additive' means that the model is a sum of tem. The predictor effects can be exarnined separately, without interactions, which is usehl when one wisbes to examine the relationship between two variables while controlling for the effects of a third variable. The estimated mode1 consists of a hinction for each of the covariates, making it usefbi as a predictive model, and for discovenng the appropriate shape of each of the covatiate effects (Hastie and Tibshirani 1990). Statistical tests of hypotheses regarding the shape of curves (linear. broken-line. smoothing spline) and diffcrences between males and femdes were made in the context of the GAM. For example. to test the hypothesis of non-linearity for variables x and y. the kst smoothing spline fit was compared to the best iinear fit through a generalized F- test. If there was a significant reduction in the residuai sum of squares from the use of the smoothing spline, then the smoothing spline was considered the better fit. in other words, the generalized F-test was used to test the improvement in fit of the spline over a straight line. Smoothing splines were compared to linear models where appropriate, Le., to test the hypothesis that r trend was linear rather than non-linear. As mentioned above, r stnight line is not always applicable to biologicai data. In the context of the generalized additive model. the hierarchy of models tried were as follows: 1) non-linear term; 2) linear terni; 3) absence of term. Each model was fit tested against its "parent" in the hienrchy in order to decide between a linear and non- linear terrn for each variable. and to decide between a linear terrn and the absence of a term for each variable. For example. in chapter 4. with BMD as the response variable. non-linear versus linear models of p;uity and age were tested. using the hierarchy of models üsted above. Through GAMs. the relationship between BMD and parity was examined, while controlling for the effects of age. These procedures were used in chapten 3 to 5 inclusive. Conditioning Plots (Co-plots) and Local Estimation Scatter Plot Smoothing (LOESS) Conditioning plots (CO-plots) and Local Estimation Scatter Plot Smoothing (LOESS)were dso used to investigate the relationships between age, parity and BMD (chapter 4). The CO-plotis a graphicd presentation of a the-dimensionai relationship. It is a series of plots of one variable versus another, with the data in each plot having similar values of a third variable (Chambers et al. 1983; Cleveland 1994). For example, in a co- plot of BMD by parity, conditioning on age, each BMD veaus parity plot contains individuals with similar values of age. Adjoining plots share a number of individuals, usually 50%; this is a default configuration of the pmgram that ensures continuity between plots and comparable sample sizes. The CO-plotis a graphical presentation of a ihree-dimensional relationship among the variables BMD, age, and parity, as follows. One should imagine the dependence of BMD on age and parity in three dimensions as a three-dimensional surface of average BMD over a grid of points of age and parity. Then. the CO-plotthat conditions on age takes slices through this surface for a number of ages. In this manner, one is able to investigate the relationship between BMD and pYity while controliing for the effects of age. if the shape of the BMD-parity curve is the same for al1 ages, then one knows the shape of the BMD-pacity curve. if it is not the same, then the effect of BMD on parity depends on age in some way (Cleveland 1994). The curves dnwn ihrough the scatter plots are fitted by LOESS, a type of scatter plot smoother associated with co-plots. At each point xi, a subset of the data near q was chosen and a line was ftted to this subset using a quaciratic equation; the value of each Yi was thus predicted, and the resulting curve was the LOESS fit (Chambers et al. 1983). -Other Statistical Procedures Gra~hicalMethods In chapter 3, a probabiiity density estimate was used to Uustrate the underlying distribution of the bone density data. Based on the histogram, this technique gives a representation of a continuous pmbability distribution in which probabüity is portrayed by areas under the density curve (Silvennan 1984). Other statistical procedures used in chapter 4 are as follows. A histogram was used to plot the frequency of different parities in this sample. A two-sample t-test was used to compare the mean BMD, BMC and area of nulliparous females to those of parous femaies of the same age. T-tests and the Mm- Whitney Rank Sum Test were also used in a senes of tests on low-parity fernales in this sample. T-tests were also used to compare the means of the osteopenic/osteoporotic groups. Box plots were used to show the relationship between parity and vertebral osteophytosis / degenerative disc disease (VO/DDD). Box-plots were also used extensively in chapter 5 to study the relationship between BMD and VO/DDD in mdes and females. In order to test the relationship between BMD and natal group affiliation (chapter 3). one-way andysis of variance (ANOVA) was used to compare average BMD in natal groups represented in the simple. Statistical data analysis was carrieci out using the S-Plus statistical software package (S-Plus for Windows. v. 3.1). Smmthing splines and broken-line regession curves were fitted using S-Plus. Some statisticai procedures were conducted with the SigrnaStat Statistical Program for Windows (v. 1.0. Jandel). Other computer programs such as Quattro Pro and Paradox for Windows (v. 5.0, Borland) were used to manage the databases. -2.4.2. VOmDD and VOA Techniques Used O Summarize the Raw Data As mentioned earlier, this study of VOlDDD and VOA Uicludes only spines with complete data, and with the normal complement of cervical, thorncic and lumbar venebrae for rhesus monkeys, i.e.. 7 cervical, 12 thoracic and 7 lumbar vertebrae. Those individuals with numerical varïability of the spine (N = 24) were not used. as the presence of additional bones or fewer bones than expected tends to complicate the computer analysis of the database. For example, some software prognms do not tolerate blank spaces in a database. Spines that hd one or two missing bones were retained in the database, and the missing articular surfaces were assigned an estimated score according to the following procedure. if the vertebrae contiguous to the missing bone had identical scores, then that score was also assigned to the missing articular surface; if the scores of the contiguous bones differed, then the lower score was assigned to the missing bone. For each spine, the raw scores of each vertebnl svm~hvsiswere summarized. to provide a single score for each intervertebnl (amphiarthrodial) joint. An intervertebral (amphiuthrodial) joint is composed of the caudd articular surface of the body of one vertebra and the cranid articuiar surface of the next one; a summary score for each joint was determined by the raw scores of VO/DDD aven CO these articular surfaces (see Figure 4). if these scores differed, the higher score was chosen to represent the surnrnary score of VOmDD for the joint, on the premise bat the higher score more adequately represents the stage of degenemtion (or joint film) exhibited by the joint. This proceduce of assessing spinal VOmDD by focusing on intervertebral joints rather than individual vertebnie is uncornmon in the Literature, and provides a more hnctional approach to quantifying spinal degeneration. Most previous studies have scored the articular surfaces of individual vertebrae, and often averaged the articular surfaces within a vertebra rather than between vertebrae, thus failing to consider the true hinctionai components of the spine - the intervertebral joint systems. Pairs of zygapophyseal (ZN)joints, connecting two vertebrae, form the other Eûnctional unit of interest in this study. Each intervertebral joint is compnsed of the caudal articulas facets of one vertebra and the cranid facets of the next, forming two intervertebrd joints. The raw scores of each pair of articulating facets were summarized in the same rnanner as described above for the centra; the higher score was chosen to represent the stage of VOA for each pair of artïcular facets. Thex two intervertebral joints were sumrnarized as one unit in ihis study (ZAP joint system), and numbered from 1 to 26; they were summvized by taking the higher score to represent the entire joint systern (see Figure 6). Thus, for both VOA and VOfDDD, the maximum expression was used to represent the level of pathology present in the joint. and hence. the degree of joint impairment. At the end of this process. each spine had a single score for VO/DDD for each of the 26 mphiarthrodial (AAR) joints. and a single score representing VOA of the zygapophyseal (ZAP) joints at each intervertebral level (ZAP 1 to 26). Since these summary scores were based on the ordinal scaling systems outlined eulier, the next step was to convert these scons to a numecical format that cm be read by statisticd software; Le., it was necessary to remove the "plus" and "minus" signs and other symbols. Both ordinal scaiing systerns were converted to a numencal system that increases by increments of one-thirci to produce a numerical scale that runs from O (Normal) to 5.33 (Ankylosis). The converted VO/DDD and VOA scores for each spine were then added up separately and averaged, to produce an index of VO/DDD and VOA for each individual. Thus, each animai was given an average score for VO/DDD and an average vaiue for VOA - two numbers which served as an index of spinal disease in each individual. The average scons for VO/DDD obtained for each individuai were plotted as a scatter plot of VO/DDD versus age for the entire sample, and for males and kmales separately. Mean VOA for the whole sample and for males and females separately were also plotted in the same muer. Smwthing splines were fitted to these scatter plots. In this part of the research project, the smoothing spline was used to summarize trends, and thus, there was no need to test the curve against a stmight line. The data for VOmDD and VOA are aiso shown as line plots or line pphs that provide a visud representation of the observed trends. The demographic variables of age, sex, pyity and natd group affiliation were tested for association with VO/DDD and VOA using the same methods described for the fitpart of this study. For both VOA and VO/DDD, a multivariate technique cdled Principal Components Analysis (PCA) was used to provide an overview of the vends observed in this sample. PCA is a data-summarizing, variance-maximizing technique that reduces the complexity of the original set of data, and simplifies the description of a set of inter-relatecl variables. Given a number of subjects, each with a number of measurements, PCA produces another set of observations which in a sense summarize the fmt set (Afifi and Clark 1990; Andemon 1990; DiUon and Goldstein 1984). PCA serves as a sumarizhg device by which muitidhensional data may be viewed in a relatively few dimensions tbat stili retain most of the originaî information. This reduction in dimensions is achieved by the elimination of comlatiow among the onginai variables - they are treated equally, i.e., they are not divided into dependent and independent variables. Reduction is achieved by a linear ~sfonnationof the original data set. which is equivalent to a simple rigid rotation of the system of axes defining the original variables. in this way, PCA transforrns the original variables into new, uncorrelated ones; the new variables are cdled the principal components (PC). Each PC is a linear combination of the original variables. The variance of each PC is a measure of the amount of information conveyed by that PC. The rotation of the axes is perfonned so that the Fust principal component variate contains the greaiest portion of the original variation, and is thus the most informative PC. Succeeding PC variates each contain less of the original variation. The PCs are arranged in order of decreasing variance. Thus, the fint principal component variate (PC i) contains most of the variation in the original sample, and subsequent principal component variates (PC U, m. IV. etc) account for successively less of the original variation. The last PC, with O variance, is a variable that does not distinguish between the members of the population. It is customary to analyze only the fmt few principal components since they will account for most of the variation in the population. PCA is also useful for testing a sample for normaiity; if the principal components are not nonnally distributed then neither are the original variables. Outliers may also be identified by PCA. Thus, PCA is very usehl as an exploratory technique (Afifi and Clark 1990; Dillon and Goldstein 1984). PCA was applied to the data in this snidy as follows. The observations for VOA and VODDD on a single spine (a single subject) were onginally written as xl, x2, x3. ... x26. The new variable consmicted by PCA is given by the equation vl = Z Wi xi, subject to the constmint that Z Wi = 1. The tint principal component results from finâing the set of Wi that gives the maximum variance for vl. In othet words, the program calculates how much weight (wi) to give to each of the measurernents in order to maximize the between-subject variance of the weighted sum of the rneasurements (Anderson 1990). The weights, shown on the Y-axis, thus indicate the location of the inter-subject variation. For the VO/DDD and VOA data, by plotting the weights against the location number on the spine, one cm see the locations where the between-animal variation will be found. The weighted sum 'v' itself can be used as a surnmary of the entire series of measurernents dong the spine. PCA indicates how much of the total between-animal variation in al1 the spinal memuremenu is captured by this single weighted sum (Anderson 1990). As before, statisticd data analysis was conducted using the S-Plus statistical software package (S-Plus for Windows, v. 3.1). and some statisticd procedures were cmied out using the SigmaStat Statisticd Prognm for Windows (v. 1.0. Jandel). Quattro Pro and Paradox for Windows (v. 5.0. Borland) were used to manage the databases. PART 1. BONE MINERAL DENSITY OF TBE SPINE CHAPTER 3 THE EFFECTS OF AGE AND SEX ON BONE MINERAL DENSITY OF THE SPINE 3.1. Resulîs: Age, Sex and Bone Minera1 Content I Bone Mineral Density Ake. Sex and Bone Mineral Densitv Table 1 provides a sumrnnry of the descriptive statistics on the sample (N = 254) by age group, in males and femdes separately. The age groups were defined according to Tumquist and Kessler (1989): sub-adults (1.0 to 5.9 yean), young adults (6.0 to 9.9 years). middle-age or prime adulthood (10.0 to 14.9 years). advanced adulthood (15.0 to 19.9 years) and aged (20+ years) (Tumquist and Kessler 1989). Looking at the entire smple. a smoothing spline fit reveais an increase in bone mineral density (BMD) with age well into adulthood, and a steady decline with increasing age after 17 years (Fig. 9). The 95% confidence band is fairly nmwacross most of the age range, but it widens across the older ages (A6yrs) due to the fact that there are relatively fewer older animals in the sample. There is iittle variation in BMD iuound the regression üne (i.e.. amund the average) among the young, age L to 7 years, and greater vuiability in BMD at every age among older animais. The main purpose of Figure 9 is to depict the data for the entire sarnple. It is best to examine males and fernales separately (set below). TABLE 1 DESCRlPTlW STATISTICS OF THE SAMPLE (N = 225) AREA BMD (cm2) (slem2) SUB-ADULT AGE GROUP (1.0 to 5.9 p.) Sam~leSize: Females = 20, Males Mean Females Males Standard Deviation Femaies Males Maximum Females Males Minimum Females Males YOUNG ADüLT AGE GROWP (6.0 to 9.9 p.) Sample Size: Females = 43, Males Mean Femaies Males Standard Deviation Females Males Maximum Females Males Minimum Females Males MIDDLE - AGE (PlUME ADULTHOOD, 10.0 to 14.9 yrs.) Sam~leSize: Females = 43, Males Mean Females Mdes Standard Deviation Females Males Maximum Females Males Minimum Femdes Males TABLE 1 COIUTINUED BMC AREA atDeath (grams) (cd) ADVANCEI Sample Size: Females = 2 1, Males Mean Females Males Standard Deviakion Femdes Males Maximum Females Males Minimum Fernales Males AGED (20 + yrs.) Sample Size: Females = 4 Mean Females Males Standard Devia tion Females Males Mnrimum Femaies Males Minimum Females Mdes Additive models testing agelsex interactions with BMD found that the shape of the curve relating age and BMD is the sme for both sexes, but the males have a consistently higher BMD for a given age (p < 0.001). Comparing smoothing splines to straight lines using F-tests revealed that the trends were not linear across di ages; there was a levelling off at maturity. Broken-üne regression was applied to the data on males and was found to give just as good a fit as the smoothing spline (p = 0.912). For the females. there was a statistically signifiant improvernent over broken-line regression when the smoothing spline was used (p = 0.035). The smoothing spline is a more Iikely representation of chimges as they occur, Le.. continuously with age. Both models are presented for comparison in some of the figures. Broken-line ngression was used to estimate the gain of bone mas per year, and the age of peak BMC and BMD in males and femaies. The smoothing spline provides a running average across the life span. Two different patterns of BMD are observed when males and femdes are examined separately. Figure 10 shows the relationship of BMD and age in females, comparing a smoothing spline and bmken-line regression; the dotted line npresents the latter. In femaies, the results from the broken-line regression of BMD by age indicate that there is an initial increase in bone density of 0.04 &rn2 pei year (standard enor for the ascending slope = 0.005 g/cm2), with a peak bone density of about 0.72 g/crn2 occumng around the age of 9.5 yem, the age break-point of the broken-Iine regression. BMD nmains constant until approximately 17.2 yean, der which there is a steady decline in BMD, clearly shom by the smoothing spline. Thus, femdes gain bone minerai density at a rate of 0.04 g/cm2 per year until peak boae density is attained. As seen in Figure 10, the younger femaies tend to have BMD close to the regression line (Le.. close to the average), whereas the older animals exhibit a great deal of variability. The oldest Rmale in this sample is 22.2 years. k The males show a different pattern of BMD. Figure 11 compares a smoothing spline and broken-line regression as applied to the data on mdes; the dotted line represents the broken-line regression, and the solid line is the smoothing spline. The two curves are close over their entire range. This graph shows that males reach a peak bone density of about 0.78 g~/cm~at an earlier age than do females, around 7 years of age, after which BMD remains relatively constant between the rges of 7 and 18.5 yean. The broken-line regression shows that males gain bone minerai density at a rate of 0.09 @cm2 pryear (standard error for the ascending slope = 0.008 g/cm2) up to age 7 yeus, when peak bone density is attained. No decrease in BMD with age is observed in males; after age 7 there is no apparent effect of age on BMD. However, the oldest maie in the siunple is 18.5 years of age. Recall that males older than 18.5 yem had to be excluded due to the presence of VO andor DDD; thecefore, it cm not be determined with certainty whether or not males experience a decrease in BMD with advancing age. Figure 11 shows that younger males dso tend to have BMD values that are close to the regression line, while older males exhibit a great deal of variability in BMD. The slope of the gnph of BMD for maies aged 1 - 7 years is steeper than that for femaies, indicating that males acquk bone mass at a faster rate during growth and development thiui do femdes (Fig. 12). Not only do males attain peak bone mass at a younger age than females, but also their peak bone mass is pater than that of femaies. Males mach a higher BMD than females at every age, except during the earliest years of development, age 1 to 3, when the accumulation of bone mass is about the sarne for both sexes (Fig. 12). The two patterns of BMD appear to diverge after sexual maturity is reached at 3 years of age. The differences in BMD that are seen between aduit males and females are sigaificant (p c 0.0001). Thus. the females are respoasible for the curvature seen in the graph of the entire sample (Fig. 9), particularly in the older age range. der 17 yean. Thus, the smoothing spline was the best model to estimate the underlying trend in the data for femaies, and the broken-line regression was the simplest model for the males. These models were useful for estimating an underlying trend, around which there is a great deal of variation. Although these are the best fitting models, they may not predict BMD very well due to variability in the sarnple; recail that prediction, another application of regression, was not a concem in this study. There may be considerable heterogeneity in the sample that is not explained by the model, but this does not invalidate the model as king a good one for the mean. It just implies that there are other factors that affect individual BMD values apart from age: among fully mature animals, there are other factors besides age that account for BMD. Age explains %MD up to a point, then other factors such as the presence of metabolic bone disease and degenerative arthritis account for much of the variability pst a certain age. Thus, age explains BMD up to the age of peak bone mass; beyond this age, other factors like disease account for much of the variation. For the early yem, most of the variation mong animals can be attributed to age, i.e., to the model. Past the plateau age. the slope against age is L relatively Bat; the mean of the BMD is used as the regession line. Figure 13 presents a different perspective on the differences in BMD between males and females. It shows box plots of BMD in males and females separately; the 'mg- plot' on the left represents the distribution of the Pmale data, and the one on the right shows the distribution of the data on males. The box plot graphs the data in a column as a box representing statistical values. The lower boundary of the box marks the 25th percentile, and the upper boundary the 75th percentile; a line within the box indicates the median. Error bars above and below the box indicate the 90th and lûth percentiles. ûutlying points are represented as lines below the 10th peseniile bracket. The median BMD value of the males (0.764 &m2) is higher than that of the femaies (0.690 g/cm2). In fernales. the 25th. 75th and 100th percentiles are 0.573glcm2. 0.772 g/cm2and 1.05 glcm2. In males, the comsponding percentiles are 0.658 glcm2, 0.838 &m2 and 1.09 g/cm2. While the distribution of the data, including the osteopenicslosteoporotics, is contained within the 10th and 25th percentiles in fernales, that of the mdes is not; the seven lines at the bottom of the male box plot represent outliers, some of the male osteopenic individuals in the sample. Refer to Table 1 for a surnrnary of descriptive statistics on maies and females in the sample, divided by age group. A t-test revealed that there is a significant difference between the rnean BMDvdue of the mdes (0.727 g/cm2) and the mean BMD of the females (0.667 @cm2) (p = 0.003). A surnmary of the main results from the DEXA study is provided by Table 2. Aae. Sex and Bone Mineral Content Bone rninenl content (BMC). measured in grams. constitutes the raw data; it follows similar patterns as bone mineral density. The following data and graphs are provided in order to show the raw data for both males and females. Analyses of the BMC data are provided for those readea interested in the study of bone mineral acquisition in primates. Note that the rrsults obtained using BMD values are given more prominence in the present study, since BMD includes a correction for bone size (projectional ma), making it a more accurate measurement of bone mass. As witb BMD,there is a wide range of BMC values in the simple of monkeys older than 7 years compareci to that of younger animds, whose BMC measurements tend to be close to the regression line. The femaie and male patterns are distinct; there is a significant sex difference (p < 0.0001) and significant non-linearity (p c O.oO 1). A t-test revealed that there is a significant difference between the mean BMC values of the males and femdes (p e 0.000 1). When the females are examined alone (Fig. 14). a peak BMC of about 1.63 grains is noted at around age 10 years from the broken-line regression (dotted line). and the second dope of the broken-iine regression shows a steady decrease in BMC with advancing age. The smoothing spline (solid line), however, shows that BMC remains constant from around age 10 to 17 yeus. after which there is a steady decline in BMC wiih increasing age (Fig. 14). The latter provides a more biologically redistic trend with age. The males show a peak BMC at around age 6.5 years, of approximately 2. L5 grams. which remains steady between the ages of 6.5 and 18.5 yean (Fig. 15). For the males, the smoothing spline and broken-line regression have vinuaily identical cuves. The males show a dnmatic. steep linear increase in BMC from age 1 to 7 yem. in contrasi to the females. who have a much shallower slope (Fig. 16). The broken-line regression shows that males acquire bone mineral at the rate of 0.36 grarns per year (standard enor for the ascending slope = 0.0326 grams) up to age 6.5 years. Femdes acquire bone mineral at the rate of 0.13 grams per year (standard error for the ascending slope = 0.0127 grams) up to 10 yem of age. These trends confirm that males tend to acquire bone mineral at a faster rate during growth and development than do females. Thus. males reach peak BMC at a younger age thiui do females, and their peak BMC is higher than in females. At every age from 1.5 years onwards, males have a higher level of bone mineral content than females (Fig. L6). Thus. BMC foiiows the sme pattern seen for BMD. Table 2 summarizes the main results for BMD, BMC and bone are& TABLE 2 SUMMARY OF DEXA lWSULTS DEXA PEAK ACE of PEAK RATE of Measurement VALUES bw INCmASE * (pet yerir, uatil age of perik) FEMALES I - 0.72 - 9.5 - 1.63 - 10.0 7.0 - 8.0 MALES BMD (g/cm2) - 0.78 - 7.0 0.09 BMC (granis) - 2.15 - 6.5 0.36 AREA (cm2) - 2.70 6.0 - 7.0 0.60 * Assessed using ascendiog dope of broken-line cegression curves. ---BMD vs. BMC Figures 17 to 19 demonstrate the strong positive correlation which exists between BMD and BMC; it is a significant non-linear relationship in both females and mûles (p É 0.0001). Both the males and females show a two-phase curve, which represents two different phenornena. Since BMD = BMC/Area, BMC and BMD are closely correlated variables. Figures 17 to 19 show that there is a lower comlation for higher values of BMC and BMD. The part of the BMD venus BMC curves that has the steeper dope in Eigures 17 to 19 is mostly comprised of younger animals, females younger than 12 yean and males younger thm 9 years of age. most of whom have not yet reached the age of peak bone density, and thus have lower BMC and BMD. Some are dso immature with respect to linear growth; in this colony, both femdes and males reach mature Iinear adult morphology at 6.5 years of age (Chevenid 198 1; Turnquist and Kessler 1989). The latter part of the smoothing splines, including the curved part in the middle of the splines, is comprised of older animais (Figs. 17 to 19). in both femaies and males, the latter part of the smoothing spline has a regioi of curvature followed by a straight line; this straight- üne portion begins just past the point where peak BMD and BMC intersec< for females and males respectively. Thus, this part of the spline represents individuais who have completed their linear growth as well as their bone minedization. These older individuals have a different relationship between %MC and BMD compared to younger animals. For example, in the males, the Iarger dope for the left portion of the cwe cepresents mostly younger animais with lower BMC and BMD. At amund BMD = 0.8 and BMC = 2.1, the respective points at which the BMD and BMC by age curves flatten out (Figs. 1 L and 15 respectively), the BMD-BMC curve begins to have a shallower dope. For the younger animais, BMC and BMD are both close to the average, i.e.. close to the regression line, with age; the close correlation of BMD with age, and of BMC with age. means that BMD and BMC will be closely comlated when age is ignored. For the older mimals, there is much more variation around the average BMC or BMD with age, and thus the correlation between BMC and BMD is smaller. This explains the two-phase cwe for BMD versus BMC. This two-phase relationship is true for both males and femdes, but the ratio of BMC to BMD is higher in males (Fig. 19). --Amvs. Ape 'Ares' denotes the 'ngion of interest' or ROI that was selected during the analysis of the DEXA scans. This region corresponds to the centrum of each vertebn. A growth curve for the centrum is generated when area is plotted venus age (Figs. 20 - 22). in this colony, endplate closure is virtually complete by approximately 7 yean of age; however. considerable variability was observed. Since there are two patterns of BMC/BMD, Le., male and female, the effect of age on area was observed in males and females separately. in females, the area of the centrum increases from age i to about age 7 to 8 years, when growth appean to end, with a peak area of about 2.10 cm2 king attained; ana then remains relatively constant with increasing age (Fig. 20). Compared to females, males show a more rapid increase in mafrom age 1 to a pedc of about 2.70 cm2 at around age 6 to 7 years, dter which area remains constant with increasing age (Fig. 21). Males attain a larger centnun area than females, they attain it more rapidly, and maintain a larger area across ai1 ages (Fig. 22). This corresponds well with the moderate sexual dimorphism which exists between rhesus males and females. -BMD b~ Natal GCOUD Macaques are organized into multi-maie, multi-female social groups in which females remain in the groups in which they are bom, while males emigrate out of their natal groups at sexual maiunty. Thus, related femdes fom the cote of the social group. Al1 individuals in the sample, and the living monkeys on Cayo Santiago, are the descendants of two onginai founding groups. A and B. As the population on Cayo Santiago increased, new social groups fissioned from these founding groups; a total of 19 new socid groups had fomed by 1988 (Kessler and Berard 1989). Thirteen natal groups are represented in this sample; the majority of animais corne from natd groups A, F, 1 and J. The natal groups of L2.2% (N = 254) of the sample are unknown; these individuals were excluded from this particular analysis. Association between BMD and natal group affiliation in this colony was tested in an effort to find evidence for a genetic cornponent to BMD. An ANOVA (Analysis of Variance) test on the average BMDs of al1 natal groups represented in the sample showed a significant difference among the groups. However, when the test was repeated excluding individuds younger than 7 years (Fig. 23), this significant difference disappeared (p = 0.376). It is concluded that these young individuds, who are expected to have lower BMD owing to their lack of completed growth. were the cause of the initial difference fouad among the groups. Among adults. no significant difference was found in average BMD across natal groups. In addition, the age ranges of the natd groups Vary greatly, which also accounts for this nsult. Further investigation. using matrilineal data, is required in order to test the hereditary component oPBMD. 3.2. Results: Osteopeda and Osteopomsis As mentioned previously. with regud to DEXA four general diagnostic categories have been established for adult women, and the following cnteria are accepted by the National Osteoporosis Foundation of the United States, the European Foundation for Osteoporosis and Bone Disease, and the World Health Organization (Kanis 1994b; Wasnich 1996): 1) Normd: a value for BMD or BMC not more than 1 SD (Standard Deviation) klow the average value of young adults; 2) Osteooenia (low bone mus): a value for BMD or BMC more than 1 SD below the young duit average, but not more than 2.5 SD below; 3) Osteo~orosis:a value For BMD or BMC more than 2.5 SD below the young adult average value: 4) Severe Osteowrosis (established osteowrosis): a value for BMD or BMC more than 2.5 SD below the young adult average value and the presence of one or more fragility fractures. These criteria were applied to the DEXA data. Although they successhilly identified the osteopenic/osteoporotic (OPEIOPO), unfnctured individuais in the sample, al1 the cases of established osteoprosis as evidenced by vertebrai compression or wedge fractures were rnissed (see bdow). Monkevs with Ostcoocnia !Osteowrosis (without fracture). Dia~nOSed& Standard There are 27 osteopenic/osteoporotic (unfractured) individuals in this sample, ranging in age fkom 7 to 22.2 years; there are 16 females and 11 males. These individuals are listed and described in Table 3. in this part of the analysis, the bones of osteoarthritic individuals were also surveyed for osteopenia and osteoprosis (N = 298). For each individual's BMO and BMC, Table 3 lis& the standard deviation units frorn the mean of young adults, and the diagnosis. based on standard criteria, is dso shown. The young adult sample to which these OPElOPO monkeys were compared is described at the bottom of Table 3. This young adult comparative group includes only individuals beyond the age of peak bone mas The frequency of OPElOPO (unfnctured) in the entire sample is 9.1% (N = 298). The individuais listed in Table 3 do not exhibit any vertebnl wedge or compression fractures. 