Lithium chloride prevents glucocorticoid-induced osteonecrosis of femoral heads and strengthens mesenchymal stem cell activity in rats.

BACKGROUND: Accumulating evidence suggests that lithium influences mesenchymal stem cell (MSC) proliferation and osteogenic differentiation. As decreased bone formation in femoral heads is induced by glucocorticoids (GCs), we hypothesized that lithium has a protective effect on GC-induced osteonecrosis of femoral heads (ONFH). METHODS: A rat ONFH model was induced by methylprednisolone (MP) and the effect of lithium chloride on the models was evaluated. Micro-computed tomography (CT)-based angiography and bone scanning were performed to analyze the vessels and bone structure in the femoral heads. Hematoxylin and eosin and immunohistochemical staining were performed to evaluate the trabecular structure and osteocalcin (OCN) expression, respectively. Bone marrow-derived MSCs were isolated from the models, and their proliferative and osteogenic ability was evaluated. Western blotting and quantitative real-time polymerase chain reaction were performed to detect osteogenic-related proteins including Runx2, alkaline phosphatase, and Collagen I. RESULTS: Micro-CT analysis showed a high degree of osteonecrotic changes in the rats that received only MP injection. Treatment with lithium reduced this significantly in rats that received lithium (MP + Li group); while 18/20 of the femoral heads in the MP showed severe osteonecrosis, only 5/20 in the MP + Li showed mild osteonecrotic changes. The MP + Li group also displayed a higher vessel volume than the MP group (0.2193 mm3vs. 0.0811 mm3, P < 0.05), shown by micro-CT-based angiography. Furthermore, histological analysis showed better trabecular structures and more OCN expression in the femoral heads of the MP + Li group compared with the MP group. The ex vivo investigation indicated higher proliferative and osteogenic ability and upregulated osteogenic-related proteins in MSCs extracted from rats in the MP + Li group than that in the MP group. CONCLUSIONS: We concluded that lithium chloride has a significant protective effect on GC-induced ONFH in rats and that lithium also enhances MSC proliferation and osteogenic differentiation in rats after GC administration.

Diminished membrane recruitment of Akt is instrumental in alcohol-associated osteopenia via the PTEN/Akt/GSK-3ß/ß-catenin axis.

Alcohol is considered a leading risk factor for osteopenia. Our previous research indicated that the Akt/GSK-3ß/ß-catenin pathway plays a critical role in the ethanol-induced antiosteogenic effect in bone mesenchymal stem cells (BMSCs). PI3K/Akt is negatively regulated by the phosphatase and tensin homolog (PTEN) phosphatase. In this study, we found that ethanol increased PTEN expression in the BMSCs and bone tissue of ethanol-treated Sprague-Dawley rats. PTEN upregulation impaired Akt recruitment to the plasma membrane and suppressed Akt phosphorylation at Ser473, thereby inhibiting Akt/GSK3ß/ß-catenin signaling and the expression of COL1 and OCN in BMSCs in vitro and in vivo. The results of in vivo assays indicated that PTEN inhibition protected bone tissue against ethanol. Interestingly, our data revealed that following ethanol stimulation, PTEN and PTEN pseudogene 1 (PTENP1) mRNA expression was increased in a time-dependent manner, resulting in an increased PTEN protein level. In addition, ethanol upregulated PTEN expression and decreased PTEN phosphorylation (p-PTEN), indicating an increase in functional PTEN levels. In summary, the ethanol-mediated transcriptional and post-transcriptional regulation of PTEN impaired downstream Akt/GSK3ß/ß-catenin signaling and BMSC osteogenic differentiation. Therefore, we propose that Akt/GSK3ß/ß-catenin activation via PTEN inhibition may be a potential therapeutic approach for preventing the development of alcohol-induced osteopenia.

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.

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.

Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease throughout the world. Exosomes derived from miR-140-5p-overexpressing synovial mesenchymal stem cells (SMSC-140s) may be effective in treating OA. We hypothesized that exosomes derived from SMSC-140 (SMSC-140-Exos) would enhance the proliferation and migration abilities of articular chondrocytes (ACs) without harming extracellular matrix (ECM) secretion. METHODS: SMSCs were transfected with or without miR-140-5p. Exosomes derived from SMSCs or SMSC-140s (SMSC-Exos or SMSC-140-Exos) were isolated and identified. Proliferation, migration and ECM secretion were measured in vitro and compared between groups. The mechanism involving alternative Wnt signalling and activation of Yes-associated protein (YAP) was investigated using lentivirus, oligonucleotides or chemical drugs. The preventative effect of exosomes in vivo was measured using Safranin-O and Fast green staining and immunohistochemical staining. RESULTS: Wnt5a and Wnt5b carried by exosomes activated YAP via the alternative Wnt signalling pathway and enhanced proliferation and migration of chondrocytes with the side-effect of significantly decreasing ECM secretion. Highly-expressed miR-140-5p blocked this side-effect via RalA. SMSC-140-Exos enhanced the proliferation and migration of ACs without damaging ECM secretion in vitro, while in vivo, SMSC-140-Exos successfully prevented OA in a rat model. CONCLUSIONS: These findings highlight the promising potential of SMSC-140-Exos in preventing OA. We first found a potential source of exosomes and studied their merits and shortcomings. Based on our understanding of the molecular mechanism, we overcame the shortcomings by modifying the exosomes. Such exosomes derived from modified cells hold potential as future therapeutic strategies.

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.

Osteonecrosis of the femoral head, nonunion and potential risk factors in Pauwels grade-3 femoral neck fractures: A retrospective cohort study.

The present study was to analyze clinical outcome of Pauwels grade-3 femoral neck fractures treated by different surgical techniques. Potential risk factors associated with nonunion and osteonecrosis of the femoral head (ONFH) were investigated as well. The retrospective study comprised of 67 sequential patients treated between January 2008 and December 2011. Patients with Pauwels grade-3 femoral neck fractures were treated by operative reduction and internal fixation. Cannulated screws (CS) were used in 46 patients, dynamic hip screw plus CS (DHS+CS) in 14, and locking compression plate (LCP) for proximal femur in 7. Reduction quality was assessed according to Haidukewych criteria. Postoperative radiographic examinations were conducted to observe fracture healing. Fracture displacement, comminution, fashion of internal fixation, and the sliding effect were analyzed, regarding the incidence of nonunion and ONFH. All patients had a follow-up of 21.6 ±â€Š6.0 months on average. The phenomenon of sliding effect was observed in 16 cases (23.9%). In terms of reduction quality, 64 cases were graded as excellent, 2 were good, and 1 was poor. ONFH was presented in 15 cases (22.4%) and nonunion was found in 8 (11.9%), with 1 patient had ONFH and nonunion concomitantly. Profound hip contour was preserved in 45 cases (67.2%). The fashion of internal fixation yielded different results regarding ONFH and nonunion, whereas the effects of fracture displacement, comminution, and the sliding effect were not significant. ONFH and nonunion were common complications following Pauwels grade-3 femoral neck fractures. Higher incidence of ONFH in DHS+CS and of nonunion in the LCP group should be noted.

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.

Transarticular fixation by hook plate versus coracoclavicular stabilization by single multistrand titanium cable for acute Rockwood grade-V acromioclavicular joint dislocation: a case-control study.

BACKGROUND: Hook plate (HP) is popularly used for acute and severely displaced acromioclavicular (AC) dislocations. However, subacromial impingement and acromion osteolysis induced by transarticular fixation are notorious. The current case-control study was to compare transarticular fixation by HP to coracoclavicular (CC) stabilization by single multistrand titanium cable (MSTC). METHODS: Between January 2006 and August 2009, 24 patients with acute AC dislocations were surgically treated by open reduction and transarticular fixation with HP. These patients were matched to a series of 24 patients, who were managed by CC stabilization with MSTC in the same period. All AC dislocations were graded as Rockwood type V. Implant was removed 8-12 months after the primary operation in all patients, and 12 months at least were needed to assess the maintenance of AC joint. Functional results were evaluated before implant removal as well as in the last follow-up based on Constant-Murley criteria. RESULTS: There were no differences of demographic data including age, dominant gender and side, injury-to-surgery interval, operation time and follow-up period. In terms of functionality, Constant score was 95.8 ± 4.1 in MSTC group, while 76.7 ± 8.0 in HP group before implant removal (P < 0.001). In detail, MSTC was superior to HP in pain, ROM and activities. Constant score was significantly improved to 86.1 ± 5.7 after hardware removal for patients in HP (P < 0.001). Degenerative change of acromioclavicular joint presented in 16 patients (66.7%) in patients treated by HP, while it was found in only 3 patients (12.5%) treated by MSTC (P < 0.001). CONCLUSIONS: MSTC is superior to HP for the treatment of Rockwood type-V acromioclavicular dislocation both before and after removal of the implant. Hardware removal is of great benefits for functional improvement in patients treated by HP.