Composições farmacêuticas com atividade osteogênica com proteínas hidrolisadas da seda: estudo in vitro e in vivo

Abstract

Bioactive molecules, such as proteins, play a fundamental role in promoting bone regeneration by directly influencing the performance of regenerative biomaterials. In this thesis, the behavior of hydrolyzed silk proteins incorporated into cement composite formulations for bone regeneration was investigated. In addition, a technology and literature survey was carried out based on scientific articles and patents. A detailed analysis of the collected data provided a comprehensive overview of the scientific and technological development related to silk proteins (fibroin and sericin), associated with bone regeneration biomaterials such as hydroxyapatite and chitosan. The results demonstrate a significant increase in publications and patents over the last two decades, reflecting the growing global interest in innovative solutions for tissue engineering and bone regeneration. Experimentally, two solid-phase formulations were prepared: one consisting of hydroxyapatite and agalmatolite (C1), and another consisting of hydroxyapatite, agalmatolite, and chitosan (C2), both combined with different concentrations of hydrolyzed silk proteins (liquid phase): 0%, 1%, 10%, and 25%. The cement composites were characterized physicochemically, in vitro, and in vivo. X-ray diffraction and Fourier-transform infrared spectroscopy analyses confirmed the incorporation of silk proteins into the formulations. Thermal stability was altered by the presence of chitosan. Scanning electron microscopy images revealed irregular and porous surfaces, with morphological changes associated with the addition of proteins. Bioactivity in simulated body fluid was confirmed by the formation of an apatite layer on the surface of the cements. Continuous Ca²⁺ ion release was observed, with lower release rates in formulations containing chitosan. The pH of all formulations remained stable at 7.4. In wettability tests, the presence of chitosan imparted a hydrophilic character to the composites, while the addition of silk proteins further enhanced this property as concentration increased. Biological tests confirmed the biocompatibility of the formulations, promoting the proliferation of co-cultures of L929 fibroblasts and MC3T3 pre-osteoblasts. Cements containing chitosan showed better viability in indirect tests, whereas cements without chitosan showed superior performance compared to those containing chitosan. Specific assays for bone regeneration markers, such as alkaline phosphatase activity, osteocalcin, nitric oxide, and mineralization, showed promising results for tissue regeneration. In the in vivo tests, C2 formulations (with chitosan) at all concentrations were evaluated, and histological analyses indicated varying levels of integration and stimulation of bone regeneration. Inflammatory infiltration was observed in all groups, with progressive reduction and presence of collagen fibers. C2-1 and C2-10 cements showed fewer TRAP-positive osteoclasts compared to C2-25, which exhibited a stronger inflammatory response. Histomorphometric analysis revealed that formulations with lower protein concentrations favored greater bone formation or achieved the same level of regeneration as higher concentrations. The results highlight the potential of the developed cement composites, especially those with lower concentrations of hydrolyzed silk proteins, for applications in bone regeneration.