骨髓间充质干细胞在骨组织工程中的应用

目的：综述具有向多种细胞分化潜能的骨髓间充质干细胞的生物学特性、分离纯化与培养技术、向骨和软骨定向诱导分化技术、细胞载体支架及其应用于骨组织工程的实验研究。 资料来源：应用计算机检索Medline数据库1990—01／2004-12期间的相关文章，检索词“mesenchymal stem cell，tissue engineer”，限定文章语言种类为英文。同时计算机检索中国期刊全文数据库、万方数据库1994—01／2004—12期间的相关文章，检索词”间充质干细胞、组织工程”，限定文章语言种类为中文。 资料选择：对资料进行初审，选取间充质干细胞特性、分离培养技术和其应用于组织工程的文献，重复研究选取代表性的、年代相对较近的文献作为纳入标准；然后筛除非骨组织工程的研究和重复研究，对剩余的文献开始查找全文。 资料提炼：共收集到78篇关于间充质干细胞应用于组织工程方面的文章，纳入31篇；排除47篇，13篇为重复研究，34篇为非骨组织工程研究。 资料综合：间充质干细胞主要存在于骨髓中，能向多种组织细胞分化，在体外可大量扩增。①间充质于细胞的分离纯化和培养目前主要有3种方法：密度梯度离心法、流式细胞仪分离法、贴壁筛选法。也有学者采用单细胞克隆技术获得纯化的间充质干细胞细胞株，但此种方法不便于大量获得种子细胞，目前应用较广泛的是密度梯度离心技术。②间充质干细胞在适宜的培养基中加入诱导剂可向骨、软骨细胞分化，其理想的载体支架应能均匀地搭载并保留细胞，支持血管快速内生，能透X射线利于观察新骨形成，新骨形成后能被吸收和替代以利骨改建，能容许或提高宿主骨的骨传导性桥接，表面与细胞相互作用以保留分化细胞的功能，组织相容性好。③间充质干细胞是理想的基因治疗的靶细胞，它可以转入生长因子和细胞因子基因，经多次分裂后体外仍表达外源蛋白。它应用于骨组织工程的动物试验中已获得了成功。 结论：以干细胞工程为代表的现代组织工程学近年来发展迅猛，但间充质干细胞组织工程学尚处于起步阶段。骨髓间充质干细胞具有易于取材、多组织分化潜能、遗传背景稳定、植入体内无排斥反应、高增殖的特性，决定了其将会成为细胞、基因治疗以及组织工程中十分有用的工具：     

成体骨髓间充质干细胞生物学特性及在组织工程和再生医学中的作用

成体骨髓间充质干细胞可向其他组织分化,称为可塑性,在组织工程和再生医学的各个领域都进行着广泛的研究.虽然骨髓间充质的生物学特性、诱导分化、调控机制等研究已取得了许多成就,但其很多功能仍不被人所知,如是否存在间充质干细胞、是否具有分化特性、骨髓间充质干细胞和其他来源干细胞的区别等.文章介绍了骨髓间充质干细胞的来源和基本特征,并研究了骨髓间充质干细胞的生长、增殖、自我更新和分化,探讨了骨髓间充质干细胞的生理功能,展望了骨髓间充质干细胞在组织工程和再生医学中的作用.     

