Effect of hormone replacement therapy on bone quality in early postmenopausal women.

HRT is an effective prophylaxis against postmenopausal bone loss. Infrared imaging of paired iliac crest biopsies obtained at baseline and after 2 years of HRT therapy demonstrate an effect on the mineral crystallinity and collagen cross-links that may affect bone quality. Several studies have demonstrated that hormonal replacement therapy (HRT) is an effective prophylaxis against postmenopausal bone loss, although the underlying mechanisms are still debated. Infrared spectroscopy has been used previously for analyzing bone mineral crystallinity and three-dimensional structures of collagen and other proteins. In the present study, the technique of Fourier transform infrared microscopic imaging (FTIRI) was used to investigate the effect of estrogen on bone quality (arbitrarily defined as mineral/matrix ratio, mineral crystallinity/maturity, and relative ratio of collagen cross-links [pyridinoline/ deH-DHLNL]) at the ultrastructural level, in mineralized, thin tissue sections from double (before and after administration of HRT regimen; cyclic estrogen and progestogen [norethisterone acetate]) iliac crest biopsy specimens from 10 healthy, early postmenopausal women who were not on any medication with known influence on calcium metabolism. FTIRI allows the analysis of undemineralized thin tissue sections (each image analyzes a 400 x 400 microm2 area with a spatial resolution of approximately 6.3 mm). For each bone quality variable considered, the after-treatment data exhibited an increase in the mean value, signifying definite changes in bone properties at the molecular level after HRT treatment. Furthermore, these findings are consistent with suppressed osteoclastic activity.     

Optimal Methods for Processing Mineralized Tissues for Fourier Transform Infrared Microspectroscopy

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) are techniques utilized in the analysis of bone mineral and matrix properties in health and disease. Since the spatial arrangement of bone tissue is conserved using FTIRM and FTIRI, quantitative data can be obtained on bone mineral (hydroxyapatite) crystalline size and composition, and on matrix structure and composition at discrete anatomic locations with a spatial resolution from approximately 7 mm (FTIRI) to 10 mm (FTIRM). To section bone for FTIRM and FTIRI, it must be preserved ("fixed") to maintain its properties, and embedded in a hard supportive material. Since most of the embedding media have components that spectrally overlap the components of mineralized tissues, it is critical to define optimal embedding and fixation protocols that have the least effect on mineral and matrix spectra. In the current study, the spectra of mouse calvaria in seven different fixatives and six different commonly used embedding media were assessed by FTIRM and FTIRI. The fixatives evaluated were absolute ethanol, 70% ethanol, glycerol, formaldehyde, EM fixative, and formalin in cacodylate or phosphate-buffered saline. The embedding media tested were Araldite, Epon, JB-4, LR White, PMMA, and Spurr. Comparisons were made to FTIR spectra obtained from unprocessed ground calvaria and to spectra of cryosections of unfixed tissue, fast-frozen in polyvinyl alcohol (5% PVA). Non-aqueous fixatives and embedding in LR White, Spurr, Araldite, and PMMA had the least effect on the spectral parameters measured (mineral to matrix ratio, mineral crystallinity, and collagen maturity) compared with cryo-sectioned calvaria and non-fixed, non-embedded calvaria in KBr pellets.     

Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.

To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. INTRODUCTION: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. MATERIALS AND METHODS: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. RESULTS: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones. CONCLUSIONS: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.     

Distribution of collagen cross-links in normal human trabecular bone.

Infrared imaging analysis of normal human iliac crest biopsy specimens shows a characteristic spatial variation in the nonreducible:reducible collagen cross-links at trabecular surfaces, depending on the surfaces' metabolic status. INTRODUCTION: Bone is a composite material consisting of mineral, collagen, non-collagenous proteins, and lipids. Bone collagen, mainly type I, provides the scaffold on which mineral is deposited and imparts specific mechanical properties, determined in part by the amount of collagen present, its orientation and fibril diameter, and the distribution of its cross-links. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections from human iliac crest biopsy specimens (N = 14) at trabecular surfaces as a function of surface activity (forming versus resorbing), with an approximately 6.3-mm spatial resolution. The biopsy specimens were obtained from patients devoid of any metabolic bone disease based on histomorphometric and bone densitometric parameters. RESULTS AND CONCLUSIONS: Distributions of collagen cross-links within the first 50 mm at forming trabecular surfaces demonstrated a progressive increase in the nonreducible:reducible collagen cross-link ratio, unlike in the case of resorbing surfaces, in which the collagen cross-links ratio (as defined for the purposes of the present report) was relatively constant.     

