Revista nº 803

10 Campos F. Hidrogeles de gelatina para ingeniería tisular Actualidad Médica · Número 803 · Enero/Abril 2018 Páginas 9 a 12 origin. Synthetic biomaterials are manufactured with a tailored architecture, and their degradation characteristics controlled by varying the polymer itself or the composition of the individual polymer including polystyrene, poly-l-lactic acid (PLLA), polyglycolic acid (PGA) and poly-dl-lactic-co- glycolic acid (PLGA) (4). However, its main drawbacks are its reduced biocompatibility and bioactivity. On the other hand, natural materials including polymers such as gelatins, silk and chitosan are biologically active molecules and typically promote excellent cell adhesion and growth (3). Gelatin is a heterogeneous mixture of peptides derived from the parent protein collagen by procedures involving the destruction of cross-linkages between the polypeptide chains along with some breakage of polypeptide bonds (5). Two types of gelatin are generally obtainable, depending on the pre- treatment procedure (prior to extraction process). Acidic pre- treatment (type A) does little affect the amide groups while the alkaline pre-treatment (type B) targets the amide groups converting many of the asparagine and glutamine residues to aspartate and glutamate (6). According to the literature, both collagen and gelatin used as scaffold components improve significantly infiltration, adhesion, spreading, and proliferation of cells on resulting scaffolds. Introduction of natural biopolymer has beneficial effect on biological recognition signals and thus cells are expected to migrate deeper into the scaffold. Additionally, improved elasticity and deformability facilitate formation of new or expansion of existing cavities for cell penetration (7). Despite the known advantages and wide applicability of biomaterials, there are several limitations that restrict their use for biomedical applications (8). Mainly, they lack adequate mechanical properties and in many instances the stability in aqueous and physiological environments required for medical applications (9). In this milieu, crosslinking techniques has been broadly used to overcome the limitations of biomaterials (10). According to this, glutaraldehyde (GA) is a well-known chemical crosslinker that can react with functional groups in both proteins and carbohydrates, and can provide materials with substantial improvement in tensile properties (11). The aim of this study is to evaluate different GA concentrations to crosslink type A gelatin in order to evaluate the robust potential of modified gelatin in tissue engineering and regenerative medicine. MATERIALS AND METHODS Preparation of gelatin hydrogels The method of hydrogel preparation is deeply described by Coester CJ et al (12). Briefly, 1,25 g of natural type A gelatin powder (from porcine skin) (Sigma-Aldrich Steinheim, Germany) was added to 25 ml of distilled water under heating. After 10 min of stirring, 500 ml of three different concentration of GA (2,5%, 5% and 8%) (Sigma-Aldrich, Steinheim, Germany) was added to crosslink the gelatin. Each group was done in triplicate. The solution was rapidly homogenized and dropped in a petri dish. Jellification process was performed at room temperature during10 minutes. Characterization of macroscopic properties Once biomaterials gelled, a macroscopic evaluation was performed to test the homogeneity, color and transparency of the gelatin. Moreover, a thermal test was performed to determine its stability at physiological temperature incubating the hydrogel at 37ºC for 24h. Characterization of microscopic properties Histological evaluation For the histological analyses a representative sample was harvested of each hydrogel. The fragments were fixed in 10% buffered formalin for 24h at room temperature. Fixed samples were routinely dehydrated and embedded in paraffin, and transverse 5 μm sections were obtained from their central parts. All samples sections were stained with hematoxylin and eosin (HE) and observed and captured using light microscopy at 200X (Nikon eclipse 90i, Japan). Structural and ultrastructural evaluation For the structural and ultrastructural evaluation, the samples were fixed in 2.5% GA in 0.05 M cacodylate buffer, pH 7.2, at 4°C for 6 h. Then the samples were washed three times in 0.05 M cacodylate buffer, pH 7.2, at 4°C and randomly assigned to scanning electron microscopy (SEM) samples were dehydrated in increasing concentrations of acetone (30–100%), subjected to the critical point method and covered with gold. Samples were transversally mounted in stabs. Analyses and imaging were carried out using a FEI Quanta 200 environmental scanning electron microscope (FEI Europe, Eindhoven, The Netherlands). RESULTS Preparation of the hydrogels The application of the previously described methods resulted in the gelation of all replicates in each group obtaining solid gelatin hydrogels. Gelation of biomaterials was GA-dependent as higher concentrations required shorter periods of gelation . However, in no cases gelation longed more than 30 min. Macroscopy characterization of crosslinked type A gelatin 2,5% GA concentration hydrogel presented a homogenous appearance according with its particular transparency and a light clear color. Although it presents a gel state at room temperature, its thermal stability was compromised when the temperature rises physiological conditions and it melts at 37ºC. Gelatin crosslinked with 5% GA exhibited some distortions in its homogeneity emerging some areas with different crosslinked properties (see arrows in Figure 1 ). Moreover, transparency was maintained through the homogeneous areas but not in the more crosslinked ones which correlate with the areas of a more intense yellow color. Furthermore, gel state was maintained in the physiological range of temperatures exhibiting a thermostable characteristic. On the contrary to the above mentioned 8% GA concentration hydrogel, showed a complete heterogeneous appearance with non-defined areas of different crosslinked properties. This hydrogel did not spread over the dish and its color turned faint yellow compromising the transparency of the gel. Finally, the thermal stability was confirmed as no differences were found between room temperature and 37ºC. Figure 1: Macroscopic analysis of crossliked gelatin hydrogels. Arrows show areas with different crosslinked properties.