骨髓间充质干细胞在骨组织工程中的应用

目的:综述具有向多种细胞分化潜能的骨髓间充质干细胞的生物学特性、分离纯化与培养技术、向骨和软骨定向诱导分化技术、细胞载体支架及其应用于骨组织工程的实验研究。资料来源:应用计算机检索Medline数据库1990-01/2004-12期间的相关文章,检索词“mesenchymalstemcell,tissueengineer”,限定文章语言种类为英文。同时计算机检索中国期刊全文数据库、万方数据库1994-01/2004-12期间的相关文章,检索词″间充质干细胞、组织工程″,限定文章语言种类为中文。资料选择:对资料进行初审,选取间充质干细胞特性、分离培养技术和其应用于组织工程的文献,重复研究选取代表性的、年代相对较近的文献作为纳入标准;然后筛除非骨组织工程的研究和重复研究,对剩余的文献开始查找全文。资料提炼:共收集到78篇关于间充质干细胞应用于组织工程方面的文章,纳入31篇;排除47篇,13篇为重复研究,34篇为非骨组织工程研究。资料综合:间充质干细胞主要存在于骨髓中,能向多种组织细胞分化,在体外可大量扩增。①间充质干细胞的分离纯化和培养目前主要有3种方法:密度梯度离心法、流式细胞仪分离法、贴壁筛选法。也有学者采用单细胞克隆技术获得纯化的间充质干细胞细胞株,但此种方法不便于大量获得种子细胞,目前应用较广泛的是密度梯度离心技术。②间充质干细胞在适宜的培养基中加入诱导剂可向骨、软骨细胞分化,其理想的载体支架应能均匀地搭载并保留细胞,支持血管快速内生,能透X射线利于观察新骨形成,新骨形成后能被吸收和替代以利骨改建,能容许或提高宿主骨的骨传导性桥接,表面与细胞相互作用以保留分化细胞的功能,组织相容性好。③间充质干细胞是理想的基因治疗的靶细胞,它可以转入生长因子和细胞因子基因,经多次分裂后体外仍表达外源蛋白。它应用于骨组织工程的动物试验中已获得了成功。结论:以干细胞工程为代表的现代组织工程学近年来发展迅猛,但间充质干细胞组织工程学尚处于起步阶段。骨髓间充质干细胞具有易于取材、多组织分化潜能、遗传背景稳定、植入体内无排斥反应、高增殖的特性,决定了其将会成为细胞、基因治疗以及组织工程中十分有用的工具。     

肌源性干细胞在组织工程研究中的现状

肌源性于细胞位于骨骼肌肌纤维膜外，属于多能干细胞，具有多细胞系分化和自我更新能力。随着组织细胞工程学和基因工程学的发展，肌源性干细胞在组织工程等方面的应用已成为研究热点。     

对成体干细胞可塑性的新认识及其在再生医学中的意义

根据个体发育过程中出现的先后次序不同 ,干细胞可分为胚胎干细胞和成体干细胞。胚胎干细胞出现在胚胎发育的胚囊内层 ,高水平表达端粒酶。1981年ＭａｒｔｉｎＥｖａｎｓ首次从小鼠胚囊中分离出小鼠胚胎干细胞 ,后被证实具有自我复制能力 ,且可分化为诸如神经细胞、心肌细胞、肝细胞、β 胰岛细胞、骨细胞、造血细胞等 2 0 0多种组织器官的原始细胞。胚胎干细胞继续分化 ,形成组织定向的多潜能干细胞 ,如多潜能造血干细胞可形成各种血细胞 ,多潜能皮肤干细胞可形成各种类型的皮肤细胞。在胎儿、儿童和成人组织中存在的这类多潜能干细胞统称…     

组织工程——现实的再生医学

1再生医学的崛起目前 ,当人类的组织或器官受到较大的损伤或缺失而丧失功能时 ,主要采取人工器官、器官移植或康复医学等的手段 ,使患者不同程度地重建、恢复或补偿已失去的功能。再生生物学的研究者们长期付出巨大努力以阐明动物组织再生的机制 ,旨在建立再生医学[1] 。自 2 0世纪 80年代末迅速发展起来的组织工程学是运用工程学和生物学、医学的原理及手段 ,以再生、维持和改善被损坏的人体组织、器官。组织工程学为再生医学的崛起开辟了崭新的道路。长期以来 ,人工器官和移植手术使众多患者受益 ,但也显露出了致命弱点 :前者与人体不相容…     

血管组织工程种子细胞在再生医学中的应用

背景:小口径人工血管替代人体小动脉和静脉一直未获得满意的效果,因此研制出一种拥有较高远期通畅率的小口径人工血管成为了一个重要的研究课题.目的:综述种子细胞在血管组织工程的研究进展.方法:以 Vascular tissue engineering, Seeding cells为检索词,应用计算机检索Pubmed 数据库1960/2009有关文章.纳入有关血管组织工程种子细胞的文献.排除原始文献设计方法简单、结果可靠性差、非英文文献及结果重复的文献,保留35篇文献做进一步分析.结果与结论:内皮细胞和平滑肌细胞是目前常用的种子细胞.内皮细胞和平滑肌细胞共同培养的体系,模拟体内环境,保持内皮细胞和平滑肌细胞具有正常的分泌功能和表型.骨髓间充质细胞可被有效的分离和扩增,在特定培养条件下可以诱导分化为多种血管细胞.在再生医学和生物组织工程方面有强大的潜力.     

