Small-Angle X-ray Scattering Demonstrates Similar Nanostructure in Cortical Bone from Young Adult Animals of Different Species.

Despite the vast amount of studies focusing on bone nanostructure that have been performed for several decades, doubts regarding the detailed structure of the constituting hydroxyapatite crystal still exist. Different experimental techniques report somewhat different sizes and locations, possibly due to different requirements for the sample preparation. In this study, small- and wide-angle X-ray scattering is used to investigate the nanostructure of femur samples from young adult ovine, bovine, porcine, and murine cortical bone, including three different orthogonal directions relative to the long axis of the bone. The radially averaged scattering from all samples reveals a remarkable similarity in the entire q range, which indicates that the nanostructure is essentially the same in all species. Small differences in the data from different directions confirm that the crystals are elongated in the [001] direction and that this direction is parallel to the long axis of the bone. A model consisting of thin plates is successfully employed to describe the scattering and extract the plate thicknesses, which are found to be in the range of 20-40 Å for most samples but 40-60 Å for the cow samples. It is demonstrated that the mineral plates have a large degree of polydispersity in plate thickness. Additionally, and equally importantly, the scattering data and the model are critically evaluated in terms of model uncertainties and overall information content.

Characterising bone material composition and structure in the ovariectomized (OVX) rat model of osteoporosis.

The ovariectomized (OVX) rat model is well established in investigations of osteoporosis and osteoporotic therapies. Advent of techniques such as Fourier-transform infrared (FTIR) spectroscopy and small angle X-ray scattering (SAXS) facilitate characterization of bone composition and mineral structure, respectively, which are key determinants of bone strength. Limited publications exist on the implementation of these techniques in the OVX rat model. At 12 weeks of age, female Sprague-Dawley rats were either sham-operated (n = 6) or ovariectomized (n = 6) and sacrificed 18 weeks later. L2 lumbar vertebrae and proximal tibiae were assessed by µCT, FTIR and SAXS. Presence of extensive trabecular deterioration in the µCT data confirmed the onset of osteoporosis. FTIR compositional parameters were determined including measures of degree of mineralization, crystallinity, collagen maturity and acid phosphate content. Mineral crystal thickness was determined from the SAXS data using two approaches available in literature. Compositionally, a decline in the heterogeneity of acid phosphate content was observed while measures of crystallinity and collagen maturity remained unaltered. Using an iterative curve fitting method, OVX-induced increases in the mineral crystal thickness of 3.8 and 7.8 % (p < 0.05) were noted in the trabecular of the vertebra and tibia, respectively. In conclusion, implementation of FTIR and SAXS techniques in the OVX rat model, identified no significant compositional changes while substantiating thickening of the mineral crystals as a general structural feature of OVX-induced osteoporosis in rats.

Mouse models of accelerated aging in musculoskeletal research for assessing frailty, sarcopenia, and osteoporosis - A review.

Musculoskeletal aging encompasses the decline in bone and muscle function, leading to conditions such as frailty, osteoporosis, and sarcopenia. Unraveling the underlying molecular mechanisms and developing effective treatments are crucial for improving the quality of life for those affected. In this context, accelerated aging models offer valuable insights into these conditions by displaying the hallmarks of human aging. Herein, this review focuses on relevant mouse models of musculoskeletal aging with particular emphasis on frailty, osteoporosis, and sarcopenia. Among the discussed models, PolgA mice in particular exhibit hallmarks of musculoskeletal aging, presenting early-onset frailty, as well as reduced bone and muscle mass that closely resemble human musculoskeletal aging. Ultimately, findings from these models hold promise for advancing interventions targeted at age-related musculoskeletal disorders, effectively addressing the challenges posed by musculoskeletal aging and associated conditions in humans.

Protocol for preparing formalin-fixed paraffin-embedded musculoskeletal tissue samples from mice for spatial transcriptomics.

Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We describe steps for tissue harvesting, sample preparation, paraffin embedding, and FFPE sample selection. We detail procedures for sectioning and placement on spatial slides prior to imaging, decrosslinking, library preparation, and final analyses of the sequencing data. The complete protocol takes ca. 18 days for mouse femora with adjacent muscle; of this time, >50% is required for mineralized tissue decalcification. For complete details on the use and execution of this protocol, please refer to Wehrle et al.1 and Mathavan et al.2.

Longitudinal in vivo monitoring of callus remodeling in BMP-7- and Zoledronate-treated fractures.

