力学因素对骨髓间充质干细胞成骨分化的影响

近年来,组织工程学领域发展突飞猛进,已被列为一种修复或再生许多组织和器官的方法。骨髓间充质干细胞具有良好的成骨向分化潜能,在骨组织工程领域中具有广阔的应用前景。骨髓间充质干细胞增殖和成骨向分化受多种力学因素的影响,且不同性质的力学刺激对其定向分化的调节作用不尽相同。目前,许多学者致力于深入探讨力学因素影响骨髓间充质干细胞成骨向分化的具体途径,但其调控机制尚未完全明确。本文综述及讨论力学因素对骨髓间充质干细胞所产生的增殖、定向成骨分化等生物学效应的影响及可能涉及的力化学信号转导通路作用机制,以期丰富研究思路     

一种新的种子细胞——滑膜间充质干细胞

滑膜间充质干细胞作为一种新的间充质干细胞在组织工程中有着广阔的应用前景。滑膜间充质干细胞能够分化为软骨、骨、脂肪、骨骼肌，具有取材方便，供区并发症少等优点。因此，滑膜间充质干细胞是软骨、骨、脂肪及骨骼肌的种子细胞的理想来源。介绍了滑膜间充质干细胞的分离、获取以及其生物学特性，并对其在组织工程中应用的研究进展做一回顾。     

CD146阳性亚群有作为软骨组织工程种子细胞的应用潜力

文题释义：CD146：也称为MUC18,MCAM,Mel-CAM或S-Endo1,是一种跨膜糖蛋白,同时属于免疫球蛋白超级家族的一员。在人体组织中,CD146阳性细胞被认为是微血管的壁细胞,其具有较强的增殖、迁移及自我更新能力。 组织工程种子细胞：组织工程包括3个关键要素——种子细胞、支架和活性因子,其中种子细胞应具有良好的细胞活性、增殖能力、分化及合成基质等生物功能。 背景：再生医学的发展、组织工程技术的出现为软骨缺损重建提供了新的解决思路。在组织工程中,间充质干细胞是应用广泛的种子细胞,然而干细胞作为一个异质性的细胞群体其不同亚群发挥着不同的功能。因此,应用间充质干细胞关键功能亚群进行软骨修复具有广泛应用前景。 目的：从人脂肪间充质干细胞中分离出CD146阳性亚群细胞,验证其生物学特性及其作为软骨组织工程种子细胞的潜力。 方法：人脂肪间充质干细胞由浙江金时代生物技术有限公司提供,通过流式细胞术对人脂肪间充质干细胞表面标志物进行鉴定,应用免疫磁珠分选方法从人脂肪间充质干细胞中分选出CD146阳性表达的细胞亚群。通过基因芯片检测技术及生物信息学分析技术揭示2种细胞的分子特性;体外诱导2种细胞成软骨分化并进行鉴定;冻存复苏前后检测2种细胞的细胞活性及凋亡情况。 结果与结论：①人脂肪间充质干细胞表达高水平干细胞相关标志物CD73、CD90,不表达造血干细胞相关标志物CD34、CD45、HLA-DR;②生物信息分析结果表明CD146阳性亚群细胞与脂肪间充质干细胞相比在炎症通路及骨骼肌肉系统疾病有不同功能;③CD146阳性亚群细胞能够成球软骨分化,并且其成软骨分化能力要优于人脂肪间充质干细胞;④CD146阳性亚群细胞复苏后凋亡情况和活性均要优于人脂肪间充质干细胞;⑤结果表明,CD146阳性亚群细胞具有良好的成软骨分化潜力,是一种具有前景的软骨组织工程种子细胞。 ORCID: 0000-0003-4210-4708(眭翔) 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     

骨髓间充质干细胞用于软骨组织工程的研究进展

细胞移植技术治疗软骨损伤已成为一项新兴的组织工程学研究热点.骨髓间充质干细胞由于其具有扩增快、便于分离提纯、可以体外诱导分化成为软骨细胞的特性,有可能成为组织工程化软骨的新型种子细胞.随着骨髓间充质干细胞应用于软骨组织工程研究的深入,结合近年的研究文献和成果,就骨髓间充质干细胞的诱导微环境和诱导方式的研究进展进行综述,探讨骨髓间充质干细胞作为种子细胞在构建组织工程软骨中的优越性.     

