应用组织工程化肌腱修复跟腱缺损

背景:间质干细胞或间质祖细胞的非造血细胞可在体内或体外分化为骨、软骨、肌肉、肌腱、脂肪及骨髓基质,这使得它们成为组织工程领域内非常有价值的种子细胞来源.目的:以骨髓间充质干细胞为种子细胞,以Ⅰ型胶原-聚羟基乙酸为支架构建组织工程化肌腱,观察其修复跟腱缺损的效果.设计,时间及地点:随机对照动物实验,于2003年在中南大学湘雅医学院进行.材料:1.5～2.5 kg健康成年家兔由中南大学湘雅医学院动物学部提供,雌雄不限.聚羟基乙酸由威高集团康利达医用制品有限公司提供.SD大鼠由中南大学实验动物学部提供.方法:提取家兔骨髓,通过离心和贴壁法培养获取骨髓间充质干细胞,将骨髓间充质干细胞进行分离、扩增.抽取SD大鼠尾腱,制备鼠尾Ⅰ型胶原溶液,并与聚羟基乙酸缝线混合培养构建胶原-聚羟基乙酸支架.将未经体外诱导的骨髓间充质干细胞接种于胶原-聚羟基乙酸支架中构建组织工程化兔肌腱模型,以不加入细胞的为对照.切开30只家兔踝部皮肤,分离出兔跟腱,造成3cm长缺损,实验组15只以自体骨髓间充质干细胞预制的组织工程化肌腱移植于缺损处,对照组15只以Ⅰ型胶原-聚羟基乙酸支架修复跟腱缺损.主要观察指标:组织工程化肌腱修复兔跟腱缺损的效果.结果:含自体骨髓间充质干细胞的Ⅰ型胶原一聚羟基乙酸支架及不含自体骨髓间充质干细胞的Ⅰ型胶原-聚羟基乙酸支架回植于体内时大体观察呈条形凝胶状.回植于体内4周后实验组和对照组均可见移植处形成条索状组织,聚羟基乙酸缝线已降解.8周时观察可见实验组组织工程化肌腱与受体肌腱组织完好连接,呈条索状.与周围其他组织无粘连,为白色、有光泽的致密腱样组织,对照组所形成的组织较实验组细小、与周围组织有粘连.结论:以骨髓间充质干细胞为种子细胞,以Ⅰ型胶原-聚羟基乙酸为支架构建的组织工程化肌腱可明显促进肌腱损伤的修复.     

Stem cell therapy for cartilage regeneration in osteoarthritis

Enhancing the regeneration potential of hyaline cartilage tissue remains a great challenge. During embryonic development, some of the cells of the inner cell mass will turn into the mesoderm. This will be the founder of the mesenchymal cells in connective tissues of adult life, such as bone, tendon, muscle, and cartilage. Some of these embryonic mesenchymal cells are believed not to differentiate, but reside in each of the tissues. These are now collectively described as adult mesenchymal stem cells, which are thought to be capable of repairing injured tissue. We will briefly summarize the current knowledge about stem cell-related cells in cartilage tissue and carefully discuss the potential of the cell population we described recently as a starting-point for a regenerative therapy for osteoarthritis. We found that repair tissue from human articular cartilage during the late stages of osteoarthritis harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPC). These exhibit stem cell characteristics combined with a high chondrogenic potential. They offer new insights into the biology of progenitor cells and may be relevant in the development of novel therapeutic approaches for a cell-based therapy for late stages of osteoarthritis.     

人骨髓间充质干细胞的培养及多能性研究

目的：培养人骨髓间充质干细胞（mesenchymal stem cells,MSC）并检测其多能性。方法：抽取人胚胎骨骨髓，Percoll分离液梯度分离后，将含MSC的低密度层培养于MSCGM培养基并扩增MSC。将MSC种植于裸鼠皮下，4周后观察MSC的分化情况。结果：成功地进行了MSC的原代和传代培养。植入裸鼠体内后MSC可分化成骨、软骨、脂肪、骨骼肌、肌腱样组织等结构。结论：人MSC是一种具有多能性的干细胞。     

成体干细胞向血管内皮细胞定向分化的研究现状

成体干细胞存在于已分化的组织器官中,具有明显的可塑性,能跨系统、跨胚层分化,在组织工程和损伤修复领域具有重要的应用价值.在血管组织工程研究领域中,成体干细胞作为种子细胞得到越来越广泛而深入的研究.目前研究表明,具有向血管内皮细胞分化的成体干细胞主要有骨髓间充质干细胞、造血干细胞、脂肪来源的干细胞以及羊膜来源的干细胞等基于此,就成体干细胞作为种子细胞在血管组织工程领域研究现状进行综述.     