95 TABLE 3 OSTEOPENIC & OSTEOPOROTIC MONKEYS IDENTIFED USING CRITERIA -- ENDoRsED- BY THE- w.H.o~? - Ca ta Age at Natal BMC BMD BMC, Diagnosis, No.' Death crpr (s) Wm2, SB! using BMD -CYm) - - S.D, unitse 1617 7.0 J 0.850 0.393 --2.28 OP0 t2133 8.0 J 0.852 0.4 15 -2.27 OP0 t2088 8.5 J 0.9 15 0.377 -2.1 1 OP0 *804 9.9 J 1.128 0.467 -1 .SS OPE "637 10.6 1 1.088 0.462 - 1.65 OPE 641 11.9 L 0.985 0.469 -1.92 OPE 2208 12.0 M 1.O39 0.554 -1.78 OPE *657 13.2 J 1.306 OS 16 - 1,O8 OPE '677 13.9 F 1.214 0.516 -1.32 OPE 2160 14.0 J 1,049 0.426 -1.76 OP0 2055 15.0 J 1.255 0.595 - 122 OPE 841 15.1 F 0.9 12 0-416 -2.12 OP0 620 16.9 F 1.627 0-613 -0.24 OPE 198 17.0 u 1.124 0.593 -1 S6 OPE 2967 22.0 F 1.140 0.545 - 1.S2 OPE * 1522 22.2 F 1.178 0,554 -1 -42 OPE 302 7.5 C 1,042 0.453 -2.54 OP0 1629 8.0 O 1.33 1 0.465 -1.86 OP0 t337 9.4 C 1.213 0.442 -2.14 OP0 247 10.0 C 1.426 0.633 - 1.64 OPE 1639 12.0 1 1.539 0.635 -1.37 OPE *Tl642 12.0 F 1.O5 1 0.405 -2.5 1 OP0 1604 13.1 1 1.486 0.57 I -1.50 OPE 348 14.0 U 1.457 0,564 -1.57 OPE 594 15.0 1 1.546 0.577 -1.36 OPE *690 18.0 J 1.747 0.584 -0.89 OPE 1229 22.0 -J - 1.687 0.654 - 1.O3 Borderline OPE Mean 13.3 - 1.229 0.5 15 -- - S.D. 4.36 LI 0.259 0.083 - - Median 13-1 - 1.178 0.516 - - Max. 22.2 - 1,747 0.654 - L hW -7.0 - U - 0.850 0,377 -U - NOTES: a CPt numbers in bold reprisent an outlier gmup of 12 osteopenic monkeys; b 16 Fedes, 11 Males = 27, Frequency = 9.1% (N = 298); U = Unknown Natai Group; Standard deviation uits hmmem of young (and primesgai) adults (see below); OPE = Osteopenia, OP0 = Osteopomsis; Table 3 Continued * Some vertebrae exhibit porosity of the cortical bone of the ventral aspect of the centrum; Cat. No. 637 is pictured in Figures 26 and 27; t The long bones of these individuais are pictured in Figures 24 & 25; $ No hctures of the spine are present in the animais listed in ibis table. d~oungand Prime Adult Sample Past the Age of Peak Bone Mass, Used in Diagnosis of Osteopenia and Osteoporosis (Standard Criteria): Sample Age Range BMD BMD BMC BMC Size (P.) Mean S.D. Mean S,D, Females 31 11 - 14 0.762 0.128 1.718 0.381 Males 71 8-14 0.780 0.121 2.127 0.428 Standard Deviation Units: (Observed BMD - Mean BMD) / Standard Deviation in Figures 9 to 12 inclusive, the OPE/OPO monkeys without fracture are represented by a number of points near the bottom of the scatter plots. Twelve of these are outliers (Figs. 9 and 12); they represent individuals with extremely low BMD, i.e., severe osteopenia. There is a mean difference in BMD of 0.264 @m2 between this group of 12 individuals and the rest of the smple. The long bones of these 12 osteopenic individuais were mdiographed with agelsex matched controls. Pronounced cortical thinning of the shaft and trabecular bone loss in the metaphyses were evident, thus supporting the osteopenic status of these individuais, and the systemic nature of the osteopenia. The long bones do not exhibit fractures, despite very severe bone loss in some cases. Figure 24 is a radiognph of the left femur and humerus of two osteopcnic females (Cat. lt2 133 and #2088), with control specimens in the center; extrernely severe bone loss is evident in the osteopenic individuals, and yet there are no fractures. Figure 25 is a radiograph of the long bones of two of the osteopenic males (Cat. #337 and #1642). with a control animal in the middle; the same pattern is observed. Figure 26 shows a photograph of the fourth and fifth lumbar vertebrae from one of these osteopenic monkeys (Cat. #637), a femde aged 10.6 yean (middle age For rhesus monkeys). In this specimen, the bone loss has progressed to such an extent that it has affected the corticai bone of the vertebinl bodies. Smdl holes are evident in the cortical bone dong the ventral aspect of the centra of the vertebne in virtually the en& spine. A laterai radiograph (Fig. 27) of the vertebral column of this specimen confms the decrease in radiodensity of bone, especiaiiy in the thoracic vertebrae; the loss of trabecular bone is infemd. Most of the endplates, which are composed of cortical bone, also appear to have been aected. A total of 5 osteopenic females and 2 males exhibit these small perforations of the ventrai aspect of some of theu vertebral bodies - a condition which indicates the presence of advanced osteopenia. A one-way ANOVA was carried out on the rnean BMDs of four of the natal groups represented among the OPUOPO individuals in Table 3: the unknowns (CI) were excluded, as were natd groups with very smdl sample sizes. The differences in the mean values among the natal groups were not statisticaily significant (p = 0.667). These negative findings may be due to either smdl sample sizes, or to the fact that age ranges vuy greatiy among the natal groups. The natal groups represented in this sample fissioned off from both of the original founding groups, A and B. and a number of matrilines are represented. Further research is required using matrilineage data in order to explore the possible genetic component to BMD and osteopenia/osteoporosis which is reflected in this sunple. Monkevs with Established Osteowrosis (Vertebral Fractures) Ten individuds with established osteoporosis occur in this sample, identified by the presence of anterior wedge fractures of the centrum. five females and five maies were found ranging in age from 9.8 to 29 years (Table 4). The standard cntena described above did not identify these individuals as having established osteoporosis; the values for BMD and BMC are greater than 2.5 SD below the young aduit average values, although fngiiity fractures of the spine are present in di individuais. Table 4 Lists the standard deviation uni& for ail subjects. with the comsponding diagnosis; four individuals were diagnosed as 'osteopenid according to the standard critena, while six were classified as 'normal'. However, the spinal hcnires of di subjects wen graded as 'moderate' or 'severe' according to the Osteoporosis Gnding Scale outlined previously (Table 4). Al1 individuals, including those diagnosed as 'nomal', exhibit classic vertebral wedge fnctures. Note that hctured vertebrae were arnong the bones scanned by DEXA. The bones selected for DEXA did not have any fnctures. which would have altered their size or shape and artificially increased mass per unit area. Thus, the individuals listed in Table 4 are either 'normal' or 'osteopenic' with respect to their BMD values, but all exhibit classic vertebrai fragility fractures. The implications of these resuits are discussed in the next section. TABLE 4 MONKEYS WTH WTABLISHED OSTEOPOROSIS: IDENTIFIED BY THE PRESENCE OF -- VERTEBRAL FRACTURES * Sex Natai BMC BMD OP0 BMC, b Crp* (s) (dcm2, S,DP Scale -- - - - F N 1.394 0.678 L3-4inciusive Severe -0.85 Noml F J 1.990 0.874 T12, LI Moderate 0.7 1 Nonnal F A 1,485 0.659 LI-3 incl. Severe -0.6 1 Normal F F 1,362 0,702 LI-3 incl. Moderate -0.93 Nonnal F L 1.434 0.685 7'9, L2-3 incl, MildIMod -0.75 Nonnal M N 2,049 O,8 14 L 1, L2-3 incl, Severe -0.M Nonnal M A 1.692 0.542 T9-1 2 incl .,L 1 ,L2 Moderate -1 ,O2 OPE M E 2,125 0,653' TB40 incl,, L7 Moderate -0.00 Borderline OPE M H 1,716 0,654 7'10-L4 incl, Severe 4.96 Borderline OPE M F -1.992 0,645 Tg-11 incl,,LI ,L3 Moderate 432 OPE Mean -* 1,724 0.69 1 S.D. c- 0.297 0.0926 Median 9- 1.704 0,669 Max, -- 2.125 0.874 Min, LI- 1,362 0.542 I - - a Cataioguc Number; b 5 Fernales, 5 Males = 10, Frequency = 3.4% (N = 298); Location of vertebral fractures (anterior wedging of the centrum); T'refers to thoracic vertebrae; 'L'refers to lumbar vertebrac; bsteoporosis Grading Scale: refer to text for description; Standard deviation units from mean of young (and prime-aged) adults (see Table 3); f~iagnosisusing standard criteria endorsed by WHO, OPE = Osteopenia; * Standard criteria did not identiQ these , individuals as having established osteoporosis (see text); t Pictured in Figures 28 and 29. O Figure 28 shows a photopph of part of the lumbar spine, LL to 5, of one of these specimens with established osteoporosis, Cat. No. 852. a 10 year-old (middle-aged) femde; fractures are clearly visible in lumbar vertebrae 3 and 4. The vertebrai bodies show severe antenor wedging. and they have fused together. This condition results in the characteristic dorsd kyphosis of the spine known as 'Dowager's hump'. The articular facets of these two vertebrae have also fused together, thus providing some stability to this fnctured region of the spine. A laterai mdiognph (Fig. 29) confimis the pnsence of wedge fractures and the loss of trabeculw bone. particularly in the thoncic vertebrae. Figure 30 shows a good example of dorsal kyphosis of the spine in one of the old femdes living on Cayo Santiago; she was about 20 years of age at the time the pictue was taken. Com~arisong& OPElOPO individuais in Tables 3 and 4 With the exception of only two individuals (Cat. No. 1229 in Table 3 and Cat. No. 643 in Table 4). the BMD values of the unfnctured, OPVOPO monkeys in Table 3 gr-g lower than the BMDs of the individuals with established osteoporosis, Le., with vertebnl fractures tisted in Table 4. This observation is supported statistically, as foLiows. Within Table 3. when the mean BMD of the osteopenic males (0.544 g/cm2) and the mem BMD of the osteopenic females (0.494 g/cm2) were compared using a t-test. it was found that there is no statistically significant difference between the sexes (p = 0.1320). This nsult. however. may be due to srnall sample sizes. For the osteoporotic individuals in Table 4, a Mann-Whitney Rank Sum Test, which was performed in place of a t-test because the data were not normally distributed, also reveaied that there is no statistically significant difference between the sexes (p = 0.0952); the Merence between the median values of the males (0.653 g/cm2) and females (0.685 &m2) in Table 4 is not significant. A number of tests compared the individuals of Table 3 to those of Table 4, thus cornparing osteopenic/osteoporotic monkeys to those that exhibit vertebral fractures. A t- test cornparing the mean BMD (0.515 g/cm2) of the OPWOPO group in Table 3 and the mean BMD (0.691 g/cm2) of the monkeys in Table 4 found that there is a statistically significant difference between these two groups (p < 0.0001). There is a difference of 0.176 g/cm2 between these groups. Fwther cornpaison revealed that the= is a statistically significant difference between the mean BMD (0.494 g/cm2) of OPBOPO females of Table 3 and mean BMD (0.720 glcm2) of the osteoporotic females with fractures in Table 4 (p < 0.01). The= is also a significant difference between the mean BMDs of males in the two groups, although not as pronounced (p = 0.0300). The mean BMD of the OPWOPO mdes in Table 3 = 0.544 &m2 and that of the osteoporotic males with fractures in Table 4 = 0.662 g/cm2. The BMD values of six of the individuals in Table 4, al1 with vertebral fractures, faIl within the range of normal variation such that the standard criteria failed to identiw them; they were found by visuai inspection and radiognphy. in contrast, probability density estimates show that both male and femaie unfractured OPUOPO subjects separate out as a group From the rest of the svnple (Figs. 3 1 and 32); the fiat two smail peaks in each gmph represent these individuals. The probability density estimate was used to illustrate the underlying distribution of the bone density data. Based on the histograrn, this technique gives a representation of a continuous probability distribution in which probability is portrayed by areas under the density cwe. Arnong fernales over age 11 years, the probability density estimate revealed that the mode of the distribution of BMD is about 0.70 &m2, with a 95% confidence interval of 0.60 to 0.90 @cm2 (Fig. 32). Among males over age 9 years. the mode was amund 0.80 g/cm2 with a 95% confidence interval of 0.65 to 1.0 g/cm2 (Fig. 3 1). A 'mg-plot' near the bottom of each gmph illustrates the distribution of data points in the sample. Individuais younger than the age of peak bone density are excluded from these two graphs. These data suggest that anot her factor. bone quality, Le., qualitative abnormaiities of the microarchitechire of the trabecular bone as well as the mineraiization of the bone. may be involved in compression or fngility fnctures. Bone quality is an important factor when assessing fracture risk; low bone density is a critical, but not necessdly sufficient. cause of fmcture. The combined frequency of OPUOPO and estrblished osteoporosis in this sample is 12.4% (N = 298). BMC Fig. 17. Bone mineral density (BMD, in glcm2) versus bone minenil content (BMC, in gram) in fernales. Solid Line = smoothing spline; brohlines indicate 95% confidence band. -'Y*' BMC Fig. 18. Bone mineral density (BMD,in &m2) versus bone mineral content (BMC, in gr=) in males. Solid Line = smwthing spline; brokn lines indicate 95% confidence band. BMC Fig. 19. Comparison of bone mineral density (BMD, in &m2) versus bone mineral content (BMC, in grams) in females and males. Lhes represent smoothbg spline fit. Females = white symbols and broken line; males = black symbols and solid line. S.. a \ *O -.oO Natal Group Fig. 23. Box plots of bone mineral density @MD, in @cm2) in natal groups representcd in the sample among individuais 7+ years of age. lndividuals of unknown natal group are excluded, as are natal groups representeà by only one member. Fig. 24. ûsteopenic haîes: Radbgmph of kR humerus and ieft mut of ml 33 (faride of hg@and m088 (right side of image) with contml specimeni, h the centet. These inâividuats aie liaed in Thle 3. Xmy taken by Dr. J. E. Tumquist, CPRC Museum. Fig. 25. Osteopenic malea: Radiogmph of leR hunecw and lett fmur of lW37 (ieft ride of image) and #1642 (MMside of image) with conûol apecimerio in the centet. Th- individuais are lisW in Tabb 3. Xiay taken by Dr. J. E. Tumqu# CPRC Murieum. Fi@ 26. Photogreph of hno lumber wibbme (14 and L5) of osteopeniic fernie M37. Note the poiwity of the cortical bons of tfm ventral aspect of the veRebral bodies. This individual is listed in Table 3. kmale W837. Note th decma~in radioâensity of bone, indikative of trabeculat bone losse This individual is hW in TaMe 3, Fii. 28. Photograph of lumbar wrbebme 1 ta S (indusîu of osteoporotic amk #852 ~otethe ~eitebra~weâge fradures in lumber wrtekae 3 and 4, with subsequent ankyfasis of wrtebrel bodies. This indMdua1 is listed in Table 4. Fig. 29. Lateral radkgfaph of veitebral column of osteopom)tic fernale W852. Note the demase in radkdensity of bone, and the vertebrai wdge fiacturss in lumbar vertebm 3 and 4.1'his individual is listd in Table 4. - stünuing dodkyphosis of the spine (~okagefs hump). BMD Fig. 32. Probability density estimate of BMD in fedes over 11 years of age; mg-plot on the X-axis represents the data for fernales. 3.3. Discussion: Effects of Age and Sex on Bone Mass -Bone Mineral Densitv and Osteowrosis Osteoporosis zdfiicu one in every four postmenopausal women in Nonh Amencri. and also poses a significant health problem among older men. It is expected that. as the proportion of the population over 65 years of age increases. the impact of this disorder will also increase in magnitude (Stini 1990: Wasnich 1996). Osteoporosis is not rnerely an affliction of modern times; age-related bone loss was also present in past populations (Allison 1988: Brickley 1997; Foldes et al. 1995; Kneissel et al. 1994; Mays. Lees. and Stevenson 1998; Mays 1996: Roberts and Wakely 1992). A complete review of bone mass and osteoporosis in ancient populations is beyond the scope of this study. However. sorne studies have demonstnted age-dependent bone loss in women and men from various human populations in antiquity, with women showing relatively more bone loss thm men. and older females exhibiting more bone loss compared to younger cohorts (Mays, Les. and Stevenson 1998: Mays 1996). These patterns are also observed ioday. Bone mineral density at any age in adulthood is the result of both the arnount of bone accurnulated during growth, and the subsequent rate of bone turnover. insufficient accumulation of skeletal mass by the tirne the age of peak bone density is reached appears to enhance the probability of fmctures later in life. when age-related bone Ioss occurs (Riggs, Peck, and Bell L99 1). According to Riggs et ai (1991). the influences of heredity and sex on the incidence of osteoporosis may reflect their effects on peak bone density; a genetically deterrnïned variation in peak bone minerai density may explain the tendency for osteoporosis to nui in families (Riggs, Peck. and Beii 1991). Thus, attainiag a high peak bone density at skeletai maturity is considend protective against age-related bone loss and the risk of subsequent fmgility fractures. Peak bone mass is ultirnately the result of a number of factors. including genetic and endocrinal effects, nutrition and physicd activity (Matkovic 1992; Recker et ai. 1992). Osteowrosis and Vertebral Fracture Patterns A nurnber of fncture patterns have been described by Mensfonh and Latimer (1989). There are many types of vertebral fracture, including central compression fracture. anterior wedge deformity. and complete collapse of the vertebrd body (Riggs and Melton iii 1988). Com~ressionfractures, also known as cmsh fractures, involve compression of the whole vertebrai body, including the posterior aspect. In wedae fnctures. the posterior height of the vertebral body is relatively preserved, but there is collapse of the antenor aspect of the ceninim. Concave or biconcave morphology of the centrum indicates collapse of the supetiot or inferior endplates. or both, with relative preservation of posterior and anterior heights of the venebd body. Biconcavity may be associated with discd herniations called 'Schmorl's nodes'. which are caused by pressure from adjacent intervertebral discs onto the thinned cortex - a feature not specific for osteoporosis (Kanis L994b). intra-discd pressure may give rise to both the biconcave morphology of the centnun and to Schmorl's nodes. Other morphological deformities include andine of the enddates and comolete cornoression fncture (Riggs and Melton üï 1988). With advancing age, both quantitative and qualitative differences occur in bone (Evans L976; Gam and Shaw 1976; Grynpas L993). Bone loss in osteoporosis is chmcterized by increased bone resorption that is most pronounced in the tnbecular component of bone. Osteoprosis cm affect trabecular bone. cortical bone, or both, depending upon the stage of the condition; trabecular bone is generally the site of its initiai appearance. due to its high surfice to volume ratio (Evans 1976; Garn and Shaw 1976). Since vertebrae contain a high proportion of trabecular bone. osteoporosis is associated with vertebnl compression fractures, and the other deformities described above. In humans. the eighth thoracic to the third lumbar vertebrae are most comrnonly involved, and advanced cases of osteoporosis rnay culminate in dorsal kyphosis of the spine. The present study found that the lower thoracic and lumbar vertebrae of rhesus monkeys are also most susceptible to compression fractures; in aged monkeys, advanced osteoporosis also causes dorsal kyphosis of the spine. Schmorl's nodes were detected in a lew of the osteoporotic specimens that exhibitrd multiple diseases at end-stage. Mensforth and Latimer have reported no statisticdly significant difference in frequency of vertebrai compression fractures between men and women in their sample (Mensforth and Latimer 1989). The present study identified more osteopenic/osteoporotic fernales thui males, 2 1 to 16, but the difference in frequency is also not significant. ----The Causes of Bone Franilitv Fncm pathogenesis is cornplex. Low bone density is a cntical. but not necessarily sufficient, cause of fracture. Bone mineral density rcounts for 75% to 85% of the variance in the ultimate strength of bone tissue (Melton III, Chao. and Lane 1988). so BMD values thus pmvide an indication of the suength of whole bones - but not a complete assessrnent of bone integrïty. Many stuclies have demoastnted that human osteoporotics with vertebrai and/or femoral neck fractures have lower bone mineml content, bone density or calcium content than normal subjects (controls) (Mazess 1981; Mazess 1983). However, some studies have shown that there is a large overlq in the bone density vdues of age- and sex- matched individu& with and without spine, hip and forearm fractures; there is overlap in bone mas values between samples of normal and osteoporotic subjects (Heaney 1992; Melton iII et al. 1989). Thus, bone density measurements do not clearly discriminate patients with osteoporotic fractures €rom those who have not yet experienced a fracture. It is thought that qualitative abnormûlities in bone structure may be contributing factoa (Heaney 1992; Heaney 1993). Therefore, factors other than bone mass may contribute to bone integrity and Fncture; low bone mass is a cause of fiagility, but this factor alone does not explain why one individual with bone loss hûs fnctures and another does not. Other factors that cm contribute to fragility include fatigue damage and reduced tnbecular connectivity. The poor bone quality that contributes to skeietai fragility includes an accumulation of unremodelled fatigue dmage and loss of critical trabecular connections - common in osteoporotic vertebral bones. Studies that cornparcd osteoporotic and normal subjects found that the former exhiiit decreased connectivity and greater fenestration of the trabecular lattice in their bones. Fatigue damage weakens the bone, changing its intrinsic strength. and trabecular disconnection weakens the structure (Heaney 19%). According to Heaney (1992), reduced bone mas is the best studied cause of bone fmgility, but whether it is the most important cause, and the extent to which it consistenilv precedes fracture, are unknown (Heaney 1992). The tact that the BMD vdues of osteopenic/osteoporotic monkeys with vertebral fractures in this study overlapped with those of osteopenic/osteoporotic non-fncnired subjects supports the view tbat low bone mass mav not aiwavs orecede ktuce. Six of the ten monkeys with vertebral fractures have nonnal BMD values. and four are classified as osteopenic or borderline osteopenic, according to the established cnteria. Al1 ten of these individuals exhibit multiple vertebral fractures of the wedge variety. It is postulated that the fiactured vertebcae with normal BMD may have experienced fatigue damage or the resorption of key trabecular structures. which compromised skeletal integrity without the loss of too much bone, thus resulting in structural fidure but normai bone mus. These results also suggest that the cnteria for diagnosing osteopenia and osteoporosis endoaed by the W.H.O. should be revised to reflect these fïndings and the data in the literature. Anatomr Vertebral Fracture: Architecture and Bone Oualitv In humans, bones like the vertebnl bodies. and the femoral and tibia1 metaphyses expenence vertical loading. Bone structures that are loaded vecticdly derive a significant portion of their structural strength from a system of horizontai cross-bracing trabeculae, which support the vertical tnbeculae. and limit lateral bowing and consequent breakage under loading (Heaney 1996). In osteopenia and osteoporosis. the horizontal trabeculae tend to be resorbed first - for unknown reasons. Studies have shown that the severance of the horizontal trabeculûr connections occurs preferentially in postmenopausal women, and is thought to be the main reason for the high prevalence of spinal osteoporosis in women compared to men (Kleerekoper et al. 1989. Thus. long unsupported vertical trabeculae are very susceptible to frachue; such fractures rnay accumulate. and compromise the cancellous structure of the vertebnl body. The presence of these structurai defects explains why prior fiactum appears to predict hinire fracture in a patient, even when bone densitv is relativelv hieh (Heaney 1996). Aithough the spines of rhesus monkeys are loaded differently fiom those of humans, it is interesting to note that X-rays of the vertebrae of osteopeniclosteoporotic monkeys reveai the same pattern of bone resorption; i.e.. the horizontal trabeculae are preferentially resorbed. Studies on the Cayo Santiago rhesus monkeys are discussed below. Cayo Santiago Rhesus Monkevs: Studies on Bone Densitv, Osteorjenia and In a single photon absorptiometry (SPA) study of radii and femora from Cayo Santiago rhesus monkeys, Aguilc5 and Cabrera (1989) found that the two sites correlate well, and that there is an age-nlated decrease in bone density. Other previous studies on Cayo Santiago macaques (Gcynpas 1992; Grynpas et al. 1989; Grynpas et al. 1993b). have shown that osteoporosis is associated with age, that fernales are prediiected, and that high parity is a protective factor. A study by Grynpas et al. (1993) found that bone density decreases with age in femaie rhesus monkeys, but not in males (Grynpas et al. 1993b). The present study, with its larger sample size (N = 254), has investigated these relationships further, and has established a baseline or standard of vertebrai density measurements in the rhesus monkey. This investigation has found two distinctive patterns of bone minerai density, one for fernales and another for maies. among the rhesus monkeys of Cayo Santiago. The BMD profile for the colony is better undeatood when the femaies and males are studied separately. In both sexes, there is little variation in BMD values among the very young (age 1 to 7 years). but pater variability among older animais at every age. in the femaies. BMD increases with age to a peak of about 0.72 g/cm2 around the age of 9.5 years, and remains constant untii 17.2 years. after which there is a steady decline in BMD with increasing age. in contrast to humans, rhesus males attain a peak BMD of about 0.78 @cm2 at an earlier age than femdes. at around 7 years of age: between the ages of 7 and 18.5 yem. there is no apparent efTect of age on BMD. Since the= are no males older than 18.5 years in this particular sample, there is insmcient evidence at this time to determine whether or not males experience a decrease in BMD with advancing age. The males show a steep linear increase in BMD from age 1 to 7 years, while femdes have a much shailower slope; this means that at every age after the age of sexud maturity (3 years). males have a higher BMD than females. Males acquire bone mass at 3 faster rate than do females. achieve a higher peak BMD. and reach this pelBMD at a younger age. In this colony, both females and males attain mature linear adult morphology at 6.5 years of age (Cheverud 198 1; Tumquist and Kessler 1989). By this time. the dentition (with the possible exception of M3) is complete and there is Ml epiphyseal closure in al1 long bones. Thus. although growth of the body is complete in femdes by 6.5 years of age. hl1 bone minerdization is not - the gin of bone mass continues until a peak BMC is attained by around 10 years of age, and a peak BMD by around 9.5 yem. By the time males complete their growth at 6.5 years of age. they aiso attain pedc BMC. which dso occurs at amund 6.5 years; peak BMD is attained somewhat Iater, by around 7 years of age. Osteopenic and osteoporotic monkeys were found in both sexes. at a combined frequency of 12.4% (371298). Thus far, no relationship between natal group affiliation and BMD has been found in this colony. Further investigation is required using matnlineage data (matemal genealogies), in order to explore. in a more direct manner, the possible genetic component to BMD. osteopenia and osteoporosis in this colony. Com~arisonwith Previous Studies of Bone Densitv and OsteowniPIOsteomn#ris Nonhuman Primates A number of studies conducted on non-human primates have demonstrated the relationship between experimentally induced menopause or hormone deficiency and bone density and osteoporosis (Jayo et al. 1990; Jerome et al. 1994; Jerome et al. 1986; Jerome et al. 1992; Longcope et al. 1989; Lundon, Dumiuiu, and Grynpas 1994; Lundon, Dumitriu, and Grynpas 1997; Lundon and Grynpas 1993). These snidies will not be discussed, as the emphasis of this thesis is on BMD and spontaneous or naturally- occurring age-related bone loss. Cross-sectional and longitudinal studies of bone mas in femde cynomolgus macaques have also ken conducted (Jayo et al. 1994; Jayo et al. 1991b). The cross- sectiond paxt of a study by Jayo et al (1994) evduated the BMC and BMD of the lumbar spine (L2-LA)and totd body of live femde monkeys. It was found that peak bone density in the lumbar spine occurs at around 9 yean of age in the female cynomolgus rnonkey. Since rhesus macaques are a larger species, a slightiy Iater age of peak bone density in rhesus monkeys (around 9.5 years for females in the present snidy) is not unexpected. Mature cynomolgus monkeys (older than 10.5 yeus) showed bone loss through time (Jayo et al. 1994). The effects of age, sex and heredity on bone mas have also been studied in pedigreed baboons (Kmerer et al. 1994; Kammerer, Sparks, and Rogers 1995; Mahaney, Kammerer, and Rogers 1997). A study by Kammerer et ai (1995) of bone mass and density by radiographie morphometry of the left second metacarpal in pedigreed baboons found that age-related changes are similar to those found in humans; di measures of bone mass used in their study changed with increasing age, and dl changes were consistent with loss of bone mass at the oldest ages (Kammerer, Sparks. and Rogers 1995). Aufdemorte et d (1993) also confi that the baboon is a good mode! of age- related systemic and oral bone loss; older animals showed a marked reduction in spinal bone mineral density, in addition to dorsal kyphosis and compression fractures (Aufdemorte et ai. 1993). There are a few earlier studies on bone density in the rhesus monkey. Pope et al (1989) studied the effects of age and sex on bone density in rhesus monkeys at the Yerkes Primate Center (Pope et al. 1989). Live animals and smples taken at necropsy were used in this study; single photon absorptiometry measurements of the humerus, third lumbar vertebra and last lumbar vertebra were taken. Age and sex differences were observed among these rhesus monkeys as well: it was reported that, in general, bone density increased with age and then reached a plateau at around 3 to 4 years in al1 bones measured. in the humenis, femaies older than 30 yem of age had a significantly lower bone density than younger fernales 4 to 24 years old, while the bone density of the humerus of older males did not decrease wiih increasing age. In the third lumbar vertebra (necropsy specimens), some evidence of age-related decreases in bone density was found in both sexes (Pope et al. 1989). However, some of the age effect in maies was due to lower bone densities in young animais under 2 years of age, and the presence of only one 17-year-old male, and one 19-year-old male, both of whom had lower densities. Femdes also showed a signifiant effect of age, due prirnarily to the younger animais and to the pnsence of one 22-year-old femaie in the smple (Pope et al. 1989). The study by Pope et al (1989) does not report on peak bone mass ages in fernales and males separately for each of the sites measured. and it is not clear how the plateau at 3 to 4 years of age, which was only reported in the abstract of thev paper, was determined Also, their data fiom the vertebral measurements are not presented graphically, thus a direct comp~sonwith the present study is not possible. Based on the fmdings of the present study. and those of a previous snidy by Chmp et al (1996), the timing of this plateau at 3 to 4 years of age for the rhesus monkeys in the Pope et ai (L989) study appears to be too Young, and may in fact be a statistical or typographical error. Graphs of BMD of the humerus versus age in the Pope et ai ( 1989) study indicate that the females attain a peak bone mais by iuound 10 - 13 years, and a decline in BMD with increasing age occus after 17 years; the males appear to attain a peak bone density in the humerus by 9 or 10 years of age. These results generdly correspond with the findings in the present study. aithough the Cayo Santiago males attain peak BMD in the lumbar spine earlier, ai around 7 years of age. A recent cross-sectional DMA study of bone density in 178 live femde rhesus monkeys at the Wisconsin Regionai Primate Research Center exarnined the effect of age on bone-area and body-weight adjusted bone mineral content of the distal radius, lumbar spine (L 1-4) and total body (Champ et ai. 1996). In many ways. this study supports the methods used in this thesis and conobomtes the results on bone density presented herein. This study used the same DEXA technology that was used in this thesis. Champ and colleagues used pediatric software for their analyses. while the data in this thesis was analyzed with software designed for small mimals, which was more appropriate for macented bones. Champ et ;il (1996) performed scans of the lumbar spine on 167 femde rhesus monkeys. ranging in age from 2.8 to 34.6 yem. Since body weight measwments were avaiiable, they were able to obtain a body-weight and bone-area adjusted BMC for each animai. The authors used multiple regression. broken-line regression. and a type of non-linear regression called Zowess', which is similar to the smoothing spline used in this thesis. Champ and colleagues found that total body BMC (N = 178) and bone mass of the lumbar spine (N = 167) increased with maturation up to age 11 years. and then remained stable with advancing age (p < 0.0001). This age of peak bone mass is similar to that reponed in this thesis for the Cayo Santiago rhesus monkey femdes (9.540 years). However, unlike the results presented in this thesis, the study by Champ et al (1996) did not find a decrease in bone mass of the lumbar spine with advancing age. A decrease in bone density with increasing age was observed only at the distd radius site. The authoa note that the presence of degenerative arthritis in the lumbar spine of these monkeys may have affected their results. Their scatter plots of aredweight-adjusted BMC by age with fitted regression lines are sirnila to the ones presented in this thesis, but their data is much more scattered, i.e., more variable, most likely due to the presence of vertebral osteophytosis. The authon also cite the limited number of postmenopausd animals in their study, and the lack of decline in weight with advancing age among these hedthy monkeys as possible sources of bias. Champ and colleagues conclude that the use of the rhesus monkey to mode1 age-relaied bone loss mûy be problematic, and ihat any reduction in bone mineral with age in these monkeys will be most apparent at the distal radius. The author of this thesis disagrees with this conclusion; the results presented herein indicate that both female and male free-ranging rhesus monkeys provide good models for the study of human bone minera1 density. Rhesus females from the Cayo Santiago population are a good mode1 of age-related bone loss, and DEXA of the lumbar venebra is a diable site of measurement provided that al1 specimens with modente to advanced vertebrai osteophytosis and degenerative disc disease are excluded from the andysis. If an entire vertebra, or a complete section of spine is to k scanned and analyzed, then it is recornmended that bones with OA of the zygapophyseal joints also be excluded. Longitudinal or cross-sectional smdies using Live animals should exclude individuals with vertebral osteophytosis, degenerative disc disease or vertebrai osteoarthritis. The skeletal remains of the free-ranging rhesus monkeys of Cayo Santiago have provided the oppominity for a population-based cross-sectional study of bone density. and naturally-occurring osteopenia and osteoporosis. The main conclusions of this pan of the study are as follows: Maies acquire bone mass at a faster rate and reach a higher peak BMD at an earlier age compared to females. in contmt to humans, rhesus males attain a peak BMD at an earlier age than rhesus females. Thus. as non-human primate models for human bone density, osteopenia and osteoporosis, these differences should be noted in future studies. Mdes have a higher BMD than femaies at every age after the age of sexuai maturity (3 years of age). i Osteopenic and osteoporotic monkeys are present, at a frequency of 12.4% (N = 298). The bone minerd density values of the osteoporotic (fractured) monkeys in this study are aenerallv hinher than those of virtudly JI of the osteopenic/osteoporotic (unfractured) monkeys. This pattern supports the view that low bone mas rnay not aiwavs orecede fracture. The curent study supports the clinical data that reveals that BMD done is not a gwd predictor of fracture risk. Other factors such as bone quaüty should aiso be considered when assessing risk of Fracture; the pattern of bone loss appears to be as important as the quantity of bone loss. While BMD gives an indication of how much bone loss has occurred, the pattern of trabecuiar resorption provides an indication of whether the bone will fracture. These results also suggest that the standard criteria for diagnosing osteopenia and osteoporosis should be revised to reflect these findings. i As a model for osteoporosis, it is recommended that only mature rhesus monkeys pst the age of peak bone mus should be used in studies of bone remodelling - males older than 7 years, and femaies over the age of 10 yeus. This study concludes that both rhesus males and femaies provide good models for the study of human bone minerai density, osteoporosis and osteopenia, provided that the limitations of the model are considered, i Vertebral bones are reliable sites of measurnent for DEXA provided that dl specimens with moderate to advanced vertebrai osteophytosis, degenerative disc disease and osteoanhntis are excluded from the iuiiilysis. THE EFFECT OF PARITY, OSTEOPENIA AND OSTEOPOROSIS ON BMD IN FE:MALES 4.1. Results: Parity and Bone Density Paritv: Frwuencies A histognm of parity (Fig. 33). shows the frequencies of parity O to 16 inclusive (N = 119) in the sarnple of females aged 4 years and older. The data consist of the total number of offspnng for each femaie in the sample; both live- and still-births are included in this andysis. Information on time since lait live-birth or time since the last perîod of lactationlnursing is not available. Twinning is rare in rhesus rnonkeys; only one female in this sample may have twinned once. A parity of 7 has the highest frequency (15) in this sample. The frequencies of parities greater thon 7 (8 to 16) are lower than those of 1 to 7 inclusive. Table 5 summarizes the descriptive statistics of this sarnple of fernales (N = 1 19). The mean age of females in this sample is 1 1.4 years. and the age range is 4.0 to 22.2 yem. The mean parity is 6.4. the median is 6.0, and parity ranges from O to 16 offspring. Paritv vs. Ape A plot of parity relative to age (Fig. 34) shows their inter-relationship across the Iife span; rhesus females reach sexual maturity at 3 years of age and usudly give birth to their fust offspring at age 4 years. A strong positive correlation between paity and age is demonstrated in Figure 34. The line on the scatter plot delineates the maximum nurnber of births that can be attined by a given year of life: it illustntes the fecundity, or biologicd potential for reproduction in this colony. At age 4 years, the maximum number of offspring is 1. and for every year beyond ihis age. the maximum number of possible offspring increases by 1; thus, Puity = (Age - 4) + 1 describes the dope of the line of potentiai parity. In this sample, the number of offspnng iunong older femdes tends to be more variable: the parity of fernales over 14 yem of age is more variable than that of younger age cohorts. In Figure 34, the data tend to deviate further from the regression line after age 14 years. This shows that from this age onward. femdes are less likely to approximate the ideal parity level indicûted by the regression line; this may be due to biological fûctoa such as degenerative diseose and/or social factors, which may have an adverse effect on parity (see below). Figure 35 shows the same scatter plot with a smooth fit based on a Poisson regression mode1 for the number of offspring as a function of age. It illusuates the running average parity across the Iife span, showing a smooth increase with advancing age, after age 4 years. Figure 35 shows a strong positive correlation between parity and age, but the curve tapers off after age 15 years; the siop of the curve is not as steep in the older age range. The oldest femde in the sample is 22.2 years of age, and the highest pacity is 16 offspring. Parity and age are closely associated variables (correlation = 0.84); age is generally a good predictor of parity and vice versa. In order to examine the ielationship be~een parity and BMD, age must be kept constant. or controlled in some rnan.net. h order to test the hypothesis that pWty has a positive effect on BMD, two nulliparous femaies 8.5 to 9 years of age were compared to parous females in the age range of 8.5 to 9.4 years (N = 13), using a two-sarnple t-test (Table 6); in the control group (parous females), parity ranges from one to six. Although the sample sizes in this malysis were smdl (Table 6), the data suggest that there is a statisticdly significant difference between the mean BMD (p = 0.008) and mean BMC (p = 0.03) values of the nulliparous and parous females; the means are significanily higher in the parous females. The mean BMD of the nulliparous females = 0.459 @cm2 and that of the parous females = 0.707 @m2; the mean BMC of the nulliparous lemdes = 1.O 1 gram and that of the parous females = 1.49 gram (Table 6). There is no significimt difference in mean bone area between the two groups (nulliparous = 2.24 cm 2. parous = 2.1 1 cm2; p = 0.38) (Table 6). It is notewonhy that one of the nulliparous females in this sample (Cat. a088 in Tables 3 and 7) is osteoporotic, and many of the low-parity females listed in Table 7 are also osteopenic or osteoporotic. The relationship between parity and BMD is examined more closely in the next section, TABLE S DESCRIPTIVE STATISTICS ON CAYO SANTIAGO FEMALES 2 4 YEARS OF AGE (N = 119) Age Parity BMC AREA BMD at Death mm@ (cm2) Wm2) 6fm Mean 11.38 6.4 1.493 2.151 0.693 Standard Deviation 4- 15 3.8 0.349 0.234 0,130 Maximum 22.2 16.0 2.540 2.840 1,050 Minimum 4.0 O 0.750 1.560 0.380 Median 11-0 6.0 1.480 2.129 0.701 TABLE 6 COMPARISON OF NULLLPAROUS AND PAROUS FEMALES IN THE SAME AGE RANGE (8.5 - 9.4 YRS.) 1 N 1 Mean BMD 1 Mean BMC 1 Mean Area Pamus Females + 13 0.707 1.49 2.1 1 P Value p = 0.008 p = 0.03 p = 0.38 Parity range: 1 - 6 inclusive. TABLE 7 Disease Age at Par@ Natal BMC AREA BMD Cat, No, Statusf Death ~roup(gram) (cm2) wem2) $ m-1 - d m 9- 12Ylepr~- 2088 OPO,VOA 8.5 O J 2179 Normal 9.0 O J 2'164 Normal 9.0 1 F 2213 Normal 9.0 L J 804 OPE 9.9 3 J 2208 OPE 12.0 4 M Median Parity, Category A 1.0 Median Parity, Control group 6.0 * Mean BMD,Category A 0.5 18 &m2 * Mean BMD, Control group 0.735 g/cm2 * 14 - 18 Yem: Cm 677 OPE 13.9 5 F 2 160 OPO,VOA,early DISH 14.0 6 J 1968 VOA, L7lS 1 VO § 14.5 7 J 2055 OPE, VOA (mild) 15.0 7 J 450 VO and VOA (mild) 16-6 7 A 670 VO (mild) 17.2 8 J 684 OPO(Fx),VOA,VO, DDD 18.0 8 A Mean Parity, Category B 6.9 Mean Parity, Control group 11.4 * Mean BMD, Category B 0.633 g/cm2 ** Mean BMD,Control group 0.763 @cm2 * * m 275 VOA, VO 21.0 11 U 1.179 2967 OPE, VOA, VO 22.0 13 F 1.140 2968 0P0(F~)JiOA(mild),V0 22.0 8 L 1.434 Mean Parity, Category C 10.7 *** Mean Parity, Control pup 153 *** Mean BMD, Category C 0.652 g/cm2 **** Mean BMD,Control group 0.650 glcrn2 **** I I *Statistically significanî, p < 0.000 1; **sîatktically signifkant, p = 0.02; O** Statistically signifiant, p = 0.04; +***Not statistically significant, p = 0.10 kJ = Unknom Natal Gmup tDbuwr: OPE = Osteopenia; OP0 = Osteoporosis; VOA = Vertebral Osteoarthritis (zygapophyseal joints), asscssod in lumbar spine only; VO = Vcrcebral ûsteophytosis, assesseci in Iumbar spuie oaly DDD = Degenerative Disc D-, assesscd in hmbar spine only; DISH = Diffulre idiopaîhic siceletai hypcrostosis; Fx = Vcrtebral wcdge bctm QVOofjoint between kt lmbirv~braand 1st sacral segment As mentioned p reviously . generalized additive models (GAMs) were used to study the relationships among the variables BMD,parity and age in the sarnple of fernales aged 4 yem and older (N = 119). Through the use of GAMs. it was possible to examine the relationship ktween parity and BMD while controlling for the effects of age, and to investigate the relationship between BMD and age after controlling for parity. With BMD as the response variable. non-Iinear versus linear models of parity and age were tested. Four models were fit to the regression of BMD on age and parity. The hierarchy of models tested was as follows: Model 1: BMD - smooth (Age) + smooth (Parity) Model 2: BMD - smooth (Age) + linear (Parity) Model 3: BMD - linear (Age) + smooth (Parîty) Model 4: BMD - linear (Age) + linear (Parity) The results show that the model that has both parity and age as smooth terms (model 1) provides a better fit than models that contain either parity or age as a linear term, or both variables as linear terms (p = O.