人类软骨糖蛋白39诱导关节软骨祖母细胞的成软骨分化

背景：有研究表明人类软骨糖蛋白39与骨关节软骨的退变与修复具有一定的关系,但其具体的作用机制并不十分明确。 目的：观察人类软骨糖蛋白39对成人膝关节软骨祖母细胞成软骨诱导分化的影响。 方法：取成人关节软骨,消化分离培养关节软骨祖母细胞;流式细胞仪检测传代细胞中能够表达CD105、CD166的细胞量并进行分离提纯。将分离的软骨祖母细胞采用单层培养法培养,传代培养至第2代后向分离培养所得的软骨祖母细胞中,分别经过含人类软骨糖蛋白39成软骨培养基及普通成软骨诱导培养基的诱导培养14 d后,通过免疫组织化学染色观察经诱导后细胞中Ⅱ型胶原的表达及通过大体组织学观察评估软骨的形成。 结果与结论：关节软骨组织中可以分离出能够表达CD105、CD166的关节软骨祖母细胞,软骨祖母细胞经过诱导分化后逐渐聚集并形成结节,经诱导后Ⅱ型胶原免疫细胞化学着色阳性,且经人类软骨糖蛋白39诱导细胞形成的结节更大,Ⅱ型胶原表达更多。结果表明,成人关节软骨中能够分离培养出具有成软骨分化能力的干细胞系细胞即软骨祖母细胞,且能够被定向诱导分化为软骨细胞,人类软骨糖蛋白39对其分化过程具有一定的促进作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Ⅱ型胶原酶消化法培养兔关节软骨细胞

背景：目前关节软骨细胞的分离培养技术已经比较成熟,但是研究中发现通过目前技术培养的软骨细胞生长周期慢,容易出现退变现象,不利于后续试验的进行。 目的：改进并探讨4周龄新西兰大白兔膝关节软骨细胞的分离与培养的方法。 方法：无菌条件下取4周龄新西兰大白兔双侧膝关节软骨,采用Ⅱ型胶原酶消化并机械吹打的方法,分离关节软骨细胞并进行原代、传代培养;采用形态学观察,甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学方法对关节软骨细胞进行鉴定;MTT法检测关节软骨细胞增殖情况。 结果与结论：倒置显微镜下见膝关节软骨分离的原代软骨细胞6 h后开始贴壁,72 h可形成单层,96 h即可传代;前3代软骨细胞表型稳定,增殖力良好;第4,5代软骨细胞增殖能力减弱,绝大部分细胞变为长梭形和不规则形状。甲苯胺蓝染色显示培养的软骨细胞细胞质染成浅蓝色,细胞核染成深蓝色;免疫组织化学显示软骨细胞Ⅱ型胶原呈黄褐色阳性表达;MTT法检测表明前3代软骨细胞增殖差异无显著性意义(P > 0.05),且第1-3代与4代软骨细胞在培养第4-7天时,吸光度值差异有显著性意义(P < 0.05),与第5代软骨细胞在培养1-7天时,吸光度值差异有显著性意义(P < 0.05)。提示采用Ⅱ型胶原酶消化并机械吹打的方法能够获得大量生长速度快且不宜退变的新西兰大白兔膝关节软骨细胞,且培养的前3代新西兰兔膝关节软骨细胞最为适宜。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

新生兔气管软骨细胞的生物学特性

 目的：探讨体外分离培养的新生兔气管软骨细胞的生物学特性。方法：通过酶消化法体外分离培养新生兔气管软骨细胞;倒置显微镜观察软骨细胞形态及生长状况;电镜观察软骨细胞超微结构;运用real-time PCR、免疫细胞化学染色和甲苯胺蓝染色检测气管软骨细胞分泌的细胞外基质成分。结果：体外分离、培养的兔气管软骨细胞呈短小三角形或不规则形贴壁生长。超微结构显示细胞较多突起,孔隙较多,胞质丰富,细胞器发达,细胞内可见大量蛋白分泌物。软骨细胞表达I、II型胶原、蛋白聚糖等,以II型胶原和蛋白聚糖表达为主。免疫细胞化学染色II型胶原和SOX9阳性,I型胶原弱阳性。甲苯胺蓝染色阳性。结论：适宜的酶消化单层培养法获得的新生兔气管软骨细胞具有分泌软骨细胞外基质成分的特性,可初步为体外构建组织工程气管治疗新生兔气管狭窄的实验研究提供种子细胞。     

