Wolf F, Candrian C, Wendt D, Farhadi J, Heberer M, Martin I, Barbero A
Department of Surgery, University Hospital, Basel, Switzerland.
Eur Cell Mater. 2008 Dec 19;16:92-9. doi: 10.22203/ecm.v016a10.
In this study, we first aimed at determining whether human articular chondrocytes (HAC) proliferate in aggregates in the presence of strong chondrocyte mitogens. We then investigated if the aggregated cells have an enhanced chondrogenic capacity as compared to cells cultured in monolayer. HAC from four donors were cultured in tissue culture dishes either untreated or coated with 1% agarose in the presence of TGFbeta-1, FGF-2 and PDGF-BB. Proliferation and stage of differentiation were assessed by measuring respectively DNA contents and type II collagen mRNA. Expanded cells were induced to differentiate in pellets or in Hyaff-11 meshes and the formed tissues were analysed biochemically for glycosaminoglycans (GAG) and DNA, and histologically by Safranin O staining. The amount of DNA in aggregate cultures increased significantly from day 2 to day 6 (by 3.2-fold), but did not further increase with additional culture time. Expression of type II collagen mRNA was about two orders of magnitude higher in aggregated HAC as compared to monolayer expanded cells. Pellets generated by aggregated HAC were generally more intensely stained for GAG than those generated by monolayer-expanded cells. Scaffolds seeded with aggregates accumulated more GAG (1.3-fold) than scaffolds seeded with monolayer expanded HAC. In conclusion, this study showed that HAC culture in aggregates does not support a relevant degree of expansion. However, aggregation of expanded HAC prior to loading into a porous scaffold enhances the quality of the resulting tissues and could thus be introduced as an intermediate culture phase in the manufacture of engineered cartilage grafts.
在本研究中，我们首先旨在确定人关节软骨细胞（HAC）在存在强软骨细胞促有丝分裂原的情况下是否以聚集体形式增殖。然后，我们研究了与单层培养的细胞相比，聚集的细胞是否具有增强的软骨形成能力。来自四名供体的HAC在组织培养皿中培养，培养皿未处理或涂有1%琼脂糖，同时存在转化生长因子β-1（TGFbeta-1）、成纤维细胞生长因子-2（FGF-2）和血小板衍生生长因子-BB（PDGF-BB）。分别通过测量DNA含量和II型胶原蛋白mRNA来评估增殖和分化阶段。将扩增的细胞诱导在微球中或Hyaff-11网片中分化，并对形成的组织进行生化分析以检测糖胺聚糖（GAG）和DNA，以及通过番红O染色进行组织学分析。聚集体培养物中的DNA量从第2天到第6天显著增加（增加了3.2倍），但随着培养时间的延长没有进一步增加。与单层扩增的细胞相比，聚集的HAC中II型胶原蛋白mRNA的表达高约两个数量级。聚集的HAC产生的微球对GAG的染色通常比单层扩增的细胞产生的微球更强。接种有聚集体的支架比接种有单层扩增的HAC的支架积累更多的GAG（1.3倍）。总之，本研究表明，HAC聚集体培养不支持显著程度的扩增。然而，在将扩增的HAC加载到多孔支架之前进行聚集可提高所得组织的质量，因此可作为工程软骨移植物制造中的中间培养阶段引入。