ûû7). This is probnbly due to the strong correlation between age and parity. The best model is the one that contains both parity and age as smooth tenns, and is depicted in Figure 36. After controlling for age by using GAMs, the results show that there is a significant positive effect of parïty on BMD. BMD increases with increosing pyity (p = 0.0006); the models tested were 1) BMD - smooth (Age) + linear (Paity) and 2) BMD - smooth (Age). After controlling for parity by GAMs, BMD decreases as animais age; the p value for comparing the two models 1) BMD - iinear (Age) + smooth (Parity) and 2) BMD - smooth (Parity) is 0.0 15. Thus. the decreasing trend in BMD with age îs statisticaily significant The increasing trend in BMD with parity is also signifcant. Figures 36 to 38 provide graphic illustrations of these relationships. Figure 36 shows the results of the GAM tests on the effects of age and parity on BMD. The two graphs show the additive fit for par@ and age, with BMD as the response variable. and two predictors - parity and age. The model depicted in this figure is BMD - smooth (Age) + smooth (Parity). Parity and age are estimated individually by a scatter plot smoother as smooth functions. With BMD as the response variable, the smooth function of p;uity is plotted versus parity, and the smooth function of age is plotted versus age. The functions are fitted jointly by itentively smoothing partial residuds, dso known as backfitting. With GAM. once the additive model is fitted to the data the roles of the predictors in modelling the response cm be examined separately. Hence, the plots in Figure 36 illustrate the roles of parity and age sepmtely. The dashed lines in each plot represent the 95% confidence band. The Y-axis in both plots shows the deviation from the ovedi mean BMD. which is why it is centered rt zero. The effects of both pacity and age appear to be non-linear. Parity has an increasing effect on BMD; the first plot shows a positive correlation between BMD and parity. Age has a decreasing effect on BMD from about 9 years of age. as shown by the second plot, which depicts a negative correlation. Thus, in the absence of aging, parity has a continuous, increasing effect on BMD. By itself, age has a decreasing effect on BMD. which begins immediately after peak bone density is attained, as seen in the second plot. Therefore, parity appears to mitigate the effects of aging on BMD; the plot of BMD versus age in females (Figure 10) shows that, in reaiity, BMD decreases with advancing age after 17 years. The relationship between BMD and parity was also explond using conditioning plots (CO-plots) and local estimation xatter plot smoothing (LOESS). As described eulier, the CO-plotailows one to examine the relationship between two variables while controllhg for the effects of a third variable; the cuves are fitted by LOESS. This method provides another graphic illustration of the relationships discussed above. --BMD vs. Pari&%Conditioned on Age !Fige 37, Codotsl Figure 37 is a CO-plotof BMD versus parity. conditioned on age. Recall that the CO-piot is a graphitai presentation of a the-dimensional relationship. and that one should imagine the dependence of BMD on age and parity in three dimensions as a 3-D surface of average BMD over a grid of points of age and parity. As described earlier. the CO-plotthat conditions on age takes slices ttuough this surface for a number of ages. Each BMD venus parity plot contains animals with similar values of age, and a number of plots or panels are usudly generated. Bar A at the top of Figure 37 represents age incnments of 4.0 to 12.5 Yeats, and bar B consists of age increments of 9 to 22.2 years. Bar A corresponds to plot A below it, and bar B corresponds to plot B. The shape of the BMD-pYity cuve is the same for al1 the ages represented in bar A. so the reldonship is surnmarized using only one plot (A). In other words, the shape of the BMD-parity curve was identical in a number of panels that took "slices" through al1 the ages represented by bar A; thus. only one panel is necessary to show this relationship. The same is tme for age bar B and plot B. Thus, only two plots are required to summarize the relationship between parity and BMD. As stated earlier, there is 50% overlap between the two plots, which is the defauli configuration of the program; it ensures that sample sizes are comparable between the gnphs, and ais0 ensures continuity between them. The association between BMD and parity is significant. The fint plot (A) shows a positive correlation between BMD and parity; among the age increments of 4 to 12.5 yem, BMD increases with increasing parity. The second plot (B) also shows that BMD increases with parity, but only up to a pacity of about 7 offspring; there is no effect of parity on BMD beyond 7 offspring (flat line). hdividuds older than 12 years of age and with parities higher than 7 have a different relationship between BMD and parity compared to younger animals. This relationship is explored further in Figure 38. --BMD vs. Age, Conditiond on Parib (Fiiz. 38. Co-~lots) Figure 38 is a CO-plotof BMD versus age, conditioned on parity. This pph also represents a three-dimensional relûtionship, this time for BMD over a grid of points of age and parity, with 'slices' taken through a number of pyities. Bar A at the top of Figure 38 represents parities O - 7, and bar B represents pdties 5 - 16. As before. bar A corresponds to plot A and bar B corresponds to plot B, and there is 50% overlap between the two plots. Due to a similacity in BMD-age curves across different parities, only two plots are required to summarize the relationship between BMD and age, conditioned on parity. Plot A shows that among the lower parities, BMD increaxs with increasing age up to about 10 yem; there is a positive correlation up to a parity of about 7. Parity greater than 7 is associated with a steep decüne in BMDwith increasing age, as shown in plot B. Thus, high parity initially has a positive effect on BMD, but parity higher than 7 may rniaiiy be detrimental (p = 0.0006). Figures 37 and 38 show that piuity has a positive effect on BMD,but only up to a certain age and parity. After a certain age. the positive effect of parity on bone density appears to be overwhelmed by the aging process. 4.2. Results: Low Parity, Osteopenia and Osteopomis Since there is a relationship between parity and BMD, this section examines the relationship between low parity, OPEIOPO and BMD. Sixteen females (28 yean of age) with Iow age-adjusted parîty occur in this sample (N = 119). They are represented by points well below the curves in the parity versus age pphs in Figures 34 and 35. Since BMD varies with age. Table 7, which lists these fernales, groups them according to three age categories; there are six low-parity females in age range 9 to 12 years (Category A), seven females in the age range fiom 14 to 18 years (Category B). and three are over 20 years of age (Category C). Since age and parity are closely associated variables, these groupings of low-parity females were compared to control groups of age-matched females. in this manner. age is controlled while parity and BMD of these low-parity females are compared with those of their age-matched cohon. Table 7 summarizes the results. There is a statistically significant difference between the median parity of Category A (1.0) and that of its control group (6.0) (Mann-Whitney Rank Sum Test. p < 0.0001). T-tests reveai that there are dso significant differences between the mean parities of Categories B (6.9) and C (10.7) and those of their respective control groups (mean parity for B's control group = 1 1.4. p < 0.000 1; menn parity for C's control group = 15.3. p = 0.04). T-tests show that there is a significant ciifference between the mean BMD of Category A (0.5 18 g/cm2) and that of its control group (0.735 @m2) (p < 0.000 1); the mean BMD of Category B (0.633 @cm2) is also significantiy different from that of its control group (0.763 @cm2) (p = 0.02). However, no significant difference was found between the mean BMDs of Category C (0.652 g/cm2) and its control group (0.650 g/cm2) (p = 0.10); both groups in Category C had relatively low BMD. Thus, the BMDs of the low-pmity females in Categories A and B are significantly lower than those of their respective control groups. It is noteworthy that three low-parity females in Category A and thein Category B have ken diagnosed with osteopenia/osteoporosis~according to the standard criteria discussed previously. An additional fernale in Category B has osteoporosis, which was determined by the presence of vertebral fnctures. There is one osteopenic and one osteoporotic (fractured) fernale in Category C. Other diseases are also pnsent among these low-parity females. as listed in Table 7; only diseases and disorders affecting the lumbar spine are recorded in this table. The above cornparisons were npeated, using oniy the low parity OPUOPO individuals in each of the age categories. T-tests showed that the mean BMD of the low-parity OPUOPO females in Category A (0.466 g,/cm2) is significantly lower than that of their control group (p = 0.0004); the mean BMD of the OPEiOPO fernales in Category B (0.549 @cm2) is also significantiy lower than that of their control group (p = 0.003). These results offer further evidence that low BMD. low parity and OPUOPO are associated variables. 4.3. Rosults: Low Parity, OsteopenialOsteopomis and Natal Group Affiliation Also noteworthy is the fact that almost half of the low-piuity females listed in Table 7 were bom in the same natal group - Group I. which also includes four of the nine osteopenic/osteoporotic individuals. Twenty percent of the entire sample over age 8 years is from Group J, yet 44% of the low-parity and the osteopenidosteoporotic individuals are fiom Group J. The representation of Group J in the sample over 8 years is simiiar to that of Group F (19%) and Group A (2146). Group I fomed in 1964 when the majority of individuals ftom two matemal genealogies split from the colony's original Group A (Sade et al. 1976). These results hint at a possible genetic component to OPE/OPO in this colony, and it warrants fiuther investigation. 4.4. Results: Parity and Vertebral Osteophytosbgenerative Dise Disease The bones with vertebnl osteophytosis (VO) and degenentive disc disease (DDD), which were originaily removed from the sarnple, were re-introduced in this section of the study in order to investigate the effect of VO/DDD on parity. This sarnple (N = 137) was divided into 3 groups based on the degree of VO/DDD (i.e., based on the presence and degne of osteophytosis and remodelling of the centrum: Group i (Normal), Group 2 (Mild VOIDDD), and Group 3 (Moderate to Severe VOIDDD). The protocol for assessing and recording VO and DDD was defined in the chapter on methods. A plot of group membenhip as a function of age and parity (Figure 39) shows that age and parity are strongiy correlated variables, and that older femdes with high parity tend to be in Groups 2 and 3; they are represented by the grey and blrk symbols nspectively in Figure 39. The youngest female in Group 2 is 9 years of age, while Group 3 includes only females 14 years of age and older. Thus, one cm roughly predict membership in the 3 groups when yeand parity are known; however, the three groups overlap. For example, of the 50 females within the age range of 14 to 24 years, there are eighteen individuais in Group 3, fourteen in Group 2 and eighteen in Group 1. The generalized additive mode1 was used to test the effect of VO/DDD group membeahip on parity in females 14 years of age and over; no group effect was evident in this older age group (p = 0.73). but age was found to be a signifcant factor (p = 0.012). Box plots of parity versus degree of osteophytosisDDD in al1 mature animûls (4+ years of age) show that the median puity of Group L (median = 5) is significanîiy lower than the median parities of Groups 2 and 3. which have similar median parities of 10 and 1 I respectively (Fig. 40). Also noteworthy is the fact that in the Gmup 3 box plot, the median and 25th percentile have the same value. The almost identical media piuities of Groups 2 and 3 confirm ihat there is some overlap in age between these two groups, as seen in Figure 39. Group 2 females range in age lrom 9 to 22 years, and range in parity from 5 to 16; Group 3 femaies have an age range of 14 to 24 years, and a parity range of 9 to 16 offsphg. Figure 39 dso shows that most of the normal individuais (Group 1. white symbols), tend to cluster, as a group, in the age range of 4 to 15 years. The mean parities of the three VOIDDD groups were compared by ANOVA. There is a significant difference between the mean parity of Group 1 (5.7) and Group 2 (10.2). and between the mean parities of Group 1 and Group 3 (1 1.7); however. there is no significant difference between the average pdties of Groups 2 and 3 (p c 0.000 1). Most of the females 14 yem of age and older have both high parity and VO/DDD (Fig. 39). Vertebnl osteophytosis and degenerative disc dixase are age-related conditions; the individuals in this satnple who exhibit severe manifestations of these conditions (black symbols in Fig. 39) are 14+ yean of age and older, which corresponds to middle ageladvanced dulthood for rhesus monkeys. These individuals. particularly those with severe osteophytosis. also have abnomdly elevated BMC and BMD compared to age-matched controls, which is a result of VODDD. There is no apparent negative effect of VOmDD on pacity, as most of the individuais with severe disease have attained high parities despite the presence of these conditions. As mentioned previously, statistical tests have confimed that there is no group effect on parity. However, there are some low-parity females who are no& with respect to lumbar VOIDDD, but bave osteopenia or osteoporosis, and two females exhibit both VOlDDD and established osteo~orosis(i.e., vertebral fractures). but have normal bone mass (Table 7). In di three age gmups, the statistically significant diffennces in BMD found between the low-parity individuals listed in Table 7 and their respective control groups cmprobably be attnbuted to osteopenia. The data suggest that low parity is related to low bone mass. It is also possible that low pmity and Iow bone mass are both related to another factor, such as membeahip in Group J. The other diseases or disorders listed in Table 7 have no apparent comlation with parity. Parity Fig. 33. Histogram of parity in femaies 2 4 years of age (N = 119). Parity of 7 has the highest fiequency in this sample. Age (years) Fig. 34. Parity versus age (years), showing maximum binhs per year in fermies 2 4 years of age OJ = 1 19). Parity = (Age - 4) +1 describes the slope of the Iine. At 4 years of age, the age of first reproduction, the maximum nurnber of offspring is 1, and for eveiy year beyond this age, the maximum number of offspring possible increases by 1. Fig. 35. Parity versus age (years), with smoothing spline fit, in females L 4 years of age (N = 1 19). The curve provides a ninoing average parity across the life span, showing a smooth increase with advancing age, after age 4 years. BMD vs Parity and A e Smooth Terms in Age an f Parity / - I Il. II. .I. . 41 tt II1 l lil Ili i I 1 I 1 O 5 10 15 Parity Fig. 36. Generalized Additive Models: The additive fit for pdty and age, with BMD as the response variable. The dashed lines represent the 95% confidence band. Given : Age Parity Fig. 37. BMD by Parity, Conditioned oa Age. in females 2 4 years (N = 119). Conditionhg plot (Co-plot) with local estimation scatter plot smoothing (LOESS). A = Aga 4.0 to 12.5 years; B = Ages 9.0 to 222 years. Given : Parity Fig. 38. BMD by Age. Conditioned on Parity, in females 2 4 years of age (N = 119). Conditionhg plot (Co-piot) with local estunation scatter plot smmthing (LOESS). A = Parities of O to 7 offsp~g;B = Parities of 5 to 16 offiring. 0 Group 1 @ Group2 Group3 Fig. 39. Parity by age and vertebrai ostwphytosis/degenerative disc disease (VOIDDD) in fernales 2 4 years of age (N = 137). Scatter plot showing parity versus age, in years. VO/DDD Group 1 = Normal (white synbols); VOIDDD Group 2 = Mild (grey synrbols); VODDD Group 3 = Moderate/Severe (bluck symbols). VODDD group membership is assessed on lumbar vertebrae used for DEXA analysis. 4.5. Discussion: Parity and BMD --Studies on Human Fernales The onset of menopause causes changes in bone mus in human femaies. An accelerated rate of bone loss from the spine occurs in perimenopausal women with irregular menses, whereas acceleration of bone loss from the radius does not begin until after the complete cessation of menses (Johnston Ir. et al. 1985). Bone loss from the spine rnay begin before the cessation of menses, and is comlated with decreases in estrogen production and increases in bone remodelling (Johnston Ir. et al. 1985). Stevenson et ai. (1989) found that ;i decrease in bone density of the spine and proximal femur is mainly attributed to menopause: in postmenopausal women, the decline in bone density is more closely related to time since postmenopause than to chronoiogical age (Stevenson et al. 1989). A cross-sectional study of lumbar spine and forem BMD in women who were 6 months to 10 yew postmenopausal found that the spine shows the highest initial rate of decline in BMD after menopause: postmenopausal bone loss is rnost pronounced in skeletal sites with the highest proportions of trabecular bone (Bjamason et al. 1995). A study by Melton IIi et al. ( 1993) found that menopausal status is a significant predictor of bone minerai density of the lumbar spine (Melton III et al. 1993). Riggs and Nelson (1985) show that some postmenopausal osteoporotic females may have senun vitmin D levels that are much lower than age-matched subjects. wbich indicates that. for some osteoporotics, reduced vitamin D activity may be partly responsible for theù bone loss (Riggs and Nelson 1985). Preenancy mi Lactation Pregnancy and lactation impose stress on the calcium homeostasis of the mother, particuiarly on the skeleton, which is the main repository of calcium in the body. Maternai cdciurn homeostasis is challenged primwily during the 1st trimester of pregnancy when the fetal skeleton is mineralized, and during the subsequent penod of lactation, when calcium dernmds are rven pater than during pregnancy. There is, however, a cornplex hormonal regulatory mechanism present in the matemal body which effectively counteracts bone loss during pregnancy and lactation by retaining excess calcium in the circulation (Kumai, Cohen, and Epstein 1980; Wieland et al. 1980). Levels of estrogen, progesterone md human placental lactogen are high during pregnancy, and levels of prolactin are high during lactation. The matemal body responds to the increased calcium demands of pregnancy and lactation by increased bone resorption, decreased bone formation, increased calcium absorption from the gut (by increasing vitamin Dj), and decreased unnary excretion of calcium. An increased level of parathyroid hormone (PTH) late in pregnancy enhances vitamin D3 synthesis and reduces calcium excretion (Kumar, Cohen, and Epstein 1980; Stevenson et d. 1979; Wieland et al. 1980). The ovedl net effect of these changes is to increase stores of calcium in the matemal body early in pregnancy, in anticipation of its increased need later on, especiaüy during lactation. Many studies have assessed and debated the impact of pregnancy and lactation on the human matemal skeleton, with rather contlicting conclusions. Some retrospective and prospective studies report ùicreased bone density with parity (Aioia et al. 1983; Fox et al. 1993; Goldsmith and Johnston 1975; Murphy et ai. 1994; Nilsson 1969; Sowen et al. 199 1; Valenzuela et al. 1987). some prospective studies show constant bone mass during pregnancy (Christiansen, R~dbro, and Heinild 1976). while others reveal significant bone loss (Drinkwater and Chesnut ID 1991; Hayslip et al. 1989; Lissner. Bengtsson, and Hansson 1991; Yamamoto et al. 1994). One prospective study by Valenzuela et al. (1987) shows that pregnancy in human femaies appears to be associated with small, but significant. increases in bone formation, which is supported by findings of an increase in biochemical indicators of bone formation even early in pregnancy (Valenzuela et al. 1987). Another report cites nulliparity as one of the factors associated with decreased bone density arnong women. which thus increases risk for osteoporosis (Stevenson et ai. 1989). With regard to the effect of lactation on bone mas, some retrospective studies show no effect on bone density (Waiker 1972). others report an increase in bone mass (Dequeker et al. 1987b; Melton iIi et al. 1993). while others show a decrease in bone density (Atkinson and West 1970; Goldsmith and Johnston 1975; Lissner, Bengtsson. and Hansson 1991; Wardlaw and Pike 1986). Hqslip et al. (1989) have shown that the extraction of calcium during lactation occun preferentially from trabecular bone sites like the spine in healthy, postpartum women (Hayslip et al. 1989). Other studies have confirmed that extraction of calcium during pregnancy and lactation occurs preferentially from trabecular bone (Kent et al. 1990; Lûmke. Brundin, and Moberg 1977). and that corticai bone appears to be unaffected by prepancy or lactation (Christiansen, Rdbro, and Heinild 1976; Fnsancho, Gam, and Ascoli 197 1; Me, Brundin, and Moberg 197î). While some snidies show that bone loss occurs with pregnancy and Lactation, othea reveal that this loss in bone mass is tmsitory, and is later recovered bv the mother (Drinkwater and Chesnut III 199 1; Kent et al. 1990; Larnke, Brundin, and Moberg 1977; Mpez et ai. 1996; Sowers et al. 1993). A radiological study that compmd bone mas in Bantu and Caucasian mothea in South Africa Coud that multiple pregnancies and long lactation in both cohorts did not result in signifkant bone loss, despite a relatively low calcium intake. Mothea of large families do not suffer from sustained cdcium depletion, and any negative calcium balances that might occur from multiple pregnancies and long lactation are not cumulative. Losses are offset by more efficient absorption of ingested calcium (Wdker, Richardson, and Walker 1972). Another study by Kent et al. (1990) confirms chat lactation in humans is associated with trabecular bone loss from the foreann and with increased bone tuniover; there is, however, rend conservation of calcium and bone mass is recovered after weaning. After weaning, the mother experiences a petiod of bone accretion mediated by an imbalance between a normal bone resorption rate and an elevated bone formation rate, which may 1st from a few months to a few yevs (Kent et ai. 1990). Sowers et al (1993) also found that changes occur in bone density with lactation, and that the extraction of calcium occurs preferentiaily from mbecular bone. This study dso confirms that, after weaning, the mother experiences a period of bone accretion which cm occur over a few rnonths or even years (Sowen et al. L993). Koetting and Wardlaw (1988) report that the bone density of women with a history of long term lactation for up to four offspring does not differ from that of nulliparous women. suggesting that recovery of bone mass took place (Koetting and Wardlaw 1988). Drinkwater and Chesnut (1991) found that Iumbar BMD values decrease dunng pregnancy, but return to pre-prepancy levels during lactation (Drinkwater and Chesnut DI 199 1). A study by Melton III et al. ( 1993) reports that. for the women in their study (N = 304). a history of breastf'eeding is associated with unchanged or slightly increased bone density at all skeletal sites measured. bcluding the lumbar spine, femur, and radius (Melton JIi et al. 1993). In this study, the presence or absence of breastfeeding, and the duration of breastfeeding are not associated with reduced bone minerai. but breastfeeding for more than eight months, which was the median duration of nursing, is associated with greater bone density at some sites, even afier age adjustment (Melton III et al. 1993). Other reproductive variables such as puity, age at menarche, age at fmt pregaancy. use of oral contraceptives or estrogen replacement therapy were also tested, but yielded no consistent effects on bone minerai. even after age adjustment. The authors found a strong protective effect of obesity (body mas index). which was dso correlated with some of the reproductive variables. They note that the positive effects of lactation have generaily been attributed to an association with parity. and with increased weight (Melton III et al. 1993). Drinkwater and Chesnut (199 1) dso cite weight gain as a factor in the changes in bone mass that occur during pregnancy and lactation (Drinkwater and Chesnut tïi 199 1). Thus, in humans, bone losses due to lactation are eventually recuperated by the mother, and while some studies show short-tenn losses in maternai bone mm,studies such as that of Koetting and Wardlaw (1988). Melton iü et al. (1993). Sowea et al. (1993). Kent et ai. (1990) and othea, suggest that there are no long-terni deletenous effects. However, Galloway (1988, cited in Galloway 1997) examined the long term effects of reproductive history on bone mineral content in women. and found that in some women the periods between cycles of pregnancy and lactation may have been insufficient for the recovery of bone, and this may be responsible for overall lower bone mineral status (Galloway 1997). Another study by Sowers et al. (1985) found that women who experienced their fint ~reancv ~norto the aee of 20 ~earshave lower than expected mid-distai radiai bone minera1 content, which suggests ihat a pregnancy dunng the teenage yem. i.e., during the years in wbich maximal bone mineraiization usudly occurs, resuits in insufficient subseauent recoverv of bone mass by the mother (Sowers, Wallace, and Lemke 1985). Fox (1993) dso noted that, compared to older mothers. the average bone density of the proximal radius is significantly lower among women who had their fust child in their teenage years. In human females, linear growth ceases at about ûge 20 years, but the bones continue to mineralize until a peak bone mss is achieved in the third decade of life (mid- to late 20's) (Matkovic 1992; Recker et al. 1992). The results obtained by Galloway and Sowen suggest that subject selection rnay account for some of the inconclusive or conflicting data in the litenture. Studies which show no correlation between parity and BMD,or a negative relationship between parity and bone density, rnay have failed to detect a positive relationship because of subject selection, choice of sites for bone measurements, small sarnple size, or failure to control for lifestyle variables (e-g. physicd activity, use of hormones, caicium supplements, smoking, alcohol). Lifestyle variables that have a negative effect on bone density include alcohol and cigarette consumption, low body weight, lack of use of orai contraceptives, and lack of regular exercise (Stevenson et al. 1989). Al1 of these factors rnay account for the inconsistent reports found in the literature. It is also possible that some bone density stuâies rnay have fGled to exclude subjects who exhibit diseases that introduce bias into a DEXA simple - such as al1 types of arthritis, and other conditions that cause the formation of bone spurs. The literature Jso records an enigmatic condition known as "pregnancy-associated osteoporosis" (Dunne et al. 1993: Khastgir and Snidd 1994; Nilsson 1969: Nordin and Roper 1995; Smith et ai. 1985; Yamamoto et al. 1994). Since plasma estradiol is high during pregnancy, the appearance of osteoporotic fractures of the spine in some pregnant women is unusud. particularly since osteoporosis is genedy associated with estrogen de ficiency. Al though vectebral compression fractures are the most common Fractures, fnctures of the hip, nis and pubic rami also occur in pregnancy-associated osteoporosis (Dunne et al. 1993). Since osteoporosis can occur even before the onset of lactation or even in its absence, lactation is discounted as a possible cause of this condition Ynong pregnant women (Dume et al. 1993). According to some reports, it appears that prrgnmcy is an accidental, not a causai factor of this idiopathic osteoporosis. The failure of hormones to prepare the maternai skeleton for the incrrased demands of pregnancy and lactation has ken cited as the probable cause of osteoporosis in these cases; for example, low levels of vitamin Dj and calcitonin have been found in these women (Smith et al. 1985). Thus. a failure in caicium accretion hm ken postulated. A study by Dunne et al. (1993) is also suggestive of an underlying abnomaiity of the skeleton ptior to pregnancy in these cases of osteoporosis; the mothea of women with pregnancy-associated osteoporosis have a significantly higher prevalence of frachues occurring at an earlier age compared to a control group. An associated genetic component to this condition is also suggested by these studies (Dunne et al. 1993). Another study by Yamamoto et ai. (1994) concludes from bone densitometric and histomorphometric data that a pre-existing pathological condition is responsible for post-pregnancy osteoporosis. Bone loss occun due to a negative calcium balance during pregnancy and lactation, and bone mas may decrease befow the fracture threshold npidly in patients with low peak bone mass (Yamamoto et ai. 1994). Thus, among these women, pregnancy and lactation highlight and exacerbate a pn-existing abnormdity of the skeleton or hormonal regulating system, leading to a form of idiopathic osteoporosis. hcrease of Bone Densitv with Parity in Human Females In women, high parity has been associated with higher bone densities due to increased absorptive efficiency during pregnancy (Stini 1990). A study by Nilsson ( L969) of bone minera1 content of the femur. metacarpal and radius in 102 women aged 45 to 71 years with an average parity of 1.9, found a tendency towarâ a positive relationship between bone mass and parity. A significant increase in bone mass with parity was found in the cortical bone of the radius and metacarpal, and in the distai femur. Virtuaily al1 women in this study had breast-fed for a period of time, and malnutrition was not a risk fmtor in this population (Nilsson 1969). Nhon (1969) cautioned that the relationship between bone mass and parity found in rhis study may not exist in populations with high birth rates and poor nutrition. The author concludes that parity neither decreases bone mass nor causes fractures later in life. Hoffman et al. (1993) also show that increasing parity may provide protection against hip fracture. and that lactation probably confers no additional risk or üdvantage; lactation has neither a positive nor a negative effect once parity is controlled. After controlling for body mus, pûnty still hm a protective effect, which suggests that there iue other factors ksides weight gain during pregnancy that protect against future fractures (Hoffman et al. 1993). A retrospective study (N = 825) by Murphy et al. (1994) ais0 reports a positive rrlationship between parity and bone mas; significantiy higher bone mineral densities are found among parous women. After adjusting for age and body mass index, mean bone minerai density among parous women aged 41 to 76 years is reported to be significantly higher at the femoral neck, intertrochanter and Ward's triangle. Parity remains a significant independent predictor of bone mineral density at ail sites after controlling forage, body rnass index. menopausai status, breast-feeding status, use of oral contraceptives and hormone replacement therapy, and cigarette smoking. An average 1.0% increase in bone density per Kve birth is reported (Murphy et ai. 1994). At ail sites except for intectrochantenc BMD, the mean bone density of wornen with lactation histories is not significantly different from that of women who had never breast-fed their infmts; there is no relationship