Bone Matrix Maturation Research Articles

Preparation of decellularized bone graft material with supercritical carbon dioxide extraction technique.

To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide. Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (conventional control group) or supercritical carbon dioxide (experimental group). Clearance rate of galactose-α-1, 3-galactose (α-Gal) and residual DNA of the two groups were analyzed to assess the immunogenicity of the xenogenic materials. Clearance rate of α-Gal was determined by enzyme-linked immunosorbent assay and residual DNA was detected by fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental group, conventional control group and positive control group. Samples were implanted over biceps femoris muscle of athymic nude mice, the explants were collected 4 weeks post implantation, hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants. The clearance rates of α-Gal antigen in the experimental group and the conventional control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental group, the conventional control group and the positive control group were (13.49±0.07) ng/mg, (15.20±0.21) ng/mg and (14.70±0.17) ng/mg, the residual DNA in the experimental group was significantly lower than that in the conventional control group (t=－13.41, P<0.01) and the positive control group (t=－11.30, P<0.01). HE staining showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, only a small number of bone formation centers were observed in the conventional control group and the positive control group, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, typeⅠcollagen and osteocalcin in the experimental group showed an increasing trend compared with those in the conventional control group and the positive control group. Compared with clinically used demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.

Decellularized Antler Cancellous Bone Matrix Material Can Serve as Potential Bone Tissue Scaffold.

Due to the limited supply of autologous bone grafts, there is a need to develop more bone matrix materials to repair bone defects. Xenograft bone is expected to be used for clinical treatment due to its exact structural similarity to natural bone and its high biocompatibility. In this study, decellularized antler cancellous bone matrix (DACB) was first prepared, and then the extent of decellularization of DACB was verified by histological staining, which demonstrated that it retained the extracellular matrix (ECM). The bioactivity of DACB was assessed using C3H10T1/2 cells, revealing that DACB enhanced cell proliferation and facilitated cell adhesion and osteogenic differentiation. When evaluated by implanting DACB into nude mice, there were no signs of necrosis or inflammation in the epidermal tissues. The bone repair effect of DACB was verified in vivo using sika deer during the antler growth period as an animal model, and the molecular mechanisms of bone repair were further evaluated by transcriptomic analysis of the regenerated tissues. Our findings suggest that the low immunogenicity of DACB enhances the production of bone extracellular matrix components, leading to effective osseointegration between bone and DACB. This study provides a new reference for solving bone defects.

Bone matrix properties in adults with osteogenesis imperfecta are not adversely affected by setrusumab-a sclerostin neutralizing antibody.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by low bone mass and frequent fractures. Children with OI are commonly treated with bisphosphonates to reduce fracture rate, but treatment options for adults are limited. In the Phase 2b ASTEROID trial, setrusumab (a sclerostin neutralizing antibody, SclAb) improved bone density and strength in adults with type I, III, and IV OI. Here, we investigate bone matrix material properties in tetracycline-labeled trans iliac biopsies from 3 groups: (1) control: individuals with no metabolic bone disease, (2) OI: individuals with OI, (3) SclAb-OI: individuals with OI after 6 mo of setrusumab treatment (as part of the ASTEROID trial). In addition to bone histomorphometry, bone mineral and matrix properties were evaluated with nanoindentation, Raman spectroscopy, second harmonic generation imaging, quantitative backscatter electron imaging, and small-angle X-ray scattering. Spatial locations of fluorochrome labels were identified to differentiate inter-label bone of the same tissue age and intra-cortical bone. No difference in collagen orientation was found between the groups. The bone mineral density distribution and analysis of Raman spectra indicate that OI groups have greater mean mineralization, greater relative mineral content, and lower crystallinity than the control group, which was not altered by SclAb treatment. Finally, a lower modulus and hardness were measured in the inter-label bone of the OI-SclAb group compared to the OI group. Previous studies suggest that even though bone from OI has a higher mineral content, the extracellular matrix (ECM) has comparable mechanical properties. Therefore, fragility in OI may stem from contributions from other yet unexplored aspects of bone organization at higher length scales. We conclude that SclAb treatment leads to increased bone mass while not adversely affecting bone matrix properties in individuals with OI.

High-fat and high-carbohydrate diets increase bone fragility through TGF-β-dependent control of osteocyte function.

