Physicochemical Characterization, Cytotoxicity, and In Vivo Evaluation of a Hydroxyapatite-Silver Composite for Bone...

This study is aimed at characterizing the physicochemical and biological properties of a hydroxyapatite-silver (HAp-Ag) composite for bone regeneration. HAp and HAp-Ag powders were analyzed using field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and fluorescence, and Fourier transform infrared spectroscopy. Cylindrical pellets obtained by powder pressing and sintering were evaluated for fracture toughness, compressive strength, relative density, porosity, and wettability. Cell viability was assessed using the Alamar Blue assay. In the in vivo experiment, an 8-mm critical-size calvarial defect was surgically created in 36 male Wistar rats randomly divided into three groups: control group (CG), untreated defect; HAp group, defect filled with HAp pellet; and HAp-Ag group, defect filled with HAp-Ag pellet. Euthanasia was performed after 90 days, and calvaria samples were analyzed by μ-CT, histology, cytokine quantification, and gene expression analysis. The powders showed an irregular, agglomerated morphology, with HAp elements and silver detected in the HAp-Ag sample. Incorporation of Ag into HAp increased fracture toughness (0.66-0.94 MPa.m^1/2) and porosity (16.43%-25.95%) and reduced compressive strength (43.17-23.99 MPa) and contact angle (36.32°-10.51°). MC3T3-E1 preosteoblastic cell viability was higher on HAp-Ag surface compared to HAp pellets at Days 1 and 7. Compared to CG, defects filled with biomaterials showed greater bone formation and better bone quality parameters. In addition, a more advanced stage of bone maturation was observed in the animals treated with HAp-Ag. HAp-Ag composite upregulated TNF-α and IL-1β levels and Procollagen 3 and eNOS gene expression compared to CG. Silver incorporation into HAp increased fracture toughness and porosity, reduced compressive strength, and improved wettability. Despite these changes, HAp-Ag enhanced cell viability and in vivo bone maturation, indicating that Ag addition does not compromise and may improve its bone regeneration potential.