Jean E. Aaron

The microanatomy of trabecular bone loss in normal aging men and women

Histomorphometric changes in normal bone were investigated using tissue from the ilium of 86 women and 98 men, aged 20-90 years. While loss of trabecular volume was common to both sexes, the histologic basis for the loss differed. Decreased formation (expressed primarily as static indices) seemed to be the principal factor in bone loss in men; in women these features remained unchanged, suggesting that increased resorption was the principal factor. Bone loss in women was principally attributable to the total removal of individual trabeculae; conversely in men, there was generalized attenuation of trabecular bone. Similar findings in bone tissues of vertebral bodies suggest that differences in bone remodelling between the sexes produce different patterns of bone loss as a sequence of aging.

Spinal osteoporosis in men

In 94 men with crush fracture, 40 were found to have primary osteoporosis. Cross-sectional measurements of a number of variables related to bone in these 40 patients were compared to the values in various groups of healthy men aged 20-96. In healthy men, metacarpal and femoral cortical area/total area, bone volume, osteoid surfaces, seam and trabecular width, plasma dihydroepiandrosterone and estrone, and radiocalcium absorption fell with age, whereas eroded surfaces, trabecular number, urine hydroxyproline and calcium/creatinine ratios, plasma alkaline phosphatase, estradiol, androstenedione, cortisol and testosterone remained constant with age. As compared with healthy men, men with primary osteoporosis had reduced femoral cortical area/total area (P less than 0.05), and Singh grade (P less than 0.001) and in seven there was a history of forearm or femoral fracture. On iliac crest biopsy, bone volume (P less than 0.001) and trabecular number (P less than 0.01) were decreased. Plasma alkaline phosphatase (P less than 0.02) was increased but urine hydroxyproline and calcium excretion were not significantly raised. Calcium balance was negative due to failure of absorption to match urinary calcium loss and radiocalcium absorption (P less than 0.01) and plasma 1,25-dihydroxyvitamin D (P less than 0.05) were reduced.

Bone histology and mineral homeostasis in human pregnancy

Summary. Mineral homeostasis was studied biochemically and histologically in patients in early pregnancy and at term. In early pregnancy there was evidence of increased and reversible resorption of bone, whereas in late pregnancy bone demonstrated active formation and rapid mineralization with minimal resorption. Gut absorption of calcium was not increased in early pregnancy. The overall findings were consistent with calcium liberation from bone in early pregnancy, and with enhanced conservation of bone calcium at term. It is proposed that the additional calcium required during pregnancy is derived largely from the skeleton during early gestation and from dietary absorption at term.

An automated method for the analysis of bone structure

Abstract Trabecular structure as well as bone mass is important in studies of bone disease and fracture. An automated method for the direct analysis of two-dimensional trabecular microanatomy and its application to human iliac crest bone biopsies is described. Compared with established methods which require expensive equipment and complex software, costs have been reduced and availability increased by using an image analyzer driven by a microcomputer. Routine histological sections are accepted and an editing function enables the removal of artifacts. An elastic window allows field expansion for large specimens. The program enables the rapid assessment of the bone volume and trabecular surface from the intact image, followed by image skeletonization and the deduction of the trabecular length, number, character, and spacing together with the number of trabecular junctions and discontinuities; the trabecular width is calculated indirectly. Images may be stored to disk or printed as permanent records for diagnostic or research purposes.

Changes in trabecular bone architecture in women during pregnancy

Objective To examine the effect of early and late pregnancy on the microarchitecture of maternal cancellous bone.

Intramembranous trabecular generation in normal bone.