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-β signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-β signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-β signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-β receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Preparation and in vivo osteogenesis of acellular dermal matrix/dicalcium phosphate composite scaffold for bone repair

To investigate the physicochemical properties, osteogenic properties, and osteogenic ability in rabbit model of femoral condylar defect of acellular dermal matrix (ADM)/dicalcium phosphate (DCP) composite scaffold. ADM/DCP composite scaffolds were prepared by microfibril technique, and the acellular effect of ADM/DCP composite scaffolds was detected by DNA residue, fat content, and α-1，3-galactosyle (α-Gal) epitopes; the microstructure of scaffolds was characterized by field emission scanning electron microscopy and mercury porosimetry; X-ray diffraction was used to analyze the change of crystal form of scaffold; the solubility of scaffolds was used to detect the pH value and calcium ion content of the solution; the mineralization experiment in vitro was used to observe the surface mineralization. Twelve healthy male New Zealand white rabbits were selected to prepare the femoral condylar defect models, and the left and right defects were implanted with ADM/DCP composite scaffold (experimental group) and skeletal gold ® artificial bone repair material (control group), respectively. Gross observation was performed at 6 and 12 weeks after operation; Micro-CT was used to detect and quantitatively analyze the related indicators [bone volume (BV), bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), bone mineral density (BMD)], and HE staining and Masson staining were performed to observe the repair of bone defects and the maturation of bone matrix. Gross observation showed that the ADM/DCP composite scaffold was a white spongy solid. Compared with ADM, ADM/DCP composite scaffolds showed a significant decrease in DNA residue, fat content, and α-Gal antigen content ( P<0.05). Field emission scanning electron microscopy showed that the ADM/DCP composite scaffold had a porous structure, and DCP particles were attached to the porcine dermal fibers. The porosity of the ADM/DCP composite scaffold was 76.32%±1.63% measured by mercury porosimetry. X-ray diffraction analysis showed that the crystalline phase of DCP in the ADM/DCP composite scaffolds remained intact. Mineralization results in vitro showed that the hydroxyapatite layer of ADM/DCP composite scaffolds was basically mature. The repair experiment of rabbit femoral condyle defect showed that the incision healed completely after operation without callus or osteophyte. Micro-CT showed that bone healing was complete and a large amount of new bone tissue was generated in the defect site of the two groups, and there was no difference in density between the defect site and the surrounding bone tissue, and the osteogenic properties of the two groups were equivalent. There was no significant difference in BV, BV/TV, BS/BV, Tb.Th, Tb.N, and BMD between the two groups ( P>0.05), except that the Tb.Sp in the experimental group was significantly higher than that in the control group ( P<0.05). At 6 and 12 weeks after operation, HE staining and Masson staining showed that the new bone and autogenous bone fused well in both groups, and the bone tissue tended to be mature. The ADM/DCP composite scaffold has good biocompatibility and osteogenic ability similar to the artificial bone material in repairing rabbit femoral condylar defects. It is a new scaffold material with potential in the field of bone repair.

Possible involvement of HtrA1 serine protease in the onset of osteoporotic bone extracellular matrix changes

High-temperature requirement A1 (HtrA1), a multidomain serine protease acting on Extracellular matrix (ECM) rearrangement, is also secreted by osteoblasts and osteoclasts. Recent and conflicting literature highlights HtrA1's role as a controller of bone remodeling, proposing it as a possible target for pathologies with unbalanced bone resorption, like Osteoporosis (OP). To add knowledge on this molecule function in bone physiopathology, here we compared HtrA1 distribution in the ECM of healthy (H) and OP bone tissue, also examining its localization in the sites of new bone formation. HtrA1 was homogeneously expressed in the mature bone ECM of H tissue showing a 55.6 ± 16.4% of the stained area, with a significant (p=0.0001) decrease in OP percentage stained area (21.1 ± 13.1). Moreover, HtrA1 was present in the endosteum and cells involved in osteogenesis, mainly in those “entrapped” in woven bone, whereas osteocytes in mature lamellar bone were negative. Based on our previous observation in OP tissue of a significantly increased expression of Decorin and Osteocalcin, both involved in bone mineralization and remodeling and equally substrates for HtrA1, we speculate that HtrA1 by controlling the proper amount of Decorin and Osteocalcin favors normal bone maturation and mineralization. Besides, we suggest that late-osteoblasts and pre-osteocytes secrete HtrA1 in the adjacent matrix whilst proceeding with their maturation and that HtrA1 expression is further modified during the remodeling from woven to the lamellar bone. Overall, our data suggest HtrA1 as a positive regulator of bone matrix formation and maturation: its reduced expression in mature OP bone, affecting protein content and distribution, could hamper correct bone remodeling and mineralization.