between BMD and dtuation of breasdeeding, and no relationship between age at fust pregnancy and BMD in this study. In addition to parity, the major determinants of BMD in the study by Murphy (1994) are age, body mass index and menopausai status (Murphy et ai. 1994). According to Murphy (1994), "...the combination of increased endogenous oestrogen, cdcitonin, intestinal calcium absorption and body weight coupled with decreased PTH levels may act in concert to produce small increases in materna1 bone mass during pregnancy" (p. 166). Thus, a possible mechanism by which parity protects against hip fracture is ihrough an increase in bone density that results from increased calcium storage during pregnancy; this increased bone mass is maintained post-partum. Parity rnay also protect against hip fracture by increased weight gain during pngnancy and weight-bearing after parturition (Hoffman et al. 1993). Funher evidence is provided by another study, which found that pregnancy is associated with increased postmenopausd bone density of the radius (Fox et al. 1993). Fox et aL(L993) compared the bone density of nullipnrous and parous women, and found that distal radial bone density was significantly lower in the nulliparous group. in the parous group, a significant positive slope for distal radius bone density was observed as births increased; bone density in the distal and proximd radius increased linearly with increasing puity. A 1.4% increase in bone density of the distd radius was observed with each additional birth (Fox et al. 1993). Aloia et al (1983) aiso report a positive correlation between number of pregnancies and distd radius and total body bone mineral content among 80 postmenopausal women (Aloia et al. 1983). It is interesting to note that a report by Stevenson (1989) cites nullioaritv as a risk factor for decreased bone density of the mine and osteo~rosisin women (Stevenson et ai. 1989). A prospective study by Sowers et al (1991) on pdty and hip BMD noted an increase in bone mass among women with lower body weight. Pregnant women who were smaller in body size at basehe were more iikely to gain bone mass at the femoral neck during pregnancy than wen their matched controls. Weight gain dunng pregnancy increases load-bearing and bone density, which are more pronounced in women with smaller body size; the femoral neck is a site where load-bearing rnay have the greatest impact in humans. Sowers et al. (1991) note that this body-size effect rnay not be observed in bone density measurements of the spine or radius, sites where the influence of additional weight loading during pregnmcy rnay be minimal (Sowers et al. 1991). This effect rnay be minimal in the spine and radius of bipedai humm fernales; however, in quadnipedal animds (such as the rhesus monkey), this additional weight rnay have a stronger influence on the bone density of these skeletd sites. Non-primate Animal Models A number of non-primate, smdl and large animal models of osteopenia have been developed (Draper 1994; Geusens 1992; Miller, Bowrnan, and Jee 1995). The ovariectomized rat model was developed for studying osteopenia caused by ovarian hormone deficiency, but this rnay not k an appropriate model of human postrnenopausal bone loss. Rats grow continuously for the duntion of the studies, and thus may be unsuitable as a model of a human disease that begins after the attainment of skeletd maturity (Kalu 199 1). Other disadvantages are evident The bone remodelling observed in rats appears to be different from that of humans; rats have more pronounced bone remodelling throughout life (Draper 1994). Rats have higher bone turnover at many skeletal sites compared to humans, and r different loading pattern on their skeletons (Kalu 1991; Mosekilde 1995). Despite these drawbacks, rats have also been used to model bone loss incurred through pregnancy and lactation. Skeletal loading dso differs between monkeys and humans. but at lest monkey models have the advantage of being based on a species that is genetically closer to humans. A number of animal models presented in Garel (1987) show that the mûximization of calcium accumulation in preparation for lactation is a strong manmalian characteristic (Garel 1987). Studies in rats confirm that pregnancy and lactation are associated with maternai bone loss from the femur (Currey and Hughes 1973; Ellinger et al. 1952; Peng et ai, 1987). Bone resorption is more prevdent during lactation; rats experience a 1525% loss of calcium from thigh bones during lactation (Gare1 L987). An early expenmental study in rats shows that lactation is associated with maternai bone loss. and that this loss is exacerbated in the presence of calcium deficiency (Rasmussen 1977). in vitamin D deficient rats. the hormone prolactin stimulates increased intestinai absorption of calcium and mobilization of calcium from bone (Pahuja and DeLuca 198 1). The beagle dog was developed as a large animal mode1 for bone loss. but it too lacks complete concordance with the human skeletd response to estrogen depletion (Miller. Bowman. and Jee 1995). Studies show that ovariectorny in beagle dogs results in 15% trakcular bone loss in the spine without compromising bone strength (Martin et al. 1987), and a cdcium deficient diet results in pronounced fernorai bone loss (Geusens 1992). --Studies on Non-human Primates Macaques as a Mode1 A study conducted on the Cayo Santiago rhesus monkeys shows that the rate of intracortical bone turnover in the macaque femur is slower than in humans (Burr 1992). but that these monkeys may be good models for some human skeletal diseases like senile osteoporosis, provided that these physiological differences are adequately considered. Macaques serve as a good mode1 for osteopenia caused by estrogen depletion; bone loss induced by estrogen depletion is well established in macaques (Jerome et al. 1994; Jerome, Lees, and Weaver 1995; Longcope et al. 1989). For example, Longcope et al. ( 1989) compared ovuiectomized femaie rhesus monkeys to intact individuds. and found that ovmiectomy results in a relatively rapid decline in vertebral bone minerai density. in rhesus females who were postmenopausal (over 30 years of age) in the Longcope study, reduction in bone density was related to estrogen deficiency. Comparative data on parity is not provided by this study (Longcope et ai. 1989). Studies show that spontaneous menopause in the fernale rhesus monkey does not occur until the second half of the third decade of life, between 25 and 30 yem of age, which is near the end of the life span. This phase of the reproductive cycle in rhesus monkeys closely resembles that of the human femaie in physiology (Hodgen et ai. 1977; Van Wagenen 1970; Walker 1995). There is a genetic component to bone mineral density (Momson et al. 1994; Salamone et al. 1996; Uitteriinden et ai. 1996; VandeBerg and Williams-Blangero 1997). S~diesof bone density in pedigreed baboons have demonstrated the heritability of bone mineral density of vertebral bones (Mahaney, Kammerer, and Rogers 1997; Mahaney et al. 1995). A study on human twins has dso suggested that there is a genetic component to bone mineral content of the spine and radius (Dequeker et al. 1987a). A relatively recent study by Momson et al. (1994) found that the main genetic component responsible for bone mass is linked to polymorphism in the Vitamin D receptor gene. This association has ken controversial in the literature, and more recent studies have shown that Vitamin D receptor polyrnorphisms me not associated with the risk of fnctun in older women (Ensmd et al. 1999: Cooper 1999). However, heredity partidly determines the peak bone mass attained by an individual during growth. Thus. a geneticdly inherited low peak BMD may lead to abnormdly low bone mas, which in tum results in osteopenia or osteoporosis, and fragility fractures. A genetic predisposition to low peak BMD rnay lead to osteopenia and susceptibility to fracture once age-related bone loss begins in affected individuals. Since osteopenia and osteoporosis are disorden of bone turnover, this abnormal physiology rnay then have an adverse effect on pregnancy outcome or fertility, and low parity or nulliparity rnay have a hirther negative effect on bone densi ty. A radiographie study by Bowden et al. (1979) of cortical thickness of metacarpais in captive macaques (Macaca fiscicularis md M. nemesuina) shows that bone mas decreases as a function of age, and that parity is associated with relatively thicker bones; mean 'percent cortical ma' is mater for high-parity (> 3) femaies than for low-parity (c 3) femaies in the same age group (Bowden et al. 1979). A study of the effect of lactation on matemal bone in laboratory-bred Afiican green monkeys (Cerco~ithecusaethio~s) reports that the BMC and BMD vaiues of lumbar vertebne gnduaiiy decreased during the penod of lactation, and then gradudly increased ;ifter weaning, but the lumbar spine did not recover completely to the values at parturition, even at 40 weeks post-partum (Hiyaoka et al. 1996). This suggests that macaques may provide a better model than aethioos for the study of human parity and bone density. A previous study of Cayo Santiago rhesus females by DeRousseau ( 1985) also found an age-related reduction in the relative thickness of the metacarpal in adult females. Furthemore, females who had given birth within three months prior to death had relatively less metacarpal bone than age-matched femaies who had not given birth the previous year (DeRousseau 198Sb). The observations of Bowden and DeRousseau suggest that, as in human females, bone loss in femde macaques may occur in relation to pregnancy or Iactation, but it is transient in nature. In conuiist, a study by Champ et al. (1996), failed to observe significmt bone loss with age in the spine and total body of femde rhesus monkeys, which they partially attribute to the smdl number of postmenopausal animals in their study (Chmp et al. 1996). They report that menses had ceased in animds older than 28 years of age, and thus continuing ovarian function in most of their females may in part explain the lack of decline in bone mass with age. They cite multiple factors, including a diet replete with vitamin D and calcium, and the relatively late occurrence of menopause in rhesus monkeys, for the lack of significant bone loss in all sites except the radius (Champ et al. 1996). However, the present study of Cayo Santiago rhesus females, which includes mostiy pre-menopausal females, shows a significant association between parity and bone mas, and a previous study (described in Chapter 3) found a significant age-related decrease in bone mass in the spines of these same females (Cerroni et ai. submitted). Aithough the diet of the Cayo Santiago monkeys is high in vitamin D and calcium, the minerai content of their bones hûs ken shown to be unaffected by diet (Gryapas et al. L993a). The presence of vertebral osteophytosis (VO) and degenentive disc disease (DDD) in the spines of older animals, and the failure to exclude them from the study, may also account for the lack of significance in the results obtained by Champ et ai. (1996) with respect to spine and total body bone mas measurements. They conclude that the use of the rhesus monkey as a mode1 for age-related bone loss may be problematic, and that any reduction in bone minerai with age will be most apparent only at radiai sites. The author of the present study disagrees with these conclusions for reasons outlined below. -The Cavo Santiago Female Rhosus Moakers increase of Bone Densitv with Paritv in Ca~oSantiago Female Monkevs The results of this work are in genenl agreement with the bone density studies conducted on human females by Murphy (1994), Aloia (1983), Nüsson (1969), Fox ( 1993) and Stevenson ( 1989), which were described eulier. Murphy et al. ( 1994) noted significantly higher bone density of the proximal lemur in parous women, and Fox et al. (1993) report higher BMD of the distal radius in parous women, as do Aloia et al. (1983). Nilsson (1969) found a positive relationship between parity and bone mass of the fernur, metacarpal and radius. Murphy et ai. (L994) report an average 1.O% increase in bone density per Live bkth mong the human femaies in their study, and Fox et ai. (1993) report a 1.4% increase, but neither study deiineates ûn upper limit on this beneficial effect of parity on BMD. The results of the present study confirm the trends observed by DeRousseau ( 1985) in her study of Cayo Santiago femde monkeys. Grynpas et al. (1993) aiso found that, among the females in this colony. parity correlates with an increase in bone density of vertebrae; it was show that bone mass increases with increasing par@, even though the bone present may be underminerdized (Grynpas et ai. 1993b). The present study on Cayo Santiago female monkeys confirms these trends. After controlling for age, there is a significant increase in BMD of the spine with increasing piuity up to 7 offspring (p = 0.0006); puity pater thon 7 is associated with a steep decline in BMD with advancing age. Thus, high pwity initially has a positive effect on BMD, but paity higher than 7 is actually detrimental. The results of the GAM testing show that the effects of parity and age are non-linear, and that parity has an increasing effect on BMD. while age has a decreasing effect from about 9 years of age. Age, as a variabie by itself, has a decreasing effect on BMD among animals older than the age of peak bone mus. In contrast, with aging held constant. parity has an increasing effect on BMD. Thus, according to the GAMs, pûrity appears to Mtigate the effects of aging on BMD. A few additionai tests were conducted to see if there is an association between nulliparity or low parity and bone mas. A statistically significant difference was found between the mean BMC (p = 0.03) and mean BMD (p = 0.008) values of the venebne of nullipuous females aged 8.5 to 9.4 years when compared to parous females in the same age range; the means are higher in the parous females. The mean BMD of the nuiliparous females = 0.459 g/cm2 and that of the parous femdes = 0.707 &m2; the mean BMC of the nulliparous females = 1.OL grams and ihat of the parous females = 1.49 grarns (Table 2). Although the sample sizes are srnail, these results indicate a trend. A totai of 16 females in this sample (N = 119) have low parities for their ages. Comparing these low-parity femdes to age-matched controls in the sample (Table 7) shows that theu parities are indeed significmtiy lower, and as a group, their mean BMD values are aiso significantly lower (with the exception of females in the 20+ year age category). Osteopenic and osteoporotic females are found in this low-parity group. Although other diseases of the spine are also represented in this group. statisticai tests show that osteopenia and osteoporosis are more likely to be associated with low parity. Nulliparity has been shown to be associated with low bone density in human females (Stevenson et al. 1989), and in the present study. the bone densities of low-parity and nulliparous lemales are significantiy lower than those of their parous cohorts. Since an average 1.0% increase in bone density per live binh is repocted in human fernales. it is reasonable to assume that a similar trend may exist in the rhesus monkey; therefon. the skeletons of low-parity and nulliparous female rhesus monkeys have not benefitted from this particular effect of multiple pregnmcies. These low parity and nulliparous fernales have not benefitted from the positive effects on bone density of increased body weight, increased estrogen, and enhanced cdciurn absorption, al1 of which are associated with pregnancy. Studies have demonstrated the positive effects of pregnancy-related weight gain on the bone density of the femod neck in human mothers (Sowers et al. 1991). A study has shown that on average, the body weights of pregnant rhesus females on Cayo Santiago tend to k higher than those of their age-matched non-pregnant or lactating cohorts (Rawlins, Kessler, and Turnquist 1984). Grynpas et al. (1993) dernonstrated a weak positive correlation between body weight and vertebral bone mass in Cayo Santiago fernales. It is plausible that pregnancy-related weight gain would exert a positive effect on the bone density of the spine in r quadnipedal animal like the rhesus monkey. Moreover. rhesus mothers continue to receive spinal load-bearing pst-panum from carrying their infants, fust in a suspended posture from the abdomen, and later on the back. ù is noteworthy that this pattern of load-bearing on the macaque spine is different from the load-beiuing experienced by the human femde spine. Pari& Age and Deeenerative Disease in Cayo Santiago Female Monkevs There is a strong positive correlation between age and parity (comldon = 0.84). A linear regression of parity on age illustrates the biological potential for reproduction in rhesus femdes from Cayo Santiago. The regression shows that at age 4 years, the maximum number of offspring for a rhesus female monkey is 1. and for every year beyond this age. the maximum number of possible offspring increves by 1. The actual piinty of femaies over 14 years of age is more variable than that of younger age cohorts; females 14 years of age and over are less likely to achieve their biologicai potential for parity, probably due to environmental or social factors. in the present study, most of the femaies 14 yem of age and older have both high parity and moderate to severe vertebrd osteophytosis/degenerative disc disease. Therefore. there is no apparent negative effect of these particular conditions on puity, as most of the individuais with severe degenentive conditions (Le.. diseases of long duntion) have attained high puïties. However, other diseaseddisorders acquired dunng the life span may have an adverse effect on pregnancy outcome and result in lower parîty. The femaie rhesus monkeys in this sample have continued to reproduce until the end of their lives. However, very old Cayo Santiago femaies tend to stop having an offspnng every year; births are spaced fiuther apûrt (Rawlins, Kessler. and Turnquist 1984). Among these females, this longer birth spacing can be attributed to a combination of degenerative àîsease and the onset of menopause. On Cayo Santiago, the average interbirth interval for females delivenng and weaning offspring in consecutive years is 372 f 33 days, and the average interbirth interval is 336 f 29 days for femaies who have stillbirths or spontaneous abortions, or whose infants die prior to weaning (Rawlins, Kessler, and Turnquist 1984). In human fernales, the average length of the interbirth interval is most influenced by the duration of postpartum amenorrhea, which is affected by lactation, and by the availability of food (Bongaarts and Potter 1983; Delgado et al. 1978). For example, snidies on poor Nnl women found that the interbirth interval was shorter in women who received nutritional supplernents compared to women whose diets wen not supplemented (Chavez and Martinez 1973). Among nonhuman primates in the wild the length of the interbirth intervd is aiso associated with nutrition; animais that have low quality àiets have longer interbirth intervais (Lee 1987). For example. in a population of baboons in Kenya. birth intervals increased over a 5 - year study period as access to food resources decreased (Stnim and Western 1982). Among the rhesus females of Cayo Santiago, however, neither diet nor prolonged lactation is the cause of a longer interbirth interval in some femdes; since these monkeys are provisioned, dl inàividuals in the colony have a well-balanced diet. Other factors must be responsible for increasing the birth interval in some females - factors such as disease and the onset of menopause. Also, for some femdes, stress caused by low position in the dominance hierarchy may also have an adverse effect on fertility - a hypothesis that should be tested in f'uture studies on this colony. Cavo Sm tiago Fernales: Heri tabilitv of Bone Mass As noted earlier, there is some evidence for a genetic component ta the pattern of osteopenia and osteoporosis seen among low-parïty females in this sample. Half of the low-parity females were born in the same natal group, Group J, which includes 4 out of 9 (44%) of the osteopenidosteoporotic individuais listed in Table 3. Group J formed in 1964 when the majority of individuals fiom two materna1 genealogies split from one of the original founding groups, Group A (Sade et al. 1976). Since femdes stay within their natal groups for life, and the core of the social group is composed of related females, this fuiding provides indirect evidence for a genetic component to the pattern of osteopenia and osteoporosis observed in this colony. These four low-parity females have osteopenia, but no vertebral compression fractures. As noted in chapter 3. these four femdes are among the 27 OPVOPO (unfnctured) individuais found in the entire sarnple ( 16 femdes and 11 males), vimiaily dl of whom exhibited BMD vaiues of the spine that are lower than those of OPE/OPO individuals with vertebrai wedge fractures. Also notewonhy is the fact that 43% of the osteopenic/osteoporotic females in the whole sarnple are found in the low-parity group. Sade (1976) noted that some social groups on Cayo Santiago were increasing in size at a faster rate thm othen. and that there is a tendency for females of higher ranking matemol genedogies to reproduce successfully at earlier ages compand to femdes of lower ranking matrilines. Higher mking matrilines were increasing at a faster rate than lower ranking ones. Since the colony is provisioned it is unlikely that differential access to food according to rank has an effect on reproduction. Sade concluded that this differential rate of increase between groups may indicate the presence of important demographic, social or genetic differences between groups. and not an association with group size or rank (Sade et ai. 1976). The present study suggests that pnetic differences between the matrilines of different social mou~smay account for the differential rate of increase between groups. Genetic differences that may have an adverse effect on reproduction may ultuaately result in differences in numencd composition between matrilines, and betwcen socid groups. This hypothesis rrquires funher testing. Since low bone mass may mn in particular matrilines, this condition may have a pater impact on the reproductive success of females boni into such matrilines than would position in the hierarchy of the social group. Fume work may test the hypothesis that low parity is conelated with low socid rank in these female rhesus monkeys, and test whether femaies that exhibit ôoth low parity and osteopenialosteoporosis are also low in social rank. Future studies rnay dso trace bone density, osteopenialosteoporosis and parity through matcilines to test for the presence of a genetic component that rnay provide more insight into some of the patterns observed in the present study. Conclusions This chapter repons the results of a cross-sectional study that compared parity and bone mass across age in female rhesus monkeys from Cayo Santiago. The main conclusions are as foilows: Studies that use ovariectomized female rhesus monkeys to study bone remoâelling of the mature skeleton and to mode1 human postmenopausal osteoporosis should only include individuais who are older than the age of peak bone mas, Le., older than 9.5 years of age. The female rhesus monkeys of Cayo Santiago reach sexual maturity at 3 years of age and have their fmt offspnng in theu fowth year. At four years of age. the maximum number of offspring for a rhesus female is 1, and for every year beyond this age, the maximum number of possible offspring increases by 1. There is a significant increase in BMD of the spine with increasing parity, up to a parity of about 7 offspring (p = 0.0006). after controlling for age using GAM. High parity initially has a positive effect on BMD. but parity pater than 7 is actudly detrimental. GAM testing shows that the effects of parity and age are non-linear, and that parity has an increasing effect on BMD, while age has a decreasing effect from about 9 yem of age. With age held constant. parity has an increasing effect on BMD; parity appears to mitigate the effects of aging on BMD. Mean BMC and mean BMD are higher in parous females compared to nulliparous females in the sarne age range (BMC:p = 0.03; BMD: p = 0.008). Mean BMD values of low-parity femdes in this sample are significantly lower than those of age-matched convois (set Table 7). Osteopenic and osteoporotic femdes are present in the low-parity group: 43% of al1 the osteopeniclosteoporotic femdes in the sample are part of this group. There is no apparent negative effect of moderate to severe vertebrai osteophytosis (VO) / degenerative disc disease (DDD) on puity. There is indirect evidence, in the fonn of natal group membership, for a genetic component to the pattern of osteopenia and osteoporosis observed in this colony. The previous chapter has show that the rhesus macaque fernales from Cayo Santiago have a pattern of skeletai maturation and acquisition of bone density of the spine which is similar to that of human femaies - including a decline in bone mus with advancing age. The resdts of this part of the DEXA study demonstrate that they are also a gocxi mode1 for the study of bone density in women as it dates to parity. Chapter 4 ihus provides normative data for bone densitometry readings of the spine in fernale rhesus macaques, and demonstrates that these free-rauging monkeys hmCayo Santiago are a good non-human primate mode1 for the study of bone density, pwity, osteopenia and osteoporosis. CHAPTER 5 BONE MINERAL DENSITY AND VERTEBRAL OSTEOPHYTOSIS (VO) I DEGENERATIVE DISC DISEASE (DDD) 5.1. Results As described eulier, for this part of the study, the entire smple was divided into three VO/DDD groupings according to the degree of osteophytosis and remodelling of the centnim: Group 1 = Normal, Group 2 = Mild VOIDDD, and Group 3 = Modente to Severe VO/DDD. The protocol for assessing and recording VO and DDD was defined in the chapter on methods. The scatter plots show the entire sample, while the box plots only include individuds 9 years of age and older. Figure 41 shows a scatter plot of BMD by age and VO/DDD group in the whole sample. The majority of Group 3 individuds (black symbols) are 15 years of age and older, anci overlap in age with Group 2 animais. Group 1 individuds tend to be more clustered in age, and the majority are younger than 15 years of age; the BMD values of these nomai monkeys also tend to be more clustered. especiaiiy in the age range of 1 to 7 yeors. Although there is some overlap among the thee groups, it is evident from the graph that the normai individuais tend to separate out more clearly as a group. Groups 2 and 3 exhibit more overlap, with the group 3 individuals showing some of the highest BMD vdues for their ages. The BMD values of severely osteophytic individuals are much more variable, and tend to be higher than those of age-matched individuais fiom the other two groups. Thus. the BMD values of Group 3 animals tend to be higher and more vanable than those of the otber groups, and most individuals with high BMD are 15 years L86 of age and older. The same trends are seen in Figure 42, which shows centrum area by VO/DDD gmup and age in the entire sample. Severely osteophytic vertebrai bones tend to have the largest centrum ares, as a result of bone remodelling and growth of marginal osteophytes - and they tend to belong to individuais who are 15 years of age and older (Fig. 42). Thus, Group 3 individuais, representing severe VOIDDD, are generdly higher in BMD and BMC, lnrger in cenuum are* and tend to be older than normal animais or those rnildly affected by this disease. As mentioned earlier, the margind osteophytes were excluded from the DEXA analysis. Since it is evident from previous analyses that there are two patterns of bone density in this population, femde and mie, the effects of VODDD group membenhip were explored funher in mdes and females sepantely. Differences between mdes and females in mean age, BMDT and area by VO/DDD group were tested for significance using t-tests, and one-way ANOVA was used to test for significant differences among groups within the sexes. Two hypotheses were iested: 1) that there is no difference in VO/DDD jgoup means between the sexes, and 2) that there is no diffeference among the the VOIDDD groups within males, and within femaies. The foollowing describes the results of these tests, and refers to a series of box plots and scatter plots that illustrate the observed trends. The scatter plots show the whole sample. divided by the three VO/DDD groups. However, the box plots and statistical tests include only individuals 9 years of age and older. in ihis sample, the earliest appearance of miid VOIDDD (Group 2) is at 9 yem of age. Thus. to control for age, individuals younger than 9 years were excluded from the statistical analyses and box plots. Ane b~ Sex and VO/DDD G~UD There is no significant difference in mean age between the sexes (p = 0.2413) by VOlDDD group in the sample aged 9+ years. Mean ages for males and females in al1 three groups are listed in Table 8. Figure 43 shows box plots of age by VO/DDD group in males and females separately. and indicates that the median age increases from Groups 1 to 3 in fernales and mdes; there is a linear trend in age for both mdes and fernales (p < 0.0001). Group 1 individuals. the nomals, tend to be younger than the memben of Groups 2 and 3. a finding that is not unexpected. given thût VODDD is an age-related disease. The 'box' in a box plot represents 50% of the data in mdes. the box representing the normal group does not overlap in age with the boxes of Groups 2 and 3. There is, however, some overlap between the boxes representing Groups 1 and 2 females. Group 3 males show the largest interquiutile range in age compared to the males of Groups 1 and 2. Aiso. the age range of Group 3 males is greater than the age range of the other two gmups, and greater thnn that of Group 3 females. The age range of Group 1 females is similar to that of Group 2 fernales. In gened, there appears to be more overlap in the age ranges of the three groups in females (Fig. 43). TABLE 8 VOlDDD GROUP* MEANS BY SEX IN SAMPLE 9+ YEARS OF AGE SEX VO/DDD VOIDDD VO/DDD CROUP 1 CROUP 2 GROUP 3 Femdes Males Females Males Females Males Females Males Females Males Vertebral Osteophytosis/DegenenitiveDisc Disuise Groups, assessed on lumbar vertebrae used in DEXA; Group 1 = Nomial, Group 2 = Mild VOIDDD; Group 3 = Moderate to Severe VO/DDD -BMD b~ --Sex and VOmDD gr ou^ Com~arisonsWithin the Sexes Within the females. a one-way ANOVA shows that the differences in mean BMD among the three VO/DDD groups are significant (p < 0.000 1 ; means are listed in Table 8). PairWise comparisons were also made, using t-tests. These tests revealed that there is a significant difference in mean BMD between groups 1 and 3 Femdes (p c 0.0001), and between groups 2 and 3 femaies (p = 0.008). There is no difference in mean BMD between groups 1 and 2 lemdes (p = 0.10). Non-pûrametric tests were required for the males. Within the males, the differences in median BMD among the three VO/DDD groups are also significant (one-way ANOVA on ranks, p = 0.023: group 1 rnedian = 0.789, group 2 median = 0.820, group 3 median = 0.903). Painvise comparisons were also made, using Mann-Whitney Rank Sum Tests. The difference in median BMD between groups 1 and 3 is significant (p = 0.007). The other pair-wise comparisons are not significant: p = 0.23 for group 2 versus group 3. and p = 0.2 1 for group 1 versus group 2. Comoarisons Between Femaies and Males With regard to BMD, the differences between the sexes are significant in group 1 animds in the sample aged 9+ years (p = 0.006); mean BMD is significantiy higher in males. There is not a significant difference in mean BMD between group 2 males and females (p = O. L 82). and between group 3 males and females (p = 0.820). The mean BMD values for males and femaies are Listed in Table 8. Figure 44 shows box plots of BMD by VO/DDD Group and sex in the sarnple of individuals 9 years of age and older; there is a significant linear trend in the data for males and females (p < 0.0001). Median BMD increases nom Group 1 to 3 in both sexes: for each VO/DDD group. the median values of males are higher than those of females. However, as noted above. some of these differences are not significmt. Some of the highest BMD values are found in Group 3 males. Group 3 females also tend to have high BMDs compared to femaies in the other two groups, as indicated by the position of the box and upper adjacent value. The Group 1 box plot for maies shows two outside dues below the lower adjacent value; these lines represent two of the osteopenic mdes in the sample. The BMDs of al1 of the osteopenic females. however, fall between the first quartile and lower adjacent value; thus, no outside values are observed below the lower adjacent value in females (Fig. 44). A scatter plot of BMD by VOmDD group and age in females shows that severely affected females have high BMD compared to age-matched individuals, and tend to be 15 years of age and older (Fig. 45). Sirnilarly in maies. a scatter plot of BMD by group and age shows that group 3 individuals have very high BMD, and also tend to be 15 years of age and older (Fig. 46). There are more group 3 males in the 20 to 30 year age range than group 3 females. In general, group 3 males outnumber group 3 femdes. and they have more variable BMD vaiues. The scatter plots in Figure 47 show a direct cornparison of BMD by age between females and males. divided by VOlDDD group; the lines represent smoothing splines. Group 1 femaies and males both show significant positive correlations between BMD and age, followed by a plateau or downward trend. A trend with age is also seen in group 2 females; a few of the older osteopenic females are part of this group. However, the smoothing splines indicate no trend with age in group 2 males and group 3 males and females. There is mon variability in BMD in group 2 and 3 maies. and more clus tering of BMD values in group 1 femdes and males (Fig. 47). As noted previously, 'area' refers to the scan area of the centmm (ROI)that was analyzed by DEXA. in the sample aged 9+ years, t-tests showed that mean area differs significantly between males and femdes in VO/DDD group 1 (p c 0.0001). group 2 (p < 0.0001) and group 3 (p < 0.0001). Area means for al1 groups are listed in Table 8. Maies and femdes are not consistently different in mean area across the VO/DDD groups; they show different trends. In the males. mean area increases from groups 1 to 3. whereas in the females, mean ûrea increases from groups 1 to 2 (p = 0.04) but remains essentially the same in group 3. In femaies, the difference between group 1 and group 3 median area is not significant (p = 0.47). and the difference in area between groups 2 and 3 is dso not significant (p = 0.88). in contrast, for the males, the difference in mean area between groups 1 and 2 is significant (p = 0.008). as is the difference between groups 2 and 3 (p = 0.0 16) and groups 1 and 3 (p c 0.000 1). Figure 48 illustrates these trends with box plots of area by sex and VO/DDD group in the sample of monkeys 9 years of age and older. The median area increases across the three groups in males; those with severe VO/DDD (group 3) exhibit the largest centrum areas due to large osteophytes and excessive remodelling of the centrum. in the females, the median maincreases from group 1 to 2, but decreases in group 3. The range of values for centrum area in group 2 femdes overlap that of group 3 femdes. with the latter showing a smaller lower adjacent value and one outlier with a high vdue. The interquartile ranges of group 3 females and males are larger than those of the other groups. Thus, among the females, osteophytosis and degenerative disc disease tend to proàuce a similar centmm area regardless of the severity of the disease. There is one exception; one fernale in group 3 has an extremely large centrum are& as shown by the ouilier above the upper adjacent value in Figure 48. Outliers, or outside values. are aiso present in nomal and group 2 mdes. in contrast to the females. centmm area tends to increase with the severity of VOIDDD in mdes. A scatter plot of centrum area by VOlDDD group and age in Fernales also shows that. with the exception of one individual, the centrum maof severely affected femaies tends to overlap that of group 1 and 2 individuals (Fig. 49). in males, a scatter plot of centrum maby group and age shows that group 3 individuals tend to have larger centrum areas compared to members of the other two groups (Fig. 50). The scatter plots in Figure 5 1 show a direct cornparison of centrum uer by age between females and maies. divided by VOlDDD group; the lines represent smwthing splines. Group 1 females and males both show significant positive correlations between area