Bone fragility and collagen cross-links.

Infrared imaging analysis of iliac crest biopsy specimens from patients with osteoporotic and multiple spontaneous fractures shows significant differences in the spatial variation of the nonreducible:reducible collagen cross-links at bone-forming trabecular surfaces compared with normal bone. INTRODUCTION: Although the role of BMC and bone mineral quality in determining fracture risk has been extensively studied, considerably less attention has been paid to the quality of collagen in fragile bone. MATERIALS AND METHODS: In this study, the technique of Fourier transform infrared imaging (FTIRI) was used to determine the ratio of nonreducible:reducible cross-links, in 2- to 4-microm-thick sections, from human iliac crest biopsy specimens (N = 27) at bone-forming trabecular surfaces. The biopsy specimens were obtained from patients that had been diagnosed as high- or low-turnover osteoporosis, as well as premenopausal women <40 years of age, with normal BMD and biochemistry, who suffered multiple spontaneous fractures. The obtained values were compared with previously published analyses of trabecular bone from normal non-osteoporotic subjects (N = 14, 6 males and 8 females; age range, 51-70 years). RESULTS AND CONCLUSIONS: Collagen cross-links distribution within the first 50 microm at forming trabecular surfaces in patients with fragile bone was markedly different compared with normal bone.     

Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia

Osteomalacia is a pathological bone condition in which there is deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The hypothesis that there are no qualitative or quantitative differences in osteomalacic bone mineral or matrix compared to disease-free bones was tested by examining unstained sections of polymethyl methacrylate (PMMA) embedded iliac crest biopsies using Fourier transform infrared imaging (FTIRI) at approximately 6-microm spatial resolution. Controls were seven female subjects, aged 36-57, without apparent bone disease. The experimental group consisted of 11 patients aged 22-72, diagnosed with osteomalacia. The spectroscopic parameters analyzed in each data set were previously established as sensitive to bone quality: phosphate/amide I band area ratio (mineral content), 1660/1690 cm(-1) peak ratio (collagen cross-links), and the 1030/1020 cm(-1) peak ratio (mineral crystallinity). The correspondence between spectroscopic mineral content (phosphate/amide I ratio) and ash weight was validated for apatite crystals of different composition and crystallite size. The FTIRI results from the biopsies expressed as color-coded images and pixel population means were compared with the nonparametric Mann-Whitney U test. There were no significant differences in the cortical parameters. Significant difference was found in the mineral content of the trabecular regions with a lower mean value in osteomalacia (P = 0.01) than in controls. Mineral crystallinity tended to be decreased in the trabecular bone (P = 0.09). This study supports the hypothesis that, in osteomalacia, the quality of the organic matrix and of mineral in the center of bone does not change, while less-than-optimal mineralization occurs at the bone surface. This study provides the first spectroscopic evaluation of whole bone mineral and matrix properties in osteomalacia, demonstrating that there are few differences in collagen cross-links between biopsies from patients with osteomalacia and from individuals without histological evidence of bone disease.     

Fourier Transform Infrared Imaging as a Tool to Chemically and Spatially Characterize Matrix-Mineral Deposition in Osteoblasts

Mineralizing osteoblasts are regularly used to study osteogenesis and model in vivo bone formation. Thus, it is important to verify that the mineral and matrix being formed in situ are comparable to those found in vivo. However, it has been shown that histochemical techniques alone are not sufficient for identifying calcium phosphate-containing mineral. The goal of the present study was to demonstrate the use of Fourier transform infrared imaging (FTIRI) as a tool for characterizing the spatial distribution and colocalization of the collagen matrix and the mineral phase during the mineralization process of osteoblasts in situ. MC3T3-E1 mouse osteoblasts were mineralized in culture for 28 days and FTIRI was used to evaluate the collagen content, collagen cross-linking, mineralization level and speciation, and mineral crystallinity in a spatially resolved fashion as a function of time. To test whether FTIRI could detect subtle changes in the mineralization process, cells were treated with risedronate (RIS). Results showed that collagen deposition and mineralization progressed over time and that the apatite mineral was associated with a collagenous matrix rather than ectopic mineral. The process was temporarily slowed by RIS, where the inhibition of osteoblast function caused slowed collagen production and cross-linking, leading to decreased mineralization. This study demonstrates that FTIRI is a complementary tool to histochemistry for spatially correlating the collagen matrix distribution and the nature of the resultant mineral during the process of osteoblast mineralization. It can further be used to detect small perturbations in the osteoid and mineral deposition process.     

Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls.

Osteonectin function in bone was investigated by infrared analysis of bones from osteonectin-null (KO) and wildtype mice (four each at 11, 17, and 36 weeks). An increase in mineral content and crystallinity in newly formed KO bone and collagen maturity at all sites was found using FTIR microspectroscopy and imaging; consistent with osteonectin's postulated role in regulating bone formation and remodeling. Mineral and matrix properties of tibias of osteonectin-null mice and their age- and background-matched wildtype controls were compared using Fourier-transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) at 10- and 7-mm spatial resolution, respectively. The bones came from animals that were 11, 17, and 36 weeks of age. Individual FTIRM spectra were acquired from 20 x 20 microm areas, whereas 4096 simultaneous FTIRI spectra were acquired from 400 x 400 microm areas. The FTIRM data for mineral-to-matrix, mineral crystallinity, and collagen maturity were highly correlated with the FTIRI data in similar regions. In general, the osteonectin-null mice bones had higher mineral contents and greater crystallinity (crystal size and perfection) than the age-matched wildtype controls. Specifically, the mineral content of the newly forming periosteal bone was increased in the osteonectin-null mice; the crystallinity of the cortical bone was decreased in all but the oldest animals, relative to the wildtype. The most significant finding, however, was increased collagen maturity in both the cortical and trabecular bone of the osteonectin-null mice. These spectroscopic data are consistent with a mechanism of decreased bone formation and remodeling.     

Effects of transforming growth factor-beta deficiency on bone development: a Fourier transform-infrared imaging analysis

Transforming growth factor-beta 1 (TGF-β1) is a cytokine member of the TGF-β superfamily involved in the control of proliferation and differentiation of various cell types. TGF-β1 plays an important role in bone formation and resorption. To determine the effect of TGF-β1 deficiency on bone mineral and matrix, tibias from mice in which TGF-β1 expression had been ablated (TGF-β1 null) were analyzed and compared with background- and age-matched wild-type (WT) control animals by Fourier transform-infrared imaging (FTIRI) and histochemistry. FTIRI allows the characterization of nondemineralized thin tissue sections at the ultrastructural level with a spatial resolution of 7 μm. The spectroscopic parameters calculated were: mineral-to-matrix ratio (previously shown to correspond to ash weight); mineral crystallinity (related to the crystallographically determined crystallite size and perfection in the apatite c-axis direction); and collagen maturity (related to the ratio of pyridinoline:deH-DHLNL collagen cross-links). Several fields were selected to represent different stages of bone development within the same specimen from the secondary ossification center to the distal diaphysis. Anatomically equivalent areas were compared as a function of age and genotype. The spectroscopic results were expressed both as color-coded images and as pixel population distributions for each of the three parameters monitored. Based on comparisons of histochemistry and FTIRI, there were distinctive age and genotype variations. At all ages examined, in the TGF-β1 null mice growth plates, alkaline phosphatase (ALP) activity and collagen maturity were reduced, but no effect on mineral content or crystallinity was noted. In the TGF-β1 null mice metaphyses, there was a persistence of trabeculae, but no significant alterations in mineral content or crystallinity. In contrast, mineral content, mineral crystallinity, and collagen maturity were reduced in the secondary ossification center and cortical bone of the TGF-β1 null mice. These results, consistent with a mechanism of impaired bone maturation in the TGF-β1 null mice, may be directly related to TGF-β1 deficiency and indirectly to increased expression of inflammatory cytokines in the TGFβ1 null mice.     

