软骨组织工程研究的新进展

软骨组织的再生能力有限，组织工程软骨的构建对修复软骨缺损意义理大。本从四方面介绍了软骨组织工程的研究的新进展，包括软骨种子细胞的研究，软骨细胞与支架的体外培养，细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

以交联透明质酸钠为支架体外构建组织工程软骨

背景：采用组织工程技术再生和重建软骨是目前修复软骨组织缺损效果最好、最有应用前景的方法。 目的：以体外培养的软骨细胞和交联透明质酸钠为支架材料,开发一套体外构建组织工程软骨的完整方案。 方法：分离新西兰兔膝关节软骨细胞,制成细胞悬液滴加于交联透明质酸钠支架上,体外复合培养21 d,提取RNA进行RT-PCR检测,制备冰冻切片进行显微观察和免疫组织化学观察。 结果与结论：软骨细胞接种于交联透明质酸钠支架材料后,可贴附于支架上生长,并且大量细胞聚集成团,在支架材料的纤维间隙中生长或呈单层细胞附着于支架材料纤维。细胞-支架复合物表达软骨组织特异性蛋白聚糖基因和Ⅱ型胶原α1基因,以及软骨组织特异性蛋白Ⅱ型胶原蛋白,可维持软骨细胞表型。表明培养的细胞-支架复合物在体外培养可形成软骨细胞外基质,有望获得组织工程软骨组织。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

气管软骨细胞和羧乙基壳聚糖-羟基磷灰石泡沫支架复合及裸鼠皮下植入

目的 探讨以兔气管软骨细胞为种子细胞在自制羧乙基壳聚糖-羟基磷灰石泡沫(NCECS-HA)支架合成组织工程气管软骨的可行性.方法 通过真空冷冻干燥法制得NCECS-HA泡沫支架.从6个月大的大耳白兔取气管软骨片段,Ⅱ型胶原酶消化,将所获得第3代软骨细胞种植于NCECS-HA三维支架上.细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周.然后取出分别进行HE染色、Ⅱ型胶原免疫组化染色和甲苯胺蓝染色,观察软骨细胞基质分泌情况.结果 8周后,构建出组织工程气管软骨示光泽良好,甲苯胺蓝染色、Ⅱ型胶原免疫组化染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌糖氨多糖和Ⅱ型胶原.结论 生物材料NCECS-HA对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架.     

人脐带Wharton胶中间充质干细胞体外无支架组织工程软骨的构建

背景:构建组织工程软骨的方法多为自体种子细胞复合天然或合成物支架,目前多存在种子细胞来源有限、支架安全性和生物相容性、以及细胞在支架中分布不均的问题。目的:探讨人脐带Wharton胶中间充质干细胞向软骨诱导分化并体外构建无支架组织工程软骨的可行性。方法:分离培养人脐带Wharton胶中的间充质干细胞,进行流式细胞学鉴定。对软骨诱导前后的细胞进行组织学和免疫组织化学染色,对其表达的葡萄糖胺聚糖和Ⅱ型胶原进行定量研究,并应用RT-PCR检测软骨诱导前后Ⅱ型胶原和Sox-9mRNA的表达。采用密集诱导培养→离心管培养→生物反应器培养,进行体外构建无支架软骨组织。结果与结论:人脐带Wharton胶富含干细胞,流式细胞仪检测结果显示这些细胞不表达造血干细胞标志,表达CD44,CD105、CD271等间充质干细胞表面标志;HLA-ABC阳性表达,HLA-DPDQDR阴性表达。未进行软骨诱导的细胞弱表达软骨细胞标志,诱导后葡萄糖胺聚糖和Ⅱ型胶原显著增高。RT-PCR结果显示人脐带Wharton胶间充质干细胞诱导前后均表达Sox-9、Ⅱ型胶原mRNA。说明人脐带Wharton胶间充质干细胞具有前软骨细胞的特性。采用密集诱导培养结合生物反应器培养,不用支架,体外可以构建成大块组织工程软骨。表明人脐带Wharton胶间充质干细胞是一种良好的构建组织工程软骨的种子细胞。     

骨髓间充质干细胞向软骨细胞分化的研究进展

骨髓间充质干细胞(BMSCs)是存在于骨髓内的一种多能干结缔组织前体细胞,具有多向分化潜能,也是最有可能成为软骨组织工程的种子细胞来源。本文从BMSCs诱导成软骨细胞的方法和研究进展做一综述,如体外细胞团聚集诱导培养、体外单层细胞诱导培养、体外三维支架环境中诱导培养、体内软骨微环境诱导培养、和软骨细胞体外共培养诱导以及基因转染诱导培养等,这也是软骨组织工程研究中不可缺少的重要环节。     

聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架与兔软骨细胞的相容性

背景：传统的支架材料存在疏水性强,材料表面缺乏细胞表面受体特异结合的生物活性分子,材料的酸性降解产物易引发无菌性炎性反应等不足。根据仿生原理及软骨真实结构和构成来选择和制备组织工程软骨支架能够获得理想效果。 目的：制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架,评价其与兔膝关节软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。 方法：采用二次相分离技术制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石复合支架,将第3代新西兰兔软骨细胞接种至复合支架材料上复合培养,倒置相差显微镜下观察细胞生长情况。细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周。 结果与结论：聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架材料经化学合成后,具有合适的三维多孔结构,孔隙率为90%,孔径300~450 μm;植入裸鼠皮下8周后Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌黏多糖和Ⅱ型胶原。提示生物材料聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架。     

胶原复合梯度磷酸三钙负载软骨细胞的体外培养

目的 观察新型三维支架材料胶原复合梯度磷酸三钙在体外与软骨细胞的相容性和黏附性,评价其作为软骨组织工程支架的可行性.方法 取8周龄新两兰大白兔膝关节软骨,以酶消化法获得高纯度软骨细胞,培养3代后与三维支架材料胶原复合梯度磷酸三钙在体外复合培养.用倒置相差显微镜、HE染色、免疫组织化学及扫描电镜观察软骨细胞形态、Ⅱ型胶原表达及成软骨能力,同时观察支架材料与软骨细胞的相容性.结果 扫描电镜观察显示支架材料具有疏松多孔结构,孔隙结构规则,孔径100～150 μm,材料内部孔与孔之间贯通良好.支架亲水性好.软骨细胞吸附于支架表面,增殖并逐渐顺孔隙迁徙至支架内部,在孔壁贴附良好,表型维持稳定,可分泌细胞外基质.结论 胶原复合梯度磷酸三钙三维支架具有良好的细胞相容性.     

壳聚糖及其衍生物在软骨组织工程中的应用

背景：作为生物型支架,壳聚糖因其独特的多孔三维结构、易于改性的特征及良好的生物相容性成为了软骨组织工程支架材料的研究热点。 目的：就壳聚糖及其衍生物的设计、改性及在软骨组织工程中的应用作一综述。 方法：应用计算机检索PubMed数据库和CNKI数据库,中文关键词为“壳聚糖,壳聚糖衍生物,支架材料,组织工程,软骨组织”,英文检索词为“chitosan;chitosan derivatives;scaffold;tissue engineering;cartilage”,检索文献时间范围为1990年1月至2015年1月。 结果与结论：壳聚糖是一种天然的生物多糖,通过化学改性、共混改性等方法可以改变壳聚糖的溶解度、机械强度、生物活性甚至生物降解性等自身特性,从而制成更为合适的生物支架材料。进一步研究表明,将壳聚糖与种子细胞进行共同体外培养可以获得正常形态的软骨细胞并能合成特异性的细胞外基质成分,在动物体内,壳聚糖支架与种子细胞所构建的组织工程软骨能够修复软骨损伤,形成与周围正常软骨相似的组织。壳聚糖及其衍生物支架材料在软骨组织工程中有较为广阔的研究前景。  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

骨、软骨组织工程种子细胞及其免疫学相关研究进展

近年来,细胞生物与生物材料学的迅速发展加快了骨、软骨组织工程的研究.骨、软骨组织工程的系列研究中,种子细胞是骨、软骨组织工程研究的核心内容.本文就骨、软骨组织工程的种子细胞成骨细胞、软骨细胞、骨髓基质细胞、间充质干细胞、胚胎干细胞及其免疫学相关研究进展作一讨论.     

环境、材料、安全、可控：组织工程技术修复关节软骨的症结*

背景：传统的软骨缺损的修复方法都有其局限性,组织工程技术的出现从根本上改变了“以创伤修复创伤”的传统治疗模式。 目的：总结分析目前组织工程技术修复关节软骨的研究进展。 方法：由第一作者检索1990年至2011年 PubMed数据及中国知网数据库有关应用组织工程技术修复关节软骨方面的文献。共检索中文187 篇,英文211 篇,最终保留49篇进入结果分析。 结果与结论：软骨组织工程的主要方法就是应用工程学和生命科学原理,在体外分离、培养、扩增所需要的种子细胞,然后将之种植于合适的生物支架材料上,将细胞支架复合体植入体内组织缺损部位,并加入一定的诱导条件,逐渐形成新的有功能的软骨组织。文章在种子细胞的选择方面重点叙述了自体软骨细胞、异体软骨细胞、胚胎干细胞、骨髓间充质干细胞的研究进展;在细胞诱导及条件培养方面重点叙述了细胞因子、细胞条件培养、转基因技术的研究进展;并对生物支架材料的选择和研究进行了相关叙述。找到最理想的种子细胞,合理联合应用细胞因子,更加真实的模拟细胞生存的微环境,基因工程安全、高效、可控转染,构建理想的支架材料,将是今后组织工程研究的重点和热点。     

