成骨诱导的兔骨髓基质干细胞成骨活性的表达及维持

目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。     

小剂量阿司匹林对去势大鼠骨髓基质干细胞成骨分化的影响

[目的]检测不同浓度低剂量阿司匹林对去势后(OVX)大鼠骨髓基质干细胞成骨分化的影响.[方法]建立SD大鼠去势模型,全骨髓贴壁法培养骨髓基质干细胞(BMSCs),传代4次后进行成骨诱导,建立对照组和不同浓度阿司匹林组(0.25、0.5、1、2及5mmol/L).于不同的培养时间点,倒置显微镜观察并进行MTT检测细胞增殖生长情况;碱性磷酸酶(ALP)试剂盒检测培养细胞上清ALP活性;于诱导7d和21d时分别行细胞碱性磷酸酶染色和茜素红钙化结节染色.[结果]阿司匹林无明显促进BMSCs增殖作用,而大剂量阿司匹林(5 mmol/L)具有抑制BMSCs增殖作用.实验组(0.25、0.5、1、2 mmol/L阿司匹林组)细胞培养上清中ALP浓度较对照组显著增强(P＜0.05);碱性磷酸酶染色(1、2 mmol/L组)阳性表达增强;茜素红染色显示,实验组(0.5、1、2mmol/L组)钙结节数量、钙化面积均高于对照组(P＜0.05).[结论]阿司匹林不能直接促进去势大鼠BMSC增殖活性,但小剂量阿司匹林可显著提高体外培养BMSC成骨活性,增强钙盐沉积,促进钙结节形成.     

不同血清对成人骨髓基质干细胞成骨诱导分化的影响

[目的]探讨三种不同来源血清对体外培养成人骨髓基质干细胞(hBMSCs)向成骨诱导分化的作用.[方法]将体外第3代hBMSCs向成骨诱导分化培养,分为胎牛血清组(对照组)、AB 血清组和自体血清组.对比观察细胞碱性磷酸酶(ALP)染色、钙结节染色.诱导培养后4、7、14 d 和 21 d,各血清组分别进行钙黄绿素法荧光显微镜动态观察矿盐沉积,检测 ALP 活性,实时荧光定量 PCR(RT-qPCR)法检测成骨基因(ALP)、骨桥蛋白(OPN)和骨钙素(OCN)的表达.[结果]ALP 染色和钙结节染色结果显示,与自体血清(AS)组、胎牛血清(FBS)组相比,AB 血清(ABS)组明显提高了 hBMSCs 的染色阳性率.荧光显微镜下观察诱导 21 d 的 hBMSCs,ABS 组较 FBS 组、AS 组呈现更多的钙盐沉积.ALP 活性结果显示,同一时间点 ABS 组的 ALP 活性均明显高于 FBS 组和 AS 组,差异有统计意义(P<0.05).RT-qPCR 结果显示,在7、14 d 和 21 d,ABS 组 ALP、OPN 和 OCN 成骨基因表达均明显高于 FBS 组、AS 组,其中,ALP 基因表达在 7 d 出现峰值,OPN 基因表达在 14 d 出现峰值,OCN 基因表达在 21 d 出现峰值,差异均有统计学意义(P<0.05).[结论]ABS 对 hBMSCs 成骨分化作用较 FBS、AS 明显增强.ABS 有望替代 FBS 建立符合骨组织工程临床应用要求的体外 hBMSCs 培养体系.     

骨髓基质干细胞成骨的研究进展

组织工程技术是目前解决大块骨与软骨组织缺损修复难题最有前景的手段之一，种子细胞是组织工程学中的重要环节。骨髓基质干细胞（bone marrow stromal cells，BMSCs）是一类具有多向分化潜能的组织干细胞，在体内外适当的诱导环境下可以分化为骨、软骨、脂肪、肌肉、神经、肌腱及韧带等多种组织细胞。该细胞群来源充足，取材方便，增殖能力强，     