and age, followed by a plateau or downwwd trend; there is dso a trend with age in group 2 females. However, the smoothing splines indicrte no trend with age in group 2 males and group 3 females and maies, confing the presence of more variability in centmm area in these groups (Fig. 5 1). VO/DDD dmu~3 Monkevs: kscri~tiveStatistics and Cornparison & Fernales and Descriptive statistics on the males and fernales assigned to VOlDDD group 3 are shown in Table 9. There is a statisticaily significant difference between the mean BMC (p = 0.0002) and mean area (p < 0.0001) of males and fernales. The mean BMC of females is 2-01 grarns. and the mean BMC olmales is 2.85 gram; the mean area of fernales is 2.27 cm2 and that of males is 3.19 cm2. There is not a significant difference. however, between the mean BMD values (p = 0.820) and median ages (p = 0.961) between the sexes. The age range differs. however, with females ranging in age from 14 to 23.9 years, and males from 10.8 to 29 years. There are 43 individuals in VOlDDD group 3, 18 females and 25 males. Some of the highest BMD values in this group are over 1.0 gkm2; the highest value recorded is 1.330 @cm2, in a male aged 13.9 years. in addition to high BMD. five individuais in group 3 also exhibit established osteoporosis; one femaie and bur males have venebral wedge fracnues. No females from the low-par@ group described exlier are pnsent in VO/DDD group 3. Figure 52 shows typical vertebrae from an individuai assigned to VOlDDD gxoup 3. Figures 53 and 54 show vertebrae from two males that exemptify the disease interaction present in this gmup. Figure 53 shows part of the lumbar spine of a 26 year old male (Cat. No. 846) who has very high BMD (0.967 &m2), DlSH and VOIDDD. in contrast, Figure 54 shows a laterai X-ray of a 24 year old male (Cat. No. 2065) with bordedine osteopenia (BMD= 0.654 @cm2), DISH, multiple vertebnl wedge hctures. and extensive osteophytosis. Both individuais were placed in VO/DDD group 3 because of the presence of extensive osteophytosis; it is notewonhy, however, that they differ pady in BMC values. The BMC of 11846 is 3.34 gram, which is not unexpected for this group; in contrast, the BMC of #2O6S is only 1.72 gram. This demonstrates that pter weight should be placed on BMC value ratber than degree of vertebrai osteophytosis when identiwng specimens with VO/DDD that may introduce bis into a DEXA sample. TABLE 9 DESCRIPTIVE STATISTICS ON VOmDD CROUP 3 MONKEYS - Age BMC BMD at Death 0 wcm2, (Ym Sample Size Females 18 -18 Males 25 25 Mean Fernales 18.4 -2,010 Males 18.9 -2.852 Standard Deviatioa Females 2.8 0,607 Males 5 .O 0.709 Maximum Females 23.9 -3.270 Males 29.0 4.500 Minimum Females 14-0 -1.196 Males 10,8 -1.687 Median Females 17-7 1.994 Males 18.0 2-97 1 P'C 2- C ObC 8'0 9'0 P-0 auua O'P Ç'Q O'C Ç'Z 0.2 Ç'C O' 1 emv Males Females Normal Mild Severe Normal Mild Severe Fige 43. Box plots of age (in years) by VOIDDD Group in males and fernales 9 years of age and over. Group I = Normal; Group 2 = Mild; Group 3 = Moderate/Severe. VO/DDD group membership is assessed on lumbar vertebrae useà for DEXA analysis. Males Females Normal Mild Severe Nomal Mild Severe Fig. 44. Box plots of BMD (in g/cm2) by VO/DDD Group in males and females 9 years of age and over. Group I = Normal; Group 2 = Mild; Gmp3 = Moderate/Severe. VO/DDD group memkrship is assessed on lumbar vertebrae used for DEXA analysis. O* c 2' C O' C 8'0 9'0 P'O awa 0 Group 1 Group2 Group 3 Fig. 46. Males: Scatter plot of BMD (in g/cm2) by age (in years) and VODDD Group. Group I = NO& (white symbols); Group 2 = Mild (grey symbols); Groq 3 = ModeraWSevere (black symbols). VODDD group membership is assessed on lumbar vertebrae wd for DEXA analysis. Females Males Fig. 47. Sc- plots of BMD (in g/cmZ) by age (m y-) in femalcs and maks, with VODDD bupsgraphed sepamtely. Gmp I = Normal; Groyp 2 = Mild; Group 3 = ModeratdSevere. Lines = smoothing spüne fit. Males Females I'J Normal Mild Severe Normal Mild Severe Fig. 48. Box plots of scan Area (in cm*) by VO/DDD Group in males and females 9 years of age and over. Group I = Normal; Group 2 = Mild; Group 3 = Moderate/Severe. VO/DDD group membership is assessed on lumbar vertebrae used for DEXA analysis. O'P 9'E O'C Ç'Z 0'2 QI O'C ew O't Ç'C O'E Ç'Z 0'2 P'L O'C -Jv Females Males Fig. 51. Scattet plots of scan Area (in cm?) by age (in years) in fernales and males, with VOmDD Gmups graphed separately. Group l = Nomial; Group 2 = Mild; Group 3 = Moderate/Sevete. Lînes = smoothing splk fit. Fig. 52. Photogmph of caudal asped of typkal lumbar vertetme hman individual in VOmOD Gmup 3 (7th lumbar vertebra in tenter). Note osteophybsis and extensive remodelling of the trabecular bone of the œnbum. VOIDDD gmup rnembership is assessed on lumbar vertebrae used for DU(A analysis. Fig. 53. Cat #û46, a mie aged 26 yean. exhibithg high BMO (OS7 #a), OlSH and VOD00. Photo of ventral aspect of spim (14 to L? inclusive) and sacnim. Fig. 54. Lateml radkgtaph d spim of Cat %!OB& a male aged 24 years, showing bordedine OPE (MD = 0.654 extensive osteophytosis, and DISH. 5.2. Discussion -Bone Mineral Densitv and Vertebral Osteo~hvtosWDePenerative Dise Disease Studies on human subjects have demonstrated that any degree of venebnl osteophytosis results in higher bone density of the spine compared to individuals without osteophytes (Jones et al. 1995). The comparisons drawn earlier between the BMC values of specimens #846 and #2065 demonstrate the importance of placing greater weight on BMC value rather than on degree of vertebral osteophytosis when identifjhng specimens with VODDD that may introduce bias into a DEXA sample. Recall that the ROI was positioned in a manner that excluded the vectebral osteophytes from the DEXA analysis; thus, the BMC and BMD values reported throughout this thesis are those of the trabecular bone of the centmm itself. The results from these two individuais show that in one case (#846), disease processes have altered the quality of the trabecular bone of the centrum and increosed its density (BMD), while in the other individual (#2065), the density of the trabecular bone has been diminished by osteoporosis. Thus, provided that the ROI excludes osteophytes from the DEXA analysis, more weight should be given to BMC values when attempting to identiS 'arthntic' specimens in a sample of vertebral bones. A previous study by Grynpas et al. (1993) used vertebrai bones denved frorn the same colony of rhesus monkeys to study bone minemlization using the technique of density fnctionation. They showed that bone minerakation of the spine increases with age in non-uthitic individuals. but decreases with aee in the presence of vertebrai 'arthritis', thus confiing that bones aected by this disease expenence changes that are distiact from the normal aging process (Grynpas et al. 1993b). Thus, vertebral bones afFected by VODDD may have abnomaiiy high bone density as determined by DEXA, but the bone present may achially be undermineralized, possibiy "...due to repair of microfractures or a defect that prevents the full mineralization of newly formed bone, or a combination of both" (Grynpas et ai. 1993b. p. 915). Techniques of bone densitometry like DEXA measure the overd quantity of bone in a given volume, but do not distinguish poorly mineraiized from highiy mineraked areas within a bone. It has been hypothesized that bone minedization, in both normal physiology and in many pathologies. is dnven by the rate of bone turnover. The matenal properties of bone are influenced by its minerdization profile, which is itself aitered by age and disease (Grynpas 1993). One study found that spinal degenerative actluitis (VODDD) in a population of women aged 50 to 85 yean is associated with a generaiized increase in BMD and a decreased rate of bone turnover, suggesting that the protective effect of this disease against osteoporosis may be mediated by decreased bone turnover (Peel et al. 1995). --The Inverse Relationshi~Between Osteoarthritis of the Peri Jeml Skeleton and The Iiterature describes an inverse relationship between osteoaahritis and osteoporosis in humans, including an inverse relationship between osteoporosis and osteophytosis of the spine (Belmonte-Serrano et ai. 1993; Dequeker 1985; Dequeker et ai. 1996; Hart et ai, 1994; Luet ai, 1997; Marcelli et ai. 1995; Peel et ai. 1995; Versmeten et al. 199 1; von der Recke et al. 1996; Yu et al. 1995). The emerging consensus is that OA affords protection against. or retards the developrnent of osteopomsis. Dequeker et al. (1995) noted that then are anthropometric, biologicai and biochemical dwerences between pnmary OA and osteoporosis, which suggest that systemic and metabolic factors are involved in theu pathophysiology, and that these cornmon diseases are not a simple consequence of aging (Dequeker. Mokassa. and Aensens 1995). Dequeker and colleagues (1983) suggested that osteoarthritis of the peripherd joints and osteoporosis are not simply phenornena of aging, and Wear and tear, but disease entities. They noted that patients with postmenopausal osteoporosis and those with OA appear to represent anthropometrically different populations (Dequeker. Goris. and Uytterhoeven 1983). They described anthropometric differences between women with OA and those with osteoporosis. showing that women with OA had an excess of body weight. skin-fold thickness. and muscle girth and strength. while osteoporotic women scored below average for these indices. rven though they were of comparable skeletal sire and age. The presence of elevated levels of subcutaneous fat in the OA group may result in an increased peripherd formation of estrogens by the conversion of androstenedione to estrone. thus preserving a better postmenopausd estrogen status in these women; this would consequently reduce the rate of bone resorption. and thus maintain bone density. In general, people with OA have better preserved bone mass for their age. even independent of body weight. In Fact. OA cases present with a significant increase in bone mass or density compared to agelsex- matched controls. even ;ifter adjustment for body weight (Dequeker et al. 1996). One of the mechanisms of initiation of OA is subchondrai bone stiffness, which is often due, in part, to inherited increased bone density (Dequeker, Mokassa, and Aerssens 1995). The quantitative and qualitative differences in OA bone mqincrease subchondral bone stiffaess, thus rendering it less capable of absorbing impact loaàs. This stiff bone transmits more force to the overlying cartilage, making it more vulnerable to damage (Radin and Paul 1970). Thus, bone aiterations usociated with OA predispose to hcular cartilage loss, but also pmvide protection against osteopomtic fractures. Studies conducted by Dequeker and his research group found that the increased bone density and bone stiffness in iliac crest bone of patients with OA is associated with increased skeletal concentrations of growth factors (insuiin-like growth factor I and II, and transforming growth factor beta) and enhanced activity of osteoblasts, thus demonstrating that OA patients have changes in bone quality in nonload-bearing areas of the skeleton apparently unaffected by the disease. Thus, the changes are systemic. These results support the hypothesis that quaiitative and quantitative differences in bone may explain the inverse relationship between osteoporosis and OA (Dequeker et ai. 1996; Dequeker, Mokassa, and Aerssens 1995). The underlying genetic determinants of this phenomenon need to be studied in more detail (Dequeker et ai. 1996). Perhaps the genetic component to this inverse relationship rnay be found mong the rhesus monkeys living on Cayo Santiago. --The Inverse Relationshi~Between Osteownid0steo~o~)~isand Vertebral Ostm~hvtosisd Oe~enerativeDise Disease Osteoporotic patients tend to exhibit lower frpquencies and milder expressions of vertebral osteophytosis and disc degenention (Dequeker 1985; Versuaeten et al. t 99 1). The data suggest that 'osteoiuthrosis' or a related factor may have a protective or retarding effect on the development of osteoporosis of the spine. in a study of 72 postmenopausal women, it was found that when both conditions coexist in the same femde patient, vertebd compression fmtures occur at a significantiy older age. and after a longer postmenopausai perîod. despite a hi@ frequency of risk factors for osteoporosis - risk fxtors like smaller stature, nulliparity. smoking and higher serum panthyroid hormone Ievels. Studies have also shown that there is a significant reduction in forearm and other fractures in patients with both kases compared to those with only vertebrai osteopomsis. In addition. the patients with both diseases experienced a milder manifestation of OA of the hip compared to those affected by OA alone (Versuaeten et al. 1991). Dequeker (1985) presents data that suggest that bone mus may be one of sevenl variables in the pathogenesis of OA, and that it may explain why osteoporotic cases develop few osteoarthritic changes of the spine and peripheral joints (Dequeker 1985). Cavo Santiago Rhesus Monkevs The results of the present study concur with the clinicai observation that patients with osteopenia/osteoporosis tend to lack. or exhibit rnilder expressions of, vertebral osteophytosis. in genenl, the osteopenic monkeys are either free of VOIDDD and VOA, or express mild forms of the disease. The outlier group of 12 osteopenic monkeys in this sample are a good example of ihis trend; predominantly osteoclastic processes are evident in the vertebrai bones. However. there are a few noteworthy exceptions in the group of monkeys with established osteoporosis. ic.. those individuals identified with vertebrai wedge fractures. The individuds in this group tend to be older monkeys that exhibit a combination of diseases a end-stage. Osteopenic/osteoporotic (unfmctured) individuals are ody found in VO/DDD group 1 (Nomal group), but the osteoporotic (fiacturrd) individuals in this simple are disiributed across the three VOlDDD groups. It is noteworthy that individuals with multiple diseases are present in VO/DDD group 3. in the fitsection of ihis thesis (chapes 3 and 4), the DEXA sample consisted of individuals in groups 1 and 2. Since the group 1 males in Figure 47 show a decrease in BMD with advancing age, it appeûrs that the males with rnild VO/DDD (group 2) are responsible For the Iack of decline in BMD shown by the sample of males in the first part of the study (chapter 3). Thus, the males in this colony mav show a decrease in BMD with advancing age, provided that g.iJ individuals with VOIDDD are excluded from the malysis. Figure 47 thus provides graphic illustration of the importance of excluding specimens with VODDD from DEXA samples used for studies on bone density and osteopenia/osteoporosis -- especiaily when the production of normative BMC and BMD vaiues is part of the god of the project. hdividuals with moderate to severe VO/DDD in this sample have abnorrnally elevated levels of bone mineral and higher bone density of the vertebral body compared to age/sex - matched controls. One may infer that the trabecular component of the bone has been aitered, both quaiitatively and quantitatively. The exact nature of these quaiitative changes should be examined in future studies. The cortical bone of the vertebrai body is also affected, as osteophytes emanate from the margins of the centmm and the endplates eventually become sclerotic. Among the femaies, vertebrai osteophytosis and degenerative disc disease tend to produce a similar centrum area (projectional area of DEXA) regardless of the severity of the disease. in males, however, the centrum area tends to increase with the severity of VO/DDD. The explmation for this ciifference rnay lie in the possibility that osteopenia and degenerative spinal disease may CO-existin some of the older femdes. Conclusions The main findings of this part of the study are as follows: Males with mild VOlDDD (group 2) are responsible for the lack of decline in BMD with advancing age in the sample of males in the Tmt part of the study. reported in chapter 3. The male rhesus monkeys of this colony may show a decrease in BMD with advancing age. provided that dl individuals with VOIDDD ae excluded from the andysis. Group 1 females and males show significant positive correlations between BMD and age, followed by a plateau or downward trend, and a trend with age is also present in group 2 femdes. The= is no trend with age in groups 2 and 3 males, and in group 3 females. There is more variability in BMD in groups 2 and 3 males. and more clustering of BMD values in group 1 for both sexes. in group 1 animals. mean BMD is significantly higher in mdes compared to group 1 femdes (p = 0.006); there is no significant difference in mean BMD between group 2 mdes and femdes (p = 0.182). and between groups 3 males md females (p = 0.820). i There is a significant difference in mean BMD between groups 1 and 3 ferndes (p < 0.0001). and between groups 2 and 3 femdes (p = 0.008). but no difference in mean BMD between groups 1 and 2 females (p = O. 10). in males, the difference in median BMD between groups 1 and 3 is significant (p = 0.07). but the other pair-wise cornparisons are not significmt. in males, mean centrum area increases from groups 1 to 3, whereas in femdes. mean area increases from groups 1 to 2, but remains essentially the same in group 3. Thus, in males. centrum area tends to increase with the severity of VO/DDDTbut not in females. i Group 3 individuals (moderate to severe VOIDDD) have abnormdly elevated BMC and BMD vdues compared to age- and sex- matched controls - even when the vertebrai osteophytes are excised digitaily by the ROI. This indicates that the irabecular bone of the centmm hûs been altered by the disease, both quantitatively and quaiitatively. i individuais in VO/DDD group 3 generaily have higher BMD and BMCT larger centrum anas, and tend to k older than their normal or mildly affected cohorts. i Some of the highest BMD vdues are Found in group 3 males. Group 3 males and kmaies tend to be 15 years of age and older. Group 3 mdes outnumber Group 3 lemdes in this sample. and their BMD and BMC vdues are more variable. i More emphasis should be placed on BMC value rather than on degree of vertebrd osteophytosis when identifying specimens with VO/DDD that may introduce bias into a DEXA sample. PART n. VERTEBRAL OSTEOPHYTOSIS (VO)I DEGENERATIVE DISC DISEASE (DDD)AND DiFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISHI VERTEBRAL OSTEOPHYTOSIS (VO)/ DEGENERATIVE DISC DISEASE (DDD) VO/DDD: Distribution Acrass the Entire Sam~le.and bv Sex This part of the analysis involves 204 spines, representing 102 femdes and 102 maies. The males range in age from 7.0 to 29.0 years, and the females from 7.0 to 23.0 years. There is a total sûmple size of 5,304 AAR joints (204 spines x 26 AAR joints). The sample was described in detail in sections 2.1.2.3 and 2.4. Figure 55 shows the results of the principal cornponents analysis (PCA) conducted on the entire sample (N = 204 spines; 5,304 AAR joints in total). The weights, shown on the Y-axis, indicate where the between-subject variation is located. For the VO/DDD data, by plotting the weights against the location number, one cm see the locations dong the spine where the between-animai variation cm be found. The program ûssigned the largest weights to AAR joints 12 to 19 inclusive. The 1st PC accounts for 69.3% of the variance, the 2nd PC accounts for only 6.3% of the variance, and the 3rd through 26th PCs account for the rest of the variation in the sample. Only the first and second PCs are shown becûuse most of the sample variance is explained by only the fiat two components; there are 26 variables, and thus 26 PCs in total. Figure 55 shows that the thoracic venebrae account for most of the variation in the sample (1st PC), 69.3% of the 220 variance - a very significant amount. The cervical and lurnbar vertebrae account for considerably less variation. Thus, most of the pathology in the sample is present in the thoracic vertebrae, from AAR joint 12 to 19 inclusive, corresponding to intervertebrai joints TSR6 to TWLl inclusive. The 2nd PC shows that the pathology in the cervical and lumbar vertebrae accounts for only 6.3% of the variance in the sample, with the highest weighted sum values assigned to AAR joint 5 (C5/C6) and AAR joint 23 (LARS). Thus, in rhesus monkeys, the thoracic region is most susceptible to developing VOIDDD. compared to the other regions of the spine. The same generai trends are observed when males and females are analyzed separately by PCA. in femaies, the 1st PC accounts for 69.9% of the variance and the 2nd PC accounts for only 8.1% (N = 102 spines; 2,652 AAR joints) (Figure 56). Most of the pathology occm in the thoracic region. from AAR joint 12 (TSK6) to AAR joint 20 (LIN); the largest weights are assigned to this section of the spine. in males, the 1st PC accounts for 70.2% of the vuiance, while the 2nd PC accounts for only 6.6% (N = 102 spines; 2,652 AAR joints) (Fig. 57); most of the pathology also occua in the thomcic region, from AAR joint 12 (TSfï6) to AAR joint 18 (T 1 l/T12). Thus, the ovedl pattern of VO/DDD is similar in males and femaies; in the femaies, however, the pattern extends to two more intervertebrai joints - AAR joints 19 and 20. In both males and fernales, an interesting 'peak' is observed at AAR joint 9 (T'A3). which is near the transitional zone between cervicai and thoracic vertebrae, Another view of the trends in the whole sample of 204 spines is provided by Figure 58. The four panels show. from AAR joints 1 to 26, the proportion of individuafs that have VOlDDD scores of 1 or greater, 2 or greater. etc (N = 204 spines; 5,304 AAR joints). Figure 59 shows these proportions by joint in females admaies separately. When multipüed by 100, these proportions yield the percentages of every AAR joint in eûch VOlDDD grade. according to the foliowing divisions: II2S, 3s and 4s. Both figures confirm that the thoracic vertebrae, the transitional thomco-lumbar vertebrae. and L7/S 1 tend to have the highest proportions of AAR joints in every grade of VODDD. Mdes and females exhibit very similar patterns across the vertebral column (Fig. 59). At AAR joints 5 and 13 to 15. 40% (N = 204) of individuals exhibit scores of 1 or greater (Fig. 58). Line graph A of Figure 59 shows that about 50% of the AAR 5 joints belonging to femaies exhibit scores of 1 or greater, while about 40% of those of males exhibit scores higher than, and equal to. 1 (N = 102 female spines. 102 male spines). A high proportion of males and females exhibit high scores in thoracic joints. The second line gnph (B) shows that around 20% of the sample of AAR joint 5 exhibit scores of 2 or mater in both males and femaies. and high scores are aiso present in the mid- to lower thoracic vertebrae (Fig. 59). Line graphs A to D show that a number of thoracic and lurnbar AAR joints in both mdes and fernales have comparatively high scores of VO/DDD. In the males. AAR joints 13 (T6/T7) to 16 (T9R 10) inclusive. 20 (LIU) and 26 (L7/S1) have a high percentage of scores 1 or pater; the femaies have a high percentage of scores greater than and equal to 1 in AAR joints 14 (Tïm8) to 19 (T12Ll) inclusive and 26 (Fig. 59. graph A). Male AAR joints 13 (T6iT7) to 16 (T9îTlO) inclusive. 19 (TlULl ), 20 (LIU) and 2 1 (W)have comparatively high percentages of scores 2. 3,4 and up, as shown in the subsequent line gnphs (B to D) in Figure 59. Femaie AAR joints 13 (T6m) to 20 (Ll/L2) inclusive and 26 (L7fSl) exhibit comparatively high percentages of these same scores, as shown in line graphs B to D (Fig. 59). The highest VO/DDD scores, 4 and 5, are found predominantly in AAR joints 13 (T6m to 22 (WU)inclusive in maies, and in AAR joints 13 to 22 inclusive and 26 (L7lS 1) in females (Fig. 59, graph D). Averanc VO/DDD b~ Joint: Overall Patterns in the Samde Figure 60 shows a line gnph of the average rating of VOlDDD at ench AAR joint in the whole sample (N = 204 spines; 5,304 AAR joints). Most of the thoncic and upper lumbar intervertebrai joints exhibit the highest average scores. particularly joints 13 (T6A"i) to 21 (WU)inclusive. Joints 4 to 6 in the cervicai vertebrae and joint 26 also exhibit high average scores. Figure 6 1 shows a line gnph of the average nting by joint in males and females separately. The overail pattern is similar in females and males, but there is one main difference: while the males show peak average values in joints 15 (T8îT9) to 19 (TlULl) inclusive. the fernale line graph shows a trough in the same ngion of the spine (Fig. 61). The gnph for femaies reveals a peak in a more cnnid direction. from joints 13 (T6/17)to 16 (T9R10) inclusive. The females show higher average values in joints 20 (LlU) to 23 (L4iL5) inclusive, compared to males. Figure 62 shows mean VO/DDD scores I2 standard enors at each AAR joint. across the entire sample (N = 204 spines; 5,304 AAR joints). This gnph is similar CO the one in Figure 60, but with the added information provided by the standard error bars. As before, the highest mean scores occur in AAR joints 4 (C4lC5) to 6 (C6/C7) inclusive. 13 (T6mto 21 (W)inclusive, and 26 (L7/S 1). The graph shows that the means decrease steadily from joint 21 to 23 inclusive, and then increûse from 24 to 26 inclusive. Figure 63 shows mean VOfDDD scores k2 standard errors at each AAR joint in males oniy, and Figure 64 shows mean VOmDD scores k2 standard errors at each AAR joint in fernales. These gnphs are similai to the ones descnid above, except for the addition of standard error bars. Overaii, the trends are similar in males and females. However, a few ciifferences are ~bserved- the pattern of VO/DDD joint distribution differs between the sexes in the manner described above. VOlDDD & Ape and Sex As described previously. the converted VO/DDD scores of each vertebrai colurnn were avenged in order to obtain an index (or summary score) of VOlDDD for each individual in the sample (N = 204 complete spines). The average VO/DDD score for each animal was then plotted venus age (Fig. 65). Figure 65 is a scatter plot of the average VO/DDD scores by age in the entire sample, with a smoothing spline fitted to the data. The curvilinear regression shows that there is a strong positive correlation between mean VO/DDD score and age; mean VO/DDD increases with advancing age. There is a definite linear trend in the data, and the line shows curvature between the ages of 7 to 15 yem. Most of the 7 to 9 par- old monkeys have normal, or virtually nomai, spines with respect to VOlDDD - mean scores of O to 0.3. A few individuds between the ages of 7.5 and 10 years have mean scores of about 0.5. Thus, vertebrai pathology begins at mund age 8 to 10 years; at this age, the venebral bodies of sorne individuals begin to show the fint signs of VO/DDD, at the incipient and fiat stages ofdevelopment (Fig. 65). A mean spinal score of 2 fust appears ai around age 11 to 12 yem in this sample. Average scores of about 2.5 to 3 begin to occur at around 15 yem of age. Spines with an average score above 3 appear at about 20 yem; spines with such hi@ mean scores are only present in the age range of 20 to 29 years. It is noteworthy that there are no individuais with completely normal spines (VOIDDD mean = O) afier the age of 15 years; beyond this age, di spines in the sample show some degree of degenerative change 65). Figure 66 shows the VODDD average scores of males and kmdes plotted separately. After the age of 15 years, both males and fernales exhibit high average scores, and the means increase with advancing age. For both sexes, the nomal individuals tend to cluster in age between 7 and 15 yem. The curves show that, compared to the femaies, the males exhibit slightly higher average scores for n given age. across the age range of the sample. However, the two curves are very close together, and show the same trend (Fig. 66). There is no signifkant difference between the overall VODDD scores of males and females (Mann-Whitney Rank Sum Test, p = 0.502). The median VOlDDD score for males is 0.34 and that for femdes is 0.21. Table 10 lists the frequencies of VOlDDD in the whole sample, and in males and females separately; these frequencies were calculated using the average ('summary') scores of each venebral column. Mdes and femaies have similar frequencies, and the overall rate of mean VO/DDD scores I to 4 inclusive is 36%. Vertebrae with typical VOIDDD are pictured in Figure 67; note the marginai osteophytosis. which is extensive in a few of the venebrae shown. TABLE 10 FREQUCNCIES OF VOmDD AND VOA, CALCULATED USING AVERAGE SCORES OF INDlVIDUAL SPINES VO/DDD Frequencies VOA Frequencies N = 102 fernale spines (VOtDDD), 1 15 fernale spines (VOA) N = 102 male spines (VOIDDD), 1O3 male spines (VOA) N = 204 spines (VOIDDD), 218 spines (VOA)in entire sample * Frequency is based on ail scom pooled VO/DDD b~ Natal G~UD For the maies, there is no statistically signif~cantdifference between the natal groups in terms of median VOlDDD (hskal-Wallis ANOVA on ranks, p = 0.17). The females aiso show no significant difference when mean VO/DDD is exarnined by natal group (Kniskal-Wallis ANOVA on ranks, p = 0.1 1). The Kruskal-Wallis Rank SmTest is a non-piuarnetric test that does not rely on nomality or equality of variances. Figure 68 shows box plots of VO/DDD average (or 'surnrnary') scores by natd group for the entire smple. and demonstrates that there is a grrat deal of overlap arnong the groups. in this sarnple, the data for natal groups are not nomdly distributed, and the viuiances are not equal. Figure 69 shows the reason for the lack of significant differences among the natal groups; there is considerable vuiation in age range among the groups. Some gmups are more variable in age than othea, even though the sample sizes rnay be similar. Some groups may have a greater range of ages, or relatively more older animais. This has the effect of obfuscating any differences in the frequency or pattern of age-related disease that may exist among the natal groups. Furthetmore. the sample sizes of the natal groups represented in this analysis dso vuy considerably. Thus, more resenrch is required. using matrilineage data, for a more direct investigation into possible genetic cffects. VOlDDD and Pari& This part of the analysis examined the VO/DDD database for females (ail 7+ years of age) for any association between parity and VO/DDD. This analysis was particularly chaiienging, shce VO/DDD is an agedated phenornenon. and parity and age are ciosely associated variables. However, with generalized additive models, the association between parity and VOIDDD cm be tested, while keeping age constant. With VO/DDD as the response variable, the effects of parity and age were tested. There is a significmt positive correlation between age and VO/DDD (p < 0.0001), and between parity and VOlDDD (p < 0.000 1). However, the latter result is due to the strong correlation between parity and age (r = 0.87), as discussed previously. in this sample, the disease in question and par@ are both age-nlated phenornena. When age is controlled in the model, the effect of puity is no longer significmt (p = 0.2 1). O 5 1O 16 20 26 Amphiarthrodial Joint 2nd PC :6.3% of Variance r O 10 15 20 Amphiarthrodtil Joint Fig. 55. VO/DDD: 1st and 2nd Principal Components in entire sample (N = 204 spines; 5,304 joints). Fernales: 1st PC :69.9% of Variance O 5 10 1s 20 2!5 Arnphiarthrodial Joint Fernales: 2nd PC :8.1 % of Variance Amphiarthrodial Joint Fig. 56. VOmDD: 1st and 2nd Principal Compnents in females (N = 102 spines; 2,652 joints). Males: 1st PC : 70.2% of Variance Amphiarthrodial Joint Males: 2nd PC :6.6% of Variance Arnphiartiuodial Joint Fig. 57. VO/DDD: 1st and 2nd Principal Components in maies (N = 102 spines; 2,652 joints). Intewertebral Joint Scores of 1 or Greater lntenrertebral Joint Scores of 2 or Greater 1 v O 5 1O 15 20 25 Inûmmbbral Joint lntewertebral Joint Scores of 3 or Greater lntewertebral Joint Swres of 4 or Gmter iO 1 Fig. 58. Proportion, by joint. of VOJDDD scores in entire sample (N = 204 spines; 5.304 joints). Percentage of Population WHIi Scores 1 or greatet Amphiarthrodial Joint Percentage of Population With Scores 2 or greater O 10 15 20 25 Arnphiarthtodial Joint Fig. 59. Proportion, by joint, of VO/DDD scores in desand females separately (Males: N = 102 spines, 2,652 joints. Femdes: N = 102 spines, 2,652 joints). Solid fine = maies; dorted line = fernales. Percentage of Population With Scores 3 or greater O 5 1O 15 20 25 Arnphiarthrodial Joint Percentage of Population With Scores 4 or greater - Males -- -- Fernalas O 5 10 15 20 25 Amphiarthrodial Joint Fig. 59 Continued. Amphiarthrodial Joint Fig. 60. Average VOlDDD rating in the entire sample at each amphiarthrodial (AAR) joint (N = 204 spines; 5,304 joints). -9-mmmmm Females - Males lntervertebral Joint Fig. 61. Average VOIDDD rating by joint in males and females separately (N = 102 male spines, 2.652 joints; 102 femaie spines, 2,652 joints). Solid line = males; dotted line = femaîes. Amphiarthrodial Joint Hg. 62. Mean VOJDDD I 2 standard emacmss the entire sample at each amphiarthrodial (AAR) joint (N = 204 spines; 5,304 joints). Fig-61. Pho~raphof kwcH thora& vert~braem-12, caudal a-) 8howing îypial VOlDDD in ikmik ageô 14 ymm (Photo A); note mrghrl orbophybs. Photo B rhom normal thora& ve-m mm a kmik agd 8 yeumm; nota mai margim of arlieulir surface8 ofthcentra. Natal Group Fig. 69. Box plots: Age (in years) by natal group of monkeys represented in this study. 6,2, Discussion Anatomv and Pathonenesis Vertebral Deeeneration Degenerative alterations in the spine involve two distinct intervertebral articular systems, the diarthodial or zygapophysed joints and the intervertebrai discs or synchondroses (Hough Ir. and Sokoloff 1989). The vertebrae are joined together by a three-joint system, comprised of the intervertebral disc anteriorly and the two zygapophyseal joints posteriorly. The spine acts as an integrated whole. and thus. injury or degeneration in one part of this joint system often affects the function and health of the other structurai components of the system (Macnab 1986). Dix degeneration and vertebrai osteophytosis are related phenornena. There are three main components to the intervertebrai disc: the mnulus fibrosus, nucleus pulposus and the hyaline cartilaginous endplate. Macnab (1986) defines the fint stage of disc degeneration as the vascularization of the aging disc - an event that leads to further anatomicai and physiological changes. Together. the nucleus pulposus and annulus fibrosus control the degree of movement permitted ktween adjacent vertebrae. A defect in the cartilage endplate may cause hemiation of material from the nucleus pulposus into the centrum; this loss of nucleus material results in excessive movement of the affected segments, with consequent damage to the annulus fibrosus, posterior joints and postenor ligaments (Macnab 1986). Dessication of the nucleus puiposus has r similv effect, by causing abnomal movements in the affected vertebrai bodies. When the fibers of the annulus fibrosus lose elasticity, they are no longer able to efficiently resist the nomial distortion of the nucleus pulposus caused by body movement or body weigbt. If the loss of elasticity is uniform, the annulus protmdes around its circumference. causing the intervertebral disc to lose height. If it is not unifom, then a localized herniation of the nucleus pulposus may occur (Macnab 1986). Thus, naturd aging resuits in Ioss of the gelatinous matenai in the intervertebrd disc. and weakening of support from the annulus fibrosus. As these degenerative changes continue. the disc loses its biomechmical integrity and stability, and the movement between adjacent vertebral segments becomes uneven, excessive and irregular (Macnab and McCulloch 1990). The effects of disc degenention cm be detected on the surrounding structures of the spine, especially on the vertebrai bones. The "tnction spur" (see below) and gas in the disc (vacuum phenornena on X-rays) are two manifestations of instability. The most cornrnon manifestation of disc degenention is üpping or osteophyte formation of the vertebral bodies. Osteophytes are bony outgrowths, described as spurs or exostoses. that develop dong the margins of vertebnl bodies. and the margins of synovial joints (van der Korst 1992). According to Macnab (1986). vertebrd osteophytes have little clinical significance, i.e., they do not produce symptoms -- unless they protrude posteriorly into the intervertebrd foramina and impinge on the nerves. However. vertebrai osteopbytes are significant in that they denote the presence of disdegeneration; they indicate that the affected vertebral segments have been unstable for some time. These conditions may also lead to degenerative changes of the yti*cular fxets, as discussed in Chapter 8. Macnab (1986) identifies a number of different types of osteophytosis, such as the 'traction spur' and 'subperiosteai ossification'. Disc degeneration is associated with excessive mobility in the affected vertebnl bodies, which applies considerable traction to the anchoring fiben of the annulus fibrosus, and traction spurs fomi as a response to this stress. This type of osteophyte projects hontontally and develops about 1 mm from the edge of the vertebrai body. Others have also described marginal osteophytes that are associated with a tear in the annulus fibrosus (François, Euldennk. and Bywaters 1995). Wben the fiben of the annulus fibrosus [ose their elasticity, causing the disc to protrude around its circumference, the longitudinal ligaments and periosteum are eventually Iifted off the vertebrai bodies. thus stimulating subperiosteal new bone formation (Macnab 1986; Macnab and McCulloch 1990). According to Macnab and McCulloch (1990). a smail traction spur indicates present instability, while a large traction spur indicates that the segment was unstable at some time in the past, but may now be stable due to fibrotic changes taking place within the disc. At a very advnnced stage of intervertebrai disc failure. the entire nucleus pulposus and most of the annulus fibrosus disappear. and eburnation of the articular surfaces of the centra may subsequentiy occur. Such changes are reminiscent of osteoarthntis of diarthrodid joints (François. Euldennk. and Bywatea 1995). A few cases of ebumation of vertebrai articular surfaces were seen in the present study. More commonly. in this sample, ebumation is found on large. curved osteophytes. Traction spun and subperiosted ossification were also observed. Macnab and McCulloch (1990) distinyish two foms of vertebrai osteophytes that ;ire associated with degenentive disc disease. 