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Aging-induced osteopenia in avian cortical bone
Bone Vol. 26, No. 4 April 2000:361–365
Aging-Induced Osteopenia in Avian Cortical Bone S. SRINIVASAN,1 S. A. KEILIN,1 S. JUDEX,2 R. C. BRAY,2 R. F. ZERNICKE,2 and T. S. GROSS1 1
Department of Orthopaedic Surgery, University of Cincinnati, Cincinnati, OH, USA McCaig Centre for Joint Injury and Arthritis, University of Calgary, Calgary, AB, Canada
2
are scarce. Secondary osteonal remodeling has been reported in sheep, but, to date, this model has been exploited primarily as a model of postmenopausal osteoporosis.26 Rodents provide a useful model for investigation of aging-associated osteopenia, especially considering their low cost, substantial compression of senescent events, and potential to utilize transgenics. However, they do not exhibit alterations in secondary osteonal remodeling and cortical porosity unless stringently provoked.18 This is an important limitation, because, although cortical porosity accounts for only a small percentage of aging-associated cortical bone loss and is often transient in nature,13 it has an enormous influence upon the mechanical properties of bone.22,24,42 The rooster represents a potentially useful and inexpensive alternative to current models of age-related osteopenia. Avian cortical bones possess the same cell types and have similar haversian and lamellar bone morphology to mammalian cortical bones. The modeling and coupled intracortical remodeling events normally observed in avian cortical bones are similar to those observed in humans during growth and adulthood. The modeling response when subject to exogenous mechanical loading,17,34 and the coupled cellular responses initiated by conditions of disuse, are similar to those observed in humans8 and dogs.15 Whereas avian bones have evolved to be lightweight for flying, functionally induced strains are similar to those observed across mammalian species.1,33 Thus, the morphological parallels between avian and mammalian bones and the similarity in the cellular response to altered physical stimuli support the use of the avian model. However, as avian species are able to rapidly mobilize (and replace) calcium from their skeleton during egg production, hens were excluded from this study. Criteria for denoting the rooster as aged, however, remain unclear. Anecdotal references suggest that roosters are capable of living up to 10 years,25 but the functional lifespan of white leghorn roosters has been found to be 5–7 years.43 Avian organ systems begin to degrade at much earlier timepoints. For example, reproductive fertility declines by 95%25 and adenylate phosphatase and adenylate deaminase activity in the heart muscle is markedly diminished by 2 years,41 and presynaptic neural degradation is pronounced by 5 years.21 The objective of this study was to therefore determine if aging induces phenotypic alterations (i.e., periosteal and endocortical expansion, elevated cortical porosity) in the cortical bone of aged roosters.
Cortical bone loss contributes substantially to the degradation of skeletal integrity associated with aging. However, animal models that closely mimic age-related alterations in cortical bone are limited. The objective of this study was to determine if aged rooster cortical bone demonstrates phenotypic alterations similar to those observed in aged human cortical bone (i.e., expansion of the endocortical and periosteal envelopes and elevated cortical porosity). When compared with young adult roosters, aged roosters demonstrated significant expansion of the endocortical (16%) and periosteal (10%) envelopes, resulting in significantly increased cross-sectional moments of inertia. In addition, aged rooster bone demonstrated significantly elevated cortical porosity (51%) and average area of porosity (83%). We conclude that rooster bone demonstrates age-related adaptations similar to those of humans at both tissue and cellular levels, and may therefore represent a relatively useful, inexpensive animal model for investigating the mechanisms of age-related bone loss. (Bone 26:361–365; 2000) © 2000 by Elsevier Science Inc. All rights reserved. Key Words: Age-related osteopenia; Rooster; Cortical bone; Bone envelope expansion; Cortical porosity. Introduction Aging-induced osteopenia poses a substantial risk to the integrity of the skeleton. Bone loss associated with aging begins near age 40 years, progresses at 0.5%–1% per year, and accounts for as much as 40% of total bone loss accumulated by age 70.27,30,32 Osteopenia associated with aging afflicts both trabecular and cortical bone,7,23 and is commonly implicated in hip, wrist, and spine fractures.10,37 In cortical bone, aging results in expanded periosteal and endocortical envelopes and in increased osteonal remodeling and cortical porosity.11,23,35 The age-related increase in cortical porosity is particularly insidious as it markedly increases skeletal fragility.22,24,42 However, the pathoetiology of aging-induced cortical bone loss is poorly understood, due in part to the lack of suitable animal models. Animal models should ideally mimic human pathophysiology as closely as possible. Nonhuman primate models satisfy this criterion, but high expense, long lifespans, and ethical issues limit their viability as a model.26 Canine models of osteopenia demonstrate altered bone envelopes,36 but reports of elevated secondary osteonal remodeling and cortical porosity in aged dogs