Cell-mediated gene therapy for bone formation and regeneration

Cell-mediated gene therapy is one of the new modalities branching out from the wide-ranging field of gene transfer and therapy. When applied to bone formation and regeneration, it has particular advantages depending on the type of cell used as a platform for gene delivery. When utilizing adult mesenchymal stem cells or osteoprogenitor cells for the expression of bone-promoting osteogenic factors, the cells not only express the factors promoting bone growth, but can respond, differentiate and participate in the bone formation process. The ability of engineered cells to respond to the transgene, as well as to other local signals in vivo, confers on them special properties that enable the formation and regeneration of large-scale bone tissue. This approach is a paradigm for the development of gene therapy strategies for other skeletal tissues. Here, we review the most recent studies related to cell-mediated gene therapy for bone formation and regeneration.     

胎儿骨髓和肝脏间充质干细胞的表型和生物学性状研究

为研究胎儿骨髓和肝脏间充质干细胞的表型和生物学性状,取胎龄为4-5个月水囊引产胎儿,将骨髓和肝脏细胞在SF（含2?S）培养基中培养,进行电镜观察,测定生长曲线,用流式细胞术对培养细胞进行表型测定。细胞周期分析。用SA方法测定Ⅰ,Ⅲ型胶原和vWF因子表达。结果表明;从胎儿骨髓和肝脏培养出的间充质干细胞,两在形态学,生长特性,免疫表型上是相似的,肝脏间充质干细胞有更好的支持造血的功能。结论提示,从胎儿骨髓和肝脏可分离培养出间充质干细胞。在体外有效扩增且保持其低分化状态。     

Tissue repairing cells that exist among mesenchymal stem cells; their potential for cell-based therapy

Although adult stem cells are generally known to generate the cell types of the tissue in which they reside, mesenchymal stem cells (MSCs) are unique in that they differentiate not only into the same mesodermal-lineage such as bone, cartilage, and adipocytes, but also into other lineages of ectodermal and endodermal cells. Furthermore, a subpopulation of MSCs are known to integrate into damaged sites, differentiate into cell types specific to the integrated tissue and contribute to the tissue repair. As MSCs comprise heterogeneous cell populations, however, the cells responsible for wide-ranging differentiation as well as for tissue repair have not been identified. Recent evidence suggests that a subpopulation of human MSCs, which were named Muse cells, were shown to have the ability to differentiate into trilineage cells and to function as tissue repairing cells in vivo. This review summarizes recent advances in MSC properties and discuss about future perspectives.     

Potential therapeutic applications of muscle-derived mesenchymal stem and progenitor cells

Importance of the field: Mesenchymal adult stem cells have properties that make them attractive for use in tissue engineering and regenerative medicine. They are inherently plastic, enabling them to differentiate along different lineages, and promote wound healing and regeneration of surrounding tissues by modulating immune and inflammatory responses, promoting angiogenesis and secreting other trophic factors. Unlike embryonic stem cells, clinical uses of mesenchymal stem cells are not encumbered by ethical considerations or legal restrictions.

Areas covered in this review: We discuss skeletal muscle as a source of mesenchymal stem and progenitor cells by reviewing their biology and current applications in tissue engineering and regenerative medicine. This paper covers literature from the last 5 – 10 years.

What the reader will gain: Skeletal muscle is a plentiful source of mesenchymal stem and progenitor cells. This tissue may be obtained via routine biopsy or collection after surgical debridement. We describe the biology of these cells and provide an overview of therapeutic applications currently being developed to take advantage of their regenerative properties.

Take home message: There is potential for stem and progenitor cells derived from skeletal muscle to be incorporated in clinical interventions, either as a cellular therapy to modify the natural history of disease or as a component of engineered tissue constructs that can replace diseased or damaged tissues.     

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.     