In vitro cartilage tissue formation by Co-culture of primary and passaged chondrocytes

Passaging chondrocytes to increase cell number is one way to overcome the major limitation to cartilage tissue engineering, which is obtaining sufficient numbers of chondrocytes to form large amounts of tissue. Because neighboring cells can influence cell phenotype and because passaging induces dedifferentiation, we examined whether coculture of primary and passaged bovine articular chondrocytes in 3-dimensional culture would form cartilage tissue in vitro. Chondrocytes passaged in monolayer culture up to 4 times were mixed with primary (nonpassaged) chondrocytes (5-40% of total cell number) and grown on filter inserts for up to 4 weeks. Passaged cells alone did not form cartilage, but with the addition of increasing numbers of primary chondrocytes, up to 20%, there was an increase in cartilage tissue formation as determined histologically and biochemically and demonstrated by increasing proteoglycan and collagen accumulation. The passaged cells appeared to be undergoing redifferentiation, as indicated by up-regulation of aggrecan, type II collagen, and SOX9 gene expression and decreased type I collagen expression. This switch in collagen type was confirmed using Western blots. Confocal microscopy showed that fluorescently labeled primary cells were distributed throughout the tissue. This coculture approach could provide a new way to solve the problem of limited cell number for cartilage tissue engineering.     

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review

Once damaged, articular cartilage has very little capacity for spontaneous healing because of the avascular nature of the tissue. Although many repair techniques have been proposed over the past four decades, none has sucessfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. Tissue engineering approaches, such as transplantation of isolated chondrocytes, have recently demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions. As such a new approach emerges, new important questions arise. One of such questions is: what kinds of biomaterials can be used with chondrocytes to tissue-engineer articular cartilage? The success of chondrocyte transplantation and/or the quality of neocartilage formation strongly depend on the specific cell-carrier material. The present article reviews some of those biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage. A new biomaterial, a chitosan-based polysaccharide hydrogel, is also introduced and discussed in terms of the biocompatibility with chondrocytes.     

Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.     

软骨组织工程中力学因素的影响及应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明,力学作用可以刺激细胞因子及激素的分泌,改变三维支架上培养的软骨细胞的新陈代谢,从而促进软骨组织的生长与重建。目前已经有诸多关于体外构建软骨组织的报道,但对于其中的力学因素的影响(包括力学因素对软骨细胞增殖的促进及力学刺激的传导机制等)还没有完全认识。就以上几方面做一综述,并简单介绍生物反应器在软骨组织工程中的应用。     

Gene expression profiling of human articular cartilage grafts generated by tissue engineering

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays. The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.     

Regenerated silk fibroin scaffold and infrapatellar adipose stromal vascular fraction as feeder-layer: a new product for cartilage advanced therapy

Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adipose-derived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer.     

Lack of oxygen in articular cartilage: consequences for chondrocyte biology

Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia‐mediated chondrocyte function in the native articular cartilage.     

Glial Fibrillary Acidic Protein Immunoreactivity of Chondrocytes in Immature and Mature Teratomas

The immunoreactivity of chondrocytes for glial fibrillary acidic protein (GFAP), other intermediate filament proteins and S-100 protein was studied in formalin-fixed paraffin-embedded sections. A total of 95 cartilage specimens were examined from five immature teratomas, 12 mature teratomas, and a teratocarcinoma. GFAP-immunoreactive chondrocytes were abundant in immature cartilages, and as the cartilages maturated, these chondrocytes decreased and became distributed peripherally. Elastic cartilage had more GFAP-immunoreactive chondrocytes than non-elastic cartilage. GFAP-immunoreactive cartilage was often located close to central nervous tissue. lmmunostaining for vimentin and S-100 protein revealed extensive distribution of immunoreactive chondrocytes in immature and mature cartilages, but in mature cartilage, chondrocytes at the center had less vimentin immunoreactivity. GFAP-immunoreactive chondrocytes also showed apparent immunostaining for vimentin. There was no difference in immunohistochemical staining for the α and α subunits of S-100 protein. The immunoreactivities of teratoma cartilage specimens were quite similar to those of respiratory tract cartilage.     

干细胞诱导形成软骨细胞研究进展

组织工程技术构建软骨需要大量的软骨细胞,成熟的软骨细胞扩增能力有限,难以满足组织构建需要。干细胞,包括胚胎干细胞、成体干细胞,均具有强大的自我更新能力及多向分化潜能。在适当的诱导条件下,这些干细胞均可被诱导分化为软骨细胞,从而满足组织工程需求。