Cell-based cartilage repair strategies such as matrixinduced 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 micromould 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 singlecells. 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 cartilagerelated genes when chondrocytes were cultured in microaggregates. In conclusion, we succeeded in controlled highthroughput formation of micro-aggregates of chondrocytes. Compared to single cell-seeded constructs, seeding of constructs with micro-aggregates greatly improved neocartilage formation. Therefore, micro-aggregation prior to chondrocyte implantation in current MACI procedures, may effectively accelerate hyaline cartilage formation.

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An accelerated negative buoyancy method has been developed to assess cell adhesion strength. This method has been used in conjunction with 3-D morphometric analysis to understand the effects of surface topology on cell response. Aligned micro-grooved surface topographies (with a range of groove depths) were produced on stainless steel 316L substrates by laser ablation. An investigation was carried out on the effect of the micro-grooved surface topography on cell adhesion strength, cell and nucleus volumes, cell phenotypic expression and attachment patterns. Increased hydrophobicity and anisotropic wettability was observed on surfaces with deeper grooves. A reduction was noted in cell volume, projected areas and adhesion sites for deeper grooves, linked to lower cell proliferation and differentiation rates and also to reduced adhesion strength. The results suggest that the centrifugation assay combined with three-dimensional cell morphometric analysis has considerable potential for obtaining improved understanding of the cell/substrate interface.

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