Abstract: BACKGROUND: Pure titanium and titanium alloy implants are widely used in the field of implant restoration due to their excellent biocompatibility and elastic modulus. However, the biological inertness of the surface of titanium-based implants leads to poor integration with surrounding bone tissues, and surface modification is required to improve the bone integration ability of titanium-based implants. 
OBJECTIVE: To fabricate hydroxyapatite/graphene oxide/interleukin-4 composite coatings on pure titanium substrates, and to investigate their physicochemical properties and biocompatibility of the coating materials.
METHODS: Hydroxyapatite/graphene oxide/interleukin-4 composite coatings were prepared on pure titanium substrates by electrochemical deposition and freeze-drying. Titanium sheets loaded with interleukin-4 and titanium sheets loaded with hydroxyapatite/graphene oxide coatings were prepared at the same time, and the physicochemical properties of pure titanium sheets and the above three titanium sheets were characterized. MC3T3-E1 cells were inoculated on the surfaces of pure titanium sheets and the above three titanium sheets, respectively. Cell proliferation was detected by CCK-8 assay and DAPI staining. Cell activity was detected by Calcein-AM/PI staining. Cell morphology and adhesion were observed by scanning electron microscopy.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and Raman spectroscopy confirmed the successful fabrication of the hydroxyapatite/graphene oxide/interleukin-4 composite coating on the titanium surface. The water contact angle of hydroxyapatite/graphene oxide/interleukin-4 group was larger than that of pure titanium group and hydroxyapatite/graphene oxide group, and smaller than that of interleukin-4 group. (2) CCK-8 assay and DAPI staining results showed that hydroxyapatite/graphene oxide/interleukin-4 coating could promote the proliferation of MC3T3-E1 cells. Calcein-AM/PI staining results showed that MC3T3-E1 cells in hydroxyapatite/graphene oxide/interleukin-4 coating group had higher activity and fewer dead cells. Scanning electron microscopy showed that MC3T3-E1 cells adhered to the surfaces of the four groups of materials with good cell morphology. Compared with the pure titanium group, the MC3T3-E1 cells in the interleukin-4 group extended more pseudopodia, the cell-cell connections were closer, and the adhesion area was larger; compared with the hydroxyapatite/graphene oxide group, the MC3T3-E1 cells in the hydroxyapatite/graphene oxide/interleukin-4 group extended more pseudopodia, the cell-cell connections were closer, and the adhesion area was larger. (3) These findings indicate that the hydroxyapatite/graphene oxide/interleukin-4 composite coating possesses favorable physicochemical and biological properties.

Key words: graphene oxide, hydroxyapatite, interleukin-4, implant composite coating, MC3T3-E1 cell, biocompatibility, engineered materials