Alterations in the microenvironment and the effects produced of TRPV5 in osteoporosis.

The pathogenesis of osteoporosis involves multiple factors, among which alterations in the bone microenvironment play a crucial role in disrupting normal bone metabolic balance. Transient receptor potential vanilloid 5 (TRPV5), a member of the TRPV family, is an essential determinant of the bone microenvironment, acting at multiple levels to influence its properties. TRPV5 exerts a pivotal influence on bone through the regulation of calcium reabsorption and transportation while also responding to steroid hormones and agonists. Although the metabolic consequences of osteoporosis, such as loss of bone calcium, reduced mineralization capacity, and active osteoclasts, have received significant attention, this review focuses on the changes in the osteoporotic microenvironment and the specific effects of TRPV5 at various levels.

Bioactive PLGA/tricalcium phosphate scaffolds incorporating phytomolecule icaritin developed for calvarial defect repair in rat model.

BACKGROUND/OBJECTIVES: For treatment of large bone defects challenging in orthopaedic clinics, bone graft substitutes are commonly used for the majority of surgeons. It would be proposed in the current study that our bioactive scaffolds could additionally serve as a local delivery system for therapeutic small molecule agents capable of providing support to enhance biological bone repair. METHODS: In this study, composite scaffolds made of poly (lactic-co-glycolic acid) (PLGA) and tricalcium phosphate (TCP) named by P/T was fabricated by a low-temperature rapid prototyping technique. For optimizing the scaffolds, the phytomolecule icaritin (ICT) was incorporated into P/T scaffolds called P/T/ICT. The osteogenic efficacies of the two groups of scaffolds were compared in a successfully established calvarial defect model in rats. Bone regeneration was evaluated by X-ray, micro-computerised tomography (micro-CT), and histology at weeks 4 and/or 8 post-implantation. In vitro induction of osteogenesis and osteoclastogenesis was established for identification of differentiation potentials evoked by icaritin in primary cultured precursor cells. RESULTS: The results of radiographies and decalcified histology demonstrated more area and volume fractions of newly formed bone within bone defect sites implanted with P/T/ICT scaffold than that with P/T scaffold. Undecalcified histological results presented more osteoid and mineralized bone tissues, and also more active bone remodeling in P/T/ICT group than that in P/T group. The results of histological staining in osteoclast-like cells and newly formed vessels indicated favorable biocompatibility, rapid bioresorption and more new vessel growth in P/T/ICT scaffolds in contrast to P/T scaffolds. Based on in vitro induction, the results presented that icaritin could significantly facilitate osteogenic differentiation, while suppressed adipogenic differentiation. Meanwhile, icaritin demonstrated remarkable inhibition of osteoclastogenic differentiation. CONCLUSION: The finding that P/T/ICT composite scaffold can enhance bone regeneration in calvarial bone defects through facilitating effective bone formation and restraining excessive bone resorption. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The osteogenic bioactivity of icaritin facilitated PLGA/TCP/icartin composite scaffold to exert significant bone regeneration in calvarial defects in rat model. It might form an optimized foundation for potential clinical validation in bone defects application.

Effect and mechanism of psoralidin on promoting osteogenesis and inhibiting adipogenesis.

BACKGROUND: Psoralidin (PL), a prenylated coumestrol, is isolated from Psoralea corylifolia L. (Fabaceae), which is frequently used for treatment of osteoporosis. PURPOSE: This study was designed to investigate the dual effects and potential mechanism of PL on promoting osteogenesis and inhibiting adipogenesis. METHODS: Bone marrow mesenchymal stem cells (BMSCs) were used to investigate the effect of PL on stimulating osteogenesis and inhibiting adipogenesis, while preosteoblast MC3T3-E1 cells and preadipocyte 3T3-L1 cells were employed to explore the potential mechanisms. Estradiol (E2) and ICI 182,780 (ICI) were used as the specific agonist and antagonist of classical estrogen receptors (ER), respectively, to interfere with classical ER signaling. Meanwhile, G-1 and G-15 were introduced as the selective agonist and antagonist of G protein coupled receptor 30 (GRP30, a membrane ER) to further clarify if membrane ER involved in PL mediating osteogenesis and adipogenesis RESULTS: PL not only promoted mineralization, but also inhibited adipocytes formation of BMSCs. In terms of osteogenesis, PL enhanced calcium nodule formation, alkaline phosphatase activity and osteocalcin levels in MC3T3-E1 cells. As for adipogenesis, PL decreased adipocyte formation in 3T3-L1 cells through down-regulating several mRNA expressions and protein synthesis of adipogenesis related factors. ICI completely blocked the effect of PL in promoting osteogenesis, but only partially suppressed its effect in inhibition of adipogenesis, while G-15 partially suppressed the effect of PL on promoting mineralization and inhibiting oil drop formation. Furthermore, during suppression of adipocyte differentiation, PL regulated protein kinase B / glycogen synthase kinase 3ß / ß-catenin signaling pathway. CONCLUSION: PL promoted osteogenesis via mediating classical ER pathway, and inhibited adipocytes formation by regulating combined classical and membrane ER pathways. PL might be a potential candidate for the treatment of postmenopausal osteoporosis by modulating the competitive relationship between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells.