Differently cross-linked and uncross-linked carboxy-terminal telopeptides of type I collagen in human mineralised bone

In bone matrix, type I collagen is stabilised by covalent cross-links formed between adjacent collagen molecules; the majority of which is believed to be immature, divalent bonds. For studying these immature forms in detail, we have developed an immunoassay for a synthetic peptide SP 4 that is analogous with and detects a linear epitope within the C-telopeptide of alpha1-chain of type I collagen. The SP 4 assay, together with the ICTP assay, which is specific for the trivalently cross-linked C-telopeptide, was used for the isolation of the differently cross-linked C-telopeptide structures of the alpha1-chain of type I collagen present in mineralised human bone. Amino acid analysis, peptide sequencing and MALDI-TOF mass spectrometry were used to identify and characterise each of the isolated structures. The cross-link content of each isolated peptide was identified. In the trivalent ICTP peptide, only 40% was cross-linked with pyridinoline, the remainder of the cross-links being currently uncharacterized. The divalent peptides contained only previously characterised cross-linking structures. Most of the divalent cross-links were dihydroxylysinonorleucine (DHLNL), with minor amounts of hydroxylysinonorleucine (HLNL). The relative proportion of the HLNL cross-link was slightly higher in the divalent alpha1Calpha2H peptide. A substantial amount of uncross-linked telopeptide structures was also found. Previous studies, where direct chemical cross-link analyses have been used to assess the maturity of cross-linking, have inferred that bone contains more divalently than trivalently cross-linked C-telopeptides. The immunochemical peptide approach used in this study may help to detect presently uncharacterized, trivalent cross-links, the presence of which is strongly suggested in this study. It also provides additional information regarding the extent and maturity of tissue type I collagen cross-linking in health and disease.     

Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.     

Examining the Relationships Between Bone Tissue Composition,Compositional Heterogeneity,and Fragility Fracture: A Matched Case‐Controlled FTIRI Study

Fourier transform infrared imaging (FTIRI) provides information on spatial distribution of the chemical composition of thin tissue specimens at ～7 µm spatial resolution. This study of 120 age‐ and bone mineral density (BMD)‐matched patients was designed to investigate the association of FTIRI variables, measured in iliac crest biopsies, with fragility fractures at any site. An earlier study of 54 women found hip BMD to be a significant explanatory variable of fracture risk for cortical bone but not for cancellous bone. In the current study, where age and BMD were controlled through matching, no such association was observed, validating the pairing scheme. Our first study of unmatched iliac crest biopsies found increases in collagen maturity (cancellous and cortical bone) and mineral crystal size (cortical bone only) to be a significant explanatory variable of fracture when combined with other covariates. The ratio for collagen maturity has been correlated to the amount of enzymatic collagen cross‐links. To assess the impact of other FTIRI variables (acid phosphate substitution, carbonate‐to‐phosphate ratio, and the pixel distribution [heterogeneity] of all relevant FTIRI variables), we examined biopsies from a matched case‐controlled study, in which 60 women with fractures were each paired with an age‐ and BMD‐matched female control. With the matched data set of 120 women, conditional logistic regression analyses revealed that significant explanatory variables of fracture were decreased carbonate‐to‐phosphate ratio in both cancellous (odds ratio [OR] = 0.580, 95% confidence interval [CI] 0.37–0.909, p = 0.0176) and cortical bone (OR = 0.519, 95% CI 0.325–0.829, p = 0.0061), and increased heterogeneity (broadened pixel distribution) of collagen maturity for cancellous bone (OR = 1.549, 95% CI 1.002–2.396, p = 0.0491). The observation that collagen maturity was no longer linked to fracture in age‐ and BMD‐matched samples suggests that age‐dependent variation in collagen maturity may be a more important contributory factor to fragility fractures than previously thought. © 2015 American Society for Bone and Mineral Research.     

The cartilage-bone interface

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.     

Comparative molecular distribution of cross-link in bone and dentin collagen. Structure-function relationships

Bone and dentin contain exclusively genetic Type I collagen. These collagens have identical amino acid sequences, cross-link precursors and cross-links yet serve different physiological functions. Complete tryptic digests of the intractable [3H]NaBH4-reduced demineralization collagen from bovine cortical bone and dentin have successfully been obtained. Chromatography of the tryptic peptides on Sephadex G-50 allowed separation of cross-link peptide fractions containing dihydroxylysinonorleucine. Chromatography of peptides of the same molecular weight distribution from each sample, which should contain identical peptides, yielded different chromatographic patterns on phosphocellulose. The phosphocellulose fractions containing the most abundant amounts of dihydroxylysinonorleucine were rechromatographed on DEAE-cellulose and yielded dissimilar profiles. It was concluded that the cross-link, dihydroxylysinonorleucine, has a different molecular distribution in bone and dentin collagen. The results demonstrate that the collagen derived from two different mineralized tissues, possess different micromolecular structures. These structural differences may be related to diverse physiological functions.     

