The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts

Mouse embryonic fibroblasts (MEFs) are multi-potent progenitor cells (MPCs), can differentiate into different lineages, such as osteogenic, and adipogenic. PTEN, a tumor suppressor, may be involved in regulating bone development through interacting with COX-2. BMP9, the most potent osteogenic BMPs, can up-regulate COX-2 in MPCs. Whether PTEN is involved in BMP9 induced osteogenic differentiation in MPCs remains unknown. The goal of this investigation is to identify the effect of PTEN on BMP9-induced osteogenic differentiation in MPCs and dissect the possible mechanism underlay this. We found that BMP9 down-regulates PTEN, and PTEN inhibitor (VO) effectively increases different osteogenic markers induced by BMP9 in MEFs. Exogenous expression of PTEN inhibits BMP9 induced ectopic bone formation apparently. Mechanistically, we found that VO can enhance BMP9 induced BMPs/Smads signaling prominently without no substantial effects on cell cycle. Further analysis indicates that VO can promote BMP9-induced expression of COX-2 in MEFs, which can be eliminated by PI3K inhibitor. Additionally, COX-2 knockdown abolishes the effect of VO on BMP9-induced ALP activities in MEFs. Our findings suggest that PTEN plays an important role in regulating BMP9 induced osteogenic differentiation in MPCs, which may be mediated by PTEN/PI3K/Akt signaling to modulate the expression of COX-2.     

Cyclic tension promotes osteogenic differentiation in human periodontal ligament stem cells

Orthodontic forces result in alveolar bone resorption and formation predominantly on the pressure and tension sides of the tooth roots, respectively. Human periodontal ligament stem cells (PDLSCs) have demonstrated the capacity to differentiate into osteoblasts, and they play important roles in maintaining homeostasis and regenerating periodontal tissues. However, little is known about how PDLSCs contribute to osteoblastogenesis during orthodontic tooth movement on the tension side. In this study, we applied a 12% cyclic tension force to PDLSCs cultured in osteoinductive medium. The osteogenic markers Runx2, ALP, and OCN were detected at the mRNA and protein levels at different time points using real-time PCR and western blot analyses. We discovered that the mRNA and protein levels of Runx2, ALP and OCN were significantly up-regulated after 6, 12 and 24 hours of mechanical loading on PDLSCs compared to levels in unstimulated PDLSCs (P < 0.05). This study demonstrates, for the first time, the effects of mechanical tensile strain on the osteogenic differentiation of PDLSCs, as examined with a Flexcell FX-4000T Tension Plus System. Our findings suggested that cyclic tension could promote the osteogenic differentiation of PDLSCs. Furthermore, the effects of orthodontic force on alveolar bone remodeling might be achieved by PDLSCs.     

DLX1联合BMP9促进人骨肉瘤细胞向成骨分化

目的探讨DLX1联合BMP9可否影响人骨肉瘤细胞MG63的成骨分化。方法用构建有DLX1和BMP9基因的重组腺病毒Ad DLX1和Ad BMP9,单独或联合感染MG63细胞,分为DLX1组(Ad DLX1+AdRFP感染组)、DLX1+BMP9组(Ad DLX1+Ad BMP9感染组)、BMP9组(Ad BMP9+AdRFP感染组)、RFP组(AdRFP感染组)。用RT-PCR和Western blot验证DLX1和BMP9的表达情况,Transwell实验检测细胞迁移、侵袭能力;碱性磷酸酶(ALP)染色和读数分析细胞早期成骨能力,茜素红染色检测细胞晚期成骨能力,Western blot检测骨桥蛋白(OPN)蛋白表达水平。结果 Ad DLX1和Ad BMP9感染MG63细胞后,DLX1和BMP9的表达水平上调(P0.05);与RFP组相比,DLX1组、DLX1+BMP9组和BMP9组细胞迁移能力和侵袭能力下降(P0.05),DLX1+BMP9组细胞迁移能力和侵袭能力下降更为明显(P0.05);ALP活性、钙盐沉积和OPN蛋白表达在DLX1+BMP9组和BMP9组增加(P0.05),且DLX1+BMP9组较BMP9组显著增强(P0.05)。结论 DLX1与BMP9联合作用可抑制骨肉瘤细胞迁移和侵袭,同时可诱导骨肉瘤细胞向成骨分化。     

Maxillofacial-derived stem cells regenerate critical mandibular bone defect

Stem cell-based bone tissue regeneration in the maxillofacial complex is a clinical necessity. Genetic engineering of mesenchymal stem cells (MSCs) to follow specific differentiation pathways may enhance the ability of these cells to regenerate and increase their clinical relevance. MSCs isolated from maxillofacial bone marrow (BM) are good candidates for tissue regeneration at sites of damage to the maxillofacial complex. In this study, we hypothesized that MSCs isolated from the maxillofacial complex can be engineered to overexpress the bone morphogenetic protein-2 gene and induce bone tissue regeneration in vivo. To demonstrate that the cells isolated from the maxillofacial complex were indeed MSCs, we performed a flow cytometry analysis, which revealed a high expression of mesenchyme-related markers and an absence of non-mesenchyme-related markers. In vitro, the MSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Gene delivery of the osteogenic gene BMP2 via an adenoviral vector revealed high expression levels of BMP2 protein that induced osteogenic differentiation of these cells in vitro and induced bone formation in an ectopic site in vivo. In addition, implantation of genetically engineered maxillofacial BM-derived MSCs into a mandibular defect led to regeneration of tissue at the site of the defect; this was confirmed by performing micro-computed tomography analysis. Histological analysis of the mandibles revealed osteogenic differentiation of implanted cells as well as bone tissue regeneration. We conclude that maxillofacial BM-derived MSCs can be genetically engineered to induce bone tissue regeneration in the maxillofacial complex and that this finding may be clinically relevant.     

Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

ABSTRACT

Aim of the study: The peripheral nervous system is involved in regulation of bone metabolism via sensory and sympathetic innervation. Substance P (SP) and calcitonin gene-related peptide (CGRP) are two sensory neuropeptides that have been associated with regulation of osteogenic differentiation. However, the interaction between SP and CGRP both with each other and the bone morphogenetic protein 2 (BMP2) in regulation of osteogenic differentiation has not been studied. Therefore, the aim of this study was to investigate the interaction between SP and CGRP on BMP2-induced bone differentiation using model progenitor cells. Materials and methods: C2C12 myoblasts and MC3T3 pre-osteoblasts were treated with SP and CGRP, both individually and in combination, in the presence of BMP2. The effects of the neuropeptides on BMP2-induced osteogenic differentiation were assessed by measuring alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic markers. Results: Both SP and CGRP enhanced BMP2 signaling, Runx2 mRNA expression, as well as mineralization in vitro. Co-stimulation with SP and CGRP resulted in down-regulation of BMP2-induced bone differentiation, suggesting potential crosstalk between the two neuropeptides in regulation of BMP2 signaling. Conclusions: Based on the results shown here, CGRP can mitigate augmenting effects of SP on BMP2 signaling and the three pathways potentially converge on Runx2 to regulate BMP2-induced bone differentiation.     

周期性牵张力介导BMP9通过PI3K/AKT信号通路调控人牙周膜成纤维细胞成骨分化

目的　研究在周期性牵张力介导下，骨形态发生蛋白9（bone morphogenetic proteins 9，BMP9）能否激活PI3K/AKT信号通路调控人牙周膜成纤维细胞（human periodontal ligament fibroblasts，hPDLFs）成骨分化。 方法　利用FlexCell系统对体外培养的hPDLFs加载正弦波、形变率10%、频率0.5 Hz的周期性牵张力，免疫荧光法检测细胞骨架蛋白的表达及分布，qPCR检测RUNX2、OCN、OPN、OSX mRNA的表达情况，免疫印迹法检测成骨标志蛋白OCN、OPN的表达，加入PI3K信号抑制剂LY294002后AKT、P-AKT以及OCN、OPN的变化。 结果　与对照组比较，6 h、12 h组的细胞骨架蛋白表达增多且呈受力方向分布，OCN、OPN表达上调，差异有统计学意义（P<0.05），qPCR检测mRNA的表达趋势与蛋白检测结果一致。 结论　周期性牵张力介导BMP9可以通过PI3K/AKT信号通路调控hPDLFs成骨分化。     

周期性牵张力介导BMP9通过PI3K/AKT信号通路调控人牙周膜成纤维细胞成骨分化

目的　研究在周期性牵张力介导下,骨形态发生蛋白9（bone morphogenetic proteins 9,BMP9）能否激活PI3K/AKT信号通路调控人牙周膜成纤维细胞（human periodontal ligament fibroblasts,hPDLFs）成骨分化。 方法　利用FlexCell系统对体外培养的hPDLFs加载正弦波、形变率10%、频率0.5 Hz的周期性牵张力,免疫荧光法检测细胞骨架蛋白的表达及分布,qPCR检测RUNX2、OCN、OPN、OSX mRNA的表达情况,免疫印迹法检测成骨标志蛋白OCN、OPN的表达,加入PI3K信号抑制剂LY294002后AKT、P-AKT以及OCN、OPN的变化。 结果　与对照组比较,6 h、12 h组的细胞骨架蛋白表达增多且呈受力方向分布,OCN、OPN表达上调,差异有统计学意义（P<0.05）,qPCR检测mRNA的表达趋势与蛋白检测结果一致。 结论　周期性牵张力介导BMP9可以通过PI3K/AKT信号通路调控hPDLFs成骨分化。     

Non-coding RNAs regulate the BMP/Smad pathway during osteogenic differentiation of stem cells

MicroRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are involved in the regulation of bone metabolism. The BMP/Smad pathway is a key signaling pathway for classical regulation of osteogenic differentiation. Non-coding RNAs (ncRNAs) and the BMP/Smad pathway both have important roles for osteogenic differentiation of stem cells, bone regeneration, and development of bone diseases. There is increasing evidence that ncRNAs interact with the BMP/Smad pathway to regulate not only osteogenic differentiation of stem cells but also progression of bone diseases, such as osteoporosis (OP), myeloma, and osteonecrosis of the femoral head (ONFH), by controlling the expression of bone disease-related genes. Therefore, ncRNAs that interact with BMP/Smad pathway molecules are potential targets for bone regeneration as well as bone disease diagnosis, prevention, and treatment. However, despite extensive studies on ncRNAs associated with the BMP/Smad pathway and osteogenic differentiation of stem cells, there is a lack of comparability. Moreover, some bone disease-associated ncRNAs with low abundance can be difficult to detect and there is a lack of mature delivery systems for their stable translocation to target sites, thus limiting their application. In this review, we summarize the research progress on interactions between ncRNAs and the BMP/Smad pathway during osteogenic differentiation of various stem cells and in the regulation of bone regeneration and bone diseases.     

NAC-PCCs载骨形态发生蛋白2基因纳米微球促大鼠骨髓间充质干细胞成骨分化

目的　构建半胱氨酸及磷酸胆碱共接枝的仿生壳聚糖载体（NAC-PCCs），并包封骨形态发生蛋白2（BMP2）基因进行诱导骨髓间充质干细胞成骨分化的研究。 方法　制备pBMP2/NAC-PCCs材料，检测其粒径、形态，绘制DNA缓释曲线，研究微球抗DNA酶降解能力。在骨髓间充质干细胞与材料共培养过程中，检测材料的转染效能、ROS清除能力、BMP2蛋白分泌水平、成骨相关基因RUNX2、OC表达、碱性磷酸酶活性等指标，以研究其对骨髓间充质干细胞成骨分化的影响。 结果　微球材料能有效避免DNA被生物酶降解，其转染效率为23.1%，ROS清除率为(36.13±0.47)%。同时实验结果显示与NAC-PCCs共培养的细胞， 其细胞内BMP2表达水平高于其余各组且成骨分化效果最佳。 结论　NAC-PCCs包封BMP2基因形成的纳米微球材料具有促进BMSC成骨分化作用。     

骨形态发生蛋白9诱导成骨分化及其在骨科中的应用

背景：骨形态发生蛋白9是一种强有力的诱导成骨分化的骨形态蛋白,但对它的研究却偏少。 目的：对近年来国内外有关骨形态发生蛋白9诱导成骨分化的研究现状以及其在骨科相关疾病治疗中的应用研究进行综述。 方法：应用计算机检索CNKI 和Pubmed 数据库中2001-01/2011-06 关于骨形态发生蛋白9的文章,以“骨形态蛋白-9;成骨分化;骨缺损;脊柱融合;肿瘤”或“bone morphogenetic protein 9, osteogenic differentiation, bone fracture, spinal fusions, tumor”为关键词进行检索。选择近5 年内关于骨形态发生蛋白9的近期发表或发表在权威杂志的文章。初检得到91 篇文献,根据纳入标准得到34 篇文献并进行系统回顾与综述。 结果与结论：迄今为止,已发现多种生长因子能够促进骨形成,其中,骨形成蛋白是骨组织形成过程中最关键的调节因子。骨形态发生蛋白9属于骨形态发生蛋白家族,不仅能强有力的诱导间充质细胞、前成骨细胞和肌肉细胞等成骨分化,而且在软骨形成中也具有重要作用。其诱导骨形成机制不完全同于传统的骨形态发生蛋白。动物实验证明其还能促进骨折愈合、诱导脊柱融合,调控肿瘤的迁徙。因此,骨形态发生蛋白9在骨科领域中具有潜在的应用前景。     

脂多糖对牙周膜干细胞生物学特性的影响

探讨脂多糖（LPS）对牙周膜干细胞（PDLSCs）生物学特性的影响.方法 分离、培养PDLSCs,并检测其间充质干细胞标志物STRO-1、CD146的表达情况.在含有LPS的培养基中培养PDLSCs,分别进行以下实验：（1）应用甲苯胺蓝染色法和Brdu掺入法分别检测PDLSCs的克隆形成率和细胞增殖率;（2）对PDLSCs进行骨向和脂肪向诱导,采用茜素红染色和油红O染色检测其骨向和脂肪向分化潜能;（3）应用ELISA法测定培养上清中IL-6的浓度;（4） Western blot检测PDLSCs磷酸化ERK1/2的表达情况.结果 PDLSCs表达STRO-1、CD146,阳性率分别为28.6％±2.3％、86.7％±3.9％.LPS对PDLSCs的克隆形成率和细胞增殖率没有影响.在含有LPS的矿化诱导液中,矿化面积明显增多,说明LPS能够促进PDLSCs骨向分化.脂肪向诱导实验发现,LPS组和无LPS组的油红染色阳性面积相近,两组无显著性差异.LPS促进IL-6的分泌和ERK1/2活化,并具有剂量依赖性.结论 LPS改变了PDLSCs的生物学特性,提示炎性状态可能影响PDLSCs介导的牙周组织再生.     

Disruption of heparan and chondroitin sulfate signaling enhances mesenchymal stem cell-derived osteogenic differentiation via bone morphogenetic protein signaling pathways

Cell surface heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans have been implicated in a multitude of biological processes, including embryonic implantation, tissue morphogenesis, wound repair, and neovascularization through their ability to regulate growth factor activity and morphogenic gradients. However, the direct role of the glycosaminoglycan (GAG) sugar-side chains in the control of human mesenchymal stem cell (hMSC) differentiation into the osteoblast lineage is poorly understood. Here, we show that the abundant cell surface GAGs, HS and CS, are secreted in proteoglycan complexes that directly regulate the bone morphogenetic protein (BMP)-mediated differentiation of hMSCs into osteoblasts. Enzymatic depletion of the HS and CS chains by heparinase and chondroitinase treatment decreased HS and CS expression but did not alter the expression of the HS core proteins perlecan and syndecan. When digested separately, depletion of HS and CS chains did not effect hMSC proliferation but rather increased BMP bioactivity through SMAD1/5/8 intracellular signaling at the same time as increasing canonical Wnt signaling through LEF1 activation. Long-term culturing of cells in HS- and CS-degrading enzymes also increased bone nodule formation, calcium accumulation, and the expression of such osteoblast markers as alkaline phosphatase, RUNX2, and osteocalcin. Thus, the enzymatic disruption of HS and CS chains on cell surface proteoglycans alters BMP and Wnt activity so as to enhance the lineage commitment and osteogenic differentiation of hMSCs.     

MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, are the most common causes of bone tissue destruction. Recently, human periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs), a population of multipotent stem cells, have been used to reconstruct tissues destroyed by chronic inflammation. However, the impact of the local inflammatory microenvironment on tissue-specific stem cells and the mechanisms controlling the effects of the local inflammatory environment remain poorly understood. In this study, we found that the multidifferentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis-affected periodontal ligament tissue (P-PDLSCs) was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue (H-PDLSCs). Inflammation in the microenvironment resulted in an inhibition of miR-17 levels, and a perturbation in the expression of miR-17 partly reversed the differentiation potential of PDLSCs in this microenvironment. Furthermore, inflammation in the microenvironment promoted the expression of Smad ubiquitin regulatory factor one (Smurf1), an important negative regulator of MSC osteogenic differentiation. Western blotting and 3' untranslated regions (3'-UTR) reporter assays confirmed that Smurf1 is a direct target of miR-17 in PDLSCs. Our data demonstrate that excessive inflammatory cytokine levels, miR-17, and Smurf1 were all involved in a coherent feed-forward loop. In this circuit, inflammatory cytokines led to direct activation of Smurf1 and downregulation of miR-17, thereby increasing degradation of Smurf1-mediated osteoblast-specific factors. The elucidation of the molecular mechanisms governing MSC osteogenic differentiation in a chronic inflammatory microenvironment could provide us with a better knowledge of chronic inflammatory disorder and improve stem cell-mediated inflammatory bone disease therapy.