兔骨髓基质干细胞与软骨细胞混和培养体内成软骨

背景:诱导因子及软骨微环境是影响骨髓基质干细胞成软骨分化及软骨形成的主要因素.目的:利用少量软骨细胞以共培养方式诱导骨髓基质干细胞体内成软骨.设计、时间及地点:2004-09/2005-03在解放军第四军医大学口腔医院病理教研室完成的随机对照动物实验.材料:选择清洁级新西兰兔15只用于细胞支架复合物的种植,随机分为混合细胞组、软骨细胞组、骨髓基质干细胞组,5只/组.取新生1～3 d龄新西兰兔5只用于骨髓基质干细胞、软骨细胞的分离培养.聚羟基乙酸无纺网支架为上海易括公司产品,材料直径15 μm,平均间距150～200 μm,孔隙率97%,厚2 mm.方法:混合细胞组将骨髓基质干细胞与软骨细胞按3:1混匀,调整细胞密度为6.0×1010 L-1,接种于经培养液预湿的塑形聚羟基乙酸 5 mm×5 mm的支架上,然后在复合物周围滴加含胎牛血清的DMEM液培养1周.软骨细胞组、骨髓基质干细胞组的细胞密度调整为6.0×1010 L-1后同法接种于支架上.各组兔麻醉后于背部一侧皮下组织植入对应的细胞-支架复合物.主要观察指标:植入后第8周行新生软骨大体观察和苏木精-伊红、Masson三色染色.结果:各组细胞与聚羟基乙酸支架黏附情况良好.植入8周后,混和培养组和软骨细胞组均形成了成熟的软骨样组织,并基本保持复合物初始的大小和形状,两组新生软骨外观及组织学特征较接近.骨髓基质干细胞组在体内培养过程中未形成软骨组织,而是形成了纤维结缔组织.结论:骨髓基质干细胞与软骨细胞按3:1混合形成的微环境,能有效诱导骨髓基质干细胞向软骨细胞分化,并促进骨髓基质干细胞的体内成软骨.     

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：模仿自然骨一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：制备明胶一硫酸软骨素一透明质酸及明胶一陶瓷化骨多孔复合支架,构建自然骨一软骨基质复合支架,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,以及苏木精一伊红染色检测。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,14d时达到稳定状态。两组苏木精一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一软骨样组织,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。     

制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为＂Cartilage tissue engineering;scaffolds;fabrication＂,中文检索词为＂软骨组织工程;制备方法;支架材料;多孔支架＂。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。     

羟基丁酸与羟基辛酸共聚物一体化骨软骨组织工程支架的制备及性能

背景:单层支架难以满足关节软骨损伤修复的要求,现提出骨软骨共同修复的一体化支架,以弥补了单一支架的部分缺陷。目的:以羟基丁酸与羟基辛酸共聚物为基础材料,羟基磷灰石等为复合材料研制一体化骨软骨组织工程支架,测试该支架的物理特性和细胞黏附性。方法:采用溶剂浇铸/颗粒沥滤法,以支架孔径、孔隙率、力学强度和细胞黏附生长率为检测指标,以羟基丁酸与羟基辛酸共聚物为连续相,通过改变致孔剂NaCl粒径和羟基磷灰石材料配比制备不同形态结构、力学强度和生物学功能的三层一体化骨软骨组织工程支架。结果与结论:致孔剂与支架材料的最佳质量配比分别为软骨层4.5/1,过渡层2.5/1,硬骨层3.5/1。扫描电镜观察显示支架的三层结构明显不同且紧密结合,其软骨层、过渡层、硬骨层的孔径分别为150~250μm,≤60μm,150~450μm;孔隙率检测结果依次为84%,60%,75%;力学强度测定依次为2.93,6.43,4.30MPa;支架对骨髓间充质干细胞无毒性,细胞黏附与生长状态良好。结果表明该一体化骨软骨组织工程支架具有仿生学特性,符合骨软骨组织工程支架的基本条件。     

