Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.     

Age-Related Changes in Collagen Properties and Mineralization in Cancellous and Cortical Bone in the Porcine Mandibular Condyle

Collagen is an important constituent of bone, and it has been suggested that changes in collagen and mineral properties of bone are interrelated during growth. The aim of this study was to quantify age-related changes in collagen properties and the degree of mineralization of bone (DMB). The DMB in cancellous and cortical bone samples from the mandibular condyle of 35 female pigs aged 0–100 weeks was determined using micro-computed tomography. Subsequently, the amount of collagen and the number of pentosidine (Pen), hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were quantified by means of high-performance liquid chromatography. The amount of collagen increased with age in cancellous bone but remained unchanged in cortical bone. The number of Pen and LP cross-links decreased in both bone types. In contrast, the number of HP cross-links decreased only in cancellous bone. The sum of the number of HP and LP cross-links decreased with age in cancellous bone only. The DMB increased in cancellous and cortical bone. It was concluded that the largest changes in the number of mature collagen cross-links and the mineralization in porcine cancellous and cortical bone take place before the age of 40 weeks. The low number of mature cross-links after this age suggests that the bone turnover rate continues to be high and thereby prevents the development of mature cross-links.     

The Essential Role of Fetuin in the Serum-Induced Calcification of Collagen

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. Our goal is to understand the mechanism of fibril mineralization, and as a first step we recently determined the size exclusion characteristics of the fibril. This study indicates that apatite crystals up to 12 unit cells in size can access the water within the fibril while molecules larger than a 40-kDa protein are excluded. We proposed a novel mechanism for fibril mineralization based on these observations, one that relies exclusively on agents excluded from the fibril. One agent generates crystals outside the fibril, some of which diffuse into the fibril and grow, and the other selectively inhibits crystal growth outside of the fibril. We have tested this mechanism by examining the impact of removing the major serum inhibitor of apatite growth, fetuin, on the serum-induced calcification of collagen. The results of this test show that fetuin determines the location of serum-driven mineralization: in fetuin’s presence, mineral forms only within collagen fibrils; in fetuin’s absence, mineral forms only in solution outside the fibrils. The X-ray diffraction spectrum of serum-induced mineral is comparable to the spectrum of bone crystals. These observations show that serum calcification activity consists of an as yet unidentified agent that generates crystal nuclei, some of which diffuse into the fibril, and fetuin, which favors fibril mineralization by selectively inhibiting the growth of crystals outside the fibril.     

Structural changes in collagen fibrils across a mineralized interface revealed by cryo-TEM

The structure of the mineralized collagen fibril, which is the basic building block of mineralized connective tissues, is critical to its function. We use cryo-TEM to study collagen structure at a well-defined hard–soft tissue interface, across which collagen fibrils are continuous, in order to evaluate changes to collagen upon mineralization. To establish a basis for the analysis of collagen banding, we compared cryo-TEM images of rat-tail tendon collagen to a model based on the X-ray structure. While there is close correspondence of periodicity, differences in band intensity indicate fibril regions with high density but lacking order, providing new insight into collagen fibrillar structure. Across a mineralized interface, we show that mineralization results in an axial contraction of the fibril, concomitant with lateral expansion, and that this contraction occurs only in the more flexible gap region of the fibril. Nevertheless, the major features of the banding pattern are not significantly changed, indicating that the axial arrangement of molecules remains largely intact. These results suggest a mechanism by which collagen fibrils are able to accommodate large amounts of mineral without significant disruption of their molecular packing, leading to synergy of mechanical properties.     