软骨细胞上清液诱导滑膜间充质干细胞微团培养成软骨

背景：滑膜间充质干细胞在体外具有多向分化的能力,有望成为软骨组织工程中治疗软骨缺损的种子细胞,在其向软骨细胞分化过程中,合适的生长因子起了重要作用。 目的：利用富含生长因子的软骨细胞上清液诱导滑膜间充质干细胞向软骨细胞分化,并对其鉴定。 方法：采用消化法分别获得SD大鼠滑膜间充质干细胞、软骨细胞。收集软骨细胞上清液离心、过滤冻存备用。培养滑膜间充质干细胞至第3代后离心成微团,并用软骨细胞上清液进行成软骨诱导分化,通过形态学观察、免疫组织化学法、RT-PCR检测进行鉴定。 结果与结论：滑膜间充质干细胞使用软骨细胞上清液成软骨诱导21 d后,微团可见似软骨样组织。免疫组化法进行Ⅱ型胶原鉴定,基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。RT-PCR结果显示诱导后的微团表达软骨特异性基因Ⅱ型胶原和蛋白聚糖。证实软骨细胞分泌的可溶性因子可以诱导大鼠滑膜间充质干细胞向软骨方向分化。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：     

力学刺激诱导骨髓间充质干细胞选择分化的研究进展

机体细胞／组织处于复杂的力学环境中，力学刺激对细胞的形态、发育和功能起着重要的调节作用。骨髓间充质干细胞的增殖、选择分化受多种力学因素的影响，而且力学刺激的大小、方式以及作用时间对骨髓间充质干细胞定向分化的调节作用不尽相同。作者就力学因素对骨髓间充质干细胞选择分化的调节作用进行简要的综述。     

脐血间充质干细胞在软骨组织工程中的应用

背景：关节软骨修复再生能力较差,软骨缺损的修复与功能重建是关节外科的一大难题,也是近年研究热点之一,而软骨组织工程技术的发展为其提供了新的思路和方法。 目的：总结和分析脐血间充质干细胞的生物学特性及其在软骨组织工程中的研究与应用。 方法：通过计算机检索中国期刊全文数据库及PubMed数据库2000-01/2010-12的有关文献资料,分别以“脐血间充质干细胞,组织工程,软骨缺损”为中文检索词,“umbilical cord blood mesenchymal stem cells,tissue engineering,cartilage repair”为英文检索词,纳入脐血间充质干细胞和软骨组织工程的相关文献,排除重复性研究,共选取33篇文献做进一步分析。 结果与结论：脐血间充质干细胞以其固有的取材方便、免疫原性较弱、分化能力强以及被病毒细菌污染率低等特有的优势,成为软骨组织工程理想的种子细胞,将在未来软骨组织工程的研究及应用中发挥重要的作用。     

转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞的分化

背景：颞下关节盘软骨缺损修复在口腔临床上仍然是较大的挑战,具有多向分化潜能的脂肪间充质干细胞为成纤维软骨类组织修复带来了希望。目前使用转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞分化的研究很少。 目的：观察转化生长因子β3对脂肪间充质干细胞生长形貌及向成纤维软骨分化的影响。 方法：采用转化生长因子β3诱导SD大鼠脂肪间充质干细胞,观察成纤维软骨细胞分化的细胞形态,组织学和免疫荧光染色等方法检测脂肪间充质干细胞产生的细胞外基质Ⅰ,Ⅱ型胶原和蛋白多糖表达情况,评价脂肪间充质干细胞作为纤维软骨组织工程种子细胞的可行性。 结果与结论：倒置荧光显微镜观察结果显示脂经转化生长因子β3生长因子诱导之后,细胞有明显的聚集生长现象,形态呈多角形、多边形,细胞外基质分泌增多。阿利新蓝染色结果表明,经转化生长因子β3诱导脂肪间充质干细胞显示明显的深蓝色,表明脂肪间充质干细胞合成了大量的糖胺聚糖。免疫染色结果表明,在转化生长因子β3 诱导下,脂肪间充质干细胞合成Ⅰ,Ⅱ型胶原细胞外基质。提示转化生长因子β3可诱导脂肪间充质干细胞向成纤维软骨样细胞分化,也意味着脂肪间充质干细胞具有作为工程化纤维软骨种子细胞的潜能。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：     

