The Contribution of Perilacunar Composition and Mechanical Properties to Whole-Bone Mechanical Outcomes in Streptozotocin-Induced Diabetes.

Osteocytes are the most abundant cell type in bone and remodel their local perilacunar matrix in response to a variety of stimuli and diseases. How the perilacunar composition and mechanical properties are affected by type 1 diabetes (T1D), and the contribution of these local changes to the decline in whole-bone functional properties that occurs with diabetes remains unclear. 12-14 week old C57/BL6 male mice were administered a series of low-dose streptozotocin injections and sacrificed at baseline (BL), 3 (D3) and 7 weeks (D7) following confirmation of diabetes, along with age-matched controls (C3, C7). Femora were then subjected to a thorough morphological (µCT), mechanical (four-point bending, nanoindentation), and compositional (HPLC for collagen cross-links, Raman spectroscopy) analysis at the whole-bone and local (perilacunar and intracortical) levels. At the whole-bone level, D7 mice exhibited 10.7% lower ultimate load and 26.4% lower post-yield work relative to C7. These mechanical changes coincided with 52.2% higher levels of pentosidine at D7 compared to C7. At the local level, the creep distance increased, while modulus and hardness decreased in the perilacunar region relative to the intracortical for D7 mice, suggesting a spatial uncoupling in skeletal adaptation. D7 mice also exhibited increased matrix maturity in the 1660/1690 cm-1 ratio at both regions relative to C7. The perilacunar matrix maturity was predictive of post-yield work (46%), but perilacunar measures were not predictive of ultimate load, which was better explained by cortical area (26%). These results show that diabetes causes local perilacunar composition perturbations that affect whole-bone level mechanical properties, implicating osteocyte maintenance of its local matrix in the progression of diabetic skeletal fragility.

External bone size identifies different strength-decline trajectories for the male human femora.

Understanding skeletal aging and predicting fracture risk is increasingly important with a growing elderly population. We hypothesized that when categorized by external bone size, the male femoral diaphysis would show different strength-age trajectories which can be explained by changes in morphology, composition and collagen cross-linking. Cadaveric male femora were sorted into narrow (n = 15, 26-89 years) and wide (n = 15, 29-82 years) groups based upon total cross-sectional area of the mid-shaft normalized to bone length (Tt.Ar/Le) and tested for whole bone strength, tissue-level strength, and tissue-level post-yield strain. Morphology, cortical TMD (Ct.TMD), porosity, direct measurements of enzymatic collagen cross-links, and pentosidine were obtained. The wide group alone showed significant negative correlations with age for tissue-level strength (R2 = 0.50, p = 0.002), tissue-level post-yield strain (R2 = 0.75, p < 0.001) and borderline significance for whole bone strength (R2 = 0.14, p = 0.108). Ct.TMD correlated with whole bone and tissue-level strength for both groups, but pentosidine normalized to enzymatic cross-links correlated negatively with all mechanical properties for the wide group only. The multivariate analysis showed that just three traits for each mechanical property explained the majority of the variance for whole bone strength (Ct.Area, Ct.TMD, Log(PEN/Mature; R2 = 0.75), tissue-level strength (Age, Ct.TMD, Log(DHLNL/HLNL); R2 = 0.56), and post-yield strain (Age, Log(Pyrrole), Ct.Area; R2 = 0.51). Overall, this highlights how inter-individual differences in bone structure, composition, and strength change with aging and that a one-size fits all understanding of skeletal aging is insufficient.

Review on material parameters to enhance bone cell function in vitro and in vivo.

Bone plays critical roles in support, protection, movement, and metabolism. Although bone has an innate capacity for regeneration, this capacity is limited, and many bone injuries and diseases require intervention. Biomaterials are a critical component of many treatments to restore bone function and include non-resorbable implants to augment bone and resorbable materials to guide regeneration. Biomaterials can vary considerably in their biocompatibility and bioactivity, which are functions of specific material parameters. The success of biomaterials in bone augmentation and regeneration is based on their effects on the function of bone cells. Such functions include adhesion, migration, inflammation, proliferation, communication, differentiation, resorption, and vascularization. This review will focus on how different material parameters can enhance bone cell function both in vitro and in vivo.

