A New Approach to Cartilage Tissue Engineering Using Human Dermal Fibroblasts Seeded on Three-Dimensional Polymer Scaffolds

Abstract

Current methods for correcting articular cartilage defects are limited by a scarcity of cartilage cells. Here we describe a novel method for the conversion of human dermal fibroblasts to chondrocyte-like cells and the potential application of this methodology to cartilage tissue engineering. Human neonatal foreskin fibroblasts were seeded on two-dimensional, tissue culture polystyrene (TCPS) in high density micromass cultures in the presence of staurosporine (50-200 nM), a protein kinase C (PKC) inhibitor, and lactic acid (40 mM) to induce functional hypoxia. Dermal fibroblasts were similarly cultured on three-dimensional polymer scaffolds composed of a non-woven polyglycolic acid (PGA) fiber mesh reinforced in a dilute solution of poly(L-lactic acid) (PLLA). At 24 hours, northern analysis revealed a staurosporine dose-dependent increase in aggrecan core protein expression in lactate-treated micromass cultures on TCPS, while type I collagen gene expression was virtually abolished in all cultures supplemented with staurosporine. The cells in these cultures displayed a rounded, cobblestone-shaped morphology typical of differentiated chondrocytes (most pronounced at 200 n.M staurosporine and 40 mM lactate), and were organized into nodules which stained positively with Alcian blue. When seeded on PGA/PLLA matrices under identical conditions as described for TCPS, a chondrocyte-like morphology was observed in cultures treated with lactate and staurosporine in contrast to the flattened sheets of fibroblast-like cells seen in untreated controls. Taken together, the above findings suggest that staurosporine treatment coupled with high density micromass culture in the presence of lactate induces chondrogenic differentiation in human dermal fibroblasts, and that these cells may be used in concert with three-dimensional polymer scaffolds for the repair of articular cartilage lesions.

Novel Fabrication of Open-Pore Chitin Matrixes

“…[14][15][16] In particular, regeneration of damaged tissues is a significant objective because of insufficient organ and tissue donors for the high demands. 17 The potential of cell transplantation has been investigated by many groups for the regeneration of several tissues such as nerve, 18,19 liver, 20,21 cartilage, [22][23][24] and bone. 25,26 The scaffolding material should be designed to have sufficient porosity, adequate mechanical stability, biocompatibility, and biodegradability or bioadsorbability.…”

“…Porous biodegradable materials are in great demand in various applications such as controlled drug release systems, − enzyme immobilization support, − molecular fractionation, , and transition-metal adsorption. − These materials are also being explored widely in tissue engineering, a field that offers an attractive approach to tissue repair and fabrication. − In particular, regeneration of damaged tissues is a significant objective because of insufficient organ and tissue donors for the high demands . The potential of cell transplantation has been investigated by many groups for the regeneration of several tissues such as nerve, , liver, , cartilage, − and bone. , The scaffolding material should be designed to have sufficient porosity, adequate mechanical stability, biocompatibility, and biodegradability or bioadsorbability.…”