Callus mineralization and maturation are delayed during fracture healing in interleukin-6 knockout mice

IL-6 is a pleiotropic cytokine involved in cell signaling in the musculoskeletal system, but its role in bone healing remains uncertain. The purpose of this study was to examine the role of IL-6 in fracture healing. Eight-week-old male C57BL/6 and IL-6 −/− mice were subjected to transverse, mid-diaphyseal osteotomies on the right femora. Sacrifice time points were 1, 2, 4, or 6 weeks post-fracture (N = 14 per group). Callus tissue properties was analyzed by microcomputed tomography (micro-CT) and Fourier transform infrared imaging spectroscopy (FT-IRIS). Cartilage and collagen content, and osteoclast density were measured histologically. In intact unfractured bone, IL-6 −/− mice had reduced crystallinity, mineral/matrix ratio, tissue mineral density (TMD), and bone volume fraction (BVF) compared to wildtype mice. This suggests that there was an underlying deficit in baseline bone quality in IL-6 −/− mice. At 2 weeks post-fracture, the callus of IL-6 −/− mice had reduced crystallinity and mineral/matrix ratio. These changes were less evident at 4 weeks. At 2 weeks, the callus of the IL-6 −/− mice had an increased tissue mineral density (TMD), an increased cartilage and collagen content, and reduced osteoclast density compared to these parameters in wildtype mice. By 4 and 6 weeks, these parameters were no longer different between the two strains of mice. In conclusion, IL-6 −/− mice had delayed callus maturity, mineralization, and remodeling compared with the callus of the wildtype mice. These effects were transient indicating that the role of IL-6 appears to be most important in the early stages of fracture healing.     

An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone

OBJECTIVE: In articular joints, the forces generated by locomotion are absorbed by the whole of cartilage, subchondral bone and underlying trabecular bone. The objective of this study is to test the hypothesis that regional differences in joint loading are related to clear and interrelated differences in the composition of the extracellular matrix (ECM) of all three weight-bearing constituents. METHOD: Cartilage, subchondral- and trabecular bone samples from two differently loaded sites (site 1, dorsal joint margin; site 2, central area) of the proximal articular surface of 30 macroscopically normal equine first phalanxes were collected. Collagen content, cross-linking (pentosidine, hydroxylysylpyridinoline (HP), lysylpyridinoline (LP)) hydroxylation, and denaturation, as well as glycosaminoglycan (GAG) and DNA content were measured in all three tissues. In addition, bone mineral density (BMD), the percentage of ash and the mineral composition (calcium, magnesium and phosphorus) were determined in the bony samples. RESULTS: For pentosidine cross-links there was an expected correlation with age. Denatured collagen content was significantly higher in cartilage at site 1 than at site 2 and was higher in trabecular bone compared to subchondral bone, with no site differences. There were significant site differences in hydroxylysine (Hyl) concentration and HP cross-links in cartilage that were paralleled in one or both of the bony layers. In subchondral bone there was a positive correlation between total (HP+LP) cross-links and Ca content. For Ca and other minerals there were corresponding site differences in both bony layers. CONCLUSIONS: It is concluded that there are distinct differences in distribution of the major biochemical components over both sites in all three layers. These differences show similar patterns in cartilage, subchondral bone and trabecular bone, stressing the functional unity of these tissues. Overall, differences could be interpreted as adaptations to a considerably higher cumulative loading over time at site 2, requiring stiffer tissue. Turnover is higher in trabecular bone than in subchondral bone. In cartilage, the dorsal site 1 appears to suffer more tissue damage.     

Impaired Bone Formation with a High-Protein Diet in Rats with Adriamycin-Induced Nephrotic Syndrome

Background/Aim: The purpose of the study was to investigate the effects of a high-protein (HP) diet on bone metabolism in rats with adriamycin (ADR)-induced nephrotic syndrome. Methods: Nephrotic syndrome was established by weekly injections of ADR (2 mg/kg, i.p.) for 6 weeks. After a final injection, we confirmed that nephrotic syndrome had developed. Then, the rats were divided into two groups for the dietary treatments, namely the HP diet (30% of calories from protein) and the low-protein (LP) diet (7% of calories from protein), and were fed an isocaloric diet for the following 5 weeks. Results: Urinary protein and phosphate excretion were significantly greater in the HP diet group than in the LP diet group (p < 0.05). Serum parathyroid hormone and osteocalcin levels were significantly higher and lower, respectively, in the HP diet group (p < 0.05). Femur weight, femur mass index and femur calcium contents were significantly lower in the HP diet group than in the LP diet group (p < 0.05). Bone mineral density was significantly lower in the HP diet group than in the LP diet group (p < 0.05); however, bone mineral content did not differ between the two groups. Conclusion: We confirmed that an HP diet negatively affects bone mineral metabolism and bone density in ADR-induced nephrotic syndrome rats.     

