Role of epidermal growth factor receptor in osteoblastic differentiation of rat bone marrow stromal cells

In an attempt to understand the role of epidermal growth factor (EGF) and its receptor (EGF-R) in osteoblastic cell differentiation, the changes in [¹²⁵I]-EGF binding capacity, synthesis of EGF-R protein, and expression of EGF-R mRNA were investigated during osteoblastic differentiation of cultured bone marrow stromal cells which were collected from the femora of young adult rats. In addition, the ability of EGF to suppress osteoblastic differentiation was also studied. Dexamethasone at a concentration of 0.1 mM increased the expression of osteoblastic markers by bone marrow stromal cells cultured in alpha-modified minimum essential medium (-MEM) con taining 1% fetal bovine serum (FBS), 50 mg/ml ascorbic acid, and 10 mM -glycerophosphate, as revealed by elevated alkaline phosphatase activity, an increase in osteopontin mRNA expression, and bone nodule formation. This osteoblastic differentiation was accompanied by a decreased expression of EGF-R mRNA, decreased synthesis of EGF-R protein, and a decreased number of EGF-binding sites without any change in affinity. When these cells were incubated with dexamethasone and EGF in combination throughout the culture, they exhibited significantly lower levels of all osteoblastic markers than did dexamethasonetreated cells, indicating suppression of osteoblastic differentiation by EGF. In contrast, EGF treatment of the cells induced expression of EGF-R mRNA. Thus, a decrease in EGF binding associated with osteoblastic differentiation could lead to decreased responsiveness of bone marrow cells to EGF, whereas the EGF-induced increase in expression of EGF-R could facilitate the inhibition of cell differentiation by EGF. These findings suggested that upregulation of EGF-R on bone marrow stromal cells antagonizes their differentiation, and thus possibly functions as a negative regulator of osteoblastic differentiation.     

Smad4促进成骨分化的机制研究

目的 探讨Smad4基因促进成骨分化的作用机制。方法 采用条件性基因敲除技术Cre/loxp,制备骨细胞特异性敲除Smad4小鼠(Smad4otcko),小鼠胚胎骨骼透明染色分析胚胎期小鼠长骨生长状况;待小鼠成长至1月龄,X-ray检测突变小鼠与对照组小鼠的骨密度差异;静态骨组织形态学分析检测突变鼠及对照鼠的骨量变化、成骨细胞数量变化等差异;实时荧光定量PCR检测Smad4突变鼠股骨成骨细胞相关因子Runx2、ALP、OSX及OCN;破骨细胞TRAP染色分析Smad4突变鼠破骨细胞形态及数量变化;qPCR检测突变鼠股骨破骨吸收标志基因RANKL、OPG,并计算RANKL/OPG比率。结果 Smad4基因敲除小鼠在胚胎期未出现长骨生长异常。X线结果显示,1月龄时,与对照组小鼠相比,Smad4突变鼠的骨密度降低（P＜0.05）,静态骨组织形态学分析表明突变鼠松质骨减少,皮质骨变薄,骨小梁数量减少（P＜0.05）;Smad4突变鼠成骨细胞标志基因表达量显著降低,成骨细胞的数量明显减少（P＜0.05）;RANKL作为破骨吸收标志物表达上调、作为其拮抗剂的OPG表达量下调,RANKL/OPG比率增高（P＜0.05）。结论 Smad4基因通过促进成骨分化,降低破骨吸收从而来维持骨稳态。     

Kinetics of polymethylmethacrylate particle-induced inhibition of osteoprogenitor differentiation and proliferation.

Periprosthetic bone loss induced by implant wear debris may be a combined effect of osteolysis and reduced bone formation resulting from particle-induced suppression of osteoprogenitor differentiation. This study investigated the time-dependent effects of polymethylmethacrylate (PMMA) particles on the osteogenic capability of bone marrow osteoprogenitor cells during the early phase of differentiation. Murine bone marrow cells were challenged with PMMA particles (0.30% v/v) on the first day of growth in osteogenic medium. Particles were removed from culture after 1, 3, and 5 days, respectively, after which cell growth in osteogenic medium was continued until the 15th day. Bone marrow osteoprogenitor cells exposed to particles during the first 5 days of differentiation showed complete, irreversible inhibition of proliferation, alkaline phosphatase expression, and mineralization. Osteoprogenitors exposed to particles for more than 5 days showed the same degree of inhibition, while those exposed to particles for less than 5 days showed a diminished inhibitory response. Conditioned medium from particle-treated cells did not suppress osteogenic development, demonstrating that suppression of osteogenesis was not due to secreted inhibitory factors. This study has shown that the early phase of osteoprogenitor differentiation is a crucial time period during which exposure to PMMA particles causes irreversible inhibition of osteogenesis.     

