Gadolinium-doped bioglass scaffolds promote osteogenic differentiation of hBMSC via the Akt/GSK3ß pathway and facilitate bone repair in vivo.

BACKGROUND: Biomaterial-induced osteogenesis is mainly related to hierarchically porous structures and bioactive components. Rare earth elements are well known to promote osteogenesis and stimulate bone repair; however, the underlying biological effects of gadolinium (Gd) element on bone regeneration are not yet known. METHODS: In this study, we successfully fabricated gadolinium-doped bioglass (Gd-BG) scaffolds by combining hollow mesoporous Gd-BG microspheres with chitosan and evaluated in vitro effects and underlying mechanisms with Cell Counting Kit-8, scanning electron microscopy, alkaline phosphatase, Alizarin red staining, and polymerase chain reaction. Cranial defect model of rats was constructed to evaluate their in vivo effects. RESULTS: The results indicated that Gd-BG scaffolds could promote the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Mechanistically, the Akt/GSK3ß signaling pathway was activated by the Gd-BG scaffolds. The enhancing effect of Gd-BG scaffolds on the osteogenic differentiation of hBMSCs was inhibited by the addition of LY294002, an inhibitor of Akt. Moreover, the in vivo cranial defect model of rats indicated that the Gd-BG scaffolds could effectively promote bone regeneration. CONCLUSION: Both in vitro and in vivo results suggested that Gd-BG scaffolds have promising applications in bone tissue engineering.

Incorporation of cerium oxide in hollow mesoporous bioglass scaffolds for enhanced bone regeneration by activating the ERK signaling pathway.

Hierarchically porous structures and bioactive compositions of artificial biomaterials play a positive role in bone defect healing and new bone regeneration. Herein, cerium oxide nanoparticles-modified bioglass (Ce-BG) scaffolds were firstly constructed by the incorporation of hollow mesoporous Ce-BG microspheres in CTS via a freeze-drying technology. The interconnected macropores in Ce-BG scaffolds facilitated the in-growth of bone cells/tissues from material surfaces into the interiors, while the hollow cores and mesopore shells in Ce-BG microspheres provides more active sites for bone mineralization. The cerium oxide nanoparticles in the scaffolds rapidly promoted the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs), as confirmed by the up-regulation of osteogenesis-related markers such as OCN, ALP and COL-1. The enhanced osteoinductivity of Ce-BG scaffolds was mainly related to the activated ERK pathway, and it was blocked by adding a selective ERK1/2 inhibitor (SCH772984). In vivo rat cranial defect models revealed that Ce-BG scaffolds accelerated collagen deposition, osteoblast formation and bone regeneration as compared to BG scaffolds. The exciting results demonstrated that the synergistic effects between hierarchically porous structures and cerium oxide nanoparticles contributed to osteogenic ability, and hollow mesoporous Ce-BG scaffolds would be a novel platform for bone regeneration.

Valproic acid prevents glucocorticoid­induced osteonecrosis of the femoral head of rats.

Glucocorticoids (GCs) are the most common cause of atraumatic osteonecrosis of the femoral head (ONFH) because their effect compromises the osteogenic capability of bone marrow­derived mesenchymal stem cells (BMSCs). Valproic acid (VPA) is a widely used anti­epileptic and anti­convulsant drug. Previous studies have reported that VPA promotes osteogenic differentiation of MSCs in vitro and osteogenesis in vivo as a histone deacetylase (HDAC) inhibitor. The purpose of the present study was to investigate the efficacy of VPA as a precautionary treatment of ONFH after GC treatment in rats. In vitro, the effect of VPA, dexamethasone or a combination treatment of the two on the proliferation and osteogenic differentiation of human BMSCs was assessed using a Cell Counting Kit­8 and apoptosis assays, and by measuring the expression of proteins associated with osteogenesis. In vivo, a GC­induced ONFH model was established in rats and VPA was added during GC treatment to investigate the preventive effect of VPA against ONFH. Rat BMSCs were also extracted to investigate the osteogenic capacity. The results of micro­computed tomography scanning, angiography of the femoral head and histological and immunohistochemical analyses indicated that 11 of 15 rats induced with methylprednisolone (MP) presented with ONFH, while only 2 of 15 rats treated with a combination of MP and VPA developed ONFH. VPA produced beneficial effects on subchondral bone trabeculae in the femoral head with significant preservation of bone volume and blood supply, as well as improved osteogenic capability of BMSCs compared with those in rats treated with GC alone. In conclusion, VPA attenuated the inhibitory effect of GC on BMSC proliferation and osteogenesis by inhibiting apoptosis and elevating the expression of proteins associated with osteogenesis, which may contribute to the prevention of GC­induced ONFH in rats.

Lanthanum-Doped Chitosan Hydrogels Promote the Apoptosis of Melanoma Cells by Bcl-2/Bax Pathway.