双腔搅拌式生物反应器提高β-磷酸三钙修复关节软骨缺损的效果

背景：前期实验自行研发了一种可进行成骨及成软骨双向诱导分化的双腔搅拌式生物反应器。 目的：探索双腔搅拌式生物反应器的力学刺激能否提高组织工程骨软骨修复山羊膝关节缺损的效果。 方法：取青山羊12只,制作双侧后肢股骨内髁骨软骨缺损,随机分组,实验组与对照组缺损处均植入在双腔搅拌式生物反应器中进行成软骨、成骨诱导2周的自体骨髓间充质干细胞与β-磷酸三钙复合体,不同的是实验组将双腔搅拌式生物反应器置于磁力搅拌仪上给予力学刺激,对照组未给予力学刺激;空白对照组不做处置。植入后12,24周进行大体观察,Masson染色、Ⅱ型胶原免疫组织化学染色和组织学评分。 结果与结论：实验组与对照组均有新生软骨与骨组织生成,实验组修复骨缺损效果明显优于对照组（P 〈 0.05）;空白对照组无新生软骨生成。表明通过双腔搅拌式生物反应器体外培养阶段的力学刺激,可以改善以山羊骨髓间充质干细胞为种子细胞、β-磷酸三钙为支架的组织工程骨软骨复合物对膝关节缺损的修复效果。     

Three‐dimensional assembly of tissue‐engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)‐terephthalate–poly(butylene terephthalate) (PEGT–PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold‐seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet‐assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone. Copyright © 2013 John Wiley & Sons, Ltd.     

胶原-壳聚糖支架与软骨细胞的生长

背景：目前软骨支架材料的种类比较多,但还没有一种材料能完全符合软骨修复的要求。目的：观察在混合材料胶原-壳聚糖支架中软骨细胞的生长情况。方法：采用冷冻干燥法将质量分数为2%胶原与3%壳聚糖混合制备胶原-壳聚糖多孔支架。将分离培养的第2代兔软骨细胞接种到胶原-壳聚糖支架上,对照组将软骨细胞接种到无支架的培养板中。观察支架的孔隙率、吸水性及内部形态结构,MTT法检测软骨细胞在支架上的增殖情况,组织切片苏木精-伊红染色,扫描电镜观察细胞在支架的生长、贴附情况,RT-PCR检测细胞支架复合物蛋白聚糖和Ⅱ型胶原mRNA表达情况。结果与结论：胶原-壳聚糖支架的吸水性为（80.0±0.55）%,孔隙率为（88.5±1.5）%,孔径为100～150μm,复合细胞培养2周后,细胞增殖活力高,软骨细胞分泌的蛋白聚糖和Ⅱ型胶原mRNA表达明显高于对照组。说明质量分数为2%胶原与3%壳聚糖的混合支架适合软骨细胞生长和快速增殖,是一种良好的修复和重建软骨载体。     

Tissue‐engineered constructs: the effect of scaffold architecture in osteochondral repair

Cartilage has a poor regenerative capacity. Tissue‐engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55 wt% poly(ethylene oxide terephthalate) and 45 wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three‐dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3 weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3 weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post‐implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM‐treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair. Copyright © 2012 John Wiley & Sons, Ltd.     

Chondrogenic,hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three‐dimensionally woven scaffolds

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three‐dimensionally woven poly(ε‐caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations—PCL alone, PCL‐bone, “point‐of‐care” seeded MSC‐PCL‐bone, and chondrogenically precultured Ch‐MSC‐PCL‐bone constructs—were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch‐MSC‐PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula‐like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.     

