A Novel Approach to Cross-Linking Gelatin Matrices

The goal of this research was the optimization of a cross-linking strategy that would yield high compressive strength, as well as increased durability of gelatin-based three dimensional porous structures, useful in tissue regeneration. A novel approach of using a dual cross-linking team i) Dimethyl Suberimidate (DMS) and ii) N-Hydroxysuccinimide (NHS) which interact with two different functional groups present in gelatin was chosen for this purpose. The hypothesis is that the DMS would form an ester with gelatin. Subsequent addition of NHS would react with the formed ester, and stabilizes cross-linking.

To test the hypothesis, experiments were performed with different concentrations of each cross-linker, the order in which cross-linkers were added, and the time allowed for cross-linking. This testing showed support for the proposed hypothesis of the DMS/NHS as it was noted that the strength and durability of the structures increased when both the cross-linkers were used. NHS then DMS showed increased strength over DMS or NHS alone, but had lower strength than DMS then NHS. To optimize the compressive properties, porous structures were cross-linked with DMS (0 to 4%) and NHS (0 to 4%) using a factorial design of experiments and tested under compression mode in hydrated conditions. A six-parameter regression model was developed using which surface plots were generated. These results showed a compressive modulus maximum (140kPa) at around 2% DMS and 2% NHS concentrations.

To better understand the increase in compressive properties, cross-linking density was assessed with Trinitrobenzenesulfonic acid labeling of primary amines, measured with UV absorption at 346nm. The percent cross-linking of the gelatin was compared to the absorption of a similar amount of an un-cross-linked gelatin control. These results showed a saturation of 50% in cross-linking density with 1 to 2% cross-linker. FTIR analysis was also performed to identify possible interactions. These results confirmed the possibility of cross-linking both the functional groups. Seven day durability studies performed at physiological conditions (37°C and hydrated condition) showed dissolution i.e., weak cross-linking by NHS. This led us to suggest the use of a suspected stronger reaction, like EDC/NHS, in place of just NHS alone. The potential here being that EDC/NHS would not rely on the formation of unstable DMS-esters to react, but instead would seek out and activate carboxyl groups independent of DMS and then would react with the primary amines left after DMS cross-linking had occurred, as well as those that are on the structure of DMS.