在纤维蛋白凝胶包裹的藻酸盐中培养软骨细胞：八周内细胞的特性
Culture of chondrocytes in alginate surrounded by fibrin gel: characteristics of the cells over a period of eight weeks.
作者信息
Almqvist K F, Wang L, Wang J, Baeten D, Cornelissen M, Verdonk R, Veys E M, Verbruggen G
机构信息
Department of Rheumatology, Ghent University Hospital, Ghent, Belgium.
出版信息
Ann Rheum Dis. 2001 Aug;60(8):781-90. doi: 10.1136/ard.60.8.781.
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 [(35)S]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.
目的
通过人关节软骨细胞在体外构建组织工程软骨，以便进一步用于体内操作治疗软骨缺损。
方法
将人关节软骨细胞分别培养于0.5%、1.0%和2.0%的海藻酸盐中，培养长达四周。确定用于细胞复制和软骨细胞合成聚集蛋白聚糖的海藻酸盐基质的最佳浓度。在培养两周和四周后测量不同培养条件下的DNA含量。在相同培养期后，通过[³⁵S]硫酸盐掺入评估聚集蛋白聚糖的合成速率及其在周围细胞外基质中的积累。为了追踪软骨细胞从海藻酸盐珠中的生长情况，将软骨细胞在0.5%或1.0%海藻酸盐中培养四周，周围包裹0.25%或0.5%的纤维蛋白凝胶。在不同培养期后测量每种培养物的DNA含量。最后，将1.0%海藻酸盐珠中的人软骨细胞包埋在0.5%纤维蛋白凝胶中培养八周。每周进行聚集蛋白聚糖、I型和II型胶原的免疫组织化学分析。
结果
在两周时，与基线值相比，0.5%和1.0%海藻酸盐培养物中每种培养物的DNA含量显著增加。这种增加在三种海藻酸盐浓度下持续到第4周。在两周时，0.5%和1.0%海藻酸盐细胞培养物中的聚集蛋白聚糖合成最高。在四周时，聚集蛋白聚糖合成速率下降，与海藻酸盐浓度无关。聚集蛋白聚糖主要积聚在区域间基质中。在整个培养期内，海藻酸盐中软骨细胞的增殖以及这些细胞在周围纤维蛋白凝胶中的生长都很明显。在培养期内，细胞周围、海藻酸盐以及纤维蛋白凝胶中聚集蛋白聚糖和II型胶原的积累逐渐增加。I型胶原在海藻酸盐和周围纤维蛋白中六周后出现。
结论
人软骨细胞在该培养系统中增殖，向周围纤维蛋白中生长，并合成软骨样基质长达八周。
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