Bone Volume Per Tissue Volume Research Articles

Endothelial Cell-Derived Exosomes Inhibit Osteoblast Apoptosis and Steroid-Induced Necrosis of Femoral Head Progression by Activating the PI3K/Akt/Bcl-2 Pathway

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) p=0.031, trabecular thickness (Tb.Th) p=0.020, and decreased separation (Tb.Sp) p=0.040 were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

Moderate static magnetic field promotes fracture healing and regulates iron metabolism in mice

BackgroundFractures are the most common orthopedic diseases. It is known that static magnetic fields (SMFs) can contribute to the maintenance of bone health. However, the effect and mechanism of SMFs on fracture is still unclear. This study is aim to investigate the effect of moderate static magnetic fields (MMFs) on bone structure and metabolism during fracture healing.MethodsEight-week-old male C57BL/6J mice were subjected to a unilateral open transverse tibial fracture, and following treatment under geomagnetic field (GMF) or MMF. The micro-computed tomography (Micro-CT) and three-point bending were employed to evaluate the microarchitecture and mechanical properties. Endochondral ossification and bone remodeling were evaluated by bone histomorphometric and serum biochemical assay. In addition, the atomic absorption spectroscopy and ELISA were utilized to examine the influence of MMF exposure on iron metabolism in mice.ResultsMMF exposure increased bone mineral density (BMD), bone volume per tissue volume (BV/TV), mechanical properties, and proportion of mineralized bone matrix of the callus during fracture healing. MMF exposure reduced the proportion of cartilage in the callus area during fracture healing. Meanwhile, MMF exposure increased the number of osteoblasts in callus on the 14th day, and reduced the number of osteoclasts on the 28th day of fracture healing. Furthermore, MMF exposure increased PINP and OCN levels, and reduced the TRAP-5b and β-CTX levels in serum. It was also observed that MMF exposure reduced the iron content in the liver and callus, as well as serum ferritin levels while elevating the serum hepcidin concentration.ConclusionsMMF exposure could accelerate fracture healing via promote the endochondral ossification and bone formation while regulating systemic iron metabolism during fracture healing. This study suggests that MMF may have the potential to become a form of physical therapy for fractures.

Axial Compressive Loading Attenuates Early Osteoarthritis by Reducing Subchondral Bone Remodeling

Background: Mechanical loading and alendronate (ALN) can be used as noninvasive physical therapy methods for osteoarthritis (OA). However, the timing and efficacy for treatments are unknown. Purpose: To determine whether the timing of mechanical loading and ALN influences the pathobiological changes of OA. Study Design: Controlled laboratory study. Methods: Mice with OA induced by anterior cruciate ligament transection were subjected to early (1-3 weeks) or late (5-7 weeks) axial compressive dynamic load or intraperitoneal injection of ALN. Changes in gait were analyzed using gait analysis system, pathobiological changes in subchondral bone, cartilage, osteophyte, and synovitis were assessed using micro–computed tomography, tartrate-resistant acid phosphatase staining, pathologic section staining, and immunohistochemistry at 1, 2, 4, and 8 weeks. Results: At 1, 2, and 4 weeks, the OA limb had lower mean footprint pressure intensity, lower bone volume per tissue volume (BV/TV) in the subchondral bone, and more osteoclasts. At 4 weeks, the early loading, ALN, and load + ALN treatments induced less cartilage destruction, with a corresponding reduction in Osteoarthritis Research Society International score and increased hyaline cartilage thickness. The treatments also resulted in fewer osteoclasts and higher BV/TV and bone mineral density of subchondral bone and suppressed inflammation and interleukin 1β– and tumor necrosis factor α-positive cells in synovium. At 8 weeks, early loading or load + ALN improved the mean footprint pressure intensity and knee flexion. At 8 weeks, early load + ALN had a synergistic effect on protecting hyaline cartilage and proteoglycans. Footprint pressure intensity and cartilage destruction were worse in late loading limbs, and no differences in BV/TV, bone mineral density, osteophyte formation, and synovium inflammation were found between the late load, ALN, and load + ALN groups and the anterior cruciate ligament transection group. Conclusion: Dynamic axial mechanical loading or ALN in the early stages of knee trauma protected against OA by suppressing subchondral bone remodeling. However, late loading promoted cartilage degeneration in advanced OA, indicating that reduced loading should be performed in the late stages of OA to avoid the acceleration of OA. Clinical Relevance: Early low-level functional exercise or antiosteoporotic drugs could clearly slow or prevent the progression of early OA. For patients with mild to severe OA, loading reduction via brace protection or maintenance of joint stability via early ligament reconstruction surgery may ameliorate OA exacerbation.

