Synthesis and Biocompatibility Characterizations of in Situ Chondroitin Sulfate-Gelatin Hydrogel for Tissue Engineering

Abstract

Novel hydrogel composed of both chondroitin sulfate (CS) and gelatin was developed for better cellular interaction through two step double crosslinking of N-(3-diethylpropyl)-N-ethylcarbodiimide hydrochloride (EDC) chemistries and then click chemistry. EDC chemistry was proceeded during grafting of amino acid dihydrazide (ADH) to carboxylic groups in CS and gelatin network in separate reactions, thus obtaining CS-ADH and gelatin-ADH, respectively. CS-acrylate and gelatin-TCEP was obtained through a second EDC chemistry of the unreacted free amines of CS-ADH and gelatin-ADH with acrylic acid and tri(carboxyethyl)phosphine (TCEP), respectively. In situ CS-gelatin hydrogel was obtained via click chemistry by simple mixing of aqueous solutions of both CS-acrylate and gelatin-TCEP. ATR-FTIR spectroscopy showed formation of the new chemical bonds between CS and gelatin in CS-gelatin hydrogel network. SEM demonstrated microporous structure of the hydrogel. Within serial precursor concentrations of the CS-gelatin hydrogels studied, they showed trends of the reaction rates of gelation, where the higher concentration, the quicker the gelation occurred. In vitro studies, including assessment of cell viability (live and dead assay), cytotoxicity, biocompatibility via direct contacts of the hydrogels with cells, as well as measurement of inflammatory responses, showed their excellent biocompatibility. Eventually, the test results verified a promising potency for further application of CS-gelatin hydrogel in many biomedical fields, including drug delivery and tissue engineering by mimicking extracellular matrix components of tissues such as collagen and CS in cartilage.

Keywords: Biocompatibility; Cartilage; Chondroitin sulfate; Gelatin; In situ hydrogel.

Fig. 1

Schematics of CS–acrylate A and gelatin–TCEP B syntheses via aminodihydrazide (ADH) grafting

Fig. 2

Chemical analyses of gelatin A and gelatin–TCEP B by ATR-FTIR

Fig. 3

Fabrications of CS–gelatin gel through two reaction mechanisms. There was an intra-crosslinking in CS–acrylate and gelatin–TCEP through ADH at the first reaction stage, and click chemistry of Michael type addition reaction has been proceeded as a second reaction

Fig. 4

Gelation times of CS–gelatin hydrogels in different concentrations in PBS at pH 7.4 and 37 °C

Fig. 5

Swelling behaviors of CS–gelatin hydrogels with different concentrations in PBS at pH 7.4 and 37 °C

Fig. 6

Surface A, B and cross-section C, D morphologies of the 10% CS–gelatin hydrogel at the magnifications of ×500 A, C and ×2000 B, D

Fig. 7

Biocompatibilities of fibroblasts both on the surface of A and inside B the 10% CS–gelatin hydrogel at day 7 by the live and dead assay

Fig. 8

Proliferation of fibroblasts on the surface of the 10% CS–gelatin hydrogel by the assay of CCK-8, where the cells were seeded at a density of 10,000 cells/cm²

Fig. 9

Cytotoxicity of the 10% CS–gelatin hydrogel measured by the assays of MTT, BrdU and Neutral red

Fig. 10

Cell compatibility of the 10% CS–gelatin hydrogel measures by the assay of direct sample covering on fibroblasts for 24 h, where A Teflon, B Latex and C 10% CS–gelatin hydrogel

Fig. 11

Inflammatory response of the 10% CS–gelatin hydrogel as measured by the nitric oxide assay