(62ac) Tissue Regeneration Using Various Structures

Objective: The objective of the research was to produce chitosan and chitosan-gelatin porous structures and proceed to colonize these structures with Human Foreskin Fibroblasts (HFF-1). Chitosan and gelatin are both biocompatible and biodegradable polymers, which is a requirement for the scaffolds. The porous structures are two dimensional (2-D), three dimensional (3-D), and hydrogels. The importance of this study is the applications in which the scaffolds can be used. The 2-D membrane is often used for wound healing, while the 3-D porous structures can be fabricated to replace multicellular membranes for organs. Hydrogels can be formed as a substitute for tissues such as cartilage using a minimally invasive procedure. Methods: As previously stated, there are three scaffolds that have been constructed for the purpose of the research. All three matrices are made with a 0.5% w/v chitosan or chitosan-gelatin solution. The two dimensional membrane is fabricated by air drying the solution in a sterile environment. The three dimensional matrix is made by freezing the solution to -20oC and then freeze drying the frozen solution. Hydrogels are formed by incubating the solution at 37oC (98.6oF). HFF-1 cells are cultured in serum containing media for more rapid growth. The cells are switched to serum-free media prior to seeding. Cells are seeded once the 2-D and 3-D scaffolds are constructed whereas the hydrogels are not formed until at the time of seeding. The seeded matrices are incubated four days changing the media every second day. The removed media is tested for collagen and elastin using Sircol Soluable Collagen Assay and Fastin Elastin Assay kits. On the fourth day the cells are stained with Actin and DAPI in order to view the cells in the different layers of the matrices. MMP-2/MMP-9 is also assessed. Results: All matrices were constructed using chitosan and chitosan-gelatin and were successfully seeded with HFF-1 cells. Standard curves for collagen and elastin assays have been achieved to fit the actual Collagen and Elastin data in the curve. Cells show spreading and attachment on chitosan-gelatin structures. Testing for MMP-2/MMP-9 must be accomplished for further analysis. Conclusions: Multiple types of matrices can be constructed using the same solution by altering the processing method without additional chemical reactions necessary.

Funding provided by: KJW was funded by the ARRA NIH REU Program, KT was funded by OK-INBRE program.