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AGE CHANGES IN HUMAN BONE: AN OVERVIEW WILLIAM D. SHARPE, M.D. Director of Laboratories Cabrini Medical Center New York, New York Clinical Associate Professor of Pathology New Jersey Medical School
That regular decay of nature which is called old age, is attended with changes which are easily detected in the dead body, and one of the principal of these is found in the bones, for they become thin in their shell and spongy in their texture. Sir Astley Cooper, 1824.
A NYONE who reviews microscopic sections of bone from patients much tolder than 60 notices subtle changes consistent enough to permit fairly accurate estimation of their ages. Bone becomes porous; haversian canals and canaliculi become plugged; many individual osteons are incompletely mineralized, hypermineralized, or hypomineralized; and the number of empty osteocyte lacunae increases. The lumina of haversian canals are often larger than normal and their blood vessels have sclerotic walls. The proportion of the skeleton composed of fragments of lamellar bone increases and microinfractions (which may seem to be artifacts, but which are quite real and contribute to the death of interstitial lamellar bone) are noted in areas of strain. After 60, the shifted balance between osteoblastic and osteoclastic activity increases the numbers of imperfectly resorbed osteons that persist as interstitial fragments and, most prominently in subendosteal areas of bone, recently formed osteons have large lumina, often containing fatty or hematopoietic marrow, imparting a trabecular pattern to this area which an observer may term osteoporosis. But these changes, in Putschar's words, ". . . blend into pathological forms of osteoporosis which either precede them in time or exceed them in degree Manuscript prepared during tenure of Contract E(l l-1)-3377, Medical Research Branch, Division of Biomedical and Environmental Research, United States Energy Research and Development Administration.
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or both."' Yet, in Dequeker's view: "Bone involution seems to be a general phenomenon and a normal manifestation of the atrophy of tissues in the process of aging."2 CELLULAR AGING Martin defines senescence as "a constellation of deteriorative changes in structure and function of an organism, generally occurring after sexual maturation, which results in a progressive decline in the efficiency of homeostasis and in the success of the reaction to injury."3 Tissue culture studies, largely of skin fibroblasts and therefore to be interpreted cautiously because adult postmitotic cells live in a matrix of other cells and cell types, suggest that large populations of normal cells and clones of cells eventually cease replication and that relative rates of clonal attenuation may contribute significantly to cellular senescence. Unlike such transformed cells as the HeLa cell, human diploid somatic cells have finite growth potentials in tissue culture, amounting to about 50 + 10 population doublings.4 Non-neoplastic, nontransformed cell colonies thus grow old and die. Martin3 cautions that collections of more and less growable cells exist in biopsy material but that the percentage of clonable cells declines consistently with the donor's age. Possibly clonal senescence is a twostage process as growth first slows and then true senescence occurs at varying rates in postreplicative cells. Holliday et al.5'6 note that aging of fibroblasts is accompanied by alterations or defects in enzymes, genes, chromosomes, DNA replication, and repair, best explained if human fibroblasts do have a defined program leading to a finite number of cell divisions. This finite lifespan can be explained if potentially immortal cells, on division, generate with a fixed probability cells programmed to become senescent and to die after a certain number of divisions. Between commitment and senescence, the uncommitted and therefore potentially immortal cells are diluted by committed cells and lost during subculturing, so that the finite growth of cultured diploid fibroblasts may be an artifact of tissue culturing techniques. Hayflick4 states that with individual variation, human physiologic functions decline at about 0.8 to 0.9% each year after age 30, and that the death of cell lines after about 50 population doublings is a property of the cells themselves, "programmed" into their original pool of genetic information and developed in orderly sequence just as are other developmental changes. Hayflick provides striking confirmation of this finite possible Bull. N.Y. Acad. Med.
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number of doublings in that fibroblasts cultured from patients with progeria (Hutchinson-Gilford's syndrome) went through only two to 10 doublings before death, when the normal would have between 20 and 40 doublings. However, Goldstein et al.7 compared skin fibroblast cultures from patients with clinically apparent or a genetic predisposition to diabetes mellitus, either of which reduces life expectancy, and from carefully defined normal subjects, and conclude that the physiologic status of the tissue donor is a more precise determinant of fibroblast replicative lifespan than is his chronologic age. HORMONAL INFLUENCES Rasmussen and Bordier8 state that hormonal and ionic factors contribute to modulation of osteocytes to bone-resorbing and bone-forming cells. Ordinarily, these osteocytic functional fluctuations affect only the tiny volumes of lacunar and canalicular bone surrounding the osteocyte and its extensions. These particular compartments of metabolically labile bone ordinarily surround the rather small percentage of the whole number of osteocytes in an area undergoing osteocytic osteolysis.9 Courpron et al.10 argue for an individual bony personality (personnalite osseuse individuelle) and that bone aging involves a parallel involution of both the bone and the hematopoietic cell lines. Dequeker2 reports that estrogens inhibit bone resorption, possibly by reducing the parathormone effect on bone, and suggests that postmenopausal increases in serum calcium levels and alkaline phosphatase activity arise from reduction of estrogenic protection against the boneresorbing action of parathormone, which would also explain the increased serum calcium levels that occur among men past 65. Laitinen'1 observes that estrogen inhibits collagenase activity in the uterus and further affects the stability and structure of bone collagen, so that it may be assumed that estrogen-induced decreases in the rate of bone resorption could be mediated by a similar mechanism. Lindsay et al.12 studied a group of castrated women, and observed a significant inverse relation between their fasting urinary hydroxyproline/creatinine ratios and plasma levels of circulating endogenous estradiol, concluding that circulating estrogen levels determine the rate of bone turnover described by this inverse correlation with urinary hydroxyproline secretion. Lindsay et al. also conclude that renal handling of phosphate ions, expressed as TmPO4Iglomerular filtration rate, depends on the endogenous estradiol level and further supports their conclusion that Vol. 55, No. 8, September 1979
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plasma estradiol levels and the rate of bone loss are inversely related. They suggest that the estradiol identified in the plasma of castrated women is largely synthesized by conversion of adrenal androstenedione in peripheral body fat. Monnier et al.13 conclude from isotopic studies of a diabetic population that age by itself does not influence the intestinal absorption of dietary calcium, but that episodes of ketosis exaggerate both intestinal absorption and urinary excretion of calcium. Marked glycosuria in a nonketotic diabetic patient reduced urinary calcium excretion by enhancing tubular calcium resorption. They conclude that no real difference exists in calcium absorption and its incorporation into bone mineral between diabetic and control groups, and that patients with diabetes mellitus are essentially at no greater risk of osteoporosis than those without diabetes. Agreement seems general14 that patients with clinical osteoporosis probably have a greater than normal rate of bone loss and may have begun life with a smaller bone mass during youth, but Laitinen" confesses that present knowledge does not provide sufficient data to define the extent to which the physiological hormonal changes of advancing age modify osteoporotic changes, although the decline in serum estrogen levels following the menopause is probably pathogenetic. He suggests that relative hypercorticosteroidism may also be important, and that changes in the concentrations of several hormones and in their action on target tissues do influence the progression and extent of senile osteoporosis. OSTEOCLASTS, OSTEOBLASTS, AND OSTEOCYTES Normally, new bone is deposited only at the sites of previous bone resorption. When calcitonin is administered to young rabbits, the number of multinucleated osteoclasts present on bone resorption surfaces decreases within 15 minutes. They are replaced by mononucleated cells that seem to be preosteoblasts and give rise to osteoblasts, so that the osteoclasts did not die but modulated into osteoblasts. The osteoblasts and preosteoblasts did not undergo mitotic division.8 Harold Frost's brilliant studies'5"6 have defined a "normal sequence" of events during endosteal bone remodeling: activation of a new group of cells, resorption of bone by the daughters of these cells, and sequential formation of new bone at the site of previous resorption. Osteoclasts and osteoblasts are not, therefore, distinct cell pools but represent differing functional states of the same cells:
Bull. N.Y. Acad. Med.
