Preparation of networks of gelatin and genipin as degradable biomaterials

https://doi.org/10.1016/j.matchemphys.2003.08.027Get rights and content

Abstract

The aim of this study is to prepare networks of Bloom number 300 gelatin and genipin as degradable biomaterials. The cross-linking ability of genipin on gelatin was studied and discs were made from the genipin-fixed gelatin. The swelling ratio, the degree of cross-linking, and the rate of degradation of the discs were measured using ninhydrin assay and differential scanning calorimetry (DSC) experiments. The results indicated that genipin is a good cross-linker for the gelatin. Additionally, varying the concentration of genipin can control the rate of degradation and the degree of cross-linking of the genipin-fixed gelatin. Finally, the concentration of genipin should exceed 0.5% of the overall weight of the gelatin-based material if a complete cross-linking reaction between gelatin and genipin molecules is required.

Introduction

Collagen is the major protein component of bone, cartilage, skin and connective tissue and also the major constituent of all extracellular matrices in animals [1]. However, collagen is of animal origin and thus must be seriously investigated before it can be used in humans because of its antigenicity. In contrast, gelatin, which is essentially denatured collagen, has relatively low antigenicity. Recently, uses of gelatin-based biomaterials in applications of artificial skin, neuron regeneration, and bone grafts have been reported [2], [3], [4]. The interest in gelatin arises mainly from the fact that this natural polymer allows the production of biocompatible and biodegradable biomaterials, which can act as temporary replacements [5], [6]. However, the main limitation of gelatin for the preparation of tissue substitutes is its rapid dissolution in aqueous environments, leading to fast degradation of grafts at body temperature [7]. Cross-linking treatments to produce the formation of molecular structures have been suggested to solve this problem. Depending on what degradable properties are desired, different cross-linking treatments, including chemical and physical methods, can be used to prolong the absorption of the gelatin.

Physical cross-linking methods include drying, heating or exposure to gamma or ultraviolet radiation. The primary advantage of physical methods is that they do not cause potential harm. However, the limitation of such methods is that obtaining the desired amount of cross-linking is difficult [8]. In chemical cross-linking methods, cross-linkers are used to bond functional groups of amino acids. Commonly used chemical cross-linkers include formaldehyde, glutaraldehyde, polyepoxy compounds, tannic acid, dimethyl suberimidate, carbodiimides and acyl azide. However, these synthetic cross-linking reagents were relatively highly cytotoxic, impairing the biocompatibility of bioprostheses [9], [10], [11]. This is the reason for the increasing demand for a cross-linking reagent that can form stable and biocompatible cross-linked products, without causing problems of cytotoxicity. Hence, a low-toxic and naturally occurring cross-linking agent, genipin, has been used as a cross-linking agent for fixing biological tissue [12], [13], [14].

Genipin and its related iridoid glucosides, extracted from the fruits of Gardenia jasminoides Ellis, have been used widely as an antiphlogistic and a cholagogue in herbal medicine [15]. Genipin can also react with amino acid or proteins to form dark blue pigments used in the fabrication of food dyes [16]. Additionally, it can form stable cross-linked products with resistance against enzymatic degradation that is comparable to that of glutaraldehyde-fixed tissue [17], [18]. Furthermore, gelatin-derived bioadhensives display higher biocompatibility and less cytotoxicity when cross-linked with genipin than with other agents, such as formaldehyde, glutaraldehyde and epoxy compounds [19]. The mechanism of the reaction of amino acids or proteins with genipin is still not well understood at present. The mechanism proposed by Touyama’s group [20], [21] for the formation of the genipin-methylamine monomer is through a nucleophilic attack by methylamine on the olefinic carbon at C-3 in genipin, followed by the opening of the dihydropyran ring and an attack by the secondary amino group on the resulting aldehyde group. The blue-pigment polymers are presumably formed through the oxygen radical-induced polymerization and dehydrogenation of several intermediary pigments. The results of these studies suggest that genipin may form intramolecular and intermolecular cross-links with cyclic structures within collagen fibrils in biological tissue [14].

This study elucidates the reaction of the gelatin and the genipin at different concentrations. The cross-linking index, the swelling ratio and the rate of degradation of the genipin-fixed gelatin were evaluated. Moreover, the thermal behavior of the genipin-fixed gelatin was examined using differential scanning calorimetry (DSC).

Section snippets

Cross-linking reactions

Type A gelatin (300 Bloom, Sigma Chemical Co., Saint Louis, MO) was extracted from porcine skin with an average molecular weight of about 50,000–100,000. The gelatin solution was prepared by dissolving 25 g of gelatin powder in 75 ml of distilled water at 70 °C in a water bath. Twenty percent genipin solution was obtained by dissolving 1 g of genipin powder (Challenge Bioproducts Co., Taichung, Taiwan) in 4 ml of 60% ethanol. The gelatin solution was then mixed with different volumes of the genipin

Evaluation of cross-linking index

Fig. 1 shows the amounts of amino acid residues in the gelatin. It shows that the OD value increased with the number of unreacted amino groups. This curve could be divided into three intervals. In the first period, the highest OD value of absorption was approximately 1.4 for the gelatin–genipin mixture when the weight percentage of the genipin solution was under 0.075 wt.%. The result indicated that the degree of cross-linking of the gelatin was very small when the concentration of the genipin

Conclusion

Mixing with a higher concentration of genipin increases the degree of cross-linking of the gelatin. Additionally, when the weight percentage of genipin exceeded 0.5 wt.%, the cross-linking reaction could be completed. Finally, at least one day was required to complete the cross-linking reaction between the Bloom number 300 gelatin and the genipin at the concentration of 0.5 wt.%.

Acknowledgements

The authors would like to thank the National Science Council of the Republic of China, Taiwan for financially supporting this research work under Contract No. NSC89-2314-B-166-001-M08.

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