Canine soft tissue sarcomas: the expression of RUNX2 and karyopherin alpha-2 in extraskeletal (soft tissues) and skeletal osteosarcomas.

Extraskeletal osteosarcoma (EOS) is a malignant tumor producing bone matrix and/or chondroid material, without direct attachment to bone or periosteum. In humans and dogs, EOS is highly infiltrating, rapidly growing, often characterized by osteoid deposition and variable ossification, similar to primary skeletal osteosarcoma (SOS). In dogs, EOS arises from visceral and soft tissue locations, occasionally in trauma or foreign body sites, or in granulomas. Few data are currently available on the phenotype of these tumors. The present study aims to assess the expression RUNX2 and Karyopherin alpha-2 in EOS, comparing it with SOS and the data available from the human counterpart. Seventeen cases of canine osteosarcoma (13 EOS and 4 SOS) were retrospectively selected and submitted to immunohistochemistry for RUNX2 and Karyopherin alpha-2. Our results showed that, in EOS, RUNX2 is expressed in a mean of 73.07 ± 5.36 neoplastic cell nuclei, in face of a mean 36.15 ± 6.25 of Karyopherin alpha-2 positive nuclei. Osteoclasts, when present, were negative for both markers. No correlation was observed among the two markers (p > 0.05), nor statistically significant difference in quantitative expression was assessed comparing EOS and SOS groups. RUNX2 is expressed in canine EOS similarly to SOS and could be used as a diagnostic marker in a larger panel. Karyopherin alpha-2 is expressed in canine EOS and SOS similarly to human SOS and could be validated in future studies as an additional diagnostic marker. Further studies should be planned to evaluate the expression of these proteins as prognostic predictive parameters.

Validierung des Lower Extremity Early Rehabilitation Score-13 (LERAS-13) – Ein neuer Fragebogen für die frühe Rehabilitationsphase nach Verletzungen der unteren Extremitäten

The performance of the porous glass ceramic doped with 10 wt% Zinc and 2 wt% TiN (46S6-10Zn), in the restoration of critical diaphyseal bone defect, was evaluated by several physicochemical methods and histological studies. The critical defect in rabbits was created and then filled with 46S6-10Zn. At different periods after implementation, animals were sacrificed. Samples were harvested for exploration. The nuclear magnetic resonance (MAS-NMR) of 31P and 29Si illustrates the progressive degradation of 46S6-10Zn in favor to of the formation and the development of biological apatite. Therefore, after one month of implementation, MAS-NMR 29Si proves the presence of Q2 (25%), Q3 (73%) and Q4 (2%). However, after six months, the disappearance of all these species was revealed and characterized by the 46S6-10Zn dissolution. Besides, MAS-NMR 31P demonstrates the presence of QC0 (4%), QHA0 (55%) and Qa0 (41%) after one month. Nevertheless, six months later, we observe the presence of QHA0 (80%) and Qa0 (20%). Histological study demonstrates an intimate contact of 46S6-10Zn surrounding bone after one month of implantation. However, after four months, mature bone matrix became calcified and the implanted 46S6-10Zn began to be degraded. Moreover, nine months later, 46S6-10Zn was nearly resorbed and replaced by a calcified tissue in the periphery and an osteoid tissue in the middle of bone defects.

Abnormal kynurenine level contributes to the pathological bone features of ankylosing spondylitis.