The ability of trabeculae to reform following localized ablation may provide further insight into the sequence of events in cancellous regeneration. Histological features of cancellous repair were examined in the iliac crest of aged female sheep at intervals after removal of a 1-cm diameter biopsy. Comparison was made with normal intramembranous trabecular formation in the foetal lamb. The first immature trabeculae to form in the defects within 3 weeks were exclusively intramembranous, not endochondral, and the systematic process was indistinguishable from that in the intact growing foetal lamb. In both the young and old skeleton, two features were prominent. First, the damaged endosteum of the sheep functioned like the intact periosteum of the lamb to produce orderly migrating arrays of discrete coarse collagenous fibres, 5-25 microns thick, which penetrated the surrounding soft tissues to form a polarised preliminary framework. Without this structure, primary trabecular development did not take place. Throughout subsequent bone apposition the preliminary framework, which bonded hard to soft tissues and new bone to old, remained largely unmineralised. Second, intratrabecular resorption channels divided the established, thickened primary bars into networks of mature secondary trabeculae. It is concluded that the two features are central and universal to trabecular proliferation and may provide a morphological basis for future trabecular restitution of the depleted elderly skeleton.

Periosteal Sharpey’s fibers: a novel bone matrix regulatory system?

Sharpey’s “perforating” fibers (SF) are well known skeletally in tooth anchorage. Elsewhere they provide anchorage for the periosteum and are less well documented. Immunohistochemistry has transformed their potential significance by identifying their collagen type III (CIII) content and enabling their mapping in domains as permeating arrays of fibers (5–25 μ thick), protected from osteoclastic resorption by their poor mineralization. As periosteal extensions they are crucial in early skeletal development and central to intramembranous bone healing, providing unique microanatomical avenues for musculoskeletal exchange, their composition (e.g., collagen type VI, elastin, tenascin) combined with a multiaxial pattern of insertion suggesting a role more complex than attachment alone would justify. A proportion permeate the cortex to the endosteum (and beyond), fusing into a CIII-rich osteoid layer (<2 μ thick) encompassing all resting surfaces, and with which they apparently integrate into a PERIOSTEAL-SHARPEY FIBER-ENDOSTEUM (PSE) structural continuum. This intraosseous system behaves in favor of bone loss or gain depending upon extraneous stimuli (i.e., like Frost’s hypothetical “mechanostat”). Thus, the birefringent fibers are sensitive to humoral factors (e.g., estrogen causes retraction, rat femur model), physical activity (e.g., running causes expansion, rat model), aging (e.g., causes fragmentation, pig mandible model), and pathology (e.g., atrophied in osteoporosis, hypertrophied in osteoarthritis, human proximal femur), and with encroaching mineral particles hardening the usually soft parts. In this way the unobtrusive periosteal SF network may regulate bone status, perhaps even contributing to predictable “hotspots” of trabecular disconnection, particularly at sites of tension prone to fatigue, and with the network deteriorating significantly before bone matrix loss.

THE IMPACT OF MAMMALIAN REPRODUCTION ON CANCELLOUS BONE ARCHITECTURE

Pregnancy and lactation make demands on maternal calcium homeostasis which may affect bone strength. Recently, changes in cancellous architecture have been described in iliac crest bone biopsies from normal pregnant women but the rarity of such human material means an animal model is essential. The microanatomy of cancellous bone was compared in uniparous and multiparous rats using undecalcified histological sections of lumbar and caudal vertebrae and also proximal femora. An automated trabecular analysis system (TAS) measured a comprehensive range of structural variables including the trabecular number, connectivity and width. In the first pregnancy cycle an early stimulation of bone formation (which quadrupled at some sites) was indicated by an increase in the skeletal uptake and spacing of double calcein labels and the immediate generation of thicker more numerous and interconnected trabeculae. A 40% increase in cancellous bone volume was observed in the lumbar spine in comparison with age‐matched virgin controls. In contrast, a rapid succession of 3 pregnancy cycles (including lactation) culminated in cancellous atrophy of 15% at the same site, with a loss in trabecular number ranging from 20% (caudal vertebra) to 30% (lumbar vertebrae). In comparison, the proximal femur lost 40% of its struts but, nevertheless, uniquely sustained its cancellous bone volume. When lactation was excluded the number of struts lost was halved although trabecular thinning then took place which was sufficient to maintain the previous 15% deficit in bone volume. It was concluded that a single pregnancy strengthens the cancellous component of the maternal skeleton while a quick succession of pregnancies weakens it. Lactation influences the pattern of bone loss but not its amount.