Gut Microbiota and Serum Metabolic Signatures of High-Fat-Induced Bone Loss in Mice.

BackgroundAccumulating evidence indicates that high-fat diet (HFD) is a controllable risk factor for osteoporosis, but the underlying mechanism remains to be elucidated. As a primary biological barrier for nutrient entry into the human body, the composition and function of gut microbiota (GM) can be altered rapidly by HFD, which may trigger abnormal bone metabolism. In the current study, we analyzed the signatures of GM and serum metabolomics in HFD-induced bone loss and explored the potential correlations of GM and serum metabolites on HFD-related bone loss.MethodsWe conducted a mouse model with HFD-induced bone loss through a 12-week diet intervention. Micro-CT, Osmium-μCT, and histological analyses were used to observe bone microstructure and bone marrow adipose tissue. Quantitative Real-Time PCR was applied to analyze gene expression related to osteogenesis, adipogenesis, and osteoclastogenesis. Enzyme-linked immunosorbent assay was used to measure the biochemical markers of bone turnover. 16s rDNA sequencing was employed to analyze the abundance of GM, and UHPLC-MS/MS was used to identify serum metabolites. Correlation analysis was performed to explore the relationships among bone phenotypes, GM, and the metabolome.ResultsHFD induced bone loss accompanied by bone marrow adipose tissue expansion and bone formation inhibition. In the HFD group, the relative abundance of Firmicutes was increased significantly, while Bacteroidetes, Actinobacteria, Epsilonbacteraeota, and Patescibacteria were decreased compared with the ND group. Association analysis showed that thirty-two bacterial genera were significantly related to bone volume per tissue volume (BV/TV). One hundred and forty-five serum metabolites were identified as differential metabolites associated with HFD intervention, which were significantly enriched in five pathways, such as purine metabolism, regulation of lipolysis in adipocyte and cGMP-PKG signaling pathway. Sixty-four diffiential metabolites were matched to the MS2 spectra; and ten of them were positively correlated with BV/TV and five were negatively correlated with BV/TV.ConclusionsThese findings indicated that the alternations of GM and serum metabolites were related to HFD-induced bone loss, which might provide new insights into explain the occurrence and development of HFD-related osteoporosis. The regulatory effects of GM and metabolites associated with HFD on bone homeostasis required further exploration.

Melatonin attenuates radiation-induced cortical bone-derived stem cells injury and enhances bone repair in postradiation femoral defect model

BackgroundThe healing of bone defects can be challenging for clinicians to manage, especially after exposure to ionizing radiation. In this regard, radiation therapy and accidental exposure to gamma (γ)-ray radiation have been shown to inhibit bone formation and increase the risk of fractures. Cortical bone-derived stem cells (CBSCs) are reportedly essential for osteogenic lineages, bone maintenance and repair. This study aimed to investigate the effects of melatonin on postradiation CBSCs and bone defect healing.MethodsCBSCs were extracted from C57BL/6 mice and were identified by flow cytometry. Then CBSCs were subjected to 6 Gy γ-ray radiation followed by treatment with various concentrations of melatonin. The effects of exogenous melatonin on the self-renewal and osteogenic capacity of postradiation CBSCs in vitro were analyzed. The underlying mechanisms involved in genomic stability, apoptosis and oxidative stress-related signaling were further analyzed by Western blotting, flow cytometry and immunofluorescence assays. Moreover, postradiation femoral defect models were established and treated with Matrigel and melatonin. The effects of melatonin on postradiation bone healing in vivo were evaluated by micro-CT and pathological analysis.ResultsThe decrease in radiation-induced self-renewal and osteogenic capacity were partially reversed in postradiation CBSCs treated with melatonin (P < 0.05). Melatonin maintained genomic stability, reduced postradiation CBSC apoptosis and intracellular oxidative stress, and enhanced expression of antioxidant-related enzymes (P < 0.05). Western blotting validated the anti-inflammatory effects of melatonin by downregulating interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels via the extracellular regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling pathway. Melatonin was also found to exhibit antioxidant effects via NRF2 signaling. In vivo experiments demonstrated that the newly formed bone in the melatonin plus Matrigel group had higher trabecular bone volume per tissue volume (BV/TV) and bone mineral density values with lower IL-6 and TNF-α levels than in the irradiation and the Matrigel groups (P < 0.05).ConclusionThis study suggested that melatonin could protect CBSCs against γ-ray radiation and assist in the healing of postradiation bone defects.

