Fabrication Of Chondrocyte-Derived Extracellular Matrix/Polycaprolactone Eelectrospun Composite Scaffolds For Chondrogenesis Of Mesenchymal Stem Cells

Extracellular matrix (ECM) plays critical roles in modulating cellular behaviors. A combination of cell-derived ECM with synthetic polymers would offer a novel strategy for manufacturing cell-instructive scaffolds in tissue engineering. In this study, we aimed at developing a chondrocyte-derived ECM coated polycaprolactone (PCL) electrospun fibrous scaffolds for directing the chondrogenic differeitation of mesenchymal stem cells (MSCs).Fibers with different diameters (1282-nm,549-nm and 285-nm) were fabricated by electro spinning PCL (16%, w/v) in a 3:1 (v/v) ratio of trichlormethane and dimethylformamide. Chondrocytes (rAC) and mesenchymal stem cells (rMSCs) were isolated from articular cartilage and bone marrow of 4-6-week-old New Zealand white rabbits, respectively. rAC at 4×104 cells/scaffold were cultured on PCL fibers for 7 days followed by decellularization to generate cartilage-like decellularized ECM coated PCL fibrous scaffolds (dECM/PCL). Surface structural features and compositions were characterized with SEM, TEM, AFM, immunofluorescence staining and biochemical analysis. rMSCs at 2×104 cells/scaffold were seeded onto dECM/PCL scaffolds and cultured for 7 days in growth medium to investigate the adhesion and proliferation of rMSCs. The capacity of dECM/PCL scaffolds to induce chondrogenesis was evaluated by seeding rMSCs at 1×105 cells/scaffold for 14 days in chondrogenic medium. Cell morphology was evaluated by F-actin staining, cell proliferation and viability was assessed with CCK-8 assay and Live & Dead staining, and gene expression was determined with RT-PCR.Surface morphology of dECM/PCL scaffolds varied significantly along with the diameter of PCL fibers based on SEM observation. For nanofiber scaffolds, a fibrous mesh structure was shown after decellularization and on 1282-nm fibrous scaffolds, the web-like ECM was elaborated with some pores. According to AFM measurement, these dECM/PCL scaffolds showed different surface roughness. In addition, it was revealed based on TEM images that more developed collagen fibers were present on dECM/PCL scaffolds with nano-fibrous PCL. Based on F-actin staining at 24 h post-seeding, MSCs showed an elongated spindle-like shape and tended to spread more on dECM/PCL scaffolds. Meanwhile, the proliferation and viability of rMSCs within 7 days was better on dECM/PCL scaffolds compared to uncoated PCL scaffolds. Gene expression analysis indicated that dECM/PCL scaffolds had a better capability of conducting chondrogenesis, especially dECM/PCL-549-nm.Collectively, dECM/PCL can stimulate the chondrogenic differentiation of MSCs and thus holds great promise in cartilage tissue regeneration.