Fascin-1 Inhibition Partially Represses Dedifferentiated Passaged Chondrocyte Phenotype

• Nikita Lachke, Human Physiology, University of Delaware

• Justin Parreno, Biological Sciences, University of Delaware

Articular cartilage has a limited capacity for self-repair, which contributes to progressive tissue degeneration and poor healing after injury. To address this, autologous chondrocyte implantation (ACI) was developed as a cell-based repair strategy. ACI expands a patient’s chondrocytes in monolayer culture before reimplantation. However, during monolayer expansion, chondrocytes dedifferentiate and acquire a fibroblast phenotype characterized by increased cell spreading, altered actin organization, and changes in matrix production, including decreased expression of chondrogenic markers and increased expression of fibroblastic markers. Because actin reorganization during monolayer expansion is a key regulator of this dedifferentiated state, identifying therapeutics that target actin dynamics may offer a strategy to improve chondrocyte quality for cartilage repair.

Many repair-oriented compounds are reported to affect the actin cytoskeleton in other musculoskeletal systems, but their effects on passaged chondrocytes remain unclear. In this study, we tested the Fascin-1 inhibitor NP-G2-044, the peptides BPC-157, and thymosin beta-4 to see whether they could reduce the dedifferentiated state.. To assess the phenotype, we examined cell morphology, including area and circularity, as well as expression of chondrogenic and dedifferentiation-associated markers using RT-PCR and capillary electrophoresis protein analysis.

Only inhibition of Fascin-1, NP-G2-044, had positive effects on the dedifferentiated phenotype. Overall, all treatments produced no changes in cell morphology. RT-qPCR analysis showed limited changes in chondrogenic marker expression across treatment groups. However, NP-G2-044 reduced the expression of dedifferentiation-associated markers type 1 collagen and alpha smooth muscle actin, a partial repression of the dedifferentiated phenotype. In contrast, BPC-157 and thymosin beta-4 had little effect under the conditions tested.

In conclusion, these findings suggest that Fascin-1 inhibition can partially influence passaged chondrocyte phenotype, but more complete redifferentiation may need long term modulation of the actin cytoskeleton. Future work will examine whether Fascin-1 inhibition can help prevent fibrocartilage formation and improve the quality of cells used for cartilage repair.