MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro

Overview: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. Materials and Methods: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. Results: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. Conclusions: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.     

人脂肪源干细胞分离培养及向成骨细胞的诱导分化

背景：脂肪源干细胞可分泌众多的免疫调节因子,不引起T细胞的细胞毒作用,并可通过调整T淋巴细胞的种类和数量。 目的：探讨人脂肪源干细胞在体外分离培养扩增的方法及向成骨细胞诱导分化的能力。 方法：以0.1%的Ⅰ型胶原酶通过组织消化的方法分离人脂肪组织中的干细胞,体外扩增培养至第2代后检测其表面抗原的表达,并在成骨诱导液中促进其向成骨细胞的分化,通过碱性磷酸酶染色、茜素红染色及对碱性磷酸酶的RT-PCR检测来明确其分化能力。 结果与结论：体外分离培养的脂肪源干细胞生长稳定,扩增速度快。流式细胞仪检测结果显示其高表达干细胞相关抗原。向成骨细胞诱导后经免疫组化染色可见矿化结节形成,RT-PCR检测发现碱性磷酸酶表达阳性。提示脂肪源干细胞在体外分离培养方法简单,扩增速度快,并具有定向分化的能力,是可靠的组织修复和细胞治疗的种子细胞来源。     

The role of epigenetic modifications in the osteogenic differentiation of adipose-derived stem cells

ABSTRACT

Over the last decade, stem cells have drawn extensive attention from scientists due to their full potential in tissue engineering, gene therapy, and cell therapy. Adipose-derived stem cells (ADSCs), which represent one type of mesenchymal stem cell (MSC), hold great promise in bone tissue engineering due to their painless collection procedure, their ability to self-renew and their multi-lineage differentiation properties. Major epigenetic mechanisms, which involve DNA methylation, histone modifications and RNA interference (RNAi), are known to represent one of the determining factors of ADSC fate and differentiation. Understanding the epigenetic modifications of ADSCs may provide a clue for improving stem cell therapy in bone repair and regeneration. The aim of this review is to present the recent advances in understanding the epigenetic mechanisms that facilitate ADSC differentiation into an osteogenic lineage, in addition to the characteristics of the main epigenetic modifications.     

脂肪干细胞成骨分化的研究进展

创伤或肿瘤导致的骨缺损是骨科、整形修复科、口腔颌面外科等科室的常见疾病,现有的治疗手段均有一定局限性。脂肪干细胞(ASC)是来自脂肪组织的多能干细胞,基于ASC的干细胞疗法和骨组织工程,为骨缺损的修复和再生提供了新的思路。ASC成骨分化是多种基因、蛋白和信号通路相互作用的复杂过程,其中Runx2和Osterix、Wnt、骨形成蛋白、Notch、成纤维细胞生长因子、环磷腺苷/蛋白激酶A、Hedgehog、丝裂原活化蛋白激酶等均扮演了重要角色。体外化学诱导ASC的成骨分化已经非常成熟,利用细胞共培养和各种支架也可诱导ASC的成骨分化。验证ASC分化成骨的方法包括茜素红染色和相关基因与蛋白的检测。影响ASC成骨分化的因素包括供体种属、年龄、脂肪获取部位等供体因素,培养液糖浓度、ASC的代数、冻存等实验因素,血管内皮细胞生长因子、生长分化因子、胰岛素样生长因子、转化生长因子β、尼尔样1型分子、LIM矿化蛋白1、低氧诱导因子1、肿瘤坏死因子α、干扰素γ、IL-6等生长因子,糖皮质激素、雌激素、胰岛素、褪黑素、瘦素等激素,一氧化氮、组蛋白H1、白藜芦醇、曲古抑菌素A、小檗碱、硼替佐米、阿司匹林、辛伐他汀等化学因子及药物,电磁场和超声波等物理因素,拉伸力和剪切力等生物力学因素,钙、锌、锂、锶、硒等金属或非金属离子,微小RNA以及富血小板血清和富血小板纤维蛋白、降钙素基因相关肽、中药和咖啡因等其他因素。本文总结了ASC成骨分化的过程,分析了相关基因和信号通路,回顾了诱导和验证方法,讨论了主要影响因素及其机制,并展望了未来研究方向。     