The surgical resection of melanoma may cause skin wounds, and the remaining melanoma cells bring a great risk of tumor recurrence. To overcome the above problem, we for the first time constructed lanthanum-doped chitosan (La-CS) hydrogels with excellent wound healing and antitumor functions. The La element was uniformly dispersed within whole hydrogels, and part of La3+ ions reacted with CS to form La-CS complex. The complexation interaction between La3+ ions and CS significantly improve the La3+ release performances of La-CS hydrogels. The as-released La3+ ions from the composite hydrogels selectively inhibited the proliferation of B-16 melanoma cells, but showed lower toxic side effects to L929 skin fibroblast cells. Moreover, the La3+ ions triggered the apoptosis of B-16 cells through Bcl-2/Bax pathway, as confirmed by the Annexin Vand PI double staining, flow cytometry, and Western blot results. The in vivo tumor models of C57 mice revealed that the La-CS hydrogels had more significant relapse-inhibition effects on B-16 melanoma cells than the pure CS hydrogels. At the same time, the in vivo wound healing was accelerated by the multifunctional hydrogels. The exciting finding provides a critical and promising strategy in the construction of La-doped hydrogels for oncotherapy.

Muscone exerts protective roles on alcohol-induced osteonecrosis of the femoral head.

Long-term alcohol abuse causes musculoskeletal disorders, among of which, alcohol-induced osteonecrosis of the femoral head (ONFH) is of concern due to its significant and severe complications. A variety of methods have been attempted to prevent alcohol-induced ONFH, and monomers extracted from Chinese herbs might benefit the disease profoundly. In the current study, muscone, the main ingredient of musk, was used to prevent alcohol-induced ONFH. In vitro, ethanol was used to affect the potential of osteogenesis and proliferation of human bone mesenchymal stem cells (hBMSCs), and beneficial role of muscone was investigated on hBMSCs. In vivo, following the establishment of alcohol-induced ONFH, muscone was employed to treat the diseased rats, which were analyzed by micro-CT scanning and a series of histologic staining. As a result, we found ethanol could significantly suppress osteogenic differentiation of hBMSCs, while muscone held the potential to promote ALP activity and mRNA expressions of COL1 and OCN under ethanol treatment. Meanwhile, imaging analysis revealed muscone could restore BV/TV ratio and bone mineral density of the necrotic femoral head, and the protective role of muscone on alcohol-induced ONFH was further confirmed by histologic examinations. Our study confirmed the protective effect of muscone against alcohol-induced ONFH both in vitro and in vivo. Therefore, muscone may be considered as a valuable therapeutic natural drug for alcohol-induced ONFH in humans.

The protective effect of PFTα on alcohol-induced osteonecrosis of the femoral head.

Epidemiologic studies have shown alcohol plays a pivotal role in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to explore the underlying mechanism of alcohol-induced ONFH and the protective effect of pifithrin-α (PFTα). In vitro, we found ethanol treatment significantly activated p53, suppressed Wnt/ß-catenin signaling and inhibited osteogenic-related proteins. Furthermore, by separating the cytoplasmic and nuclear proteins, we found ethanol inhibited osteogenesis by impairing the accumulation of ß-catenin in both the cytoplasm and nucleus in human bone mesenchymal stem cells (hBMSCs), which resulted from activating glycogen synthase kinase-3ß (GSK-3ß). Therefore, PFTα, a p53 inhibitor, was introduced in this study to block the ethanol-triggered activation of p53 in hBMSCs and alcohol-induced ONFH in a rat model. In vivo, we established alcohol-induced ONFH in rats and investigated the protective effect of PFTα. Hematoxylin & eosin (H&E) staining combined with TdT-mediated dUTP nick end labeling (TUNEL), cleaved caspase-3 immunohistochemical staining, and micro-CT images revealed substantial ONFH in the alcohol-administered rats, whereas significantly less osteonecrosis developed in the rats injected with PFTα. Osteogenic-related proteins, including osteocalcin, osteopontin and collagen I, were significantly decreased in the alcohol-administered rats, whereas these results were reversed in the PFTα-injected rats. Fluorochrome labeling similarly showed that alcohol significantly reduced the osteogenic activity in the rat femoral head, which was blocked by the injection of PFTα. In conclusion, PFTα had an antagonistic effect against the effects of ethanol on hBMSCs and could be a clinical strategy to prevent the development of alcohol-induced ONFH.

The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head.

BACKGROUND: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH), in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. METHODS: Human bone mesenchymal stem cells (hBMSCs) were identified by multi-lineage differentiation. Alkaline phosphatase (ALP) activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E) staining and micro-computerized tomography (micro-CT) were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemical staining was carried out to detect OCN and COL1. RESULTS: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/ß-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. CONCLUSION: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

Novel Akt activator SC-79 is a potential treatment for alcohol-induced osteonecrosis of the femoral head.