辛伐他汀对老年大鼠骨髓基质干细胞成骨和成脂分化的影响

目的 从细胞和基因水平探讨辛伐他汀对老年大鼠骨髓基质干细胞成骨和成脂分化的影响.方法 18月龄雄性SD大鼠的骨髓基质干细胞进行成骨和成脂诱导培养,培养介质中加入辛伐他汀(10-6、10-7、10-8 mol/L和10-9 mol/L),同时设溶剂对照组.成骨检测:碱性磷酸酶(ALP)染色和定量,茜素红矿化染色和定量,ALP和骨钙素(OC)基因分析;成脂检测:油红脂肪细胞染色和定量,脂蛋白脂酶(LPL)和过氧化物酶增殖活化受体(PPARγ2) 基因分析.结果 在含有低剂量地塞米松的成骨诱导条件下,辛伐他汀随浓度增加促进了细胞基质的矿化,增强了ALP的活性及染色,提高了ALP和OC的基因表达(若无地塞米松,辛伐他汀则无法单独诱导成骨,此结果 未展示);同时,辛伐他汀随浓度增加减弱了脂肪细胞的油红染色,抑制了LPL 和PPARγ2的基因表达.显著性差异皆发生于10-6 mol/L和10-7 mol/L辛伐他汀组.结论 辛伐他汀随浓度增加抑制了老年骨髓基质干细胞的成脂分化,并中等强度地促进了老年骨髓基质干细胞的成骨分化.这表明辛伐他汀具有促进骨合成代谢的作用,可以用于治疗常见的骨代谢疾病,如增龄性的骨质疏松症.     

骨髓基质干细胞成骨-成脂分化及补肾中药对其影响的研究进展

骨髓基质干细胞在不同条件的诱导下,可以分化为成骨细胞、软骨细胞、成纤维细胞、脂肪细胞、神经细胞等多种细胞系,在骨髓微环境中骨和脂肪形成之间的关系是复杂的,成骨细胞与脂肪细胞均来源于骨髓基质干细胞 (MSCs),并且在其向成骨细胞和脂肪细胞分化之间存在相互逆转的关系和很大程度的可塑性。骨质疏松患者骨量减少与骨髓腔中脂肪组织增加有关,这可能与骨髓中MSCs分化失衡,过多向脂肪细胞分化有关。中医从“肾主骨”、“髓生骨”理论出发,认为肾精不足所致的骨髓空虚是骨质疏松症发病的关键,补肾中药通过影响骨髓基质干细胞成骨-成脂分化,从而防止骨质疏松症的发生。     

冲击波诱导人骨髓基质干细胞向成骨细胞分化及其机制研究

目的 观察在体外培养条件下冲击波诱导健康成年人骨髓基质干细胞(BMSCs)向成骨细胞分化特点,研究适宜冲击波作用强度,并探讨其机制. 方法 抽取健康成年人骨髓血,采用Percoll法进行分离,进行体外培养传代,免疫组织化学鉴定,选择处于对数生长期的BMSCs,应用不同强度冲击波进行诱导,40 d后进行碱性磷酸酶(ALP)钙-钴法染色、茜素红染色及Ⅰ型胶原免疫组化染色,应用图文分析系统,通过BMSCs向成骨细胞分化程度确定最佳冲击波作用强度.采用RT-PCR的方法检测c-fos和c-jun基因表达量的变化.结果 最佳作用强度为8.5 kV,120次,其能量密度是(0.230±0.015)mJ/mm2.成骨作用明显优于对照组(P＜0.01).采用最佳强度冲击波作用后,c-fos和c-jun基因表达量较对照组明显增加,45 min达到高峰. 结论 低能冲击波在适宜强度下可诱导BMSCs向成骨细胞分化,c-foe和c-jun基因表达增加.     

辛伐他汀在骨髓基质干细胞向成骨细胞分化过程中的作用

目的 通过对小鼠骨髓干细胞体外培养的观察，研究辛伐他汀在骨髓基质干细胞向成骨细胞定向分化过程中的作用。方法 取雄性6周ICR小鼠股骨骨髓基质细胞进行原代和传代培养，应用组织化学及yon Kossa方法检测细胞碱性磷酸酶染色和细胞外基质矿化；在细胞培养早期加入辛伐他汀(实验组)或保持基础培养条件(对照组)，应用半定量RT-PCR方法分别检测两组Ⅰ型胶原蛋白(COL1)、碱性磷酸酶(ALP)、转录因子CBFA1和Osterix(OSX)在成骨细胞分化过程中的表达。结果 小鼠骨髓基质细胞经体外诱导后分化为具备碱性磷酸酶活性和矿化细胞外基质的成熟成骨细胞。实验组COL1、ALP和CBFA1表达在细胞培养第3，5天均高于对照组，OSX表达差异不明显。结论 辛伐他汀在成骨细胞分化过程中促进其相关基因的表达。     

