Caracterização física e avaliação biológica in vivo de um compósito de hidroxiapatita-prata para regeneração óssea

Abstract

Introduction: Repairing large bone defects in the craniofacial region is a major challenge in the context of tissue regeneration. Inherent limitations of autogenous grafts render the use of synthetic grafts more convenient, with a special emphasis on hydroxyapatite (HAp), a calcium phosphate that is similar to natural bone. The association of HAp with other materials seeks to overcome the limited mechanical properties of HAp, which may compromise its performance in extensive lesions or those susceptible to mechanical stress. Silver (Ag) is a metal with a wellestablished antimicrobial activity and has been demonstrating promising osteogenic performance. Objective: To characterize the physical properties of a novel Hap-Ag composite and evaluate its biological performance in an experimental model of critical-size calvarial defect. Methodology: HAp and HAp-Ag powders were obtained and analyzed for morphology by a Field Emission Scanning Electron Microscope (FE-SEM) and evaluated by X-ray diffraction (XRD) to characterize the crystalline phases. Cylindrical pellets obtained from the pressing and sintering of the powders were subjected to the Vickers microhardness test to analyze the fracture toughness, and to wettability analysis to evaluate the hydrophilicity. For the in vivo experiment, 36 male Wistar rats were subjected to surgery to create an 8 mm criticalsize calvarial defect and randomly distributed into three groups (n = 12/group): Control group (CG), untreated defect; HAp group, defect filled with HAp pellet; and Hap-Ag group, defect filled with Hap-Ag pellet. After 90 days of surgery, the animals were euthanized, and calvaria samples were collected and underwent evaluation by computerized microtomography (µ-CT), histological staining with hematoxylin and eosin (H&E), quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6), and analysis of relative gene expression (BMP-2, RUNX2, Procollagen types I and III, TGF-β, Osterix, and eNOS). Data obtained were evaluated by analysis of variance (ANOVA) followed by Tukey's post-test (normal distribution) or by the Kruskall-Wallis test followed by Dunn's post-test (non-normal distribution), with a significance level set at 5%. Results: FE-SEM images revealed powders with irregular and agglomerated morphology, and the elemental analysis by EDS confirmed the elements of HAp formation and the presence of silver in the HAp-Ag sample. Based on XRD analysis, it was possible to observe the formation of HAp with a hexagonal crystalline structure, while for the HAp-Ag pellets there was a reduction in the intensity of the main peaks without the formation of a new phase. The incorporation of Ag into HAp provided an increase in fracture toughness (HAp: 0.66 MPa.m1/2; HAp-Ag: 0.94 MPa.m1/2) and a reduction in the contact angle (36.32° for HAp, and 10.51° for HAp-Ag). Compared to the Control group, defects filled with both biomaterials showed greater bone formation (BV/TV%). The new bone showed less porosities, and a greater number of trabeculae and less trabecular separation. Histological analysis revealed a more advanced stage of bone maturation in the animals in the Hap-Ag group compared to the GC (p < 0.05). Higher levels of TNF-α were observed in the Hap-Ag group, in relation to the GC (p < 0.05), and of IL-1β in relation to the other groups (p < 0.05). A higher gene expression of the pro-collagen 3 (p < 0.05) and eNOS (p < 0.01) markers was observed in the animals treated with the Hap-Ag composite in comparison to the GC group. Conclusion: Within the limitations of the present study, it can be concluded that the incorporation of Ag into HAp provided the production of a biomaterial with better physical properties without compromising its biological potential for new bone formation.