Multi-Targeting DKK1 and LRP6 Prevents Myeloma-Induced Bone Disease

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodeling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting their quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. Therapeutic strategies aimed at promoting bone formation are therefore required to overcome the loss of skeletal integrity and subsequent fractures in MM patients.Therapeutic agents that target the canonical Wnt signaling pathway, a potent regulator of bone formation, have the potential to address these skeletal complications, where they could rebuild lost bone and improve bone strength in affected individuals. We have demonstrated a novel anti-LRP6 agent, which potentiates Wnt signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. However, since MM patients present with both increased bone resorption and decreased bone formation, we hypothesised that combining anti-LRP6 with the bone anabolic anti-DKK1 (100mg/kg twice weekly intravenously) would lead to more robust improvements in bone structure than single treatment approaches.MicroCT analysis demonstrated a 74% increase in femoral trabecular bone volume per tissue volume (BV/TV) in naïve, non-tumour bearing mice given the combination treatment compared to control agents (p<0.0001). Mice injected with 5TGM1eGFP murine myeloma cells had a 34% reduction in femoral BV/TV compared to naïve controls (p<0.0001). Combination treatment drastically improved BV/TV in 5TGM1-bearing mice by 111% (p<0.0001), compared to control, and this improvement with the combination treatment strategy was 25% greater than anti-LRP6 single treatment approaches (p<0.001). MicroCT analysis in L4 lumbar vertebrae demonstrated similar bone structural changes in 5TGM1-bearing mice treated with the combination strategy. Consequently, this combination significantly improved resistance to fracture in L4 vertebrae in 5TGM1eGFP-bearing mice compared to their controls (p<0.001), and it provided greater protection against fracture compared to anti-LRP6 single agent treatment. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1eGFP-bearing mice treated with the combination strategy (p<0.001) compared to control. Importantly, tumour activity was not altered with either single or combination Wnt-promoting treatment strategies.This study defines a novel therapeutic strategy, which will reduce fractures and improve quality of life in patients with MM when used in combination with tumour-targeted treatments. [Display omitted] DisclosuresCong: Novartis Institutes for Biomedical Research: Current Employment. Daley: Novartis Institutes of Biomedical Research: Current Employment.

Bone healing around titanium implants in a preclinical model of bile duct ligation-induced liver injury.

ObjectivesChronic liver disease increases the risk for periodontal disease and osteoporotic fractures, but its impacts on bone regeneration remain unknown. Herein, we studied the impact of liver cirrhosis on peri‐implant bone formation.Material and MethodsA total of 20 male Wistar rats were randomly divided into two groups: one with the common bile duct ligated (BDL) and the respective sham‐treated control group (SHAM). After four weeks of disease induction, titanium mini‐screws were inserted into the tibia. Successful induction of liver cirrhosis was confirmed by the presence of clinical symptoms. Another four weeks later, peri‐implant bone volume per tissue volume (BV/TV) and bone‐to‐implant contact (BIC) were determined by histomorphometric analysis.ResultsPeri‐implant bone formation was not significantly different between the SHAM and BDL groups. In the cortical compartment, the median percentage of peri‐implant new bone was 10.1% (95% CI of mean 4.0–35.7) and 22.5% (13.8–30.6) in the SHAM and BDL groups, respectively (p = .26). Consistently, the new bone in direct contact with the implant was 18.1% (0.4–37.8) and 23.3% (9.2–32.8) in SHAM and BDL groups, respectively (p = .38). When measuring the medullary compartment, the new bone area was 7.1% (4.8–10.4) and 10.4% (7.2–13.5) in the SHAM and BDL groups, respectively (p = .17). Medullary new bone in direct contact with the implant was 10.0% (1.2–50.4) and 20.6% (16.8–35.3) in SHAM and BDL groups, respectively, and thus comparable between the two groups (p = .46).ConclusionsBile duct ligation has no significant impact on the early stages of peri‐implant bone formation.