Evaluation of methods for the construction of collagenous scaffolds with a radial pore structure for tissue engineering

Type I collagen is used widely as a biomaterial. The structure of collagenous biomaterials, including pore sizes and general architecture, can be varied by a number of techniques. In this study, we developed a method to construct flat fibrillar type I collagen scaffolds, 6 cm in diameter and with a radially orientated pore structure, by the use of directional freezing. Different methodologies were tested, the optimal one being freezing of a collagen suspension inside-out, using a centrally positioned liquid nitrogen-cooled tube. Pore sizes could be varied by the use of different tube materials. Use of aluminium tubes resulted in radial scaffolds with a pore size of 20-30 μm, whereas use of stainless steel produced radial scaffolds with 70-100 μm pore sizes. Brass- and copper-based tubes produced scaffolds with less homogeneous radial pores, pore sizes being 90-100 and 50-80 μm, respectively. Fibreglass tubes gave even less uniformity (pore size 100-150 μm). Scaffolds were free of cracks, except in case of aluminium. Scaffolds with a radial inner structure may be especially suitable for tissue engineering of organs with a radial scaffold structure, such as the diaphragm.     

In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells,employing a two‐chambered co‐culture well design

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk–RADA peptide scaffold in a specially designed two‐chambered co‐culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two‐chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co‐culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage‐like and subchondral bone‐like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single‐step approach that highlights the feasibility of rabbit BMSCs as a single‐cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage

The repair of articular cartilage lesions remains a clinical problem. Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns. We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage. Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls. Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo. Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen. IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity. The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage. This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone.     

Cell yield,chondrogenic potential,and regenerated cartilage type of chondrocytes derived from ear,nasoseptal, and costal cartilage

Functional reconstruction of large cartilage defects in subcutaneous sites remains clinically challenging because of limited donor cartilage. Tissue engineering is a promising and widely accepted strategy for cartilage regeneration. To date, however, this strategy has not achieved a significant breakthrough in clinical translation owing to a lack of detailed preclinical data on cell yield and functionality of clinically applicable chondrocytes. To address this issue, the current study investigated the initial cell yield, proliferative potential, chondrogenic capacity, and regenerated cartilage type of human chondrocytes derived from auricular, nasoseptal, and costal cartilage using a scaffold‐free cartilage regeneration model (cartilage sheet). Chondrocytes from all sources exhibited high sensitivity to basic fibroblast growth factor within 8 passages. Nasoseptal chondrocytes presented the strongest proliferation rate, whereas auricular chondrocytes obtained the highest total cell amount using comparable cartilage sample weights. Importantly, all chondrocytes at fifth passage showed strong chondrogenic capacity both in vitro and in the subcutaneous environment of nude mice. Although some significant differences in histological structure, cartilage matrix content and cartilage type specific proteins were observed between the in vitro engineered cartilage and original tissue; the in vivo regenerated cartilage showed mature cartilage features with high similarity to their original native tissue, except for minor matrix changes influenced by the in vivo environment. The current study provides detailed preclinical data for choice of chondrocyte source and thus promotes the clinical translation of cartilage regeneration approach.     