'traction spurs' and 'claw spurs' (Macnab and McCulloch 1990). The claw-type osteophyte develops from the rim of the vertebrd body and curves over the outer fibers of the intenrertebral disc; it curves as it grows anxtnd the bulging intervertebnl disc. The results of one study conducted on human lumbar spines showed that claw osteophytes are more common than traction osteophytes. that both may coexist in a single vertebrd body, and that these osteophytes represent diEferent stages of the same pathologie process (Pate et ai. 1988). The r~sultsof the present study on rhesus monkeys confum that both claw and traction osteophytes may coexist in the same vertebrae. Both the horizontaily-oriented traction spurs and the vertical'y-oriented claw osteophytes were observed in the present simple of rhesus monkey spines. An extensive description of the ordinal scaling method used in this study is provided in Chaptkr 2. The ordinal scale shows that traction spurs, claw osteophytes, and disc degeneration with its concomitant changes in vertebral centra, are al1 part of the same process. Radiogmphic criteria for the diagnosis of established degenerative disc disease include nmwed intervertebrai disc space. sclerosis of adjacent vertebral margins and osteophytosis (Macnab 1986). The latter two features were observed on the X-rays of spines from animais with DDD in this study. It is not possible to mess disc space narrowing directly in macerated spines; it is usually inferred from the presence of other features. Depcnerative SpinPl Disease in the Rhesus Monkevs of Ca~oSantiaw: Vertebral Osteo~hvtasisJVO) and Dononerative Dise Disease (DDD) Age !Sex Distribution. Prevalence and Joint Distribution VODDD DeRousseau and other researchen investigated the prevalence of sjmntmeous degenerntive spinai disease (vertebnl osteophytosis) among the rhesus monkeys on Cayo Santiago (DeRousseau 198%; DeRousseau, Bito, and Kaufman 1986). DeRousseau and colleagues (1986) found th, in the extant population. vertebral osteophytosis is most prominent in the thoracic region of the spine, and is weU advanced by the age of 25 yem. They observed that degenerative changes in the vertebrai bodies increased in frequency with advancing age, becoming very comnin male and fernale monkeys over the age of 20 yem. This study ceported no significant diffeiences berneen males and femaies in the kquency of degenentive spinal disease. DeRousseau (1985) examined ndiographs of the spine from monkeys living on Cayo Santiago, and averaged the scores for vertebral osteophytosis (which she cdsdegenerative joint disease or DJD) for al1 the intervertebrai symphyses from the lower spine. She reported that the average 'DJD'score for joints in the lower spine is significantly comlated with age, and that pronounced and widespread changes do not occur before the age of 20 years. There is minimal degeneration of the spine before the age of 10 years. No differences were found between the sexes. The eariiest degenentive changes in the lower spine were noted at the thoraco-lumbar junction (DeRousseau 198%). To the writer's knowledge, the present study is the fint extensive, detailed analysis of the patterning, distribution of joint involvement, and prevaience of vertebnl osteophytosis/degenentive disc disease (VOIDDD) in the vat skeletd collection derived from the rhesus monkey colony of Cayo Santiago. The present study complements the previous work by DeRousseau and her colleagues, and confims that VO/DDD has a predilection for the thoracic region of the spine in rhesus monkeys. The thoncic vertebne account for a very significant proportion of the variation in the total sample of 5,304 AAR joints - 69.346 of the variance. Most of the pathology in the sample occurs in intervertebnl joints T5/T6 to TiULl inclusive. Males and females show a very sirnilar pattern of joint disuibution of VOIDDD; in both sexes, 70% of the variance in the sample is explaincd by the thoracic vertebrae. An AAR joint near the cervical-thoracic junction appears to be particularly susceptible to VO/DDD in both males and females - AAR joint 9 (Tm3). The location of this intervertebral joint is at the end of the cervical lordosis (dorsdly concave curvanire), and at the start of the thomco-lumbar (dorsally convex) curve of the spine. h a spine that functions biomechanicdy üke a cantilever, the vertebral joints lucated in the transitional mafrom neck to thorax, and from thorax to lumbar section. may experience particulas stresses that predispose hem to developing degenentive changes over time. in both femdes and males, the thoracic vertebrae, the tmnsitiond thonco-lumbar region and the joint between L7 and S 1 tend to exhibit the highest frequencies of al1 grades of VO/DDD. Unlike the lumbar vertebrae. the movement of the thoracic spine is constrained by the ribs. In a quadrupedal spine, this anatomical difference is significant in terms of the biomechanical stresses experienced by the AAR joints during locomotion and other activities. According to Sullivan ( 1933), the thoracic region of the spine in the rhesus monkey is capable of free flexion. extension and lateral movement, but not of great range. Torsion is present. but it is limited. In the lurnbar section. there is limited flexion and extension, and considenbly more lateral movement is permitted. but torsion is minimal. The shape and size of the supenor and infenor articular processes and facets determine the type and degree of motion admissible within each section of the spine (Sullivan 1933). Located at the apex of curvature in the macaque spine. the thonco- lumbar junction is likely subjected to unique biomechanical stresses. These vertebrae are situated at the point where a change in mobility of the spine is observed, from the thom to the less constrained lumbar joints. The L7/Sl joint demmates another change in the matornical shape and structure of the macaque axial skeleton. b VOIDDD: Comparinn Females and Males For each spine in the sarnple, an average score of VO/DDD was caiculated, providing a measure of the extent of the disease in each individual. As shown in Table 10.36% of the monkeys in the total sample (N = 204) exhibit sorne degree of VODDD, from an average score of 1 (mild) to an average score of 4 (severe); 3446 of females (N = 102) and 38% of males (N = 102) have VOtDDD. The number of affected males and femaies is companble. Exvnining average scores of VOIDDD by intervertebrai joint in the entire sample (N = 204 spines; 5,304 AAR joints), and in males and females separately, confinns that most of the thoracic and upper lumbar joints (T6 to W)show the highest scores, as does joint 26 (L7/S 1). While the overail pattern of joint distribution of VO/DDD is similar in males and females, the thoracic distribution reveals one main difference between them. in mdes. the most affected joints are those from T8IT9 to TlULl inclusive. while in females. the most affected part of the thoracic spine lies in a more cranial orientation - from T6/T7 to T9/TlO inclusive. The reason for this difference between males and femaies is not known; however, one possible explmation cornes to mind. Females carry their infants on their back or suspended from their abdomen. and this behaviour may stress the thoraco-lumbar spine in a manner that is unique to their sex. This may also explain why fernales exhibit higher average values in joints LlR2 to LAILS than do males. The present study found a strong positive correlation between mem VOIDDD and age; VO/DDD increases in seventy with advancing age in the entire sample (N = 204 spines). The same trend is observed when males and femaies are examined separately. Most of the individuais younger dian 10 yem of age show normal, or vinually normal spines with respect to VOmDD. The present study found that degenerative changes of the vertebrai bodies begin at about 8 to 10 years of age. Al1 individuals over the age of 15 years exhibit some degree of degenentive change. Mer age 15. both males and femaies show high mean spinai scores. Mean spinal scores of about 2.5 to 3 are fust seen at about 15 years of age. Scores above 3 are observed in individuals who are 20 years of age and older. The mdes exhibit slightly higher average scores compared to the females, mss the age range of the sarnple; however, the differences between the sexes are not significant (p = 0.502). The observations from the present study are consistent with DeRousseau's findings on the live colony. Paritv and VO/DDD A previous study examined knee joint tissue for spontaneous osteoarthritis in 35 rhesus monkeys derived From the Cayo Santiago colony, and assessed the histology and relationship of OA to the epidemiologicd factors of age, sex, parity, body weight and caging history (Chateauvert et al. 1990; Pritzker et ai. 1989). Cornparisons were made between four groups, 'young-nomai', 'old-normal'. 'young-OA' and 'old-OA'. and the sample sizes are listed as 6,5. 9. and 21 respectively. The authors note that obesity may be a relevant factor in younger animais, and that caging appears to retard the progression of OA. They conclude that the frequency of knee joint OA increases with age, and is more prevalent in females, especially in those with high parity. They compare the parity of femdes in the 'old-normal' group to that of femdes in the 'old-OA' group, md find that the diffennces are significant (p = 0.03): the mean ages of these two groups are similar (Chateauvert et al. 1990). However, the authors note that the fernales in both normal groups are significantly below the exmcted ~acitvfor their aee (p < 0.05); a sipificant proportion of monkeys are either nulliparous or below average in number of offspring. They also state that the parities of the females in the OA group are within their expected range (Chateauvert et ai. 1990). This suggests that OA did not affect the capacity of these femdes to reproduce successfully. The fact that the parities of the normal females are not at expected levels suggests a problem with sampüng, or perhaps the presence of an unknown condition that had a detrimental effect on parity. Thus, the significant result they report between parity and OA may actually be attributed to the nomai individuals who have lower par@ than expected. and to whom the femdes with OA were compared. Srna simple sizes for the groups may have affected their results. In addition, since both parity and OA are strongiy age-related. it is a challenge to investigate the relationship between these two variables while controlling for age. in the present study, no relationship between parity and VO/DDD could be demonstmted When the effects of age are controlled through the use of generalized additive models (GAM), there is no significant association between VODDD and parity (p = 0.21). When the statisticd test was performed without controlling for age, a significant positive conelation was found between parity and VOtDDD (p c 0.0001). This demonstrates the strong effect of age. and the importance of accounting For such effects in any mode1 that involves parity. Although the present investigation did not find evidence for a genetic component to VO/DDD mong these rhesus monkeys. there is evidence in the literature to support a genetic etiology for this disease (Postacchini, Lami, and Pugliese 1988; Scapinelli 1993; Simmons Ir. et ai. 1996). The resuits of a study by Postacchi~et ai (1988) point to a strong familial predisposition to discogenic low back pain, suggesting that the etiology of DDD is related to both genetic and environmental factors. Scapinelli (1993) observed lumbar disc hemiation in eight siblings with a positive family history for degenerative disc disease. The maximum incidence occmd in males of the third pneration, who cequired surgicd intervention, while other membea of the family suffered from chronic low back pain. The author postdates a genetic predisposition to early disc degeneration to explain this cluster of DDD cases. A study by Simmons Jr. et al (1996) found that, of 65 patients who had undergone surgery for degenerative disc disease. 44.6% had a positive family history for the disease, compared to 25.456 of patients in a control group. They conclude that a familial predisposition to DDD cm exist dong with other risk factors. These studies indicate that it would be fniitful to pursue an investigation of a possible genetic predisposition to VOlDDD mong the rhesus monkeys of Cayo Santiago. using a different approach - comparing data from matrilines. Vertebral Osteoahvtosis JVO) and Denenerative Dhc Disease (DDD)& Non-human Primates and Other Animals Spontaneous degenerative joint disease of the spine also occurs among rhesus monkeys in other primate research centers (DeRousseau 1985a). and in other research settings (Sokoloff, Snell, and Stewart 1968). The results from the present study bave recently been corrobonted by a raâiographic study of thonco-lumbar degenerative disc disease (DDD) in a group of 108 colony-raised, live female pig-tail macaques (Macaca. nemesuina), aged 5.2 to 29.2 yevs (Newell-Moms et al. 1999). Degenerative disc disease does not occur in pig-tail macaques younger than 12 years of age, and the disease is ubiquitous in anirnals older than 17 yem. The initial signs of disc space nmowing are fit observed in animais as young as 5.5 yem, and the earliest age at which osteophytosis occurs is 9.3 years. Thus. among these female macaques, disc space narrowing is present at younger ages than is osteophytosis. The authors note that the initial evidence of disc space narrowing is randomly distniuted throughout the spine, while initial osteophytosis is nstricted to the thoracic vertebrae. Eighty-three percent of individuais with osteophytosis exhibit osteophytes at the T9/10 and Tl2Ll joint spaces. A high incidence of vertebral osteophytosis has been reported in many species of monkeys and apes. Studies have shown that gorillas, chimpanzees and orangutans exhibit relatively higher frequencies than do monkeys (Clauser 1981). Various studies have reveaied that vertebrai osteophytosis of the amphiarthrodiai joints of the spine and osteoarthritis of the anicular filcets vary in fiequency across the primate genera, with the highest kquency recorded in gorillas. followed by orangutans and chimpanzees (Fox 1939; Jurmain 1989a; Love11 1987; Lovell 1990; Schultz 1956). These differences have been attributed to the greater body weight of gonllas. compared with other primate genera (Fox L939). However. a study on OA of the hip, knee and ankle in chimpanzees and gorillas found a weak correlation between body size and OA (Woods 1986, cited in Jurmain. 1989). The relationship between body weight, biomechanics of the spine. and degenentive arthritis should be explored mer. but this subject is beyond the scope of the prexnt study. It is especiaily noteworthy that, although vertebrnl osteophytosis is very cornmon in human and mountain gorilla skeletal populations. it is quite rare in chimpanzees (Jurmain 1989a; Love11 1987; Zihlman, Morkck, and Gd11990). Schultz (L969) also noted the rarity of severe vertebnl arthritis in chimpanzees. lurmain (1989) studied the skeletons of 15 chimpanzees denved From Gombe National Park, and found a complete lack of vertebral osteophytosis (N = 344 intervertebd joints), even mong the older animais, thought to be over 30 years of age at death. The vertebral joint surfaces and mûrgins are completely normal, even in the very old animals. He aiso found OA in only a few vertebral articular facets (out of 738 in totd), and recorded a low incidence of OA in peripheral joints. These results stand in contrast to the pattern seen in most human populations (see below), where this disease is almost ubiquitous among individuals 40 years of age and older. However, Iurmain suggests that this discrepancy may be due to the smaii sample size of the study. In conuast, mother study on a srnall group of 11 chimpanzees found a relatively high frequency of vertebral osteophytosis in the lower lumbar spine (21% of centra) and a very high frequency of VOA (52% of articular facets) (Cook et al. 1983). A comparative biomechanical analysis of osteoarthntis of the hip. knee and ankle in humans. gorillas and chimpanzees actually found that chimpanzees were more affected by degenerative disease than were gorillas (Woods 1986, cited in lurmain, 1989). DeRousseau et ai (1980) note th* vertebral osteophytosis in macaques and orangutans is most frequent in the lower thoracic vertebrae, while the predilected site in gibbons, chimpimzees and humans is the lumbar region of the spine. in gorillas, vertebral osteophytosis is more evenly distributed throughout the thoracic and lumbar spine. Evidence of disc hemiation and vertebnl osteophytosis have been reported in orangutans, with degenerative disc disease (DDD) king most predorninant in the thoracic region of the spine (Fox 1939; Lovell 1987). DeRousseau notes that marked anterior expansion of mid-thoracic vertebrai centra is most pronounced in the macaque, but also occurs to a lesser degree in chimpanzees and humans (DeRousseau et ai. 1980). Love11 (1990) describes spinal degenerative joint dwase (DDD) in a group of mountain gorillas (the Fossey series), a group that dso exhibits extensive appendicular joint degenemtion. scoliosis. osteornyelitis, trauma, neoplasia, periodontal disease, and carious lesions. However, the primary Miction of the skeleton is arthritis, predominantiy spinal degenerative disease (vertebrai osteophytosis) (Lovell 1990). Schultz (1969) aiso describes arthritic changes in the skeletons of old 'wild-shot' eastern godias. He found severe chronic arthntis in some of these specirnens, and noted marked vertebral osteophytosis of the lumbar vertebrae (Schultz 1969). In Schultz's survey of the skeletal remains of 118 wild adult gibbons (mostîy Hvlobates hrJ, he found 6 cases of 'arthritis' of the vertebrai column (Schultz 1939). in gibbons. the vertebral column is most frequently affected by arthritic changes compared to other skeletai sites. It is not clear whether this 'arthritis' was vertebral osteophytosis or osteoarthntis of the articular facets (or both). Schultz concludes that chronic arthritis is probably limited to catarrhines, with the highest frequencies occming in gibbons. orangutans and gorillas. He notes that the disease increases in frequency with advancing age in al1 three groups (Schultz 1956). Krumrine ( 1999) studied the spines of 88 gorillas, 46 chimpanzees. 13 orangutans and 40 gibbons to examine the expression and distribution of vertebral osteophytosis. This study reports that, like humans, the seventy of the osteophytes is greatest in the lumbar region of the spine in al1 genera. with the notable exception of the gibbons. which were negative for osteophytosis. Chimpanzees exhibit the second most severe osteophytosis in the thoracic region, while for the orangutans and gonllas, the cervical vertebrae show the second most severe osteopbytes. Krumrine notes that the chimpanzee pattern of osteophytosis is consistent with the expected pattern for bipeds. suggesting a similar distribution of this condition in humons and apes. KNmrine maintains that these results suggest that locomotion and activity patterns may not be the only causai mechanisrns for the development of vertebnl osteophytosis (KruInrine 1999). Fox (1939) conducted a museum survey of over 1,70 skeletons derived from wild rnammals, looking for evidence of 'iuthritis' (Fox 1939). He found that naturally-occumng arthntic lesions of the spine and appendicular skeleton are extremely common in mamrnals. and comparable to the human counterpart of the disease. The most numerous lesions occurred in the vertebral columns hmthis series of skeletons. Fox examined a number of godias, orangutans and baboons, and found that the mid-cervical. upper thoracic, lower thoracic and mid-lumbar regions of the spine have the greatest number of -tic joints. In baboons, vertebral osteophytosis most fiequently affects the mid- and lower cervical vertebrae (Fox 1939; Schultz 1956). Degenerative disc disease and vertebral osteophytosis are widely distributed among quadrupeda1 species in other fnmilies. Fox also noted a high distribution of thoncic osteophytosis among the carnivores. bovids. cervids, and suids included in his study. Noteworthy is the complete lack of arthritic disease in the dents, bats, sloths. mnadillos and cebid monkeys surveyed by Fox. According to Fox. it appears that arthritis-bearing animals tend have a larger body size (and weight) than non-arthritics; however. he notes some exceptions to ibis trend. The rhinoceros and the came1 were the only heavy animals in his study that did not exhibit arthritis (Fox 1939). Hansen (1959, cited in Sokoloff, 1960) has described many cases of degenerative disc disease in domestic dogs. and noted chat domestic cats are less prone to this disease compared to dogs. Hansen also found cases of DDD in cattle, pigs and camels that were corûined to isolated intervertebral spaces. in cmels. he found that the lesions were most predorninant in the cemico- thoncic region of the spine, an ares of transition in terms of curvature and morphology. in al1 cases of DDD among these animais. Hansen noted destruction of the intervertebral discs and cartilage plates. followed by prolific marginal osteophytosis and reactive derosis of bone in the vertebrai bodies (Sokoloff 1960). Vertebral osteophytosis and degenerative disc disease have also been noted in fernale pigs (Doige 1979). bous (Doige 1980), rabbits (Green,Fox, and Sokoloff 1984) and sand rats (Moskowitz et al. 1990). VOIDDD Humans, Past and Present Degenerative changes of the spine have a high pnvalence in extant human populations. and are almost ubiquitous in older individuals (Hadler 1993; Iayson 1988). The prevalence of degenemtive disc disease shows a linear incnase with advancing age. particularly after the age of 30 years. An autopsy study of 4.000 vertebral columns found that vertebrai osteophytosis was present in 60% of women and 80% of men by the age of 50 years (Schrnorl and Iunghanns 1971). Another investigation of 60 lumbar intervertebral discs from 273 cadavers representing ail age groups (up to 96 years) found that male discs wen mon degenerated than lemaie discs across most ages, most significantly in the second, fifth, sixth and seventh decades (Miller, Schmatz, and Schultz 1988). On average, ciiscs at the U/L4 and LAN levels exhibited more degeneration than discs between other lumbar vertebrae. Males showed earlier and more severe disc degeneration than did fernales, but by age 50 yem. 97% of ail lumbar discs in the sample exhibited degeneration. The authors speculate that greater compression loading may predispose the human mde intervertebral disc to earlier and more severe degeneration (Miller, Schmatz, and Schultz 1988). Butler (1990) conikms that W/L5 and L5/S 1 are the joints most susceptible to disc degeneration. Newell-Morris et al (1999) compared their results on DDD in pig-rail macaques to a group of 39 normal women aged 60 to 78 years, who were participants in a survey of osteoporosis. Sixty-nine percent of these women exhibited the typical chancteristics of DDD on radiographs, Le., disc space nurowing, compared with 100% of the macaques in an equivaient age range (older than 15 years). However, the prevalence of osteophytosis was 95% in the macaques and lOWb in the wown, suggesting to the authors that disc nmwing and osteophytosis may develop and interact differently in the two species - in tenns of biomechanics md senescence in joint structures (Newelî-Moms et al. 1999). A high incidence of vertebral osteophytosis was also found in the present study on rhesus monkeys from Cayo Santiago. The vast litenture on the paleopathology of VO is too extensive to review hem, and is beyond the scope of this thesis; but, suffice it to Say, vertebrai osteophytosis (degenerative dix disease) md vertebral osteoarthntis have alfected humPnkind since antiquity. Exarnples of skeletd lesions attributed to these conditions have been described in skeletons from a wide range of cultural affiliations, and temporal and geographic distributions (Bridges 1994; Chapman 1969; Clabeaux 1976; Kelley 1982; Knüsei, Goggel, and Lucy 1997; Lovell 1994; Maat, Mastwijk, and van der Velde 1995; Merbs 1983; Molto 1986; Onner and Putschar 1985; Rogen, Watt, and Dieppe 198 1; Rogen, Watt, and Dieppe 1985). These studies reved that VOlDDD and VOA were as common in ancient people as they are in modem populations. Rogers and colleagues (1985) conducted an extensive survey of spinai osteophytosis, DISH and ankylosing spondylitis in 560 adult skeletons and several thousand disarticulated venebrae from individuds representing various cultures and historical periods, from a 2 1st dynasty Egyptian mummy to a mid-19th century skeleton. They found that vertebrai osteophytes were present in about half of the specimens in their study. In fact, osteophytes associated with degenentive disc disease and vertebrai osteophytosis were the most common observation. Forty-six percent (N = 81) of individuds of Romano-British affiliation exhibited vertebral osteophytosis, 69% (N = 121) of individuds of Saxon denvation were aftected, and 37% (N = 303) of mediaeval skeletons had osteophytosis of the spine. Maat et al (1995) exmined the skeletal remains of 176 adult indîviduals from the Iate mediaevai city of Dordrecht in Hoiland for the distribution of degenentive changes in VO, VOA. DISH and peripheral OA. The authors compared their results to other studies in the literature. and found that their pattems of frequencies with respect to age and sex were essentiaily the same as those of a modem Dutch suburban village population. in the ancient human skeletal population. high frequencies for vertebnl osteophytosis (70.9%) were almost twice those for VOA (36.88), and both diseases increased with age in both sexes. Vertebral osteophytosis is present in individuais older than 20 years of age. and is very prevalent in ai1 older age intervals. The overall frequencies of VO and VOA for the Dutch mediaeval population fdl within modem ranges. and are similar to those of laie mediaevd Britain. as described by Rogers (1981. 1985). From observations of the individual patterns of VO and VOA, Maat et al (1995) assert that each disease progresses as a sepante entity from the other. In VO/DDD, stresses on the intervertebral discs are primmily the result of the weight-beming function of these joints, whereas in VOA, body movements are the main cause of stress on the articular facets. This functiond difference between the anterior and postenor joints of the spine is the underlying reason for the dissimilar patterns of joint degeneration. A functionai difference between anterior and posterior joints also explains the results obtained in the present study. Two distinct pattems of spinal 'arthntis' exist in the macaque spine - to be discussed in chapter 8. In the ancient Dutch sample studied by Maat, osteoarthritis iippears to be a systemic disease. affecting synovial joints in general. including those of the spine - the articular facets. This study dso found that, with increasing age. bone spurs associated with dix degeneration may become hidden anatomically by the para-vertebral enthesophytes of DISH (Maat. Mastwijk. and van der Velde 1995). This phenomenon was also observed in the rhesus rnonkeys of the present study. and is discussed in chapter 7. Another study of ancient human remains supports the conclusions of Maat and coIIeagues (1995). Knüsel et al ( L997) examined the intervertebral and zygapophyseal joints of the spine in 81 aduii. male skeletons denved from a mediaevai cemetery in England. For ench spine, a single measure of 'DJD'(their term for VO and VOA) seventy was determined by summing the lesion scores for each joint, for a total of 49 joints (24 intervertebrai joints and 25 zygapophyseal joints). As described earlier, a very sirnilar technique for obtaining a summary score of VODDD and VOA for a vertebrai column was developed independently for the present study on rhesus monkeys, which makes the results of the present study directly comparable to those of Knüsel et al (1997). The authors found that the pattern of vertebrai osteophytosis differed from the pattern of vertebrai osteoarthntis. The anterior and postenor joints show dissimilu pattems of spinal joint degeneration, which confirms the observations of Maat et al (1995), and the results of the present study. According to Knüsel et al (1997). the two types of joints appear to exhibit an almost inverse pattern of severity. They note that the most severely affected intervertebrai joints (AAR joints) occur in C5/C6, C61C7, T8n9 to Tl lm12 inclusive and W. The joints least affected by 'DJD' are C7 to Tl, Tlnland L5/S 1. The highest degree of VOA occun in facet joints CX3to C4KS inclusive, C7A'l, T lO/Tll, and U/L4 to LSIS 1, and the lowest scores are found in the occipital condyles to CI, C5/C6 to C6/C7, and the joints from TI to T9 inclusive. They found that the articular facets exhibit progressively increasing seventy in the lower thoracic and lumbar vertebrae, with no reduction in severity in the LWS1 joint. The authors specdate that these observed differences between anterior and postenor joints are caused by joint response to the stresses of erect posture during bipedal locomotion, reflecting normal vertebnl curvatwes rather than occupational stress (Knüsel, Goggel, and Lucy 1997). From her study of vertebral arthritis in the prehistoric southeastem United States, Bridges (1994) also concluded that the pattems of VO and VOA in her sample of ancient humans reflaft the stresses imposed by spinal cwature and weight-bearing due to habituai erect posture. She noted the highest frequencies of VO and VOA in the lumbar vertebrae, followed by the cervicd and thoracic segments. The results of the present study on VO/DDD and VOA (see chapter 8) ais0 appear to reflect the normal vertebral curvatures of the macaque spine. With respect to rhesus monkey and human VO/DDD, there is r stnking similarity in the overall shape of the graph of mean VOIDDD scores by joint in male rhesus (see Fig. 62) cornpared to male humans, as shown by Knüsel et ai in their 1997 paper - but a few key differences exist. The scores for VO/DDD in cervical joints increase in both rhesus monkeys and humans, and a break occurs between cervicai and thoracic vertebrae h both species; there is a sharp drop in vdue at the C7Rl joint. reflecting the change in curvature at this junction. The VO/DDD values then increase incrementaily throughout the thoracic joints in the human maies. and a srnalier break is discernible at the TluLl joint, where there is a slight drop in value at this junction. However, VO/DDD for the lumbar joints remains relatively stable at high vaiues in the human spines. The data for rhesus monkeys shows a different pattern. The values for VO/DDD increase incrementally from the lower thoncic joints through TlUL1, then gradudly decrease from joint to joint in the lurnbar spine dom to URS. &ter which the values increase sharply from joint to joint to L7/S 1 (the highest value). These results mflect the vertebral curvatures and biornechiuiical stresses of a spine from a habituai qudmped with an upnght sitting posture as part of its positional behaviour. Conclusions The main findings of this part of the study are as follows: VO/DDD is present in relatively high frequencies in this sample of rhesus monkeys. Thirty-six percent of al1 individuals (N = 204) exhibit some degree of VO/DDD, from a spinai average score of 1 (mild) to 4 (severe), and there is no statisticdly significant difference in fnquency between the sexes. VO/DDD has a predilection for the thoracic spine in rhesus monkeys. The thoracic vertebrae account for a very signilicant proportion of the variance (69.3%) in the total sample of 204 spines, 5,304 AAR joints altogether. Most of the pathology in the total sample occurs in intervertebrai joints T5n6 to T 12L1 inclusive. Maies and femdes have a very similar pattern of joint distribution of VOIDDD, and in both sexes, 70% of the variance is explained by the thoracic vertebrae. While the overall pattern of joint distribution of VO/DDD is similar in females and males, there is one main difference in thoncic distribution. The most affected put of the thoracic spine in femdes lies in a more cranid ilction compared to that of males. Ferniiles aiso exhibit relatively higher average values in joints LlL2 to U/LS than do males. It is postulated that these differences may be explained by a behaviour unique to females - carrying infants on the back or suspended from the abdomen. In both femdes and males, the thoracic vertebrae, the transitionid thoraco-lumbar region and the joint between L7 and SI tend to exhibit the highest frequencies of al1 grades of VODDD. VOIDDD in thk study appears to reflect the normal vertebral curvatures of the macaque spine. The boundaries between the different types of vertebrae (cervical, thoracic. lumbar) are subjected to high levels of biomechanical stress. These areas demarcate transitions in matornical structure and function. Thus, the thoraco-lumbar junction is likely subjected to unique biomechanicd stresses due to its location at the apex of curvature of the vertebrai colurnn. This site also exhibits some of the greatest frequencies and highest scores of VO/DDD. i There is a strong positive correlation between VO/DDD and age in this sample of rhesus monkeys. VO/DDD increases in severity with advancing age in both males and females (N = 204). i Most individuals younger than 10 years of age have normal spines. Degenerative changes of the vertebral bodies begin at about 8 to 10 yem of age. Al1 individuals over the age of 15 yeûrs exhibit some degree of VO/DDD. i There is no significmt association between parity and VOlDDD in this sûmple. No relationship between natal group affiliation and VO/DDD could be detected. This part of the maiysis should be repeated in a tùture study. replacing the natal group variable with matrilineage information. i Researchers using the rhesus monkey as a mode1 for human VO/DDD should be aware of the biomechanical differences between the human and macaque spine, and should note that the pattern of joint distribution of the disease differs between them. Aiso noteworthy is the fact that human mdes have a higher prevalence of disc degeneration than do human females. in contrasi, the male and femde rhesus monkeys in the present study exhibit comparable Irequencies. and similar patterns of VO/DDD with respect to age and sex. It is advised that mearchers take these differences into account when utilizing the rhesus macaque as a mode1 for buman spinal degenerative disease. CHAPTER 7 DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS (DISH) ui this sample, there are two definite cases of DISH and the cases of probable DISH, according to the standard cntena described in chapter 2. The frequency of DiSH in this sample is 2.2% (9228). However, this may not represent the actual prevdence of DISH in this colony of rhesus monkeys: recall that some individuals in this sample were excluded from the analyses because a number of their vertebrae were missing as a result of destructive sampling by a previous investigator. Such individuals could not be evaluated properly For the pnsence of DISH. The two definite cases of DISH are males (Cat. #2065 and #846); the former was aged 24 yem at the time of death, and the latter was 26 years of age. The spines of these individuals show dl the characteristics of classic DISH. They exhibit prolifentive enthesopathy, new bone formation, and continuous ossification of the anterior longitudinal ligament (AU) of at least four contiguous vertebral bodies. in X-rays, radiolucent zones are present between the deposited bone and the anterior surface of the centrum. Figures 70 and 71 show photographs of some of these uaits in Cat. #2065. The absence of îùsion in the zygapophyseal and sacroiliac joints distinguishes this disorder from ankylosing spondylitis. Then is essentially normal disc space height between the vertebrae co~ectedby ossified ALL, except in the presence of vertebral wedge fractures. It is noteworthy that multiple diseases, at end- stage, are present in the spines of these two monkeys. For example, the spine of Cat. m065 exhibits osteopenia, multiple vertebrai wedge factures, advanced DISH (enthesophytes) and vertebrai osteophytosis. In Cat. #2065, metabolic bone disease eventuaiiy led to signifiicant bone loss and vertebral wedge ktures, despite the presence of DISH and osteophytosis. It is probable that the osteopenia developed after parts of the spine became immobilized by advanced DISH; this condition eventually led to wedge fractures of the centra, and to fractures of the enthesophytes as well. The fractures in the ossified AU. in various puts of the spine may also explain the presence of advanced remodelling of the articular surfmes of the centra in those areas. as shown in Figure 7 1. The fractures in the ossified W are visible in Figure 70: such fracturing causes additionai stress to the affected vertebrae, thus promoting intervertebral disc degeneration. A lateral X-ray of the spine of this specimen was shown previously, in Figure 54, chapter 5. The various conditions affecting this spine, including osteopenia (borderline, BMD = 0.654 g/cm2). are cleiuly visible in the X-ray. In specimen W846, DISH involves the lower thoracic (from Tg) and lumbar vertebrae. in specimen #2065, dl of the thoracic and lumbar spine are involved in DISH; complete fusion via ossifïed ALL occurs in the lower thoracic and lumbar vertebrae* A number of vertebral articular surfices are completely remodelled, an example of which is shown in Figure 7 1; some of the vertebrae even exhibit Schmorl's nodes. The three cases of 'probabk DISH' are ail females, aged 14, 16, and 24 years, al1 showing the eariy signs of DISH in two contiguous vertebrai bodies, Le., ossification of the ALL connecting two contiguous centra. Very large, curved enthesophytes are also present in other vertebrae, but they do not connect contiguous vertebral bodies. The 24- year old femaie may have had a more developed case of DISH, but this could not be ascertained due to a number of missing vertebrae. Thus, she was assigned the diagnosis of 'probable DISH'. Fig. 70. Phobgraph of the Meml aspect of the vertebrai cokimn damonkey (Cet.