不同代次兔髓核细胞的形态学特征和生物学性状

背景：椎间盘退变是个慢性、复杂的过程,然而椎间盘退变其发生机制尚未完全阐明,很难自行修复。近年来研究细胞移植治疗椎间盘退行性变尚处在实验室阶段。研究髓核细胞的生物学性状可为研究椎间盘退变机制、组织工程构建椎间盘、基因治疗等提供理论依据。 目的：研究兔不同代次髓核细胞的生物学特性,旨在找出合适的种子细胞去治疗椎间盘退变性疾病。 方法：从新西兰大耳白兔椎间盘髓核组织中,分离并培养髓核细胞同时进行培养传代,对原代及第3,4代髓核细胞进行苏木精-伊红染色观察细胞形态学变化;甲苯胺蓝染色和免疫细胞化学法检测髓核细胞内聚集蛋白聚糖和Ⅱ型胶原的表达;反转录PCR法测定Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平,观察各代髓核细胞生物学特性的变化。 结果与结论：兔椎间盘髓核细胞可以在体外培养并进行传代,原代髓核细胞一般需7 d左右贴壁,形状呈类圆形或多角形,原代和第3代髓核细胞都呈圆形或多角形,活力较强,苏木精-伊红染色后细胞核被染成均一蓝黑色,胞浆呈现淡粉色;髓核细胞经过甲苯胺蓝染色后,胞浆内呈现天蓝色,通过Ⅱ型胶原免疫组织化学染色后,胞浆内表现为黄褐色沉淀。到第4代细胞出现退变,Ⅱ型胶原和聚合蛋白聚糖mRNA的表达水平较前几代细胞显著下降。前3代的髓核细胞代谢旺盛,表型一致,聚集蛋白聚糖和Ⅱ型胶原表达正常,传第4代后髓核细胞开始出现衰老、退变。     

骨髓间充质干细胞向软骨细胞的分化

背景：以骨髓间充质干细胞构建组织工程气管尚缺乏理想的特异性表面标志物,对其鉴定主要依赖细胞形态学、细胞表型及诱导分化的功能进行分析。 目的：体外分离培养、鉴定兔骨髓间充质干细胞,观察在特定条件下向气管软骨细胞分化的潜能。 方法：无菌环境取兔骨髓,经全骨髓贴壁筛选法分离培养细胞至第2代,流式细胞术鉴定第1、第2代细胞表面抗原CD44、CD45的表达。无菌环境取气管,经酶消化法分离培养气管软骨细胞,甲苯胺蓝染色鉴定软骨细胞蛋白聚糖的合成。在使用转化生长因子β1的基础上,将骨髓间充质干细胞与气管软骨细胞通过Transwell小室非接触式共培养,倒置显微镜观察细胞形态,甲苯胺蓝染色鉴定蛋白聚糖的合成,荧光实时定量PCR鉴定Ⅱ型胶原和蛋白聚糖 mRNA的表达。 结果与结论：分离、培养的细胞呈长梭形、不规则形聚集生长,传代后细胞生长速度明显增快,呈鱼群状聚集生长。第1代有96.97%的细胞表达CD44、13.72%的细胞表达CD45,第2代有99.11%的细胞表达CD44、8.54%的细胞表达CD45。气管软骨细胞甲苯胺蓝染色阳性。在诱导后,骨髓间充质干细胞形态逐渐由长梭形变为三角形或不规则形,表达软骨细胞特异性Ⅱ型胶原和蛋白聚糖 mRNA基因,甲苯胺蓝染色示阳性。结果表明全骨髓贴壁筛选法可成功分离培养骨髓间充质干细胞,第2代纯度较高,且在特定诱导条件下具有分化为气管软骨细胞的潜能。     