胶原/羟基磷灰石复合支架负载软骨细胞构建组织工程软骨

背景:新型复合软骨组织工程支架克服了传统支架的诸多不足,如组织相容性差、生物力学性能不足、降解过快、材料单一、难与关节软骨的分层结构相匹配等,为软骨组织工程的发展提供新的途径。目的:观察具有柱状分层结构的胶原/羟基磷灰石复合支架对软骨细胞的吸附作用,以及对软骨细胞生物学性状的影响,评价其作为软骨组织工程支架的可行性与价值。设计:开放性实验。单位:中山大学附属第三医院骨科,华南理工大学材料学院。材料:实验于2004-06/11在中山大学附属第三医院动物实验中心完成。选取2周龄普通级健康新西兰白兔1只,雄性,饲养环境温度20℃,湿度40%。方法:①在羟基磷灰石中加入少量的去离子水,分散后加入I型胶原溶液搅拌复合,并按一定比例加入碳二亚氨,调配形成不同比例的胶原/羟基磷灰石复合物,并逐层加入模具,最上层采用纯胶原,底层为纯羟基磷灰石。将制备好的柱状分层的胶原/羟基磷灰石复合材料冷冻干燥后,经环氧乙烷气体消毒后备用。②体外培养幼兔关节软骨细胞并扩增,吸附于多孔胶原/羟基磷灰石复合支架上三维立体培养,通过相差倒置显微镜、组织学、扫描电镜及免疫组织化学检测支架对软骨细胞的表型、增殖及功能的影响。主要观察指标:①胶原/羟基磷灰石多孔支架的形貌观察。②体外培养的软骨细胞生物学性状观察。③胶原/羟基磷灰石多孔支架材料与软骨细胞相容性的观察。结果:①胶原/羟基磷灰石多孔支架的形貌观察:胶原/羟基磷灰石为具有一定力学强度的柱状分层的三维多孔支架,最上层为纯胶原,底层为纯羟基磷灰石,中间为胶原与羟基磷灰石复合。扫描电镜可见支架内具有不规则的通孔结构,孔径较均匀,相互连通,平均孔径约为147μm。②体外培养的软骨细胞生物学性状观察:刚分离的软骨细胞为球形,活细胞率达95%。24h后细胞开始贴壁,逐渐伸展为三角或多角形,内含分泌颗粒。细胞保持单层生长,传代后增殖速度加快,4d铺满培养瓶。随着传代次数的增加,增殖速度开始减慢,当传到第6代时,细胞分裂能力明显下降,细胞变形明显,体积变大,梭形为主,边沿模糊,折光性弱,表现出细胞老化和向成纤维细胞反分化的趋势。③胶原/羟基磷灰石多孔支架材料与软骨细胞相容性的观察:多孔胶原/羟基磷灰石复合支架亲水性好,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,分泌胞外基质。结论:柱状分层结构的胶原/羟基磷灰石复合支架具有良好的细胞相容性,较单纯胶原力学性能更强,是比较理想的软骨组织工程支架材料。     

Macromolecular crowding as a means to assess the effectiveness of chondrogenic media

Chondrocyte‐based tissue engineering requires in vitro cell expansion, which is associated with phenotypic losses, decrease in Collagen Type II synthesis and increase in Collagen Type I synthesis. Another major obstacle in clinical translation of chondrocyte‐based therapies is the lack of extracellular matrix (ECM) in the engineered cartilage substitutes. Various research and commercially available media claim that they can maintain chondrogenic phenotype, whereas macromolecular crowding (MMC) has been shown to increase tissue‐specific ECM deposition and maintain cell phenotype in vitro. Herein, we hypothesised that the combination of chondrogenic media with MMC will enable chondrogenic phenotype maintenance during in vitro expansion and increase cartilage‐specific ECM deposition, enabling that way the development of a tissue‐engineered cartilage substitute. Immunocytochemistry analysis of Passage 3 human chondrocytes in normal media in monolayer revealed that MMC significantly increased Collagen Type I deposition, whereas no statistical difference was observed in Collagen Type II deposition. When Passage 3 human chondrocytes were cultured in normal media and alginate beads, immunocytochemistry analysis revealed that MMC increased, albeit not significantly, both Collagen Type I and Collagen Type II deposition. Subsequently, human chondrocytes were expanded up to Passage 6 in either fetal bovine serum or human serum and redifferentiated using commercially available chondrogenic media in either monolayer or alginate beads. Immunocytochemistry analysis revealed that MMC, independently of the serum used, significantly increased Collagen Type I deposition in human‐redifferentiated monolayer and alginate bead chondrocyte cultures, whereas almost no Collagen Type II was detected. These data clearly illustrate that an optimal chondrogenic medium is still elusive.     