骨髓间充质干细胞诱导分化成骨方法的研究与进展

背景：骨髓间充质干细胞具有良好的成骨分化潜能,移植入体内后可分化为骨、软骨、肌肉、脂肪、肝脏和神经等多种细胞。目的：综述大鼠骨髓间充质干细胞诱导分化成骨最新方法进展。方法：分别以“骨髓间充质干细胞,成骨细胞,诱导分化”、“bone mesenchymal stem cells,osteoblast cells,osteogenic differentiation”为检索词,应用计算机检索CHKD期刊全文数据库,PubMed数据库和万方数据库1995至2011年有关文章。纳入有关骨髓间充质干细胞的文献。排除内容重复和缺乏原创性文献。保留34篇文献做进一步分析。结果与结论：骨髓间充质干细胞在适当的生长因子诱导下可以快速地向成骨细胞分化和增殖。在体外诱导骨髓问充质干细胞向成骨细胞分化需要特定的诱导条件、优良的诱导剂以及合适的剂量。骨髓间充质干细胞向成骨细胞分化受许多自身调控因素（控制基因表达的转录因子）和环境调控因素（分泌因素、细胞外基质,化学因素、细胞与细胞间的相互作用）的影响,这些调控因素并不是孤立存在的,而是相互联系、相互作用,共同调控骨髓间充质干细胞向成骨细胞分化。,     

Epigenetic approaches to regeneration of bone and cartilage from stem cells

Introduction: Embryonic stem cells (ESCs) or adult stem cells, especially mesenchymal stem cells (MSCs), have been intensively studied for skeletal tissue regeneration including bone and cartilage. Epigenetic mechanisms play essential roles in stem cell maintenance and differentiation. However, little is known about the epigenetic regulation of osteogenesis and chondrogenesis of stem cells.

Areas covered: In this review, features of ESCs and adult stem cells, epigenetics and chromatin structure, as well as epigenetic mechanisms, such as chromatin remodeling, DNA methylation and histone modifications, polycomb group (PcG) proteins and microRNAs are described. Epigenetic researches of stem cell are introduced.

Expert opinion: Epigenetic alterations of stem cell during the in vitro differentiation can be controlled for clinical applications. MSCs are effective resources for skeletal tissue regeneration in both undifferentiated and differentiated states. Understanding epigenetic signatures of MSC is crucial to maintain the stemness. In addition, investigation of epigenetic changes in the differentiation of MSCs is very important to develop methods or chemicals to promote efficient differentiation of MSCs. Inhibition of PcG protein enhancer of zeste (Ezh2) a chromatin modifier, could be a promising candidate to improve MSC differentiation by decreasing Ezh2-mediated H3K27me3.     

Tenogenic differentiation of stem cells for tendon repair—what is the current evidence?

Tendon/ligament injuries are very common in sports and other rigorous activities. Tendons regenerate and repair slowly and inefficiently in vivo after injury. The limited ability of tendon to self-repair and the general inefficiencies of current treatment regimes have hastened the motivation to develop tissue-engineering strategies for tissue repair. Of particular interest in recent years has been the use of adult mesenchymal stem cells (MSCs) to regenerate functional tendons and ligaments. Different sources of MSCs have been studied for their effects on tendon repair. However, ectopic bone and tumour formation has been reported in some special circumstances after transplantation of MSCs. The induction of MSCs to differentiate into tendon-forming cells in vitro prior to transplantation is a possible approach to avoid ectopic bone and tumour formation while promoting tendon repair. While there are reports about the factors that might promote tenogenic differentiation, the study of tenogenic differentiation is hampered by the lack of definitive biomarkers for tendons. This review aims to summarize the cell sources currently used for tendon repair as well as their advantages and limitations. Factors affecting tenogenic differentiation were summarized. Molecular markers currently used for assessing tenogenic differentiation or neotendon formation are summarized and their advantages and limitations are commented upon. Finally, further directions for promoting and assessing tenogenic differentiation of stem cells for tendon repair are discussed.     