Methods A group of young adult (age 1 year, mean mass 2 kg, n ⫽ 12) and aged (age 5.5 years, mean mass 3 kg, n ⫽ 5) white leghorn roosters of the same strain and seed colony were utilized in this study. The aged roosters represented the survivors of an original colony of young adults. Upon killing, cortical bone cross sections
Address for correspondence and reprints: Sundar Srinivasan, Ph.D., Department of Orthopaedic Surgery, P.O. Box 670212, University of Cincinnati, Cincinnati, OH 45267-0212. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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(250 m thickness) were obtained from the right ulnae of the animals at identical sites located 33% of the diaphyseal length from the distal metaphysis. Sections were then hand ground to 75 m and toluidine blue stained. With the identity of the sections blinded, the cross sections were imaged using an Olympus light microscope fitted with a Polaroid digital camera. The digital images were then used to assess morphological adaptations in cross-sectional areal properties and cortical porosity. Cross-Sectional Morphology To determine morphological changes in cortical bone crosssectional areal properties, all cross sections were imaged at ⫻20 magnification, and digital images were obtained at a resolution of 130 pixels/mm (on average, 12 images were required per bone cross section). A composite image reconstituted the cross section into one digital image (Figure 1). With the identity of the sections blinded, image analysis of the composite image was then performed utilizing PV-WAVE (Visual Numerics Inc., Boulder, CO). Similar to previous studies,11,23 pixel-counting routines determined cortical bone area (Ct.Ar), endocortical envelope area (Ec.Ar), and periosteal envelope area (Ps.Ar) (Figure 1). Cortical width (Ct.Wi) was measured as the average distance between the periosteal perimeter and the endocortical perimeter along 360 equiangular rays originating from the centroid of the bone cross section. Using numerical integration, the cross-sectional moments of inertia about the x (Ixx) and y (Iyy) axes and polar moment of inertia (J) were determined. As a control for the pixel-counting technique, composite images (from two arbitrarily selected bone cross-sections) were scanned at twice the scanning density (260 pixels/mm) and the areal properties were determined. Doubling the scanning density (from 130 to 260 pixels/ mm) resulted in minimal alterations in Ct.Ar (0.6%), Ec.Ar (0.6%), Ps.Ar (0.08%), Ct.Wi (0.1%), Ixx (0.8%), Iyy (0.7%), and J (0.8%). Cortical Porosity To determine morphological changes in bone cortical porosity, we analyzed ulnar cross sections of young (n ⫽ 6, selected at random from the original group of 12) and aged (n ⫽ 5) roosters. Similar to previous work,23 a finite number of sampling sites was selected to access indices of cortical porosity in both groups. With the sections blinded, 12 equiangular locations at both endocortical and periosteal sites around the middiaphyseal cortex (i.e., a total of 24 sampling sites per cross section) were imaged at ⫻400, and digital images were obtained at a resolution of 2600 pixels/mm (Figure 1). Using NIH IMAGE software, particle analysis was performed on each digital image. Briefly, each grayscale digital image was thresholded based on the gray-scale histograms of arbitrarily selected lacunae. All particles within the image that represented osteocyte lacunae were then excluded from the thresholded image, similar to previous work.23 Next, for voids representing a cortical porosity (i.e., particles other than those representing osteocyte lacunae), the total numbers of pixels falling within individual voids were counted. The average porosity area (Po.Ar) was determined as the ratio of total number of pixels falling within the porosities to total number of porosities (Po.N). The cortical porosity (Ct.Po) was calculated as a percentage of the total number of pixels falling within the porosities to the total pixels representing the imaged tissue area. As a control for the image analysis technique, 24 arbitrarily selected images were thresholded based on the gray-scale histograms of alternatively selected lacunae. The mean (⫾SE) variability of Ct.Po was minimal (2.74 ⫾ 0.5%).
Figure 1. Schematic of a right ulnar cross section composite digital image. (a) Cortical bone area (Ct.Ar) was calculated as the number of pixels falling within the cortex (black region). Endocortical area (Ec.Ar) was calculated as the number of pixels falling within the endocortical envelope (gray region). Periosteal area (Ps.Ar) was calculated as the sum of Ct.Ar and Ec.Ar. (b) Around the ulnar cortex, images (original magnification ⫻400) were obtained at 12 equiangular locations and at both endocortical (E) and periosteal (P) sites (total of 24 sampling locations per cross section) to examine age-related alterations in cortical porosity. (c) A sample image (original magnification ⫻400).
Statistical Analysis The Mann–Whitney nonparametric test of two independent samples was used to assess between-group cross-sectional morphological differences. For morphological indices at the level of cortical porosities, both between-group (young vs. aged; Mann– Whitney) as well as within-group (endocortical vs. periosteal
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Figure 3. (a) Digital images (original magnification ⫻400) from the ulnae of aged (5.5 years) and (b) young (1 year roosters with cortical porosities noted [white arrows]). (c) Across all sampling locations, aging induced significant (asterisks) increases in Ct.Po and Po.Ar.
