Osthole stimulates bone formation,drives vascularization and retards adipogenesis to alleviate alcohol‐induced osteonecrosis of the femoral head

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol‐induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β‐catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well‐constructed rat model of ethanol‐induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro‐CT scanning, haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol‐induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.     

High-Fat Diet/Low-Dose Streptozotocin-Induced Type 2 Diabetes in Rats Impacts Osteogenesis and Wnt Signaling in Bone Marrow Stromal Cells

Bone regeneration disorders are a significant problem in patients with type 2 diabetes mellitus. Bone marrow stromal cells (BMSCs) are recognized as ideal seed cells for tissue engineering because they can stimulate osteogenesis during bone regeneration. Therefore, the aim of this study was to investigate the osteogenic potential of BMSCs derived from type 2 diabetic rats and the pathogenic characteristics of dysfunctional BMSCs that affect osteogenesis. BMSCs were isolated from normal and high-fat diet+streptozotocin-induced type 2 diabetic rats. Cell metabolic activity, alkaline phosphatase (ALP) activity, mineralization and osteogenic gene expression were reduced in the type 2 diabetic rat BMSCs. The expression levels of Wnt signaling genes, such as β-catenin, cyclin D1 and c-myc, were also significantly decreased in the type 2 diabetic rat BMSCs, but the expression of GSK3β remained unchanged. The derived BMSCs were cultured on calcium phosphate cement (CPC) scaffolds and placed subcutaneously into nude mice for eight weeks; they were detected at a low level in newly formed bone. The osteogenic potential of the type 2 diabetic rat BMSCs was not impaired by the culture environment, but it was impaired by inhibition of the Wnt signaling pathway, likely due to an insufficient accumulation of β-catenin rather than because of GSK3β stimulation. Using BMSCs derived from diabetic subjects could offer an alternative method of regenerating bone together with the use of supplementary growth factors to stimulate the Wnt signaling pathway.     

The effect of combined regulation of the expression of peroxisome proliferator-activated receptor-γ and calcitonin gene-related peptide on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells

Studies have shown that alcohol can upregulate the expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in bone marrow mesenchymal stem cells (BMSCs). High expression of PPARγ can promote adipogenic differentiation of BMSCs, and reduce their osteogenic differentiation. Abnormal proliferation of adipocytes and fatty accumulation in osteocytes can result in high intraosseous pressure and disturbance of blood circulation in the femoral head, which induces osteonecrosis of the femoral head (ONFH). Downregulation of PPARγ is efficient in inhibiting adipogenesis and maintaining osteogenesis of BMSCs, which might potentially reduce the incidence of ONFH. Calcitonin gene-related peptide (CGRP) is a neuropeptide gene which has been closely associated with bone regeneration. In this study, we aimed to observe the effect of combined regulation of the expression of PPARγ and CGRP genes on alcohol-induced adipogenic differentiation of BMSCs. Our results demonstrated that simultaneous downregulation of PPARγ and upregulation of CGRP was efficient in suppressing adipogenic differentiation of BMSCs and promoting their osteogenic differentiation. These findings might enlighten a novel approach for the prevention of ONFH.     

Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of β‐catenin‐BMP2 signalling pathway

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti‐resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up‐regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β‐catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β‐catenin inhibitor DKK1. Silencing the β‐catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β‐catenin was silenced, although the knockout of BMP2 did not affect β‐catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β‐catenin‐BMP signalling, providing a novel strategy in the treatment of osteoporosis.     

Activation of aldehyde dehydrogenase 2 protects ethanol‐induced osteonecrosis of the femoral head in rat model

ObjectivesOsteonecrosis of the femoral head (ONFH) is a devastating disease characterized by destructive bone structures, enlarged adipocyte accumulation and impaired vascularization. The aldehyde dehydrogenase 2 (ALDH 2) is the limiting enzyme for ethanol metabolism with many physiological functions. The aim was investigated the potential protective role of activated ALDH 2 by Alda‐1 for ethanol‐induced ONFH.Materials and MethodsThe ethanol‐induced ONFH in rat was performed to explore the protective of Alda‐1 by various experimental methods. Subsequently, the effect of Alda‐1 and ethanol on the osteogenic and adipogenic differentiation was investigated via multiple cellular and molecular methods. Finally, the effect of Alda‐1 and ethanol on the neo‐vascularization was detected in Human umbilical vein endothelial cells (HUVECs) and ONFH model.ResultsFirstly, radiographical and pathological measurements indicated that alda‐1 protected ethanol‐induced ONFH. Moreover, ethanol significantly inhibited the proliferation and osteogenic differentiation of BMSCs, whereas Alda‐1 could distinctly rescue it by PI3K/AKT signalling. Secondly, ethanol remarkably promoted the lipid vacuoles formation of BMSCs, while Alda‐1 significantly retarded it on BMSCs by AMPK signalling pathway. Finally, ethanol significantly inhibited proliferation and growth factor level resulting in reduced angiogenesis, whereas Alda‐1 could rescue the effect of ethanol. Additionally, Alda‐1 significantly reduced the occurrence of ONFH and promoted vessel number and distribution in alcoholic ONFH.ConclusionsAlda‐1 activation of ALDH 2 was highly demonstrated to protect ethanol‐induced ONFH by triggering new bone formation, reducing adipogenesis and stimulating vascularization.

Alda‐1 activation of ALDH 2 was highly demonstrated to protect ethanol‐induced ONFH by triggering new bone formation, reducing adipogenesis and stimulating vascularization. Therefore, ALDH 2 may be a potential therapeutic target and small molecule Alda‐1 may be promising pharmacotherapeutic for ONFH in the future.     

Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.     

Periostin regulates osteogenesis of mesenchymal stem cells from ovariectomized rats through actions on the ILK/Akt/GSK-3β Axis

Osteoporosis is a condition of the skeleton that mainly results from estrogen deficiency. Periostin is a matricellular component in bone that is involved in osteoblast differentiation. However, how Periostin promotes osteogenesis remains largely unknown. Here, we isolated bone marrow skeletal stem cells (BMSCs) derived from an ovariectomy (OVX)-induced osteoporosis rat model and the effects of periostin on BMSCs derived from OVX rats (OVX-BMSCs) were assessed. Overexpression of periostin enhanced alkaline phosphatase (ALP) and alizarin red staining in OVX-BMSCs as well as the osteogenic genes OCN, BSP and Runx2. ILK is a downstream effector of signals from the extracellular matrix and participates in bone homeostasis. Overexpression of periostin also increased expression of protein levels for ILK, as well as the downstream targets pAkt and pGSK3β. Suppression of ILK or Akt partially suppressed the enhancement of osteogenic ability induced by periostin overexpression in OVX-BMSCs. Thus, periostin may promote the osteogenic ability of OVX-BMSCs through actions on the ILK/Akt/GSK3β axis.     

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.     

METTL3‐m6 A methylase regulates the osteogenic potential of bone marrow mesenchymal stem cells in osteoporotic rats via the Wnt signalling pathway

ObjectivesBone marrow mesenchymal stem cells (BMSCs) hold a high osteogenic differentiation potential, but the mechanisms that control the osteogenic ability of BMSCs from osteoporosis (OP‐BMSCs) need further research. The purpose of this experiment is to discuss the osteogenic effect of Mettl3 on OP‐BMSCs and explore new therapeutic target that can enhance the bone formation ability of OP‐BMSCs.Materials and MethodsThe bilateral ovariectomy (OVX) method was used to establish the SD rat OP model. Dot blots were used to reveal the different methylation levels of BMSCs and OP‐BMSCs. Lentiviral‐mediated overexpression of Mettl3 was applied in OP‐BMSCs. QPCR and WB detected the molecular changes of osteogenic‐related factors and Wnt signalling pathway in vitro experiment. The staining of calcium nodules and alkaline phosphatase detected the osteogenic ability of OP‐BMSCs. Micro‐CT and histological examination evaluated the osteogenesis of Mettl3 in OP rats in vivo.ResultsThe OP rat model was successfully established by OVX. Methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro experiment, overexpression of Mettl3 could upregulate the osteogenic‐related factors and activate the Wnt signalling pathway in OP‐BMSCs. However, osteogenesis of OP‐BMSCs was weakened by treatment with the canonical Wnt inhibitor Dickkopf‐1. Micro‐CT showed that the Mettl3(+) group had an increased amount of new bone formation at 8 weeks. Moreover, the results of histological staining were the same as the micro‐CT results.ConclusionsTaken together, the methylation levels and osteogenic potential of OP‐BMSCs were decreased in OP‐BMSCs. In vitro and in vivo studies, overexpression of Mettl3 could partially rescue the decreased bone formation ability of OP‐BMSCs by the canonical Wnt signalling pathway. Therefore, Mettl3 may be a key targeted gene for bone generation and therapy of bone defects in OP patients.

In this study, the osteoporosis rat model was successfully established by OVX. OP‐BMSCs were successfully isolated and cultured from the femur of OP rat. Lentiviral‐mediated overexpression of Mettl3 could partially rescue the impaired osteogenic ability of OP‐BMSCs by activating the canonical Wnt signalling pathway in vitro and in vivo .     

Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of β-catenin-BMP2 signalling pathway

Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti-resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up-regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β-catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β-catenin inhibitor DKK1. Silencing the β-catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β-catenin was silenced, although the knockout of BMP2 did not affect β-catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β-catenin-BMP signalling, providing a novel strategy in the treatment of osteoporosis.     

High-mobility group box 1 accelerates distraction osteogenesis healing via the recruitment of endogenous stem/progenitor cells

Background aimsWhile distraction osteogenesis (DO) achieves substantial bone regeneration, prolonged fixation may lead to infections. Existing stem cell and physical therapies have limitations, requiring the development of novel therapeutic approaches. Here, we evaluated high-mobility group box 1 (HMGB1) as a novel therapeutic target for DO treatment.MethodsMicro-computed tomography (Micro-CT) analysis and histological staining of samples obtained from tibial DO model mice was performed. Transwell migration, wound healing, and proliferation assays were also performed on cultured human mesenchymal stem cells (hMSCs) and human umbilival vein endothelial cells (HUVECs). Tube formation assay was performed on HUVECs, whereas osteogenic differentiation assay was performed on hMSCs.ResultsMicro-CT analysis and histological staining of mouse samples revealed that HMGB1 promotes bone regeneration during DO via the recruitment of PDGFRα and Sca-1 positve (PαS⁺) cells and endothelial progenitor cells. Furthermore, HMGB1 accelerated angiogenesis during DO, promoted the migration and osteogenic differentiation of hMSCs as well as the proliferation, migration and angiogenesis of HUVECs in vitro.ConclusionsOur findings suggest that HMGB1 has a positive influence on endogenous stem/progenitor cells, representing a novel therapeutic target for the acceleration of DO-driven bone regeneration.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

MiR-19b-3p accelerates bone loss after spinal cord injury by suppressing osteogenesis via regulating PTEN/Akt/mTOR signalling

Rapid and extensive bone loss, one of the skeletal complications after spinal cord injury (SCI) occurrence, drastically sacrifices the life quality of SCI patients. It has been demonstrated that microRNA (miRNA) dysfunction plays an important role in the initiation and development of bone loss post-SCI. Nevertheless, the effect of miR-19b-3p on bone loss after SCI is unknown and the accurate mechanism is left to be elucidated. The present work was conducted to explore the role of miR-19b-3p/phosphatase and tensin homolog deleted on chromosome ten (PTEN) axis on osteogenesis after SCI and further investigates the underlying mechanisms. We found that miR-19b-3p level was increased in the femurs of SCI rats with decreased autophagy. The overexpression of miR-19b-3p in bone marrow mesenchymal stem cells (BMSCs) targeted down-regulation of PTEN expression, facilitated protein kinase B (Akt) and mammalian target of rapamycin (mTOR) phosphorylation, and thereby suppressing BMSCs osteogenic differentiation via autophagy. Besides, the inhibiting effects of miR-19b-3p on osteogenic differentiation of BMSCs could be diminished by autophagy inducer rapamycin. Meanwhile, bone loss after SCI in rats was also reversed by antagomir-19b-3p treatment, suggesting miR-19b-3p was an essential target for osteogenic differentiation via regulating autophagy. These results indicated that miR-19b-3p was involved in bone loss after SCI by inhibiting osteogenesis via PTEN/Akt/mTOR signalling pathway.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.