Influence of nutritional factors on bone collagen fibrils in ovariectomized rats

The influence of both vitamin D(3) and Ca:P ratio on bone collagen fibrils was investigated in ovariectomized rats. Six weeks after ovariectomy the rats were maintained for 80 days with diets containing vitamin D(3) and calcium supplementation. Age-matched ovariectomized animals were fed a normal diet. When vitamin D(3) was increased in the diet, although no effect in fibril organization was observed in relation to that from ovariectomized rats with the normal diet, a highly significant effect in fibril diameter was detected. When the calcium:phosphorus (Ca:P) ratio was increased from 1:1 to 2:1 (without vitamin D(3) supplementation) both structural fiber parameters were significantly affected. The results were closer to normal (i.e., collagen fibrils from animals without ovariectomy) when vitamin D(3) and Ca:P ratios were combined.     

Large Deformation Mechanisms,Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content

Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom‐up approach. By conserving the three‐dimensional structure and the entanglement of the molecules, we were able to construct finite‐size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ～21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease.     

Structural alterations in rat skin and bone collagen fibrils induced by ovariectomy

In this study, the influence of ovariectomy in rat skin and bone (trabecular and cortical) collagen fibrils is examined using electron microscopy. Structural changes (fibril architecture and diameter) were detected, at the ultrastructural level, in skin and bone specimens from ovariectomized rats. The overall collagen fibril architecture was disturbed as compared with normal animals. Treated collagen fibrils' mean diameter values were significantly smaller than those from controls, in all tissues examined. The banding patterns of fibrils were normal in all cases; however, measurements by a computerized method of measuring axial periodicity of fibrils indicated significantly lower values for treated samples than untreated samples. Our results show a correlation between the effects induced by ovariectomy in skin and bone collagen. But, the question of whether these changes play a role in the pathogenesis of ovarian hormone deficiency in osteoporosis remains to be demonstrated.     

Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross-linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44-88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross-links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross-links, that is, hydroxylysyl-pyridinoline (HP), lysyl-pyridinoline (LP), and pyrrole cross-link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross-link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.     

Changes with age in the urinary excretion of lysyl- and hydroxylysylpyridinoline, two new markers of bone collagen turnover

Two intermolecular cross-linking amino acids, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), are promising markers in urine of collagen resorption because their levels in urine should reflect only collagen resorption and, unlike hydroxyproline, should not be influenced by degradation of either newly synthesized collagen molecules or noncollagenous proteins. Changes with age in the urinary excretion of HP and LP were studied in 24 h collections of urine from a group of 22 male and 27 female healthy subjects aged from 2 to 70 years. The pyridinolines were quantitated, utilizing their natural fluorescence, after resolution by reversed-phase HPLC. Levels of both pyridinolines were higher in children than in adults, but in adults no evidence of age or sex variations were observed except in the 20-30 year age group. Mean values of HP/Cr and LP/Cr in 37 adults (21-70 years) were 27.2 +/- 1.9 and 8.8 +/- 0.8 mumols/mol, respectively; in the 12 children (2-15 years) the mean values were 14.4 and 12.4 times higher than the respective adult values. Making certain assumptions, the mean amount of bone resorbed in normal adults was tentatively estimated at 1.9 g per 24 h. The finding that differences between children and adults in these relatively specific markers were greater than with hydroxyproline suggests that hydroxyproline values may considerably underestimate the actual amount of bone turnover occurring in growing children or overestimate the adult turnover rate.     