骨髓基质干细胞成脂分化的调控及其对骨质疏松的影响

研究发现随着年龄的增长和绝经期后骨质疏松,骨髓腔内的脂肪细胞逐渐增多,取代了成骨细胞,因此抑制骨髓基质干细胞成脂分化是目前治疗骨质疏松的新途径。应用各种成脂诱导剂建立体外定向诱导骨髓基质干细胞成脂分化模型是研究新途径的基础。除了通过光学显微镜观察成脂分化的能力,还可运用分子生物学的方法定性、定量检测成脂分化的程度。过氧化物酶增殖子激活受体(PPARγ)是脂肪细胞的分化过程中起关键性作用的调节因子,组蛋白去乙酰化酶(SIRT1)被激活后能抑制PPARγ的表达,减少脂肪细胞的生成,起到预防和治疗骨质疏松的作用。笔者就以上方面和对减少骨髓脂肪分化的策略作了综述。     

辛伐他汀在人骨髓基质干细胞向成骨细胞分化过程中的作用

目的观察辛伐他汀对体外培养的人骨髓基质干细胞（Human Bone Marrow Stromal cells,hBMCs）成骨分化功能的影响,探讨其刺激成骨的作用机制。方法体外培养来自于外伤所致股骨颈骨折患者的骨髓基质干细胞,传代后实验组加入1×10^-7mol/L的辛伐他汀,在不同时间点采用ELISA检测核转录因子1（Core Binding Factor1,Cbfa1）与DNA的结合活性,碱性磷酸酶（Alkaline Phosphatase,ALP）比活性,及放射免疫法检测骨钙素（Osteocalcin,OCN）含量。结果在辛伐他汀作用后,实验组与对照组比较,实验组Cbfa1因子与DNA的结合活性增高,ALP比活性增高且骨钙素含量增加。结论1×10^-7mol/L辛伐他汀能够促进人骨髓基质干细胞成骨分化,此种促进作用可能与辛伐他汀增加其分化过程中相关转录因子的活性有关。     

Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation

Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8–80 years, with no systemic illness, yielded a mean of 92 ± 65 × 10⁶ nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 ± 28 alkaline phosphatase-positive colonies per 10⁶ nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D₃ synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.     

冲击波诱导人骨髓基质细胞成骨分化及机制的研究

目的 观察出生后人骨髓基质细胞(hIMSCs)在体外培养条件下增殖与分化的特点；研究适宜能量冲击波对出生后hMSCs成骨分化的作用及机制。方法 抽取健康自愿者髂骨骨髓，采用密度梯度离心法进行hMSCs体外培养。设冲击波组(SW组)与对照组，应用不同能量级冲击波对SW组原代细胞进行处理，根据细胞活力测定与集落形成数量确定适宜的冲击波能量值。应用适宜的冲击波能量处理hMSCs原代细胞并传代培养，采用倒置显微镜观察、细胞增殖活力测定、ELISA法检测细胞分泌TGF-81、茜素红染色、钙钴法染色、四环素荧光标记、细胞分泌碱性磷酸酶测定和逆转录一聚合酶链反应(RT-PCR)检测骨钙素mRNA表达等方法，对SW组和对照组的各代细胞形态、增殖与分化及其机制进行探讨。结果冲击波处理体外原代培养hMSCs的适宜能量为10kV(500)。SW组细胞在冲击波处理后早期分泌TGF-B1显著高于对照组(P＜0．001)。SW组与对照组细胞在形态学方面第3代前无明显差别；SW组各代细胞分泌碱性磷酸酶显著高于对照组(P＜0．01)；茜素红染色、钙钴法染色、四环素荧光标记等显示SW组细胞的成骨作用明显优于对照组；SW组细胞经冲击波处理后第10天应用RT-PCR方法可以检测到骨钙素mRNA的表达，与对照组比较差异有统计学意义(P＜0．001)。结论 冲击波对体外培养的出生后人骨髓基质细胞具有促进成骨分化的作用，适宜能量为10kv(500)，其机制之一为TGF-B1介导的促hMSCs成骨分化作用。10kV(500)能量级的冲击波对体外培养的hMSCs增殖无影响，大于该能量的冲击波具有抑制细胞增殖的作用。     

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.     