Figure 2. (a) The ulnar middiaphyseal cross section from aged (5.5 years) and (b) young (1 year) roosters. (c) Aging induced significant (asterisks) increases in Ec.Ar and Ps.Ar, but not in Ct.Ar. (d) The ulnar cross-sectional moments of inertia, Ixx, Iyy, and J, were all increased significantly in the aged roosters.
sites; Wilcoxon signed-rank test) comparisons were performed. All analyses were performed using SPSS (SPSS Inc., Chicago, IL) with p ⫽ 0.05, and results reported as mean ⫾ SE. Results
Ec.Ar (11.9 ⫾ 0.7 vs. 10.3 ⫾ 0.3 mm2, p ⫽ 0.05) (Figure 2c). The age-related increase in both Ps.Ar (10%) and Ec.Ar (16%) resulted in an increase in the cross-sectional moments of inertia as well. Aging significantly increased Ixx (30.5 ⫾ 2.03 vs. 25.9 ⫾ 1.06 mm4, p ⫽ 0.03), Iyy (55.8 ⫾ 4.04 vs. 46.9 ⫾ 1.96 mm4, p ⫽ 0.05), and J (63.6 ⫾ 4.5 vs. 53.6 ⫾ 2.2 mm4, p ⫽ 0.04) (Figure 2d). As a percentage, aging significantly increased Ixx by 18%, Iyy by 19%, and J by 19%. Neither the total cortical bone area, Ct.Ar (13.7 ⫾ 0.5 vs. 12.9 ⫾ 0.3 mm2, p ⫽ 0.16), nor the cortical width, Ct.Wi (0.9 ⫾ 0.03 vs. 0.9 ⫾ 0.02 mm, p ⫽ 0.45), were altered significantly by aging.
Cross-Sectional Morphology
Cortical Porosity
Cross-sectional bone envelope areas were increased with age (Figure 2). Compared with the young adult roosters, the cortical bone cross section of the aged bones displayed significantly greater Ps.Ar (25.6 ⫾ 0.9 vs. 23.2 ⫾ 0.5 mm2, p ⫽ 0.03) and
Avian cortical bone became increasingly porotic with age (Figure 3). Across sampling locations, there was a significant agerelated increase in both Ct.Po (4.25 ⫾ 0.49 vs. 2.82 ⫾ 0.26%, p ⫽ 0.01) and in Po.Ar (466 ⫾ 101 vs. 254 ⫾ 30 m2, p ⫽ 0.01)
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Figure 4. Cortical porosity at endocortical and periosteal sampling locations in the ulnae of aged (5.5 years) and young (1 year) roosters. Aging induced significant (asterisks) increases in (a) Ct.Po and (b) Po.Ar only at endocortical sampling locations.
(Figure 3c). In fact, aging elevated Ct.Po by 51% and Po.Ar by 83%. In contrast, Po.N (124.7 ⫾ 7.8 vs. 128.9 ⫾ 3.0/mm2, p ⫽ 0.47) was not significantly increased in aged bones. In both age groups, there was significantly more Ct.Po (4.52 ⫾ 0.52 vs. 2.43 ⫾ 0.22%, p ⬍ 0.01) and Po.Ar (475 ⫾ 96 vs. 225 ⫾ 42 m2, p ⫽ 0.01), but significantly less Po.N (118.6 ⫾ 3.9 vs. 135.4 ⫾ 5.1/mm2, p ⫽ 0.02) at endocortical vs. periosteal sampling locations. At endocortical sampling locations, there was a significant age-related increase in Ct.Po (5.73 ⫾ 0.84 vs. 3.51 ⫾ 0.26%, p ⬍ 0.01) and Po.Ar (657 ⫾ 185 vs. 323 ⫾ 24 m2, p ⫽ 0.01), but not in Po.N (114.6 ⫾ 8.0 vs. 121.9 ⫾ 3.1/mm2, p ⫽ 0.33) (Figure 4). As a percentage, age-related increases in Ct.Po and Po.Ar were 63% and 103%, respectively, at endocortical sampling locations. However, at periosteal sampling locations, aging-associated alterations in Ct.Po (2.78 ⫾ 0.28 vs. 2.14 ⫾ 0.29%, p ⫽ 0.06), Po.Ar (274.91 ⫾ 79.68 vs. 190.69 ⫾ 43.96 m2, p ⫽ 0.06), and Po.N (134.7 ⫾ 11.18 vs. 136.04 ⫾ 3.49/mm2, p ⫽ 0.33) were not statistically significant. Discussion Age-related morphological changes in cortical bone were determined in adult roosters. One means of defining an aged population is the timepoint at which 50% mortality is reached. Within our original rooster colony, 54% mortality was observed by 5 years of age. Young adults were assessed at 1 year (an age at which long bone growth plates are closed19). Both the periosteal (Ps.Ar) and endocortical (Ec.Ar) bone envelopes were significantly increased by aging. The larger bone envelopes associated with aged rooster bones resulted in a significant increase in the bending (Ixx, Iyy) and in the twisting (J) moments of inertia. These increases are similar to what has been observed between the third and seventh decades in human cortical bone.11,23 The lack of a significant aging-associated
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change in cortical bone area (Ct.Ar) and cortical width (Ct.Wi) reported here is also similar to that observed in humans in the eight decade and beyond.11,23 At the continuum level, an age-related increase in bone envelopes may result in greater resistance to the bending and twisting loads encountered during functional activity. Utilizing calibration strain-gauge values in conjunction with beam theory estimates, we have previously established the end-loading boundary conditions in the avian ulna subject to dynamic bending loads.14 Under these boundary conditions (similar to those induced during maximal wing flap), preliminary calculations suggest a 20% reduction in induced strains due to age-related expansion of cortical bone envelopes observed in this study. Similar reductions in induced strains during functional activity would result in a concomitant increase in structural strength, at least at the