Investigation of angiogenesis in bioactive 3-dimensional poly(d,l-lactide-co-glycolide)/nano-hydroxyapatite scaffolds by in vivo multiphoton microscopy in murine calvarial critical bone defect.

UNLABELLED: Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. The continuous release of bioactive lentiviral vectors (LV-pdgfb) from the scaffolds could be detected for 5days in vitro. In vivo, the released LV-pdgfb transfected adjacent cells and expressed PDGF-BB, facilitating angiogenesis and enhancing bone regeneration. The expression of both pdgfb and the angiogenesis-related genes vWF and VEGFR2 was significantly increased in the pdgfb gene-carrying scaffold (PHp) group. In addition, microCT scanning and histomorphology results proved that there was more new bone ingrowth in the PHp group than in the PLGA/nHA (PH) and control groups. MicroCT parameters, including BMD, BV/TV, Tb.Sp, and Tb.N indicated that there was significantly more new bone formation in the PHp group than in the other groups. With regard to neovascularization, 8weeks post-implantation, blood vessel areas (BVAs) were 9428±944µm(2), 4090±680.3µm(2), and none in the PHp, PH, and control groups, respectively. At each time point, BVAs in the PHp scaffolds were significantly higher than in the PH scaffolds. To our knowledge, this is the first use of multiphoton microscopy in bone tissue-engineering to investigate angiogenesis in scaffolds in vivo. This method represents a valuable tool for investigating neovascularization in bone scaffolds to determine if a certain scaffold is beneficial to neovascularization. We also proved that delivery of the pdgfb gene alone can improve both angiogenesis and bone regeneration Acronyms. STATEMENT OF SIGNIFICANCE: Reconstruction of critical size bone defects remains a major clinical challenge because of poor bone regeneration, which is usually due to poor angiogenesis during repair. Satisfactory vascularization is a prerequisite for the survival of grafts and the integration of new tissue with existing tissue. In this work, we investigated angiogenesis in 3D scaffolds by in vivo multiphoton microscopy during bone formation in a murine calvarial critical bone defect model and evaluated bone regeneration 8weeks post-implantation. To verify that pdgfb-expressing vectors carried by the scaffolds can promote angiogenesis in 3D-printed scaffolds in vivo, we monitored angiogenesis within the implants by multiphoton microscopy. To our knowledge, this is the first study to dynamically investigate angiogenesis in bone tissue engineering scaffolds in vivo.

Bone defect animal models for testing efficacy of bone substitute biomaterials.

Large bone defects are serious complications that are most commonly caused by extensive trauma, tumour, infection, or congenital musculoskeletal disorders. If nonunion occurs, implantation for repairing bone defects with biomaterials developed as a defect filler, which can promote bone regeneration, is essential. In order to evaluate biomaterials to be developed as bone substitutes for bone defect repair, it is essential to establish clinically relevant in vitro and in vivo testing models for investigating their biocompatibility, mechanical properties, degradation, and interactional with culture medium or host tissues. The results of the in vitro experiment contribute significantly to the evaluation of direct cell response to the substitute biomaterial, and the in vivo tests constitute a step midway between in vitro tests and human clinical trials. Therefore, it is essential to develop or adopt a suitable in vivo bone defect animal model for testing bone substitutes for defect repair. This review aimed at introducing and discussing the most available and commonly used bone defect animal models for testing specific substitute biomaterials. Additionally, we reviewed surgical protocols for establishing relevant preclinical bone defect models with various animal species and the evaluation methodologies of the bone regeneration process after the implantation of bone substitute biomaterials. This review provides an important reference for preclinical studies in translational orthopaedics.