Phage Display to Augment Biomaterial Function.

Biomaterial design relies on controlling interactions between materials and their biological environments to modulate the functions of proteins, cells, and tissues. Phage display is a powerful tool that can be used to discover peptide sequences with high affinity for a desired target. When incorporated into biomaterial design, peptides identified via phage display can functionalize material surfaces to control the interaction between a biomaterial and its local microenvironment. A targeting peptide has high specificity for a given target, allowing for homing a specific protein, cell, tissue, or other material to a biomaterial. A functional peptide has an affinity for a given protein, cell, or tissue, but also modulates its target's activity upon binding. Biomaterials can be further enhanced using a combination of targeting and/or functional peptides to create dual-functional peptides for bridging two targets or modulating the behavior of a specific protein or cell. This review will examine current and future applications of phage display for the augmentation of biomaterials.

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition.

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling.

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1(-/-)) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1(-/-) mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1(-/-) mice had decreased trabecular bone volume/total volume and that both male and female Wisp1(-/-) mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1(-/-) mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1(-/-) mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1(-/-) mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1(-/-) bone marrow stromal cells had reduced expression of ß-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1(-/-) mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones.

Characterizations of Bone-Like Apatite Powder Fabricated Using Modified Simulated Body Fluid.

The objective of this study is to fabricate bone-like apatite (BLAp) powder using the modified simulated body fluid (SBF). The SBF2X and SBF4X groups were prepared by increasing the concentration of inorganic ions by two and four times, respectively, to that of the standard SBF. The mSBF4X group was prepared by particularly increasing the concentrations of calcium and phosphate ions in SBF. Bovine serum albumin (BSA) was added for SBF2X-BSA, SBF4X-BSA, and mSBF4X-BSA groups. BLAp powders were precipitated in these SBFs while being kept at 60 °C. Micro-morphology of BLAp powders showed tens of micrometers-sized rounded clusters which composed with sheet-like nano crystallites. The radius of BLAp clusters were decreased by increasing the concentration of inorganic ions and by incorporating the BSA. The hydroxyapatite crystalline structure was dominant for all sample groups. Further, octacalcium phosphate structure was detected in the mSBF4X group. However, these peaks were decreased in mSBF4X-BSA. FT-IR spectra demonstrated that BSA was co-precipitated in BLAp crystallites, and the amount of BSA was higher in the mSBF4X-BSA group than in the SBF4X-BSA group.

Dual-functioning phage-derived peptides encourage human bone marrow cell-specific attachment to mineralized biomaterials.

Cell instructive mineralized biomaterials are a promising alternative to conventional auto-, allo-, and xenograft therapies for the reconstruction of critical sized defects. Extracellular matrix proteins, peptide domains, and functional motifs demonstrating cell and mineral binding activity have been used to improve cell attachment. However, these strategies vary in their tissue regeneration outcomes due to lack of specificity to both regenerative cell populations and the material substrates. In order to mediate cell-specific interactions on apatite surfaces, we identified peptide sequences with high affinity towards apatite (VTKHLNQISQSY, VTK) and clonally derived human bone marrow stromal cells (DPIYALSWSGMA, DPI) using phage display. The primary aims of this study were to measure apatite binding affinity, human bone marrow stromal cell (hBMSC) adhesion strength, and peptide specificity to hBMSCs when the apatite and cell-specific peptides are combined into a dual-functioning peptide. To assess binding affinity to hydroxyapatite (HA), binding isotherms were constructed and peptide binding affinity (K1) determined. HBMSC, MC3T3 and mouse dermal fibroblast (MDF) adhesion strength on biomimetic apatite functionalized with single- and dual-functioning peptide sequences were evaluated using a centrifugation assay. DPI-VTK had the highest binding strength towards hBMSCs (p < 0.01). DPI-VTK, while promoting strong initial attachment to hBMSCs, did not encourage strong adhesions to MC3T3s or fibroblasts (p < 0.01). Taken together, phage display is a promising strategy to identify preferential cell and material binding peptide sequences that can tether specific cell populations onto specific biomaterial chemistries.