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Flanagan and Nichols17 estimated the cellularity of human bone samples using DNA content as an index, and observed that cortical bone contained approximately one quarter as many cells as did mixed cortical and trabecular bone, with a clear reduction in the number of cells with advancing age. They regard the number rather than the relative metabolic activity of bone cells as the most important factor controlling the overall metabolic rate of bone in any particular part of the normal skeleton. The metabolic rates of bone samples from a variety of ages and skeletal sites were closely similar when calculated on the basis of sample DNA content despite variation in cell numbers. Osteopenic bone typically has no more osteoclasts than normal, but does have increased resorptive surfaces and decreased bone-forming surfaces. Osteoprogenitor cells of older people, however, form osteoclasts with fewer than the normal number of nuclei so that after fission, modulation may be delayed or some of the modulated cells may fail to become functional osteoblasts. Inactive or arrested resorption surface increases as the area of active bone-forming surface decreases and, for these reasons, Rasmussen and Bordier8 attribute osteoporosis to osteoblast failure. The percentage of Howship's lacunae which contain osteoclasts decreases up to the fifth decade and slightly increases thereafter, and the percentage of empty osteoclastic lacunae is minimal between ages 30 and 40.18 Delling describes certain age-related changes on the inner surface of bone: after the fifth decade, the percentage of large and presumably bone-resorbing osteocytes rises while that of small and presumably bone-forming osteocytes falls, as empty osteocyte lacunae increase in number with advancing years.18 Melsen et al. found that the mean size of periosteocytic lacunae is not affected by age.19 BONE COLLAGEN
Laitinen1' states that about three quarters of the total volume of bone is organic matrix, more than 95% of the fat-free organic material is collagen,
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and the remainder is mucopolysaccharides and mucoproteins. Schmidt, Kalbe, and SielafF20 observe that with advancing age, soluble bonecollagen fractions and metabolism decrease only slightly and that the collagen half-life increases proportionately. Both the insoluble bone collagen half-life and the insoluble share of the total bone collagen increase, but its metabolic activity decreases. This shift in the proportions of soluble and insoluble bone collagen and increased half-lives are apparently associated with collagen fiber interlacing through cross-linkage and hardening, and corresponds to what is known of other connective tissue collagens with aging. Bone collagen is not inert even in old age, but with aging, insoluble collagen is withdrawn from resorption and therefore from metabolic turnover. The elimination of hydroxyproline, and especially of free hydroxyproline, is reduced by about half from the third to the seventh decades and is related to reduction in the soluble collagen fraction. Whether apolarly attacking collagen peptidases also undergo a decrease with age is not known. Fujii, Kuboki, and Sasaki2I report a marked association with age in the decrease in dihydroxylysinonorleucine and hydroxylysinonorleucine with significantly increased lysinonorleucine in both bone and articular cartilage collagen. Simultaneously, the collagen crosslink precursors hexitollysine and dihydroxynorleucine increased. They suggest that both collagens become less soluble with age through in vivo reduction of crosslinks or transformation to unknown forms of nonreducible linkages. Grant and Prockop22 suggest that complete cross-linking of collagen may require months or years, and that the duration of this process and stiffening of connective tissues with age raise the possibility that increased cross-linking of collagen may be critical in aging, although no evidence suggests that inhibition of this cross-linking retards the effects of aging. Dequeker2 concurs that with advancing age no striking qualitative changes have been found in the collagen or mineral composition of bone, although collagen saline solubility decreases with age, probably because of the increased number of collagen crosslinks. Laitinen1' describes a labile portion of recently synthesized bone collagen that loses its metabolic activity rapidly with advancing age, and states that the rate at which collagen matures also declines with age, a decrease most pronounced in recently formed bone. Laitinen summarizes the steps in the metabolism of collagen that are affected by aging as: the rate of both synthesis and catabolism of collagen decreases with age, a general change Bull. N.Y. Acad. Med.
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most pronounced in bone; the maturation of collagen through the synthesis of the inter- and intramolecular crosslinks of the triple helical tropocollagen molecules slows; the formation, of stable "mature" collagen is retarded; and, despite the decreased rate of collagen maturation, the number of inter- and intramolecular collagen crosslinks increases with age. He suggests that increased numbers of collagen crosslinks may explain the decrease in the rate of collagen catabolism with aging. Laitinen11 suggests that both experimental and clinical disturbances in calcium metabolism cause changes in skeletal metabolism that seem to be initiated and mediated largely through changes in the collagen matrix of bone. He reports that in both experimental hyperthyroidism and hyperparathyroidism, the rate whereby the collagen matrix of bone is degraded acts as a moderator or "final messenger" in hormone-induced bone resorption.
GROUND SUBSTANCE
Schmidt, Kalbe, and Sielaff20 report that proteoglycane synthesis in the ground substance of bone is reduced with age apace generally reduced rates of bone-protein synthesis and rapidly decreased sulphate radical incorporation rate. With aging, the hexosamine and uronic acid content and hexosamine-hydroxyproline ratios decrease, proteoglycane metabolic activity decreases, and hydroxyproline content remains the same or very slightly increases. Not only do the metabolic rates of osteoblasts and osteoclasts decline with age, but deposition of hydroxyapatite in the collagenous matrix of bone depends largely on the chemical state of the interstitial substance, especially its proteoglycane content and pattern. These changes lead to a bone matrix that is inherently less and less mineralizable. Hayflick4 describes a physiological decrease with age in normal human fibroblasts in vitro in mucopolysaccharide synthesis, collagen synthesis, collagen synthesis and collagenolytic activity, alkaline phosphatase production, and the number of cells in the proliferating pool.
OSTEOID Ultrastructural examination of the layer of bone matrix between the osteoblasts and the mineralized bone, called osteoid, demonstrates that osteoid is a structured sequential modification of collagen fibrils and mucopolysaccharides that undergoes modification during certain abnormal Vol. 55, No. 8, September 1979
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states.23 Whereas fibroblasts form collagen fibers all around themselves,18 osteoblasts characteristically secrete collagen precursors and ground substance in only one direction, the collagen precursors passing through the osteoblast cell membrane and, external to the osteoblast, polymerizing with the ground substance to yield osteoid. The calcium concentration within the osteoid seam increases from that region of the osteoid nearest the marrow cavity to that nearest the fully mineralized bone, and hydroxyapatite forms along the mineralization front. Inspection of actively forming bone reveals some areas of osteoid covered by large hexagonal or cuboidal osteoblasts, deep to which rapid primary mineralization takes place, but other areas where osteoid is covered by flat, resting osteoblasts and where secondary mineralization occurs, apparently independently of osteoblastic activity. Most cancellous bone surfaces physiologically lack osteoid seams and Howship's lacunae, and constitute a neutral resting surface bordering the marrow space. Whether this resting surface is completely covered by mesenchymal cells is not yet clear, but these flat cells remain connected to osteocyte processes and seem to be important in the regulation of serum calcium and phosphorus levels. The osteocyte's high metabolic activity, which includes regulation of bone mineral and water content and exchange with extracellular fluid, is fostered by interconnections between osteocyte processes, osteoblasts, and osteoclasts.18 The osteocyte lacunar wall, which consists of collagen fibers, ground substance, and bone mineral, approximates about 20 sq.mm. for each cubic mm. of cortical bone, but the canalicular surface containing osteocyte cell processes has an area of about 200 sq.mm. for the same volume. (Some lacunae have a delicate membranous wall, the function and importance of which remain obscure.) Hydroxyapatite crystals cover the collagen fibers in a very regular manner to make up an optimal mechanical and metabolic system. Delling18 reports that the percentage of nonmineralized bone in relation to a unit volume of bone tissue remains nearly constant after the first two decades, and, although the bone surface covered by osteoid increases with age, thickness of the osteoid decreases. (Significantly thickened osteoid seams at any age suggest disturbed bone formation.) These seams cover 10 to 25% of the total bone surface, are minimal between ages 40 and 50, and increase with age. Delling18 notes: "In contrast to the total extent of seams, the percentage of active seams (osteoid with osteoblasts) decreases from 22.4 + 8.1% in the first decade to 0.9 + 0.6% in the eighth." He Bull. N.Y. Acad. Med.
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states that the percentage of inactive to total bone surface remains constant from the 10th to the 90th years, although the absolute value decreases as bone mass diminishes. Melsen et al.19 found no significant sex difference or change with increasing age in osteoid surface area, although the relative osteoid volume decreased slightly but insignificantly with increasing age, a decrease more pronounced as the amount of trabecular bone decreased with increasing age. In contrast to the decrease in active osteoid seams with age, the surface covered by inactive seams (osteoid without osteoblasts) increases from the fifth or sixth decade. Delling18 suggests that this is related to renal arteriosclerosis and reduction in available renal parenchyma, the site of hydroxylation of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D for bone tissue. Hence, the reduction in the quantity of active osteoid and its eventual mineralization may be secondary to the pathologic or physiologic age changes in other organs, just as some individuals seem to suffer a decline in the efficiency of intestinal calcium absorption with age. Melsen et al.'9 agree that the quantity of both trabecular and cortical bone decreases with advancing age in both men and women, but state that the extent, volume, and width of osteoid seams are independent of age. They observe that osteoclastic resorption in cortical bone in men increased with advancing age.
COMPACT BONE
Kerley24 describes fairly consistent changes in the shafts of long bones associated with advancing age, and most marked in the inner third of the cortex, the outer third being least affected. Description of these changes requires certain definitions. Osteons are vascular channels surrounded by concentric lamellae containing rather evenly spaced osteocytes and bordered by a peripheral growth reversal line. Fragments are portions of old osteons that may surround the peripheries of newer osteons. The number of osteon fragments increases as more and more osteons are laid down until, in extreme old age, most of the complete osteons are surrounded by fragments of older osteons. Circumferential lamellar bone consists of evenly spaced, generally parallel or concentric bone lamellae in the outer subperiosteal portion of the cortex, and its collagenous matrix consists of long, parallel, fairly uniform fibers. Nonhaversian canals contain primary vascular channels formed by incorporation of small, peripheral, originally Vol. 55, No. 8, September 1979
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periosteal vessels into the bone deposited as the cortex expands. These have no osteonal pattern, lack growth-reversal lines, and represent unremodeled bone, just as osteonal bone is formed by osteoclastic remodeling. At the tissue level, bone remodeling is continuous.15'25 Within lamellar bone, cutting cones produce cavities of the order of 200 micra in diameter and as much as several millimeters long. This "cutting head" of osteoclasts is followed by a fibrocapillary leash, and the cavity cut by the osteoclasts is filled in centripetally by osteoblasts which arise from the capillary perithelial cells or are modulated in situ to produce the secondary osteon. Osteoblasts deposit the bone of secondary osteons in a definite, organized pattern wherein the collagen matrix fibers change course in each layer, again leading to maximum flexibility and physical strength. Space between these secondary osteons is occupied by interstitial lamellar bone, and if this interstitial bone cannot be adequately nourished by the canaliculi, it dies. The relative proportion of dead to living interstitial bone increases steadily with age. Kerley24 describes a life cycle for cortical bone. The number of osteons increases from birth to old age, the proportion of circumferential bone decreases with age, and nonhaversian canals disappear during the sixth decade, indicating a very radical decrease in subperiosteal bone deposition by that time. The earliest phase of bone deposition is of radial spicules of coarsely woven bone with irregularly arranged collagen fibers having no particular relation to the large vascular spaces which they enclose. Later, the vascular spaces are occupied by new bone apposited on their walls to become the primary nonhaversian osteons.26 As the cortex and medulla expand, small subperiosteal blood vessels running along the long axis of the bone are incorporated by the expanding peripheral cortex to form nonhaversian canals. Finally, osteoclastic cuffing cones form secondary osteons that are typically themselves partially destroyed by subsequent remodeling. Pankovich, Simmons, and Kulkarni27 studied microradiographs of rib sections from patients eight to 80 years old, and found changes in the proportions of osteons with low, intermediate, and high mineral density as the skeleton aged. They describe an osteon characteristic of old age as having low to intermediate mineral density, wide haversian canals, and hypercalcified borders. They note a trend toward increasing percentages of osteons with incomplete growth-arrest lines in older patients, and suggest that intraosteonal remodeling and growth retardation play increasingly Bull. N.Y. Acad. Med.