Ankylosing spondylitis (AS) exhibits paradoxical bone features typically characterized by new bone formation and systemic bone loss. Although abnormal kynurenine (Kyn), a tryptophan metabolite, has been closely linked to the disease activity of AS, the distinct role of its pathological bone features remains unknown. Kynurenine sera level was collected from healthy control (HC; n=22) and AS (n=87) patients and measured by ELISA. In the AS group, we analyzed and compared the Kyn level based on the modified stoke ankylosing spondylitis spinal score (mSASSS), MMP13, and OCN. Under osteoblast differentiation, the treatment with Kyn in AS-osteoprogenitors conducted cell proliferation, alkaline phosphatase activity, bone mineralization-related alizarin red s (ARS), von kossa (VON), hydroxyapatite (HA) staining, and mRNA expression markers (ALP, RUNX2, OCN, and OPG) for bone formation. TRAP and F-actin staining was used for osteoclast formation of mouse osteoclast precursors. Kyn sera level was significantly elevated in the AS group compared to the HC. In addition, Kyn sera level was correlated with mSASSS (r=0.03888, p=0.067), MMP13 (r=0.0327, p=0.093), and OCN (r=0.0436, p=0.052). During osteoblast differentiation, treatment with Kyn exhibited no difference in cell proliferation and alkaline phosphate (ALP) activity for bone matrix maturation but promoted ARS, VON, and HA staining for bone mineralization. Interestingly, osteoprotegerin (OPG) and OCN expressions of AS-osteoprogenitors were augmented in the Kyn treatment during differentiation. In growth medium, Kyn treatment of AS-osteoprogenitors resulted in induction of OPG mRNA, protein expression, and Kyn-response genes (AhRR, CYP1b1, and TIPARP). Secreted OPG proteins were observed in the supernatant of AS-osteoprogenitors treated with Kyn. Notably, the supernatant of Kyn-treated AS-osteoprogenitors interrupted the RANKL-mediated osteoclastogenesis of mouse osteoclast precursor such as TRAP-positive osteoclast formation, NFATc1 expression, and osteoclast differentiation markers. Our results revealed that elevated Kyn level increased the bone mineralization of osteoblast differentiation in AS and decreased RANKL-mediated osteoclast differentiation by inducing OPG expression. Out study have implication for potential coupling factors linking osteoclast and osteoblast where abnormal Kyn level could be involved in pathological bone features of AS.

Craniotomy Simulator with Force Myography and Machine Learning-Based Skills Assessment.

Craniotomy is a fundamental component of neurosurgery that involves the removal of the skull bone flap. Simulation-based training of craniotomy is an efficient method to develop competent skills outside the operating room. Traditionally, an expert surgeon evaluates the surgical skills using rating scales, but this method is subjective, time-consuming, and tedious. Accordingly, the objective of the present study was to develop an anatomically accurate craniotomy simulator with realistic haptic feedback and objective evaluation of surgical skills. A CT scan segmentation-based craniotomy simulator with two bone flaps for drilling task was developed using 3D printed bone matrix material. Force myography (FMG) and machine learning were used to automatically evaluate the surgical skills. Twenty-two neurosurgeons participated in this study, including novices (n = 8), intermediates (n = 8), and experts (n = 6), and they performed the defined drilling experiments. They provided feedback on the effectiveness of the simulator using a Likert scale questionnaire on a scale ranging from 1 to 10. The data acquired from the FMG band was used to classify the surgical expertise into novice, intermediate and expert categories. The study employed naïve Bayes, linear discriminant (LDA), support vector machine (SVM), and decision tree (DT) classifiers with leave one out cross-validation. The neurosurgeons' feedback indicates that the developed simulator was found to be an effective tool to hone drilling skills. In addition, the bone matrix material provided good value in terms of haptic feedback (average score 7.1). For FMG-data-based skills evaluation, we achieved maximum accuracy using the naïve Bayes classifier (90.0 ± 14.8%). DT had a classification accuracy of 86.22 ± 20.8%, LDA had an accuracy of 81.9 ± 23.6%, and SVM had an accuracy of 76.7 ± 32.9%. The findings of this study indicate that materials with comparable biomechanical properties to those of real tissues are more effective for surgical simulation. In addition, force myography and machine learning provide objective and automated assessment of surgical drilling skills.

Clinical, Radiographic, and Histological Evaluation of the Mineralized Plasmatic Matrix/Xenograft Mixture in Maxillary Sinus Floor Augmentation (A Randomized Controlled Clinical Trial)

Introduction: Maxillary sinus pneumatization and alveolar ridge resorption following the extraction of posterior teeth make the installation of dental implants in the maxillary posterior region challenging. The direct sinus lift procedure proved to be a viable treatment option for such conditions. Aim of the study: to evaluate the mineralized plasmatic matrix/xenograft mixture in sinus elevation surgery. Materials and Methods: Eighteen patients were selected and randomly allocated into two groups; study group received a mineralized plasmatic matrix/xenograft mixture, while the control group received xenograft alone following sinus lifting. Results: The early wound healing index score showed a non-significant difference between both groups. Also, bone height was evaluated at the 6-month follow-up period, and there was a non-statistically significant difference. Core biopsies were taken for histological examination by H&amp;E from both groups, revealing the presence of a more mature bone matrix in relation to the test group. Conclusion: The addition of mineralized plasmatic matrix to xenograft can speed up bone formation, thus reducing treatment duration.