Peripheral canalicular branching is decreased in streptozotocin-induced diabetes and correlates with decreased whole-bone ultimate load and perilacunar elastic work.

Osteocytes, the most abundant cell type in bone, play a crucial role in mechanosensation and signaling for bone formation and resorption. These cells reside within a complex lacuno-canalicular network (OLCN). Osteocyte signaling is reduced under diabetic conditions, and both type 1 and type 2 diabetes lead to reduced bone turnover, perturbed bone composition, and increased fracture risk. We hypothesized that this reduced bone turnover, and altered bone composition with diabetes is associated with reduced OLCN architecture and connectivity. This study aimed to elucidate: (1) the sequence of OLCN changes with diabetes related to bone turnover and (2) whether changes to the OLCN are associated with tissue composition and mechanical properties. Twelve- to fourteen-week-old male C57BL/6 mice were administered streptozotocin at 50 mg/kg for 5 consecutive days to induce hyperglycemia, sacrificed at baseline (BL), or after being diabetic for 3 (D3) and 7 (D7) wk with age-matched (C3, C7) controls (n = 10-12 per group). Mineralized femoral sections were infiltrated with rhodamine, imaged with confocal microscopy, then the OLCN morphology and topology were characterized and correlated against bone histomorphometry, as well as local and whole-bone mechanics and composition. D7 mice exhibited a lower number of peripheral branches relative to C7. The total number of canalicular intersections (nodes) was lower in D3 and D7 relative to BL (P < 0.05 for all), and a reduced bone formation rate (BFR) was observed at D7 vs C7. The number of nodes explained only 15% of BFR, but 45% of Ct.BV/TV, and 31% of ultimate load. The number of branches explained 30% and 22% of the elastic work at the perilacunar and intracortical region, respectively. Collectively, the reduction in OLCN architecture and association of OLCN measures with bone turnover, mechanics, and composition highlights the relevance of the osteocyte and the OLCN and a potential therapeutic target for treating diabetic skeletal fragility.

Using polymeric materials to control stem cell behavior for tissue regeneration.

Patients with organ failure often suffer from increased morbidity and decreased quality of life. Current strategies of treating organ failure have limitations, including shortage of donor organs, low efficiency of grafts, and immunological problems. Tissue engineering emerged about two decades ago as a strategy to restore organ function with a living, functional engineered substitute. However, the ability to engineer a functional organ is limited by a limited understanding of the interactions between materials and cells that are required to yield functional tissue equivalents. Polymeric materials are one of the most promising classes of materials for use in tissue engineering, due to their biodegradability, flexibility in processing and property design, and the potential to use polymer properties to control cell function. Stem cells offer potential in tissue engineering because of their unique capacity to self-renew and differentiate into neurogenic, osteogenic, chondrogenic, and myogenic lineages under appropriate stimuli from extracellular components. This review examines recent advances in stem cell-polymer interactions for tissue regeneration, specifically highlighting control of polymer properties to direct adhesion, proliferation, and differentiation of stem cells, and how biomaterials can be designed to provide some of the stimuli to cells that the natural extracellular matrix does.

Connexin 43 as a signaling platform for increasing the volume and spatial distribution of regenerated tissue.