The ultrastructure of slam-frozen bone mineral

SummaryBone salt may be altered by preparative procedures. ‘Slam’ freezing can usefully be applied to bone mineral because it minimizes preparation and preserves the tissue chemistry. The structure and composition of the mineral in ‘slam’-frozen neonatal mouse calvaria (which require neither previous slicing nor manipulation) was examined by transmission electron microscopy and X-ray microanalysis in unstained sections, 0.25 μm thick (i.e. unusually thick). Comparison was made with fresh intact calvaria and with ‘snap’-frozen histological sections of mature rat femora. Under the optical microscope, calcified microspheres, up to 1 μm in diameter, were evident within ‘young’ osteocytes and within the extracellular matrix of both immature and mature, unstained and von Kossa-stained bone. In the electron microscope, microspheres of similar dimension and distribution were observed after ‘slam’ freezing and were divided into two groups. One group, found inside and outside cells, had a substructure of closely packed, electron-dense, rounded bodies 30–40 nm in diameter; despite their unusual stability in EDTA, X-ray microanalysis indicated high levels of both calcium and phosphorus. The other group was found at the calcification front and, although similar to the first group in size and chemical composition, these microspheres had a substructure of clusters of 5-nm-thick electron-dense filaments containing mineral that was characteristically EDTA labile. The 30- to 40-nm dense bodies did not appear to be mitochondrial and were absent from customary fixed and resin-embedded, ultrathin, stained preparations. They were not observed singly and their aggregation into arrangements of microspheres, sometimes linked by bridges, may be an important preliminary step in the development of the filamentous clusters. Needle-shaped and plate-like crystals of bone mineral were absent. It was concluded that ‘slam’ freezing preserves both intracellular and extracellular bone salt in the form of microspheres within which the mineral may modulate from dense bodies into filamentous arrays of variable density with maturity

Effect of deproteination on bone mineral morphology: Implications for biomaterials and aging

Bone mineral morphology is altered by processing and this is rarely considered when preparing bone as a bioimplant material. To examine the degree of transformation, a commercial, coarsely particulate bone mineral biomaterial produced by prolonged deproteination, defatting, dehydration, and heating (donor material) was compared with similar particles of human bone (recipient material) prepared optimally by low-temperature milling. The two powders were freeze-substituted and embedded without thawing in Lowicryl K4M before sectioning for transmission electron microscopy (TEM) (other aliquots were processed by traditional TEM methods). To maximize resolution, electron micrographs were image-enhanced by digitization and printed as negatives using a Polaroid Sprint Scan 45. In addition to their morphology, the particles were examined for antigenicity (specific by reference to fluorescein isothiocyanate [FITC]-conjugated fibronectin, and nonspecific by reference to general FITC-conjugated immunoglobulins). Results showed that the optimally prepared human bone fragments stained discretely for fibronectin with negligible background autofluorescence. In contrast, the bioimplant fragments stained extensively with this and any other FITC-conjugated antibody and, unlike fresh bone, it also autofluoresced a uniform yellow. This difference was also expressed structurally and, although the bioimplant mineral consisted of rhomboidal plates up to 200 nm across and 10 nm thick, the optimally prepared bone mineral was composed of numerous clusters of 5-nm-wide sinuous calcified filaments of variable density and indeterminate length (which became straight needles 50 nm long and 5 nm thick following traditional chemical TEM fixation/staining). It was concluded that the inorganic phase of bone is both morphologically and immunologically transmutable and that, in biomaterials, the transformation is apparently so great that a broad indigenous antigenicity is unmasked, increasing the likelihood of resorption or rejection. This marked change may also provide preliminary insight into a more modest natural aging phenomenon with the localized lateral fusion of calcified filaments into less flexible, more immunologically reactive fenestrated plates.