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important roles in bone turnover in the aged skeleton, and contribute to the increased porosity and lower mean osteonal mineral density characteristic of old age. Intraosteonal remodeling may preferentially affect older osteons because the oldest peripheral lamellae are typically highly mineralized and this process, which does not seem to affect interstitial bone, appears to be a mechanism whereby osteon mineral density is reduced and skeletal porosity increased among the aged. Trotter and Hixon28 examined the weight, density, and percentage ashed weight of 426 dry, fat-free bony skeletons of both sexes, black and white, from the 16th week of gestation to 100 years old, and found a gradual loss in bone weight and density (but not volume) that began during the fourth decade, and averaged 15.6 grams yearly. Change in weight was accompanied by change in ashed weight. Race and sex differences emerged by the second decade: black skeletons exceeded white, and male skeletons exceeded female skeletons in mean weight and density and, although to a smaller extent, in percentage ashed weight. Smith, Khairi, and Johnston,29 by longitudinal studies, documented that individuals lose bone mineral at unequal rates with age. Bone mineral content, using the radius as a standard, had a normal statistical distribution at all ages, variation from the norm did not increase with age, and because no bimodal distribution emerged from their data, a distinct population of "rapid bone losers" (which exists, according to some workers) could not be identified. Hence, the rate of individual bone mineral loss is not constant, or the rate of bone mineral loss is proportionate to the amount of mineral present at maturity, or both. They suggest that the bone mineral content of patients identified as osteoporotic falls at the lower end of a normal curve, probably because they have had a low bone mineral content since maturity. BONE Loss
Dequeker2 summarizes the problem of skeletal aging as decreased skeletal weight added to decreased physical density (weight/volume) of individual bones. Similar changes occur in paleopathologic material, so this is not a modern phenomenon. Although good statistical relations exist between changes in the peripheral and axial skeletons, changes in one particular bone do not accurately predict changes in another. It has been known forever that old people have more fragile bones than young people, and Dequeker suggests that bone strength, which measures compliance Vol. 55, No. 8, September 1979
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and bone brittleness, which measures breakability, may be different things, and that old people may have "bad bone" in terms of brittleness. During adult life, bone is gained or lost at one or any combination of four sites: periosteal surfaces, surfaces of vascular channels within cortical bone, cortical endosteal surfaces, and trabecular endosteal surfaces. Even after bones stop growing in length, cancellous and cortical bones undergo constant resorption of existing bone and deposition of new bone. Changes in this balance underlie every disease affecting the skeleton. Periosteal bone growth is not merely compensation for endosteal bone loss, because subperiosteal deposition in old age fails to keep pace with subendosteal resorption, nor is continuing bone deposition invariably related to weightbearing or flexion stress. Loss of bone after the fifth decade arises from three processes: slightly increased osteoclasia which reduces the amount of cancellous bone, although increases in resorption surfaces are distinct only among women 50 to 59;19 simultaneous decrease of osteoblastic activity which accentuates this rarefaction by decreased formation of new bone; and delayed mineralization of inactive osteoid surfaces. (Some osteoblasts thicken main bone trabeculae in a form of compensatory hypertrophy without, however, affecting the net process.) Even in physiological osteoporosis,11 bone mineral metabolism itself remains within normal limits or is only slightly decreased. The total bone tissue, excluding marrow, constitutes about 20% of cancellous bone, a proportion which is fairly constant until about the fifth decade, after which loss of bone mass occurs as rarefaction of bone is accompanied by progressive encroachment of marrow spaces filled by fat and nests of hematopoietic marrow.18'19 Dequeker2 argues that the endosteal cavity expands in response to endosteal bone loss, and that this process accelerates after 50 as endosteal surfaces enlarge faster than the periosteal surfaces. Melsen et al.19 describe the loss of cortical bone as a decrease in mean cortical width and, for women, an increase in cortical
porosity. Because inactivity so quickly leads to osteoporosis, Dunnill, Anderson, and Whitehead30 studied changes in the bones of patients of various ages who died in accidents, and found that the proportion of bone occupied by osseous tissue decreased steadily with age as, with advancing years, fatty marrow steadily increased to replace both bone and hematopoietic marrow. They agree that an overlap exists between the ordinarily increased porosity Bull. N.Y. Acad. Med.
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of skeletal senescence and osteoporosis. Takahashi and Frost31 found that women begin life with significantly less bone than men, primarily because their bones are smaller, and both sexes have maximum amounts of bone between 15 and 25 but have lost about a quarter of this maximum by 60. They could find no correlation between a bone's total cross-section area and that of its cortical component from puberty to the menopause, and suggest that if osteoporosis is related to the menopause, the mechanism is extra-ovarian. Increase in total bone cross-section area occurs after 60, more prominently among women than among men, and, although new bone formation declines to a basal level, it does not cease. The periosteal surfaces are never in negative balance, that is, the space inside the periosteum never shrinks. Smith, Khairi, Norton, and Johnston32 found that the mean rate of mineral loss is not constant with aging, is slower in the elderly than during earlier postmenopausal years, and that factors other than decreased physical activity are more significant in determining the rates of mineral loss. Gergely et al.33 compared the bone mineral content of 436 old people to that of 198 healthy people between 21 and 50, and found that the bone mineral content of postmenopausal women declined steadily with age but that of men began to decrease only after 70. Bone width and age at menopause seemed to influence bone mineral content, but previous physical activity seemed to have no effect on the bone mineral content of the aged. Laitinen"1 regards decreasing physical activity as only a secondary cause of age-related osteoporosis, although Nokso-Koivisto et al." maintain that bone mineral is lost in some chronic diseases mainly because of physical inactivity. Regression analyses of measurements by Melsen et al.19 document significant falls in the quantity of bone with increasing age for both sexes and that the quantity of trabecular bone among the youngest group was higher in women than in men, the more severe bone loss by women between ages 50 and 59 resulting in equalization of amounts of trabecular bone among both sexes in old age. Dequeker et al. 14 suggest that heterogeneity between persons explains variations in bone loss, but that errors in measurement can account for considerable variation, and many individuals maintain essentially normal trabecular structure until the eighth decade. Rasmussen and Bordier8 state that slow but progressive bone loss occurs throughout life although transverse diameters of bones increase from a net Vol. 55, No. 8, September 1979
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positive balance of subperiosteal and a net negative balance of subendosteal bone. From 20 to 50 or 55, women lose bone slightly more rapidly than men, but undergo an abrupt loss of skeletal mass between 50 or 55 and 65, after which the rate of bone loss again stabilizes.14 LaitinentI holds that under normal circumstances the breakdown of the organic collagen matrix is the chief factor regulating calcium resorption from bone, and that other factors such as exchange of mineral substances within the hydroxyapatite crystals play only a minor role in the process. Current views as to the pathophysiology of bone loss may be summarized2'" as a senescent imbalance between bone deposition and bone resorption in the latter direction. Parathormone plays a major, but indirect, part in enhanced bone resorption by changing end organ sensitivity, particularly to changes in the androgenic/estrogenic balance. Currently, dietary calcium deficiency is not considered a major factor in bone loss because human beings seem to adapt quite comfortably to low calcium diets, absent pregnancy, and lactation-which are rarely geriatric problems. However, a peculiar calcium deficiency may arise from primary or secondary hypercalciuria which lowers the serum ionized calcium content and results in increased parathormone levels and, therefore, enhanced bone resorption. Frost", has documented that bone formation and resorption tend to change in the same direction with a variable time lag between the two phases, thus, treatment may reduce resorption, but the organism will then reduce bone formation to match. MECHANICAL EFFECTS
Burstein, Reilly, and Martens35 studied the tension, torsion, and compression responses of bone from a population of patients between 21 and 86, and found no significant differences in the mechanical properties of bone from normal, osteoporotic, and adrenocortiscosteroid-treated individuals. They report that the ultimate strain at the point of fracture consistently decreased 8.4% for each decade, and comment: . . That this decrease with age was seen in both femoral and tibial bone tissue suggests that the ultimate strain reflects a property or quality which depends on the age of the individual at the time that the bone tissue under test was originally deposited. Hence, age effects on ultimate strain are determined by the age of the organism rather than the time that the tissue has been in situ. ...
