Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan–gelatin porous structures

Abstract

Influence of mechanical characteristics and matrix architecture of substrates used in cell culture is an important issue to tissue engineering. Chitosan-based materials have been processed into porous structures, injectable gels and membranes, and are investigated to regenerate various tissues. However, the effect of these structures on cell growth and matrix production in accordance with each of the differing scaffolds has not been examined. We investigated the influence of porous structures, hydrogels, and membranes on the growth of normal human fibroblasts and their matrix production in a serum-free system. We used chitosan alone and in combination with gelatin. Injectable hydrogels were prepared using 2-glycerol phosphate. From the same solution, porous scaffolds and membranes were formed using controlled rate freezing and lyophilization, and air-drying, respectively. Fibroblast growth was evaluated on the 4th and 10th days using flow cytometry and CFDA-SE pre-staining. Cell morphology was assessed using actin and nucleus staining. Total protein content, collagen, tropoelastin, and MMP2/MMP-9 activity in the media supernatant were assessed by BCA, Sircol™, Fastin Elastin, and fluorogeneic peptide assays. Collagen accumulated in the matrix was assessed by Sircol™ assay after pepsin/acetic acid digestion and by Masson's Trichrome staining. These results showed increased viability of fibroblasts on chitosan-gelatin porous scaffold with decreased proliferation relative to tissue culture plastic (TCP) surface despite the cells showing spindle shape. The total protein, collagen, and tropoelastin contents were higher in the spent media from chitosan-gelatin porous scaffolds compared to other conditions. MMP2/MMP9 activity was comparable to TCP. An increase in collagen content was also observed in the matrix, suggesting increased matrix deposition. In summary, matrix production is influenced by the form of chitosan structures, which significantly affects the regenerative process.

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