A comparison of the osteogenic potential of adult rat mesenchymal stem cells cultured in 2-D and on 3-D collagen glycosaminoglycan scaffolds.

Adult mesenchymal stem cells (MSCs) have the capability to differentiate along several lineages including those of bone, cartilage, tendon and muscle, thus offering huge potential for the field of tissue engineering. The purpose of this study was to characterise the differentiation capacity of rat MSCs cultured on standard plastic coverslips in 2 dimensions and on a novel collagen glycosaminoglycan scaffold in the presence of a standard combination of osteoinductive factors. Cells were cultured for 3, 7, 14 and 21 days and several markers of osteogenesis were analysed. While the initial response of the cells in 3-D seemed to be faster than cells cultured in 2-D, as evidenced by collagen type I expression, later markers showed that osteogenic differentiation of MSCs took longer in the 3-D environment of the collagen GAG scaffold compared to standard 2-D culture conditions. Furthermore, it was shown that complete scaffold mineralisation could be evoked within a 6 week timeframe. This study further demonstrates the potential use of MSC-seeded collagen GAG scaffolds for bone tissue engineering applications.     

Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway

Human mesenchymal stem cells (hMSCs) are a population of multipotent bone marrow cells capable of differentiating along multiple lineages, including bone. Our recently published proteomics studies suggest that focusing of gene expression is the basis of hMSC osteogenic transdifferentiation, and that extracellular matrix proteins play an important role in controlling this focusing. Here, we show that application of a 3-5% tensile strain to a collagen I substrate stimulates osteogenesis in the attached hMSCs through gene focusing via a MAP kinase signaling pathway. Mechanical strain increases expression levels of well-established osteogenic marker genes while simultaneously reducing expression levels of marker genes from three alternate lineages (chondrogenic, adipogenic, and neurogenic). Mechanical strain also increases matrix mineralization (a hallmark of osteogenic differentiation) and activation of extracellular signal-related kinase 1/2 (ERK). Addition of the MEK inhibitor PD98059 to reduce ERK activation decreases osteogenic gene expression and matrix mineralization while also blocking strain-induced down-regulation of nonosteogenic lineage marker genes. These results demonstrate that mechanical strain enhances collagen I-induced gene focusing and osteogenic differentiation in hMSCs through the ERK MAP kinase signal transduction pathway.     

骨髓间充质干细胞复合多肽凝胶及成软骨生成因子修复兔关节软骨缺损

背景：多肽水凝胶因为其具有良好的可塑型性,能够与损伤部位很好的无缝隙结合,所以采用该材料作为支架是骨、软骨组织工程中一种可行的探索。 目的：骨髓间充质干细胞联合新型可注射多肽凝胶及成软骨生成因子修复兔关节软骨缺损,观察其修复效果。 方法：首先分离培养兔骨髓间充质干细胞,兔左侧膝关节处制备直径5 mm,深3 mm的全层骨-软骨缺损模型;右侧造模后空置作为对照。实验分为3组,单纯自组装多肽凝胶移植组,自组装多肽凝胶+成软骨因子组和自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组。采用的成软骨因子包括转化生长因子β1,地塞米松和胰岛素样生长因子1,三者混合后加入到自组装多肽凝胶或骨髓间充质干细胞中。于处理后12周时处死动物行大体及组织学观察、X射线摄片、免疫组织化学法进行组织学评分评估修复情况。 结果与结论：单纯自组装多肽凝胶移植在12周后显示出非常好的修复效果,可见番红O染色,Ⅱ型胶原蛋白免疫组织化学染色强度以及组织学评分明显高于其他组(P < 0.05)。自组装多肽凝胶+成软骨因子组修复效果较好,与自组装多肽凝胶组相似,但其修复区域蛋白聚糖表达比对照组明显升高(P < 0.01)。自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组修复效果不佳,12周未能完全修复缺损区域,与单纯自组装多肽凝胶组比较骨赘的形成有所增加。结果表明,单纯自组装多肽凝胶能够在原位修复骨软骨缺损并促进软骨修复,提示以自组装多肽凝胶支架移植有望提高目前修复软骨缺损的效果。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：     

Adult Stem Cell Driven Genesis of Human-Shaped Articular Condyle

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.     

