Immobilization of chitosan film containing semaphorin 3A onto a microarc oxidized titanium implant surface via silane reaction to improve MG63 osteogenic differentiation

Kaixiu Fang,^1,* Wen Song,^2,* Lifeng Wang,¹ Sen Jia,³ Hongbo Wei,¹ Shuai Ren,¹ Xiaoru Xu,¹ Yingliang Song<sup>1

1</sup>State Key Laboratory of Military Stomatology, Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China; ²State Key Laboratory of Military Stomatology, Department of Prosthetic Dentistry, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China; ³State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, People’s Republic of China

*These authors contributed equally to this work

Abstract: Improving osseointegration of extensively used titanium (Ti) implants still remains a main theme in implantology. Recently, grafting biomolecules onto a Ti surface has attracted more attention due to their direct participation in the osseointegration process around the implant. Semaphorin 3A (Sema3A) is a new proven osteoprotection molecule and is considered to be a promising therapeutic agent in bone diseases, but how to immobilize the protein onto a Ti surface to acquire a long-term effect is poorly defined. In our study, we tried to use chitosan to wrap Sema3A (CS/Sema) and connect to the microarc oxidized Ti surface via silane glutaraldehyde coupling. The microarc oxidization could formulate porous topography on a Ti surface, and the covalently bonded coating was homogeneously covered on the ridges between the pores without significant influence on the original topography. A burst release of Sema3A was observed in the first few days in phosphate-buffered saline and could be maintained for >2 weeks. Coating in phosphate-buffered saline containing lysozyme was similar, but the release rate was much more rapid. The coating did not significantly affect cellular adhesion, viability, or cytoskeleton arrangement, but the osteogenic-related gene expression was dramatically increased and calcium deposition was also abundantly detected. In conclusion, covalent bonding of CS/Sema could strongly improve osteogenic differentiation of osteoblasts and might be applied for Ti implant surface biofunctionalization.

Keywords: titanium, semaphorin 3A, silane reaction, microarc oxidation, osteogenic differentiation