Materials Used in Cranial Reconstruction: A Systematic Review and Meta-Analysis

Cranioplasty is a common neurologic procedure, with complication rates ranging from 20% to 50%. It is hypothesized that the risks of various complications are affected by which material is used for cranioplasty. To evaluate the literature comparing rates of complications after cranioplasty using different materials including autologous bone, hydroxyapatite, methyl methacrylate, demineralized bone matrix, polyetheretherketone, titanium, or composite materials. The PubMed/MEDLINE database was searched for relevant articles published between 2010 and 2020. After screening, 35 articles were included. Outcomes included infection, wound problems, poor cosmesis, overall complications, duration of surgery, and length of stay. For each outcome, a frequentist network meta-analysis was conducted to compare materials used. The risk of infection was 1.62 times higher when methyl methacrylate was used compared with autologous bone (relative risk, 1.62; 95% confidence interval [CI], 1.07-2.45). Length of stay after cranioplasty was on average 3.62 days shorter when titanium was used compared with autologous bone (95% CI, 6.26 to -0.98). The networks constructed for other outcomes showed moderate to substantial between-study heterogeneity, wide CIs, and no significant differences between materials. The quality of existing literature on this topic is relatively poor, almost exclusively comprising single-center retrospective studies. There is not strong enough evidence available to make comprehensive conclusions regarding the risk profiles of various cranioplasty materials across multiple outcomes. Prospective randomized trials are necessary to confirm the significant results found in this analysis and to further elucidate the differential risks of various cranioplasty materials.

The Signalling Effects of Photobiomodulation on Osteoblast Proliferation, Maturation and Differentiation: A Review.

Proliferation of osteoblasts is essential for maturation and mineralization of bone matrix. Ossification, the natural phase of bone-forming and hardening is a carefully regulated phase where deregulation of this process may result in insufficient or excessive bone mineralization or ectopic calcification. Osteoblasts can also be differentiated into osteocytes, populating short interconnecting passages within the bone matrix. Over the past few decades, we have seen a significant improvement in awareness and techniques using photobiomodulation (PBM) to stimulate cell function. One of the applications of PBM is the promotion of osteoblast proliferation and maturation. PBM research results on osteoblasts showed increased mitochondrial ATP production, increased osteoblast activity and proliferation, increased and pro-osteoblast expression in the presence of red and NIR radiation. Osteocyte differentiation was also accomplished using blue and green light, showing that different light parameters have various signalling effects. The current review addresses osteoblast function and control, a new understanding of PBM on osteoblasts and its therapeutic impact using various parameters to optimize osteoblast function that may be clinically important. Graphical Abstract.

Expression of tartrate-resistant acid phosphatase and cathepsin K during osteoclast differentiation in developing mouse mandibles.

The present study was designed to test the hypothesis that osteoclasts appear after or at the same time as the initiation of bone mineralization in developing intramembranous bones. We examined mineral deposition via Von Kossa staining to determine when bone mineralization begins, tartrate-resistant acid phosphatase (TRAP) activity and cathepsin K immunoreactivity to identify the presence of osteoclasts, and their mRNA expression levels to assess osteoclastic differentiation in the embryonic mouse mandible. Cathepsin K-immunopositive cells were detected around the same time as the onset of bone mineralization, whereas TRAP-positive cells appeared prior to bone mineralization. Cathepsin K protein was expressed only in multinucleated osteoclasts, whereas TRAP activity was identified in both mono- and multinucleated cells. During bone development, TRAP-positive cells altered their morphology, which was related to the number of their nuclei. The elevated mRNA levels of TRAP and cathepsin K were consistent with the increased percentage of multinucleated osteoclasts and the progression of bone development. Our study revealed that TRAP-positive cells appear prior to bone mineralization, and TRAP- and cathepsin K-positive multinucleated osteoclasts appear at the same time as the initiation of bone mineralization in embryonic mouse mandibles, suggesting that osteoclasts contribute to bone matrix maturation during intramembranous ossification.