胶原海绵上诱导骨髓间充质干细胞向软骨细胞分化的体外实验研究

研究在胶原海绵上间充质干细胞(M SC s)向软骨细胞的定向诱导,以及其对M SC s细胞增殖力、细胞周期的影响,为临床应用胶原海绵作为M SC s支架、修复软骨缺损奠定基础。取大鼠股骨骨髓,经梯度离心法和贴壁法分离M SC s,用加有转化生长因子1β10 ng/m l、地塞米松1-0 7m o l/L、转铁蛋白3 m g/m l、胰岛素2 m g/m l的诱导因子培养基分别在胶原海绵、培养板中进行诱导,14 d后免疫组化检测II型胶原分泌情况,四甲基偶氮唑蓝(M TT)法比较两者之间细胞增殖情况,流式细胞仪(FCM)检测各组细胞周期。14 d后,胶原海绵上及培养板中均诱导出表达II型胶原的软骨细胞,M TT法检测胶原海绵诱导组,和培养板诱导组细胞增殖值分别为0.9213±0.0312和0.5875±0.0258,两者间有显著性差异(P<0.05),FCM检测胶原海绵各组S期 G2/M期百分率,发现其与培养板中同期细胞相比有显著性差异(P<0.05),各组均未发现异倍体。结果表明在胶原海绵上,M SC s能定向诱导为软骨细胞,细胞增值能力更强,M SC s细胞DNA合成、细胞分裂增加,无异倍体产生,可作为一种较为理想的组织工程学支架。     

Dermis isolated adult stem cells for cartilage tissue engineering

Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor β1 (TGF-β1) or bone morphogenetic protein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-β1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.     

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.     

体外构建组织工程骨-软骨复合组织

背景：设计一体化、具有过渡结构的双层支架材料,复合软骨细胞、骨髓间充质细胞,有利于新生的骨与软骨组织之间形成良好界面。 目的：模仿自然骨-软骨基质构建复合支架,以软骨细胞和骨髓间充质干细胞为种子细胞,体外观察复合组织的成软骨及成骨能力。 方法：制备明胶-硫酸软骨素-透明质酸及明胶-陶瓷化骨多孔复合支架,构建自然骨-软骨基质复合支架,复合兔软骨细胞与骨髓间充质干细胞,分未成骨诱导与成骨诱导两组培养,并进行MTT、糖胺多糖含量、碱性磷酸酶活性检测,以及苏木精-伊红染色检测。 结果与结论：未成骨诱导与成骨诱导两组骨髓间充质干细胞增殖及糖胺多糖含量差异无显著性意义。未成骨诱导组碱性磷酸酶活性缓慢上升,成骨诱导组诱导后碱性磷酸酶活性迅速上升,14 d时达到稳定状态。两组苏木精-伊红染色结果无明显区别,均已形成含有双层组织的类似骨-软骨样组织,其间可见未降解支架形态,但由于基质形成不完善及支架未完全降解,此种结构不成熟,细胞分布不均匀,支架内部可见散在无细胞区域。证实采用两种细胞与双层结构的支架经体外分层复合能够形成组织工程骨软骨复合组织。     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

骨髓间充质细胞复合胶原-壳聚糖材料联合骨搬移修复胫骨缺损：随机对照实验方案

背景：骨髓间充质干细胞发挥成骨作用需要支架材料的辅助,一方面支架材料不仅可将细胞运载至骨缺损区域,另一方面还可作为新骨生长的框架结构。胶原-壳聚糖复合材料是骨组织工程较为理想的支架材料之一,同时其具有骨诱导性,比常规支架材料更优越的成骨能力。骨搬移技术在临床上在修复长段骨缺损方面已得到广泛应用,但也存在成骨慢、外固定时间长、骨不连等缺憾。如何进一步加快骨形成速度,减少并发症发生,已成当前亟待解决的问题。实验假设：骨髓间充质干细胞复合胶原-壳聚糖支架移植能提高胫骨缺损骨搬移修复效果。 方法/设计：随机对照动物实验。分为体外和体内实验两部分。体外实验中取月龄一两个月的新西兰大白兔股骨骨髓,提取骨髓间充质干细胞,培养至第3代,将细胞悬液滴于胶原-壳聚糖支架材料,构建骨髓间充质干细胞复合胶原-壳聚糖支架。体内实验选用24只三四月龄新西兰大白兔,被随机分配接受如下干预：骨搬移、支架植入、骨搬移联合支架植入。研究的主要观察指标为植入材料与骨缺损界面的生长情况、X射线检测的缺损区骨修复情况、苏木精-伊红染色及扫描电镜观察缺损区成骨情况、免疫组织化学染色检测成骨区Ⅰ型胶原蛋白的表达情况、扫描电子显微镜观察移植材料与宿主骨的界面键合情况、超微结构及新骨的生成。 讨论：实验结果将有助于确定对骨缺损进行骨搬移治疗过程中,应用骨髓间充质干细胞复合胶原-壳聚糖支架移植促进骨缺损再生修复效果的可行性。 实验方案获基金支持情况：获辽宁省科学技术计划项目资助(2012225019)。 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     

Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold

Functional engineering of musculoskeletal tissues generally involves the use of differentiated or progenitor cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. We have previously shown that electrospun, three-dimensional nanofibrous scaffolds that morphologically resemble collagen fibrils are capable of promoting favorable biological responses from seeded cells, indicative of their potential application for tissue engineering. In this study, we tested a three-dimensional nanofibrous scaffold fabricated from poly(epsilon-caprolactone) (PCL) for its ability to support and maintain multilineage differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in vitro. hMSCs were seeded onto pre-fabricated nanofibrous scaffolds, and were induced to differentiate along adipogenic, chondrogenic, or osteogenic lineages by culturing in specific differentiation media. Histological and scanning electron microscopy observations, gene expression analysis, and immunohistochemical detection of lineage-specific marker molecules confirmed the formation of three-dimensional constructs containing cells differentiated into the specified cell types. These results suggest that the PCL-based nanofibrous scaffold is a promising candidate scaffold for cell-based, multiphasic tissue engineering.     