间充质干细胞促进关节软骨的修复与再生

背景：关节软骨损伤后,软骨组织几乎没有修复能力,关节软骨损伤的修复一直是临床工作的难点。 目的：探讨修复关节软骨损伤的干细胞种类及其生物学特性,明确干细胞在修复关节软骨损伤中的作用及优缺点。 方法：由第一作者检索1998至2015年PubMed数据及CNKI中国期刊全文数据库,英文检索词“Articular cartilage injury,Mesenchymal stem cells ,regeneration”;中文检索词“关节软骨损伤,间充质干细胞,再生”,纳入47篇文献进行分析。 结果与结论：关节软骨损伤最有效的修复方案是以细胞为基础的治疗方法,来源于骨髓、脂肪及脐血的间充质干细胞均有较强的成软骨特性和克隆能力。骨髓间充质干细胞具有更高的分化潜能,对软骨缺损有修复作用,来源于脐血的间充质干细胞致瘤性低,脂肪源性干细胞的生长增殖速度更快。干细胞复合天然载体材料如胶原、明胶、纤维蛋白和藻酸盐等可促进细胞黏附、分化和增殖,以此构建组织工程软骨将有效修复关节软骨缺损。 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      

不同来源间充质干细胞生物学特性差异

背景：间充质干细胞在体内或体外特定的诱导条件下,可分化为软骨、肌肉、肌腱等。间充质干细胞进行的临床试验主要包括组织损伤修复,如骨、软骨、关节损伤的修复,心脏、肝脏、脊髓损伤和神经系统疾病的治疗。 目的：比较各种来源的间充质干细胞的生物学特性。 方法：检索1987到2015年PubMed数据库和中国知网数据库收录的与间充质干细胞来源,间充质干细胞生物学特性相关的文献。从细胞表面标记物,增殖、分化、迁移能力,以及功能方面进行分析总结,探讨了各种来源的间充质干细胞的优缺点。 结果与结论：不同来源的间充质干细胞的增殖潜力和表面标记物存在差异。不同组织来源的间充质干细胞免疫活性可能与间充质干细胞处于不同组织中的活化状态、种属差异、组织来源和培养条件的不同有关,从而导致不同源性间充干细胞免疫活性也不完全相同。深入认识影响不同组织来源间充质干细胞迁移的因素和机制,可以增强不同源性间充质干细胞靶向迁移的能力,提高其在创伤愈合、组织修复和再生中的治疗效率。  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     

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

Mechanical properties of hyaline and repair cartilage studied by nanoindentation

Articular cartilage is a highly organized tissue that is well adapted to the functional demands in joints but difficult to replicate via tissue engineering or regeneration. Its viscoelastic properties allow cartilage to adapt to both slow and rapid mechanical loading. Several cartilage repair strategies that aim to restore tissue and protect it from further degeneration have been introduced. The key to their success is the quality of the newly formed tissue. In this study, periosteal cells loaded on a scaffold were used to repair large partial-thickness cartilage defects in the knee joint of miniature pigs. The repair cartilage was analyzed 26 weeks after surgery and compared both morphologically and mechanically with healthy hyaline cartilage. Contact stiffness, reduced modulus and hardness as key mechanical properties were examined in vitro by nanoindentation in phosphate-buffered saline at room temperature. In addition, the influence of tissue fixation with paraformaldehyde on the biomechanical properties was investigated. Although the repair process resulted in the formation of a stable fibrocartilaginous tissue, its contact stiffness was lower than that of hyaline cartilage by a factor of 10. Fixation with paraformaldehyde significantly increased the stiffness of cartilaginous tissue by one order of magnitude, and therefore, should not be used when studying biomechanical properties of cartilage. Our study suggests a sensitive method for measuring the contact stiffness of articular cartilage and demonstrates the importance of mechanical analysis for proper evaluation of the success of cartilage repair strategies.     

运动性关节软骨损伤修复材料的选择及其生物力学特征

背景：关节软骨是无血管、淋巴管和神经的组织,通常情况下软骨细胞不能进行有丝分裂,这导致自身修复能力有限。生理负荷下,关节软骨经常处在应力环境中。根据软骨自身的结构和特点,作为人工软骨的替代材料应具有良好的生物力学性能。 目的：总结运动性关节软骨损伤修复材料的应用进展及其生物替代材料的生物力学特征。 方法：以“关节软骨,生物材料,生物力学”为中文关键词,以“ tissue enginneering, articular cartilage, scaffold material, biomechanics” 为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库1993-01/2010-10相关文章。纳入与运动有关的关节软骨损伤修复、目前常用于修复关节软骨损伤的生物材料以及生物替代材料的生物力学特征研究文章;排除重复研究或Meta分析类文章。以20篇文献为主重点对运动性关节软骨缺损修复材料的生物力学特征进行讨论。 结果与结论：关节软骨是一种各向异性、非均质、具有黏弹性并充满液体的可渗透物质,具有独特的力学性能。损伤的关节软骨在生物力学方面均与原来的软骨不同,且极易退变。骨软骨柱移植力学性能近期效果最佳;脱细胞软骨基质、小肠黏膜下基质具有一定的力学强度;普通聚乙烯醇水凝胶的最大缺陷是力学性能的不足;聚乙烯醇材料其良好的柔韧性和高弹性能,具有与人关节软骨相似的力学性能;n-HA浆料与聚酰胺66在溶剂中复合,无论在力学性能还是化学组成上都与自然骨相似。提示在众多关节软骨替代材料中,无论是人工合成材料、天然材料、复合材料其生物力学性能各有不同,且目前还无法再造与天然生成的软骨具有相同力学性能的软骨组织。      

