Histomorphometric evaluation, SEM, and synchrotron analysis of the biological response of biodegradable and ceramic hydroxyapatite-based grafts: from the synthesis to the bed application.

Abstract

This study aimed to analyze the physicochemical and histological properties of nanostructured hydroxyapatite and alginate composites produced at different temperatures with and without sintering, and implanted in rabbit tibiae. Hydroxyapatite-alginate (HA) microspheres (425-600µm) produced at 90 and 5 ºC without (HA90 and HA5) or with sintering at 1000ºC (HA90S and HA5S) were characterized by XRD, FT-IR, SEM, and in vitro degradation assay; also were implanted in bone defects on rabbit's tibiae (n=12). The animals were randomly divided into five groups (blood clot, HA90S, HA5S, HA90, and HA5) and euthanized after 7 and 28 days. The samples were assessed by light (histomorphometry) and polarized light microscopy, SEM-BSE microscopy, EDS-SEM elemental analysis, and synchrotron radiation X-ray microfluorescence. The non-sintered biomaterials had a lower crystallinity than sintered materials, being more degradable in vitro and in vivo. However, the sinterization of HA5 led to the apatite phase's decomposition into tricalcium phosphate (TCP). Histomorphometric analysis showed the highest (p<0.01) bone density in the blood clot group, similar bone levels among HA90S, HA90, and HA5, and significantly less bone in the HA5S. HA90 and HA5 groups presented higher degradation and homogeneous distribution of the new bone formation onto the surface of biomaterials fragments, compared to HA90S, presenting bone only around intact microspheres (p<0.01). EDS-SEM and μXRF-SR mapping techniques showed the elemental distribution of Ca, P, and Zn in the newly formed bone similar to the cortical bone indicating the bone maturity at 28 days. The synthesized biomaterials are biocompatible and osteoconductive. The heat treatment directly influenced the material's behavior, where non-sintered HA90 and HA5 showed higher degradation allowing a better distribution of the new bone onto the surface of the biomaterials fragments compared to HA90S presenting the same level of new bone, but only on the surface of the intact microspheres, potentially reducing the bone-biomaterial interface.