小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

晚期骨关节炎患者膝关节滑膜间充质干细胞的体外成骨分化

背景：间充质干细胞是组织工程中常用的种子细胞,而来源于人膝关节滑膜组织的间充质干细胞能否作为骨组织修复与再生中合适的种子细胞还需要进一步验证。 目的：观察晚期骨关节炎患者膝关节滑膜组织来源的间充质干细胞体外向成骨细胞定向分化的潜能,对所诱导细胞的成骨特性进行鉴定。 方法：无菌条件下从行全膝关节置换的晚期骨关节炎患者膝关节腔内获取滑膜组织,Ⅰ型胶原酶消化分离获得有核细胞。以最适密度接种(200个有核细胞/cm2),挑选单细胞克隆,筛选出滑膜间充质干细胞。基础培养基培养,传至第3代时改换为含地塞米松、β-甘油磷酸钠和抗坏血酸的成骨诱导培养基诱导培养。 结果与结论：体外分离培养的滑膜间充质干细胞早期可形成葵花样细胞集落,克隆样生长。传代至第3代,细胞呈成纤维细胞样生长,形态均一。滑膜间充质干细胞成骨诱导后呈典型的“铺路石样”成骨细胞形态,诱导至第7天碱性磷酸酶染色呈强阳性,碱性磷酸酶活性表达也在诱导7 d时达最高峰;诱导21 d茜素红染色可见大量钙结节形成;同时反转录PCR检测到Ⅰ型胶原、Runx2、骨结合蛋白和骨桥蛋白的表达在诱导后增加,21 d时表达最高。从晚期骨关节炎患者膝关节滑膜组织分离获得的滑膜间充质干细胞,在体外可成功诱导分化为成骨细胞,所诱导生成的细胞具有典型的成骨细胞特性。提示滑膜间充质干细胞有望成为骨组织工程理想的种子细胞来源,在骨组织的再生修复中发挥重要作用。     

Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cells by enhancing expression of cartilage extracellular matrix genes

In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-beta (TGF-beta), or DEX plus TGF-beta. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-beta-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrix-sulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GRalpha, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GRbeta, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-beta, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GRbeta dramatically inhibited promoter activity, suggesting that GRalpha is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.     

In Vitro Osteogenesis of Synovium Mesenchymal Cells Induced by Controlled Release of Alendronate and Dexamethasone from a Sintered Microspherical Scaffold

In vitro osteogenesis was successfully achieved with synovium-derived mesenchymal stem cells (SMSCs), which intrinsically have a strong chondrogenic tendency, by in situ release of alendronate (AL) and dexamethasone (Dex) from poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) sintered microspherical scaffold (PLGA/HA-SMS). Cumulative release profiles of AL and Dex from PLGA/HA-SMS and the influence on SMSCs osteogenic commitment were investigated. SMSCs seeded in Al-/Dex-loaded PLGA/HA-SMS (PLGA/HA-Com-SMS) exhibited significant osteogenic differentiation, as indicated by high yields of alkaline phosphatase (ALP) and bone calcification. In addition, mechanical properties (compressional) of PLGA/HA-Com-SMSs were also evaluated and approved. In conclusion, by promoting osteogenic commitment of SMSCs in vitro, this newly designed controlled-release system opens a new door to bone reparation and regeneration.     

Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-β3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7 days, but not 14 days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28 days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.     

Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells

Mesenchymal stem cells are pluripotent cells from bone marrow, which can be differentiated into the osteogenic, chondrogenic, and adipogenic lineages in vitro and are a source of cells in bone and cartilage tissue engineering. An improvement in current tissue-engineering protocols requires more detailed insight into the molecular cues that regulate the distinct steps of osteochondral differentiation. Because Wnt signaling has been widely implicated in mesenchymal differentiation, we analyzed the role of Wnt signaling in human mesenchymal stem cell (hMSC) biology by stimulation of the pathway with lithium chloride and Wnt3A-conditioned medium. We demonstrate a role for low levels of Wnt signaling in proliferation of uncommitted hMSCs and confirm that Wnt signaling controls osteoprogenitor proliferation. On the other hand, at high Wnt levels we observed a block in adipogenic differentiation and an increase in the expression of alkaline phosphatase, suggesting a role in the initiation of osteogenesis. The results of this study suggest that bone tissue engineering could benefit from the activation of critical levels of Wnt signaling at defined stages of differentiation. Moreover, our data suggest that hMSCs provide a valid in vitro model to study the role of Wnt signaling in mesenchymal biology.     

Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow

We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.     

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage.