海藻酸钠复合载体长期培养软骨细胞的生物学稳定性

背景：近年研究发现在海藻酸钠三维培养体系中软骨细胞存活时间延长,细胞外基质分泌旺盛。　 目的：观察海藻酸钠复合载体对体外长期培养软骨细胞生物学稳定性的影响。 方法：将兔软骨细胞接种至以海藻酸钠、透明质酸、壳聚糖、纤维连接蛋白、碱性成纤维细胞生长因子为主要成分的复合载体三维培养体系中,以平面培养作为对照。定期观察在两种不同培养体系中软骨细胞形态学改变,细胞生长曲线差异,细胞上清液糖胺多糖含量差异。 结果与结论：软骨细胞在海藻酸钠复合载体中生长良好,生长旺盛,并形成球状细胞团,细胞分裂增殖活跃;培养2 d时复合载体培养软骨细胞增殖稍高于平面培养软骨细胞;培养4 d时可见复合载体培养软骨细胞增殖明显加快;培养6~14 d时复合载体培养的仍保持较稳定增殖,而平面培养的软骨细胞增殖逐渐降低;复合载体细胞外液糖胺多糖含量明显高于平面培养软骨细胞。说明海藻酸钠复合载体可以长期培养软骨细胞并保持其生物学稳定性。     

SIS重塑为海绵状后对BMSCs诱导的软骨细胞增殖分化的影响

目的观察大鼠骨髓干细胞(BMSCs)诱导分化的软骨细胞,在具有三维孔隙结构的海绵状猪小肠粘膜下层(SIS)表面的生长情况,探讨SIS由薄膜状重塑为海绵状后对软骨样细胞增殖分化的作用,为软骨组织工程提供新型的天然生物衍生材料。方法原代培养SD大鼠BMSCs,流式术检测细胞表面抗原表达;诱导BMSCs分化为软骨细胞,甲苯胺蓝染色鉴定。按Abraham法将猪近段空肠制备成脱细胞的SIS,再将薄膜状的SIS经液氮低温研磨制成微粒,交联后采用冷冻干燥技术重塑形为具有三维孔隙结构的海绵状SIS。将软骨细胞与海绵状SIS共培养:扫描电镜观察细胞生长状态;Western blotting检测共培养组织ColⅡ的表达;DMMB法测量葡萄糖胺聚糖(GAG)表达量。应用材料浸提液培养软骨细胞,相差显微镜观察细胞形态,MTT法检测细胞的增殖。结果原代培养的BMSCs表达干细胞相关抗原,并可分化为软骨细胞,甲苯胺蓝将糖胺多糖染成蓝色。脱细胞的SIS未见有核物质,海绵状的SIS具有大量较均匀的三维孔隙。软骨细胞能在材料孔隙内良好生长,软骨分化指标ColⅡ与GAG表达量明显增加;浸提液培养的软骨细胞增殖能力强。结论重塑形后具有三维孔隙结构的海绵状SIS促进BMSCs来源的软骨细胞增殖和分化,为软骨组织工程提供了新型的天然生物衍生材料。     

三维培养诱导人脂肪间充质干细胞微球的成软骨分化能力

背景：关节软骨损伤后修复结果不满意,需要新的手段,而脂肪间充质干细胞较适宜做种子细胞诱导软骨,然而怎么能够使诱导的软骨具有功能需要研究。 目的：采用三维培养体系诱导人脂肪间充质干细胞微球向软骨分化。 方法：无菌切取吸脂术后脂肪组织,分离培养人脂肪间充质干细胞,传至第3代进行流式细胞术分析,成骨成脂肪诱导等鉴定,同时也给予合适的培养条件用三维培养的方式向软骨细胞诱导,并行阿利辛蓝染色鉴定糖胺多糖的合成,苏木精-伊红染色进行组织学分析,免疫荧光检测Ⅱ型胶原表达,称质量测量软骨硬度。 结果与结论：分离的人脂肪间充质干细胞CD105,CD44,CD29均高表达,而 CD45,CD34低表达,并且成骨成脂诱导后细胞茜素红染色和油红O染色均为阳性。三维培养法诱导的软骨细胞可表达大量糖胺多糖及Ⅱ型胶原。结果证实,三维培养法诱导人脂肪间充质细胞向软骨分化后,具有软骨细胞的特性。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：     

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。     

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.     

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1010 L -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。     

以壳聚糖-胶原共混膜为三维支架同种异体工程化软骨的构建

目的探讨以壳聚糖-胶原共混膜为三维支架材料的同种异体软骨细胞构建组织工程化软骨的能力。方法将分离、培养、扩传兔软骨细胞，接种在壳聚糖-胶原共混膜上，倒置显微镜下观察细胞在共混膜上的生长情况。体外培养7d后，将细胞-材料复合物种植在新西兰兔皮下，6周取材，对获得的同种异体工程化软骨进行组织学评价。结果兔软骨细胞接种于壳聚糖-胶原共混膜上4h后有贴壁现象出现，细胞呈梭形。培养48h后，软骨细胞分裂增殖越来越多并向周围延伸，培养第7天取材，HE染色示细胞生长良好，呈梭形。体内培养6周取材，HE染色、Masson染色为均一的成熟软骨组织，且共混膜已降解。结论以壳聚糖-胶原共混膜为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨。     

与软骨细胞共培养和条件培养液诱导骨髓间充质干细胞向软骨细胞分化的比较

背景：骨髓间充质干细胞体外转化很大程度上依赖于合适的培养条件。 目的：比较与软骨细胞共培养和条件培养液2种不同的诱导方案诱导骨髓间充质干细胞向软骨细胞分化的特点。 方法：分离培养大鼠骨髓间充质干细胞和耳软骨细胞,采用骨髓间充质干细胞与软骨细胞共培养及条件培养液诱导成软骨的方法,诱导骨髓间充质干细胞向软骨细胞分化。以MTT法及流式细胞仪检测细胞活性及周期,糖胺多糖、甲苯胺蓝以及免疫组化染色检测细胞生物学特性,以RT-PCR法检测诱导后的软骨细胞Ⅱ型胶原RNA表达情况。 结果与结论：采用共培养方式诱导的软骨细胞,其生物学特性与采用条件培养液诱导的软骨细胞相比,前者优于后者,如分泌糖胺多糖的能力以及基质分泌量均较高。提示共培养方式诱导的软骨细胞更接近正常软骨细胞,更有利于作为组织工程软骨的种子细胞。     

低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响

背景：多项体内外研究表明低氧和共培养均促进干细胞向软骨细胞方向分化。目的：观察低氧对脂肪干细胞和关节软骨细胞三维共培养成软骨能力的影响。方法：脂肪干细胞和关节软骨细胞二者按3∶1比例混合,以5×10¹⁰ L^-1接种于聚乳酸-羟基乙酸共聚物/明胶支架上,分别在常氧(体积分数为20% O₂)、低氧(体积分数为5% O₂)环境下培养6周。苏木精-伊红染色进行组织学分析,阿尔新蓝染色鉴定糖胺多糖的合成,免疫组化鉴定Ⅱ型胶原的表达,并测定各组支架-细胞复合物的DNA、糖胺聚糖、羟脯氨酸含量。结果与结论：低氧组苏木精-伊红染色显示大量细胞及细胞外基质生成,阿尔新蓝染色显示有大量糖胺多糖生成,免疫组化显示Ⅱ型胶原表达强阳性,且DNA、糖胺聚糖、羟脯氨酸等各项指标均高于常氧组。表明低氧促进脂肪干细胞和关节软骨细胞共培养成软骨分化。 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：     

三维动态培养构建注射型组织工程软骨远期修复效果的实验研究

目的三维诱导自体细胞软骨分化构建注射型组织工程软骨，探讨动物模型的远期修复效果。方法 分离培养兔自体骨髓间充质干细胞，分别三维动态诱导及二维平面培养诱导，诱导软骨细胞分化并鉴定，比较分化效果。分别收获两种方法诱导后的细胞，与蛋白胶混合制备注射型组织工程软骨，注射修复兔关节软骨缺损。随机设定三维动态培养组、二维平面培养组及空白对照组3组，24、48周后，观察大体和组织学形态，组织学评分比较远期效果。结果二维平面培养诱导后只有低水平的蛋白多糖沉积和Ⅱ型胶原等标志物表达，三维动态培养后的分化质量明显提高，大量表达蛋白多糖及Ⅱ型胶原。在动物模型中，三维动态培养组：24、48周后缺损基本修复，表面光滑坚韧，修复组织与周围软骨无界限，仍保持类透明软骨形态。二维平面培养组：24、48周后明显退化，失去软骨组织形态。空白对照组：无明显修复，缺损长期残留。结论三维动态培养显著改善自体MSC注射型组织工程软骨的修复效果，为提高关节软骨的微创修复提供新思路。     

Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed.

Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.07±0.14 days) comparable to T-flask expansion (1.20±0.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte–polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5±29.2 μg) than those with T-flask-expanded cells (164.0±28.7 μg). Total collagen content was similar between the two groups.

This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches.     

Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell? filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.     

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.     

Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity

To treat a cartilage defect with tissue-engineering techniques, multiplication of donor cells is essential. However, during this multiplication in monolayer expansion culture chondrocytes will lose their phenotype and produce matrix of inferior quality (dedifferentiation). Dedifferentiation occurs more extensively with low seeding densities and passaging. To obtain cartilage of good quality it is important that the multiplicated cells regain their cartilaginous phenotype (redifferentiation capacity). A "gold standard" for the multiplication of chondrocytes in monolayer, with respect to seeding density and passaging, is lacking. In numerous available studies, various cell densities have been used, making comparison of the results of these studies difficult. Therefore, we performed a comparative study to gain insight concerning the effect of seeding density and passaging on the capacity of cells to redifferentiate. From the resulting data we deduced the seeding density in monolayer culture for which cell expansion is both sufficient and fast, while the cells retain a capacity to redifferentiate. As a guideline we calculated that, at minimum, 20-fold multiplication is needed to fill an average cartilage defect of 4 cm(2) with the amount of donor chondrocytes we obtained. For this study we used isolated ear chondrocytes from five children. Four different seeding densities in monolayer culture were used, ranging from 3500 to 30000 cells/cm(2). The cells were cultured for four passages. The capacity of the expanded chondrocytes to redifferentiate (redifferentiation capacity) was studied after an additional 3-week culture in alginate beads and was assessed by glycosaminoglycan production and immunohistochemical stainings for collagen type I, collagen type II, elastin, and a fibroblast marker (11-fibrau). In general, we found that both passaging and decreasing seeding density yielded an increase in expanded chondrocytes, but at the same time decreased the dedifferentiation capacity. In further analyzing our data according to the proposed guidelines we found that with lower seeding densities sufficient multiplication (20 times) was reached in less time and with less passaging than at higher seeding densities. Importantly, the redifferentiation capacity of these chondrocytes was preserved. It was equal to or even surpassed that of chondrocytes multiplied 20 times at higher seeding densities, which required more time and more passages in monolayer culture. Thus, for cartilage tissue-engineering purposes we propose that expansion culture with low seeding densities is preferable.     

Expansion of bovine chondrocytes on microcarriers enhances redifferentiation

Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of collagen synthesis from type II to type I. The aim of this study was to evaluate the postexpansion chondrogenic potential of microcarrier-expanded bovine articular chondrocytes in pellet cultures. A selection of microcarriers was screened for initial attachment of chondrocytes. On the basis of those results and additional selection criteria related to clinical application, Cytodex-1 microcarriers were selected for further investigation. Comparable doubling times were obtained in T-flask and microcarrier cultures. During propagation on Cytodex-1 microcarriers, cells acquired a spherical-like morphology and the presence of collagen type II was detected. Both observations are indicative of a differentiated chondrocyte. Pellet cultures of microcarrier-expanded cells showed cartilage-like morphology and staining for proteoglycans and collagen type II after 14 days. In contrast, pellets of T-flask-expanded cells had a fibrous appearance and showed abundant staining only for collagen type I. Therefore, culture of chondrocytes on microcarriers may offer useful and cost-effective cell expansion opportunities in the field of cartilage tissue engineering.     

Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.     

Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.     

Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering

Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes.     

A cell-seeded biocomposite for cartilage repair.

Chondrocytes in monolayer cultures lose their phenotype and capability to express type-II collagen, they dedifferentiate into a fibroblastic cell type. Using three-dimensional culture systems a redifferentiation of these cells may occur. In the present study we investigated the morphology and biosynthetic activity of human articular chondrocytes seeded on porous matrices of type I/III collagen (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland). Microscopical examinations showed that chondrocytes adhere firmly to a collagen-I/III-membrane exhibiting their characteristic spherical cell shape. Cell numbers after enzymatic digestion of the membrane showed a 93% recovery of seeded cells. Immunohistological examination revealed positive staining for type-II collagen in some areas. The generated biocomposite withstands mechanical stress, keeps its size and design and does not shrink in culture. It is therefore easy to handle, can be sutured, glued or fixed with pins. This study shows, that in vitro production of autologous cartilage-like tissue could be established using a bilayer collagen type I/III fleece. This biocomposite carries active chondrocytes and is currently being evaluated in vivo in a sheep model as well as in a clinical trial for the repair of localized cartilage defects in the knee.     

Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.