Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, Enschede, The Netherlands.
Eur Cell Mater. 2012 Jun 5;23:387-99. doi: 10.22203/ecm.v023a30.
Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression over single cells and micro-aggregates of 50 and 200 cells. Matrix metalloproteinases 1, 9 and 13 mRNA levels were decreased in micro-aggregates compared to single-cells. Histological and biochemical analysis demonstrated enhanced matrix deposition in constructs seeded with micro-aggregates cultured in vitro and in vivo, compared to single-cell seeded constructs. Whole genome microarray analysis and single gene expression profiles using human chondrocytes confirmed increased expression of cartilage-related genes when chondrocytes were cultured in micro-aggregates. In conclusion, we succeeded in controlled high-throughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neo-cartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.
基于细胞的软骨修复策略，如基质诱导的自体软骨细胞植入（MACI），可以通过增强细胞性能得到改善。我们假设，在植入缺陷前在高通量条件下生成的软骨细胞微聚集体可以刺激软骨基质的沉积和重塑。为了解决这个问题，我们设计了一个微模具，以实现微聚集体的可控高通量形成。评估了人源和牛源软骨细胞微聚集体的形态、稳定性、基因表达谱和软骨生成潜力，并与微孔和 3D 培养的单细胞以及包封在葡聚糖-酪胺（Dex-TA）水凝胶中的 3D 培养的单细胞进行了比较，体外和体内。我们成功地在 24 小时内形成了具有高度可控大小、稳定性和活力的人源和牛源软骨细胞微聚集体。100 个细胞的微聚集体在 Collagen type I 和 Collagen type II 基因表达方面比单细胞和 50 个和 200 个细胞的微聚集体具有更好的平衡。与单细胞相比，微聚集体中基质金属蛋白酶 1、9 和 13 的 mRNA 水平降低。与单细胞接种的构建体相比，在体外和体内培养的微聚集体接种的构建体中，基质沉积增强。使用人软骨细胞进行全基因组微阵列分析和单个基因表达谱分析证实，当软骨细胞在微聚集体中培养时，软骨相关基因的表达增加。总之，我们成功地实现了软骨细胞的高通量微聚集体形成。与单细胞接种的构建体相比，微聚集体接种的构建体极大地改善了新软骨的形成。因此，在当前的 MACI 程序中，在软骨细胞植入前进行微聚集，可能会有效地加速透明软骨的形成。
