作者:Ursula Anderer、Jeanette Libera
DOI:10.1359/jbmr.2002.17.8.1420
日期:——
A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro‐engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three‐dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage‐like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline‐specific proteins collagen type II and S‐100, as well as the synthesis of hyaline‐specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage‐like tissue where medium supplements were restricted to human serum.
组织工程学领域的一个挑战是体外生成人体软骨。为了达到体外工程软骨的标准,例如防止免疫反应以及与周围组织在结构和功能上的整合,我们建立了一个三维细胞培养系统,无需添加外源生长因子或支架。人体软骨细胞以球状培养。通过球状冷冻切片组织学和免疫组织化学研究,对组织形态和蛋白质表达进行了分析。当球状体在仅添加人血清的培养基中培养时,会产生类似于天然软骨的软骨样组织。这种体外组织的特征是透明质酸特异性蛋白胶原蛋白II型和S-100的合成,以及透明质酸特异性黏多糖的合成,其合成量随着培养时间的延长而增加。3个月后,体外组织内部的细胞数量减少,仅为天然软骨的两倍。此外,球状体迅速附着并迁移到玻璃片和人体髁软骨上。在自体球状体培养物中添加抗生素会抑制基质蛋白的合成。值得注意的是,用胎牛血清代替人血清会导致球状体内部破坏,表面仅保留有存活的细胞边缘。这些结果表明,球状体培养首次实现了自体体外工程化人体软骨样组织,其中培养基仅限于人血清。