Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking

Abstract

The mechanical, thermal, swelling and release properties of glutaraldehyde (GTA) crosslinked gelatin films have been investigated in order to verify the influence of GTA concentration on the stability of the films. Air-dried films were submitted to treatment with GTA solutions at concentrations ranging from 0.05 to 2.5 wt%. At the smallest GTA concentration, the crosslinking degree, determined by trinitrobenzensulfonic acid assay, amounts to about 60% and increases up to values near 100%, obtained with GTA concentrations ⩾1 wt%. Simultaneously, the deformability of the films decreases, whereas the stress at break, σ_b, and the Young's modulus, E, increase. A crosslinking degree of about 85%, obtained using 0.25% GTA, is enough to prevent gelatin release in buffer solution and to provoke a significant reduction of the swelling in physiological solution. Furthermore, crosslinking greatly affects the thermal stability of the samples, as indicated by the results of differential scanning calorimetry (d.s.c.) investigation carried out on wet and air-dried films. The data suggest that the use of GTA at low concentration, which is desiderable to prevent toxicity, allows to modulate the physico-chemical properties of gelatin films, in order to obtain stable materials with a wide range of possible biomedical applications.

Introduction

Thermal denaturation or physical and chemical degradation of collagen involves the breaking of the triple-helix structure to give gelatin, a biodegradable, biocompatible and nonimmunogenic product, suitable for medical applications [1], [2], [3]. At a temperature of about 40°C, gelatin aqueous solutions are in the sol state and form physical thermoreversible gels on cooling. During gelling, the chains undergo a conformational disorder–order transition and tend to recover the collagen triple-helix structure [4], [5]. Gelatin is widely used in the pharmaceutical industry as well as in the biomedical field: hard and soft capsules, microspheres, sealants for vascular prostheses, wound dressing and adsorbent pad for surgical use are among its most frequent applications [2], [3], [6], [7], [8]. With respect to collagen, which is also known to have wide biomedical applications, gelatin does not express antigenicity in physiological conditions, and it is much cheaper and easier to obtain in concentrate solutions. On the other hand, gelatin exhibits poor mechanical properties which limit its possible applications as a biomaterial. Noticeable improvements of the mechanical properties of gelatin films in the direction of deformation have been obtained by inducing segmental orientation in gelatin films through uniaxial stretching and successive air drying at constant elongation [9]. The improvement of the mechanical properties of drawn gelatin films has been related to the renaturation level of the protein, evaluated through differential scanning calorimetry (d.s.c.) [9], [10]. Since gelatin is soluble in aqueous solution, gelatin materials for long-term biomedical applications must be submitted to crosslinking, which improves both the thermal and the mechanical stability of the biopolymer. Among the chemical crosslinking agents, glutaraldehyde (GTA) is by far the most widely used, due to its high efficiency of collagenous materials stabilization. Crosslinking of collagenous samples with GTA involves the reaction of free amino groups of lysine or hydroxylysine amino acid residues of the polypeptide chains with the aldehyde groups of GTA [11]. GTA is easily available, inexpensive and its aqueous solutions can effectively crosslink collagenous tissues in a relatively short period [12]. The success of thousands of bioprosthetic implants demonstrated in the last tens of years indicates that glutaraldehyde crosslinking has been clinically acceptable and has many merits in spite of the reports on its cytotoxicity [13]. Furthermore, GTA crosslinked gelatin microspheres have been successfully employed as microcarriers for the growth and propagation of fibroblasts and endothelial cells [14]. However, if released into the host due to biodegradation, GTA is toxic [11], [15]. Thus, several agents, including carbodiimides, epoxy compounds and genipin were recently tested to crosslink collagen-based materials [16], [17], [18]. Although some of these agents seem to be preferable in order to reduce the risk of cytotoxicity, they cannot equal GTA in collagen stabilization [18], [19]. The biocompatibility of GTA crosslinked collagenous materials can be improved by lowering the concentration of GTA solutions, as reported for anionic collagenous membranes [20]. In order to verify the possibility to stabilize gelatin films by crosslinking with GTA at low concentration, we have studied the effect of GTA concentration on the mechanical and thermal properties, as well as on the swelling and release, of crosslinked gelatin films.