Abstract: BACKGROUND: As nanomaterials and medical devices containing nanomaterials have unique characteristics such as small size and large specific surface area, which may lead to special biological effects, their biological safety has become the focus of attention. 
OBJECTIVE: To evaluate the biosafety of poly(lactic acid)/nano-hydroxyapatite composite microspheres.
METHODS: Poly(lactic acid)/nano-hydroxyapatite composite microspheres were prepared by emulsion solvent evaporation. (1) Cytotoxicity test: Mouse fibroblasts were co-cultured with poly(lactic acid)/nano-hydroxyapatite composite microspheres extract. Blank control (complete medium), negative control (high-density polyethylene resin extract) and positive control (zinc diethyldithiocarbamate extract) were set. The cytotoxicity was evaluated by MTT assay. (2) Sensitization reaction: Polar and non-polar extraction of the sample, intradermal induction and excitation test were conducted, and the results were recorded. (3) Intradermal reaction: Polar extraction and non-polar extraction of composite microspheres were performed. Immediately, 24, 48 and 72 hours after intradermal injection, the status of each injection site was observed. (4) Acute systemic toxicity: The composite microspheres were extracted by polar extraction and non-polar extraction. After intravenous and intraperitoneal injection into the body of KM mice, the changes in body weight of mice were observed. (5) Pyrogen reaction: After polar extraction, the composite microspheres were injected through the ear vein into the rabbit to record changes in body temperature. (6) Bone implantation test: Cylindrical composite microspheres with high-density polyethylene rods were implanted into the tibial defects of rabbits for gross observation and local histopathological observation. 
RESULTS AND CONCLUSION: (1) The poly(lactic acid)/nano-hydroxyapatite composite microspheres had no cytotoxicity, sensitization, intradermal irritation, or acute systemic toxicity. The pyrogen reaction was in line with the test regulations. After the composite microspheres were implanted into the tibial defect of rabbits, no lesions such as hematoma, edema or inflammation were observed at the implantation site, and the histological observation showed that the composite microspheres had a certain ability of bone tissue repair. (2) Bone results show that poly(lactic acid)/nano-hydroxyapatite composite microspheres had good biosafety. 

Key words: nano-hydroxyapatite, poly(lactic acid), composite material, cytotoxicity, sensitization reaction, intradermal reaction, acute systemic toxicity, pyrogen reaction, bone implantation test