Pharmacological interventions that combine pro-anabolic and anti-catabolic drugs to treat recalcitrant fractures have shown remarkable efficacy in augmenting the regenerative response. Specifically, in rodent models of fracture repair, treatment with BMP-7 and Zoledronate (ZA) has almost uniformally resulted in complete union. However, delayed remodeling may be problematic for ZA-treated fractures. The increase in newly formed bone is substantial but if translated in humans, delayed remodeling may delay functional recovery. Our objective was to determine if, and to what extent, bone morphogenetic protein (BMP) (in synergistically administered BMP-7 + ZA) can modulate the delayed hard callus remodeling caused by ZA. Callus remodeling in BMP-7-only and BMP-7 + ZA-treated osteotomies were monitored using in vivo µCT to follow the progression of healing at 6-week intervals over 24 weeks in an open femoral fracture rat model. None of the groups recovered baseline cortical bone volumes within 24 weeks post-osteotomy. Treatment prolonged the remodeling phase but the kinetics of remodeling appeared to differ between BMP and BMP + ZA groups. However, the mechanical characteristics were largely restored. Callus/bone volumes in BMP-only treated fractures peaked as early as week 3 suggesting that remodeling is stimulated prematurely. However, this rate of remodeling was not maintained as BMP-7 was found to exhibit negligible changes in callus/bone volumes between weeks 6 and 18, whereas declines in callus/bone volumes were present at these time points in the BMP-7 + ZA group. Our findings suggest that inclusion of ZA as an anti-catabolic agent may not be detrimental to the regenerative process despite a prolonged remodeling phase.

18F-fluoride as a prognostic indicator of bone regeneration.

Positron emission tomography (PET) is a form of nuclear imaging, which quantitatively assesses the metabolic activity through the uptake of radioactive tracers. 18F-fluoride is a positron-emitting isotope with high affinity for bone. Despite its potential as a non-invasive measure of bone metabolism, quantitative 18F-fluoride PET has only been used sparsely in orthopaedic applications. It has been speculated that 18F-fluoride PET characterizes cellular activity of bone forming cells in the early stages of the regenerative process and therefore precedes the mineralization detected by conventional computed tomography (CT). Our aim was thus to combine in vivo PET and CT to map the spatiotemporal course of bone regeneration during fracture healing using an open femur fracture model in the rat and characterize regeneration in untreated and pharmacologically treated fractures using both imaging modalities. We hypothesized that PET 18F-fluoride tracer activity at an earlier time point is predictive of CT measured bone formation at a later time point. On the basis of the RMSE and R2 metrics of linear regression models it was conceivable for bone volumes to be predicted up to three weeks in advance in a rodent model (RMSE: 14 mm3-18 mm3, R2: 0.79-0.82). Moreover, the data suggested that 18F-fluoride positron-emitting activity had the potential to separate bone formation from resorption and thus could be of interest across a wide array of orthopaedic applications. Based on this data, we conclude that 18F-fluoride positron-emitting activity is strongly correlated to bone formation and could potentially predict the volume of bone regenerated at fracture sites. The volume of bone regenerated at a fracture site can be interpreted as a measure of the healing response and 18F-fluoride should be further investigated as a predictive diagnostic tool to identify if bone fractures will heal successfully or result in delayed healing or non-union. STATEMENT OF SIGNIFICANCE: We aimed to combine in vivo PET and CT imaging to map the spatiotemporal course of bone regeneration during fracture healing using an open femur fracture model in the rat and characterize regeneration in untreated and pharmacologically treated fractures using both imaging modalities. We hypothesized that PET 18F-fluoride tracer activity at an earlier time point is predictive of CT measured bone formation at a later time point. Our data suggest that 18F-fluoride positron-emitting activity can separate bone formation from resorption and thus could be of interest across a wide array of orthopaedic applications including as a predictive diagnostic tool to identify if fractures will heal successfully or result in delayed healing or non-union.

The compositional and nano-structural basis of fracture healing in healthy and osteoporotic bone.

Osteoporosis, a prevalent metabolic bone disorder, predisposes individuals to increased susceptibility to fractures. It is also, somewhat controversially, thought to delay or impair the regenerative response. Using high-resolution Fourier-transform infrared spectroscopy and small/wide-angle X-ray scattering we sought to answer the following questions: Does the molecular composition and the nano-structure in the newly regenerated bone differ between healthy and osteoporotic environments? And how do pharmacological treatments, such as bone morphogenetic protein 7 (BMP-7) alone or synergistically combined with zoledronate (ZA), alter callus composition and nano-structure in such environments? Cumulatively, on the basis of compositional and nano-structural characterizations of newly formed bone in an open-osteotomy rat model, the healing response in untreated healthy and ovariectomy-induced osteoporotic environments was fundamentally the same. However, the BMP-7 induced osteogenic response resulted in greater heterogeneity in the nano-structural crystal dimensions and this effect was more pronounced with osteoporosis. ZA mitigated the effects of the upregulated catabolism induced by both BMP-7 and an osteoporotic bone environment. The findings contribute to our understanding of how the repair processes in healthy and osteoporotic bone differ in both untreated and treated contexts and the data presented represents the most comprehensive study of fracture healing at the nanoscale undertaken to date.