Expresión del marcador óseo CD44 en hueso alveolar previamente injertado.

Objetive: To determine the expressions of the bone surface marker CD44 in samples of alveolar bone pr iously regenerated with allograft, xenograft, and mixed, using the technique of guided bone regeneration. Material and Methods: This exploratory study was approved by the institutional research and ethics committee. By means of intentional sampling and after obtaining informed consent for tissue donation, 20 samples of alveolar bone previously regenerated with guided bone regeneration therapy with particulate bone graft and membrane were taken during implant placement. The samples were stained with hematoxylin-eosin for histological analysis, and by immunohistochemistry for the detection of CD44. Results: Sections with hematoxylin-eosin showed bone tissue with the presence of osteoid matrix and mature bone matrix of usual appearance. Of the CD44+ samples, 80% were allograft and 20% xenograft. The samples with allograft-xenograft were negative. There were no differences in the intensity of CD44 expression between the positive samples. The marker was expressed in osteocytes, stromal cells, mononuclear infiltrate, and some histiocytes. Eighty percent of the CD44+ samples and 100% of the samples in which 60 or more cells were labelled corresponded to allografts (p=0.000). A total of 67% of the samples from the anterior sector, and 40% from the posterior sector were CD44+ (p=0.689). Conclusion: This study shows for the first time that guided bone regeneration using allografts is more efficient for the generation of mature bone determined by the expression of CD44, compared to the use of xenografts and mixed allograft-xenograft, regardless of the regenerated anatomical area.

Calcimimetics Alter Periosteal and Perilacunar Bone Matrix Composition and Material Properties in Early Chronic Kidney Disease.

ABSTRACTChronic kidney disease (CKD) affects 15% of Americans and greatly increases fracture risk due to elevated parathyroid hormone, cortical porosity, and reduced bone material quality. Calcimimetic drugs are used to lower parathyroid hormone (PTH) in CKD patients, but their impact on bone matrix properties remains unknown. We hypothesized that tissue‐level bone quality is altered in early CKD and that calcimimetic treatment will prevent these alterations. To test this hypothesis, we treated Cy/+ rats, a model of spontaneous and progressive CKD‐mineral and bone disorder (CKD‐MBD), with KP‐2326, a preclinical analogue of etelcalcetide, early in the CKD disease course. To measure tissue‐level bone matrix composition and material properties, we performed colocalized Raman spectroscopy and nanoindentation on new periosteal bone and perilacunar bone using hydrated femur sections. We found that CKD and KP treatment lowered mineral type B carbonate substitution whereas KP treatment increased mineral crystallinity in new periosteal bone. Reduced elastic modulus was lower in CKD but was not different in KP‐treated rats versus CTRL. In perilacunar bone, KP treatment lowered type B carbonate substitution, increased crystallinity, and increased mineral‐to‐matrix ratio in a spatially dependent manner. KP treatment also increased reduced elastic modulus and hardness in a spatially dependent manner. Taken together, these data suggest that KP treatment improves material properties on the tissue level through a combination of lowering carbonate substitution, increasing mineral crystallinity, and increasing relative mineralization of the bone early in CKD. As a result, the mechanical properties were improved, and in some regions, were the same as control animals. Therefore, calcimimetics may help prevent CKD‐induced bone deterioration by improving bone quality in new periosteal bone and in bone tissue near osteocyte lacunae. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

3-D macro/microporous-nanofibrous bacterial cellulose scaffolds seeded with BMP-2 preconditioned mesenchymal stem cells exhibit remarkable potential for bone tissue engineering

Bone repair using BMP-2 is a promising therapeutic approach in clinical practices, however, high dosages required to be effective pose issues of cost and safety. The present study explores the potential of low dose BMP-2 treatment via tissue engineering approach, which amalgamates 3-D macro/microporous-nanofibrous bacterial cellulose (mNBC) scaffolds and low dose BMP-2 primed murine mesenchymal stem cells (C3H10T1/2 cells). Initial studies on cell-scaffold interaction using unprimed C3H10T1/2 cells confirmed that scaffolds provided a propitious environment for cell adhesion, growth, and infiltration, owing to its ECM-mimicking nano-micro-macro architecture. Osteogenic studies were conducted by preconditioning the cells with 50 ng/mL BMP-2 for 15 min, followed by culturing on mNBC scaffolds for up to three weeks. The results showed an early onset and significantly enhanced bone matrix secretion and maturation in the scaffolds seeded with BMP-2 primed cells compared to the unprimed ones. Moreover, mNBC scaffolds alone were able to facilitate the mineralization of cells to some extent. These findings suggest that, with the aid of ‘osteoinduction’ from low dose BMP-2 priming of stem cells and ‘osteoconduction’ from nano-macro/micro topography of mNBC scaffolds, a cost-effective bone tissue engineering strategy can be designed for quick and excellent in vivo osseointegration.