They attribute the declining mechanical strength of bone to changes in the structure of the bone collagen matrix. Bull. N.Y. Acad. Med.
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Burstein, Zika, Heiple, and Klein36 elegantly tested wet bovine haversian bone as though they were testing the plastic behavior of a metal, and found by sequential decalcification that as bone mineral content decreased, the tension yield point and ultimate stress decreased progressively without changing yield strain or ultimate strain short of complete decalcification. Their findings "are consistent with an elastic-perfectly plastic model for the mineral phase of bone tissue in which the mineral contributes the major portion of the tension yield strength." They suggest that collagen itself plays only a minor role in producing bone's tension yield strength. Thus, when a bone is subjected to tensile loading, there is an initial elastic response in which the load deformation curve is essentially reversible, followed by a plastic-or permanent-deformation of the bone. Under tensile stress, then, the bone mineral deforms in a plastic fashion and acts as a metal, not as though it were a synthetic plastic composite. Mineralized bone31; develops numerous tiny cracks and dislocations once a critical mechanical stress is reached and, during stress-induced plastic deformation, these cracks propagate a limited distance in the bone until they are interrupted. Added deformation causes additional cracks or dislocations, and once the strain reaches the point that structural interruption no longer can stop the propagation of these microfractures, a 'catastrophic crack' spreads and the tissue fails. Weaver and Chalmers37 studied changes in bone strength with aging, and found that most of the fractures which increase with age occur in bones wherein trabecular bone provides a significant portion of support, and that the compression strength of cancellous bone is closely enough related to bone mineral content that compression failure strength proves a reliable function of mineral content regardless of age or sex. Bone strength and mineral content diminish steadily with age. Before age 45 or 50 there is no sex difference, but because bone loss begins earlier and is more profound in women than in men, by the seventh decade women's vertebral bone ashed weight is 20% less than men's. (Activity, of course, influences cancellous bone strength and mineral content, an influence exerted through the orientation and distribution of trabeculae.) Nokso-Koivisto et al.34 found good correlation between bone strength, age, and bone mineral density, and tested the force required to break bone trabeculae, concluding that trabecular bone strength averaged 15% higher in men than in women.
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SUMMARY
The human skeleton steadily changes structure and mass during life because of a variety of internal and external factors. Extracellular substance and bone cells get old, characteristic structural remodeling occurs with age, and these age-related changes are important in the discrimination between pathological and physiological changes during old age. Perhaps 20% of the bone mass is lost between the fourth and the ninth decades, osteoblasts function less efficiently, and gradual loss of bone substance is enhanced by delayed mineralization of an increased surface area of thin and relatively less active osteoid seams. After the fifth decade, osteoclasia and the number of Howship's lacunae increase and, with age, the number of large osteolytic osteocytes increases as the number of small osteocytes declines and empty osteocyte lacunae become more common. The result is greater liability to fracture and diminished healing or replacement of injured bone. REFERENCES 1. Putschar, W. G. J.: General Pathology of the Musculo-Skeletal System. In: Handbuch der Allgemeinen Pathologie, Bichner, F., Letterer, E., and Roulet, F., editors. Berlin, Springer-Verlag, 1960, vol. III.2, pp. 359-488. 2. Dequeker, J.: Bone and aging. Ann. Rheum, Dis. 34:100-15, 1975. 3. Martin, G. M.: Cellular aging-Clonal senescence. Am. J. Pathol. 89:484-511, 1977. 4. Hayflick, L.: The cell biology of human aging. N. Engl. J. Med. 295:1302-08, 1976. 5. Holliday, R., Huschtscha, L.I., Tarrant, G.M., and Kirkwood, T.B.L.: Testing the commitment theory of cellular aging. Science 198:366-72, 1977. 6. Ageing at the cellular level (editoral). Lancet /:79, 1978. 7. Goldstein, S., Moerman, E. J., Soeldner, J. S., et al.: Chronologic and physiologic age affect replicative lifespan offibroblasts from diabetic, prediabetic and normal donors. Science 199:781-82, 1978. 8. Rasmussen, H. and Bordier, P.: The cellular basis of metabolic bone disease. N.
Engl. J. Med. 289:25-32, 1973. 9. Bohatirchuk, F.: Calciolysis as the initial stage of bone resorption. Am. J. Med. 4/:836-46, 1966. 10. Courpron, P., Meunier, P., Vignon, G., et al.: Donnees Histologiques quantitives sur le Vieillissement osseus humain. Lyon MMdi. 226:755-66, 1971. 11. Laitinen, O.: Relation to osteoporosis of age- and hormone-induced changes in the metabolism of collagen and bone. Isr. J. Med. Sci. /2:620-37, 1976. 12. Lindsay, R., Hart, D. M., Sweeney, A., et al.: Endogenous oestrogen and bone loss following oophorectomy. Calcif. Tiss. Res. 22:213-16, 1977. 13. Monnier, L., Chevallet, M., Huh, K. B., et al.: Etude de la Densit6 et de 1' Absorption intestinale du Calcium en Fonction de 1'Age et du Sexe par des Methodes isotopiques. Probl. Actuels Endocrinol. Nutr. 20:307-16, 1976. 14. Dequeker, J., Burssens, A., Creytens, G., and Bouillon, R.: Ageing of bone-Its relation to osteoporosis and osteoarthrosis in post-menopausal women. Front. Horm. Res. 3:116-30, 1975. 15. Frost, H. M.: BoneRemodelingDynamBull. N.Y. Acad. Med.
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ics. Springfield, Ill., Thomas, 1963. 16. Frost, H. M.: Metabolism of bone. N. Engl. J. Med. 289:864-65, 1973. 17. Flanagan, B. and Nichols, G., Jr.: Metabolic studies of human bone in vitro. I. Normal bone. J. Clin. Invest. 44:178894, 1965. 18. Delling, G.: Age-related bone changes. Curr. Top. Pathol. 58:117-47, 1973. 19. Melsen, F., Melsen, B., Mosekilde, L., and Bergmann, S.: Histomorphometric analysis of normal bone from the iliac crest. Acta Path. Microbiol. Scand., Sect. A, 86:70-81, 1978. 20. Schmidt, U. J., Kalbe, I., and Sielaff, F.: Bone Aging. In: Cell Impairment in Aging and Development, Cristofalo, V. J. and Holeckovi, E., editors. New York and London, Plenum, 1975, pp. 371-74. (Adv. Exp. Med. Biol. 53:37174, 1975.) 21. Fujii, K., Kuboki, Y., and Sasaki, S.: Aging of human bone and articular cartilage collagen-Changes in the reducible crosslinks and their precursors. Calcif. Tiss. Res. 15:165-66, 1974. 22. Grant, M. E. and Prockop, D. J.: The biosynthesis of collagen. N. Engl. J. Med. 286:194-99, 242-49, 291-99, 1972. 23. Fornasier, V. L.: Osteoid-An ultrastructural study. Human Pathol. 8:24354, 1977. 24. Kerley, E. R.: The microscopic determination of age in human bone. Am. J. Phys. Anthropol. 23:149-64, 1965. 25. Johnson, L. C.: Morphologic Analysis in Pathology-The Kinetics of Disease and General Biology of Bone. In: Bone Biodynamics, Frost, H. M., editor. Boston, Little Brown, 1964, pp. 543-654. 26. Smith, J. W.: The arrangement of collagen fibers in human secondary osteons. J. Bone Joint Surg. 42B:588-605, 1960. 27. Pankovich, A. M., Simmons, D. J., and Kulkarni, V. V: Zonal osteons in cortical
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bone. Clin. Orthop. /00:356-63, 1974. 28. Trotter, M. and Hixon, B. B.: Sequential changes in weight, density and percentage ash weight of human skeletons from an early fetal period through old age. Anat. Record /79:1-18, 1974. 29. Smith, D. M., Khairi, M. R. A., and Johnston, C. C., Jr.: The loss of bone mineral with aging and its relationship to risk of fracture. J. Clin. Invest. 56:31 118, 1975. 30. Dunnill, M. S., Anderson, J. A., and Whitehead, R.: Quantitative histological studies on age changes in bone. J. Path. Bact. 94:275-91, 1967. 31. Takahashi, H. and Frost, H. M.: Age and sex related changes in the amount of cortex of normal human ribs. Acta Orthop. Scand. 37:122-30, 1966. 32. Smith, D. M., Khairi, M. R. A., Norton, J., and Johnston, C. C., Jr.: Age and activity effects on rate of bone mineral loss. J. Clin. Invest. 58:716-21, 1976. 33. Gergely, I., Krasznai, I., Horvith, T., et al.: Bone mineral content of the healthy aged. Aktuel. Gerontol. 8:109-12, 1978. 34. Nokso-Koivisto, V. -M., Alhava, E. M., and Olkkonen, H.: Measurement of cancellous bone strength correlations with mineral density, ageing and disease. Ann. Clin. Res. 8:399-402,1976. 35. Burstein, A. H., Reilly, D. T., and Martens, M.: Aging of bone tissueMechanical properties. J. Bone Joint Surg. 58A:82-86, 1976. 36. Burstein, A. H., Zika, J. M., Heiple, K. G., and Klein, L.: Contribution of collagen and mineral to the elastic-plastic properties of bone. J. Bone Joint Surg. 57A:956-61, 1975. 37. Weaver, J. K. and Chalmers, J.: Cancellous bone-Its strength and changes with aging. J. Bone Joint Surg. 48A:289-308. 1966.