Bacterial inhibition potential of 3D rapid-prototyped magnesium-based porous composite scaffolds--an in vitro efficacy study.

Bone infections are common in trauma-induced open fractures with bone defects. Therefore, developing anti-infection scaffolds for repairing bone defects is desirable. This study develoepd novel Mg-based porous composite scaffolds with a basal matrix composed of poly(lactic-co-glycolicacid) (PLGA) and tricalcium phosphate (TCP). A unique low-temperature rapid prototyping technology was used to fabricate the scaffolds, including PLGA/TCP (PT), PLGA/TCP/5%Mg (PT5M), PLGA/TCP/10%Mg (PT10M), and PLGA/TCP/15%Mg (PT15M). The bacterial adhesion and biofilm formation of Staphylococcus aureus were evaluated. The results indicated that the Mg-based scaffolds significantly inhibited bacterial adhesion and biofilm formation compared to PT, and the PT10M and PT15M exhibited significantly stronger anti-biofilm ability than PT5M. In vitro degratation tests revealed that the degradation of the Mg-based scaffolds caused an increase of pH, Mg(2+) concentration and osmolality, and the increased pH may be one of the major contributing factors to the antibacterial function of the Mg-based scaffolds. Additionally, the PT15M exhibited an inhibitory effect on cell adhesion and proliferation of MC3T3-E1 cells. In conclusion, the PLGA/TCP/Mg scaffolds could inhibit bacterial adhesion and biofilm formation, and the PT10M scaffold was considered to be an effective composition with considerable antibacterial ability and good cytocompatibility.

Src blockage by siRNA inhibits VEGF-induced vascular hyperpemeability and osteoclast activity - an in vitro mechanism study for preventing destructive repair of osteonecrosis.

INTRODUCTION: Destructive repair is the pathological feature of ONFH characterized with the elevated vascular permeability and persistent bone resorption, which is associated with higher VEGF expression, activated c-Src, and vascular leakage. Activated c-Src also participates in mediating endothelial permeability and osteoclasts activity. However, the molecular mechanism of the VEGF and c-Src contributing to the destructive repair process remains unknown. The purpose of this study is to delineate the role of VEGF and c-Src in triggering destructive repair of osteonecrosis in vitro, as well as to elucidate if VEGF mediating vascular permeability and osteoclastic bone resorption are Src dependent. METHODS: We employed pharmacological VEGF to induce higher endothelial permeability and osteoclasts activity for simulating related pathological features of destructive repair in vitro. Src specific pp60(c-src)siRNA was used for determining the contribution of VEGF and Src to destructive repair. The primary endothelial cells and osteoclasts were treated with 50ng/ml VEGF and/or transfected with the pp60(c-src)siRNA, while equivalent PBS and non-targeting siRNA were treated in the control groups. RESULTS: VEGF enhanced Src bioactivity through promoting dephosphorylation of Src at Y527 and phosphorylation of Src at Y416. Meanwhile, Src specific pp60(c-src)siRNA significantly reduced Src expression in both cells. VEGF destroyed the junctional integrity of endothelial cells resulting in higher endothelial permeability. However, Src blockade significantly relieved VEGF induced actin stress and inhibited caveolae and VVOs formation, meanwhile further stabilized the complex ß-catenin/VE-cadherin/Flk-1 through decreasing phosphorylation of VE-cadherin, ultimately decreasing VEGF-mediating higher vascular permeability. In addition, VEGF promoted osteoclasts formation and function without affecting the adhesion activity and cytoskeleton. We further found that Src blockade significantly impaired cytoskeleton resulting in a lower adhesion activity through down-regulation of phosphorylation of Src, Pyk2 and Cbl, and ultimately inhibited osteoclasts formation and function. CONCLUSIONS: These findings provide a new insight into VEGF and c-Src mode of reaction in triggering destructive repair of osteonecrosis and further indicate that VEGF mediating vascular permeability and osteoclasts activity are Src-dependent. Blockade of Src may have great potential as an effective therapy targeting destructive repair in osteonecrosis.