Deletion of Cx43 from Osteocytes Results in Defective Bone Material Properties but Does Not Decrease Extrinsic Strength in Cortical Bone

Deletion of connexin (Cx) 43 from osteoblasts and osteocytes (OCN-Cre;Cx43(fl/-) mice) or from osteocytes only (DMP1-8kb-Cre;Cx43(fl/fl) mice) results in increased cortical, but not cancellous, osteocyte apoptosis and widening of the femoral midshaft without changes in cortical thickness. Despite the consequent larger moment of inertia, stiffness and ultimate load, measures of mechanical strength assessed by three-point bending, are not higher in either model of Cx43 deficiency due to reduced Young's modulus, a measure of the stiffness of the material per unit of area. In OCN-Cre;Cx43(fl/-) mice, this was accompanied by a reduced ratio of nonreducible/reducible collagen cross-links as assessed by Fourier transformed infrared imaging (FTIRI) in the femoral diaphysis. On the other hand, DMP1-8kb-Cre;Cx43(fl/fl) mice did not show a significant reduction in collagen maturation in the same skeletal site, but a small decrease in mineralization was detected by FTIRI. Remarkably, both osteoblastic and osteocytic cells lacking Cx43 expressed lower mRNA levels of lysyl oxidase, a crucial enzyme involved in collagen maturation. These findings suggest that Cx43 expression in osteoblasts is involved in maintaining the quality of the bone matrix in cortical bone through the maturation of collagen cross-links. Osteocytic Cx43 expression is important also to maintain the stiffness of the bone material, where Cx43 deficiency results in local reduction in mineralization, possibly due to osteocyte apoptosis.     

Compositional mapping of the mature anterior cruciate ligament‐to‐bone insertion

The anterior cruciate ligament (ACL)‐to‐bone interface constitutes a complex, multi‐tissue structure comprised of contiguous ligament, non‐mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL‐to‐bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non‐mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513–2523, 2017.

     

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and β-APN treated animals were fed additionally with β-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2-L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that β-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of β-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, β-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of β-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.     

Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus

Collagen cross-linking, a major post-translational modification of collagen, plays important roles in the biological and biomechanical features of bone. Collagen cross-links can be divided into lysyl hydroxylase and lysyl oxidase-mediated enzymatic immature divalent cross-links, mature trivalent pyridinoline and pyrrole cross-links, and glycation- or oxidation-induced non-enzymatic cross-links (advanced glycation end products) such as glucosepane and pentosidine. These types of cross-links differ in the mechanism of formation and in function. Material properties of newly synthesized collagen matrix may differ in tissue maturity and senescence from older matrix in terms of cross-link formation. Additionally, newly synthesized matrix in osteoporotic patients or diabetic patients may not necessarily be as well-made as age-matched healthy subjects. Data have accumulated that collagen cross-link formation affects not only the mineralization process but also microdamage formation. Consequently, collagen cross-linking is thought to affect the mechanical properties of bone. Furthermore, recent basic and clinical investigations of collagen cross-links seem to face a new era. For instance, serum or urine pentosidine levels are now being used to estimate future fracture risk in osteoporosis and diabetes. In this review, we describe age-related changes in collagen cross-links in bone and abnormalities of cross-links in osteoporosis and diabetes that have been reported in the literature.     

Comparative molecular distribution of cross-links in bone and dentin collagen. Structure-function relationships

Summary Bone and dentin contain exclusively genetic Type I collagen. These collagens have identical amino acid sequences, cross-link precursors and cross-links yet serve different physiological functions. Complete tryptic digests of the intractable [³H]NaBH₄-reduced demineralized collagen from bovine cortical bone and dentin have successfully been obtained. Chromatography of the tryptic peptides on Sephadex G-50 allowed separation of cross-link peptide fractions containing dihydroxylysinonorleucine. Chromatography of peptides of the same molecular weight distribution from each sample, which should contain identical peptides, yielded different chromatographic patterns on phosphocellulose. The phosphocellulose fractions containing the most abundant amounts of dihydroxylysinonorleucine were rechromatographed on DEAE-cellulose and yielded dissimilar profiles. It was concluded that the cross-link, dihydroxylysinonorleucine, has a different molecular distribution in bone and dentin collagen. The results demonstrate that the collagen derived from two different mineralized tissues, possess different micromolecular structures. These structural differences may be related to diverse physiological functions.     

Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal ghost in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.