软骨细胞微载体（Cytodex-3）平板三维培养扩增的实验研究

目的通过在微载体上进行平板培养扩增软骨细胞,并结合液态壳聚糖构建组织工程软骨。方法比较兔软骨细胞在单层培养与微载体上进行三维培养扩增软骨细胞的再分化能力。通过酶解法消化幼兔膝关节软骨后,得到种子细胞,分别进行单层和微载体三维培养扩增。通过评价细胞活性,倍增时间分析培养效果。并进行体外球型培养评价软骨细胞再分化能力,进行了糖胺多糖的定量生化分析。三维培养扩增软骨细胞与壳聚糖复合构建组织工程软骨,培养21天后通过组织学特种染色鉴定构建组织特性。结果微载体培养的软骨细胞可以保持良好活力和再分化能力,与单层培养体系相比较,糖胺多糖的定量生化分析（30．417±1．116ugGAG／mg样本）和（45．122±1．239ugGAG／mg样本）的差异具有统计学意义（P〈0．05）。结论在微载体上进行三维培养扩增软骨细胞可以加强细胞再分化能力。软骨细胞与壳聚糖合成后,可以在体外形成形态稳定的组织工程软骨。     

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。     

骨髓基质干细胞与软骨细胞体外共培养向软骨细胞转化的实验研究

目的 探讨骨髓基质干细胞（BMSCs）与软骨细胞体外共培养成软骨的可行性,以及能有效促进BMSCs的软骨向分化混合培养的比例.方法 以全骨髓法及梯度密度离心法分离幼兔BMSCs、梯度密度离心法分离软骨细胞,并分别对这2种细胞进行培养、扩传,将P2 BMSCs与P3软骨细胞进行体外共培养,分为：BMSCs/软骨细胞为2/1及4/1的2个共培养组,软骨细胞组,BMSCs组,分别于第1,3,5,7,9 d以MTT法检测细胞的增殖情况;分别于第1、2、3周对4组的细胞进行甲苯胺蓝染色及以RT-PCR方法检测蛋白多糖和Ⅱ型胶原表达的变化.结果 2种分离培养方法所得BMSCs及软骨细胞增殖旺盛,细胞形态正常,各组增殖情况良好,1,3,5,7,9 d时各组之间的差异具有统计学意义（P＜0.05）,但2/1与4/1共培养组之间无显著性差异;2/1及4/1共培养组在3周时以软骨样细胞为主,细胞呈均一异染,细胞被染成紫色,核仁染成深蓝色;蛋白多糖及Ⅱ型胶原基因表达：在1,2,3周时各组之间的差异具有统计学意义（P＜0.05）,共培养组和软骨细胞组3组之间蛋白多糖及Ⅱ型胶原表达水平无显著性差异,且均与BMSCs组之间有显著性差异,具有统计学意义（P＜0.05）.结论 将BMSCs与软骨细胞体外共培养,可以被有效地诱导为软骨细胞,软骨微环境在BMSCs分化为软骨细胞的过程中起到了很重要的作用.     

脱细胞半月板细胞外基质对半月板细胞的影响

目的探讨脱细胞半月板细胞外基质(dMECM)对传代半月板细胞增殖、细胞活性以及细胞表型的影响。方法用CCK-8法检测dMECM对半月板细胞增殖的影响;将P3代的内侧半月板纤维软骨细胞种植在d MECM修饰盖玻片上,以未修饰盖玻片为对照,体外培养7、14d后进行细胞活性检测,糖胺多糖、胶原分泌含量测定并用RT-PCR检测半月板细胞特异性基因mRNA表达变化。结果 CCK-8结果证实,与对照组比较,生长在dMECM修饰盖玻片上的P3代兔半月板纤维软骨细胞具有更好的细胞增殖特性(P0.05);细胞死/活染色结果证实dMECM组可维持更好的细胞活性;糖胺多糖和胶原定量检测结果证实dMECM组在第7、14天时,比对照组分泌更多的糖胺多糖和胶原(P0.05)。RT-PCR的检测结果证实体外培养7、14 d时,dMECM组II型胶原mRNA表达显著上调(P0.05)。结论 dMECM可以很好地促进半月板纤维软骨细胞的增殖、分化以及细胞活性的维持,可能是未来半月板组织工程领域非常有前景的支架材料之一。     