干细胞在再生医学领域的临床应用现状及其前景

干细胞在一定条件下,可以定向分化成机体内的功能细胞,形成任何类型的组织和器官,文章叙述了干细胞与再生医学的概念与分类,探讨了干细胞在糖尿病、心肌细胞损伤性疾病、神经损伤性疾病治疗方面的应用,分析并总结了干细胞与再生医学所面临的问题.分析处理资料显示,干细胞已经广泛应用于心血管系统疾病、神经系统疾病、骨骼肌肉相关疾病,糖尿病等多种疾病的临床试验中,并且取得初步疗效,展示了广泛的临床应用前景.     

牙源和非牙源性干细胞在组织工程再生牙研究中的作用

背景：牙齿缺失是人类最为常见的器官缺失,近年组织工程学器官的研究日渐成熟,各类成体干细胞被用在再生牙的研究中。目的：对各类成体干细胞的特性进行阐述,探讨各类成体干细胞在再生牙研究过程中的作用及新进展。方法：由作者应用计算机检索1979至2012年PubMed数据库及CNKI数据库的文献,在英文标题和摘要中以“stemcell”和“toothmgenemtion”检索,中文文献检索以“干细胞,牙再生,组织工程学牙,种子细胞”为关键词,选择内容与成体干细胞、再生牙与牙组织工程相关的文章,同一领域文献则选择近期发表或发表在权威杂志文章,共纳入53篇文献。结果与结论：牙齿生长发育条件特殊,易缺失且自体难以再生,再生组织工程牙的研究为牙缺失的治疗提供了一个新的治疗手段,在大量的实验研究中发现,不同的成体干细胞无论在体内诱导还是体外重建再生牙的过程中都可起到不同的作用,且各有优缺点,相比之下,脐血干细胞的优势就较为突出,在今后的再生牙研究中可能将发挥重要的作用。     

牙源干细胞参与组织再生修复的作用

背景：随着干细胞技术的不断发展,牙源性干细胞的研究也不断深入。它们是十分理想的种子细胞来源,在牙组织工程的应用上具有无可比拟的优势。目的：综述牙源干细胞的相关研究进展。方法：由第一作者检索PubMed数据库、Highwire数据库及中国知网（CNKI）数据库。英文检索词为＂dental stem cells,tissue engineering,application＂,中文检索词为＂牙源干细胞,组织工程,应用＂。初步检索文献量总计156篇,选择牙源性干细胞,包括牙髓干细胞和牙周膜干细胞等在组织工程中的基础研究及临床应用相关报道,排除陈旧及重复实验文章,最终纳入30篇符合标准的文献。结果与结论：多种牙源干细胞,都具有较强自我更新能力和多向分化的能力,与目前研究较为深入的骨髓间充质干细胞有很大的相似性,因此在组织工程,尤其是牙组织工程上有良好的应用前景。目前虽然有关牙源干细胞在牙组织再生的应用上有一定报道,但研究不多,且仅处于动物模型阶段。相信随着科技进步,在不久的将来,各种牙源干细胞有望真正进入临床,为解决人类各种口腔疾病提供可能。     

Dental stem cells: recent progresses in tissue engineering and regenerative medicine

Since the disclosure of adult mesenchymal stem cells (MSCs), there have been an intense investigation on the characteristics of these cells and their potentialities. Dental stem cells (DSCs) are MSC-like populations with self-renewal capacity and multidifferentiation potential. Currently, there are five main DSCs, dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP), periodontal ligament stem cells (PDLSCs) and dental follicle precursor cells (DFPCs). These cells are extremely accessible, prevail during all life and own an amazing multipotency. In the past decade, DPSCs and SHED have been thoroughly studied in regenerative medicine and tissue engineering as autologous stem cells therapies and have shown amazing therapeutic abilities in oro-facial, neurologic, corneal, cardiovascular, hepatic, diabetic, renal, muscular dystrophy and auto-immune conditions, in both animal and human models, and most recently some of them in human clinical trials. In this review, we focus the characteristics, the multiple roles of DSCs and its potential translation to clinical settings. These new insights of the apparently regenerative aptitude of these DSCs seems quite promising to investigate these cells abilities in a wide variety of pathologies.

• Key messages

• Dental stem cells (DSCs) have a remarkable self-renewal capacity and multidifferentiation potential;

• DSCs are extremely accessible and prevail during all life;

• DSCs, as stem cells therapies, have shown amazing therapeutic abilities in oro-facial, neurologic, corneal, cardiovascular, hepatic, diabetic, renal, muscular dystrophy and autoimmune conditions;

• DSCs are becoming extremely relevant in tissue engineering and regenerative medicine.