Microstructural and mechanical recovery of bone in ovariectomized rats: The effects of menaquinone-7

The loss of bone quantity and quality in postmenopausal female patients can be a problem for dental treatment. A sufficient intake of nutrients such as calcium, magnesium, and vitamins D and K is likely correlated with the mechanical properties of bone. In particular, vitamin K2, also called menaquinone (MK), inhibits bone loss in postmenopausal women. Here we demonstrate the microstructural and mechanical properties of bone recovery in ovariectomized (OVX) rats during MK-7 administration. Bilateral ovariectomy and a sham operation were performed on 14-week-old female SPF Wistar rats. MK-4 and -7 were orally administered at 30 mg/kg daily for 12 weeks. The femur was used for the 3-point bending test and microstructural analysis of the cancellous bone by micro-CT, and the mandibular cortical bone for the evaluation of mechanical properties on a nanoscale. Micro-computed tomography revealed irregular trabecular architecture, hollow marrow cavities, and sparse trabecular bone in the femurs of the OVX group. Trabecular bone structure analysis showed that the MK-7 group had greater bone volume per tissue volume (BV/TV) and a higher trabecular number than the OVX group. The bulk-scale 3-point bending test did not allow the mechanical properties between OVX and OVX/MK7 groups to be discerned, yet at the smallest level, the elastic–plastic transition point of the nanoindentation stress–strain curve of the mandibular cortical bone was higher in the MK-7 group than in the OVX group. These findings suggest that MK-7 enables bone microstructural and mechanical recovery in the OVX model.

Genistein acts in bone metabolism and improves peri-implant healing in rats with estrogen deficiency

The present study aimed to evaluate the peri-implantar bone healing in the presence of genistein treatment in ovariectomized rats. Thirty female rats with 4 months old were divided into 3 groups according to the experimental condition and the drug treatment: SHAM (rats submitted to the fictional surgery and gavage with 0.9% saline solution); OVX (rats submitted to bilateral ovariectomy and gavage with 0.9% saline solution); OVX GEN (rats submitted to bilateral ovariectomy and gavage with 1mg/day of genistein). 60 implants were installed, with two implants in each animal. The calcified group was subjected microcomputerized tomography and the parameters analysed was bone volume per tissue volume (BV/TV) and connective density (Cnn.Dn). The decalcified samples were evaluated through immunolabeling analysis, in order to detect the presence of RUNX2, Alkaline Phosphatase, Osteocalcin, Osteopontin and TRAP. All the quantitative data were submitted to the normality curve to determine the most adequate test. The significance level of p&lt;0.05 was considered for all tests. The morphometric analysis of the OVX GEN group showed higher percentage of bone volume and lower connective density when compared with OVX. Immunohistochemical analysis favors expression. For the markers that positively label osteoblastic activity. This study shows that genistein therapy improves peri-implant bone healing in ovariectomized rats.