continuum level. However, any structural benefit of the larger cortical bone cross sections and elevated moments of inertia would be tempered by the significant increase in cortical porosity (51%) observed in our aged rooster population. In fact, an elevation in cortical porosity from 4% to 10% has been estimated to result in a 20% decrease in bone’s elastic moduli.22 Furthermore, at the microstructural level, cortical porosities act as stress risers and have a profound influence on the bone’s strength and fracture toughness.24,42 For example, a change in absolute cortical porosity from 5% to 10% can result in a 50% reduction in bone’s fracture toughness.42 Ultimately, any structural benefit of the larger bone envelopes would appear to be mitigated by these considerations and may simply reflect the lifelong accumulation of cellular activity at the bone surfaces. Similar to previous reports,5,39 the increased cortical porosity in the aged bones was primarily due to the substantial increase in the average size of each porosity (86%). One possible explanation for this result is that active remodeling was dramatically enhanced in aged bones. However, a preliminary analysis (via blinded counting of number of scalloped voids at ⫻20) indicated that, of an average of 127 porosities/mm2, there was no difference in the small percentage of sites undergoing active remodeling (0.1% young vs. 0.2% aged, p ⫽ 0.16). This observation suggests that our measure of cortical porosity (size, number) was influenced minimally by the transient nature of porosities or the remodeling state of the skeleton (i.e., the ulnae of both groups of animals were quiescent at time of killing). The age-related increase in porosity size suggests that the accrued deficit in bone mass was due to accumulated alterations in the balanced coupled resorptive process,6,12 impotent osteoblastogenesis, and/or ineffective in-filling by osteoblasts.16,20 However, other reports have suggested that the age-related increase in cortical porosity may be due primarily to an increase in the total number of porosities,23,28 potentially via increased osteoclastic activity.29 Although our results support the former observations, they do not offer a conclusive validation of either pathway, due to the limited timepoints considered. Regardless of the mechanism involved, elevated cortical porosity occurred primarily at the endocortical sites (via increased area of each porosity). The age-related increase in cortical porosity near the endocortical surface is similar to that observed in human bones23 and may reflect proximity to osteoclast progenitor pools.31 Alternatively, other processes have recently been hypothesized as potential initiators of intracortical bone loss, including osteocyte apoptosis40 and hypoxia,9 and may display an envelope-specific prevalence as well. Although the age-related increase in cortical porosity observed in this study models that observed in humans, the magnitude of cortical porosity, per se, was low (4.3%). This is relevant given that cortical porosity in human bones at middiaphyseal sites is on the order of 7.5%–20%.11,23,28 and between 10%–25% at sites of fracture, such as the forearm and the
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femur3,4,38 Interestingly, while cortical porosity at fracture sites in these studies was no different from that found in age-matched controls, increased size of porosities and the presence of abnormally large porosities were more closely related to risk of skeletal fragility. Thus, it remains unclear if cortical porosities must be in the 20% range, or if the size of porosity (in proportion to the cortical width) is a more important determinant of fracture risk. It may be possible to address the issue of age-related skeletal fragility in the avian model by exacerbating the extent of cortical porosities in aged skeletons via induction of hormonal deficits (e.g., gonadal hormone deficiency2) or treatment with parathyroid hormones and prostaglandins, such as in dogs.36 In summary, we observed significant ageing-induced cortical bone adaptation in the ulna of aged roosters. The adaptations observed here were similar to phenotypic cortical bone morphological adaptations previously reported in aged humans and nonhuman primate models. In particular, morphological indices at the tissue (endocortical, periosteal expansion, and increased moments of inertia) and cellular (increased cortical porosities) levels reflected agerelated adaptations. These results suggest that the rooster is a suitable, inexpensive animal model for investigation of aging-related osteopenia. Our future work will utilize this avian model to examine the cellular mechanisms responsible for the morphological alterations observed in the present study.
Acknowledgment: This study was funded in part by NIH Grant AG 14881.