New Surgical Model for Bone-Muscle Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout Mouse.

Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the vastus lateralis (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone–muscle crosstalk studies.

Osteoporosis Treatments Affect Bone Matrix Maturation in a Rat Model of Induced Cortical Remodeling.

ABSTRACTTo test how osteoporosis drugs affect bone matrix maturation during cortical bone remodeling, 72 pregnant rats were switched from a 0.4% to a 0.01% calcium diet at parturition for a 23‐day lactation period. At weaning, eight dams were sacrificed to establish baseline values, while the remaining dams were returned to 0.4% calcium and treated with vehicle (saline), sodium fluoride (NaF), zoledronic acid (ZA), or sclerostin antibody (Scl‐Ab) for either 7 or 28 days (eight animals per group per time point). Femora were examined by μCT, dynamic histomorphometry, Fourier transform infrared imaging, and three‐point bending of notched specimens. Cortical porosity decreased in all groups from baseline to day 28. Intracortical mineralizing surface (MS/BS) and mineral apposition rate (MAR), as well as the mineral‐to‐matrix ratio were unaffected by treatment, but intracortical crystallinity was increased in the ZA group at day 10 compared with vehicle. Cortical area increased in all groups over 28 days mainly because of an addition of bone at the endocortical surface. Endocortical MS/BS did not vary among the groups, but endocortical MAR was suppressed in the NaF group at day 2 and elevated in the Scl‐Ab group at day 4 compared with vehicle. Endocortical mineral‐to‐matrix ratio was increased at days 5 and 10 following NaF treatment and endocortical crystallinity was increased at day 5 following ZA treatment compared with vehicle. Fracture toughness did not differ among the groups. Thus, the treatments affected matrix maturation more strongly at the endocortical then intracortical envelope. In this model of induced remodeling, the bone formation phase is synchronized at multiple sites, facilitating study of the effects of drugs or other bone‐targeting agents on matrix maturation independent of their effects on the initiation of remodeling. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Effects of ionizing radiation on woven bone: influence on the osteocyte lacunar network, collagen maturation, and microarchitecture.

Evaluate the effects of ionizing radiation on microarchitecture, the osteocyte lacunar network, and collagen maturity in a bone repair site. Bone defects were created on tibias of 20 New Zealand rabbits. After 2weeks, the animals were randomly divided into (n = 10) NoIr (nonirradiated group) and Ir (irradiated group). In the Ir, the animals received single-dose irradiation of 30Gy on the tibia and were euthanized after 2weeks. Bone microarchitecture parameters were analyzed by using micro-CT, and the osteocyte lacunar network, bone matrix, and collagen maturation by histomorphometric analysis. The data were analyzed using unpaired Student's t test (α = 0.05). Trabecular thickness in Ir was lower than that in NoIr (P = 0.028). No difference was found for bone volume fraction and bone area. Lacunae filled with osteocytes were more numerous (P < 0.0001) in NoIr (2.6 ± 0.6) than in Ir (1.97 ± 0.53). Empty lacunae were more prevalent (P < 0.003) in Ir (0.14 ± 0.10) than in NoIr (0.1 ± 0.1). The mean osteocyte lacunae size was higher (P < 0.01) in Ir (15.4 ± 4.41) than in NoIr (12.7 ± 3.7). Picrosirius red analysis showed more (P < 0.05) mature collagen in NoIr (29.0 ± 5.3) than in Ir (23.4 ± 4.5). Immature collagen quantification revealed no difference between groups. Ionizing radiation compromised bone formation and an impairment in bone repair in irradiated woven bone was observed. Before radiotherapy, patients usually need surgical intervention, which may be better performed, if clinicians understand the repair process in irradiated bone, using novel approaches for treating these individuals.