AGE CHANGES IN HUMAN BONE: AN OVERVIEW WILLIAM D. SHARPE, M.D. Director of Laboratories Cabrini Medical Center New York, New York Clinical Associate Professor of Pathology New Jersey Medical School
That regular decay of nature which is called old age, is attended with changes which are easily detected in the dead body, and one of the principal of these is found in the bones, for they become thin in their shell and spongy in their texture. Sir Astley Cooper, 1824.
A NYONE who reviews microscopic sections of bone from patients much tolder than 60 notices subtle changes consistent enough to permit fairly accurate estimation of their ages. Bone becomes porous; haversian canals and canaliculi become plugged; many individual osteons are incompletely mineralized, hypermineralized, or hypomineralized; and the number of empty osteocyte lacunae increases. The lumina of haversian canals are often larger than normal and their blood vessels have sclerotic walls. The proportion of the skeleton composed of fragments of lamellar bone increases and microinfractions (which may seem to be artifacts, but which are quite real and contribute to the death of interstitial lamellar bone) are noted in areas of strain. After 60, the shifted balance between osteoblastic and osteoclastic activity increases the numbers of imperfectly resorbed osteons that persist as interstitial fragments and, most prominently in subendosteal areas of bone, recently formed osteons have large lumina, often containing fatty or hematopoietic marrow, imparting a trabecular pattern to this area which an observer may term osteoporosis. But these changes, in Putschar's words, ". . . blend into pathological forms of osteoporosis which either precede them in time or exceed them in degree Manuscript prepared during tenure of Contract E(l l-1)-3377, Medical Research Branch, Division of Biomedical and Environmental Research, United States Energy Research and Development Administration.
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or both."' Yet, in Dequeker's view: "Bone involution seems to be a general phenomenon and a normal manifestation of the atrophy of tissues in the process of aging."2 CELLULAR AGING Martin defines senescence as "a constellation of deteriorative changes in structure and function of an organism, generally occurring after sexual maturation, which results in a progressive decline in the efficiency of homeostasis and in the success of the reaction to injury."3 Tissue culture studies, largely of skin fibroblasts and therefore to be interpreted cautiously because adult postmitotic cells live in a matrix of other cells and cell types, suggest that large populations of normal cells and clones of cells eventually cease replication and that relative rates of clonal attenuation may contribute significantly to cellular senescence. Unlike such transformed cells as the HeLa cell, human diploid somatic cells have finite growth potentials in tissue culture, amounting to about 50 + 10 population doublings.4 Non-neoplastic, nontransformed cell colonies thus grow old and die. Martin3 cautions that collections of more and less growable cells exist in biopsy material but that the percentage of clonable cells declines consistently with the donor's age. Possibly clonal senescence is a twostage process as growth first slows and then true senescence occurs at varying rates in postreplicative cells. Holliday et al.5'6 note that aging of fibroblasts is accompanied by alterations or defects in enzymes, genes, chromosomes, DNA replication, and repair, best explained if human fibroblasts do have a defined program leading to a finite number of cell divisions. This finite lifespan can be explained if potentially immortal cells, on division, generate with a fixed probability cells programmed to become senescent and to die after a certain number of divisions. Between commitment and senescence, the uncommitted and therefore potentially immortal cells are diluted by committed cells and lost during subculturing, so that the finite growth of cultured diploid fibroblasts may be an artifact of tissue culturing techniques. Hayflick4 states that with individual variation, human physiologic functions decline at about 0.8 to 0.9% each year after age 30, and that the death of cell lines after about 50 population doublings is a property of the cells themselves, "programmed" into their original pool of genetic information and developed in orderly sequence just as are other developmental changes. Hayflick provides striking confirmation of this finite possible Bull. N.Y. Acad. Med.
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number of doublings in that fibroblasts cultured from patients with progeria (Hutchinson-Gilford's syndrome) went through only two to 10 doublings before death, when the normal would have between 20 and 40 doublings. However, Goldstein et al.7 compared skin fibroblast cultures from patients with clinically apparent or a genetic predisposition to diabetes mellitus, either of which reduces life expectancy, and from carefully defined normal subjects, and conclude that the physiologic status of the tissue donor is a more precise determinant of fibroblast replicative lifespan than is his chronologic age. HORMONAL INFLUENCES Rasmussen and Bordier8 state that hormonal and ionic factors contribute to modulation of osteocytes to bone-resorbing and bone-forming cells. Ordinarily, these osteocytic functional fluctuations affect only the tiny volumes of lacunar and canalicular bone surrounding the osteocyte and its extensions. These particular compartments of metabolically labile bone ordinarily surround the rather small percentage of the whole number of osteocytes in an area undergoing osteocytic osteolysis.9 Courpron et al.10 argue for an individual bony personality (personnalite osseuse individuelle) and that bone aging involves a parallel involution of both the bone and the hematopoietic cell lines. Dequeker2 reports that estrogens inhibit bone resorption, possibly by reducing the parathormone effect on bone, and suggests that postmenopausal increases in serum calcium levels and alkaline phosphatase activity arise from reduction of estrogenic protection against the boneresorbing action of parathormone, which would also explain the increased serum calcium levels that occur among men past 65. Laitinen'1 observes that estrogen inhibits collagenase activity in the uterus and further affects the stability and structure of bone collagen, so that it may be assumed that estrogen-induced decreases in the rate of bone resorption could be mediated by a similar mechanism. Lindsay et al.12 studied a group of castrated women, and observed a significant inverse relation between their fasting urinary hydroxyproline/creatinine ratios and plasma levels of circulating endogenous estradiol, concluding that circulating estrogen levels determine the rate of bone turnover described by this inverse correlation with urinary hydroxyproline secretion. Lindsay et al. also conclude that renal handling of phosphate ions, expressed as TmPO4Iglomerular filtration rate, depends on the endogenous estradiol level and further supports their conclusion that Vol. 55, No. 8, September 1979
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plasma estradiol levels and the rate of bone loss are inversely related. They suggest that the estradiol identified in the plasma of castrated women is largely synthesized by conversion of adrenal androstenedione in peripheral body fat. Monnier et al.13 conclude from isotopic studies of a diabetic population that age by itself does not influence the intestinal absorption of dietary calcium, but that episodes of ketosis exaggerate both intestinal absorption and urinary excretion of calcium. Marked glycosuria in a nonketotic diabetic patient reduced urinary calcium excretion by enhancing tubular calcium resorption. They conclude that no real difference exists in calcium absorption and its incorporation into bone mineral between diabetic and control groups, and that patients with diabetes mellitus are essentially at no greater risk of osteoporosis than those without diabetes. Agreement seems general14 that patients with clinical osteoporosis probably have a greater than normal rate of bone loss and may have begun life with a smaller bone mass during youth, but Laitinen" confesses that present knowledge does not provide sufficient data to define the extent to which the physiological hormonal changes of advancing age modify osteoporotic changes, although the decline in serum estrogen levels following the menopause is probably pathogenetic. He suggests that relative hypercorticosteroidism may also be important, and that changes in the concentrations of several hormones and in their action on target tissues do influence the progression and extent of senile osteoporosis. OSTEOCLASTS, OSTEOBLASTS, AND OSTEOCYTES Normally, new bone is deposited only at the sites of previous bone resorption. When calcitonin is administered to young rabbits, the number of multinucleated osteoclasts present on bone resorption surfaces decreases within 15 minutes. They are replaced by mononucleated cells that seem to be preosteoblasts and give rise to osteoblasts, so that the osteoclasts did not die but modulated into osteoblasts. The osteoblasts and preosteoblasts did not undergo mitotic division.8 Harold Frost's brilliant studies'5"6 have defined a "normal sequence" of events during endosteal bone remodeling: activation of a new group of cells, resorption of bone by the daughters of these cells, and sequential formation of new bone at the site of previous resorption. Osteoclasts and osteoblasts are not, therefore, distinct cell pools but represent differing functional states of the same cells:
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Flanagan and Nichols17 estimated the cellularity of human bone samples using DNA content as an index, and observed that cortical bone contained approximately one quarter as many cells as did mixed cortical and trabecular bone, with a clear reduction in the number of cells with advancing age. They regard the number rather than the relative metabolic activity of bone cells as the most important factor controlling the overall metabolic rate of bone in any particular part of the normal skeleton. The metabolic rates of bone samples from a variety of ages and skeletal sites were closely similar when calculated on the basis of sample DNA content despite variation in cell numbers. Osteopenic bone typically has no more osteoclasts than normal, but does have increased resorptive surfaces and decreased bone-forming surfaces. Osteoprogenitor cells of older people, however, form osteoclasts with fewer than the normal number of nuclei so that after fission, modulation may be delayed or some of the modulated cells may fail to become functional osteoblasts. Inactive or arrested resorption surface increases as the area of active bone-forming surface decreases and, for these reasons, Rasmussen and Bordier8 attribute osteoporosis to osteoblast failure. The percentage of Howship's lacunae which contain osteoclasts decreases up to the fifth decade and slightly increases thereafter, and the percentage of empty osteoclastic lacunae is minimal between ages 30 and 40.18 Delling describes certain age-related changes on the inner surface of bone: after the fifth decade, the percentage of large and presumably bone-resorbing osteocytes rises while that of small and presumably bone-forming osteocytes falls, as empty osteocyte lacunae increase in number with advancing years.18 Melsen et al. found that the mean size of periosteocytic lacunae is not affected by age.19 BONE COLLAGEN
Laitinen1' states that about three quarters of the total volume of bone is organic matrix, more than 95% of the fat-free organic material is collagen,
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and the remainder is mucopolysaccharides and mucoproteins. Schmidt, Kalbe, and SielafF20 observe that with advancing age, soluble bonecollagen fractions and metabolism decrease only slightly and that the collagen half-life increases proportionately. Both the insoluble bone collagen half-life and the insoluble share of the total bone collagen increase, but its metabolic activity decreases. This shift in the proportions of soluble and insoluble bone collagen and increased half-lives are apparently associated with collagen fiber interlacing through cross-linkage and hardening, and corresponds to what is known of other connective tissue collagens with aging. Bone collagen is not inert even in old age, but with aging, insoluble collagen is withdrawn from resorption and therefore from metabolic turnover. The elimination of hydroxyproline, and especially of free hydroxyproline, is reduced by about half from the third to the seventh decades and is related to reduction in the soluble collagen fraction. Whether apolarly attacking collagen peptidases also undergo a decrease with age is not known. Fujii, Kuboki, and Sasaki2I report a marked association with age in the decrease in dihydroxylysinonorleucine and hydroxylysinonorleucine with significantly increased lysinonorleucine in both bone and articular cartilage collagen. Simultaneously, the collagen crosslink precursors hexitollysine and dihydroxynorleucine increased. They suggest that both collagens become less soluble with age through in vivo reduction of crosslinks or transformation to unknown forms of nonreducible linkages. Grant and Prockop22 suggest that complete cross-linking of collagen may require months or years, and that the duration of this process and stiffening of connective tissues with age raise the possibility that increased cross-linking of collagen may be critical in aging, although no evidence suggests that inhibition of this cross-linking retards the effects of aging. Dequeker2 concurs that with advancing age no striking qualitative changes have been found in the collagen or mineral composition of bone, although collagen saline solubility decreases with age, probably because of the increased number of collagen crosslinks. Laitinen1' describes a labile portion of recently synthesized bone collagen that loses its metabolic activity rapidly with advancing age, and states that the rate at which collagen matures also declines with age, a decrease most pronounced in recently formed bone. Laitinen summarizes the steps in the metabolism of collagen that are affected by aging as: the rate of both synthesis and catabolism of collagen decreases with age, a general change Bull. N.Y. Acad. Med.