Linking multiscale deformation to microstructure in cortical bone using in situ loading, digital image correlation and synchrotron X-ray scattering.

The incidence of fragility fractures is expected to increase in the near future due to an aging population. Therefore, improved tools for fracture prediction are required to treat and prevent these injuries efficiently. For such tools to succeed, a better understanding of the deformation mechanisms in bone over different length scales is needed. In this study, an experimental setup including mechanical tensile testing in combination with digital image correlation (DIC) and small/wide angle X-ray scattering (SAXS/WAXS) was used to study deformation at multiple length scales in bovine cortical bone. Furthermore, micro-CT imaging provided detailed information about tissue microstructure. The combination of these techniques enabled measurements of local deformations at the tissue- and nanoscales. The orientation of the microstructure relative to the tensile loading was found to influence the strain magnitude on all length scales. Strains in the collagen fibers were 2-3 times as high as the strains found in the mineral crystals for samples with microstructure oriented parallel to the loading. The local tissue strain at fracture was found to be around 0.5%, independent of tissue orientation. However, the maximum force and the irregularity of the crack path were higher when the load was applied parallel to the tissue orientation. This study clearly shows the potential of combining these different experimental techniques concurrently with mechanical testing to gain a better understanding of bone damage and fracture over multiple length scales in cortical bone. STATEMENT OF SIGNIFICANCE: To understand the pathophysiology of bone, it is important to improve our knowledge about the deformation and fracture mechanisms in bone. In this study, we combine several recently available experimental techniques with mechanical loading to investigate the deformation mechanisms in compact bone tissue on several length scales simultaneously. The experimental setup included mechanical tensile testing in combination with digital image correlation, microCT imaging, and small/wide angle X-ray scattering. The combination of techniques enabled measurements of local deformations at the tissue- and nanoscales. The study clearly shows the potential of combining different experimental techniques concurrently with mechanical testing to gain a better understanding of structure-property-function relationships in bone tissue.

Biomechanical properties of bone in a mouse model of Rett syndrome.

Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2(stop/y) male mice in which Mecp2 is silenced in all cells and female Mecp2(stop/+) mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies.

Cannabidiol, a Major Non-Psychotropic Cannabis Constituent Enhances Fracture Healing and Stimulates Lysyl Hydroxylase Activity in Osteoblasts.

Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non-psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid-femoral fractures. The maximal load and work-to-failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ(9) -tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work-to-failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro-computed tomography (µCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes.

Investigating the synergistic efficacy of BMP-7 and zoledronate on bone allografts using an open rat osteotomy model.

Bone grafts are well-established in the treatment of fracture non-unions but union is still not always achieved. Harvesting autograft is associated with donor site morbidity and the available amount of bone is limited. Allograft is more easily obtained and available in greater quantities but lacks the osteoinductive characteristics of autograft. We have previously shown a synergistic effect of bone morphogenetic protein (BMP-7), systemic bisphosphonates and autograft. In the present study we hypothesized that the combination of allograft+BMP-7+systemic ZA is more effective than autograft alone, which is currently the most frequently used aid in augmenting fracture and non-union healing. Femoral osteotomies were performed on 82 male Sprague Dawley rats and fixed with intramedullary K-wires. The rats were randomized into 7 groups: (i) saline, (ii) autograft, (iii) allograft, (iv) allograft+BMP-7, (v) autograft+zoledronate (ZA), (vi) allograft+ZA and (vii) allograft+BMP-7+ZA. Autografts were harvested from the contralateral tibia. Allografts were obtained from donor rats and frozen. BMP-7 was administered locally in the form of a putty placed circumferentially around the osteotomy. At 2 weeks, the rats were injected with a single dose of either saline or ZA. The rats were sacrificed at 6 weeks and the femurs were evaluated using radiography, histology, µCT and three-point bending tests. Complete radiological healing was seen in all rats in the BMP-7 groups. The callus volume was larger and the calluses were denser with allograft+BMP-7+ZA than in all other groups (µCT, p<0.001). Mechanical testing yielded a substantially higher peak force with the allograft+BMP-7+ZA combination than all other groups (p<0.01, p<0.001). This was further reinforced in the 59% increase in the peak force observed in the osteotomized femurs of the allograft+BMP-7+ZA group compared to the control femurs (p<0.01), whereas significant decreases of 22-27% were observed in the saline or bone-graft alone groups (p<0.01, p<0.05). Thus our results suggest that allograft combined with the anabolic effect of BMP-7 and the anti-catabolic effect of zoledronate is more efficient than autograft alone.