同种异体脱钙骨与骨髓间充质干细胞关节腔内共培养:与同腔软骨性状的对比简

背景：临床发现膝关节内游离体能长期存在于关节腔内并能保持一定的软骨组织学特性和生理学特性,因此大胆提出假设：关节腔环境可能是软骨细胞生长、发育的较佳环境并提出“腔内培养,腔内移植”的理念。目的：观察兔骨髓间充质干细胞复合同种异体脱钙骨基质体外培养或关节腔内培养组织工程软骨与同腔软骨的性状差异。 方法：实验分3组进行,体外培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙骨基质体外复合培养;腔内培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙骨基质以筋膜包裹,复合培养于成年新西兰兔膝关节腔内,以同腔内正常软骨为对照。 结果与结论：培养12周后：①体外培养组苏木精-伊红染色见软骨细胞少量增生,胞核蓝染;甲苯胺蓝染色见软骨细胞排列无序,少量周围基质包绕;Masson染色阳性区域小,细胞排列无序;Ⅱ型胶原免疫组织化学见软骨细胞胞浆及胞外基质少量黄色颗粒。②腔内培养组苏木精-伊红染色见软骨细胞增生,胞核蓝染;甲苯胺蓝染色见软骨细胞成串排列,软骨陷窝形成,周围基质包绕;Masson染色阳性,软骨细胞多,基质蓝染,按一定应力方向排列;Ⅱ型胶原免疫组织化学见细胞外基质中出现较多棕黄色颗粒,Ⅱ型胶原染色阳性。说明骨髓间充质干细胞与同种异体脱钙骨基质复合物可在体外及膝关节腔内培养出组织工程软骨,关节腔内培养的软骨比体外培养的软骨更接近正常软骨。     

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.     

Scaffold‐free 3D cellulose acetate membrane‐based cultures form large cartilaginous constructs

Scaffold‐free three‐dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold‐free 3D membrane‐based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane‐based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin‐O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α₁ (COL2A1). Membrane‐based cultures (P1) formed flexible disc‐like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin‐O and collagen type II staining was found in membrane‐based and pellet cultures at P1–3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane‐based cultures at P1. After 21 days the membrane‐based cultures did not express COL2A1. We concluded that membrane‐based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane‐based cultures provided the highest COL2A1 expression and a large, smooth and cartilage‐like surface. As these are appropriate features for clinical applications, we assume that membrane‐based cultures might be of use in cartilage regeneration if they displayed similar results in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损

背景:脐带Wharton胶富含透明质酸,糖胺多糖及胶原等,成分与天然软骨细胞外基质类似,因此由人脐带提取的Wharton胶很可能是一种较为理想的软骨组织工程支架材料。目的:评价自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损的效果。方法:将终浓度为1010L-1、成软骨方向诱导后的兔自体脂肪间充质干细胞与人脐带Wharton胶支架复合,继续培养1周构建组织工程软骨,对兔膝关节全层软骨缺损进行修复(实验组),并与单纯支架修复的对照组及空白组进行比较。术后3个月对修复组织行大体观察、组织学检测、糖胺多糖、总胶原定量检测及生物力学测定。结果与结论:实验组的缺损多为透明软骨修复,对照组以纤维组织修复为主,空白组无明显组织修复。提示脂肪间充质干细胞作为软骨组织工程种子细胞具有可行性;实验构建的组织工程软骨能有效的修复关节软骨缺损,人脐带Wharton胶可作为软骨组织工程良好的支架材料。     

State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective

Scaffold-based bone tissue engineering aims to repair/regenerate bone defects. Such a treatment concept involves seeding autologous osteogenic cells throughout a biodegradable scaffold to create a scaffold-cell hybrid that may be called a tissue-engineered construct (TEC). A variety of materials and scaffolding fabrication techniques for bone tissue engineering have been investigated over the past two decades. This review aims to discuss the advances in bone engineering from a scaffold material point of view. In the first part the reader is introduced to the basic principles of bone engineering. The important properties of the biomaterials and the scaffold design in the making of tissue engineered bone constructs are discussed in detail, with special emphasis placed on the new material developments, namely composites made of synthetic polymers and calcium phosphates. Advantages and limitations of these materials are analysed along with various architectural parameters of scaffolds important for bone tissue engineering, e.g. porosity, pore size, interconnectivity and pore-wall microstructures.