Loading icariin on titanium surfaces by phase-transited lysozyme priming and layer-by-layer self-assembly of hyaluronic acid/chitosan to improve surface osteogenesis ability

Yunjia Song,1,2 Aobo Ma,1 Jia Ning,1 Xue Zhong,1 Qian Zhang,1 Xu Zhang,1 Guang Hong,3,4 Ying Li,1 Keiichi Sasaki,2 Changyi Li1 1School of Dentistry, Stomatological Hospital, Tianjin Medical University, Tianjin, China; 2Division of Advanced Prosthetic Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan; 3Liaison Center for Innovative Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Japan; 4Faculty of Dental Medicine, Airlangga University, Surabaya, Indonesia Purpose: Icariin (ICA) is one of the main active constituents of Herba Epimedii for improving osteogenesis. It is necessary to create a simple and efficient method to load ICA onto the surface of titanium (Ti) implant. The purpose of this study was to establish a local ICA delivery system via a layer-by-layer (LbL) self-assembly system on phase-transited lysozyme (PTL)-primed Ti surface.Materials and methods: A PTL nanofilm was first firmly coated on the pristine Ti. Then, the ICA-loaded hyaluronic acid/chitosan (HA/CS) multilayer was applied via the LbL system to form the HA/CS-ICA surface. This established HA/CS-ICA surface was characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurement. The ICA release pattern of the HA/CS-ICA surface was also examined. MC3T3-E1 osteoblast culture test and a rat model were used to evaluate the effects of the HA/CS-ICA surface in vitro and in vivo.Results: SEM, XPS and contact angle measurement demonstrated successful fabrication of the HA/CS-ICA surface. The HA/CS-ICA surfaces with different ICA concentrations revealed a controlled release profile of ICA during a 2-week monitoring span. Osteoblasts grown on the coated substrates displayed higher adhesion, viability, proliferation and ALP activity than those on the polished Ti surface. Furthermore, in vivo histological evaluation revealed much obvious bone formation in the ICA-coated group by histological staining and double fluorescent labeling at 2 weeks after implantation.Conclusion: The present study demonstrated that ICA-immobilized HA/CS multilayer on the PTL-primed Ti surface had a sustained release pattern of ICA which could promote the osteogenesis of osteoblasts in vitro and improve early osseointegration in vivo. This study provides a novel method for creating a sustained ICA delivery system to improve osteoblast response and osseointegration. Keywords: icariin, controlled release, phase-transited lysozyme, layer-by-layer self-assembly, surface modification of titanium, osteogenesi