Gap junction intercellular communication (GJIC) is ubiquitous in the majority of vertebrate cells and is required for the proper development of most tissues. The loss of gap junction-mediated cell-to-cell communication leads to compromised development in many tissues and organs. Because cells constantly interact through gap junctions to coordinate tissue functions and homeostasis, we hypothesized that increasing cell-to-cell communication, via genetically engineering cells to overexpress gap junction proteins, could enhance cell differentiation in the interior regions of 3D tissue equivalents, thereby increasing the ability to regenerate larger and more uniform volumes of tissue. To test this hypothesis, we used bone as a model tissue because of the difficulty in achieving spatially uniform bone regeneration in 3D. In bone marrow stromal cells (BMSC), GJIC and osteogenic differentiation were compromised in 3D cultures relative to 2D monolayers and in the core of 3D cultures relative to the surface. Overexpression of connexin 43 (Cx43) via transduction of BMSCs with a lentivirus overcame this problem, enhancing both the magnitude and spatial distribution of GJIC and osteogenic differentiation markers throughout 3D constructs. Transplantation of cells overexpressing Cx43 resulted in an increased volume fraction and spatial uniformity of bone in vivo, relative to nontransduced BMSCs. Increased GJIC also enhanced the effect of a potent osteoinductive agent (BMP-7), suggesting a synergism between the soluble factor and GJIC. These findings present a platform to improve cell-to-cell communication in 3D and to achieve uniformly distributed tissue regeneration in 3D.

Calcium and phosphorus supplemented diet increases bone volume after thirty days of high speed treadmill exercise in adult mice.

Weight-bearing exercise increases bone mass and strength. Increasing bone loading frequency during exercise can strengthen bone. Combining exercise with a calcium- and phosphorus-supplemented diet increases cortical area more than exercise alone in mice. Thus, we hypothesized that combining high-speed treadmill exercise while feeding mice a mineral-supplemented diet would lead to greater cortical area than high-speed exercise on a standard diet and low-speed exercise on a supplemented diet. Fifteen-week old male C57BL/6 mice were assigned to seven groups-(1) baseline, (2) non-exercise fed a control diet, (3) non-exercise fed a supplemented diet, (4) low-speed exercise fed a control diet, (5) low-speed exercise fed a supplemented diet, (6) high-speed exercise fed a control diet, and (7) high-speed exercise fed a supplemented diet. Mice exercised thirty days for 20 min/day at 12 m/min or 20 m/min. Tibiae were assessed by micro-CT and 4-point bending. Cortical area fraction and trabecular bone volume fraction (BV/TV) were significantly increased by the supplemented diet. High-speed exercised mice had significantly lower body weight, with no detrimental effects to bone health. Increasing running speed can decrease body weight while maintaining the benefits of exercise and nutrition on bone health. Running can lower body weight without harming bone health.

Collagen cross-link profiles and mineral are different between the mandible and femur with site specific response to perturbed collagen.

Compromises to collagen and mineral lead to a decrease in whole bone quantity and quality in a variety of systemic diseases, yet, clinically, disease manifestations differ between craniofacial and long bones. Collagen alterations can occur through post-translational modification via lysyl oxidase (LOX), which catalyzes enzymatic collagen cross-link formation, as well as through non-enzymatic advanced glycation end products (AGEs) such as pentosidine and carboxymethyl-lysine (CML). Characterization of the cross-links and AGEs, and comparison of the mineral and collagen modifications in craniofacial and long bones represent a critical gap in knowledge. However, alterations to either the mineral or collagen in bone may contribute to disease progression and, subsequently, the anatomical site dependence of a variety of diseases. Therefore, we hypothesized that collagen cross-links and AGEs differ between craniofacial and long bones and that altered collagen cross-linking reduces mineral quality in an anatomic location dependent. To study the effects of cross-link inhibition on mineralization between anatomical sites, beta-aminoproprionitrile (BAPN) was administered to rapidly growing, 5-8 week-old male mice. BAPN is a dose-dependent inhibitor of LOX that pharmacologically alters enzymatic cross-link formation. Long bones (femora) and craniofacial bones (mandibles) were compared for mineral quantity and quality, collagen cross-link and AGE profiles, and tissue level mechanics, as well as the response to altered cross-links via BAPN. A highly sensitive liquid chromatography/mass spectrometry (LC-MS) method was developed which allowed for quantification of site-dependent accumulation of the advanced glycation end-product, carboxymethyl-lysine (CML). CML was ∼8.3× higher in the mandible than the femur. The mandible had significantly higher collagen maturation, mineral crystallinity, and Young's modulus, but lower carbonation, than the femur. BAPN also had anatomic specific effects, leading to significant decreases in mature cross-links in the mandible, and an increase in mineral carbonation in the femur. This differential response of both the mineral and collagen composition to BAPN between the mandible and femur highlights the need to further understand how inherent compositional differences in collagen and mineral contribute to anatomic-site specific manifestations of disease in both craniofacial and long bones.

