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(575e) Optimization Of Different Cross-Linkers In Generating Gelatin Porous Scaffolds

Madihally, S. V., Oklahoma State University
Castleberry, S., Oklahoma State University
Weirick, T., Oklahoma State University

Gelatin is a proteinaceous biocompatible, and biodegradable polymer derived from the hydrolysis of collagen. It has a long history of safe use in pharmaceuticals, cosmetics, as well as food products. It is also used in maintaining mouse embryonic stem cells in conjunction with Leukemia Inhibitory Factor. The large number of pendant functional groups in the gelatin structure aid in chemical cross-linking and derivatization. The goal of this research was to investigate alternative cross-linkers (besides Glutaraldehyde) for generating stable gelatin porous matrices useful in tissue regeneration applications. In this regard, effectiveness of Dimethyl Suberimidate (DMS) and N-hydroxysuccinimide (NHS) in cross-linking gelatin was tested either alone, together, or in sequence: i) cross-linking first with NHS then DMS, and ii) cross-linking first with DMS then NHS. Testing was performed using 2% gelatin solution and porous structures formed by freeze-drying (pre-freezing at -20degreeC); 2% gelatin solution was chosen based on initial cross-linking experiments with various concentrations of glutaraldehyde which showed physicochemical stability and pore size (as assessed by scanning electron microscopy) large enough for mammalian cell infiltration. These results showed that cross-linking porous structures first with DMS and then with NHS was stronger and produced matrices with open pore. To optimize the cross-linking protocol, porous structures were cross-linked with DMS (0 to 4%) and NHS (0 to 4%) using factorial design of experiments and tested i) under compression mode in hydrated conditions, and ii) for cross-linking density with TNBS (Trinitrobenzenesulfonic acid) which labels only primary amines, and UV spectroscopy. A six-parameter regression model was developed using which surface plots were generated. These results showed an optimum around 2% DMS and 2% NHS concentration with a compression modulus of 140kPa. In vitro cell culture experiments were performed using mouse embryonic fibroblasts for four days which showed that the matrices are not toxic and support cell growth as measured by Resazurin assay. Gelatinases (MMP-2 and MMP-9) were also assessed in the spent medium using fluorogeneic peptides. These results have shown promising potential of using gelatin-based matrices in tissue regeneration strategies.