Utilização de agente de reticularização em matriz descelularizada (SCAFFOLD) de pele de tilápia (Oreochromis niloticus) para o desenvolvimento de biomaterial com aplicação em medicina regenerativa

Abstract

Medical devices derived from extracellular matrix (ECM) of animal origin available on the market are expensive and inaccessible to the low-income population. To meet the criteria of a good material, tests are needed that guarantee resistance and are promising in terms of cost/benefit for patients. Recognizing the great therapeutic value of tilapia skin and the already developed scaffold, it is necessary to optimize this matrix and modify the structural properties so that they acquire durability, resistance and biocompatibility for purposes in internal and reconstructive applications of regenerative medicine. Such properties can be improved through reticularization, that is, the addition of crosslinks in the protein structure of the extracellular matrix of the skin. Therefore, this work aimed to develop a protocol for the production of reticular decellularized protein matrix (scaffold) derived from tilapia skin (Oreochromis niloticus). For this purpose, the scaffolds were produced and then submitted to decellularized matrix modification protocols by adding the chemical crosslinking agent Glutaraldehyde (GTA). The experimental design consisted of: 4 protocols (4 concentrations - 0.05%, 0.25%, 0.625% and 1% and 2 different incubation times - 24h and 72h), totaling 8 test groups compared to the scaffold without treatment and fresh tilapia skin. All samples were submitted to histological analysis (Hematoxylin-Eosin - HE and Picrosirius Red), tensiometric, biochemical (degradation and hydrolysis) and cytotoxic analysis following ISO 10993-5:2009. Quantitative data were analyzed using the ANOVA test (specify post tests), with a significance level of 95%, using the GraphPad Prism® software. The results demonstrate that crosslinking protocol at lower concentration (0.05%) and incubation time (24h) was the most promising for future in vivo tests. The addition of cross-links enabled a lower level of enzymatic biodegradation (DH), optimized fibrillar organization both in thickness and in quantification of total collagen, types I and III and the proportion I/>III. The structural characteristic of the biomaterial (scaffold without treatment) was preserved in all protocols, except for the highest concentration (1%) regardless of the incubation time. After reticularization, the biomaterial showed greater resistance to traction, demonstrating a greater degree of rigidity and elasticity of the matrix, corroborating the histological findings. All tested protocols passed the cytotoxicity test. All protocols demonstrate similarity with fresh tilapia skin in terms of mechanical resistance and in vitro biocompatibility. Regarding time optimization, the biggest advantage is the cost/benefit ratio in the biomaterial production process; because, at lower concentrations and shorter contact time between the scaffold and the GTA, greater physical resistance (elasticity), biochemical resistance (less enzymatic lysis action) and in vitro biocompatibility are obtained. Finally, the decellularized and reticularized ECM showed better biological behavior and collagen stability with resistance capacity to the in vitro biodegradable enzymatic action at lower concentrations and contact times with the GTA agent. Future in vivo tests will help to understand the long-term durability of this biomaterial for internal and reconstructive surgeries in regenerative medicine.