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Date Received: September 22, 1999 Date Revised: December 13, 1999 Date Accepted: December 14, 1999
Aging-Induced Osteopenia in Avian Cortical Bone S. SRINIVASAN,1 S. A. KEILIN,1 S. JUDEX,2 R. C. BRAY,2 R. F. ZERNICKE,2 and T. S. GROSS1 1
Department of Orthopaedic Surgery, University of Cincinnati, Cincinnati, OH, USA McCaig Centre for Joint Injury and Arthritis, University of Calgary, Calgary, AB, Canada
2
are scarce. Secondary osteonal remodeling has been reported in sheep, but, to date, this model has been exploited primarily as a model of postmenopausal osteoporosis.26 Rodents provide a useful model for investigation of aging-associated osteopenia, especially considering their low cost, substantial compression of senescent events, and potential to utilize transgenics. However, they do not exhibit alterations in secondary osteonal remodeling and cortical porosity unless stringently provoked.18 This is an important limitation, because, although cortical porosity accounts for only a small percentage of aging-associated cortical bone loss and is often transient in nature,13 it has an enormous influence upon the mechanical properties of bone.22,24,42 The rooster represents a potentially useful and inexpensive alternative to current models of age-related osteopenia. Avian cortical bones possess the same cell types and have similar haversian and lamellar bone morphology to mammalian cortical bones. The modeling and coupled intracortical remodeling events normally observed in avian cortical bones are similar to those observed in humans during growth and adulthood. The modeling response when subject to exogenous mechanical loading,17,34 and the coupled cellular responses initiated by conditions of disuse, are similar to those observed in humans8 and dogs.15 Whereas avian bones have evolved to be lightweight for flying, functionally induced strains are similar to those observed across mammalian species.1,33 Thus, the morphological parallels between avian and mammalian bones and the similarity in the cellular response to altered physical stimuli support the use of the avian model. However, as avian species are able to rapidly mobilize (and replace) calcium from their skeleton during egg production, hens were excluded from this study. Criteria for denoting the rooster as aged, however, remain unclear. Anecdotal references suggest that roosters are capable of living up to 10 years,25 but the functional lifespan of white leghorn roosters has been found to be 5–7 years.43 Avian organ systems begin to degrade at much earlier timepoints. For example, reproductive fertility declines by 95%25 and adenylate phosphatase and adenylate deaminase activity in the heart muscle is markedly diminished by 2 years,41 and presynaptic neural degradation is pronounced by 5 years.21 The objective of this study was to therefore determine if aging induces phenotypic alterations (i.e., periosteal and endocortical expansion, elevated cortical porosity) in the cortical bone of aged roosters.
Cortical bone loss contributes substantially to the degradation of skeletal integrity associated with aging. However, animal models that closely mimic age-related alterations in cortical bone are limited. The objective of this study was to determine if aged rooster cortical bone demonstrates phenotypic alterations similar to those observed in aged human cortical bone (i.e., expansion of the endocortical and periosteal envelopes and elevated cortical porosity). When compared with young adult roosters, aged roosters demonstrated significant expansion of the endocortical (16%) and periosteal (10%) envelopes, resulting in significantly increased cross-sectional moments of inertia. In addition, aged rooster bone demonstrated significantly elevated cortical porosity (51%) and average area of porosity (83%). We conclude that rooster bone demonstrates age-related adaptations similar to those of humans at both tissue and cellular levels, and may therefore represent a relatively useful, inexpensive animal model for investigating the mechanisms of age-related bone loss. (Bone 26:361–365; 2000) © 2000 by Elsevier Science Inc. All rights reserved. Key Words: Age-related osteopenia; Rooster; Cortical bone; Bone envelope expansion; Cortical porosity. Introduction Aging-induced osteopenia poses a substantial risk to the integrity of the skeleton. Bone loss associated with aging begins near age 40 years, progresses at 0.5%–1% per year, and accounts for as much as 40% of total bone loss accumulated by age 70.27,30,32 Osteopenia associated with aging afflicts both trabecular and cortical bone,7,23 and is commonly implicated in hip, wrist, and spine fractures.10,37 In cortical bone, aging results in expanded periosteal and endocortical envelopes and in increased osteonal remodeling and cortical porosity.11,23,35 The age-related increase in cortical porosity is particularly insidious as it markedly increases skeletal fragility.22,24,42 However, the pathoetiology of aging-induced cortical bone loss is poorly understood, due in part to the lack of suitable animal models. Animal models should ideally mimic human pathophysiology as closely as possible. Nonhuman primate models satisfy this criterion, but high expense, long lifespans, and ethical issues limit their viability as a model.26 Canine models of osteopenia demonstrate altered bone envelopes,36 but reports of elevated secondary osteonal remodeling and cortical porosity in aged dogs
