Abstract
The main limitation of autologous chondrocyte implantation techniques is the necessity for in vitro cell expansion, which is associated with phenotypic drift and loss of extracellular matrix synthesis. Although media supplements (e.g. TGF-β) are extensively used to mitigate the tendency of de-differentiation, the lack of extracellular matrix is still one of the major obstacles to obtaining engineered cartilage substitutes with long-term clinical efficacy. Macromolecular crowding (MMC) is a biophysical phenomenon that increases tissue-specific extracellular matrix deposition. This study aimed to test whether MMC can be used to enhance hyaline-like ECM deposition in human chondrocyte culture: this hypothesis was tested in cells at P2 and at P7. Cells at P2 were cultured using a standard medium (DMEM/F12) in monolayer or alginate beads, whilst cells at P7 were cultured and re-differentiated using the system Clonetics™ of Lonza in the presence of 5 % HS or 5 % FBS, in monolayer and alginate beads. Macromolecular crowding medium was added 14 days after the start of re-differentiation. Collagen deposition was evaluated after 2, 5 and 10 days using SDS-PAGE and immunocytochemistry. MMC enhanced matrix deposition in all the conditions tested. However, although cells at P7 were cultured using a commercially available system, their deposited matrix was richer in collagen type I, whilst collagen type II was barely detectable. This was even more evident for cells in monolayer in HS and indicates that cells acquired a fibroblastic phenotype. To conclude, we showed that MMC increased matrix deposition in chondrocyte culture and that, unfortunately, commercially available systems are not always able to maintain chondrogenic phenotype. Since ECM produced is often undetectable and collagen expression and synthesis are not always correlated with its secretion, we propose to use MMC to assess chondrocyte phenotype maintenance and effectiveness of re-differentiation media.