Specific Changes of Urinary Excretion of Cross-Linked N-Telopeptides of Type I Collagen in Pre- and Postmenopausal Women: Correlation with Other Markers of Bone Turnover

Urinary excretion of cross-linked N-telopeptide of type I collagen (NTx) has been reported to be a specific marker of bone resorption [18]. We assessed a new immunoassay for NTx as an indicator of changes in bone resorption caused by spontaneous menopause and compared cross-sectionally the levels of urinary NTx, hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), hydroxyproline (OH-Pr), other serum biochemical indices, and lumbar spine and proximal femur bone mineral density (BMD). Eighty-one Japanese women aged 22–77 participated in this study; 36 were premenopausal and 45 were postmenopausal. Urinary HP, LP, and NTx stayed at low levels in the premenopausal period and rose 21%, 30%, and 67% in the postmenopausal period, respectively. The rise in LP and NTx was statistically significant (P < 0.01), suggesting that NTx is mostly released from bone matrix when bone resorption is accelerated. When premenopausal women were divided into two age groups and postmenopausal women were divided into two groups according to years since menopause (YSM) there were significant differences in LP and NTx between women <4 YSM and women aged <40 and those women aged 41+ (P < 0.01 and P < 0.05, respectively). A significant 110% increase in urinary NTx and a 48% increase in urinary LP were observed in postmenopausal women compared with age-matched premenopausal women aged 45–55. All biochemical markers other than serum PTH correlated significantly with each other (r = 0.243–0.858, P < 0.05–0.0001). Urinary NTx inversely correlated with lumbar spine BMD. When postmenopausal women were divided into three groups, the correlation between bone resorption and formation markers in women 0-1 YSM was greater than in women 2–10 YSM and in women 11 + YSM, indicating that resorption and formation are coupled at the early postmenopausal period. We conclude that urinary NTx is responsive to changes in bone metabolism caused by estrogen deficiency and may be a more sensitive and specific marker than HP, LP, or OH-Pr in the early postmenopausal years. Received: 15 February 1995 / Accepted: 18 October 1996     

Detection of Mature Collagen in Human Dental Enamel

Mature dental enamel is the most mineralized of all mammalian tissues and considered to be free of collagen. Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are two nonreducible cross-links of mature collagen. Hydroxyproline (Hyp) is an amino acid that is believed to be indicative of the presence of collagen. We set out to assess the concentrations of Hyp, HP, and LP in dental enamel and dentin (control) to clarify whether there was minor collagen content in dental enamel. We studied 17.53 g of enamel and 22.12 g of dentin gained from 120 extracted human teeth. Enamel and dentin (control) were separated with a diamond dental drill under microscopic control by wasting a margin of enamel (Ca. 2 mm) at the dentin-enamel border. Collagen alpha-chains were analyzed by Sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) after decalcification and collagen extraction. Concentrations of HP and LP where measured by using high-performance liquid chromatography (HPLC). Hyp was analyzed by a spectrophotometric method. The pooled probe of enamel contained 0.23 mug/g of Hyp. This concentration was 49 times lower than that in dentin. Concentrations of HP and LP in enamel were 0.07 nmol/g and 0.02 nmol/g, respectively being 605.57 (HP) and 251.50 (LP) times lower in enamel as compared to dentin. Collagen type I was found in enamel; collagen types I and V were found in dentin samples. In reports of many studies and textbooks, collagen is considered to be completely absorbed in the course of the mineralization and maturation of dental enamel. We show that this is not the case. However, the concentration of collagen in enamel was considerably lower as compared to that in dentin.     

Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.     

Accretion of bone quantity and quality in the developing mouse skeleton.