In vivo effects of short- and long-term MAPK pathway inhibition against neuroblastoma

Background/purpose

It was reported that almost 80% of relapsed neuroblastomas showed MAPK pathway mutations. In our previous study, both trametinib (MEK inhibitor) and CH5126766 (RAF/MEK inhibitor) showed in vitro antitumor effects on neuroblastoma cells with ERK phosphorylation (pERK). In this study, we analyzed the in vivo effects of MAPK pathway inhibition in neuroblastoma xenografts.

Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.     

骨形成蛋白-2基因在人骨髓基质细胞中的表达及对其成骨分化的作用

目的利用构建的人骨形成蛋白-2(BMP2)真核表达载体pcDNA3／BMP2，检测其转染人骨髓基质细胞后的表达及对其成骨分化的影响。方法酶切鉴定构建的真核表达载体pcDNA3／BMP2，利用脂质体介导的转染技术，将所构建的载体导入骨髓基质细胞中，体外单层培养。分别于转染后48h和4周采用原位杂交、免疫组化和碱性磷酸酶、钙化学染色方法检测BMP2的基因蛋白表达以及对骨髓基质细胞成骨分化的影响。结果pcDNA3／BMP2酶切片段的大小与理论相符。转染后细胞能检测到BMP2基因和BMP2蛋白表达，并促进成骨转化。结论pcDNA3／BMP2转染骨髓基质干细胞中可获得短暂和长期表达，并加强骨髓基质细胞的成骨分化能力。     

脂联素通过调节BMP信号通路促进骨髓干细胞成骨分化

目的 研究脂联素（adiponectin, ApN）对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化的作用,并探讨其可能的机制。方法 体外培养BMSCs,构建ApN过表达质粒及干扰质粒,转染至BMSCs中。将BMSCs随机分为5组：对照组、过表达组、过表达空载组、干扰组和干扰空载组。茜素红染色观察各组细胞钙化沉积。碱性磷酸酶（alkaline phosphatase, ALP）染色观察各组细胞成骨分化能力。qRT-PCR检测ApN受体、骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路及成骨相关基因表达情况。结果 与对照组相比,过表达组BMSCs中钙化沉积和ALP阳性表达增多,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著升高（P<0.05）;干扰组BMSCs中钙化沉积和ALP阳性表达减少,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著降低（P<0.05）。结论 ApN可能通过上调BMP信号通路促进BMSCs成骨分化。     

COMP‐Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway

Mesenchymal stem cells (MSCs) are pleiotrophic cells that differentiate to chondrocytes, osteoblasts, or adipocytes, as a result of crosstalk by specific signaling pathways including MAPK pathway. Recently cartilage oligomeric matrix protein angiopoietin1 (COMP‐Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms. Our results clearly demonstrated that MSCs cultured in each induction medium with COMP‐Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5‐ and 2‐fold, respectively) as well as up‐regulate each gene, except for adipogenic differentiation. Accordingly, we found that phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts. Therefore, our findings suggest that COMP‐Ang1 present a portal to promote MSCs differentiation to chondrocytes and osteoblasts through Ang1/Tie2 signaling pathway and provide insights into novel therapies for bone diseases. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1920–1928, 2013     

Strontium ranelate promotes osteoblastic differentiation and mineralization of murine bone marrow stromal cells: involvement of prostaglandins.

Strontium ranelate is a new anti-osteoporosis treatment. This study showed that strontium ranelate stimulated PGE(2) production and osteoblastic differentiation in murine marrow stromal cells, which was markedly reduced by inhibition of COX-2 activity or disruption of COX-2 gene expression. Hence, some anabolic effects of strontium ranelate may be mediated by the induction of COX-2 and PGE(2) production. INTRODUCTION: Strontium ranelate is an orally active drug that reduces vertebral and hip fracture risk by increasing bone formation and reducing bone resorption. Strontium ranelate effects on bone formation are the result of increased osteoblastic differentiation and activity, but the mechanisms governing these effects are unknown. Based on previous work, we hypothesized that strontium ranelate increases cyclooxygenase (COX)-2 expression and that, consequently, the prostaglandin E(2) (PGE(2)) produced could mediate some effects of strontium ranelate on osteoblasts. MATERIALS AND METHODS: Marrow stromal cells (MSCs) from COX-2 wildtype (WT) and knockout (KO) mice were cultured with and without low-dose dexamethasone. Osteoblastic differentiation was characterized by alkaline phosphatase (ALP) activity, real-time PCR for ALP and osteocalcin (OCN) mRNA expression, and alizarin red staining for mineralization. Medium PGE(2) was measured by radioimmunoassay or enzyme immunoassay. RESULTS AND CONCLUSIONS: In MSCs from COX-2 WT mice, strontium ranelate significantly increased ALP activity, ALP and OCN mRNA expression, and mineralization after 14 or 21 days of culture. A short treatment at the beginning of the culture (0-7 days) with strontium ranelate was as effective as continuous treatment. Strontium ranelate (1 and 3 mM Sr(+2)) dose-dependently increased PGE(2) production, with maximum PGE(2) production occurring during the first week of culture. NS-398, a selective COX-2 inhibitor, blocked the strontium ranelate stimulation of PGE(2) production and significantly inhibited the strontium ranelate stimulation of ALP activity. In MSCs from COX-2 KO mice, the strontium ranelate stimulation of ALP and OCN mRNA expression and mineralization were markedly reduced compared with COX-2 WT cultures. Similar effects of strontium ranelate on osteoblastic markers and on PGE(2) production were seen when MSCs were cultured with or without low-dose dexamethasone (10 nM). We conclude that PGE(2) produced by the strontium ranelate induction of COX-2 expression plays a role in strontium ranelate-induced osteoblastic differentiation in MSCs in vitro.     