Alcohol is a leading risk factor for osteonecrosis of the femoral head (ONFH). We explored the molecular mechanisms underlying alcohol-induced ONFH and investigated the protective effect of the novel Akt activator SC-79 against this disease. We found that ethanol inhibited expression of the osteogenic genes RUNX2 and OCN, downregulated osteogenic differentiation, impaired the recruitment of Akt to the plasma membrane, and suppressed Akt phosphorylation at Ser473, thereby inhibiting the Akt/GSK3ß/ß-catenin signaling pathway in bone mesenchymal stem cells. To assess SC-79's ability to counteract the inhibitory effect of ethanol on Akt-Ser73 phosphorylation, we performed micro-computerized tomography and immunofluorescent staining of osteopontin, osteocalcin and collagen type 1 in a rat model of alcohol-induced ONFH. We found that SC-79 injections inhibited alcohol-induced osteonecrosis. These results show that alcohol-induced ONFH is associated with suppression of p-Akt-Ser473 in the Akt/GSK3ß/ß-catenin signaling pathway in bone mesenchymal stem cells. We propose that SC-79 treatment to rescue Akt activation could be tested in the clinic as a potential therapeutic approach to preventing the development of alcohol-induced ONFH.

Protective effect of VK2 on glucocorticoid-treated MC3T3-E1 cells.

Glucocorticoids (GCs) contribute to the increased incidence of secondary osteoporosis and osteonecrosis, and medications for the prevention and treatment of these complications have been investigated for many years. Vitamin K2 (VK2) has been proven to promote bone formation both in vitro and in vivo. In this study, we examined the effects of VK2 on dexamethasone (DEX)-treated MC3T3-E1 osteoblastic cells. We observed that VK2 promoted the proliferation and enhanced the survival of dexamethasone-treated MC3T3-E1 cells. In addition, VK2 upregulated the expression levels of osteogenic marker proteins, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin, which were significantly inhibited by dexamethasone. On the whole, our findings indicate that VK2 has the potential to antagonize the effects of GCs on MC3T3-E1 cells, and may thus prove to be a promising agent for the prevention and treatment of GC-induced osteoporosis and osteonecrosis.

Decreased extracellular pH inhibits osteogenesis through proton-sensing GPR4-mediated suppression of yes-associated protein.

The pH of extracellular fluids is a basic property of the tissue microenvironment and is normally maintained at 7.40 ± 0.05 in humans. Many pathological circumstances, such as ischemia, inflammation, and tumorigenesis, result in the reduction of extracellular pH in the affected tissues. In this study, we reported that the osteogenic differentiation of BMSCs was significantly inhibited by decreases in the extracellular pH. Moreover, we demonstrated that proton-sensing GPR4 signaling mediated the proton-induced inhibitory effects on the osteogenesis of BMSCs. Additionally, we found that YAP was the downstream effector of GPR4 signaling. Our findings revealed that the extracellular pH modulates the osteogenic responses of BMSCs by regulating the proton-sensing GPR4-YAP pathway.

Vitamin K2 Prevents Glucocorticoid-induced Osteonecrosis of the Femoral Head in Rats.

Glucocorticoid medication is one of the most common causes of atraumatic osteonecrosis of the femoral head (ONFH), and vitamin K2 (VK2) has been shown to play an important and beneficial role in bone metabolism. In this study, we hypothesized that VK2 could decrease the incidence of glucocorticoid-induced ONFH in a rat model. Using in vitro studies, we investigated how bone marrow-derived stem cells in the presence of methylprednisolone proliferate and differentiate, specifically examining osteogenic-related proteins, including Runx2, alkaline phosphatase and osteocalcin. Using in vivo studies, we established glucocorticoid-induced ONFH in rats and investigated the preventive effect of VK2. We employed micro-CT scanning, angiography of the femoral head, and histological and immunohistochemical analyses, which demonstrated that VK2 yielded beneficial effects for subchondral bone trabecula. In conclusion, VK2 is an effective antagonist for glucocorticoid on osteogenic progenitors. The underlying mechanisms include acceleration of BMSC propagation and promotion of bone formation-associated protein expression, which combine and contribute to the prevention of glucocorticoid-induced ONFH in rats.

Continuous hypoxia regulates the osteogenic potential of mesenchymal stem cells in a time-dependent manner.

The effects of hypoxia on the osteogenic potential of mesenchymal stem cells (MSCs) have been previously reported. From these studies, possible factors affecting the association between hypoxia and the osteogenic differentiation of MSCs have been suggested, including hypoxia severity, cell origin and methods of induction. The effect of the duration of hypoxia, however, remains poorly understood. The aim of the present study was to investigate the effect of continuous hypoxia on the induced osteogenesis of MSCs. Rat MSCs were isolated and cultured in vitro. Once the cells had been cultured to passage three, they were switched to 1% oxygen and cultured either with or without osteogenic medium, while cells in the control groups were cultured under normoxia in corresponding conditions. Four osteogenic differentiation biomarkers, runt-related transcription factor 2, osteopontin, osteocalcin and alkaline phosphatase, were analyzed by quantitative polymerase chain reaction and western blotting at defined intervals throughout the culture period. In addition, Alizarin Red staining was used to assess changes in mineralization. The results showed that 1% hypoxia was able to enhance and accelerate the osteogenic ability of the MSCs during the initial phases of differentiation, and the protein expression of certain associated biomarkers was upregulated. However, continuous hypoxia was shown to impair osteogenesis in the latter stages of differentiation. These findings suggest that hypoxia can regulate the osteogenesis of MSCs in a time-dependent manner.

miR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia.

The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body's response. We found miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia.