Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks
Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks
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DOI：
10.1136/ard.60.8.781
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发表时间：
2001-08-01
影响因子：
27.4
通讯作者：
Verbruggen, G
中科院分区：
文献类型：
--
作者：
Almqvist, KF;Wang, L;Verbruggen, G
体外培养人关节软骨细胞，制备组织工程软骨，用于软骨缺损的治疗。方法将人关节软骨细胞分别在0.5%、1.0%和2.0%的藻酸盐中培养4周。确定了用于细胞复制和软骨细胞聚集蛋白聚糖合成的藻酸盐基质的最佳浓度。在两周和四周后测量不同培养条件下的DNA含量。在相同的培养时间后，通过[S-35]硫酸盐掺入评估聚集蛋白聚糖合成速率和周围细胞外基质中的积累。为了跟踪软骨细胞从藻酸盐珠的生长，将软骨细胞在0.25或0.5%纤维蛋白凝胶包围的0.5或1.0%藻酸盐中培养四周。在不同的培养时间后测量每个培养物的DNA含量。最后，将1.0%藻酸盐珠中的人软骨细胞包埋在0.5%纤维蛋白凝胶中8周。免疫组化分析蛋白聚糖，I型和II型胶原蛋白进行weekly.Results-at两周的DNA含量在每个文化显着增加，在0.5和1.0%的藻酸盐文化与基线值相比。在三种藻酸盐浓度下，这种增加持续到第4周。两周时聚集蛋白聚糖的合成在0.5%和1.0%藻酸盐细胞培养物中最高。在四周聚集蛋白聚糖合成率下降独立的藻酸盐浓度。聚集蛋白聚糖主要聚集在区域间基质中。软骨细胞在藻酸盐中的增殖和这些细胞在周围纤维蛋白凝胶中的生长在整个培养期间是明显的。聚集蛋白聚糖和II型胶原蛋白的细胞周围的积累，在藻酸盐以及在纤维蛋白凝胶，逐渐增加在培养期间。I型胶原蛋白出现在六个星期后，在藻酸盐和周围的fibrin.Conclusion-Human软骨细胞增殖在这个培养系统中，显示出生长到周围的纤维蛋白，并合成软骨样基质长达八周。
Objective-To produce tissue engineered cartilage by human articular chondrocytes in vitro for further use in in vivo manipulations for the treatment of cartilage defects.Methods-Human articular chondrocytes were cultured in 0.5%, 1.0%, and 2.0% of alginate for up to four weeks. The optimal concentration of an alginate matrix for cell replication and for aggrecan synthesis by chondrocytes was determined. DNA content in the different culture conditions was measured after two and four weeks. Aggrecan synthesis rates and accumulation in the surrounding extracellular matrix were assessed by [S-35]sulphate incorporation after the same periods of culture. To follow the outgrowth of chondrocytes from the alginate beads, chondrocytes were cultured for four weeks in 0.5 or 1.0% alginate surrounded by 0.25 or 0.5% fibrin gel. DNA content of each culture was measured after different culture periods. Finally, human chondrocytes in 1.0% alginate beads were embedded in 0.5% fibrin gel for eight weeks. Immunohistochemical analysis for aggrecan, type I and II collagen was performed weekly.Results-At two weeks the DNA content in each culture significantly increased in 0.5 and 1.0% alginate cultures in comparison with baseline values. This increase continued until week 4 at the three alginate concentrations. Aggrecan synthesis at two weeks was highest in 0.5 and 1.0% alginate cell cultures. At four weeks aggrecan synthesis rates decreased independently of the alginate concentrations. Aggrecan mainly accumulated in the interterritorial matrix. Proliferation of chondrocytes in alginate and outgrowth of these cells in the surrounding fibrin gel were evident throughout the culture period. The accumulation of aggrecan and type II collagen around the cells, in alginate as well as in fibrin gel, gradually increased over the culture period. Type I collagen appeared after six weeks in alginate and in the surrounding fibrin.Conclusion-Human chondrocytes proliferate in this culture system, show an outgrowth into the surrounding fibrin, and synthesise a cartilage-like matrix for up to eight weeks.