牵张应力调控Notch1信号通路促进大鼠BMSCs增殖和成骨分化

目的 探讨牵张应力调控Notch1促进骨髓间充质干细胞(BMSCs)增殖和成骨分化的机制。方法 制备并鉴定大鼠BMSCs。细胞分组设置为：0%拉伸幅度组、0%+DAPT组、5%拉伸幅度组、5%+DAPT组。运用CCK-8法测定BMSCs增殖;采用碱性磷酸酶、茜素红和油红O染色法检测细胞成骨和成脂分化;通过Western bloting检测Notch1和Jagged1蛋白表达。结果 5%拉伸幅度牵张应力可显著提高BMSCs增殖、碱性磷酸酶表达及钙化结节形成,抑制BMSCs成脂分化(P<0.05或P<0.01),同时显著提高Notch1和Jagged1蛋白表达(P<0.01);DAPT可显著逆转牵张应力对BMSCs增殖、碱性磷酸酶表达、钙化结节形成、成脂分化及Notch1和Jagged1蛋白表达的影响(P<0.05或P<0.01)。结论 5%拉伸幅度的牵张应力促进了BMSCs增殖和成骨分化,Notch1信号通路可能是牵张应力促进BMSCs增殖和成骨分化的靶点。     

辛伐他汀对人牙髓干细胞增殖和分化的影响

目的:研究辛伐他汀对人牙髓干细胞(dental pulpstem cells,DPSCs)增殖和成骨分化的影响。方法:将第3代人DPSCs在矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(1×10-5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,碱性磷酸酶试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,茜素红染色鉴定成骨分化。结果:各浓度辛伐他汀均抑制人DPSCs增值,辛伐他汀浓度为1×10-5mol/L时,抑制作用最明显。适宜浓度辛伐他汀(1×10-6mol/L、1×10-7mol/L、1×10-8mol/L)促进人DPSCs向成骨细胞分化,其中,1×10-7mol/L的辛伐他汀促进ALP活性的作用最明显。结论:辛伐他汀抑制人DPSCs的增值,适宜浓度的辛伐他汀可有效促进人DPSCs的成骨分化。     

p38MAPK与 ERK1/2的协同效应及对成骨细胞分化的调控

 目的探讨骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化过程中 p38MAPK与 ERK1/2的协同效应及其机制。方法以成骨细胞分化添加剂诱导小鼠 BMSCs向成骨细胞分化,测定碱性磷酸酶活性和钙沉积量。检测磷酸化 p38MAPK和磷酸化 ERK1/2(p-ERK1/2)的表达水平评估通路的激活状况。以 SB203580或 PD98059阻断 p38MAPK或 ERK1/2通路,观察对成骨细胞分化的影响。以 SB203580或亚砷酸钠阻断或激活 p38MAPK通路,观察 p-ERK1/2的变化。以冈田酸抑制蛋白磷酸酯酶 2A(protein phosphatases type 2A,PP2A)活性,观察 p-ERK1/2的变化及对成骨细胞分化的影响。通过免疫共沉淀实验观察 PP2A和 ERK1/2间的结合及 SB203580对结合的影响。结果成骨细胞分化添加剂诱导 BMSCs向成骨细胞分化的过程伴有 ERK1/2和 p38MAPK通路的激活, SB203580剂量±赖性抑制成骨细胞分化,PD98059剂量±赖性增强成骨细胞分化。 SB203580使 p-ERK1/2表达增加,亚砷酸钠减弱其表达。冈田酸使 p-ERK1/2表达增加,并使成骨细胞分化受到抑制。 PP2A可直接与 ERK1/2结合,SB203580使 PP2A与 ERK1/2的结合减弱。结论 p38MAPK可通过 PP2A与 ERK1/2产生协同效应,并调节 BMSCs向成骨细胞分化。     

Matrix vesicles are carriers of bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), and noncollagenous matrix proteins

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.     