脂肪源性干细胞成骨分化过程中成骨相关miRNA的表达模式

背景：miRNA在细胞分化等众多生理病理过程中起重要调控作用,可调节脂肪来源干细胞成骨分化。 目的：筛选成骨分化相关miRNA,分析miRNA在脂肪来源干细胞成骨分化过程中的表达模式。 方法：从皮下脂肪组织中分离、培养人脂肪来源干细胞。应用基因芯片技术筛选脂肪来源干细胞成骨诱导前后表达差异显著的miRNA,采用RT-PCR技术检测所筛选的miRNA在成骨诱导第7,14,21天上调/下调的相对表达强度,以ELISA试剂盒检测相应时间点成骨相关蛋白的表达情况。 结果与结论：①倒置显微镜下观察,传3代后可获得均一性较高的脂肪来源干细胞,一定诱导条件下具有成骨、成脂、成软骨分化能力。②基因芯片技术筛选出成骨分化前后表达差异变化明显的9个miRNA,其中5个上调,4个下调。③成骨诱导第7天,miR-106a表达上调1.58倍(P < 0.05)。第14天时,9个miRNA均表达上调。第21天时,5个miRNA表达上调,4个表达下调。④成骨相关蛋白骨钙素、碱性磷酸酶、Ⅰ型胶原及骨唾液酸蛋白的质量浓度在诱导成骨分化第7天即明显升高,第14天达到峰值,第21天略有下降。     

miR-302对脂肪间充质干细胞向成脂和成骨分化的调控

背景：间充质干细胞具有自我更新能力,在一定条件下能够分化为一定谱系的细胞,但很多机制至今未明。 目的：探究miR-302b对脂肪间充质干细胞向成脂和成骨分化的调控作用。 方法：miR-302模拟物转染作用于脂肪间充质干细胞进行成骨成脂诱导,对照组转染miR-302阴性对照模拟物miR-NC。采用碱性磷酸酶染色及活性分析、茜素红染色、油红O染色和萃取实验观察miR-302上调对脂肪间充质干细胞成骨和成脂分化的影响,以及Western blot检测miR-302上调后成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在脂肪间充质干细胞中的表达。 结果与结论：①碱性磷酸酶沉淀物在miR-302过表达细胞中产生的量均明显少于对照组,进一步发现miR-302过表达实验组碱性磷酸酶活性明显低于对照组(P < 0.05)。②miR-302过表达明显抑制了矿物质沉积钙结节的形成,miR-302上调实验组橘红色的钙结节明显少于对照组。③miR-302过表达实验组油红O染色阳性的细胞数明显高于对照组,进一步表明实验组细胞萃取得到的油红O吸光度值明显上升(P < 0.05)。④成骨诱导第6天时成骨分化转录因子Runx2和成骨早期标志物碱性磷酸酶在miR-302过表达的细胞中都有不同程度的下降。⑤以上结果表明miR-302的上调能够抑制脂肪间充质干细胞的成骨分化,同时促进其向成脂分化。miR-302在间充质干细胞向成脂和成骨分化平衡发挥了双向调控作用。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

力学因素对间充质干细胞成骨分化的影响

背景：随着间充质干细胞的广泛研究,间充质干细胞能够向成骨方向分化已得到充分证实。近年来,许多学者致力于研究力学因素对体外培养间充质干细胞增殖和定向诱导分化的影响。 目的：综述力学因素对间充质干细胞成骨分化产生的影响。 方法：应用计算机检索PubMed,ScienceDirect,ISI-SCIE,中国期刊全文数据库,中国期刊全文数据库_世纪期刊,中国博士学位论文全文数据库,中国优秀硕士学位论文全文数据库数据库中1999-01/2012-01关于力学因素对间充质干细胞成骨分化影响的文章,在标题和摘要中以“力学因素, 间充质干细胞,成骨分化”或“mechanical factors,Mesenchymal Stem Cell,Osteogenic Differentiation”为检索词进行检索。选择文章所述内容涉及力学因素对间充质干细胞中成骨分化的作用,并且选择近期发表或发表在权威杂志文章。初检索到文献104篇,最终入选30篇文献进行综述。 结果与结论：近年的各项实验都证明,力学因素对间充质干细胞的增殖和分化、形态、发育和功能起着重要的调节作用。而力学刺激的大小、方式以及作用时间对间充质干细胞定向分化的调节、功效作用不尽相同。应选择最佳的力学刺激促使间充质干细胞的成骨分化,以达到最佳的临床治疗效果。     

