Efeito do tratamento hidrotérmico com esponja de colágeno impregnadas com nanopartículas de hidroxiapatita na proliferação e ativação de osteoblastos e avaliação de sua biocompatibilidade (in vitro/in vivo)

Abstract

The biocomposites of collagen and hydroxyapatite have been widely used in bone defects treatment, allowing the drug delivery and growth factors, as well as serving as scaffold. They also present osteogenic activity by mechanisms not yet clarified. We evaluated the effect of collagen sponges impregnated with different concentrations of nanohydroxyapatite developed by our research group on murine osteoblasts in culture, as well as the biocompatibility of these materials on the back of Wistar rats. Cell viability and proliferation were evaluated by the MTT assay and Ki67 immunolabeling, respectively, after 24 and 48 hours of the osteoblasts incubation with the collagen sponges impregnated with different concentrations of nanohydroxyapatite (CHAP2h and CHAP5h).Osteoblasts incubated with commercially used collagen sponges (C) and collagen sponges without hydroxyapatite prepared by our group (COL and CHAP0h) were used as control groups. The quantification of bone alkaline phosphatase (FAO) levels in the culture medium after 24h, 48h, 5 days and 7 days of incubation was one of the parameters used to investigate the activity of osteoblasts, besides the mineralization test by Von Kossa, after 21 days of cell culture. For the investigation of the mechanisms involved in cell activation, the protein expression of BMP-2 was investigated by Western Blot after 7 days of incubation. The cell morphology was evaluated by scanning electronic microscopy. In the in vivo studies for the evaluation of biocompatibility investigated the inflammatory response induced by the implantation of sponges on the back of male Wistar rats at different observation times. The animals were divided into 3 experimental groups of 12, 12 and 6 animals, respectively, based on different observation periods (24 hours, 7 and 15 days), and the groups were: Control: commercial collagen sponges; (2) Collagen sponges produced by our group; (3) collagen sponges produced by our group carried with nano particles of Hydroxyapatite. A total of 30 animals were added. The following parameters were considered: number of inflammatory cells around the sponges, quantification of myeloperoxidase concentrations (MPO) and cytokines IL-1β and TNF-α in tissues collected by ELISA and immunohistochemistry, as well as quantification of collagen, through the J image software in the subcutaneous tissue around the sponges. After 48 hours of incubation, we observed a significant increase in the number of viable and Ki-67 positive cells in the CHAP5h group.This same group showed more intense mineralization compared to the control groups, besides a significant increase of the FAO activity and expression of BMP-2 after 7 days of incubation. SEM also reveals the presence of hexagonal crystals of hydroxyapatite with high phosphorus content. In vivo, the CHAP5h group showed less migration of inflammatory cells, quantified 1 and 7 days after implantation, and lower IL-1β immunolabeling on the 7th day, associated with the increase in IL-1β collagen synthesis, evaluated 15 days after sponge implantation. The results suggest a positive effect of the CHAP5h group on the proliferation, viability and activation of osteoblasts, possibly due to increased expression of BMP-2 and FAO. The association of the in vitro data and the biocompatibility found in the in vivo study strongly suggests the use of this biomaterial in the regeneration of bone defects.