Methods A group of young adult (age 1 year, mean mass 2 kg, n ⫽ 12) and aged (age 5.5 years, mean mass 3 kg, n ⫽ 5) white leghorn roosters of the same strain and seed colony were utilized in this study. The aged roosters represented the survivors of an original colony of young adults. Upon killing, cortical bone cross sections
Address for correspondence and reprints: Sundar Srinivasan, Ph.D., Department of Orthopaedic Surgery, P.O. Box 670212, University of Cincinnati, Cincinnati, OH 45267-0212. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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(250 m thickness) were obtained from the right ulnae of the animals at identical sites located 33% of the diaphyseal length from the distal metaphysis. Sections were then hand ground to 75 m and toluidine blue stained. With the identity of the sections blinded, the cross sections were imaged using an Olympus light microscope fitted with a Polaroid digital camera. The digital images were then used to assess morphological adaptations in cross-sectional areal properties and cortical porosity. Cross-Sectional Morphology To determine morphological changes in cortical bone crosssectional areal properties, all cross sections were imaged at ⫻20 magnification, and digital images were obtained at a resolution of 130 pixels/mm (on average, 12 images were required per bone cross section). A composite image reconstituted the cross section into one digital image (Figure 1). With the identity of the sections blinded, image analysis of the composite image was then performed utilizing PV-WAVE (Visual Numerics Inc., Boulder, CO). Similar to previous studies,11,23 pixel-counting routines determined cortical bone area (Ct.Ar), endocortical envelope area (Ec.Ar), and periosteal envelope area (Ps.Ar) (Figure 1). Cortical width (Ct.Wi) was measured as the average distance between the periosteal perimeter and the endocortical perimeter along 360 equiangular rays originating from the centroid of the bone cross section. Using numerical integration, the cross-sectional moments of inertia about the x (Ixx) and y (Iyy) axes and polar moment of inertia (J) were determined. As a control for the pixel-counting technique, composite images (from two arbitrarily selected bone cross-sections) were scanned at twice the scanning density (260 pixels/mm) and the areal properties were determined. Doubling the scanning density (from 130 to 260 pixels/ mm) resulted in minimal alterations in Ct.Ar (0.6%), Ec.Ar (0.6%), Ps.Ar (0.08%), Ct.Wi (0.1%), Ixx (0.8%), Iyy (0.7%), and J (0.8%). Cortical Porosity To determine morphological changes in bone cortical porosity, we analyzed ulnar cross sections of young (n ⫽ 6, selected at random from the original group of 12) and aged (n ⫽ 5) roosters. Similar to previous work,23 a finite number of sampling sites was selected to access indices of cortical porosity in both groups. With the sections blinded, 12 equiangular locations at both endocortical and periosteal sites around the middiaphyseal cortex (i.e., a total of 24 sampling sites per cross section) were imaged at ⫻400, and digital images were obtained at a resolution of 2600 pixels/mm (Figure 1). Using NIH IMAGE software, particle analysis was performed on each digital image. Briefly, each grayscale digital image was thresholded based on the gray-scale histograms of arbitrarily selected lacunae. All particles within the image that represented osteocyte lacunae were then excluded from the thresholded image, similar to previous work.23 Next, for voids representing a cortical porosity (i.e., particles other than those representing osteocyte lacunae), the total numbers of pixels falling within individual voids were counted. The average porosity area (Po.Ar) was determined as the ratio of total number of pixels falling within the porosities to total number of porosities (Po.N). The cortical porosity (Ct.Po) was calculated as a percentage of the total number of pixels falling within the porosities to the total pixels representing the imaged tissue area. As a control for the image analysis technique, 24 arbitrarily selected images were thresholded based on the gray-scale histograms of alternatively selected lacunae. The mean (⫾SE) variability of Ct.Po was minimal (2.74 ⫾ 0.5%).
Figure 1. Schematic of a right ulnar cross section composite digital image. (a) Cortical bone area (Ct.Ar) was calculated as the number of pixels falling within the cortex (black region). Endocortical area (Ec.Ar) was calculated as the number of pixels falling within the endocortical envelope (gray region). Periosteal area (Ps.Ar) was calculated as the sum of Ct.Ar and Ec.Ar. (b) Around the ulnar cortex, images (original magnification ⫻400) were obtained at 12 equiangular locations and at both endocortical (E) and periosteal (P) sites (total of 24 sampling locations per cross section) to examine age-related alterations in cortical porosity. (c) A sample image (original magnification ⫻400).
Statistical Analysis The Mann–Whitney nonparametric test of two independent samples was used to assess between-group cross-sectional morphological differences. For morphological indices at the level of cortical porosities, both between-group (young vs. aged; Mann– Whitney) as well as within-group (endocortical vs. periosteal
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Figure 3. (a) Digital images (original magnification ⫻400) from the ulnae of aged (5.5 years) and (b) young (1 year roosters with cortical porosities noted [white arrows]). (c) Across all sampling locations, aging induced significant (asterisks) increases in Ct.Po and Po.Ar.
