RESUMO
Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.
RESUMO
OBJECTIVE: Cartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. The current study identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. DESIGN: Chondrocytes were isolated from juvenile bovine knee joints and cultured with (primed) or without (unprimed) a growth factor cocktail. Gene expression, cell morphology, cell adhesion, cytoskeletal protein distribution, and cell mechanics were assessed. Following passage 5, cells were embedded into agarose hydrogels to evaluate functional properties of engineered cartilage. RESULTS: Priming cells during expansion culture altered cell phenotype and chondrogenic tissue production. Unbiased ribonucleic acid-sequencing analysis suggested, and experimental studies confirmed, that growth factor priming delays dedifferentiation associated changes in cell adhesion and cytoskeletal organization. Priming also overrode mechanobiological pathways to prevent chondrocytes from remodeling their cytoskeleton to accommodate the stiff, monolayer microenvironment. Passage 1 primed cells deformed less and had lower yes associated protein 1 activity than unprimed cells. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5. CONCLUSIONS: Priming suppresses mechanobiologic cytoskeletal remodeling to prevent chondrocyte dedifferentiation, resulting in more cartilage-like tissue-engineered constructs.