骨髓间充质干细胞生物学特性及其在组织修复中的应用

学术背景：骨髓间充质干细胞是具有多向分化能力的非造血多能干细胞，是细胞工程及基因治疗理想的靶细胞。 目的：综述骨髓间充质干细胞的生物学特性及其在组织修复中应用的研究进展。 检索策略：应用计算机检索PubMed 1997—01／2007—07期间相关文章，检索词为“mesenchymal stem cells，bone marrow，biological characterization，mesenchymal stemcells．bone marrow，tissue engineering”，并限定文章语言种类为English，同时检索中国期刊全文数据库2001—01／2007-03期间相关文章，检索词为“骨髓间充质干细胞，组织工程”。对资料进行初审，并查看每篇文献后的引文。纳入标准：文章所述内容应与骨髓问充质干细胞的生物学特性及其在组织修复中应用的研究进展相关。排除标准：重复研究或Meta分析类文章。共收集到313篇相关文献，26篇文献符合纳入标准，排除的287篇为内容陈旧或重复文献。 文献评价：从检索到的313篇文献中选择符合要求的相关文献26篇。符合纳入标准的26篇文献中，12篇涉及骨髓间充质干细胞的生物学特性，14篇涉及在组织修复中的研究进展。 资料综合：骨髓间充质干细胞主要存在于骨髓、软骨膜、骨膜、骨骼肌、骨小梁中，以骨髓组织中含量最为丰富。骨髓间充质干细胞具有多向分化潜能，在特定的条件下能向多种细胞系分化，具有向损伤局部集中的趋化性及“局部专一诱导性分化”、免疫调节、支持造血等生物学特性，目前已广泛应用于骨和软骨、心脏、皮肤、神经系统等多种组织修复的实验及临床研究，展现出良好的应用前景。 结论：随着对骨髓间充质干细胞研究、认识的深入，其在组织修复领域将有着广阔的临床应用前景。     

Osteogenic differentiation of human adipose-derived stem cells: comparison of two different inductive media

Human mesenchymal stem cells (MSCs) have the potential to differentiate into cells of connective tissue lineages, including bone, cartilage, fat, muscle and also neurons. In our study we have examined the phenotypic profile of human adipose tissue-derived stem cells (hASCs) and compared different osteogenic-inductive media to assess hASC differentiation. Cells were enzymatically isolated from adipose tissues derived by liposuction from several adult human donors, purified and then expanded in culture. We obtained an abundant yield of hASCs with a constant proliferative trend, a doubling time of about 68 h and a mild variable clonogenic capacity. At passage 4, hASCs expressed MSC-related cell surface antigens (CD13, CD105, CD54, CD90, CD44), and subsequently hASCs were induced to differentiate into the osteogenic lineage for at least 3 weeks of culture in two distinct media, OM1 and OM2, differing in dexamethasone and ascorbic acid concentrations. Osteogenic differentiation of OM1- and OM2-cultured cells was assessed by evaluating cell morphology, osteopontin expression, alkaline phosphatase activity and calcium deposition. OM2 medium showed a higher osteogenic potential than OM1, as assessed by increased levels of calcium deposition, alkaline phospatase activity and osteopontin expression in comparison with OM1-differentiated cells. We conclude that hASCs efficiently differentiate into osteogenic lineage, particularly when cultured in inductive medium supplemented with 10 nM dexamethasone and 150 microM ascorbic acid.     

干细胞参与运动损伤的组织修复

背景：既往多项研究已证实间充质干细胞具备多向分化潜力,并应用到多个领域,同样干细胞疗法对解决组织损伤修复将是一种革命性进步。目的：对国内外应用干细胞治疗软组织损伤的现状及新进展作一综述。方法：应用计算机检索CNKI和Elsevier数据库中2000-01/2010-09关于干细胞参与软组织损伤治疗的文章,在标题和摘要中以＂干细胞,治疗,损伤＂或＂stem cells,treatment,Soft tis sues injure＂为检索词进行检索。选择文章内容与干细胞治疗有关者,同一领域文献则选择近期发表或发表在权威杂志文章。最终选择关于干细胞治疗组织损伤的36篇文献进行综述。结果与结论：体育运动过程中导致的肌腱,骨骼损伤,难以根治,常反复发作,严重影响到运动员的竞技状态。干细胞技术的研究对治疗运动员的损伤无疑具有重大意义。目前以间充质干细胞为基础的细胞疗法正成为再生医学研究领域中的热点和最前沿,因此间充质干细胞作为最重要的成体干细胞来源,具有极其重要的应用前景。     

Plasticity of marrow-derived stem cells

Many exciting discoveries reported over the past 3 years have caused us to expand the paradigm for understanding somatic stem cell plasticity. Within adult organs, there are not only specific stem cells that are capable of producing functional cells of one organ system, but also cells with the flexibility to differentiate into multiple other cell types. In the bone marrow, for example, in addition to hematopoietic stem cells and supportive stromal cells, there are cells with the potential to differentiate into mature cells of the heart, liver, kidney, lungs, GI tract, skin, bone, muscle, cartilage, fat, endothelium and brain. A subpopulation of cells in the brain can differentiate into all of the major cell types in the brain and also into hematopoietic and skeletal muscle cells. In this brief overview, several of these recent findings are summarized.     

