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.     

Overexpression of IGF-Binding Protein 5 Alters Mineral and Matrix Properties in Mouse Femora: An Infrared Imaging Study

The anabolic effects of insulin-like growth factors (IGFs) are modulated by a family of IGF-binding proteins (IGFBPs). Among the six known IGFBPs, IGFBP-5 is considered to play a role in bone formation. To investigate the effects of IGFBP-5 on bone mineral and matrix properties, femurs from transgenic mice overexpressing IGFBP-5 under the control of the osteocalcin promoter were evaluated by Fourier Transform Infrared Imaging (FTIRI). Analyses were done at the time of maximal osteocalcin expression (5 weeks). The spectroscopic parameters monitored were mineral-to-matrix ratio (indicative of the relative amount of mineral present), mineral crystallinity (index of the mineral crystal size and perfection) and collagen maturity (reflecting the ratio of non-reducible and reducible collagen cross-links). Multiple fields were selected for each femur, ranging from epiphysis to diaphysis. Previously, we showed that these transgenic mice display decreased osteoblastic function and osteopenia. In the present work, FTIRI showed that transgenic mice as compared to wild types have a different pattern of bone mineralization and matrix maturation. Specifically, cortical bone, primary spongiosa, and secondary ossification centers had lower values for mineral-to-matrix ratio and collagen maturity. Differences were not statistically significant in all cases although the trends were consistent. The mineral crystallinity did not vary significantly between the two groups, implying that the crystal maturation of mineral was not affected by IGFBP-5 overexpression. This study demonstrates that femurs from transgenic mice over expressing IGFBP-5 under the control of the osteocalcin promoter have modest alterations in mineral and matrix distribution, consistent with a role of IGF in osteoblast maturation.     

DSPP effects on in vivo bone mineralization

Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (micro-CT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r(2)=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bones was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.     

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.     

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.     

Effects of locally applied transforming growth factor-β1 on distraction osteogenesis in a rabbit limb-lengthening model

In this study we tested the effect of locally applied transforming growth factor-β1 (TGF-β1) on distraction osteogenesis in rabbits. A total of 61 rabbits were studied. Seven days after tibial osteotomy, distraction was started at a rate of 0.25 mm/12 h for 3 weeks. Starting with distraction, TGF-β1 was applied in four different dosages (0, 10, 20, and 40 ng/day) at the site of osteotomy through a catheter connected to a subcutaneously implanted miniosmotic pump. Rabbits were killed at the end of the distraction period or 3 weeks later, and histological, densitometric, and biomechanical parameters were assessed. TGF-β1 treatment had no detectable effect on bone mineral density or histologically determined bone volume in the distraction gap but it increased the amount of fibrous tissue in the callus region. Load to failure in uniaxial tension tended to be lower in TGF-β1-treated animals. In conclusion, TGF-β1 treatment during distraction osteogenesis did not have a beneficial effect in this model.     

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.     

Transforming growth factor-β administration modifies cyclosporine A-induced bone loss

Cyclosporine A (CsA), a potent immunosuppressant used in transplantation, induces increased formation with excess resorption in the rat with resultant osteopenia. These findings are confirmed in the human model. Transforming growth factor-β (TGF-β) is reported to be involved in the coupling of bone formation with resorption and in vivo and in vitro stimulates osteoblasts, and in vitro inhibits osteoclasts. CsA stimulates secretion of TGF-β1 in humans, which, while improving immunosuppression, may also contribute to renal toxicity. This study was performed determine whether exogenously administered TGF-β would modify the bone effects of CsA. Male Sprague-Dawley rats, 6 months of age, were randomized to receive: TGF-β and CsA vehicle (group A); TGF-β 5 μg/kg three times per week and CsA vehicle (group B); TGF-β vehicle and CsA 10 mg/kg (group C); or TGF-β 5 μg/kg three times per week and CsA 10 mg/kg (group D). These were compared with control over 28 days. CsA, but not TGF-β, increased serum 1,25(OH)₂D levels throughout the study. CsA increased osteocalcin (BGP), but TGF-β negated this effect. Histomorphometry confirmed the known effects of CsA, whereas TGF-β alone had no effect. However, in combination, TGF-β blocked CsA’s effect and increased osteoblast recruitment and activity, as reflected by increased percent mineralizing surface, percent osteoid perimeter, bone formation rate (bone volume referent), and activation frequency. Thus, it appears as if TGF-β administration may have potential in modulating the deleterious bone effects of CsA.     

Use of FTIR Spectroscopic Imaging to Identify Parameters Associated With Fragility Fracture

BMD does not entirely explain an individual's risk of fracture. The purpose of this study was to assess whether specific differences in spatially resolved bone composition also contribute to fracture risk. These differences were assessed using Fourier transform infrared spectroscopic imaging (FTIRI) and analyzed through multiple logistic regression. Models were constructed to determine whether FTIRI measured parameters describing mineral content, mineral crystal size and perfection, and collagen maturity were associated with fracture. Cortical and cancellous bone were independently evaluated in iliac crest biopsies from 54 women (32 with fractures, 22 without) who had significantly different spine but not hip BMDs and ranged in age from 30 to 83 yr. The parameters that were significantly associated with fracture in the model were cortical and cancellous collagen maturity (increased with increased fracture risk), cortical mineral/matrix ratio (higher with increased fracture risk), and cancellous crystallinity (increased with increased fracture risk). As expected, because of its correlation with cortical but not cancellous bone density, hip BMD was significantly associated with fracture risk in the cortical but not the cancellous model. This research suggests that additional parameters associated with fracture risk should be targeted for therapies for osteoporosis.     

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.     

Bone mineral and collagen quality in humeri of ovariectomized cynomolgus monkeys given rhPTH(1-34) for 18 months.

A recent study of ovariectomized monkeys, treated with recombinant human parathyroid hormone (rhPTH)(1-34) at 1 or 5 mg/kg/day for 18 months or for 12 months followed by 6 months withdrawal from treatment, showed significant differences in the geometry and histomorphometry of cortical bone of the midshaft humerus. To determine the extent to which the rapid bone turnover and cortical porosity induced by rhPTH(1-34) in ovariectomized monkeys modified mineral content, mineral crystal maturity and collagen maturity (cross-link distribution) in the cortical periosteal and endosteal regions, cross-sections of the cortical bone of the mid-humerus, were examined using Fourier transform infrared imaging (FTIRI). FTIRI analyses demonstrated that rhPTH(1-34) altered bone mineral and collagen properties in a dose-dependent manner. Mineral crystal maturity and collagen cross-link ratio (pyridinoline/dehydro-dihydroxylysinonorleucine) on both endosteal and periosteal surfaces decreased relative to ovariectomized animals, consistent with new bone formation. These changes were partially sustained after withdrawal of the higher dose of rhPTH(1-34), suggesting a prolonged after-effect on bone properties for at least two bone remodeling cycles. In conclusion, treatment of ovariectomized monkeys with rhPTH(1-34) had significant effects on cortical bone mineral-to-matrix ratio, mineral crystal maturity, and collagen cross-link ratio. These were fully reversible when the 1-microg rhPTH(1-34) treatment was withdrawn, but only partially reversed when the 5-microg rhPTH(1-34) dose was withdrawn.     

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.     

Bone Mineral and Organic Properties in Postmenopausal Women Treated With Denosumab for Up to 10 years

In postmenopausal women with osteoporosis, denosumab (DMAb) therapy through 10 years resulted in significantly higher degree of mineralization of bone, with a subsequent increase from years 2–3 to year 5 and no further difference between years 5 and 10. Our aim was to assess the variables reflecting the quality of bone mineral and organic matrix (Fourier transform infrared microspectroscopy), and the microhardness of bone (Vickers microindentation). Cross-sectional assessments were performed in blinded fashion on iliac bone biopsies from osteoporotic women (72 from FREEDOM trial, 49 from FREEDOM Extension trial), separately in cortical and cancellous compartments. After 2–3 years of DMAb, mineral/matrix ratio and microhardness of cortical bone were significantly higher compared with placebo, whereas mineral maturity, mineral crystallinity, mineral carbonation, and collagen maturity were not different in both bone compartments. Through 5 years of DMAb, mineral carbonation was significantly lower and mineral/matrix ratio, mineral maturity, and crystallinity were significantly higher versus 2–3 years and were not different between 5 and 10 years, with the exception of mineral maturity in cancellous bone. These data support a transition of mineral to more mature crystals (within physiological range) and the completeness of secondary mineralization within 5 years of DMAb treatment. Microhardness in cortical and cancellous compartments was significantly lower at 5 years of DMAb versus 2–3 years and was not different from years 5 to 10. The lower microhardness at years 5 and 10 is likely the result of maturation of the organic matrix in a persistently low state of bone remodeling over 5 and 10 years. © 2022 American Society for Bone and Mineral Research (ASBMR).     

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.     

Transforming growth factor-β enhances fracture healing in rabbit tibiae

The ability of exogenous Transforming Growth Factor-β (TGF-β) to stimulate bone formation in fracture healing was investigated. TGF-β was continuously applied in doses of 1 and 10 μg/day for 6 weeks to 2 groups of adult rabbits with unilateral plated midtibial osteotomies. A group receiving solvent without TGF-β served as control. Fracture healing was evaluated by mechanical tests, bone morphometry and bone densitometry. Increased maximal bending strength and callus formation were demonstrated in the groups receiving TGF-β. TGF-β had no effect on bending-stiffness, bone mineral content, cortical thickness or haversian canal diameter. We conclude that local application of exogenous TGF-β may enhance fracture healing in rabbits.     

Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-β1 where it acts as a downstream mediator of TGF-β1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-β1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-β1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-β1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-β1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-β1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-β1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-β1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-β in some cell types, we determined its role in TGF-β1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-β1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-β1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-β1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (− 787 to + 1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-β1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-β1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-β1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-β1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.     

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ‐null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ‐null mice. The antagonist‐induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle‐treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ‐null mice, and increases in pSmad1/5/8 levels and Id1‐luc activity were observed in RARγ antagonist‐treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096–1105, 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.     

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 of femoral neck bone: Reduced heterogeneity of mineral‐to‐matrix and carbonate‐to‐phosphate and more variable crystallinity in treatment‐naive fracture cases compared with fracture‐free controls

After the age of 60 years, hip fracture risk strongly increases, but only a fifth of this increase is attributable to reduced bone mineral density (BMD, measured clinically). Changes in bone quality, specifically bone composition as measured by Fourier transform infrared spectroscopic imaging (FTIRI), also contribute to fracture risk. Here, FTIRI was applied to study the femoral neck and provide spatially derived information on its mineral and matrix properties in age‐matched fractured and nonfractured bones. Whole femoral neck cross sections, divided into quadrants along the neck's axis, from 10 women with hip fracture and 10 cadaveric controls were studied using FTIRI and micro‐computed tomography. Although 3‐dimensional micro‐CT bone mineral densities were similar, the mineral‐to‐matrix ratio was reduced in the cases of hip fracture, confirming previous reports. New findings were that the FTIRI microscopic variation (heterogeneity) of the mineral‐to‐matrix ratio was substantially reduced in the fracture group as was the heterogeneity of the carbonate‐to‐phosphate ratio. Conversely, the heterogeneity of crystallinity was increased. Increased variation of crystallinity was statistically associated with reduced variation of the carbonate‐to‐phosphate ratio. Anatomical variation in these properties between the different femoral neck quadrants was reduced in the fracture group compared with controls. Although our treatment‐naive patients had reduced rather than increased bending resistance, these changes in heterogeneity associated with hip fracture are in another way comparable to the effects of experimental bisphosphonate therapy, which decreases heterogeneity and other indicators of bone's toughness as a material. © 2013 American Society for Bone and Mineral Research