Divergent mechanical properties of older human male femora reveal unique combinations of morphological and compositional traits contributing to low strength.

Bone strength is generally thought to decline with aging and prior work has compared traits between younger and older cohorts to identify the structural and compositional changes that contribute to fracture risk with age. However, for men, the majority of individuals do not fracture a bone in their lifetime. While fracture occurrence is multifactorial, the absence of fracture in the majority of males suggests that some individuals maintain bone strength or do not lose enough strength to fracture, whereas others do lose strength with aging. Consequently, not all structural and material changes observed with age may lead to strength-decline. We propose that consideration of different subgroups of older individuals will provide a more precise understanding of which structural and material changes directly contribute to strength-decline. We identified subgroups using latent profile analysis (LPA), which is a clustering-based algorithm that takes multiple continuous variables into account. Human cadaveric male femoral diaphyses (n = 33, 26-89 years) were subjected to whole bone and tissue-level mechanical tests. Morphological traits, porosity, and cortical tissue mineral density (Ct.TMD) were obtained, as were measures of enzymatic cross-links and the advanced glycation end product, pentosidine (PEN). A univariate analysis first identified a younger cohort (YNG, n = 11) and older cohort (n = 22). LPA was then conducted using three mechanical traits (whole bone strength, tissue-level strength, and tissue-level post-yield strain), resulting in a further stratification of the older group into two similarly aged groups (p = 0.558), but one with higher (OHM, n = 16) and another with lower mechanical properties (OLM, n = 6). The OLM group exhibited lower whole bone strength (p = 0.016), tissue-level strength (p < 0.001), and tissue-level post-yield strain (p < 0.001) compared to the YNG group. Meanwhile, the OHM only exhibited significantly lower tissue-level post-yield strain (p < 0.001), compared to the YNG group. Between the two older groups, the OHM group exhibited higher whole bone strength (p = 0.037), tissue-level strength (p = 0.006), and tissue-level post-yield strain (p = 0.012) than the OLM group. Probing the morphological and compositional relationships among the three groups, both OHM and OLM exhibited increased PEN content (p < 0.001, p = 0.008 respectively) and increased Log(cortical pore score) relative to YNG (p = 0.003, p < 0.001 respectively). Compared to the OHM group, the OLM also exhibited increased marrow area (p = 0.049), water content (p = 0.048), and decreased Ct.TMD (p = 0.005). The key traits that were unique to the OLM group compared to YNG were decreased Ct.TMD (p < 0.001) and increased Log(porosity) (p = 0.002). There were many properties that differed between the younger and older groups, but not all were associated with reduced mechanical properties, highlighting the relative importance of certain age-related traits such as porosity, Ct.TMD, water content, and marrow area that were unique to the OLM group. Overall, this work supports the hypothesis that there are subgroups of men showing different strength-decline trajectories with aging and establishes a basis for refining our understanding of which age-related changes are directly contributing to decreased strength.

