High throughput generated micro-aggregates of chondrocytes stimulate cartilage formation in vitro and in vivo

Abstract

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.

Effects of Serum and Compressive Loading on the Cartilage Matrix Synthesis and Spatiotemporal Deposition Around Chondrocytes in 3D Culture

“…In a recent study by Moreira et al, it is stated that chondrocytes cultured in microaggregates showed lower MMP-1, 9, and 13 expression levels and enhanced matrix deposition in constructs compared to single-cellseeded constructs. 24 Additionally, due to the low cell density in constructs, the quantity of newly synthesized matrix is too small to improve gross material properties, which would not make our samples suitable for material testing.…”

From Skeletal Development to Tissue Engineering: Lessons from the Micromass Assay

“…Recent studies describe a possible solution by maintaining small high-density spheres using chondrocytes or adult human MSC in culture for a certain amount of time while inducing cartilage tissue phenotype, before merging several spheres into a larger construct. 93,94 Alternatively, 3D printing could be exploited to place small cellular aggregates into a large porous structure. 95,96 Such an approach would facilitate sufficient diffusion and provide structural support while maintaining a high cell density, and thus meet both the microscopic and macroscopic requirements of a cartilage tissue-engineering construct.…”

Repair of Osteochondral Defects with Rehydrated Freeze-Dried Oligo[Poly(Ethylene Glycol) Fumarate] Hydrogels Seeded with Bone Marrow Mesenchymal Stem Cells in a Porcine Model

“…Seeding BMSCs after OPF hydrogel scaffold fabrication instead of during scaffold fabrication enabled cellular condensation to occur and could be a contributing factor to the formation of the zonal arrangement, as enhanced matrix deposition that could stimulate cartilage remodeling had been reported in constructs seeded with microaggregates compared to single dissociated cells. 40 The presence of hyaline tissue indicates differentiation, but it could have been preceded by cell proliferation, as tissue repair outgrowth was observed. The concurrent proliferation and differentiation were also noted in MSCs seeded into a nanofibrous scaffold.…”