Biological response of chondrocytes cultured in three-dimensional nanofibrous poly(epsilon-caprolactone) scaffolds

Abstract

Nanofibrous materials, by virtue of their morphological similarities to natural extracellular matrix, have been considered as candidate scaffolds for cell delivery in tissue-engineering applications. In this study, we have evaluated a novel, three-dimensional, nanofibrous poly(epsilon-caprolactone) (PCL) scaffold composed of electrospun nanofibers for its ability to maintain chondrocytes in a mature functional state. Fetal bovine chondrocytes (FBCs), maintained in vitro between passages 2 to 6, were seeded onto three-dimensional biodegradable PCL nanofibrous scaffolds or as monolayers on standard tissue culture polystyrene (TCPS) as a control substrate. Gene expression analysis by reverse transcription-polymerase chain reaction showed that chondrocytes seeded on the nanofibrous scaffold and maintained in serum-free medium supplemented with ITS+, ascorbate, and dexamethasone continuously maintained their chondrocytic phenotype by expressing cartilage-specific extracellular matrix genes, including collagen types II and IX, aggrecan, and cartilage oligomeric matrix protein. Specifically, expression of the collagen type IIB splice variant transcript, which is indicative of the mature chondrocyte phenotype, was up-regulated. FBCs exhibited either a spindle or round shape on the nanofibrous scaffolds, in contrast to a flat, well-spread morphology seen in monolayer cultures on TCPS. Organized actin stress fibers were only observed in the cytoplasm of cells cultured on TCPS. Histologically, nanofibrous cultures maintained in the supplemented serum-free medium produced more sulfated proteoglycan-rich, cartilaginous matrix than monolayer cultures. In addition to promoting phenotypic differentiation, the nanofibrous scaffold also supported cellular proliferation as evidenced by a 21-fold increase in cell growth over 21 days when the cultures were maintained in serum-containing medium. These results indicate that the biological activities of FBCs are crucially dependent on the architecture of the extracellular scaffolds as well as the composition of the culture medium, and that nanofibrous PCL acts as a biologically preferred scaffold/substrate for proliferation and maintenance of the chondrocytic phenotype. We propose that the PCL nanofibrous structure may be a suitable candidate scaffold for cartilage tissue engineering.