鹿茸多肽诱导骨髓间充质干细胞体外向软骨表型的分化

背景：实验证实鹿茸多肽可以促进体外培养软骨细胞的增殖和细胞外基质糖胺多糖、Ⅱ型胶原、Aggrecan蛋白的表达。 目的：通过对体外培养的兔骨髓间充质干细胞在特定培养液作用下向软骨细胞表型分化的研究,探讨鹿茸多肽对其软骨分化的影响。 方法：将第3代兔骨髓间充质干细胞随机分为空白对照组、诱导组、鹿茸多肽组,分别采用普通培养液、诱导培养液、含10 mg/L鹿茸多肽的诱导培养液于离心管内进行培养;并取兔的关节软骨细胞作为关节软骨组。分别于1,2,3周后取材,通过组织学、生物化学和RT-PCR技术,对离心管内构建的软骨组织进行形态学和细胞功能状态的观察。 结果与结论：空白对照组培养2周后,细胞团块逐渐崩解,无法进行苏木精-伊红染色。诱导组、鹿茸多肽组细胞团块除有轻度收缩外,呈白色半透明状;苏木精-伊红染色发现部分细胞为圆形或卵圆形,表层细胞密度大;诱导组、鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达随培养时间延长而增多,各时间点诱导组、鹿茸多肽组含量均高于空白对照组(P < 0.05);各时间点鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达均高于诱导组,但低于关节软骨组 (P< 0.05)。提示骨髓间充质干细胞在特定培养条件下能向软骨细胞表型分化,且鹿茸多肽对其定向软骨分化有明显促进作用。虽然在体外可以构建出软骨组织,但其与关节软骨质量相比仍有很大差距。     

TGF-β1诱导分化的兔BMSCs复合左旋聚乳酸\β-磷酸三钙多孔支架材料体外研究实验

目的 研究体外培养rBMSCs经TGF-β1诱导分化的软骨细胞复合左旋聚乳酸\β-磷酸三钙(PLLA\β-TCP)多孔支架材料体外构建仿生人工软骨. 方法 低温挤出成形法制备成PLLA\β-TCP复合多孔支架材料,体外分离、培养rBMSCs至第3代,利用含有TGF-β1特殊诱导系统诱导其向软骨细胞分化,诱导14d后用甲苯胺蓝染色及Ⅱ型胶原免疫组化进行鉴定后与PLLA\β-TCP多孔支架材料体外复合培养,并取第7、14、21d细胞复合材料进行电镜扫描观察细胞贴附、生长、增殖状况,同时消化收集贴附支架第7、14、21d的细胞,行RT-PCR检测分化软骨细胞相关基因aggrecan、Co12A1在mRNA水平的表达,Western-bolt检测Ⅱ型胶原蛋白的分泌情况.结果 rBMSCs经诱导后向软骨细胞分化,甲苯胺蓝染色见分化软骨细胞分泌糖胺聚糖(glycosaminoglycan,GAG),Ⅱ型胶原免疫组织化学染色呈阳性;电镜扫描见分化细胞在支架材料分布均匀,黏附良好;RT-PCR及Westem-bolt检测示7、14、21daggrecan、Co12A1在mRNA水平、Ⅱ型胶原蛋白均有不同程度表达. 结论 利用含有TGF-β1特殊诱导系统诱导rBMSCs分化的软骨细胞复合到PLLA\β-TCP多孔支架材料上,细胞生长良好,并能正常分泌软骨细胞特异细胞外基质,体外成功构建了组织工程软骨.     

胰岛素样生长因子1促进创伤性关节炎软骨细胞的体外增殖

背景：早期受损的软骨细胞在体外培养时容易产生去分化,表型不稳,常需添加一定的生长因子。 目的：观察胰岛素样生长因子1对成年兔创伤性关节炎早期关节软骨细胞体外增殖的促进作用。 方法：采用改良Hulth法制备成兔创伤性关节炎模型,造模成功后无菌条件下片状切取股骨远端及胫骨近端,用消化培养法培养软骨细胞。将软骨细胞随机分为2组,对照组加入含体积分数10%胎牛血清因子的DMEM培养液培养;实验组在对照组的基础上加入100 μg/L的胰岛素样生长因子1。通过细胞形态学、细胞计数、细胞活性检测胰岛素样生长因子1对创伤性关节炎关节软骨细胞增殖的影响。 结果与结论：成功培养出早期创伤性关节炎兔软骨细胞,细胞多数为小细胞,形态有小梭形、小圆形及小多边形。苏木精-伊红染色显示实验组细胞数量多于对照组,MTT实验证实实验组细胞的吸光度值大于对照组   (P < 0.01)。结果提示,胰岛素样生长因子1能促进早期创伤性关节炎模型兔软骨细胞的体外增殖。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

同种异体血清体外培养兔膝关节软骨细胞的增殖*◆

背景：兔关节软骨细胞体外单层培养采用胎牛血清培养基易去分化,有必要寻找合适培养基来提高兔关节软骨细胞培养质量。 目的：观察同种异体兔血清对体外培养的兔膝关节软骨细胞增殖能力的影响。 方法：以0.4%链霉蛋白酶和0.025%Ⅱ型胶原酶分离成年兔膝关节软骨细胞,将获得的软骨细胞随机分为实验组和对照组。实验组以体积分数10%异体兔血清+DMEM/F12培养;对照组以体积分数10%胎牛血清+DMEM/F12培养,传代培养至4代。 结果与结论：实验组前4代软骨细胞增殖较对照组慢,但软骨细胞形态未发生明显改变而对照组软骨细胞出现去分化现象。提示体积分数10%异体兔血清培养利于维持软骨细胞增殖和形态的稳定,是较好的获取大量优良软骨细胞的体外培养方式。      

自体激活富血小板血浆干预兔骨髓间充质干细胞体外成软骨分化的研究

背景：自体富血小板血浆激活后可释放多种生长因子,可以促进骨髓间充质干细胞的增殖与分化。 目的：观察自体激活富血小板血浆对体外培养的兔骨髓间充质干细胞向成软骨细胞分化的影响。 方法：取兔股骨骨髓,全骨髓贴壁法分离培养骨髓间充质干细胞;取第3代骨髓间充质干细胞,分别应用体积分数10%自体激活富血小板血浆和体积分数10%胎牛血清培养液进行体外培养,观察其向成软骨细胞分化情况。 结果与结论：分离培养的兔骨髓间充质干细胞呈长梭形,传代后细胞生长迅速。流式细胞仪检测发现第3代细胞高表达CD29、CD44,而低表达CD45。免疫荧光细胞化学染色显示经自体激活富血小板血浆诱导的骨髓间充质干细胞表达Ⅱ型胶原;实时荧光定量PCR检测发现经自体激活富血小板血浆诱导的骨髓间充质干细胞Ⅱ型胶原α1链基因和聚集蛋白聚糖基因表达明显高于经胎牛血清诱导的骨髓间充质干细胞(P < 0.01)。可见自体激活富血小板血浆具有促进兔骨髓间充质干细胞向软骨细胞方向分化的潜能。     

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.     

Rabbit articular chondrocytes seeded on collagen-chitosan-GAG scaffold for cartilage tissue engineering in vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.     