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most pronounced in bone; the maturation of collagen through the synthesis of the inter- and intramolecular crosslinks of the triple helical tropocollagen molecules slows; the formation, of stable "mature" collagen is retarded; and, despite the decreased rate of collagen maturation, the number of inter- and intramolecular collagen crosslinks increases with age. He suggests that increased numbers of collagen crosslinks may explain the decrease in the rate of collagen catabolism with aging. Laitinen11 suggests that both experimental and clinical disturbances in calcium metabolism cause changes in skeletal metabolism that seem to be initiated and mediated largely through changes in the collagen matrix of bone. He reports that in both experimental hyperthyroidism and hyperparathyroidism, the rate whereby the collagen matrix of bone is degraded acts as a moderator or "final messenger" in hormone-induced bone resorption.
GROUND SUBSTANCE
Schmidt, Kalbe, and Sielaff20 report that proteoglycane synthesis in the ground substance of bone is reduced with age apace generally reduced rates of bone-protein synthesis and rapidly decreased sulphate radical incorporation rate. With aging, the hexosamine and uronic acid content and hexosamine-hydroxyproline ratios decrease, proteoglycane metabolic activity decreases, and hydroxyproline content remains the same or very slightly increases. Not only do the metabolic rates of osteoblasts and osteoclasts decline with age, but deposition of hydroxyapatite in the collagenous matrix of bone depends largely on the chemical state of the interstitial substance, especially its proteoglycane content and pattern. These changes lead to a bone matrix that is inherently less and less mineralizable. Hayflick4 describes a physiological decrease with age in normal human fibroblasts in vitro in mucopolysaccharide synthesis, collagen synthesis, collagen synthesis and collagenolytic activity, alkaline phosphatase production, and the number of cells in the proliferating pool.
OSTEOID Ultrastructural examination of the layer of bone matrix between the osteoblasts and the mineralized bone, called osteoid, demonstrates that osteoid is a structured sequential modification of collagen fibrils and mucopolysaccharides that undergoes modification during certain abnormal Vol. 55, No. 8, September 1979
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states.23 Whereas fibroblasts form collagen fibers all around themselves,18 osteoblasts characteristically secrete collagen precursors and ground substance in only one direction, the collagen precursors passing through the osteoblast cell membrane and, external to the osteoblast, polymerizing with the ground substance to yield osteoid. The calcium concentration within the osteoid seam increases from that region of the osteoid nearest the marrow cavity to that nearest the fully mineralized bone, and hydroxyapatite forms along the mineralization front. Inspection of actively forming bone reveals some areas of osteoid covered by large hexagonal or cuboidal osteoblasts, deep to which rapid primary mineralization takes place, but other areas where osteoid is covered by flat, resting osteoblasts and where secondary mineralization occurs, apparently independently of osteoblastic activity. Most cancellous bone surfaces physiologically lack osteoid seams and Howship's lacunae, and constitute a neutral resting surface bordering the marrow space. Whether this resting surface is completely covered by mesenchymal cells is not yet clear, but these flat cells remain connected to osteocyte processes and seem to be important in the regulation of serum calcium and phosphorus levels. The osteocyte's high metabolic activity, which includes regulation of bone mineral and water content and exchange with extracellular fluid, is fostered by interconnections between osteocyte processes, osteoblasts, and osteoclasts.18 The osteocyte lacunar wall, which consists of collagen fibers, ground substance, and bone mineral, approximates about 20 sq.mm. for each cubic mm. of cortical bone, but the canalicular surface containing osteocyte cell processes has an area of about 200 sq.mm. for the same volume. (Some lacunae have a delicate membranous wall, the function and importance of which remain obscure.) Hydroxyapatite crystals cover the collagen fibers in a very regular manner to make up an optimal mechanical and metabolic system. Delling18 reports that the percentage of nonmineralized bone in relation to a unit volume of bone tissue remains nearly constant after the first two decades, and, although the bone surface covered by osteoid increases with age, thickness of the osteoid decreases. (Significantly thickened osteoid seams at any age suggest disturbed bone formation.) These seams cover 10 to 25% of the total bone surface, are minimal between ages 40 and 50, and increase with age. Delling18 notes: "In contrast to the total extent of seams, the percentage of active seams (osteoid with osteoblasts) decreases from 22.4 + 8.1% in the first decade to 0.9 + 0.6% in the eighth." He Bull. N.Y. Acad. Med.
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states that the percentage of inactive to total bone surface remains constant from the 10th to the 90th years, although the absolute value decreases as bone mass diminishes. Melsen et al.19 found no significant sex difference or change with increasing age in osteoid surface area, although the relative osteoid volume decreased slightly but insignificantly with increasing age, a decrease more pronounced as the amount of trabecular bone decreased with increasing age. In contrast to the decrease in active osteoid seams with age, the surface covered by inactive seams (osteoid without osteoblasts) increases from the fifth or sixth decade. Delling18 suggests that this is related to renal arteriosclerosis and reduction in available renal parenchyma, the site of hydroxylation of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D for bone tissue. Hence, the reduction in the quantity of active osteoid and its eventual mineralization may be secondary to the pathologic or physiologic age changes in other organs, just as some individuals seem to suffer a decline in the efficiency of intestinal calcium absorption with age. Melsen et al.'9 agree that the quantity of both trabecular and cortical bone decreases with advancing age in both men and women, but state that the extent, volume, and width of osteoid seams are independent of age. They observe that osteoclastic resorption in cortical bone in men increased with advancing age.
COMPACT BONE
Kerley24 describes fairly consistent changes in the shafts of long bones associated with advancing age, and most marked in the inner third of the cortex, the outer third being least affected. Description of these changes requires certain definitions. Osteons are vascular channels surrounded by concentric lamellae containing rather evenly spaced osteocytes and bordered by a peripheral growth reversal line. Fragments are portions of old osteons that may surround the peripheries of newer osteons. The number of osteon fragments increases as more and more osteons are laid down until, in extreme old age, most of the complete osteons are surrounded by fragments of older osteons. Circumferential lamellar bone consists of evenly spaced, generally parallel or concentric bone lamellae in the outer subperiosteal portion of the cortex, and its collagenous matrix consists of long, parallel, fairly uniform fibers. Nonhaversian canals contain primary vascular channels formed by incorporation of small, peripheral, originally Vol. 55, No. 8, September 1979
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periosteal vessels into the bone deposited as the cortex expands. These have no osteonal pattern, lack growth-reversal lines, and represent unremodeled bone, just as osteonal bone is formed by osteoclastic remodeling. At the tissue level, bone remodeling is continuous.15'25 Within lamellar bone, cutting cones produce cavities of the order of 200 micra in diameter and as much as several millimeters long. This "cutting head" of osteoclasts is followed by a fibrocapillary leash, and the cavity cut by the osteoclasts is filled in centripetally by osteoblasts which arise from the capillary perithelial cells or are modulated in situ to produce the secondary osteon. Osteoblasts deposit the bone of secondary osteons in a definite, organized pattern wherein the collagen matrix fibers change course in each layer, again leading to maximum flexibility and physical strength. Space between these secondary osteons is occupied by interstitial lamellar bone, and if this interstitial bone cannot be adequately nourished by the canaliculi, it dies. The relative proportion of dead to living interstitial bone increases steadily with age. Kerley24 describes a life cycle for cortical bone. The number of osteons increases from birth to old age, the proportion of circumferential bone decreases with age, and nonhaversian canals disappear during the sixth decade, indicating a very radical decrease in subperiosteal bone deposition by that time. The earliest phase of bone deposition is of radial spicules of coarsely woven bone with irregularly arranged collagen fibers having no particular relation to the large vascular spaces which they enclose. Later, the vascular spaces are occupied by new bone apposited on their walls to become the primary nonhaversian osteons.26 As the cortex and medulla expand, small subperiosteal blood vessels running along the long axis of the bone are incorporated by the expanding peripheral cortex to form nonhaversian canals. Finally, osteoclastic cuffing cones form secondary osteons that are typically themselves partially destroyed by subsequent remodeling. Pankovich, Simmons, and Kulkarni27 studied microradiographs of rib sections from patients eight to 80 years old, and found changes in the proportions of osteons with low, intermediate, and high mineral density as the skeleton aged. They describe an osteon characteristic of old age as having low to intermediate mineral density, wide haversian canals, and hypercalcified borders. They note a trend toward increasing percentages of osteons with incomplete growth-arrest lines in older patients, and suggest that intraosteonal remodeling and growth retardation play increasingly Bull. N.Y. Acad. Med.