     

Current opportunities and challenges in skeletal muscle tissue engineering

The purpose of this article is to give a concise review of the current state of the art in tissue engineering (TE) of skeletal muscle and the opportunities and challenges for future clinical applicability. The endogenous progenitor cells of skeletal muscle, i.e. satellite cells, show a high proneness to muscular differentiation, in particular exhibiting the same characteristics and function as its donor muscle. This suggests that it is important to use an appropriate progenitor cell, especially in TE facial muscles, which have a exceptional anatomical and fibre composition compared to other skeletal muscle. Muscle TE requires an instructive scaffold for structural support and to regulate the proliferation and differentiation of muscle progenitor cells. Current literature suggests that optimal scaffolding could comprise of a fibrin gel and cultured monolayers of muscle satellite cells obtained through the cell sheet technique. Tissue‐engineered muscle constructs require an adequate connection to the vascular system for efficient transport of oxygen, carbon dioxide, nutrients and waste products. Finally, functional and clinically applicable muscle constructs depend on adequate neuromuscular junctions with neural cells. To reach this, it seems important to apply optimal electrical, chemotropic and mechanical stimulation during engineering and discover other factors that influence its formation. Thus, in addition to approaches for myogenesis, we discuss the current status of strategies for angiogenesis and neurogenesis of TE muscle constructs and the significance for future clinical use. Copyright © 2009 John Wiley & Sons, Ltd.     

The suitability of human adipose‐derived stem cells for the engineering of ligament tissue

Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports‐related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament‐derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose‐derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.     

Novel method for the isolation of adipose stem cells (ASCs)

Adipose stem cells (ASCs) represent a cell population with great potential for tissue engineering applications. Several articles have been published showing the proliferation and differentiation potential, the markers and the wide range of potential applications of these cells. In the majority of these studies the ASCs are isolated using a basic enzymatic procedure, which results in a quite heterogeneous cell population that compromises their proliferation and differentiation. This paper reports the development and optimization of a new isolation/purification method that allows populations of ASCs to be obtained, which significantly reduces (and eventually eliminates) the ‘contamination’ of other cell types. This method is based on the use of immunomagnetic beads coated with specific antibodies. The first part of the study described here analysed the expression of marker genes for stem cells and the colony‐forming unit (CFU) capacity of the cells isolated, while the second part is dedicated to the osteogenic differentiation potential of the isolated cells. The results showed that, using the isolation method based on immunomagnetic beads, it was possible to obtain ASCs and also underline the existence of several subpopulations of stem cells in the adipose tissue. Copyright © 2008 John Wiley & Sons, Ltd.     

Graphene nanomaterials as biocompatible and conductive scaffolds for stem cells: impact for tissue engineering and regenerative medicine

The discovery of the interesting intrinsic properties of graphene, a two‐dimensional nanomaterial, has boosted further research and development for various types of applications from electronics to biomedicine. During the last decade, graphene and several graphene‐derived materials, such as graphene oxide, carbon nanotubes, activated charcoal composite, fluorinated graphenes and three‐dimensional graphene foams, have been extensively explored as components of biosensors or theranostics, or to remotely control cell–substrate interfaces, because of their remarkable electro‐conductivity. To date, despite the intensive progress in human stem cell research, only a few attempts to use carbon nanotechnology in the stem cell field have been reported. Interestingly, most of the recent in vitro studies indicate that graphene‐based nanomaterials (i.e. mainly graphene, graphene oxide and carbon nanotubes) promote stem cell adhesion, growth, expansion and differentiation. Although cell viability in vitro is not affected, their potential nanocytoxicity (i.e. nanocompatibility and consequences of uncontrolled nanobiodegradability) in a clinical setting using humans remains unknown. Therefore, rigorous internationally standardized clinical studies in humans that would aim to assess their nanotoxicology are requested. In this paper we report and discuss the recent and pertinent findings about graphene and derivatives as valuable nanomaterials for stem cell research (i.e. culture, maintenance and differentiation) and tissue engineering, as well as for regenerative, translational and personalized medicine (e.g. bone reconstruction, neural regeneration). Also, from scarce nanotoxicological data, we also highlight the importance of functionalizing graphene‐based nanomaterials to minimize the cytotoxic effects, as well as other critical safety parameters that remain important to take into consideration when developing nanobionanomaterials. Copyright © 2014 John Wiley & Sons, Ltd.     