膝关节腔内培养骨髓间充质干细胞复合脱钙骨的组织工程软骨

背景：如何更好地以组织工程学方法修复关节软骨缺损并达到良好的远期疗效目前尚无公识。 目的：创新性地在膝关节腔内培养兔骨髓间充质干细胞复合同种异体脱钙骨的组织工程软骨。  方法：采用全骨髓贴壁筛选法分离培养兔骨髓间充质干细胞,DMEM/F12完全培养基培养,成软骨诱导条件培养基诱导分化。取同种异体兔的髂骨和椎体骨制作成脱钙骨支架,诱导后的骨髓间充质干细胞种植于脱钙骨支架上,培养1 d后将细胞-支架复合物用筋膜包裹置于兔左膝关节腔内培养,单纯脱钙骨支架筋膜包裹置入右膝关节腔。于培养第4,8,12周分别取材,行大体观察并制成石蜡切片,采用苏木精-伊红染色、甲苯胺蓝染色,Ⅱ型胶原免疫组化染色方法进行组织学观察。 结果与结论：培养4,8周,细胞-支架组标本Ⅱ型胶原免疫组化的平均吸光度值(A)分别为0.263±0.031,0.340±0.052,单纯支架组标本分别为0.147±0.027,0.165±0.030,两组比较差异有显著性意义(P < 0.05);培养12周细胞-支架组标本Ⅱ型胶原免疫组化A值平均为0.362±0.037,标本类似正常软骨外观,Ⅱ型胶原免疫组化反应呈阳性;而单纯支架组脱钙骨支架降解。培养12周细胞-支架组苏木精-伊红染色结果显示细胞数量多,脱钙骨支架基本被吸收;而甲苯胺蓝染色结果显示有被染成紫红色的异染性基质形成。结果提示兔骨髓间充质干细胞复合同种异体脱钙骨可在兔膝关节腔内培养出组织工程软骨。     

Differentiation Capacity of Human Chondrocytes Embedded in Alginate Matrix

Healing capacity of cartilage is low. Thus, cartilage defects do not regenerate as hyaline but mostly as fibrous cartilage which is a major drawback since this tissue is not well adapted to the mechanical loading within the joint. During in vitro cultivation in monolayers, chondrocytes proliferate and de-differentiate to fibroblasts. In three-dimensional cell cultures, de-differentiated chondrocytes could re-differentiate toward the chondrogenic lineage and re-express the chondrogenic phenotype. The objective of this study was to characterize the mesenchymal stem cell (MSC) potential of human chondrocytes isolated from articular cartilage. Furthermore, the differentiation capacity of human chondrocytes in three-dimensional cell cultures was analyzed to target differentiation direction into hyaline cartilage. After isolation and cultivation of chondrogenic cells, the expression of the MSC-associated markers: cluster of differentiation (CD)166, CD44, CD105, and CD29 was performed by flow cytometry. The differentiation capacity of human chondrocytes was analyzed in alginate matrix cultured in Dulbecco’s modified eagle medium with (chondrogenic stimulation) and without (control) chondrogenic growth factors. Additionally, the expression of collagen type II, aggrecan, and glycosaminoglycans was determined. Cultivated chondrocytes showed an enhanced expression of the MSC-associated markers with increasing passages. After chondrogenic stimulation in alginate matrix, the chondrocytes revealed a significant increase of cell number compared with unstimulated cells. Further, a higher synthesis rate of glycosaminoglycans and a positive collagen type II and aggrecan immunostaining was detected in stimulated alginate beads. Human chondrocytes showed plasticity whilst cells were encapsulated in alginate and stimulated by growth factors. Stimulated cells demonstrated characteristics of chondrogenic re-differentiation due to collagen type II and aggrecan synthesis.     

Chondrocyte phenotypes on different extracellular matrix monolayers

Chondrocytes undergo a process of dedifferentiation in monolayer culture that is characterized by a transition to a fibroblast-like phenotype. This behavioral change poses a challenge for tissue-engineered cartilage constructs, as approaches using autologous cells require expansion in vitro. Because chondrocytes express a variety of integrin receptors specific to different adhesive proteins, we hypothesized that chondrocytes expanded on various underlying protein monolayers would have different phenotypic responses. Bovine articular chondrocytes were cultured for up to 2 weeks on tissue culture plastic, fibronectin, collagen type I or collagen type II substrate in the presence or absence of ascorbate. Contrary to our hypothesis, the extracellular matrix protein substrates used in this study did not significantly alter the changes in chondrocyte morphology, gene expression, matrix formation, or cytoskeletal organization. Cells on all substrates assembled equivalent matrices, which may have subsequently regulated cell behavior. In cultures with ascorbate, populations of round and spread cells emerged after 1 week, with round cells expressing collagen type II and the differentiated phenotype and spread cells dedifferentiating. In cultures without ascorbate, chondrocytes rapidly adhered and spread onto organized fibronectin matrices via the ₅β₁ integrin, which has been associated with survival and proliferation of chondrocytes in vitro. These findings indicate that expanding chondrocytes on protein monolayers may not be an effective solution to preventing dedifferentiation and improving autologous chondrocyte transplantation.     

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：     

Engineering cartilage with human nasal chondrocytes and a silanized hydroxypropyl methylcellulose hydrogel

Tissue engineering strategies, based on developing three-dimensional scaffolds capable of transferring autologous chondrogenic cells, holds promise for the restoration of damaged cartilage. In this study, the authors aimed at determining whether a recently developed silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel can be a suitable scaffold for human nasal chondrocytes (HNC)-based cartilage engineering. Methyltetrazolium salt assay and cell counting experiments first revealed that Si-HPMC enabled the proliferation of HNC. Cell tracker green staining further demonstrated that HNC were able to form nodular structures in this three-dimensional scaffold. HNC phenotype was then assessed by RT-PCR analysis of type II collagen and aggrecan expression as well as alcian blue staining of extracellular matrix. Our data indicated that Si-HPMC allowed the maintenance and the recovery of a chondrocytic phenotype. The ability of constructs HNC/Si-HPMC to form a cartilaginous tissue in vivo was finally investigated after 3 weeks of implantation in subcutaneous pockets of nude mice. Histological examination of the engineered constructs revealed the formation of a cartilage-like tissue with an extracellular matrix containing glycosaminoglycans and type II collagen. The whole of these results demonstrate that Si-HPMC hydrogel associated to HNC is a convenient approach for cartilage tissue engineering.     

Chondrogenesis of aged human articular cartilage in a scaffold-free bioreactor

Chondrogenesis of aged human articular chondrocytes was evaluated under controlled in vitro conditions, using a rotating bioreactor vessel. Articular chondrocytes isolated from 10 aged patients (median age, 84 years) were increased in monolayer culture. A single-cell suspension of dedifferentiated chondrocytes was inoculated in a rotating wall vessel, without the use of any scaffold or supporting gel material. After 90 days of cultivation, a three-dimensional cartilage-like tissue was formed, encapsulated by fibrous tissue resembling a perichondrial membrane. Morphological examination revealed differentiated chondrocytes ordered in clusters within a continuous dense cartilaginous matrix demonstrating a strong positive staining with monoclonal antibodies against collagen type II and articular proteoglycan. The surrounding fibrous membrane consisted of fibroblast-like cells, and showed a clear distinction from the cartilaginous areas when stained against collagen type I. Transmission electron microscopy revealed differentiated and highly metabolically active chondrocytes, producing an extracellular matrix consisting of a fine network of randomly distributed cross-banded collagen fibrils. Chondrogenesis of aged human articular chondrocytes can be induced in vitro in a rotating bioreactor vessel using low shear and efficient mass transfer. Moreover, the tissue-engineered constructs may be used for further in vitro studies of differentiation, aging, and regeneration of human articular cartilage.     

Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using "bead-in-bead" polysaccharide capsules

Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.     

组织工程软骨与柚皮苷联合修复兔膝关节软骨缺损的组织学证实

背景：目前临床上虽有多种方法用于治疗软骨缺损,但没有从根本上解决关节软骨缺损修复问题。 目的：通过组织学研究进一步评价柚皮苷结合组织工程软骨修复兔关节软骨缺损的效果。 方法：取兔骨髓间充质干细胞体外增殖后,复合于改建后的脱细胞真皮基质载体上,制成组织工程软骨,植入到兔膝关节软骨缺损,并以柚皮苷汤灌胃,于 4,8周后分别对修复组织进行苏木精-伊红、Masson三色染色、甲苯胺蓝染色、Ⅱ型胶原染色、Ⅹ型胶原染色等组织学检查。 结果与结论：术后8周, 柚皮苷结合干细胞复合体组缺损处修复组织变成乳白色,半透明光滑组织,缺损修复组织与周围正常软骨已基本难区分,表面光滑。组织学检查发现修复缺损处基本为新生软骨填充。结果证实,柚皮苷结合组织工程软骨能提高家兔膝关节软骨缺损的修复质量。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：