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important roles in bone turnover in the aged skeleton, and contribute to the increased porosity and lower mean osteonal mineral density characteristic of old age. Intraosteonal remodeling may preferentially affect older osteons because the oldest peripheral lamellae are typically highly mineralized and this process, which does not seem to affect interstitial bone, appears to be a mechanism whereby osteon mineral density is reduced and skeletal porosity increased among the aged. Trotter and Hixon28 examined the weight, density, and percentage ashed weight of 426 dry, fat-free bony skeletons of both sexes, black and white, from the 16th week of gestation to 100 years old, and found a gradual loss in bone weight and density (but not volume) that began during the fourth decade, and averaged 15.6 grams yearly. Change in weight was accompanied by change in ashed weight. Race and sex differences emerged by the second decade: black skeletons exceeded white, and male skeletons exceeded female skeletons in mean weight and density and, although to a smaller extent, in percentage ashed weight. Smith, Khairi, and Johnston,29 by longitudinal studies, documented that individuals lose bone mineral at unequal rates with age. Bone mineral content, using the radius as a standard, had a normal statistical distribution at all ages, variation from the norm did not increase with age, and because no bimodal distribution emerged from their data, a distinct population of "rapid bone losers" (which exists, according to some workers) could not be identified. Hence, the rate of individual bone mineral loss is not constant, or the rate of bone mineral loss is proportionate to the amount of mineral present at maturity, or both. They suggest that the bone mineral content of patients identified as osteoporotic falls at the lower end of a normal curve, probably because they have had a low bone mineral content since maturity. BONE Loss
Dequeker2 summarizes the problem of skeletal aging as decreased skeletal weight added to decreased physical density (weight/volume) of individual bones. Similar changes occur in paleopathologic material, so this is not a modern phenomenon. Although good statistical relations exist between changes in the peripheral and axial skeletons, changes in one particular bone do not accurately predict changes in another. It has been known forever that old people have more fragile bones than young people, and Dequeker suggests that bone strength, which measures compliance Vol. 55, No. 8, September 1979
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and bone brittleness, which measures breakability, may be different things, and that old people may have "bad bone" in terms of brittleness. During adult life, bone is gained or lost at one or any combination of four sites: periosteal surfaces, surfaces of vascular channels within cortical bone, cortical endosteal surfaces, and trabecular endosteal surfaces. Even after bones stop growing in length, cancellous and cortical bones undergo constant resorption of existing bone and deposition of new bone. Changes in this balance underlie every disease affecting the skeleton. Periosteal bone growth is not merely compensation for endosteal bone loss, because subperiosteal deposition in old age fails to keep pace with subendosteal resorption, nor is continuing bone deposition invariably related to weightbearing or flexion stress. Loss of bone after the fifth decade arises from three processes: slightly increased osteoclasia which reduces the amount of cancellous bone, although increases in resorption surfaces are distinct only among women 50 to 59;19 simultaneous decrease of osteoblastic activity which accentuates this rarefaction by decreased formation of new bone; and delayed mineralization of inactive osteoid surfaces. (Some osteoblasts thicken main bone trabeculae in a form of compensatory hypertrophy without, however, affecting the net process.) Even in physiological osteoporosis,11 bone mineral metabolism itself remains within normal limits or is only slightly decreased. The total bone tissue, excluding marrow, constitutes about 20% of cancellous bone, a proportion which is fairly constant until about the fifth decade, after which loss of bone mass occurs as rarefaction of bone is accompanied by progressive encroachment of marrow spaces filled by fat and nests of hematopoietic marrow.18'19 Dequeker2 argues that the endosteal cavity expands in response to endosteal bone loss, and that this process accelerates after 50 as endosteal surfaces enlarge faster than the periosteal surfaces. Melsen et al.19 describe the loss of cortical bone as a decrease in mean cortical width and, for women, an increase in cortical
porosity. Because inactivity so quickly leads to osteoporosis, Dunnill, Anderson, and Whitehead30 studied changes in the bones of patients of various ages who died in accidents, and found that the proportion of bone occupied by osseous tissue decreased steadily with age as, with advancing years, fatty marrow steadily increased to replace both bone and hematopoietic marrow. They agree that an overlap exists between the ordinarily increased porosity Bull. N.Y. Acad. Med.
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of skeletal senescence and osteoporosis. Takahashi and Frost31 found that women begin life with significantly less bone than men, primarily because their bones are smaller, and both sexes have maximum amounts of bone between 15 and 25 but have lost about a quarter of this maximum by 60. They could find no correlation between a bone's total cross-section area and that of its cortical component from puberty to the menopause, and suggest that if osteoporosis is related to the menopause, the mechanism is extra-ovarian. Increase in total bone cross-section area occurs after 60, more prominently among women than among men, and, although new bone formation declines to a basal level, it does not cease. The periosteal surfaces are never in negative balance, that is, the space inside the periosteum never shrinks. Smith, Khairi, Norton, and Johnston32 found that the mean rate of mineral loss is not constant with aging, is slower in the elderly than during earlier postmenopausal years, and that factors other than decreased physical activity are more significant in determining the rates of mineral loss. Gergely et al.33 compared the bone mineral content of 436 old people to that of 198 healthy people between 21 and 50, and found that the bone mineral content of postmenopausal women declined steadily with age but that of men began to decrease only after 70. Bone width and age at menopause seemed to influence bone mineral content, but previous physical activity seemed to have no effect on the bone mineral content of the aged. Laitinen"1 regards decreasing physical activity as only a secondary cause of age-related osteoporosis, although Nokso-Koivisto et al." maintain that bone mineral is lost in some chronic diseases mainly because of physical inactivity. Regression analyses of measurements by Melsen et al.19 document significant falls in the quantity of bone with increasing age for both sexes and that the quantity of trabecular bone among the youngest group was higher in women than in men, the more severe bone loss by women between ages 50 and 59 resulting in equalization of amounts of trabecular bone among both sexes in old age. Dequeker et al. 14 suggest that heterogeneity between persons explains variations in bone loss, but that errors in measurement can account for considerable variation, and many individuals maintain essentially normal trabecular structure until the eighth decade. Rasmussen and Bordier8 state that slow but progressive bone loss occurs throughout life although transverse diameters of bones increase from a net Vol. 55, No. 8, September 1979
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positive balance of subperiosteal and a net negative balance of subendosteal bone. From 20 to 50 or 55, women lose bone slightly more rapidly than men, but undergo an abrupt loss of skeletal mass between 50 or 55 and 65, after which the rate of bone loss again stabilizes.14 LaitinentI holds that under normal circumstances the breakdown of the organic collagen matrix is the chief factor regulating calcium resorption from bone, and that other factors such as exchange of mineral substances within the hydroxyapatite crystals play only a minor role in the process. Current views as to the pathophysiology of bone loss may be summarized2'" as a senescent imbalance between bone deposition and bone resorption in the latter direction. Parathormone plays a major, but indirect, part in enhanced bone resorption by changing end organ sensitivity, particularly to changes in the androgenic/estrogenic balance. Currently, dietary calcium deficiency is not considered a major factor in bone loss because human beings seem to adapt quite comfortably to low calcium diets, absent pregnancy, and lactation-which are rarely geriatric problems. However, a peculiar calcium deficiency may arise from primary or secondary hypercalciuria which lowers the serum ionized calcium content and results in increased parathormone levels and, therefore, enhanced bone resorption. Frost", has documented that bone formation and resorption tend to change in the same direction with a variable time lag between the two phases, thus, treatment may reduce resorption, but the organism will then reduce bone formation to match. MECHANICAL EFFECTS
Burstein, Reilly, and Martens35 studied the tension, torsion, and compression responses of bone from a population of patients between 21 and 86, and found no significant differences in the mechanical properties of bone from normal, osteoporotic, and adrenocortiscosteroid-treated individuals. They report that the ultimate strain at the point of fracture consistently decreased 8.4% for each decade, and comment: . . That this decrease with age was seen in both femoral and tibial bone tissue suggests that the ultimate strain reflects a property or quality which depends on the age of the individual at the time that the bone tissue under test was originally deposited. Hence, age effects on ultimate strain are determined by the age of the organism rather than the time that the tissue has been in situ. ...