软骨寡聚基质蛋白对BMP 2诱导骨髓间质干细胞分化的影响

 目的 探讨过表达软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）对BMP-2诱导骨髓间质干细胞成骨及成软骨分化的影响。方法 用BMP-2诱导骨髓间质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间质干细胞过表达COMP,空载质粒作为对照。以RT-PCR检测成骨相关基因Ⅰ型胶原、RUNX2、骨桥蛋白、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖的表达变化;通过碱性磷酸酶染色观察成骨过程中的碱性磷酸酶活性,茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果 COMP组目的基因COMP mRNA的表达显著升高;骨桥蛋白mRNA表达水平较对照组低,呈现出一致的下调趋势（P<0.05）;Ⅰ型胶原、RUNX2、骨钙蛋白mRNA表达水平在诱导早期均高于对照组（P<0.05）,但在诱导晚期均明显低于对照组（P<0.05）;成软骨指标（Ⅱ型胶原、SOX9、蛋白聚糖）的基因表达水平强于对照组,呈一致的上调趋势（P<0.05）;SOX9 mRNA表达水平高于对照组,仅在第7天时差异具有统计学意义（P<0.05）;细胞成骨染色（碱性磷酸酶染色、茜素红染色）均弱于对照组,而阿利新蓝染色强于对照组。结论 COMP能抑制BMP-2诱导骨髓间质干细胞成骨分化,促进BMP-2诱导骨髓间质干细胞成软骨分化。     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

激活BMP信号的骨细胞对骨髓基质细胞成骨及成脂分化的作用研究

目的激活骨细胞系MLO-Y4细胞中BMP信号检测培养上清对骨髓基质细胞系ST2成骨、成脂分化的影响,进一步探讨其机制。方法 0.5‰DMSO,0.5μmol/L BMP激动剂FK506分别处理MLO-Y4 24 h后,使用CCK-8检测细胞活力变化,实时荧光定量PCR检测BMP下游靶基因ID1及ID2 mRNA表达变化;用20%MLO-Y4培养上清与80%新鲜培养基混合后培养ST2细胞,分为DMSO组、FK506组。碱性磷酸酶染色代表其成骨分化能力,通过实时荧光定量PCR检测碱性磷酸酶(ALP)、骨钙蛋白(OCN)、骨唾液酸蛋白(BSP)、Runx2等成骨细胞标志基因,过氧化物酶体增殖剂激活受体γ(PPARγ)和C/EBP成脂分化标志基因。免疫印迹试验(Western blotting)检测ST2细胞内Wnt信号下游β-catenin、BMP信号下游p-smad5蛋白表达水平。结果与DMSO作用的MLO-Y4细胞相比,FK506激动的MLOY4细胞内BMP信号靶基因ID1、ID2上调,但不影响细胞活力。FK506组ST2细胞同DMSO组对比,成骨分化相关标志物,包括ALP、OCN、BSP、Runx2(P0.001)均显著升高;成脂分化标志物PPARγ及C/EBP表达则显著降低(P0.001); ST2细胞内β-catenin蛋白表达量上调(P0.05)。结论 BMP信号激动后MLO-Y4细胞上清可以促进ST2细胞成骨分化、抑制成脂分化,其成骨能力增强与细胞内Wnt信号增强有关。     

p44/42和p38 MAPKs在骨髓间充质干细胞向成骨细胞分化中发挥不同的功能

目的 观察p44／42MAPK、p38MAPK通路在维生素C(Vit C)、β-磷酸甘油(β-GP)诱导骨髓间充质干细胞(BMSCs)向成骨细胞分化过程中的作用。方法 用[^3H]-甲基胸腺嘧啶掺入率法反映细胞增殖情况；洲定碱性磷酸酶活性与钙沉积积反映细胞向成骨细胞分化状态；有Western-blotting法反映MAPK的表达情况。结果 与溶剂对照组相比．在促成骨细胞分化剂Vit C，β-GP作用下，骨髓间充质干细胞(BMSCs)p44／42MAPK、p38MAPK通路均提前5d激活。p44／42MAPK通路阻断剂(PID8059)明显减少[^3H]-甲基胸腺嘧啶掺入率，抑制BMSCs的增殖；而p38MAPK通路的阻断剂(SB203580)则显著降低ALP活性及钙沉积量．抑制BMSCs向成骨细胞的分化。结论 p44／42MAPK通路在BMSCs的增殖过程中起苇要作用，而p38MAPK通路可能与BMSCs向成骨细胞分化调节有关。