Updates on stem cells and their applications in regenerative medicine

Stem cells have the capacity for self-renewal and capability of differentiation to various cell lineages. Thus, they represent an important building block for regenerative medicine and tissue engineering. These cells can be broadly classified into embryonic stem cells (ESCs) and non-embryonic or adult stem cells. ESCs have great potential but their use is still limited by several ethical and scientific considerations. The use of bone marrow-, umbilical cord-, adipose tissue-, skin- and amniotic fluid-derived mesenchymal stem cells might be an adequate alternative for translational practice. In particular, bone marrow-derived stem cells have been used successfully in the clinic for bone, cartilage, spinal cord, cardiac and bladder regeneration. Several preclinical experimental studies are under way for the application of stem cells in other conditions where current treatment options are inadequate. Stem cells can be used to improve healthcare by either augmenting the body's own regenerative potential or developing new therapies. This review is not meant to be exhaustive but gives a brief outlook on the past, present and the future of stem cell-based therapies in clinical practice.     

骨组织工程中干细胞的研究与应用

背景：是否可以通过改进对已知的可以分化成骨的干细胞的培养方式或者寻找到新的干细胞,为骨组织工程找到更为合适的种子细胞.目的：从干细胞的分离培养、生物学特性及标志物等方面对各类干细胞的在骨组织工程中的应用进行综述.方法：以英文检索词为“bone tissue engineering,seed cel s,stem cel ,osteoblast differentiation”及中文检索词为“骨组织工程,种子细胞,干细胞,成骨分化”,由第一作者检索1995年至2012年PubMed 数据库及中国知网中文科技数据库,查阅近年种子细胞相关文献,最终保留50篇文献,从分离培养方法、基本特性及在骨组织工程中的应用3方面进行综述.结果与结论：文章分别对各类干细胞（包括：胚胎干细胞、骨髓间充质干细胞、滑膜间充质干细胞、脐带间充质干细胞和脐带血间充质干细胞、外周血来源的多潜能间充质干细胞、脂肪干细胞、子宫内膜基质干细胞、人羊膜基质细胞、牙髓干细胞）的分离方法、生物学特性、标志物等相关实验研究进行了探讨.目前用于分离纯化骨髓间充质干细胞的方法主要有4种：密度梯度离心法、全骨髓贴壁培养法、流式细胞仪分离法和免疫磁珠法.研究证实各类干细胞在特定条件下均有分化成骨的能力.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

牙髓干细胞在组织工程研究中的应用

背景：牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能.通过对牙髓干细胞的深入研究,有助于人类为组织工程研究提供可能的细胞来源,进而指导临床相关治疗与预防.目的：就牙髓干细胞的研究现状及其在组织工程中的应用作一综述.方法：应用计算机检索CNKI和Pubmed数据库中2000年1月至2012年5月关于牙髓干细胞的文章,在标题和中以“牙髓干细胞,诱导,培养,分化”或“dental pulp stem cel s,culture,induced, differentiation”为检索词进行检索.选择文章内容与牙髓干细胞有关者,同一领域文献则选择近期发表或发表在权威杂志文章.初检得到205篇文献,根据纳入标准选择关于牙周局部缓释剂的44篇文献进行综述.结果与结论：相比较其他成体干细胞,牙髓干细胞的研究尚面临许多问题.但随着科学技术的日益进步,随着这些问题的深入探讨和逐步解决,牙髓干细胞的研究将日趋完善.牙髓干细胞有望成为牙组织工程、骨组织工程和神经组织工程的种子细胞,在牙髓再生、牙体的修复等方面有着广阔的应用前景.     

Skeletal tissue engineering using embryonic stem cells

Various cell types have been investigated as candidate cell sources for cartilage and bone tissue engineering. In this review, we focused on chondrogenic and osteogenic differentiation of mouse and human embryonic stem cells (ESCs) and their potential in cartilage and bone tissue engineering. A decade ago, mouse ESCs were first used as a model to study cartilage and bone development and essential genes, factors and conditions for chondrogenesis and osteogenesis were unravelled. This knowledge, combined with data from the differentiation of adult stem cells, led to successful chondrogenic and osteogenic differentiation of mouse ESCs and later also human ESCs. Next, researchers focused on the use of ESCs for skeletal tissue engineering. Cartilage and bone tissue was formed in vivo using ESCs. However, the amount, homogeneity and stability of the cartilage and bone formed were still insufficient for clinical application. The current protocols require improvement not only in differentiation efficiency but also in ESC‐specific hurdles, such as tumourigenicity and immunorejection. In addition, some of the general tissue engineering challenges, such as cell seeding and nutrient limitation in larger constructs, will also apply for ESCs. In conclusion, there are still many challenges, but there is potential for ESCs in skeletal tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.