Region-specific associations among tissue-level mechanical properties, porosity, and composition in human male femora.

Region-specific differences in age-related bone remodeling are known to exist. We therefore hypothesized that the decline in tissue-level strength and post-yield strain (PYS) with age is not uniform within the femur, but is driven by region-specific differences in porosity and composition. Four-point bending was conducted on anterior, posterior, medial, and lateral beams from male cadaveric femora (n = 33, 18-89 yrs of age). Mid-cortical porosity, composition, and mineralization were assessed using nano-computed tomography (nanoCT), Raman spectroscopy, and ashing assays. Traits between bones from young and elderly groups were compared, while multivariate analyses were used to identify traits that predicted strength and PYS at the regional level. We show that age-related decline in porosity and mechanical properties varied regionally, with highest positive slope of age vs. Log(porosity) found in posterior and anterior bone, and steepest negative slopes of age vs. strength and age vs. PYS found in anterior bone. Multivariate analyses show that Log(porosity) and/or Raman 1246/1269 ratio explained 46-51% of the variance in strength in anterior and posterior bone. Three out of five traits related to Log(porosity), mineral crystallinity, 1246/1269, mineral/matrix ratio, and/or hydroxyproline/proline (Hyp/Pro) ratio, explained 35-50% of the variance in PYS in anterior, posterior and lateral bones. Log(porosity) and Hyp/Pro ratio alone explained 13% and 19% of the variance in strength and PYS in medial bone, respectively. The predictive performance of multivariate analyses was negatively impacted by pooling data across all bone regions, underscoring the complexity of the femur and that the use of pooled analyses may obscure underlying region-specific differences.

Sex and External Size Specific Limitations in Assessing Bone Health From Adult Hand Radiographs.

Morphological parameters measured for the second metacarpal from hand radiographs are used clinically for assessing bone health during growth and aging. Understanding how these morphological parameters relate to metacarpal strength and strength at other anatomical sites is critical for providing informed decision-making regarding treatment strategies and effectiveness. The goals of this study were to evaluate the extent to which 11 morphological parameters, nine of which were measured from hand radiographs, relate to experimentally measured whole-bone strength assessed at multiple anatomical sites and to test whether these associations differed between men and women. Bone morphology and strength were assessed for the second and third metacarpals, radial diaphysis, femoral diaphysis, and proximal femur for 28 white male donors (18-89 years old) and 35 white female donors (36-89+ years old). The only morphological parameter to show a significant correlation with strength without a sex-specific effect was cortical area. Dimensionless morphological parameters derived from hand radiographs correlated significantly with strength for females, but few did for males. Males and females showed a significant association between the circularity of the metacarpal cross-section and the outer width measured in the mediolateral direction. This cross-sectional shape variation contributed to systematic bias in estimating strength using cortical area and assuming a circular cross-section. This was confirmed by the observation that use of elliptical formulas reduced the systematic bias associated with using circular approximations for morphology. Thus, cortical area was the best predictor of strength without a sex-specific difference in the correlation but was not without limitations owing to out-of-plane shape variations. The dependence of cross-sectional shape on the outer bone width measured from a hand radiograph may provide a way to further improve bone health assessments and informed decision making for optimizing strength-building and fracture-prevention treatment strategies. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Natural-abundance 43Ca solid-state NMR spectroscopy of bone.

Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Radiation-induced changes to bone composition extend beyond periosteal bone.

BACKGROUND: Cancer patients receiving radiotherapy for soft tissue sarcomas are often at risk of post-irradiation (post-RTx) bone fragility fractures, but our understanding of factors controlling radiation-induced bone injury is limited. Previous studies have evaluated post-RTx changes to cortical bone composition in the periosteum of irradiated tibiae, but have not evaluated effects of irradiation in deeper tissues, such as endosteal or mid-cortical bone, and whether there are differential spatial effects of irradiation. In this study, we hypothesize that post-RTx changes to cortical bone composition are greater in endosteal compared to mid-cortical or periosteal bone. METHODS: A pre-clinical mouse model of limited field hindlimb irradiation was used to evaluate spatial and temporal post-RTx changes to the metaphyseal cortex of irradiated tibiae. Irradiation was delivered unilaterally to the hindlimbs of 12-wk old female BALB/cJ mice as 4 consecutive daily doses of 5 Gy each. RTx and non-RTx tibiae were obtained at 0, 2, 4, 8, and 12 wks post-RTx (n = 9 mice/group/time). Raman spectroscopy was used to evaluate spatial and temporal post-RTx changes to cortical bone composition in age-matched RTx and non-RTx groups. RESULTS: Significant early spatial differences in mineral/matrix and collagen crosslink ratios were found between endosteal and periosteal or mid-cortical bone at 2-wks post-RTx. Although spatial differences were transient, mineral/matrix ratios significantly decreased and collagen crosslink ratios significantly increased with post-RTx time throughout the entire tibial metaphyseal cortex. CONCLUSIONS: Irradiation negatively impacts the composition of cortical bone in a spatially-dependent manner starting as early as 2-wks post-RTx. Long-term progressive post-RTx changes across all cortical bone sites may eventually contribute to the increased risk of post-RTx bone fragility fractures.

Loss of BMP signaling mediated by BMPR1A in osteoblasts leads to differential bone phenotypes in mice depending on anatomical location of the bones.

Bone morphogenetic protein (BMP) signaling in osteoblasts plays critical roles in skeletal development and bone homeostasis. Our previous studies showed loss of function of BMPR1A, one of the type 1 receptors for BMPs, in osteoblasts results in increased trabecular bone mass in long bones due to an imbalance between bone formation and bone resorption. Decreased bone resorption was associated with an increased mature-to-immature collagen cross-link ratio and mineral-matrix ratios in the trabecular compartments, and increased tissue-level biomechanical properties. Here, we investigated the bone mass, bone composition and biomechanical properties of ribs and spines in the same genetically altered mouse line to compare outcomes by loss of BMPR1A functions in bones from different anatomic sites and developmental origins. Bone mass was significantly increased in both cortical and trabecular compartments of ribs with minimal to modest changes in compositions. While tissue-levels of biomechanical properties were not changed between control and mutant animals, whole bone levels of biomechanical properties were significantly increased in association with increased bone mass in the mutant ribs. For spines, mutant bones showed increased bone mass in both cortical and trabecular compartments with an increase of mineral content. These results emphasize the differential role of BMP signaling in osteoblasts in bones depending on their anatomical locations, functional loading requirements and developmental origin.

Time-resolved dehydration-induced structural changes in an intact bovine cortical bone revealed by solid-state NMR spectroscopy.

Understanding the structure and structural changes of bone, a highly heterogeneous material with a complex hierarchical architecture, continues to be a significant challenge even for high-resolution solid-state NMR spectroscopy. While it is known that dehydration affects mechanical properties of bone by decreasing its strength and toughness, the underlying structural mechanism at the atomic level is unknown. Solid-state NMR spectroscopy, controlled dehydration, and H/D exchange were used for the first time to reveal the structural changes of an intact piece of bovine cortical bone. (1)H spectra were used to monitor the dehydration of the bone inside the rotor, and high-resolution (13)C chemical shift spectra obtained under magic-angle spinning were used evaluate the dehydration-induced conformational changes in the bone. The experiments revealed the slow denaturation of collagen due to dehydration while the trans-Xaa-Pro conformation in collagen remained unchanged. Our results suggest that glycosaminoglycans in the collagen fiber and mineral interface may chelate with a Ca(2+) ion present on the surface of the mineral through sulfate or carboxylate groups. These results provide insights into the role of water molecules in the bone structure and shed light on the relationship between the structure and mechanics of bone.