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：     

Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响CSCD

背景:实验证明胶原蛋白底物具有刺激成软骨的作用,但关于不同类型胶原蛋白刺激成软骨作用的能力仍存在争议。目的:观察Ⅰ、Ⅱ型胶原蛋白对体外培养人软骨细胞生物学特性的影响。方法:将P3代人软骨细胞分别加入普通培养板、Ⅰ型胶原蛋白包被培养板、Ⅱ型胶原蛋白包被培养板继续培养。培养10 d内,MTT法绘制细胞生长曲线;培养28 d后,采用ELISA法、聚合酶链反应、二甲基亚甲基蓝比色等方法检测3种培养板中软骨细胞分泌Ⅰ胶原蛋白、Ⅱ型胶原蛋白及糖胺多糖的量。结果与结论:Ⅱ型胶原蛋白包被培养板中软骨细胞数量最多,增殖速度为Ⅰ型胶原蛋白包被培养板的2倍、普通培养板的5倍。Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅰ型胶原蛋白最少,与普通培养板板检测结果差异有显著性意义(P<0.01),与Ⅰ型胶原蛋白包被培养板检测结果差异无显著性意义;Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅱ型胶原蛋白、糖胺多糖最多,与其他两种培养板检测结果差异有显著性意义(P<0.01)。表明胶原蛋白包被培养板培养软骨细胞优于普通培养板,其中Ⅱ型胶原蛋白包被培养板在培养软骨细胞时更能维持细胞形态,延长去分化现象出现的时间,更利于细胞再分化。     

海藻酸钠支架复合组织工程化软骨细胞修复兔佐剂性关节炎膝关节软骨缺损的实验研究

目的 探讨海藻酸钠支架复合组织工程化软骨细胞对兔佐剂性关节炎膝关节软骨缺损修复的影响.方法 将新西兰白兔分为正常对照组、佐剂性模型组和海藻酸盐支架治疗组.模型组和治疗组兔膝关节内注入弗氏佐剂建立佐剂性关节炎模型.抽取治疗组兔左后健侧肢体的骨髓,用Percoll法分离出自体骨髓间充质干细胞(ABMSC),体外培养传代至P3代并诱导分化为软骨细胞.将软骨细胞与海藻酸钠支架混合注入治疗组的病变膝关节.对照组和模型组关节注射等量的生理盐水.海藻酸钠支架复合组织工程化软骨细胞治疗1个月后处死兔,应用Ⅱ型胶原免疫组织化学法染色和HE染色,观察各组兔的膝关节关节软骨缺损的修复情况.结果 诱导成功软骨细胞且生长良好,呈现明显的纺锤体形,细胞成活率达98.5％.软骨细胞与海藻酸钠复合后体外培养显示细胞生长旺盛,增殖成株状或岛状,株状细胞团周围有类似的软骨陷窝形成,细胞排列密集,核圆形.组织形态学结果显示,治疗组与模型组比较,治疗组关节炎症反应明显减轻,关节Ⅱ型胶原阳性软骨细胞显著增高,软骨缺损得到明显修复.结论 海藻酸钠支架复合组织工程化软骨细胞对兔佐剂性关节炎膝关节软骨缺损有一定的修复作用.     

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-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-硫酸软骨素-透明质酸及明胶-陶瓷化骨多孔复合支架,构建自然骨-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-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,14 d时达到稳定状态。两组苏木精-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-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。     

Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响

背景:实验证明胶原蛋白底物具有刺激成软骨的作用,但关于不同类型胶原蛋白刺激成软骨作用的能力仍存在争议。目的:观察Ⅰ、Ⅱ型胶原蛋白对体外培养人软骨细胞生物学特性的影响。方法:将P3代人软骨细胞分别加入普通培养板、Ⅰ型胶原蛋白包被培养板、Ⅱ型胶原蛋白包被培养板继续培养。培养10 d内,MTT法绘制细胞生长曲线;培养28 d后,采用ELISA法、聚合酶链反应、二甲基亚甲基蓝比色等方法检测3种培养板中软骨细胞分泌Ⅰ胶原蛋白、Ⅱ型胶原蛋白及糖胺多糖的量。结果与结论:Ⅱ型胶原蛋白包被培养板中软骨细胞数量最多,增殖速度为Ⅰ型胶原蛋白包被培养板的2倍、普通培养板的5倍。Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅰ型胶原蛋白最少,与普通培养板板检测结果差异有显著性意义(P0.01),与Ⅰ型胶原蛋白包被培养板检测结果差异无显著性意义;Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅱ型胶原蛋白、糖胺多糖最多,与其他两种培养板检测结果差异有显著性意义(P0.01)。表明胶原蛋白包被培养板培养软骨细胞优于普通培养板,其中Ⅱ型胶原蛋白包被培养板在培养软骨细胞时更能维持细胞形态,延长去分化现象出现的时间,更利于细胞再分化。     