Figure 2. (a) The ulnar middiaphyseal cross section from aged (5.5 years) and (b) young (1 year) roosters. (c) Aging induced significant (asterisks) increases in Ec.Ar and Ps.Ar, but not in Ct.Ar. (d) The ulnar cross-sectional moments of inertia, Ixx, Iyy, and J, were all increased significantly in the aged roosters.
sites; Wilcoxon signed-rank test) comparisons were performed. All analyses were performed using SPSS (SPSS Inc., Chicago, IL) with p ⫽ 0.05, and results reported as mean ⫾ SE. Results
Ec.Ar (11.9 ⫾ 0.7 vs. 10.3 ⫾ 0.3 mm2, p ⫽ 0.05) (Figure 2c). The age-related increase in both Ps.Ar (10%) and Ec.Ar (16%) resulted in an increase in the cross-sectional moments of inertia as well. Aging significantly increased Ixx (30.5 ⫾ 2.03 vs. 25.9 ⫾ 1.06 mm4, p ⫽ 0.03), Iyy (55.8 ⫾ 4.04 vs. 46.9 ⫾ 1.96 mm4, p ⫽ 0.05), and J (63.6 ⫾ 4.5 vs. 53.6 ⫾ 2.2 mm4, p ⫽ 0.04) (Figure 2d). As a percentage, aging significantly increased Ixx by 18%, Iyy by 19%, and J by 19%. Neither the total cortical bone area, Ct.Ar (13.7 ⫾ 0.5 vs. 12.9 ⫾ 0.3 mm2, p ⫽ 0.16), nor the cortical width, Ct.Wi (0.9 ⫾ 0.03 vs. 0.9 ⫾ 0.02 mm, p ⫽ 0.45), were altered significantly by aging.
Cross-Sectional Morphology
Cortical Porosity
Cross-sectional bone envelope areas were increased with age (Figure 2). Compared with the young adult roosters, the cortical bone cross section of the aged bones displayed significantly greater Ps.Ar (25.6 ⫾ 0.9 vs. 23.2 ⫾ 0.5 mm2, p ⫽ 0.03) and
Avian cortical bone became increasingly porotic with age (Figure 3). Across sampling locations, there was a significant agerelated increase in both Ct.Po (4.25 ⫾ 0.49 vs. 2.82 ⫾ 0.26%, p ⫽ 0.01) and in Po.Ar (466 ⫾ 101 vs. 254 ⫾ 30 m2, p ⫽ 0.01)
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Figure 4. Cortical porosity at endocortical and periosteal sampling locations in the ulnae of aged (5.5 years) and young (1 year) roosters. Aging induced significant (asterisks) increases in (a) Ct.Po and (b) Po.Ar only at endocortical sampling locations.
(Figure 3c). In fact, aging elevated Ct.Po by 51% and Po.Ar by 83%. In contrast, Po.N (124.7 ⫾ 7.8 vs. 128.9 ⫾ 3.0/mm2, p ⫽ 0.47) was not significantly increased in aged bones. In both age groups, there was significantly more Ct.Po (4.52 ⫾ 0.52 vs. 2.43 ⫾ 0.22%, p ⬍ 0.01) and Po.Ar (475 ⫾ 96 vs. 225 ⫾ 42 m2, p ⫽ 0.01), but significantly less Po.N (118.6 ⫾ 3.9 vs. 135.4 ⫾ 5.1/mm2, p ⫽ 0.02) at endocortical vs. periosteal sampling locations. At endocortical sampling locations, there was a significant age-related increase in Ct.Po (5.73 ⫾ 0.84 vs. 3.51 ⫾ 0.26%, p ⬍ 0.01) and Po.Ar (657 ⫾ 185 vs. 323 ⫾ 24 m2, p ⫽ 0.01), but not in Po.N (114.6 ⫾ 8.0 vs. 121.9 ⫾ 3.1/mm2, p ⫽ 0.33) (Figure 4). As a percentage, age-related increases in Ct.Po and Po.Ar were 63% and 103%, respectively, at endocortical sampling locations. However, at periosteal sampling locations, aging-associated alterations in Ct.Po (2.78 ⫾ 0.28 vs. 2.14 ⫾ 0.29%, p ⫽ 0.06), Po.Ar (274.91 ⫾ 79.68 vs. 190.69 ⫾ 43.96 m2, p ⫽ 0.06), and Po.N (134.7 ⫾ 11.18 vs. 136.04 ⫾ 3.49/mm2, p ⫽ 0.33) were not statistically significant. Discussion Age-related morphological changes in cortical bone were determined in adult roosters. One means of defining an aged population is the timepoint at which 50% mortality is reached. Within our original rooster colony, 54% mortality was observed by 5 years of age. Young adults were assessed at 1 year (an age at which long bone growth plates are closed19). Both the periosteal (Ps.Ar) and endocortical (Ec.Ar) bone envelopes were significantly increased by aging. The larger bone envelopes associated with aged rooster bones resulted in a significant increase in the bending (Ixx, Iyy) and in the twisting (J) moments of inertia. These increases are similar to what has been observed between the third and seventh decades in human cortical bone.11,23 The lack of a significant aging-associated
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change in cortical bone area (Ct.Ar) and cortical width (Ct.Wi) reported here is also similar to that observed in humans in the eight decade and beyond.11,23 At the continuum level, an age-related increase in bone envelopes may result in greater resistance to the bending and twisting loads encountered during functional activity. Utilizing calibration strain-gauge values in conjunction with beam theory estimates, we have previously established the end-loading boundary conditions in the avian ulna subject to dynamic bending loads.14 Under these boundary conditions (similar to those induced during maximal wing flap), preliminary calculations suggest a 20% reduction in induced strains due to age-related expansion of cortical bone envelopes observed in this study. Similar reductions in induced strains during functional activity would result in a concomitant increase in structural strength, at least at the continuum level. However, any structural benefit of the larger cortical