(761e) Influence of Hydrogel and Chondrogeneic Soluble Factors On Fibroblast Differentiation

Recent advances in stem cell technologies demonstrate the reprogramming capability of mature fibroblasts into induced stem cells by genetic engineering.  However, the effect of matrix elements and soluble factors on differentiation is not well understood.  The objective of the research was to test the differentiation of Human Foreskin Fibroblasts (HFF-1) into chondrocytes using 3D structures and soluble factors.  Mesenchymal stem cell (MSC) differentiation to chondrocytes was used as a positive control and same conditions were used for fibroblasts.  Chitosan-gelatin hydrogels and porous structures were formed using techniques established in the literature.

HFF-1 cells are initially cultured in serum containing media for more rapid growth. Once confluent the cells were seeded in pellet form into the hydrogels and differentiated using Complete Chondrogenic Medium. Hydrogels were formed by incubating the chitosan-gelatin solution at 37^oC (98.6^oF).  The seeded hydrogels were cultured in for one week as well as twenty eight days changing the media every three to four days, similar to MSC cultures.  At the time of inoculation the cells were stained with CFDA for a visual confirmation of suspension of cells in the hydrogel and spreading characteristics via fluorescent microscopy.  The cells harvested after one week were used for flow cytometry analysis and supernatants of all cultures were preserved for collagen type II analysis.  Cells were stained with anti-CD44-PE antibody and anti-CD151-FITC antibody and analyzed by flow cytometry.  The retrieved media from the MSC and fibroblast cultures were tested for Collagen Type II using an ELISA. Histology and immunohistochemical analyses were performed on the regenerated structures after twenty eight days for visual confirmation of the production of Collagen Type II.  Masson’s Trichrome and Alcian blue were used for the presence of cartilage and proteoglycans.

These results show that the fibroblasts show rounded morphology in the hydrogels compared to spindle shape on porous structures and tissue culture plastic.  Both conditions showed reduced cell growth relative to tissue culture plastic.   ELISA analysis suggested Collagen Type II production in the first two in fibroblast cultures, similar to MSC cultures.  Flow cytometry profiles demonstrate alternations in CD44 and CD151 expression.  Analysis of the histology showed an increase in matrix accumulation in the structures.