They attribute the declining mechanical strength of bone to changes in the structure of the bone collagen matrix. Bull. N.Y. Acad. Med.
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Burstein, Zika, Heiple, and Klein36 elegantly tested wet bovine haversian bone as though they were testing the plastic behavior of a metal, and found by sequential decalcification that as bone mineral content decreased, the tension yield point and ultimate stress decreased progressively without changing yield strain or ultimate strain short of complete decalcification. Their findings "are consistent with an elastic-perfectly plastic model for the mineral phase of bone tissue in which the mineral contributes the major portion of the tension yield strength." They suggest that collagen itself plays only a minor role in producing bone's tension yield strength. Thus, when a bone is subjected to tensile loading, there is an initial elastic response in which the load deformation curve is essentially reversible, followed by a plastic-or permanent-deformation of the bone. Under tensile stress, then, the bone mineral deforms in a plastic fashion and acts as a metal, not as though it were a synthetic plastic composite. Mineralized bone31; develops numerous tiny cracks and dislocations once a critical mechanical stress is reached and, during stress-induced plastic deformation, these cracks propagate a limited distance in the bone until they are interrupted. Added deformation causes additional cracks or dislocations, and once the strain reaches the point that structural interruption no longer can stop the propagation of these microfractures, a 'catastrophic crack' spreads and the tissue fails. Weaver and Chalmers37 studied changes in bone strength with aging, and found that most of the fractures which increase with age occur in bones wherein trabecular bone provides a significant portion of support, and that the compression strength of cancellous bone is closely enough related to bone mineral content that compression failure strength proves a reliable function of mineral content regardless of age or sex. Bone strength and mineral content diminish steadily with age. Before age 45 or 50 there is no sex difference, but because bone loss begins earlier and is more profound in women than in men, by the seventh decade women's vertebral bone ashed weight is 20% less than men's. (Activity, of course, influences cancellous bone strength and mineral content, an influence exerted through the orientation and distribution of trabeculae.) Nokso-Koivisto et al.34 found good correlation between bone strength, age, and bone mineral density, and tested the force required to break bone trabeculae, concluding that trabecular bone strength averaged 15% higher in men than in women.
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SUMMARY
The human skeleton steadily changes structure and mass during life because of a variety of internal and external factors. Extracellular substance and bone cells get old, characteristic structural remodeling occurs with age, and these age-related changes are important in the discrimination between pathological and physiological changes during old age. Perhaps 20% of the bone mass is lost between the fourth and the ninth decades, osteoblasts function less efficiently, and gradual loss of bone substance is enhanced by delayed mineralization of an increased surface area of thin and relatively less active osteoid seams. After the fifth decade, osteoclasia and the number of Howship's lacunae increase and, with age, the number of large osteolytic osteocytes increases as the number of small osteocytes declines and empty osteocyte lacunae become more common. The result is greater liability to fracture and diminished healing or replacement of injured bone. REFERENCES 1. Putschar, W. G. J.: General Pathology of the Musculo-Skeletal System. In: Handbuch der Allgemeinen Pathologie, Bichner, F., Letterer, E., and Roulet, F., editors. Berlin, Springer-Verlag, 1960, vol. III.2, pp. 359-488. 2. Dequeker, J.: Bone and aging. Ann. Rheum, Dis. 34:100-15, 1975. 3. Martin, G. M.: Cellular aging-Clonal senescence. Am. J. Pathol. 89:484-511, 1977. 4. Hayflick, L.: The cell biology of human aging. N. Engl. J. Med. 295:1302-08, 1976. 5. Holliday, R., Huschtscha, L.I., Tarrant, G.M., and Kirkwood, T.B.L.: Testing the commitment theory of cellular aging. Science 198:366-72, 1977. 6. Ageing at the cellular level (editoral). Lancet /:79, 1978. 7. Goldstein, S., Moerman, E. J., Soeldner, J. S., et al.: Chronologic and physiologic age affect replicative lifespan offibroblasts from diabetic, prediabetic and normal donors. Science 199:781-82, 1978. 8. Rasmussen, H. and Bordier, P.: The cellular basis of metabolic bone disease. N.
Engl. J. Med. 289:25-32, 1973. 9. Bohatirchuk, F.: Calciolysis as the initial stage of bone resorption. Am. J. Med. 4/:836-46, 1966. 10. Courpron, P., Meunier, P., Vignon, G., et al.: Donnees Histologiques quantitives sur le Vieillissement osseus humain. Lyon MMdi. 226:755-66, 1971. 11. Laitinen, O.: Relation to osteoporosis of age- and hormone-induced changes in the metabolism of collagen and bone. Isr. J. Med. Sci. /2:620-37, 1976. 12. Lindsay, R., Hart, D. M., Sweeney, A., et al.: Endogenous oestrogen and bone loss following oophorectomy. Calcif. Tiss. Res. 22:213-16, 1977. 13. Monnier, L., Chevallet, M., Huh, K. B., et al.: Etude de la Densit6 et de 1' Absorption intestinale du Calcium en Fonction de 1'Age et du Sexe par des Methodes isotopiques. Probl. Actuels Endocrinol. Nutr. 20:307-16, 1976. 14. Dequeker, J., Burssens, A., Creytens, G., and Bouillon, R.: Ageing of bone-Its relation to osteoporosis and osteoarthrosis in post-menopausal women. Front. Horm. Res. 3:116-30, 1975. 15. Frost, H. M.: BoneRemodelingDynamBull. N.Y. Acad. Med.
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ics. Springfield, Ill., Thomas, 1963. 16. Frost, H. M.: Metabolism of bone. N. Engl. J. Med. 289:864-65, 1973. 17. Flanagan, B. and Nichols, G., Jr.: Metabolic studies of human bone in vitro. I. Normal bone. J. Clin. Invest. 44:178894, 1965. 18. Delling, G.: Age-related bone changes. Curr. Top. Pathol. 58:117-47, 1973. 19. Melsen, F., Melsen, B., Mosekilde, L., and Bergmann, S.: Histomorphometric analysis of normal bone from the iliac crest. Acta Path. Microbiol. Scand., Sect. A, 86:70-81, 1978. 20. Schmidt, U. J., Kalbe, I., and Sielaff, F.: Bone Aging. In: Cell Impairment in Aging and Development, Cristofalo, V. J. and Holeckovi, E., editors. New York and London, Plenum, 1975, pp. 371-74. (Adv. Exp. Med. Biol. 53:37174, 1975.) 21. Fujii, K., Kuboki, Y., and Sasaki, S.: Aging of human bone and articular cartilage collagen-Changes in the reducible crosslinks and their precursors. Calcif. Tiss. Res. 15:165-66, 1974. 22. Grant, M. E. and Prockop, D. J.: The biosynthesis of collagen. N. Engl. J. Med. 286:194-99, 242-49, 291-99, 1972. 23. Fornasier, V. L.: Osteoid-An ultrastructural study. Human Pathol. 8:24354, 1977. 24. Kerley, E. R.: The microscopic determination of age in human bone. Am. J. Phys. Anthropol. 23:149-64, 1965. 25. Johnson, L. C.: Morphologic Analysis in Pathology-The Kinetics of Disease and General Biology of Bone. In: Bone Biodynamics, Frost, H. M., editor. Boston, Little Brown, 1964, pp. 543-654. 26. Smith, J. W.: The arrangement of collagen fibers in human secondary osteons. J. Bone Joint Surg. 42B:588-605, 1960. 27. Pankovich, A. M., Simmons, D. J., and Kulkarni, V. V: Zonal osteons in cortical
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bone. Clin. Orthop. /00:356-63, 1974. 28. Trotter, M. and Hixon, B. B.: Sequential changes in weight, density and percentage ash weight of human skeletons from an early fetal period through old age. Anat. Record /79:1-18, 1974. 29. Smith, D. M., Khairi, M. R. A., and Johnston, C. C., Jr.: The loss of bone mineral with aging and its relationship to risk of fracture. J. Clin. Invest. 56:31 118, 1975. 30. Dunnill, M. S., Anderson, J. A., and Whitehead, R.: Quantitative histological studies on age changes in bone. J. Path. Bact. 94:275-91, 1967. 31. Takahashi, H. and Frost, H. M.: Age and sex related changes in the amount of cortex of normal human ribs. Acta Orthop. Scand. 37:122-30, 1966. 32. Smith, D. M., Khairi, M. R. A., Norton, J., and Johnston, C. C., Jr.: Age and activity effects on rate of bone mineral loss. J. Clin. Invest. 58:716-21, 1976. 33. Gergely, I., Krasznai, I., Horvith, T., et al.: Bone mineral content of the healthy aged. Aktuel. Gerontol. 8:109-12, 1978. 34. Nokso-Koivisto, V. -M., Alhava, E. M., and Olkkonen, H.: Measurement of cancellous bone strength correlations with mineral density, ageing and disease. Ann. Clin. Res. 8:399-402,1976. 35. Burstein, A. H., Reilly, D. T., and Martens, M.: Aging of bone tissueMechanical properties. J. Bone Joint Surg. 58A:82-86, 1976. 36. Burstein, A. H., Zika, J. M., Heiple, K. G., and Klein, L.: Contribution of collagen and mineral to the elastic-plastic properties of bone. J. Bone Joint Surg. 57A:956-61, 1975. 37. Weaver, J. K. and Chalmers, J.: Cancellous bone-Its strength and changes with aging. J. Bone Joint Surg. 48A:289-308. 1966.