bone cross sections and elevated moments of inertia would be tempered by the significant increase in cortical porosity (51%) observed in our aged rooster population. In fact, an elevation in cortical porosity from 4% to 10% has been estimated to result in a 20% decrease in bone’s elastic moduli.22 Furthermore, at the microstructural level, cortical porosities act as stress risers and have a profound influence on the bone’s strength and fracture toughness.24,42 For example, a change in absolute cortical porosity from 5% to 10% can result in a 50% reduction in bone’s fracture toughness.42 Ultimately, any structural benefit of the larger bone envelopes would appear to be mitigated by these considerations and may simply reflect the lifelong accumulation of cellular activity at the bone surfaces. Similar to previous reports,5,39 the increased cortical porosity in the aged bones was primarily due to the substantial increase in the average size of each porosity (86%). One possible explanation for this result is that active remodeling was dramatically enhanced in aged bones. However, a preliminary analysis (via blinded counting of number of scalloped voids at ⫻20) indicated that, of an average of 127 porosities/mm2, there was no difference in the small percentage of sites undergoing active remodeling (0.1% young vs. 0.2% aged, p ⫽ 0.16). This observation suggests that our measure of cortical porosity (size, number) was influenced minimally by the transient nature of porosities or the remodeling state of the skeleton (i.e., the ulnae of both groups of animals were quiescent at time of killing). The age-related increase in porosity size suggests that the accrued deficit in bone mass was due to accumulated alterations in the balanced coupled resorptive process,6,12 impotent osteoblastogenesis, and/or ineffective in-filling by osteoblasts.16,20 However, other reports have suggested that the age-related increase in cortical porosity may be due primarily to an increase in the total number of porosities,23,28 potentially via increased osteoclastic activity.29 Although our results support the former observations, they do not offer a conclusive validation of either pathway, due to the limited timepoints considered. Regardless of the mechanism involved, elevated cortical porosity occurred primarily at the endocortical sites (via increased area of each porosity). The age-related increase in cortical porosity near the endocortical surface is similar to that observed in human bones23 and may reflect proximity to osteoclast progenitor pools.31 Alternatively, other processes have recently been hypothesized as potential initiators of intracortical bone loss, including osteocyte apoptosis40 and hypoxia,9 and may display an envelope-specific prevalence as well. Although the age-related increase in cortical porosity observed in this study models that observed in humans, the magnitude of cortical porosity, per se, was low (4.3%). This is relevant given that cortical porosity in human bones at middiaphyseal sites is on the order of 7.5%–20%.11,23,28 and between 10%–25% at sites of fracture, such as the forearm and the
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femur3,4,38 Interestingly, while cortical porosity at fracture sites in these studies was no different from that found in age-matched controls, increased size of porosities and the presence of abnormally large porosities were more closely related to risk of skeletal fragility. Thus, it remains unclear if cortical porosities must be in the 20% range, or if the size of porosity (in proportion to the cortical width) is a more important determinant of fracture risk. It may be possible to address the issue of age-related skeletal fragility in the avian model by exacerbating the extent of cortical porosities in aged skeletons via induction of hormonal deficits (e.g., gonadal hormone deficiency2) or treatment with parathyroid hormones and prostaglandins, such as in dogs.36 In summary, we observed significant ageing-induced cortical bone adaptation in the ulna of aged roosters. The adaptations observed here were similar to phenotypic cortical bone morphological adaptations previously reported in aged humans and nonhuman primate models. In particular, morphological indices at the tissue (endocortical, periosteal expansion, and increased moments of inertia) and cellular (increased cortical porosities) levels reflected agerelated adaptations. These results suggest that the rooster is a suitable, inexpensive animal model for investigation of aging-related osteopenia. Our future work will utilize this avian model to examine the cellular mechanisms responsible for the morphological alterations observed in the present study.
Acknowledgment: This study was funded in part by NIH Grant AG 14881.
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Date Received: September 22, 1999 Date Revised: December 13, 1999 Date Accepted: December 14, 1999