NOVEL GELATIN-BASED HYDROGELS FOR TISSUE ENGINEERING APPLICATION

Introduction: Biomaterials play pivotal roles in modern strategies of tissue engineering as designable biophysical and biochemical milieus that control cell fate and function. The key strategy relies on the optimum combination of cells with a suitable biodegradable matrix that could support the cell viability and remodelling of tissues. In tissue engineering, hydrogels, 3D network of hydrophilic polymers, have received much attention due to their biocompatibility, biodegradability, structural similarity to the extracellular matrix (ECM). Driven by enormous potential of hydrogels, we have developed a novel gelatin (G)-based hydrogel with tunable mechanical, degradation and biological properties. Chitosan (CH) and hydroxyethyl cellulose (HEC) were added to better match the native ECM composition and mechanical properties as well as to tailor the degradation resistance and available cell binding motifs. The effects of different material composition on physico-chemical properties, mechanical behaviour, cell adhesion and viability were evaluated. Materials and methods: G-PEG hydrogel was prepared in aqueous solutions following a synthetic procedure which involves the reaction between gelatin amino-groups and the functional end groups of poly(ethylene glycol) (PEG). In order to obtain adducts with a number of reactive end groups able to produce crosslinking, an excess of PEG over protein amino-groups was employed. Moreover, in order to obtain insoluble materials with good mechanical properties, improved water resistance and controlled degradation rate, a specific crosslinking agent, i.e. ethylene diamine, able to react with unreacted epoxy groups, was added. G-PEG-HEC hydrogel and G-PEG-CH hydrogel were prepared adding the proper amount of HEC or CH solution to a G-PEG aqueous solution obtained starting from a 9% (w/v) solution of G. Results: Hydrogels have been fully characterized by FTIR spectroscopy that confirmed the expected structure. Mechanical tensile tests were performed and swelling and weight loss were also monitored over a period of about 30 days. Interestingly, G-PEG hydrogel as well as G-PEG-HEC and G-PEG-CH, with a G/PEG ratio of about 3.6:1, display good stiffness, flexibility and extensibility. They show non-linear J-shaped stress-strain curves, similar to those found for ECM, with initial elastic modulus and strain at break over the range of 1.5-6.5 MPa and 30-70%, respectively. The swelling test revealed a wide range of equilibrium swelling rate (200-450%). Hydrogels showed no significant change in dimension and shape during degradation tests carried out at 37oC, and the resistance to hydrolytic degradation was longer than 30 days for all the formulations. All the hydrogels showed good cell viability during long term culture of a human fibroblast cell line. Discussion: Functionalized PEG was chosen to verify the possibility of grafting on gelatin and chitosan to obtain hydrogel that could eventually undergo crosslinking in the presence of a suitable curing agent. The final purpose to obtain adequately stiff and strong biomaterials, which possessed at the same time limited solubility and degradation rate in comparison with pure gelatin or chitosan, was attained. This is in fact a perfect condition to obtain a suitable biodegradable/resorbable matrix promoting cell viability and remodelling of tissue.