Effects of treatment with a bone-targeted prostaglandin E2 receptor 4 agonist C3 (Mes-1007) in a mouse model of severe osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually caused by mutations affecting collagen type I synthesis in osteoblasts. Bisphosphonates are widely used to decrease fracture rate but are only partially effective. Bone anabolic compounds, such as prostaglandin E2 receptor 4 (EP4) agonists may be an alternative treatment approach. Here we assessed the effect of Mes-1007, a novel bone-targeted EP4 agonist in Jrt mice, a model of severe OI. Experimental study. Male 8-week old wild type (WT) and OI mice were randomly assigned to 4weeks of three intraperitoneal injections per week with Mes-1007 (25mg per kg body mass), phosphate-buffered saline, zoledronate (5μg per kg), or a combination treatment of zoledronate and Mes-1007. Treatment with Mes-1007 alone did not lead to higher trabecular bone volume per tissue volume (BV/TV) in the distal femur or lumbar vertebra 4 in either WT or OI mice. Treatment with zoledronate alone was associated with a significant increase in distal femur and vertebra BV/TV in both genotypes. In zoledronate-treated WT and OI mice, Mes-1007 increased bone formation rate in vertebral trabecular bone and had an additive effect on BV/TV. Vertebral BV/TV in OI mice that received zoledronate or Mes-1007/zoledronate combination treatment was similar to untreated WT mice (p=0.25). At the femoral midshaft, Mes-1007/zoledronate combination treatment increased cortical thickness in both genotypes and led to higher periosteal diameter in OI mice. Three-point bending tests of femurs showed that Mes-1007/zoledronate combination treatment increased the stiffness, load at yield and maximal load in WT but not in OI mice. Dosing Mes-1007 in combination with zoledronate improved the bone properties in a manner that is consistent with a mechanism of action of EP4 agonists on bone and additive to effects of anti-resorptives typified by zoledronate.

Bone healing and reactivation of remodeling under asfotase alfa therapy in adult patients with pediatric-onset hypophosphatasia

Hypophosphatasia (HPP) is a hereditary musculoskeletal disorder caused by inactivating variants in the ALPL gene and subsequently reduced serum tissue-nonspecific alkaline phosphatase (TNSALP) activity. This inborn error of metabolism results in decreased bone quality, accumulations of osteoid, and reduced bone mineralization. Increased incidence of fractures and prolonged bone healing are characteristic features for HPP. Available enzyme replacement therapy (asfotase alfa), was reported to recover bone mineralization and bone quality in adult HPP patients. Moreover, it was shown that asfotase alfa improved fracture healing of former nonunions in two adult HPP patients. We hypothesized that the nonunions are filled partially with osteoid, offering great potential to benefit from the treatment with asfotase alfa to promote bone healing. In the present study, we report three adult patients with pediatric-onset HPP and detected ALPL-mutations with prolonged bone healing after arthrodesis, tibial stress fracture, and osteotomy. After the initiation of asfotase alfa, immediately increased levels of alkaline phosphatase (ALP) and bone-specific ALP, as well as decreased levels of pyridoxal-5-phosphate (PLP), were detected in biochemical analysis. Importantly, even after up to 5years of non-healing, a progredient consolidation was shown, assessed by a custom three-dimensional evaluation of repeated cone-beam computed tomography (CBCT) images, characterized by rapidly increasing levels of bone volume per tissue volume (BV/TV) within the volume of interest (i.e., the region of the non-healing bone). These radiographical findings were in line with the reported restoration of functional ability and pain-free full weight-bearing, as well as increased neuromuscular parameters (e.g., improved muscle strength). Taken together, our findings indicate that asfotase alfa improves the osseous consolidation of nonunions likely due to re-mineralization of osteoid tissue filling the former gap and improving the functional ability in adult HPP patients, characterized by increasing levels of BV/TV assessed via an innovative three-dimensional evaluation of CBCT images.

Components of the Gut Microbiome That Influence Bone Tissue-Level Strength.

Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n=7-10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (-28%, p=0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissue-level strength of bone independent of bone quantity and without noticeable changes in bone turnover. © 2021 American Society for Bone and Mineral Research (ASBMR).

Microstructural volumetric analysis of vertical alveolar ridge augmentation using autogenous tooth roots.

