Fibronectin Coating of Tissue Culture Polystyrene to Improve Superficial Zone Chondrocyte Expansion

• John Caputo, Biological Sciences, University of Delaware
• Thomas Manzoni, Biological Sciences, University of Delaware

• Justin Parreno, Biological Sciences, University of Delaware

Articular cartilage plays a critical role in joint function by providing a lubricated surface for frictionless movement, achieved through zone-specific protein production. Superficial zone chondrocytes (SZC) produce lubricin/proteoglycan-4 (PRG4), lubricating the cartilage surface and reducing friction. Cartilage injuries present significant challenges due to the limited regenerative capacity of native chondrocytes. Passaged full-thickness chondrocytes are FDA approved for cartilage repair, though lack successful long-term repair capabilities due to chondrocyte dedifferentiation throughout passaging. Additionally, redifferentiation of full-thickness chondrocytes does not yield an adequate superficial zone. Passaging SZC on chondrocyte derived-decellularized extracellular matrices (CD-ECM) improves SZC attachment and proliferation, while limiting dedifferentiation. CD-ECM contains abundant fibronectin (FN), a matrix protein known to regulate chondrocyte adhesion, proliferation, and differentiation. It is unknown if FN coatings can improve SZC expansion. We hypothesize: FN coated polystyrene will increase primary SZC attachment and proliferation, while limiting dedifferentiation throughout passaging. To test our hypothesis primary bovine SZC were seeded and cultured on uncoated polystyrene, FN coated polystyrene or CD-ECM. Primary SZC had greater cell attachment when seeded on FN compared to polystyrene, and similar to SZC seeded on CD-ECM. Furthermore, SZC passaged on FN and CD-ECM reached confluency faster than SZC cultured on uncoated polystyrene. At P0, there were no differences in cell area or circularity. By P2, SZCs on CD-ECM had significantly smaller area than on FN and polystyrene, indicating reduced cell spreading which is more typical of chondrocytes. At P2, chondrogenic mRNA levels (aggrecan, collagen-II, PRG4) were similar across all conditions. However, SZCs on FN and polystyrene showed increased collagen-I and α-SMA mRNA, markers of fibroblastic and contractile phenotypes, indicating greater dedifferentiation compared to CD-ECM. Overall, our initial hypothesis was not fully supported. While passaging SZC on FN coated polystyrene improves SZC attachment and proliferation, FN does not limit the effect of differentiation throughout passaging.