骨髓间充质干细胞的研究前景

除造血干细胞以外,骨髓中还含有另一类干细胞,被称为间充质干细胞（ｍｅｓｅｎｃｈｙｍａｌ ｓｔｅｍ ｃｅｌｌ,ＭＳＣ）在不同的诱导条件下,这类细胞可分化为多种造血以外组织特别是中胚层和神经外胚层来源组织细胞,如成骨细胞、成软骨细胞、脂肪细胞、成肌细胞和星形神经胶质细胞等。骨髓间充质干细胞易于分离和培养扩增,还易于外源基因的导入和表达,有望成为一种新的细胞治疗和基因治疗的靶细胞,在多种造血以外组织的缺     

骨髓间充质干细胞在软骨缺损修复中的应用

学术背景:骨髓间充质干细胞由于其易于获取,并可以在体外短期内大量扩增,是目前最有希望应用于软骨组织工程的干细胞.目的:对骨髓问充质干细胞在软骨缺损修复中的应用状况进行综述.检索策略: 由该论文的研究人员应用计算机检索1982-10/2006-12Pubmed数据库与骨髓间充质干细胞修复软骨缺损相关文献,检索词"Marrow;Mesenchymal stem cells:cartilage defect",限定语言种类为English,同时检索1982-10/2006-12中国科技成果数据库检索词"骨髓;间充质干细胞;软骨缺损",并限定文章语言种类为中文.共检索到126篇文献,对资料进行初审,纳入标准:①综述类及实验类文章;②中文核心期刊收录文献.排除标准:重复性研究.文献评价:文献的来源主要来自Pubmed数据库及中国科技成果数据库.文献类型为综述类及实验研究.资料综合:骨髓间充质干细胞在体内具有分化产生软骨的能力已被证明,而在体外培养条件下软骨表型的分化却是一个受多种因素限制的复杂过程,目前调控机制仍不明确.动物实验表明,骨髓间充质干细胞能够修复具有临床意义的骨和软骨缺损.虽然近年来对骨髓间充质干细胞及其在软骨组织工程方面的研究已取得较大进展,但对骨髓间充质干细胞分化各阶段细胞标志物、骨髓间充质干细胞的增殖、分化的控制及基因转染技术及临床应用的评估结果都有待进一步研究.结论:动物实验表明,骨髓间充质干细胞能够修复具有临床意义的骨和软骨缺损,虽然近年来对骨髓间充质干细胞及其在软骨组织工程方面的研究已取得较大进展,但对骨髓间充质干细胞的基础和临床研究中仍存在诸多问题需要解决.     

Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro.

Adult mesenchymal stem cells (MSCs) have the capacity to differentiate into various connective tissues such as cartilage and bone following stimulation with certain growth factors. However, less is known about the capacity of these cells to undergo chondrogenesis when these proteins are delivered via gene transfer. In this study, we investigated chondrogenesis of primary, bone marrow-derived MSCs in aggregate cultures following genetic modification with adenoviral vectors encoding chondrogenic growth factors. We found that adenoviral-mediated expression of TGF-beta1 and BMP-2, but not IGF-1, induced chondrogenesis of MSCs as evidenced by toluidine blue metachromasia and immunohistochemical detection of type II collagen. Chondrogenesis correlated with the level and duration of expressed protein and was strongest in aggregates expressing 10-100 ng/ml transgene product. Transgene expression in all aggregates was highly transient, showing a marked decrease after 7 days. Chondrogenesis was inhibited in aggregates modified to express >100 ng/ml TGF-beta1 or BMP-2; however, this was found to be partly due to the inhibitory effect of exposure to high adenoviral loads. Our findings indicate that parameters such as these are important functional considerations for adapting gene transfer technologies to induce chondrogenesis of MSCs.