In vitro growth and osteoblastic differentiation of human bone marrow stromal cells supported by autologous plasma

Autologous bone marrow stromal cells have been proposed as an adjuvant in the treatment of bone nonunion. This cell therapy requires the establishment of culture conditions that permit the rapid expansion of these cells ex vivo while retaining their potential for further differentiation. Several culture models have been proposed, all of them using fetal calf serum (FCS) as a source of growth factors. This is problematic for subsequent autologous implantation because of possible disease transmission. Here we report the establishment and characterization of a cell culture system in which standard FCS has been replaced by autologous plasma recovered from bone marrow (APM). Short-term cultures of human bone marrow stromal (HBMS) cells grown in mineralizing conditions with APM exhibited a significantly higher number of ALP-positive colonies than those grown with FCS, indicating an enhanced ability of APM to recruit osteoprogenitor cells for culture. Analyses of long-term cultures showed that the use of APM did not affect cell proliferation as cell number at confluence and proliferation rate were similar whether primary cultures had been maintained with APM or FCS. In first-passage cultures, an osteoblastic differentiation was observed in both cases as the cells expressed ALP and formed mineralized bone-like nodules. We noted that the age of donor had a negative effect on the number of osteoprogenitor cells recruited for culture. This effect had an impact on proliferation rate in primary cultures performed with APM, although the cell number obtained after expansion remained independent of age. Our study shows that proliferative capacity and osteoblastic differentiation potential of HBMS cells are maintained when cultured with APM. Thus, this cell culture system could provide a new and safer tool to elaborate an autologous cell therapy designed to enhance osteogenesis.     

辛伐他汀促进大鼠骨髓基质细胞的成骨分化

目的观察辛伐他汀体内给药对去卵巢大鼠骨髓基质细胞(BMSC)增殖和分化的影响,探讨辛伐他汀的成骨作用机制.方法24只12周龄雌性SD大鼠随机分成4组,每组6只假手术组(G1)和去卵巢组(G2)每天蒸馏水灌胃;去卵巢大鼠10mg·kg-1·d-1辛伐他汀灌胃(G3);去卵巢大鼠20mg·kg-1·d-1辛伐他汀灌胃(G4).实验持续4周,第29天处死所有大鼠取骨髓细胞培养.用Cell Counting Kit-8(CCK-8)法检测成骨细胞的增殖能力;细胞培养第14天用半定量RT-PCR和western blot检测Runx2/Cbfal mRNA和蛋白的表达、第16天检测碱性磷酸酶活性(ALP)和第21天检测茜素红染色.结果辛伐他汀不能促进去卵巢大鼠成骨细胞增殖.G3组和G4组的Runx 2/Cbfa1 mRNA表达水平和ALP活性都显著高于G2组,给药两组之间无显著差别.G4组的Runx 2/Cbfal蛋白表达水平比G2组和G3组显著增加,G2组与G3组无明显差别.茜素红染色表明辛伐他汀能促进成骨细胞的矿化能力,两种剂量组之间无明显差别.结论辛伐他汀体内给药能够促进去卵巢大鼠BMSC向成骨细胞分化,但是不能促进细胞增殖.     

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.     

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and -glycerophosphate, under 0.8%–15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1h and 6h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.     

Cooperative actions of hepatocyte growth factor and 1,25-dihydroxyvitamin D3 in osteoblastic differentiation of human vertebral bone marrow stromal cells

Bone formation and remodeling require continuous generation of osteoprogenitor cells from bone marrow stromal cells (MSC), which generate and respond to a variety of growth factors with putative roles in hematopoiesis and mesenchymal differentiation. In this study we examine the interaction of two such factors on the maturation of skeletal components. We previously reported that these factors, hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D(3) (vitD(3)), act together to increase alkaline phosphatase in chondroblasts. We now describe the cooperative effect of these agents on MSC isolated and cultured from human vertebral bone marrow. MSC (passages 3-9) isolated from bone marrow cells of human vertebrae (T1-L5) from 22-36-year-old normal donors were first expanded in vitro and then plated in the presence or absence of 10 ng/mL HGF and/or 10 nmol/L vitD(3), for 7-18 days. HGF treatment increased cell proliferation 2.5-fold, with no effect on alkaline phosphatase activity. Whereas vitD(3) treatment inhibited cell growth by 50%, alkaline phosphatase activity was stimulated eightfold, although no mineralization was observed. HGF together with vitD(3) increased cell proliferation 1.5-fold and alkaline phosphatase activity 13-fold over untreated control. Moreover, mineralization was detected only with this combination. Our findings provide evidence that HGF in concert with vitamin D may promote growth and differentiation of human MSC into osteogenic cells.