To volumetrically assess the bone microstructure following vertical alveolar ridge augmentation using differently conditioned autogenous tooth roots (TR) and second-stage implant placement. The upper premolars were bilaterally extracted in n = 4 beagle dogs and randomly assigned to either autoclavation (TR-A) or no additional treatment (TR-C). Subsequently, TR were used as block grafts for vertical alveolar ridge augmentation in both lower quadrants. At 12 weeks, titanium implants were inserted and left to heal 3 weeks. Microcomputed tomography was used to quantify bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp) at vestibular (v) and oral (o) aspects along the implant and in the augmented upper half of the implant, respectively. Median BV/TV [TR-C: 51.33% (v) and 70.42% (o) vs TR-A: 44.05% (v) and 64.46% (o)], Tb.th [TR-C: 0.22 mm (v) and 0.27 mm (o) vs TR-A: 0.23 mm (v) and 0.29 mm (o)] and Tb.Sp [TR-C: 0.26 mm (v) and 0.13 mm (o) vs TR-A: 0.29 μm (v) and 0.15 mm (o)] values were comparable in both groups. Both TR-C and TR-A grafts were associated with a comparable bone microstructure within the grafted area.

The Impact of Compaction Force on Graft Consolidation in a Guided Bone Regeneration Model.

Compaction of particulated grafts is done manually; thus, the effect of compression force on bone regeneration remains unclear. The aim of this study was to evaluate the impact of two different compression forces on the consolidation of particulated bovine hydroxyapatite. Two titanium cylinders were fixed on the calvarium of eight New Zealand rabbits. Both defects were filled with particulated bovine hydroxyapatite subjected to a compression force of 0.7 kg/cm2 or 1.6 kg/cm2 before being covered with a resorbable collagen membrane. A handheld device that uses a spring to control the compression force applied by the plugger was used. At 6 weeks, histomorphometry of the area immediately adjacent to the calvaria bone and to the collagen membrane was performed. It was shown that next to the calvaria, the bone volume per tissue volume (BV/TV) was 29.0% ± 8.8% and 27.6% ± 8.2% at low and high compression force, respectively; the bone-to-biomaterial contact (BBC) was 58.2% ± 25.0% and 69.3% ± 22.9%, respectively (P > .05). In the corresponding area next to the collagen membrane, BV/TV was 4.9% ± 5.1% and 5.7% ± 4.7%, and the BBC was 18.3% ± 20.8% and 20.1% ± 15.9%, respectively (P > .05). In addition, the number and area of blood vessels were not significantly affected by compression force. Both compression forces applied resulted in similar consolidation of bovine hydroxyapatite expressed by new bone formation and vascularization based on a rabbit calvaria augmentation model.

Suppression of Inflammation, Osteoclastogenesis and Bone Loss by PZRAS Extract.

Panax ginseng has a wide range of activities including a neuroprotective effect, skin protective effects, enhanced DNA repairing, anti-diabetic activity, and protective effects against vascular inflammation. In the present study, we sought to discover the inhibitory effects of a mixture of natural products containing Panax ginseng, Ziziphus jujube, Rubi fructus, Artemisiae asiaticae and Scutellaria baicalensis (PZRAS) on osteoclastogenesis and bone remodeling, as neither the effects of a mixture containing Panax ginseng extract, nor its molecular mechanism on bone inflammation, have been clarified yet. PZRAS upregulated the levels of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSH-R) and glutathione peroxidase (GSH-Px) and reduced malondialdehyde (MDA) in LPS-treated RAW264.7 cells. Moreover, treatment with PZRAS decreased the production of IL-1β and TNF-α. PZRAS also inhibited osteoclast differentiation through inhibiting osteoclastspecific genes like MMP-2, 9, cathepsin K, and TRAP in RANKL-treated RAW264.7 cells. Additionally, PZRAS has inhibitory functions on the RANKL-stimulated activation of ERK and JNK, which lead to a decrease in the expression of NFATc1 and c-Fos. In an in vivo study, bone resorption induced by LPS was recovered by treatment with PZRAS in bone volume per tissue volume (BV/TV) compared to control. Furthermore, the ratio of eroded bone surface of femurs was significantly increased in LPStreated mice compared to vehicle group, but this ratio was significantly reversed in PZRAS-treated mice. These results suggest that PZRAS could prevent or treat disorders with abnormal bone loss.