In this work, we found that bone mineral formation proceeded very rapidly in mice by 1 day of age, where the degree of mineralization, the tissue mineral density, and the mineral crystallinity reached 36%, 51%, and 87% of the adult values, respectively. However, even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of its adult value, indicating that the intrinsic stiffening of the bone lags considerably behind the initial mineral formation. INTRODUCTION: To meet the mechanical challenges during early development, the skeleton requires the rapid accretion of bone quality and bone quantity. Here, we describe early bone development in the mouse skeleton and test the hypothesis that specific compositional properties determine the stiffness of the tissue. MATERIALS AND METHODS: Tibias of female BALB mice were harvested at eight time-points (n = 4 each) distributed between 1 and 40 days of age and subjected to morphometric (muCT), chemical (Fourier transform infrared microspectroscopy), and mechanical (nanoindentation) analyses. Tibias of 450-day-old mice served as fully mineralized control specimens. RESULTS: Bone growth proceeded very rapidly; at 1 day of age, the degree of mineralization (phosphate/protein ratio), the density of mineralized bone (TMD), and mineral crystallinity had reached 36%, 51%, and 87% of the adult (450 days) values, respectively. Spatially, the variability in mineralization across the mid-diaphysis was very high for the early time-points and declined over time. In contrast to the notable changes in mineralization, carbonate substitution into the mineral lattice (carbonate/phosphate ratio) and collagen cross-linking did not show any significant changes over this time period. Even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of the adult value and increased to 89% (of its adult value) after 40 days. Between samples of different time-points, significant positive correlations were observed between the elastic modulus and TMD (r(2) = 0.84), phosphate/protein ratio (r(2) = 0.59), and crystallinity (r(2) = 0.23), whereas collagen cross-linking showed a small but significant negative correlation (r(2) = 0.15). CONCLUSIONS: These data indicate that specific chemical and morphometric properties modulate bone's stiffness during early growth. The intrinsic stiffening of the bone, however, lags considerably behind the initial mineral formation, emphasizing the importance of bone mineral quality for optimizing matrix integrity.     

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy

In the bone, collagen fibrils form a lamellar structure called the “twisted plywood-like model.” Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.     

Callus formation in femur and tibia during leg lengthening:7 patients examined with DXA

We examined the callus formation during leg lengthening in 7 achondroplastic patients who underwent 3 bilateral femoral and 4 bilateral tibial lengthenings. Bone mineral content and bone mineral density (BMD) in the lengthened callus space were evaluated every 1 or 2 weeks for 10 weeks after the start of distraction using dual energy X-ray absorptiometry.

The mean rate of callus mineralization in femurs (0.64 g/wk) was higher than in tibias (0.22 g/wk). The mean BMD at 10 weeks after the start was 0.35 g/ cm² in the femur and 0.14 g/cm² in the tibia. Different rates of callus formation in different kinds of long tubular bones have not been reported previously.     

The loci of mineral in turkey leg tendon as seen by atomic force microscope and electron microscopy

Transmission electron micrographs of fully mineralized turkey leg tendon in cross-section show the ultrastructure to be more complex than has been previously described. The mineral is divided into two regions. Needlelike-appearing crystallites fill the extrafibrillar volume whereas only platelike crystallites are found within the fibrils. When the speciment is tilted through a large angle, some of the needlelike-appearing crystallites are replaced by platelets, suggesting that the needlelike crystallites are platelets viewed on edge. If so, these platelets have their broad face roughly parallel to the fibril surface and thereby the fibril axis, where the intrafibrillar platelets are steeply inclined to the fibril axis. The projection of the intrafibrillar platelets is perpendicular to the fibril axis. The extrafibrillar volume is at least 60% of the total, the fibrils occupying 40%. More of the mineral appears to be extrafibrillar than within the fibrils. Micrographs of the mineralized tendon in thickness show both needlelike-appearing and platelet crystallites. Stereoscopic views show that the needlelike-appearing crystallites do not have a preferred orientation. From the two-dimensional Fourier transform of a selected area of the cross-sectional image, the platelike crystallites have an average dimension of 58 nm. The needlelike-appearing crystallites have an average thickness of 7 nm. The maximum length is at least 90 nm. Atomic force microscopy (AFM) of unstained, unmineralized turkey leg tendon shows collagen fibrils very much like shadow replicas of collagen in electron micrographs. AFM images of the mineralized tendon show only an occasional fibril. Mineral crystallites are not visible. Because the collagen is within the fibrils, the extrafibrillar mineral must be embedded in noncollagenous organic matter. When the tissue is demineralized, the collagen fibrils are exposed. The structure as revealed by the two modalities is a composite material in which each component is itself a composite. Determination of the properties of the mineralized tendon from the properties of its elements is more difficult than considering the tendon to be just mineral-filled collagen.