Mechanical quality of tissue engineered cartilage: results after 6 and 12 weeks in vivo

Traumatic events are a primary cause for local lesions of articular cartilage. If treated early, restoration of the initial joint geometry and integrity may be achieved. In large defects, sufficient material is not available to bridge the affected area. Heterologeous transplantation is not well accepted due to the risk of infection and immune response. Alternatives are cartilage-like structures, which may be cultured in vitro and transplanted into the defect site. Critical to the success of these new tissues are their mechanical properties. Goals of this study were to generate a hyaline-like cartilage structure, to evaluate its performance in vivo and to verify that its cellular and material properties meet those of native cartilage. Hyaline-like cartilage specimens were generated in vitro and implanted in the backs of nude mice. Specimens were explanted after 6 and 12 weeks, mechanically tested using an indentation test and histologically examined. In mechanical testing, stiffness and failure load significantly increased between weeks 6 and 12. At 12 weeks, mechanical properties of the hyaline-like cartilage were comparable to those of native nasal septal cartilage. Compared to native articular cartilage, the engineered tissue achieved up to 30-50% in strength and mechanical stiffness. In histological examination, specimens showed neocartilage formation. The mechanical testing procedure proved to be sufficiently sensitive to identify differences in properties between cartilage specimens of different origin and at different stages of healing. As an adjunct to histological analysis, mechanical testing may be a valuable tool for judging the utility of engineered cartilage prior to a broad clinical usage.     

Functional biomaterials for cartilage regeneration

The injury and degeneration of articular cartilage and associated arthritis are leading causes of disability worldwide. Cartilage tissue engineering as a treatment modality for cartilage defects has been investigated for over 20 years. Various scaffold materials have been developed for this purpose, but has yet to achieve feasibility and effectiveness for widespread clinical use. Currently, the regeneration of articular cartilage remains a formidable challenge, due to the complex physiology of cartilage tissue and its poor healing capacity. Although intensive research has been focused on the developmental biology and regeneration of cartilage tissue and a diverse plethora of biomaterials have been developed for this purpose, cartilage regeneration is still suboptimal, such as lacking a layered structure, mechanical mismatch with native cartilage and inadequate integration between native tissue and implanted scaffold. The ideal scaffold material should have versatile properties that actively contribute to cartilage regeneration. Functional scaffold materials may overcome the various challenges faced in cartilage tissue engineering by providing essential biological, mechanical, and physical/chemical signaling cues through innovative design. This review thus focuses on the complex structure of native articular cartilage, the critical properties of scaffolds required for cartilage regeneration, present strategies for scaffold design, and future directions for cartilage regeneration with functional scaffold materials.     

Designing zonal organization into tissue-engineered cartilage

Cartilage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to the native zonal organization of articular cartilage. The objective of this study was to use bilayered photopolymerized hydrogels to organize zone-specific chondrocytes in a stratified framework and study the effects of this three-dimensional coculture system on the properties of the engineered tissue. Superficial and deep zone chondrocytes from bovine articular cartilage were photoencapsulated in separate hydrogels as well as in adjacent layers of a bilayered hydrogel. Histology, mechanical testing, and biochemical analysis was performed after culturing in vitro. To evaluate the influence of coculture on tissue properties, the layers were separated and compared to constructs containing only superficial or deep cells. In the bilayered constructs, deep cells produced more collagen and proteoglycan than superficial cells, resulting in cartilage tissue with stratified, heterogeneous properties. Deep cells cocultured with superficial cells in the bilayered system demonstrated reduced proliferation and increased matrix synthesis compared to deep cells cultured alone. The bilayered constructs demonstrated greater shear and compressive strength than homogenous cell constructs. This study demonstrated that interactions between zone-specific chondrocytes affect the biological and mechanical properties of engineered cartilage. Strategies aimed to structurally organize zone-specific cells and encourage heterotypic cell interactions may contribute to improved functional properties of engineered cartilage.     

Engineering lubrication in articular cartilage

Despite continuous progress toward tissue engineering of functional articular cartilage, significant challenges still remain. Advances in morphogens, stem cells, and scaffolds have resulted in enhancement of the bulk mechanical properties of engineered constructs, but little attention has been paid to the surface mechanical properties. In the near future, engineered tissues will be able to withstand and support the physiological compressive and tensile forces in weight-bearing synovial joints such as the knee. However, there is an increasing realization that these tissue-engineered cartilage constructs will fail without the optimal frictional and wear properties present in native articular cartilage. These characteristics are critical to smooth, pain-free joint articulation and a long-lasting, durable cartilage surface. To achieve optimal tribological properties, engineered cartilage therapies will need to incorporate approaches and methods for functional lubrication. Steady progress in cartilage lubrication in native tissues has pushed the pendulum and warranted a shift in the articular cartilage tissue-engineering paradigm. Engineered tissues should be designed and developed to possess both tribological and mechanical properties mirroring natural cartilage. In this article, an overview of the biology and engineering of articular cartilage structure and cartilage lubrication will be presented. Salient progress in lubrication treatments such as tribosupplementation, pharmacological, and cell-based therapies will be covered. Finally, frictional assays such as the pin-on-disk tribometer will be addressed. Knowledge related to the elements of cartilage lubrication has progressed and, thus, an opportune moment is provided to leverage these advances at a critical step in the development of mechanically and tribologically robust, biomimetic tissue-engineered cartilage. This article is intended to serve as the first stepping stone toward future studies in functional tissue engineering of articular cartilage that begins to explore and incorporate methods of lubrication.     

Mechanical Modulation of Cartilage Structure and Function During Embryogenesis in the Chick

The mechanical behavior of cartilage is intimately related to its biochemical composition, and tissue composition is known to be influenced by its local mechanical loading environment. Although this phenomenon has been well-studied in adult cartilage, few investigations have examined such structure-function relationships in embryonic cartilage. The goal of this work was to elucidate the role of mechanical loading on the development of cartilage composition during embryogenesis. Using an embryonic chick model, cartilage from the tibiofemoral joints of immobilized embryos was compared to that of controls. The normal time course of changes in glycosaminoglycan/DNA and hydroxyproline/DNA were significantly influenced by loading history, with the most pronounced effects observed between days 9 and 14 during the period of most rapid increase in motility in control embryos. Stress-relaxation tests conducted on samples from day 14 indicate that the effects of embryonic immobilization on cartilage matrix composition have direct consequences for the mechanical behavior of the tissue, resulting in compromised material properties (e.g. 50% reduction in E(inst)). Because embryogenesis provides a unique model for identifying key factors which influence the establishment of functional biomechanical tissues in the skeleton, these data suggest that treating mechanical loading as an in vitro culture variable for tissue engineering approaches to cartilage repair is likely to be a sound approach.     

Prediction of the optimal mechanical properties for a scaffold used in osteochondral defect repair

The optimal mechanical properties of a scaffold to promote cartilage generation in osteochondral defects in vivo are not known. During normal daily activities cartilage is subjected to large cyclic loads that not only facilitate nutrient transport and waste removal through the dense tissue but also act as a stimulus to the chondrocytes. In contrast, cartilage tissue is commonly engineered in vitro in a static culture; hence, in many cases, the properties of scaffolds have been tailored to suit this in vitro environment. In this study, a mechanoregulation algorithm for tissue differentiation was used to determine the influence of scaffold material properties on chondrogenesis in a finite element model of an osteochondral defect. It is predicted that increasing the stiffness of the scaffold increases the amount of cartilage formation and reduces the amount of fibrous tissue formation in the defect, but this only holds true up to a certain threshold stiffness above which the amount of cartilage formed is reduced. Reducing the permeability of the scaffold was also predicted to be beneficial. Considering a nonhomogeneous scaffold, an optimal design was determined by parametrically varying the mechanical properties of the scaffold through its depth. The Young's modulus reduced nonlinearly from the superficial region through the depth of the scaffold, while the permeability of the scaffold was lowest in the superficial region. As tissue engineering moves from a science toward a product, engineering design becomes more relevant, and predictive models such as that presented here can provide a scientific basis for design choices.