Age-related changes of micro-morphological subchondral bone properties in the healthy femoral head

Alterations in the subchondral bone (SCB) are likely to play a decisive role in the development of osteoarthritis (OA). Since aging represents a major risk factor for OA, the aim of the current study was to assess the microstructural changes of the subchondral bone in the femoral head during aging. Femoral heads and matched iliac crest biopsies of 80 individuals (age 21-99 years) were collected post-mortem. The bone microstructure of the subchondral trabecular bone as well as the cartilage thickness (Cg.Th) and subchondral bone plate thickness (SCB.Th) were quantified using histomorphometry. The different subregions of the SCB were also imaged by quantitative backscattered electron imaging (qBEI) in 31 aged cases to assess the bone mineral density distribution (BMDD). The detected linear decline of bone volume per tissue volume (BV/TV) in the femoral head with aging (Slope, 95% CI:-0.208 to-0.109 %/yr.) was primarily due to a decrease in trabecular thickness (Tb.Th, Slope, 95% CI:-0.774 to-0.343μm/yr). While SCB.Th declined with aging (Slope, 95% CI:-1.941 to-0.034μm/yr), no changes in Cg.Th were detected (Slope, 95% CI:-0.001 to 0.005mm/yr). The matrix mineralization of the subchondral bone was lower compared to the trabecular bone and also decreased with aging. Regular changes of the SCB during aging primarily involve a reduction of Tb.Th, SCB.Th and matrix mineralization. Our findings facilitate future interpretations of early and late OA specimens to decipher the role of the SCB in OA pathogenesis.

MiRNA-21 deficiency impairs alveolar socket healing in mice.

BackgroundMicroRNAs (miRNAs) are small noncoding RNAs demonstrated as critical post‐transcriptional modulators in dental tissues and bone regeneration, particularly miR‐21‐5p. However, the role of miR‐21‐5p in the healing of alveolar sockets following tooth extraction remains unknown. In this study we evaluated the influence of miR‐21‐5p in the healing of alveolar socket after tooth extraction.MethodsEight miR‐21‐5p knockout mice and eight littermate controls underwent tooth extraction of the upper right incisor. After a healing period of 14 days microCT and histological analyses were performed.ResultsMicroCT analysis showed that the percentage of bone in the extraction socket was significantly higher in the control group than in the miR‐21 knockout mice; either in the coronal (39.0%, CI 31.8 to 48.0 versus 23.0%, CI 17.8 to 35.2, P = 0.03) or in the middle part of the alveolar socket (56.0%, CI 50.9 to 62.5 versus 43.5% CI 28.6 to 54.6, P = 0.03). These differences were not noted in the apical part of the extraction socket. Histological analysis supported the microCT findings. Newly bone volume per tissue volume (BV/TV) was significantly higher in the control group when compared to miR‐21 knockout mice, 27.4% (CI 20.6 to 32.9) versus 19.0% (CI 14.7 to 21.5, P < 0.05), respectively. Surprisingly, no evident signs of buccal bone resorption were observed in both groups.ConclusionDespite the limitation of one observation period, these findings suggest that miR‐21‐5p delays the early healing of alveolar socket following tooth extraction. Whether miR‐21‐5p is essential for healing of alveolar sockets remains to be elucidated.

EphrinB2 overexpression enhances osteogenic differentiation of dental pulp stem cells partially through ephrinB2-mediated reverse signaling