##2û65) wWi diffime idiopathic steletel hypembsis (DISH). This specîmen is a male, aged 24 pars. A radiraph of this spine is depidsd in Figure 54. Fig. 71. Photograph of the caudal aspect of a vertebra from he spine wiüi DlSH pichrd in Figure 70. This pickire shows the ossification of the anterkr kngitudinal ligament (AU), a spaœ between the anterior surfkice uf the centrum and the ossiîïeâ ML(radiolucent zone in radbgraphs). Note aie remodelled articular surface of the 7.2. Discussion -Diffuse Idio~athic Skeletal Hviicrostosis(DISH): kcri~tion DUlerential Not al1 bone spurs near joints and intervertebrai discs are classilied as true osteophytes. Osteophytes that develop in the bony attachment sites (insertions) of ligaments, tendons and capsules are temed entheso~hytes. True vertebral osteophytes grow outwards horizontdly f'om the margins of the vertebral bodies, whereas enthesophytes mise in the insertions of ligaments and tendons, and cwve as they grow dong these structures (François. Eulderink, and Bywaters 1995; van der Koat 1992). Thus, the osteophytes of DISH are enthesophytes. DISH was identified as a distinct ankylosing disorder of the spine in 1950 (Forestier and Rotes-Querol 1950). As described earlier, DlSH is a disorder characterized by flowing, exuberant calcification and ossification dong the anterior, and to a lesser extent, lateral, aspects of the spine; this calcification occua in the ALL. There is prolifentive enthesopathy at the site of attachent of the AU. to the anterior surface of the vertebrai body. DISH is characterized by entheseal hyperostoses at sites of ligament, tendon or capsular insertions of both the axid and appendicular skeleton (Resnick et al. 1978). The diagnostic criteria for DISH were described in the Methods section. As mentioned earlier, the main criterion for the diagnosis of DISH is the pnsence of osseous bridging of at least four contiguous vertebral bodies. This bridging is the result of ossification andlor calcification into the ALL - a distinguishing featm of this disorder. This criterion serves to differentiate DISH fmm so-cded 'spondylosis' (vertebral osteophytosis), which is a degenerative process of the anulus fibrosus (Resnick 1988; Resnick et al. 1978). hdividuds with ossification between only two or three vertebral centra are considered to have possible DISH: they rnay later develop more diffke hyperostosis throughout the skeleton. In this study, had the monkeys with probable DISH lived longer, they rnay have developed more pronounced manifestations of DISH. Another cnterion for the diagnosis of DISH is the absence of intra-articular osseous ankylosis of the sacroiiiac and zygapophyseal joints: the absence of these features distinguishes DISH from ankylosing spondylitis (AS) (Resnick 1988). Finally, another important diagnostic cntenon for DISH is the relative preservation of intervertebrai disc height. which differentiates DISH from degenerative disc disease (DDD) (Resnick 1988). However, it is important to note that DISH and DDD may occur together in the same spine. Disc degeneration rnay occur in DISH after the ossified ALL has ankylosed sections of the spine - if portions of the ossified ALL sustain fractures (see below). The new bone formation in DISH predorninates on the right anterolaterd aspect of the thoracic vertebral bodies and intervertebrai discs, creating a bumpy spinal contour with large irreplar excrescences, particularly overlying the intervertebral discs (Resnick and Niwayama 1976; Resnick et al. 1978). The restriction to the right side of the thoracic spine is thought to be due to the pulsations of the descending aorta, which is on the left. The gross morphological appearance of this new bone formation is reminiscent of àripping candie wax. in radiographs, a radiolucent line between the deposited bone and the anterior vertebrai surface provides another critical diagnostic due, since it is not seen in most cases of ankylosing spondylitis (AS). Furthemore, in AS the degree of new bone formation is less intensive, and the consequent spinal contour is generally smooth and Iess stikiag in appearmce. In contrast, the appearance of a spine afflicted with AS is remiluscent of bamboo. The ossification observed in DISH is much greater than that manifested in AS, psoriasis or Reiter syndrome; the osteophytes in the latter disorders tend to be thin and slender by cornparison (Resnick 1985). There are also extraspinal manifestations of DISH wbich serve as diagnostic clues. They include extensive bone spurs (osteophytosis). calcification and ossification at the bony insertion sites of tendons and ligaments in the pelvis. calcaneous. ulnar olecranon. patella and other sites (Doyle and Littlejohn 1986; Resnick et al. 1978; Resnick. Shaul, and Robins 1975). in the appendicular skeleton, the diagnosis of DISH is not usually problematic. as there are no erosions or periosted proliferations as seen in the inflammatory enthesopathies; the bone formation is well-defined and distinct from the linear calcification obsewed in calcium pyrophosphate crystal deposition disease (CPPD)(Doyle and Littlejohn 1986). Some sirnilarities between DISH and vertebral osteophytosis (VO) exist. and have been a source of confusion in the literature (François, Euldennk, and Bywaters 1995). The range of spinal motion is relatively preserved in both DISH and VO, probably due to sparing of the zygapophyseal joints (Paley et al. 1991). However, the combination of anomalies in DISH is specific, and the new bone formation has a unique appearance; with careful observation, the osteophytes of VO rnay be distinguished from the enthesophytes of DISH. An association of DISH with ossification of the posterior longinidinai ligaments has been postulated, although instances of both disorders occumng simultaneously are inf~quent(Hukuda et al. 1983; Resnick 1985). DISH tends to modify the pathogenesis and course of other spinal diseases such as rheumatoid arthritis, venebd osteoarthntis, osteoporosis and vertebral osteophytosis (Doyle and Littlejohn 1986; Hutton 1989). For example, DISH may delay the onset of osteoporosis and the vectebnl fractures associated with it It has also ken suggested that DISH may protect against zygapophyseal OA (Hutton 1989). According to some investigators. DISH may not be a pathology. but rather a protective hyperostotic reaction (Hutton 1989; Rothschild 1988). Fractures and DISH Fractures of the ossified ALL tend to destabilize the vertebral column at the affected sites. The disc at the affected intervertebral segment tends to be subjected to increased stress as a result of the fracture, and thereby undergoes degenerative changes. Cat. a065 is a case in point (Figs. 70 and 71); the ossified W sustained severai fractures during the lifetime of this individual, and the articular surfaces of the affected vertebrae exhibit extensive bone remodelling. Figure 7 1 shows the remodeiled Yticular surface of a vertebra involved in one of the regions of the spine affected by fractures in the ossified ALL. Reports of fnctures of the spine (Le.. of the vertebral bodies) in DISH are rare in the medical literature (Paiey et al. 199 1). The enthesophytes of DISH connect vertebral bodies. and are particularly thick at the level of the disc space. These enthesophytes attach at the proximal and distd thirds of the vertebral bodies, and thus. the rniddle of the centrum has compantively little hyperostosis. This phenomenon. combined with the relative preservation of the fibrous tissue of the intervertebral disc, predisposes the areas above and below the attachment of the enthesophytes CO fncture. The preseace of osteopenia is an additionai compounding factor in older subjects (Paley et al. 1991). Despite the relatively high incidence of DISH (see below), spinai fractures associated with this condition are rarely reported. However, a few cases of fractures associated with DISH have also ken observed in human patients (Paley et ai. 1991). Two types of fracture patterns were recognized in the group of patients snidied by Pdey and colleagues. The fiat type occuned through the rnidportion of an ankylosed segment of the vertebrd column, and involved the vertebral body. The second type of fracture occurred at the top or bottom of a hsed segment of spine (Paley et al. 199 1). Both types of fiactures occuned in this sample of rhesus monkeys. in addition to vertebral wedge fractures caused by osteoporosis. Other researchers have noted that spinal ankylosis in DISH can predispose the spine to abnormal stresses and fncture. A case study in a human maie reveals that a fracture through an ankylosed segment with continued motion at the site of fracture rnay result in pseudoarthrosis. Pseudoarthrosis may also develop at the junction of the fùsed and mobile spine, secondary to chronic abnormal stresses, leading to single-level disc space nanowing. vertebrai endplate erosions, vertebrai sclerosis and excessive osteophytosis (Quagliano, Hayes. and Palmer 1994). in the present study, pseudoarthrosis was mled out in Cat. #2065 because DDD is distributed tbroughout the thoracic spine, and a few lumbar vertebrae are also affected. DISH in Humans: E~idemiolonv -0 DISH is a disorder of midde-aged and elderly people, and is more frequent in men than in women. It is a common, genecaily asymptomatic disorder of unknown etiology. Its frequency in patients over 65 years of age is approxirnately 5% to 10% (Hadier 1993: Resnick 1988). The incidence of DISH is 7 in every 100 men, and 4 in every 10 women older than 30 years of age (Pdey et al. 1991). This disorder is sometimes assoa-ated with diabetes and obesity (Rogers and Waldron 1995). Patients with DISH may not necessarily exhibit symptoms, but clinical manifestations usudly relate to restricted range of motion and stifhess in the affected spinal segments, and to tendonitis in extraspinal sites. The clinicd effects are generaily minor compared with the sinking mocphological consequences of this condition (Resnick 1988). However, DISH is not aiways benign; senous complications such as spind stenosis and parapiegia have been reported (Johnsson et ai. 1983; Reisner, Stiles, and Tindall 1990). Such cases illustrate that significant morbidity, although unusual, can be associated with DISH. One study concluded that there is no significant difference in frequency of back pain in patients with DISH compared to controls. provided that complications such as spinal stenosis are absent (Schlapbach et al. 1989). Although the thoncic spine is the most predilected section (Kerr and Resnick 1984). extensive ossification is dso common in the cervicd and lumbar regions of vertebral colurnns affected by DISH. In the latter. DISH tends to be expressed as large osteophytes. in ail sections, there is preservation of intervertebrai disc height, according to the standard criteria for diagnosing DISH. in human spines. DISH is most prevalent in the thoracic spine, especially from the 7th to the 1 lth vertebne (Kerr and Resnick 1984). DISH has been recognized as n distinct disorder in human paleopathology; it existed in antiquity in many different populations from al1 archaeological periods. For example, DISH is present in a Nemdertd skeleton (Shanidar 1) (Cnibézy and Tnnkaus 1992). and in skeletal remains from ancient Egyptian, British, Saxon and Mediaeval populations (Maat, Mastwijk, and van der Velde 1995; Rogers and Waldron 1989; Rogers and Waldron 1995; Rogers, Watt. and Dieppe 198 1; Rogers, Watt. and Dieppe 1985). In the paleopathological literature before 1950, skeletons with DISH were fquently misdiagnosed as having AS; thus, part of this Literature requires revision. In general, the prevalence of this disorder in skeletai populations is similar to that observed in modem populations (Rogers and Waldron 1995). DISH in Animals: E~idemiolonv -0 Non-human Primates Among the rhesus monkeys in this study, DISH is prevalent in both the thoncic and lumbar regions of the spine. The distribution of DISH in the macaque spine is similar to the pattern observed in humans. The low frequency of DISH in this sample (2.2%) is probably not repcesentative of the prevaience of this disorder among the monkeys on Cayo Santiago. More data should be collected, on both the live monkeys and the skeletal collection. The pnsent study reports fractures of tk ossified ALL and osteoporotic fnctures in the sme individual afflicted with DISH (Cat. #2065); low bone rniiss was determined using DEXA. The spine of this monkey also exhibits degenerative disc disease in the regions of the spine associated with the fractures in the ALL. To the writer's knowledge. there is no pnor report of this particular constellation of traits associated with DISH. DISH occun in other species of macaques; for example, this disorder was noted in Macaca svlvanus, the so-cailed "barbary ape" (Kandel et ai. 1983). DlSH also occurs in gonllas (Rothschild and Woods 1988). Schultz (1969) noted "arthntic" changes in the spines of many species of primates collected from the wild, including "...large exostoses and tusion between some vertebrae in an old male proboscis monkey..." (p. 190); a drawing of this specimen depicts the distinctive flowing enthesophytes of DISH. Schultz observed "sevece vertebnl exostoses" in many gorilias and orangutans, but only in a few chimpamees (Schultz L969); it is possible that these specimcns also represent cases of DISH in wild apes. In another publication by Schuitz (L956), images of bone disease of the spine in various primates, described as typical examples of "arthritic changestt, may actually be cases of probable DISH; early DISH may be present in a female gibbon and a female baboon. However, the only way to ascertain this diagnosis is to examine the actud specimens. Parts of the spine from the same male proboscis monkey with DISH are also depicted in this particular study, but described as showing "arthntic" changes (Schultz 1956). Schultz apparentiy did not distinguish DISH as a sepmte disorder from "nrthritis", and thus, large bridging enthesophytes were diagnosed as a manifestation of "severe arthritis" of the spine. Therefore. his work on "acthritis" of the spine should be reappraised. Fox (1939) did not distinguish DISH as a separate disorder when he conducted an extensive survey of arthcitis in wild mammals, but he did note the presence of vertebral ankylosis "dong the anterior ligament" in a male muidrill. Two old mde hamadryas baboons and an aged female Japanese macaque are describing as exhibiting "rigidity of spine" and ossified Ligaments and discs - which is probably DISH or ankylosing spondylitis (AS) (Fox 1939). The collection of gonlla skeletons (the Fossey series) surveyed by LoveIl (1990) may also contain a case of DISH (or AS); a photognph of part of the lumbar spine and sacrum from a femde gorilla exhibits clear ligarnentous ossifications (Lovell 1990). However, Lovell does not provide a diagnosis for this individual, but oniy describes the presence of osteophytes bridging contiguous vertebrae. -Other Animals Lagier (1989) notes that DISH is present in many different kinds of marnmals, independent of mode of locomotion and environment; for example, this disorder is found in homes, dogs and whales (Lagier 1989). Fox ( 1939) noted a disorder, w hich appears to be DISH, in the spines of two buffdos representing different species; he describes ossification of the anterior longitudinal ligament causing ankylosis of three vertebrae. DISH may also have been present in a specimen of tapir examined by Fox (Fox 1939). DlSH is aiso present in the fossil record. Spinal longitudinal ligament calcification occurs in many species of dinosaurs and euly mammals (Rothschild 1987: Rothschild 1988). Exarnples of DISH are found in various species of mastodons, primitive camels, early horses, saber-toothed cab, fossil whaies. and other organisms. L was noted that the sites of vertebrai fusion in these creatures tended to occur at stress points in the spine. suggesting that the ossified ligaments served to buttress and stabilize the vertebrai column. For example, in dinosaun, ossified W at particular stress points butmssed the spine in a manner that kept the tail off the gmund, and allowed it to be used as a whip- like organ for defence. These observations have led some investigators to suggest that DISH is a protective bone-forming phenornenon. and not a disorder of the spine (Hutton 1989: Rothschild 1987; Rothschild 1988). The frequency of DISH in this panicular sample is 2.2% (5/228). This may not represent the actual prevdence of DISH in the rhesus monkeys of Cayo Santiago; a more complete investigation should be conducted on both the skeletal collection and the colony. This study reports a case of DISH and vertebral osteopomtic fracture occwing together, at end-stage, in an aged male rhesus monkey. This study dso reports a case of degenerative disc disease (DDD)and DISH occming together in the same individual mentioned above; DDD was initiated by fractures in the ossified anterior longitudinal ligament. These observations suggest that there rnay be notable exceptions to the standard criteria for DISH; intervertebral disc space rnay not necessarily be pnserved. especidly when complications an present, or when multiple diseases at end-stage co- exist in aged individuals. investigators should pay specid attention to extra-spinal manifestations of DISH when investigating skeletal populations for this disorder. The present study shows that the pathology of DISH in the rhesus monkey is very similar to the human counterpart of this disorder, indicating that the rhesus monkeys of Cayo Santiago rnay provide a good non-human primate mode1 for this condition. The chacacteristics of this unique colony suggest a number of avenues for further nsearch. In humans, DISH is sometimes associated with diabetes and obesity: these associations can be tested using the monkeys of Cayo Santiago, among whom both obesity (Schwartz 1989) and diabetes (Howard,Kessler, and Schwartz 1989) have been recorded. The occurrence of DISH can be traced through matrilines and patrilines in order to test for a genetic component to this disorder. This provides the opponunity CO test Rothschild's hypothesis that DISH is a not a disorder, but a protective osteoblastic mechanism of the spine. If DISH is found to occur in specifk matrilines or patrilines, then it suggests a disorder of genetic etiology, not simply of biomechanics. A genetic propensity for bone forming may be present in certain individuals, and if so, trauma or stress to vertebral bones rnay precipitate a profuse osteophytic phenornenon like DISH. This hypothesis should also be tested. CHAPTER 8 VERTEBRAL OSTEOARTHRITIS (VOA) -VOA: Distribution Across the Entire Samele and bv Sex The totd number OF vertebrai columas in this part of the analysis is 218. representing 115 femdes and 103 males. The males range in age from 7.0 to 29.0 years. and the females from 7.0 to 23.0 years. There is a totd sample size of 5.668 zygapophyseal (ZAP)joint systems. There are 2.990 ZAP joint systems for the femdes. and 2,678 for the males. The sample was described in detail in sections 2.1.2.3 and 2.4. Figure 72 shows the results of the principal components andysis (PCA) conducted on the entire sample (N = 118 spines: 5,668 joints). As before, the weights indicate the locations of the between-subject variation. The program assigned the largest weights to only three ZAP joints; a great deai of vviability exists in ZAP joints 8 (Tlm). 17 (TIOlTl 1) and 20 (LliL2). The 1 st PC accounts for 54.2% of the variance. the 2nd PC accounts for only 8% of the variance. and the 3rd ihrough 26th PCs account for the rest of the variation in the sample; as before. only the fmt and second PCs are required to explain the variation. The pattern shown by the fmt PC (Fig. 72) indicates that VOA is generally distributed throughout the spine; no particuiar region contains most of the variation in the sample. In the second PC, most of the variance is found in the lower thoracic and lumbar ZAP joints, from ZAP 17 (TLO/Tll) to 26 (L7lS 1) inclusive, but recall that this is only 8% of the sample variance. When the females and males an studied separately (Figs. 73 and 74 respectively), different patterns are evident in the fmt PC. VOA is generally distributed throughout the ZAP joints in both males and females, but the mdes show the highest peaks (i.e.. greatest variability) at ZAP joints 8, 16 and 20. The pattern br females is more homogeneous, although joints 17, 20 and 24 display considerable variation. as indicated by the peaks on the graph (Fig. 73). Another view of the trends in the whole sample is provided by Figure 75. which shows the proportion of ZAP joints in various categories of VOA scores (N = 2 18 spines; 5,668 ZAP joints). Figure 76 shows these proportions by joint in females and males separately. These graphs show that the proportions of every categoty of VOA score are quite evenly distributed across the vertebnl column - in the sample as a whole. and in males and females. In both sexes, ZAP joint 8 (TIIIT) shows r distinctive peak across the levels of VOA. The frequencies of the various levels of VOA rross the joints are remarkably simiiar in fernales and males; the patterns depicted by the line graphs are virtually identical (Fig. 76). It is notable that the females have slightly higher proportions, by joint, of VOA scores of 2 or greater. Average VOA bv ZAP Joint Svstem: Ovemll Patterns in the Sam~k When the average rating of VOA is plotted across al1 the ZAP joint systems of the whole sample (Fig. 77). it is seen that, with the exception of only a couple of joints, the level of VOA is fairly homogeneous thughout the spine. ZAP joints 8 (TlIIT), 17 (TIOml 1) anci 23 (IAILS) show the highest mean scores. In general, the thoraco-lumbar joints have relatively higher mean VOA scores than those of the cervical region. Likewise, the pattern of mean VOA scores across the spine in males is comparable to thn of the females (Fig. 78); the peaks and troughs of the line graphs are in similar areas of the spine. Both males and females exhibit a sharp peak at the joint between Tl and T2. In sevenl ZAP joint systems, mostly in the cervical. mid-thoracic and lumbar regions. the mean VOA scores of the Fernales are relatively higher than those of the males. Figure 78 demonstrates that, in the rhesus monkey, VOA commonly affects al1 regions of the spine to a similar degree, and males and femdes show similar patterns across the joints. Figure 79 shows rnean VOA scores S standard errors at each ZAP joint system, across the entire sample (N = 218 spines; 5,668 ZAP joints). This pphis siMlar to the one in Figure 77, but with the additional information provided by the standard error bars. The score for ZAP joint 8 (TllT2) stands out as an outlier in this gnph. This graph confirms that the VOA means are quite comparable across the whole spine -- with the notable exceptions of ZAP joint 8 (Tl/"),which has a very high mean score. and joint 1 (Cl/C2), which exhibits an exuemely low VOA average. Figure 80 shows mean VOA scores f2 standard errors at each ZAP joint in males only, and Figure 81 shows mean VOA scores 52 S.E. at each ZAP joint in females. These figures confirm that similar patterns of VOA exist in mdes and females, with joints 1 and 8 exhibiting the lowest. and highest, means respectively. It is also evident that the VOA averages of the mdes are more variable compared to those of the females. -VOA bv Age and Sex As described earlier, the converted VOA scores of each vertebral colum were averaged in order to obtain an index (or summary score) of VOA for each individual in the sample (N = 218 complete spines). The average VOA score for each monkey was then plotted venus age (Fig. 82). Figure 82 is a scatter plot of the mean VOA scores by age in the entire sample, with a smoothing spline fitted to the data The curvilinear regression shows that there is a strong positive correlation between mean VOA score and age; mean VOA increases with advancing age in the whole sample. There is a definite linear trend in the data. Most of thc 7 to 10 year- old monkeys have normal, or vinually normal. spines with respect to VOA, as indicated by mean scores below 1.0. After age 10 years. the graph reveds numerous individuals with mean VOA scores between 1.0 and 2.5. A mean score of 2.5 first appears at around age 13 years. in a femde monkey. Mean VOA scores higher than 3.0 occur &ter the age of 17 yeûrs. After the age of 15 yem. there are very few (only 6) individuals with average VOA scores of less than 1.0 (Fig. 82). Figure 83 shows the VOA mean scores for males and females plotted separately. After the age of 17 years. both males and femaies exhibit high average scores. and the means incwse with advancing age in both sexes. The curves show that. compared to the males. the females exhibit higher average scores for a given age, after the age of 1 1 years. Note that the oldest female in this panicular sample is 23 yean old. and the oldest male is 29 years of age. The two curves show the same basic trends across the age range (Fig. 83). There is no significant difference between the overall VOA scores of males and females (Mann-WhitneyRank Sum Test. p = 1.0); the median score for females is 0.97, and that for males is 0.93. Table 10. in cbapier 6. also lis&the fmquencies of VOA in the whole smple, and in femaies and males separately. These rates were assessed using the average VOA scores of individual vertebrai columns. Maies and femdes exhibit simila. frequencies of VOA, and the overall rate of mean scores 1 to 3 inclusive is 465. Vertebrae with typical VOA are pictured in Figure 84: lipping, pitting and eburnation an visible in the ;trticular facets of these vertebrae, -VOA b~ Natal G~OUD Figure 85 shows box plots of VOA average (or 'surnrnary') scores by natd group in the whole sarnple. As before, the sarnple sizes and variance of the natal groups differ, so it is difficult to test for signifiant differences in VOA. However, when a non-pametric equivalent of ANOVA. the Kniskal-Wallis test, was applied to the data, it gave a p-value just over the usual significance level (p = 0.06). indicating that there may be a trend. There may be diffennces in VOA between some natal groups. But, as with VODDD in the previous analysis, this trend disappears once age differences are accounted for (p = 0.57). boking ai males and femdes sepyately dso reveded that natal group is not a good predictor of VOA (p = 0.07 for males, p = 0.2 for females). Therefore, as mentioned previously, funher analysis is required, using matrilineage data instead of natal groups. In this analysis, the VOA database for bmdes (al1 7+ years of age) was tested for a possible association between par@ and VOA. As before, since VOA is an age-related disease, and pmity and age are closely associated variables, testing for associations between parity and VOA is a challenge. Using generaiized additive models, the association between parity and VOA was tested, with and without age in the models. With VOA as the response variable, the effects of parity and age were examined. There is a significant positive correlation between age and VOA (p c 0.0001). and between parity and VOA (p < 0.0001), but this is due to the strong association with age (correlation of parity and age = 0.88). When age is accounted for, the effect of parity is no longer significant (p = 0.52). 1st PC :54.2% of Variance O 5 10 15 20 25 ZygapophyseaC Joint 2nd PC : 8% of Variance Fig. 72. VOA: 1st and 2nd Principal Components in entire sampk (N = 218 spines; 5,668 ZAP joint systems). Females: 1st PC : 58.6% of Variance O 5 1O 15 20 25 Zygapophyseal Joint Females: 2nd PC :8.2% of Variance Zygapophyseai Joint Fig. 73. VOA: 1st and 2nd Principal Components in fernales (N = 115 spines; 2,990 ZAP joint systerns). Males: 1st PC :51.7% of Variance Males: 2nd PC :8.6% of Variance Fig. 74. VOA: 1st and 2nd Principal Components in males (N = 103 spines; 2,678 ZAP joint systems). Proportion of Population with Proportion of Population with ZAP scores of 1 or Greater 2AP scores of 2 or Greatet Q)d 71Ae Joint LAe Joint ZAP scores of 3 or Greater W scores of 4 or Greater Q 1 O 5 10 15 20 25 ZAe Joint Fig. 75. hoponion, by joint, of VOA scores in entire sample (N = 218 spines; 5,668 ZAP joint systems). ZAP scores of 1 or Greater ZAP scores of 2 or Gmater - --- baies ZAP Joint ZAP Joint ZAP scores of 3 or Greater ZAP scores of 4 or Greater a -- 2 y - Males - Mabs 2 - Femaka O 5 10 15 20 25 ZAe Jotnt ZAF Jotnt Fig. 76. Proportion, by joint, of VOA scores in males and females separately vernales: N = 115 spines, 2,990 ZAP joint systerns; Males: N = 103 spines, 2,678 ZAP joint systems). Solid line = males; d~edline = females. ZAP Joint Fig. 77. Average VOA rating in the entin sample at each ZAP joint system (N = 218 spines; 5,668 ZAP joint systems). Males O ,.. ,...... Females Fig. 83. Average VOA score versus age (in years) in males and females separately. Lines = smoothing splines. Males = black synrbols and solid line; females = white synrbols and dotted line (N = 1 15 female spines and 103 male spines). Fi. 84. Photograph ofthoradc vertebrae showing typical VOA in cranial aiacular aœts (A) and cauâal attkular faœb (8) of aie same bones. VMekee are hma femaîe aged 8.5 yeen (Cet. Wûûû);note Hpping, pitting and ebumation. NmIarticuler fBcets are picaired in Figum 7. 8.2. Discussion Denewration of the Articular Facets of the S~ine The zygapophyseal joints are synovial joints that allow simple gliding movements. The outermost fibers of the annulus fibrosus are the main stnictures that restrain the movements of these joints, and some suppon is also provided by the supraspinous ligaments and the ligmentum flavurn. As mentioned previously, according to Macnab's work, there is a relationship between degenerative disc disease and degeneration of the postenor joints. Osteoarthritis of the zygapophyseal joints and degenerative disc disease often occur together (Butler et al. 1990; Jayson 1988; Macnab 1986; Macnab and McCulloch 1990). According to Macnab ( 199O), segmental instability of vertebnl bodies rnay lead to posterior joint subluxation at that vectebral level. The spine is vulnerable to trauma when disc degeneration and segmental instability are present; under these conditions, the posterior joints are susceptible to excessive strain or subluxation. When the anterior fibea of the annulus fibrosus lose theu elasticity, the affected vertebnl body is allowed to hyperextend, thus resulting in subluxation of the related articular facets. As the intervertebral discs lose height during the course of disc degeneration, the postenor joints tend to ovemde and subluxate, leading to degenerative changes or osteoarthritis of the acticular facets. In the human spine, the facet joints normally carry from 12 to 2596 of the load imposed by body weight. However, in the presence of disc nanowing from degenerative disc disease, the load on the anicuiar facets rnay increase to about 70%. Such excessive Ioads on these joints lead to degenerative diseûse in the zygapophyseal joints (Builough 1992). Nerve root compression may occur as a result of this subIw fibrosus, predisposes the related articular facets to excessive suain or subluxation. and ultimately to osteoarthntic changes. Mmab notes. however, ihat vertebrai osteophytosis rnay exist without associated degenerative changes in the articular facets (Macnab and McCulloch 1990). A study by Butler et al (1990) confims that disc degeneration tends to occur pnor to facet joint OA. Macnab (1986) also notes that instability of the spine predisposes the articulûr facets to fncture. Chip fmctures on the mugins. and fissure fractures across the surface of facets have ken observed. Other disorders of vertebral bodies are known to affect the posterior joints. For example. a vertebral compression fncture rnûy lead to maialignment of the postenor joints of the affected vertebra. resulting in instability or subluxation of those joints (Macnab and McCulloch 1990). Excessive strain or subluxation of the posterior joints induces a number of degenentive changes. The joint capsule of the articulas lacet stretches, and eventuaily the tip of the inferior facet rnay impinge on the subjacent lamina, inducing an osseous reaction: a bony ridge rnay form on the pars interarticularis. The earliest phase of degeneration of the acticular facet is fibrillation of the anicular ciutilage. Eventuaily, the cartilage is pushed up amund the edge of the joint. resulting in a 'corona', which provides the precursor for Iater osteophytic lipping. The cartilage overlying the joint surface is eventuaily worn away, exposing the underlying bone (Macnab 1986). In the late stages of vertebrai osteoarthntis, the surface of the anicular facet exhibits excessive pitting, sclerosis and ebumation, and the joint surface rnay become enluged. The margins of the joint become irreplar in contour, and exhibit lipping (osteophytes). Joint space narrowing, bone ebmation and osteophytosis are discernible on radiograpbs. Although rare, intra-articula bone fusion reminiscent of ankylosing spondylitis may occur, particularly in the cervical region of the spine (Resnick 1985). The gross morphologie features of OA that are observed in other diuthrodid joints are ai1 present in vertebrai osteoarthritis - syaovitis, capsule laxity. carulage fibrillation. loss of cartilage with ebumation of subchondral bone. and the formation of marginal osteophytes (Bullough 1992). In the human spine. the articular processes of venebrae C3 to C7 have Bat, ovd articular facets that lie in an oblique coronal plane. in the thoracic spine. the supetior Yticular facets are oriented in a dorsal. laterd and slightly cranial position. and oppose infenor ;irticular facets that face in the opposite direction. In the lumbar spine, the superior Ûnicular facets face posterioriy and medially, whenas the infenor facets are positioned in an anterior and laterd orientation (Resnick 1985). Such differences in orientation of the articular processes are related to the various functions of each region of the spine wiih respect to bdancing the requirements of mobility and siability. The relationship between facet geometry (joint angle and asymmetry in facet joint angle) and disc degeneration has been explored in a nurnber of studies (Boden et ai. 1996; Malmivaan et ai. 1987; Noren et ai. 1991). It was noted that subjects with lacet joint tcopism (facet joint mgie asyrnmeuy) in the lumbar spine have a significantly higher prevaience of degeneration of the associated discs; the risk of disc degeneration is increased in the presence of tropism (Nom et al. 1991). A "pathoanatomic" association between facet onentation and degenentive changes in the human spine was dso noted by Mdmivaara et ai (1987). These authors noted marked variation in the onentation of articuiar facets at the TL lm12 joint - the transitional area between thoracic-type and Iurnbar-type anicular facets. At this joint. both anterior and posterior degeneration occurred - disc degenention, VO, Schmori's nodes, and VOA. At the T12nl joint, degeneration of the posterior joints (VOA) was predominant, whiie the TlO~lljoint exhibited mainly anterior degeneration, namely, disc degenention, VO and Schmorl's nodes (Mnlmivaan et ai. 1987). Recentiy, Scott and col1eagues (1996) have suggested that increased facet angles represent natufal variations in anatomy, and that the definition of excessive asymmeiry or tropism has been arbitnry in the past. The results of their study showed that more sagittdly oriented facet joints at the level of the fourth and fifth lumbar vertebrae in the human spine are highly associated with herniated discs and degenerative spondylolisthesis. An earlier study of VO and DDD in the lumbar spine of the boar found asyrnrnetry in lumbar articular facets accompanied by large osteophytes around the margins of the facets: the same spines also exhibited disc degeneration (Doige 1%O). in monkeys, the articular facets in the lower thoracic vertebrae assume a lumbar orientation (and hence, function) beginning with the caudal facets of TlO. Thus, the articular facets of the lower thoracic vertebrae in the spine of the rhesus monkey function like lumbar-type facets. Future studies using the present data on the rhesus monkeys of Cayo Santiago may contribute to this discourse, and may offer more insight into the relationship between facet asymrnetry and joint degeneration. Vertebml Osteoarthritis POAI in the Rhesus Monkevs of Cavo Santiaeo Age/Sex Distribution. Prevaience, and Joint Distribution of VOA Vertebral osteoacthritis is genedy distributed throughout the macaque spine. The principai components analysis shows that no pdcuiar region of the spine accounts for most of the variation in the sample - uniike the resdts for VOIDDD, where the thoracic vertebrae clearly show most of the variance. The maies show the greatest variability in ZAP joints 8. 