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。     

Chondrocytes culture in three-dimensional porous alginate scaffolds enhanced cell proliferation, matrix synthesis and gene expression

For the limited availability of autologous chondrocytes, a cultured system for expansion in vitro until sufficient cells are obtained must be developed. These cells must maintain their chondrocyte phenotype in vitro as well as in vivo, following implantation to ensure that differentiated chondrocytes synthesize a normal hyaline cartilage matrix and not a fibro-cartilage matrix. This study uses porous three-dimensional (3-D) alginate scaffolds within a perfusion system to culture low-density (5 x 10(5) cells) primary porcine chondrocytes for 1-4 weeks to study their proliferation and differentiation. The results of RT-PCR reveal that most cells could maintain their differentiation state for up to 4 weeks of culturing. Chondrocytes proliferated to 3 x 10(7) cells after 4 weeks in culture. Alginate scaffolds induced the formation of chondrocyte clusters and stimulated the synthesis of matrix, which effects were evaluated using histology and electron microscopy. These findings demonstrate that culturing chondrocytes in alginate scaffolds may effectively prevent the dedifferentiation and improve autologous chondrocyte transplantation.     

Effect of dynamic compression on in vitro chondrocyte metabolism

BACKGROUND: Chondrocytes can detect and respond to the mechanical environment by altering their metabolism. This study was designed to explore the effects of dynamic compression on chondrocyte metabolism. METHODS: Chondrocytes were harvested from newborn Wistar rats. After 7 days of expansion, chondrocytes embedded in agarose discs underwent uniaxial unconfined dynamic compression loads at different amplitudes (5%, 10%, and 15%) and frequencies (0.5 Hz, 1.0 Hz, 2.0 Hz, and 3.0 Hz) with a duration of 24 hours. The delayed effects on the chondrocytes were studied at 1, 3, and 7 days after the experiment. RESULTS: The results showed that at 10% strain, higher-frequency compression pressure can enhance the proliferation of chondrocytes. The synthesis of glycosaminoglycan (GAG) increased at 10%-15% strain and a 1-Hz load. The synthesis of nitric oxide (NO) increased at the 0.5-Hz load; while decreasing at the 15% strain. With 10% strain, 1 Hz dynamic compression, the proliferation of chondrocytes and GAG synthesis increased and persisted for 7 days, and NO synthesis decreased at the third and seventh days of culture. CONCLUSIONS: This study showed that chondrocytes respond metabolically to compressive loading, which is expected to modulate the growth and the resultant biomechanical properties of these tissue-engineered constructs during culture.     

Behavior of human articular chondrocytes derived from nonarthritic and osteoarthritic cartilage in a collagen matrix

The purpose of this study was to evaluate the morphologic and biochemical behavior and activity of human chondrocytes taken from nonarthritic and osteoarthritic cartilage and seeded on a three-dimensional matrix consisting of collagen types I, II, and III. Human articular chondrocytes were isolated from either nonarthritic or osteoarthritic cartilage of elderly subjects, and from nonarthritic cartilage of an adolescent subject, seeded on collagen matrices, and cultured for 12 h, 7 days, and 14 days. Histological analysis, immunohistochemistry, and biochemical assays for glycosaminoglycans (GAGs) and DNA content were performed for cell-seeded and unseeded matrices. Chondrocytes of nonarthritic cartilage revealed a larger number of spherical cells, consistent with a chondrocytic phenotype. The biochemical assay showed a net increase in GAG content in nonarthritic chondrocytes, whereas almost no GAGs were seen in osteoarthritic cells. The DNA results suggest that more osteoarthritic cells than chondrocytes from nonarthritic cartilage attached to the matrix within the first week. Human articular chondrocytes isolated from osteoarthritic cartilage seem to have less bioactivity after expansion and culture in a sponge consisting of type I, II, and III collagen compared with chondrocytes from nonarthritic cartilage.     

Effect of stratified culture compared to confluent culture in monolayer on proliferation and differentiation of human articular chondrocytes

With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue.With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.     

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold)

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.     

Influence of various alginate brands on the redifferentiation of dedifferentiated bovine articular chondrocytes in alginate bead culture under high and low oxygen tension

We examined the influence of various alginates on the redifferentiation of dedifferentiated articular chondrocytes in alginate bead culture under low (5%) and (21%) high oxygen supply. Isolated bovine articular chondrocytes were dedifferentiated and multiplied by 2-week monolayer culture under 21% oxygen. They were subcultured at a density of 10(7) cells/mL in six different commercially available sodium alginates (1.2%, w/v) and held under 21 or 5% oxygen for 3 weeks. Proliferation (DNA measurement on days 0 and 21 of culture), collagen type II production (immunocytochemistry and Western blotting), and [(3)H]proline and [(35)S]sulfate incorporation were monitored. Collagen type II production was significantly stronger under 5% oxygen compared with 21% oxygen in two alginates (three other alginates nearly reached the significance level). However, alginate-based differences proved not to be significant. [(3)H]Proline incorporation was not influenced by alginate but showed strong oxygen dependency (up to 3-fold higher under 5% oxygen). For [(35)S]sulfate incorporation oxygen dependency was even stronger (up to 8-fold higher under 5% oxygen) and significant alginate-dependent differences were found for several alginates. The effects of the different alginates did not correlate with their pH, viscosity, or guluronic:mannuronic acid ratio. Thus, the type of alginate and even more, the oxygen supply, influence the redifferentiation and matrix production of dedifferentiated bovine articular chondrocytes.     

Cell yield, proliferation, and postexpansion differentiation capacity of human ear, nasal, and rib chondrocytes

Human ear, nasal, and rib chondrocytes were compared with respect to their suitability to generate autologous cartilage grafts for nonarticular reconstructive surgery. Cells were expanded for two passages in medium containing 10% fetal bovine serum without (control) or with transforming growth factor beta(1) (TGF-beta(1)), fibroblast growth factor 2 (FGF-2), and platelet-derived growth factor bb (PDGF-bb) (TFP). Expanded cells were cultured as three-dimensional pellets in chondrogenic serum-free medium containing insulin, dexamethasone, and TGF-beta(1). Chondrocytes from all three sources were successfully isolated, increased their proliferation rate in response to TFP, and dedifferentiated during passaging. Redifferentiation by ear and nasal, but not rib, chondrocytes was enhanced after TFP expansion, as assessed by the significant increase in glycosaminoglycan (GAG)/DNA content and collagen type II mRNA expression in the resulting pellets. TFP-expanded ear and nasal chondrocytes generated pellets of better quality than rib chondrocytes, as assessed by the significantly higher GAG/DNA content and collagen type II mRNA expression, and by the relative stain intensities for GAG and collagen types I and II. In conclusion, postexpansion cell yields suggest that all three sources investigated could be used to generate autologous grafts of clinically relevant size. However, ear and nasal chondrocytes, if expanded with TFP, display superior postexpansion chondrogenic potential and may be a preferred cell source for cartilage tissue engineering.     

Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants

For cartilage engineering a variety of biomaterials were applied for 3-dimensional chondrocyte embedding and transplantation. In order to find a suitable carrier for the in vitro culture of chondrocytes and the subsequent preparation of cartilage transplants we investigated the feasibility of a combination of the well-established matrices fibrin and alginate. In this work human articular chondrocytes were embedded and cultured either in alginate, a mixture of alginate and fibrin, or in a fibrin gel after the extraction of the alginate component (porous fibrin gel) over a period of 30 days. Histomorphological analysis, electron microscopy, and immunohistochemistry were performed to evaluate the phenotypic changes of the chondrocytes, as well as the quality of the newly formed cartilaginous matrix. Our experiments showed that a mixture of 0.6% alginate with 4.5% fibrin promoted sufficient chondrocyte proliferation and differentiation, resulting in the formation of a specific cartilage matrix. Alginate served as a temporary supportive matrix component during in vitro culture and can be easily removed prior to transplantation. The presented tissue engineering method on the basis of a mixed alginate-fibrin carrier offers the opportunity to create stable cartilage transplants for reconstructive surgery.     

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.