BackgroundAlveolar bone loss is a frequent occurrence. Dental pulp stem cells (DPSCs) which have invasive accessibility and high osteogenic potential is a promising source for cell-based bone regeneration. EphrinB2 is involved in bone homeostasis and osteogenesis. The aim of this study was to investigate the effect and mechanism of ephrinB2 overexpression on osteogenic differentiation of DPSCs and bone defect repair.MethodsEphrinB2 expression was analyzed during osteogenic induction of human DPSCs (hDPSCs). Endogenous ephrinB2 expression in hDPSCs was then upregulated using EfnB2 lentiviral vectors. The effect of ephrinB2 overexpression on osteogenic differentiation capacity of hDPSCs was investigated in vitro, and activation of ephrinB2-EphB4 bidirectional signaling in ephrinB2-overexpressing hDPSCs was detected. In vivo, a canine alveolar bone defect model was established and canine DPSCs (cDPSCs) were cultured, characterized, EfnB2-tranfected, and combined with a PuraMatrix scaffold. Micro-CT analysis was performed to evaluate the therapeutic effect of ephrinB2-overexpressing cDPSCs on bone defect repair.ResultsEphrinB2 was upregulated after osteogenic induction of hDPSCs. EphrinB2 overexpression enhanced osteogenic differentiation capacity of hDPSCs in vitro. Moreover, p-ephrinB2 instead of p-EphB4 was upregulated by ephrinB2 overexpression, and activation of ephrinB2-mediated reverse signaling promoted osteogenic differentiation of hDPSCs. In a canine bone defect model, ephrinB2 overexpression in cDPSCs significantly improved trabecular bone volume per tissue volume (BV/TV) and trabecular thickness, as demonstrated by radiographic analysis.ConclusionsEphrinB2 overexpression enhanced osteogenic potential of DPSCs partially via upregulation of ephrinB2-mediated reverse signaling and effectively promoted alveolar bone defect repair.

Implantable electrical stimulation bioreactor with liquid crystal polymer-based electrodes for enhanced bone regeneration at mandibular large defects in rabbit.

The osseous regeneration of large bone defects is still a major clinical challenge in maxillofacial and orthopedic surgery. Previous studies demonstrated that biphasic electrical stimulation (ES) stimulates bone formation; however, polyimide electrode should be removed after regeneration. This study presents an implantable electrical stimulation bioreactor with electrodes based on liquid crystal polymer (LCP), which can be permanently implanted due to excellent biocompatibility to bone tissue. The bioreactor was implanted into a critical-sized bone defect and subjected to ES for one week, where bone regeneration was evaluated four weeks after surgery using micro-CT. The effect of ES via the bioreactor was compared with a sham control group and a positive control group that received recombinant human bone morphogenetic protein (rhBMP)-2 (20μg). New bone volume per tissue volume (BV/TV) in the ES and rhBMP-2 groups increased to 132% (p < 0.05) and 174% (p < 0.01), respectively, compared to that in the sham control group. In the histological evaluation, there was no inflammation within the bone defects and adjacent to LCP in all the groups. This study showed that the ES bioreactor with LCP electrodes could enhance bone regeneration at large bone defects, where LCP can act as a mechanically resistant outer box without inflammation. Graphical abstract To enhance bone regeneration, a bioreactor comprising collagen sponge and liquid crystal polymer-based electrode was implanted in the bone defect. Within the defect, electrical current pulses having biphasic waveform were applied from the implanted bioreactor.

Possible osteoprotective effects of myricetin in STZ induced diabetic osteoporosis in rats

Myricetin is a flavonoid which has many pharmacological effects. However, to date there is no evidence study on the effect of myricetin in diabetic condition. This study was aimed to investigate whether myricetin could protect against diabetic osteoporosis in streptozotocin induced rats.Female Wistar rats were randomly allocated to four equal groups: diabetic group (DG), diabetic group with myricetin (50 mg per kilogram per day), (D) diabetic group with myricetin (100 mg/kg/day) and normal control group (CG). Body weight was recorded once a week. After treatment with myricetin for 12 weeks, serum biochemical analyses, the microarchitecture of femora, and histological changes were evaluated.We found that the bone mineral density (BMD) of myricetin (100 mg per kilogram per day)treatment group significantly increased than in the diabetic group (P < 0.05). The alkaline phosphatase and osteocalcin were markedly blocked in diabetic rats relative to normal control group (P < 0.05); however, the inhibition was prevented by the myricetin treatment group. Results also showed that myricetin treatment could dramatically improve trabecular bone microarchitecture through increasing bone mass such as trabecular number (Tb.N), bone volume per tissue volume (BV/TV), and decreasing that of structure model index (SMI) and trabecular separation (Tb.Sp), comparing with the control group. We also found that myricetin could significantly lower the oxidative damage and up-regulate the activity of superoxide dismutase (SOD) and catalase activity.In summary, we showed that myricetin can effectively improve abnormal bone metabolism in streptozotocin induced rats, which may provide a beneficial medicine on diabetic bone disease.