Effective clinical treatments of cartilage lesions in affected joints require large numbers of viable chondrogenic cells generated through in vivo stimulation or ex vivo expansion of chondrocytes isolated from small biopsy specimens. Conventional passaging of chondrocytes in culture provides sufficient cells for treatments but these cells usually lose their differentiated phenotype. This leads to the formation of fibrocartilaginous tissue due to a malfunctioning repair process. Biostimulation of passaging chondrocytes with low level laser irradiation (LLLI) may theoretically produce more functional chondrocytes for cell-based repair of cartilage defects. Molecular and cellular analyses, cytochemistry, cell cultivation, and microscopy showed that LLLI treatments were found to (1) increase chondrocyte viability, (2) promote secretion of matrix proteins, (3) upregulate expression of chondrogenic genes, and (4) downregulate gene expression of cell destructive proteases and genes coding for mediators involved in the extrinsic apoptosis signaling pathway. Furthermore, LLLI attenuated induction of genes associated with cell death and matrix breakdown induced by IL-1β, some of which was seen at the protein level, with verification of effects on gene expression in the C28/I2 human chondrocyte line. LLLI treatments during culture generated larger numbers of viable chondrocytes compared to untreated cultures. Moreover, LLLI-treated chondrocytes in culture also rectified and simultaneously maintained their differentiated phenotype. Cultured chondrocytes treated with LLLI are a promising cell source for repairing cartilage lesions in vivo and restoration of articular function using tissue engineering strategies.

A major challenge in cartilage regeneration is maintaining the hyaline cartilage phenotype and matrix over time while limiting hypertrophy and fibrosis. Here, type II collagen (Col2)-hyaluronic acid (Col2H) and type I collagen-hyaluronic acid (Col1H) sponges were compared using long-term three-dimensional chondrocyte culture, cross-species transcriptomic analysis, and functional experiments. Col2H supported longer-term chondrocyte phenotype and matrix retention than Col1H. Cytl1 was prioritized because it ranked highly in Col2H-associated analysis and was consistently downregulated during chondrocyte dedifferentiation in human and rat datasets. Functional assays, ChIP-qPCR, and co-immunoprecipitation supported involvement of an Itgb8-Nov-Tp63-Cytl1 regulatory network in the Col2-associated response. Col2H also showed preliminary cartilage-repair potential in a rabbit defect model. In human osteoarthritis cartilage, a five-gene signature comprising Cytl1, Tp63, Nov, Itgb8, and TIMP3 showed internal stability and an association with histological severity. These findings support a material-associated regulatory role for Col2H in chondrocyte phenotype and matrix maintenance.