生物支架材料诱导脂肪来源干细胞成骨分化的最新热点

文题释义： 脂肪干细胞的优势：脂肪来源干细胞具有以下特点：高增殖能力和分泌活性;兼具多向分化潜能;通过其免疫调节能力,能够提高移植后的愈合效果;来源丰富,可在自体或异体上迅速提取,以上优势使其成为骨组织工程的理想种子细胞。 骨组织工程支架材料的分类：主要分为3类,无机材料、天然高分子材料、合成高分子材料,无机材料包括羟基磷灰石、磷酸三钙、生物活性玻璃、钛金属、镁金属,天然高分子材料包括胶原、丝素蛋白、壳聚糖,合成高分子材料包括聚己内酯、聚乳酸、聚乙醇酸及其共聚物-聚乳酸-羟基乙酸共聚物。 背景：脂肪来源干细胞获取便捷且具有显著的成骨分化能力,被认为是骨缺损修复的理想种子细胞。然而骨组织工程学的研究进展揭示,生物支架材料改性能够直接调控干细胞的成骨分化。 目的：综述能够调控脂肪来源干细胞成骨分化效果的各种生物支架材料。 方法：由第一作者通过检索中国知网、万方、维普、PubMed、Embase和Web of Science数据库2016年1月至2019年5月发表的相关文献,检索词为“脂肪干细胞,支架材料,成骨,金属,钛;Adipose derived stem cells,scaffold,osteogenic,metal,Ti”,最终选取符合标准的文献62篇。 结果与结论：用于骨组织工程的支架材料分为无机材料、天然高分子材料、合成高分子材料3类,无机材料包括羟基磷灰石、磷酸三钙、生物活性玻璃、钛金属、镁金属,天然高分子材料包括胶原、丝素蛋白、壳聚糖,合成高分子材料包括聚己内酯、聚乳酸、聚乙醇酸及其共聚物-聚乳酸-羟基乙酸共聚物。设计能与细胞相互作用以指导其生物反应和骨分化的材料研究一直层出不穷,但如何营造更安全、更合理、更贴近生物体内的细胞的生长微环境仍然面临着很多困难。对生物支架材料的改性能够直接调控干细胞的成骨分化,同时成骨诱导之外的血管化及植入后的感染也是需要关注的问题。 ORCID: 0000-0003-4520-3031(张圣敏) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The isolation and differentiation of human adipose-derived stem cells using membrane filtration

Human adipose-derived stem cells (hADSCs) were purified from a suspension of human adipose tissue cells (stromal vascular fraction) by the conventional culture method and by membrane filtration through polyurethane (PU) foam membranes. hADSCs can be obtained from a suspension of human adipose tissue cells using the membrane filtration method in less than 30?min, whereas the conventional culture method requires 5-12 days. hADSCs that express the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the recovery solution from the PU membranes; no hADSCs were isolated in the permeate. After filtration, the cells expressing the mesenchymal stem cell markers were 3-4.5 times more concentrated than in the initial suspension of human adipose tissue cells, with the level of concentration depending on the surface modification of the PU membrane. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the recovery solutions, whereas CD34(+) cells could not be purified by the conventional culture method. The hADSCs in the recovery solution demonstrated a superior capacity for osteogenic differentiation than did the cells in the suspension of human adipose tissue cells. These results suggested that the hADSCs with the capability for osteogenic differentiation adhered to the PU membranes.     

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.     