16 and 20 (N = 103 spines; 2.678 ZAP joint systems). while the fernales exhibit relatively more variation in ZAP joints 17.20 and 24 (N = 115 spines; 2.990 ZAP joint systems). Although VOA scores are quite evenly distributed across the spine in both femaies and mdes. ZAP joint 8 (Tl/T2) shows consistentiy high frequencies of dl degrees of VOA in both sexes. The frequencies of al1 levels of VOA across the joints. and the overail pattern of the disease, is very sirnilar in males and females. The patterns portnyed by the Iine graphs in Figure 76 are virtually identical. The mean scores of VOA are fairly consistent across dl the ZAP joints of the spine in the whole sample, and in males and females separately. in both males and fernales, the articular facets joining Tl and T2 (ZAP joint 8) exhibit the highest mean scores of VOA. ZAP joint 1 (C I/C2) exhibits an extremely Iow VOA average for both sexes. There is o strong positive comlation between mean spinal VOA score and age, with mean values increasing with advancing age in the whole svnple (N = 218 spines), and in males and females separately. Animais younger than IO years of age tend to be nomai with respect to VOA. and above this age. moderate to severe cases of VOA are common. Severe cases of VOA are present in both female and male monkeys older than 17 years of age. Femdes exhibit relatively higher average scores for r given age, after the age of 11 years, than do males, but the differences are not statistically significant. --VOA and Parîty / VOA bv Natal Grouv There is no significant association between phty and VOA in the females of ihis sample (p = 0.52). Although previous studies (Chateauvert et ai. 1990; Ritzker et al. 1989) have reported a sigaifiant association between parïty and knee joint OA in this colony, there may have been a problem with sampling or with the presence of an unknown condition that had a detrimental effect on the parities of the normal cohort of females (see Chapter 6). Vectebnl osteoarthritis does not appear to have an effect on pûnty in these rhesus monkeys. This study found that natal group affiiation is not a gwd predictor of VOA (p = 0.57). Further andysis is required. using matrifines instead of natal groups, to study the hentability of VOA in these rhesus monkeys. Osteoarthntis is thought to have a multifactorial etiology (Hough Jr. and Sokoloff 1989; Moskowitz and Goldberg 1988). and to have a genetic component in humans (Ala-Kokko et d. 1990). Future research may provide data in support of a genetic component to the etiology of VOA. in the form of more direct evidence from matrilined affiliation. VOA in Non-human Primates and Other Animals _I- Sokoloff (1960) found that osteoarthritis of the articular facets of the spine had not been studied systematicaily at the time of his comparative survey; he does not mention any cases. The extensive survey conducted by Fox (1939) revealed that naturaily- occurring arthritic disease of the spine and appendicular joints is common in mammals with a range of body sizes, but is especially common in Iarger animais. He describes many cases of 'chronic arthritis' in wild mammds. Among the primates in the skeletal collection, he noted marked &tic changes of the spine in baboons (especially in a mandrill), and in many of the ormgutans and gorilias ihat were studied (Fox 1939). Schultz observed that sponianeous chronic aahntis of synovial joints is common in goriiias and ormgutans, but is rare in chimpanzees. and absent in platynhines and prosimians (Schultz 1969). However, as noted in chapter 6, other studies have found wlatively hi@ frequencies of arthritis in chimpanzees (see chapter 6). As mentioned in chapter 6, studies have shown that the frequencies of OA of the anicuiar facets of the spine (VOA) and vertebrai osteophytosis vary across the primate genera, with gonllas showing the highest frequency, followed by ormgutans and chimpanzees (Fox 1939; Junnain 1989a; Lovell 1987; Lovell 1990: Schultz 1956). Jumain (1989) noted OA in only a few vertebral anicular facets (out of 738) in his study of the skeletal remains of chimpanzees from Gombe, and he also recorded a low incidence of OA in their peripheral joints. In conuast, Cook et al (1983) found a very high frequency of VOA (5246 of articular facets) among the chimpanzees included in their study. As mentioned eulier, a comparative biomechanicd study on OA of the hip, knee and ûnkle in humans. godlas and chimpanzees discovered that chimpanzees were actually more affected by degeneraiive disease than were gorillas. This study also reports a weak comlation between body size iuid OA (Woods L986, cited in Junain, 1989). VOA in Humans, Past and -nt -0 The zygapophyseal joints of the human spine are a cornmon site of degeneration. Vertebd osteoarthritis may affect any level of the spine. but it has a panicular affinity for the middle and lower cervical spine, upper and mid-thoracic and lower lumbar spine (Ken and Resnick 1984). Studies of cadaveric spines have shown that osteoarthritis of the zygapophyseal joints is alrnost universal in subjects older than 60 years of age (Resnick 1985). Both disc degeneration anci facet osteoarthritis increase with advancing age in men and women. and there is no ~ign~cantciifference between the sexes with regard to OA of the articular facets (Butler et al. 1990). Vertebral osteoarthritis (VOA) is also a common finding in paleopathology. The shidy by Rogers et al (1985) on the paleopathology of DISH. vertebral osteophytosis and ankylosing spondylitis. which was discussed earlier, also assessed the articular facets of the spines for vertebral osteouthntis. The authors note that VOA was present in their extensive sample, which included skeletons from Romano-British, Saxon. Mediaeval md Egyptian affiliations. but they do not provide frequencies. A previous study by the sarne authoa showed that nearly 40% of a total of 400 Romano-British, Sûxon and mediaeval skeletons hd osteoarthritis of the large peripheral joints. These ancient people also exhibited both spinal osteophytosis and OA of the zygapophysed joints in high frequencies (47% of Saxon, 49% of mediaeval skeletons) (Rogers. Watt. and Dieppe 198 1). in the study by Mmet ai (1995) conducted on a mediaeval siceletal population in Holland, the authoa found that dl cases of VOA were accompanied by venebral osteophytosis, but only about half of the cases of VO were associated with VOA. They found that spines flicted with VO may or may not exhibit VOA. However, once an individual has developed VOA, it is likely that VO will also be present. They found high frequencies For vertebral osteophytosis (70.9%) that were almost twice those for VOA (36.8%), and both diseases increased with age in both sexes. In this sample of ancient human skeletons. osteoanhritis appears to be a systemic disease, affecting synovial joints in pnerai. including those of the spine - the articular facets. The authors found that the hquencies of peripherai OA of aii joints appeared to be higher in individuals with VOA chan in those with VO. As described in chapter 6. the study by Knüsel et al (1997) of 'DJD' of the spine in ancient mediaeval men supports the conclusions of Mat and colleagues (1995). The authors demonstrated that the anterior and posterior joints of the spine show dissimilx patterns of spinal joint degeneration. confing the observations of Maat et ai (1995). According to ffiüsel et al (1997). the pattern of vertebrai osteophytosis in their sample diffea from the pattern of vestebral osteosli2hritis; the two types of joints appear to exhibit an almost inverse pattern of seventy. As described in chapter 6 (and repeated here for emphasis) they note that the most severely affected intervertebd (AAR) joints are WC6. C6/C7, T8/19 to Tl lm12 inclusive, and W. The joints leut affected by 'DJD' (VODDD) are those from C7 to Tl, T 1x1md LYS 1. The highest degree of VOA occurs in facet joints CUC3 to C4K5 inclusive, C7R1, T lO/T1 1. and in UL4 to LYS 1 inclusive. while the lowest scores are found in the occipital condyles to Cl, C5K6 to C6/C7. and in the joints from Tl to T9 inclusive. They found that the articular facets exhibit progressively increasing seventy in the lower thomcic and lumbar venebrae. with no reduction in severity in the LS/Sl joint. The authors speculate that these observed differences between antenor and posterior joints are caused by joint response to the stresses of erect posture during bipedai locomotion, reflecting nonnd vertebral curvatures raiher than occupationai stress (Knüsel, Goggel, and Lucy 1997). in her study of vertebral arthritis in the prehistoric southenstem United States, Bridges ( L994) noted the highest frequencies of VO and VOA in the lumbar vertebne, followed by the cervical and thoracic segments. She found that the variability between the segments with regard to VOA was less suiking than was the expression of VO. She also concluded that the patterns of VO and VOA in her sample retleci the stresses imposed by spinai curvahue and weight-bearing due to habitua1 erect posnue. The spine is the central sinichue of the axial skeleton in both humans and quadrupeds like the rhesus rnonkey. This structure "...must balance movement with support, flexibility with fixation" (Knüsel. Goggel, and Lucy 1997, p. 493). The various regions of the vertebral column - cervical. thoracic. lumbar and sacrai - differ in function. thus permitting a balance between movement and support. The muscles and ligaments of the spine dso play a vital role in this dual function. As Knüsel and colleagues ;iffilm. the two types of joints in the spine reflect this balance between movement and support; the joints between the vertebral bodies provide support. while the zygapophyseal joints are designed to permit varying degrees of movement ûccording to the region of the spine. According to Knüsel et al (1997). this functional difference is reflected in the inverse pattem of 'DJD between zygapophyseal and intervertebrai (vertebral centra) joints in humans. Relationshio between VOA and VOlDDD Rhesus Monkevs Just as in the study by Maat et d (1995). virtually dl cases of VOA in the rhesus spines were associated with VODDD in the sarne subjects. However, it was common to observe VO/DDD without VOA in this sample. Once an individual hm established (severe) VOA, its spine is likely to be affiicted with VO/DDD as well. As mentioned eulier. KnüseI et al (1997). noted that the pattern of vertebral osteophytosis in their sample differs fiom the pattern of vertebral osteoarthritis. The mtenor and posterior joints of the spiae appear to exhibit an alrnost inverse pattern of severity. This inverse pattern of degenerative disease is also present in the current study of VODDD and VOA in rhesus macaques, as dernonstrated by the graph of VOmDD in Figure 62 and that of VOA in Figure 79, and by a combined graph (Fig. 86) which compares average VO/DDD and VOA rating by joint in the enth sample. For example, AAR joint 8 (TIIIT) has a low mean score of VO/DDD and a high mean score of VOA in the entire sample. While the cervical vertebrae show increasing scores of mean VOlDDD from C 1 to C6 (C6K7). the sme joints exhibit decrensing scores of mean VOA from CZC3 to C6/C7. While mean VOlDDD scores decrease sharply from C6K7 to C7/rl,the posterior joints at this level exhibit an increase in mean VOA. There is also a sharp decrease in mean VOA from ZAP joint 17 (TIOR11) to joint 18 (T 1 l/T12) (Fig. 79. Fig. 86). It is interesting to note that this "break" corresponds to the point in the lower thoracic spine where the curved, lumbar-type facets begin; the pattern of lumbor VOA starts at this junction. The females and males show similar inverse patterns between anterior and posterior joint degenention. Figures 63 and 64 show that. in both males and femdes. the anterior joints exhibit a shift in pattem of VOIDDD nt AAR joints 17 and 18. exrtly where the inferior fûcets of T 10 assume a lumbar-type morphology. In the entire sample, and in both mdes and females, AAR joint 23 (UN)has a low mean VO/DDD score, while the posterior joints at this level exhibit high mean scores of VOA. Both femaies and mdes show very high mean VO/DDD scores at AAR joint 26 (L7/S 1). and low mean VOA scores in the corresponding postenor joints - another "break" in the pattern of degeneration, and in skeletal morphology. It is noteworthy that these shifts in patterns occur at sites of change in gross skeletd morphology and function, and tend to follow the curvatures of the macaque spine corresponding to the points of greatest stress, such as the base of the neck where ceficd vertebrae join the thoracic spine, and the articulation between the last lumbûr bone and the sacrum. Thus, as in humans, the pattems of VOA and VO/DDD in the rhesus macaque are consvained by biology, and refîect the anatomy and function of the v~ousregions of the spiae. - - VOA - JO/DDD Joint Fig. 86. Cornparison of average VODDD and VOA rating in the entire sample, by interveitebral joint (N = 5,304 AAR joints, 204 spines; N = 5,668 ZAP joint systems, 218 spines). Solid line = VO/DDD; dashed line = VOA. The patterns shown by the human and macaque spine have a few points in common. For example, the human C7îT1 AAR joint shows a very low severity of VOIDDD, but its posterior joints are among the most severely flected by VOA; this is a very stressed joint with respect to movement (Knüsel et d. 1997). The same trend is exhibited by the macaque spine. Tbere is a shift in pattern from the cervical to the thoncic spine in both humans and macaques. in the neck. the C5K6 joint shows the highest mean VO/DDD of the cervicd vertebrae in both monkeys and humans, and in both species, C5K6 and C6K7 exhibit low mean vaiues of VOA. The main differences between macaque and human anterior spinal joint degeneration occur in the lower thoncic and lumbar vertebrae. In general, there is a remarkable sirnilvity in the overall pattem of VOA of the postenor joints in the macaque and human spine. Both show a rather uniform distribution of the disease across the postenor joints; no particular region of the spine appears to be disproportionately susceptible to VOA. Conclusions Both male and female rhesus monkeys have high frequencies of al1 severity levels of VOA (47% br fernales. 448 for males). Both sexes show great sirnilarity in the overall pattem of VOA, and in the frequency of the disease across the posterior joints of the spine. Aithough the macaque and human vertebrai column differ in oved matornical shape and function, they both show an inverse pattem of VO/DDD and VOA across the joints of the spine. Knüsel and colleagues (1997) assert that, in humans, the spine may not be suitable for the study of activity-related skeletai alterations, unless the forces involved are of sufficient magnitude that dow these lesions to be discriminated fiam those of biological origin. The authors note that theu results contribute to the cumnt viewpoint that the upper limbs and pectoral girdle shouid be the focus of studies devoted to markea of occupational stress. The present study on rhesus monkeys Iends support to this concept. and illustrates the extent to which anatomy and hinction influence the pathogenesis of joint degeneration in the spine. PART IV. SYNOPSIS, IMPLICATIONS, AND DIRECTIONS FOR FUTURE MSEARCH DEGENERATIVE SPINAL DISEASE, BONE MINERAL DENSITY AND OSTEOPENIA / OSTEOPOROSIS: SYNOPSIS, IMPLICATIONS AND DIRECTIONS FOR FUTURE RESEARCH 9.1. VO/DDD and VOA: Fiai Conciusions VODDD and VOA are present in relatively high frequencies in rhis skeletal collection, and there are no statisticdly significant differences between males and females. VO/DDD and VOA show an inverse pattern across the joints of the spine, in both maie and Female rhesus monkeys. VOA is quite uniformiy distributed across the posterior joints of the spine in both maies and females; no particular region is dispropoctionately susceptible to VOA. For VOA, shifts in pattern of joint degeneration correspond to changes in gros morphology and function of the spine, especiaily to sites of high biomechanical stress. Vinually al1 cases of VOA are associated with VO/DDD, but it is common to observe VO/DDD without the presence of VOA. However, once an individual hasevere or established VOA, its spine will likely be afflicted with antenor joint degeneration as well. In both females and males. the thoracic vertebrae, the transitional thoraco-lumbar region and the motion segment between L7 and S1 tend to exhibit the highest frequencies of ail grades of VOIDDD. In rhesus monkeys, VOiDDD has a strong prediiection for the thoracic spine; thoracic vertebme account for a very significant proportion of the variance in the total sarnple. The joint distribution of VO/DDD is similar in both sexes, and the thoracic spine accouats for 70% of the vasiance in both males and fernales. Both VOA and VO/DDD are strongly correlated with age in both sexes. and both diseases increase in severity with advmcing age. Most individuals under the age of 10 yem have normal spines with respect to VOIDDD and VOA, and aii monkeys over the age of 15 years show some degree of VOIDDD. Moderate to severe cases of VOA are common in individuais over the age of 10 years; severe cases are observed in monkeys oldet than 17 years. The fernales show no association between parity and VO/DDD, and no relationship between parity and VOA. Natd group affiliation. in this simple, is also not associated with VOlDDD or VOA. VO/DDD in this study reflects the normal curvatures of the macaque spine. i.e.. the hinctionai stresses of a cantilever. The thoraco-lumbar region of the spine, located ai the apex of curvature and subjected to significant biomechmicd stress, exhibits some of the highest frequencies and levels of VOIDDD. Knüsel et al (1997) concluded from their study that the pattern of osteoarthritis of the facet joints in humans appears to reflect the stresses produced in the vertebral column by movements, through reducing or accentuating the vertebral curvatures. They state that the pattern of VOlDDD severity reflects the spinai curvatures, exhibiting the highest severity where the curvatures are fdest away from the line of gravity. and the lowest expression where the spine passes through it (p. 493). in regards to VOIDDD, this mode1 is not totdlv consistent with the present data nor is it consistent with the data on deeenerative s~inddisease in humans. Due to habitual upright posture, the weight supported by the vertebrae increases progressively from the cervical to the lumbiu sections of the human spine. The unique double4 curvam of the spine of bipeds is explained by the need to dissipate body weight efficiently, and to balance the upright body on two limbs. Thus, in the human spine, the iine of gravity passes hughthe cervical and lumbar vertebrae (Bullough and Boachie-Adjei 1988). Studies have shown thût although human VODDD can occur in any spinal segment. it most often affects the more mobile portions of the spine - the cervical and lumbar sections. VODDD is most frequent, and most pronounced, in the motion segments of the lower cervical and lower lurnbar spine, due to the high biomechanical demaads on these regions (Bullough 1992; Krher 1990). Thus, it appears that VO/DDD does reflect the spinal cwatures, but it exhibits the lowest seventy where the curvatures are fmhest awav from the line of gravity, and the hichest severitv where the spine Passes through it. This pattern also applies to the spine of the macaque. A spine designed for quadmpedai posture and locomotion assumes a cantilever-like shape and function, with the apex of curvature of the rhesus monkey spine occumng in the lower thoracic vertebrae. in quadnipedd primates, the maximum curvature of the spine occurs in the lower thoracic region (T9- 12). The line of gravity passes through this region of the spine, which is also rnost cornmonly affected by severe degenentive arthritis (VOIDDD). The upper thoracic and upper lurnbar venebne exhibit the lowest mean scores of VO/DDD (Figs. 62 to 64). and they are situated away from the line of pvity. The center of gravity in the human spine lies just antenor to the second sacrai vertebra, which explains the high prevalence of severe lower lumbar VO/DDD. The center of gravity is located in a more cranid orientation in the macaque spine, which explains the high prevalence of severe lower thoracic VOIDDD in the monkey. However, in its habitua1 sitting posture. the center of gravity would pass through the pelvis of the rhesus macaque. This would account for the high incidence of severe degenerative disc disease that was noted in the motion segment between the 1st lumbar vertebra and the sacrum. The sacrum is positioncd at a swctural intersection between the vertebrai column and the caudal segments. and between the pelvis and the lower extremities (Ankel 1972). Those who use the rhesus monkey as a mode1 for human VOlDDD should be awve of the biomechanicd differences between the macaque and human spine, and should note that the pattern of joint degeneration diffea between them. The present study shows that male and female rhesus monkeys have comparable frequencies and similar age-related patterns of VO/DDD. In contrast, human males have a higher prevdence of disc degeneration than do human fernales. Therefore, it is advised that investigatoa account for these differences when utilizing the rhesus macaque as a mode1 for human spinal degenerative disease. Venebnl osteophytosis / degenerative disc disease (anterior spinal joint degeneration) should be distinguished from vertebral osteoarthntis (posterior spinal joint degenention). The results reported herein fidysupport this view. and ûflirm the use of distinctive tenninology to distinguish ihese conditions (François, Euldennk. and Bywaten 1995). In contrast, Hough Jr. and Sokoloff (1989) and others have suggested that the mechanisms involved in spinal osteophytosis or degenerative disc disease are not quaiitatively different from those that operate in the synovial joints. They note that the discovertebral and synovial articulations are composed of andogous structures: both have cartilage, subchondrai bone and surrounding fibrous tissue - annulus fibrosus in the former, fibrous capsule in the latter. Moreover, they propose that the semigelatinous nucleus pulposus of the intervertebnl disc is analogous to the yticular fluid of the synovial joint (Hough Ir. and Sokoloff 1989; Resnick and Niwayama 1988). Although these two different joint systems may have analogous anatomical structures, they differ in hinction. and this snidy demonstrates that they also vary in pattern of joint degeneration. To acknowledge these distinctions, separate tennhology would be usefd in reference to these diseases. This study found that vertebral bones assessed with moderate to severe VOlDDD have abnomdly elevated BMC and BMD values compared to age- and sex- matched controls, even though the vertebral osteophytes were excised digitally by the DEXA cornputer prognm. This indicates that the tcabecular bone of the centrurn bas been altered by the disease, both quantitatively and qualitatively. Thus, it is advised that more emphasis be placed on BMC value nther than on degree of vertebrai osteophytosis in order to identify bones with VO/DDD that may introduce bias into a DEXA simple. It is recommended that specimens with moderate to severe VOIDDD or VOA be excluded from studies of bone density and osteoporosis, and ftom snidies designed to estûblish normative data on BMD. 9.2. Bone Mineral Density and Osteopenia / Osteoporosis: Final Conclusions Fracture Patterns in the Vertebral Column As mentioned above, there are biomechanical differences between the spines of Macaca and Homo, such as different vertebral loading due to dissimilar habitua1 modes of locomotion. In humans, the pattern of vertebd fracture is dictated by the effects of vertical loadiing on the spine, coupled with the force of gravity. The line of gravity is positioned anterior to the thoracic vectebne, but it passes through the lumbar spine. Thus, fractures in the human thoncic spine tend to be wedge-shaped anteciorly, while fnctures in the lumbar venebrae tend to be biconcave (Bullough and Boachie-Adjei 1988). Complete compression fracture is a common fonn of vertebral kturein humans affected with osteoporosis. It is noteworthy that corndete com~ressionfnctures are not present in the osteowmtic macaaue mines of the current snidy; only antenor vertebral wedging was observed. Vertical loading is not the nom for the macaque spine, and therefore. biconcave fractures do not occur. Also, the tmnk is supported by an inverted piirabolic structure under tension, fomed by the sternum and abdominal muscles (Napier and Napier 1967); such forces Iücely contribute to the anterior wedge type of vertebral fncture. Vertebral osteoporotic fractures are generdy more comrnon in humans. A relatively low fmquency of established osteoporotic fractures was observed in these rhesus monkeys. -Bone Density This study demonstrates that vertebrae are reliable sites of measurement for DEXA provided that al1 specimens with moderate to advanced VO/DDD and VOA are excluded from the analysis. Maie rhesus monkeys acquire bone mineral density at a faster rate. and reach a higher peak BMD at an exlier age compared to female monkeys. Thus. in contrat to humans, rhesus males attain a peak BMD at an earlier age than do femaies. Researchers who use the rhesus monkey as a primate model for human bone density and osteopenia/osteoporosis should note this difference. For such studies. it is recommended that only mature rhesus monkeys past the age of attainrnent of peak bone mass be used in studies of bone remodelling - males older than 7 years and females over the age of 9.5 yem. Maie and female rhesus monkeys are good models for the study of human bone density, osteopenia and osteoporosis, provided that the limitations of the model ye considered. in females, there is a significant increase in BMD of the spine with increasing parity after controlîing for age, up to a parity of about 7 offspnng. Parity has an increasing effect on BMD, whiie age has a decreasing effect fiom around 9 years of age. Parity appears to mitigate the effects of aging on BMD. The degenerative disease VOlDDD does not appear to bave a negative effect on parïty in these females. This study has shown that the fernale rhesus monkeys frorn Cayo Santiago have a pattern of skeletal maturation and acquisition of bone minerai density that is similar to that of human females - a pattem that includes a decline in bone density of the spine with advancing age. The bone minerai density values of the osteoporotic (fractured) monkeys in this study are nenerally higher than those of virtuaily al1 of the osteopenic/osteoporotic (unfractured) monkeys. This observation supports the view that low bone mass mav not alwavs ~recedefncture. There are the factors that contribute to skeletal fragility: decreased bone mus. accumulated fatigue damage and loss of trabecular connectivity. The current study only investigated the first variable, bone mineral density. This study supports the clinical data that demonstrates that BMD alone is not a good predictor of fracture risk. Other factors such as bone quality (i.e., trûbecular connectivity. fatigue damage) should also be considered when assessing risk of fracture. The pattem of bone Ioss aDDears to be as irnwrtant as the auantitv of bone loss. While BMD gives an indication of how much bone loss hos occurred, the pattern of trabecular resorption provides an indication of whether or not the bone wili fracture. The results of this study dso suggest that the standard criteria for diagnosing osteopenia and osteoporosis endorsed by the WHO should be revised to nflect these findings. The standard diagnostic criteria based on BMD measurements, endorsed by the WHO,should be revised for another reason. The WHO endorsed the definition of four thresholds for diagnosing osteoporosis that are based on reference populations of healthy young women. The BMD of women of al1 ages is compared to those with peak bone mass, using a 2-score. With this system. women will be labei3ed 'at risk' or 'diseased' if they do not maintain their bone mass at -yak ievels throughout theu life span. Thus, the definitions of osteopenia and osteoporosis employed by the WHO do not account for normal age-related bone loss, and the bioloaical variation that exists amonP: healthv adults (Green et al. 1997). The present study demonstrates that there is also normal variation in BMD among fully mature rhesus monkeys, and age-related bone loss in fernales. 93. DISE?: Final Conclusions This study demonstrates that the pathology of DISH in the rhesus monkey is similar to that of humans. A case of DISH with concomitant vertebral fractures was reported in an aged male rhesus monkey. The saine individual also exhibits degenentive disc disease. It is postulated that the DDD in this case was initiated by fractures in the ossified antenor longitudinal ligament. These results suggest that there may be notable exceptions to the standard criteria for DISH. For exmple. intervertebral disc space may not necessarily be preserved, given the presence of complications, or the cosxistence of multiple diseases at end-stage in aged individuds. In such cases, the extra-spinal manifestations of DISH should be emphasized as part of the diagnosis. The extra-spinal cnteria for DISH should also be given more emphasis in popuiation surveys for this disorder, particularly in paleopathology. 9.4. Degeaemtive Di- and Rimate Behaviour As they age, the monkeys on Cayo Santiago spend a greater proportion of time on the gsound, wallcing quadrupedaily and feeding from standing or sitting positions. Older animais are less inciined to engage in acrobatie or suspensory activity in the mes compared to younger individu& (DeRousseau. 1988). These age-related modifications in positionai behaviour are Iikely related to degenerative diseases such as the age-related disorders of the spine reported in this study, and OA of the peripheral joints. Such degenerative conditions impair the fuaftion of these skeletal elements, necessitating changes in behaviour. Skeletal changes due to aging not only affect the locomotor behaviour of aged individuds. but they mq also restrict that of Sected young adults (DeRousseau, Bito, and Kaufinan 1986). That certain age-related changes in the behaviour of primates may be rooted in biology or degenerative disease should be of interest to those who study the social behaviour of primates, panicularly those pnmatologists who use the Cayo Santiago colony to test theories of social behaviour. 9.5. Final Considerations Cavo Santiago Rhesus Monkevs: Non-human Primate Models of Disease As noted earlier, due to their protective environment, the rhesus macaques of Cayo Santiago survive to advanced ages despite the physicai impairment caused by degenerative disease. Thus, a number of spontaneous diseases and disorders that are cornrnon in human populations also occur in this unique colony, as described at the beginning (Chateauvert et al. 1990; Dawson et ai. 1989; DeRousseau 1985; DeRousseau 1985b; Grynpas 1992; Grynpas et al. 1989; Howard, Kessler, and Schwartz 1989; Priaker et al. 1989; Schwartz 1989; Turnquist 1986). Congenital anomalies (Rawlins and Kessler 1983), and healed fractures (Buikstra 1975) dso occur. and these monkeys develop spinal osteophytosis. vertebral osteoarthritis and degenerative disc disease spontaneously with advancing age, simiiar to humans (Cerroni 1992; DeRousseau 1988; DeRousseau, Bito, and Kaufman 1986). This thesis demonstrates that these diseases are present in high hrquencies in this colony, increasing in incidence with advancing age. Another age-related disorder, DISH, is also present in this colony, and closely resembles the human form of the disorder in gross morphology. This study shows that bone loss in the spine, bone loss in the appendicular skeleton, and the morphology of vertebral wedge fractures closely resemble their human counterputs, suggesting that the monkeys of Cayo Santiago provide a very good mode1 for bone minerai density, osteopenia and osteoporosis in humons. Directions For Future Research The severely osteopenic individuals found in this study, and their ancestors and descendants, should be investigated more extensively. Future research should test the hypothesis that low parity in the femde rhesus monkeys of Cayo Santiago is correlated with low social rank. and mess whether femdes that exhibit both low parity and osteopenia/osteoporosis are also Iow in the sociai hienrchy. Subsequent studies should dso trace bone density and osteopenia/osteoporosis through matrilines (matemal lineages) and patnlines (prtemd lineages) to test for the presence of a genetic component that may provide mon insight into the patterns observed in this study. This colony also presents an oppominity to study the hentability of bone minera1 density, by investigating whether there is a tendency for peak BMD values to Vary by matdine. A Mer direction for the bone density research is to study bone minerai density in the extant monkeys of the colony, and to relate BMD to demographic parameten such as age, sex, p&y, body weight and matdineal I pavilined affiiliation. The dtimate objective would be to establish a profde of bone density measurements, a database of normative bone mass, for the Living monkeys on the island. A more extensive study of bone mass would fulfill another objective - to determine whether or not bone mineral density decreases with age in male rhesus monkeys. Due to the necessary elimination of rnost of the arthritic older males fiom the DEXA sample of the present study. it was not possible to ascenain with absolute certainty whether or not such a trend exists. A study of bone density in male rhesus monkeys from both the skeletal collection and the live colony should provide valuable new data. Matrilineal / patrilineal mdysis should also be applied to the data on VO/DDD, VOA and DISH to iissess the heritability of these degenerative conditions. Future studies using the present data on VOA and VOlDDD in these rhesus monkeys should examine the relationship between articular facet asymmetry and joint degeneration. 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