Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

Project description:Objectives: Articular cartilage damage has become a universal health burden. Despite the recent progresses in cell-based cartilage regeneration, chondrocyte dedifferentiation has compromised the clinical outcomes, which is frequently seen in chondrocyte expansion and osteoarthritis, involving functional phenotype loss. However, its concept remains elusive with unknown intermediate process and mechanisms. Here we demonstrated a time-lapse atlas of chondrocyte dedifferentiation, to provide molecular details and informative biomarkers for clinical chondrocyte evaluation. Methods: Single-cell RNA sequencing (scRNA-seq) was employed to dissect the chondrocyte dedifferentiation and identify distinct cellular subpopulations. To validate the findings, live-cell metabolic assay, high-resolution imaging and ¡°assay for transposase-accessible chromatin with high throughput sequencing¡± (ATAC-seq) were conducted. Using a chemical inhibitor BTB06584 affecting on mitochondrial F1F0ATPase, we revealed the recovery potential of early and late dedifferentiated chondrocytes. Results:Our findings shape a biphasic model consist of early and late dedifferentiation stages. Early dedifferentiated chondrocytes uniquely obtained a transient glycolytic phenotype with an activation of metabolic and anti-oxidant genes; late dedifferentiated chondrocytes exhibited ultrastructural changes including mitochondrion damages and stress-associated chromatin remodeling. Using a chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinguished recovery potential from functional phenotype loss. BTB06584 treatment ameliorated early phenotype loss through inhibiting MAPK pathway. Notably, this two-stage transition was validated in human chondrocyte culture. An image-based approach was established for clinical utilization, to efficiently predict chondrocytes plasticity with stage-specific biomarkers. Conclusion: Thus this study lays a foundation for future clinical chondrocyte quality regulation and provides insights for deeper understanding of chondrocyte dedifferentiation.

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Involvement of ERK, Wnt and BMP2 signaling in human articular chondrocyte dedifferentiation in monolayer culture

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