脂肪干细胞诱导分化的现状及前景

背景：脂肪干细胞是由中胚层发育而来的多能干细胞,在特殊的生长因子和环境等诱导培养条件下,可以向不同的谱系分化。 目的：详细阐述脂肪干细胞诱导分化的条件及鉴定方法。 方法：应用计算机检索万方数据库及PubMed数据库2005至2014年10年间的文献,中文检索词为“脂肪干细胞,诱导,分化”;英文检索词为“adipose derived stem cells,differentiation”。依据纳入排除标准选择37篇文献进行归纳总结。 结果与结论：脂肪干细胞在抗坏血酸、胰岛素、地塞米松、转化生长因子β作用下可向软骨细胞分化;成脂诱导液的配方包括3-异丁基-1-甲基黄嘌呤(IBMX)、胰岛素、地塞米松、吲哚美辛;成骨分化常用的诱导剂包含地塞米松或维生素D3、抗坏血酸,β-甘油磷酸钠;碱性成纤维细胞生长因子、表皮生长因子及维生素B27可联合应用诱导脂肪干细胞成神经分化;向心肌细胞分化普遍应用的诱导因子是5-氮杂胞苷;血管内皮生长因子和碱性成纤维细胞生长因子共同作用可以诱导脂肪干细胞向血管内皮细胞分化。随着分子生物学和细胞生物学的迅速发展,脂肪干细胞的分化研究也会更加深入,在目前对脂肪干细胞诱导分化现象观察的基础上,应加强对其内在的分子机制及调控脂肪干细胞可塑性的基因和蛋白的研究。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics

This study investigated the in vitro effects of akermanite, a new kind of Ca-, Mg-, Si-containing bioceramic, on the attachment, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). Parallel comparison of the cellular behaviors of hASCs on the akermanite was made with those on beta-tricalcium phosphate (beta-TCP). Scanning electron microscope (SEM) observation and fluorescent DiO labeling were carried out to reveal the attachment and growth of hASCs on the two ceramic surfaces, while the quantitative assay of cell proliferation with time was detected by DNA assay. Osteogenic differentiation of hASCs cultured on the akermanite and beta-TCP was assayed by ALP expression and osteocalcin (OCN) deposition, which was further confirmed by Real-time PCR analysis for markers of osteogenic differentiation. It was shown that hASCs attached and spread well on the akermanite as those on beta-TCP, and similar proliferation behaviors of hASCs were observed on the two ceramics. Both of them exhibited good compatibility to hASCs with only minor cytotoxicity as compared with the tissue culture plates. Interestingly, the osteogenic differentiation of hASCs could be enhanced on the akermanite compared with that on the beta-TCP when the culture time was extended to approximately 10 days. Thus, it can be ascertained that akermanite ceramics may serve as a potential scaffold for bone tissue engineering.     

Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo

Human, rat, and mouse studies have demonstrated the existence of a population of adipose-derived adult stem (ADAS) cells that can undergo multilineage differentiation in vitro. However, it remains unclear whether these cells maintain their multilineage potential in vivo. The aim of this study was to examine the in vitro and in vivo characteristics and behavior of a potential population of murine ADAS (muADAS) cells isolated from the visceral fat of the abdominal cavity of C57BL/10J mice. We used flow cytometry to examine the cells' expression of CD29, CD31, CD45, CD34, CD44, CD144, CD146, Flk1, and Sca-1. The isolated cell population was CD45 negative, which precludes contamination by hematopoietic cells, but was partially positive for Sca-1 and CD34: 2 stem-cell markers. After induction in conditioned medium, the muADAS cells gained the ability to undergo adipogenic, osteogenic, chondrogenic, myogenic, and hematopoietic differentiation in vitro. The muADAS cells readily differentiated to form bone and cartilage in vivo for up to 24 weeks, but their ability to regenerate muscle or reconstitute bone marrow was found to be limited.     

人脂肪源性干细胞多潜能分化特性

目的原代分离培养人脂肪干细胞(ADSCs),探讨细胞增殖及多向分化,间充质干细胞相关特性。方法取人吸脂术的脂肪组织通过酶消化法分离培养ADSCs,观察细胞形态,MTT法检测细胞增殖活性,流式细胞术检测细胞周期和表面抗原的表达,用茜素红、甲苯胺蓝和油红O染色鉴定向骨、软骨及脂肪诱导分化。结果人ADSCs呈长梭形、旋涡状生长,传至第15代的ADSCs仍具较强的增殖能力,ADSCs具有干细胞特性,高表达标记CD90和CD44的间充质干细胞,不表达内皮细胞标记CD31,而CD49d低表达,CD106阴性。成骨诱导后可见钙结节形成并被茜素红染成紫红色,软骨诱导后分泌大量蛋白多糖,被甲苯胺蓝染成蓝色,成脂诱导后细胞内可见大量脂滴被油红O染色红色。结论人脂肪干细胞可分化成间充质干细胞,具有稳定增殖和成骨、成软骨和成脂等多向分化潜能。     

Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells

Adipose-derived human mesenchymal stem cells (hMSCs) will be more valuable for tissue engineering applications if they can be extensively subcultured without loss of phenotype and multilineage differentiation ability. This study examined the effects of serial passaging on growth rate, gene expression, and differentiation potential of adipose-derived hMSCs. Differentiation was assessed by analyzing changes in messenger RNA (mRNA) expression of osteogenic and adipogenic marker genes and by determining production of calcium deposits and lipid vacuoles. Cells cultured in osteogenic medium for 2 weeks upregulated expression of alkaline phosphatase mRNA relative to cells in growth medium, and deposited calcium. Calcium deposition decreased in cells from passages 4 to 6 but returned to levels near or above those of primary cells by passage 10. Cells cultured in adipogenic medium upregulated expression of lipoprotein lipase and peroxisome proliferator activated receptor-gamma mRNA relative to cells in growth medium, and formed lipid vacuoles at all passages. By passage 8, however, cells in adipogenic medium also deposited calcium. Growth rate was stable through passage 5, then decreased. The results of this study indicate that adipose-derived hMSCs are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings) but that osteogenic differentiation may start to dominate at later passages.     

马血清在脂肪干细胞成肌诱导中的作用

背景：利用5-氮杂胞苷诱导脂肪干细胞成肌实验对培养技术以及细胞活性要求较高,诱导成肌时间较长。倘若某种添加剂可缩短诱导时间,将具有重要意义。 目的：评估马血清在5-氮杂胞苷诱导脂肪干细胞成肌细胞实验中的作用。 方法：将第1代长满100 mm培养皿的脂肪干细胞传代至3个6孔板,实验组培养基中加入体积分数为5%马血清+10 μmol/L 5-氮杂胞苷;对照组培养基中单纯添加10 μmol/L的5-氮杂胞苷;空白组为单纯低糖DMEM培养基,余培养、传代及鉴定所需的条件相同。每日光镜观察记录形态,在培养的第7,14,28,35天行免疫荧光和流式细胞仪检测肌蛋白表达差异。 结果与结论：免疫荧光检测成肌特异性胞质蛋白表达以及流式细胞检测相应蛋白表达率提示,加入马血清后的实验组脂肪干细胞的成肌速度和诱导成肌所需时间明显比单纯加入5-氮杂胞苷的对照组更有优势。实验初步表明马血清在促进脂肪干细胞诱导成肌方面起着缩短诱导成肌所需时间的作用。     

大鼠腹腔脂肪干细胞的分离培养及诱导成骨

背景：脂肪干细胞取材方便、增殖旺盛、具有多向分化潜能,有望取代骨髓间充质干细胞而成为新一代组织工程的种子细胞。然而,脂肪干细胞的分离培养仍存在诸多困难与不足。 目的：优化脂肪干细胞的分离培养方法,并鉴定其成骨分化潜能。 方法：选取200 g成年雌性大鼠1只,无菌环境下切取大鼠肾脏、子宫周围及腹后外侧白色脂肪组织,多次胶原酶消化法分离消化,低糖培养基培养脂肪干细胞,倒置显微镜下观察脂肪干细胞的形态变化及增殖能力。采用成骨诱导培养液对第3代细胞进行成骨诱导,并采用碱性磷酸酶、茜素红染色方法进行鉴定。 结果与结论：脂肪干细胞形态以长梭形为主,增殖活跃呈旋涡状生长。经成骨诱导10 d后,碱性磷酸酶染色为阳性;成骨诱导28 d后,茜素红染色阳性。提示采用多次酶消化法得到的大鼠腹腔源性脂肪干细胞在体外易于分离培养,可以稳定传代。在一定条件诱导下,可以向成骨细胞分化。采用以上方法进行脂肪干细胞的分离培养可以为组织工程提供大量、优质的种子细胞。     

Cyclic tensile stretch modulates osteogenic differentiation of adipose-derived stem cells via the BMP-2 pathway

Introduction

Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals.

Material and methods

We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µɛ) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µɛ). Then osteogenic related genes were analysed by real-time PCR.

Results

The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h’ duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP.

Conclusions

We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.