TiO2 Nanotube Implants Modified with Silk Fibroin and Mesoporous Silica Nanocomposite Coatings Enable Efficient Drug Release to Promote Osteogenesis

Abstract

Enhanced bone healing within 1 week after post-titanium (Ti) dental implant surgery especially contributes to the subsequent long-term osseointegration, and the commonly used drug-loaded TiO₂ nanotubes (TNTs) can promote osteogenesis yet still face the challenge of burst drug release that makes it difficult to maintain long-term effective drug concentrations and good osseointegration. Here, we prepared a double drug loading/release system of silk fibroin/mesoporous silica nanoparticles (SF/MSN) nanocomposite coating modified TNTs (TAMA) with AZD2858 (Wnt/β-catenin pathway agonist for promoting osteogenesis) as the therapeutic drug, realizing a long-term stable drug release and better osteogenesis. The increased β-sheet content of SF reduced the degradation rate of the SF/MSN coating, thus avoiding the AZD2858 burst release. The adsorption of MSN maintained the effective drug concentration more than 1 week that was especially critical for early bone healing. Under the protection of SF/MSN coating, the TAMA implant showed a well-organized spatial release of AZD2858, well enabling the osteogenic differentiation and mineralization at cellular level for up to 21 days. Animal experiments further demonstrated that the slow release of AZD2858 in the TAMA implant effectively activated the Wnt/β-catenin pathway, enabling rapid bone healing in the early stage of implantation and finally achieving the best osseointegration efficacy. Thus, this study proposed an efficient strategy for developing high-performance dental implants via the construction of a biodegradable SF/MSN coating.

Keywords: TiO2 nanotubes; coating; dental implant; drug delivery; osseointegration.

1

Loading the SF/MSN nanocomposite coating onto the surface of drug-loaded TNTs, which continuously release AZD2858 for more than 1 week around the implant to promote osseointegration.

2

Characterization of different specimens. (a) Loading the SF/MSN nanocomposite coating on the surface of AZD2858-loaded TNTs. (b) FE-SEM and TEM images of surface and cross-sectional morphologies of different specimens. (c) The pore size distribution analysis of MSN. (d) The particle size distribution analysis of MSN. (e) The nitrogen adsorption–desorption isotherm of MSN. (f) Nanoscratch results for TAMA. (g) Water contact angle of different specimens.

3

Drug release and degradation behavior of the coatings. (a,b) Coatings secondary structure analysis by FTIR spectroscopy. (c,d) The drug release of AZD2858 from different specimens. (e,f) The degradation behavior of the coating of TAMA. Figure a: Created in BioRender. Mu, Y. (2025) https://BioRender.com/opi22ox.

4

In vitro biocompatible and osteogenic assessment of MC3T3-E1 cells. (a) Surface morphology (FE-SEM) of cells adhered on different specimens after incubation for 4 h. (b) Cell proliferation ability evaluated through CCK-8 for 1, 3, and 5 days. (c and d) ALP quantitative and staining results of cells incubated on the different specimens for 7 and 14 days of osteogenic induction. (e and f) Alizarin red staining and quantitative results of the cells seeded on different specimens at 21 days of osteogenic induction. (g) mRNA levels of osteogenic differentiation-related genes (β-catenin, AXIN2 and ALP) in cells cultured on the different specimens at 14 days after seeding in osteogenic medium. (h) Western blotting assay of β-catenin, RUNX2 and OCN protein expression of MC3T3-E1 cells cultured on different specimens for 7 days, and (i) relative density quantification was normalized to β-actin.

5

In vivo osteogenic assessment. (a and b) Representative 3D-reconstructed micro-CT images of the bone around each group of implants after 4 and 8 weeks. (c) Quantitative statistics of BV/TV, Tb.Th, Tb.N and Tb.Sp according to the micro-CT images after 4 and 8 weeks. (d and e) Histological analysis of peri-implant new bone formation by methylene blue/acid fuchsin and H&E staining after 4 and 8 weeks (*P < 0.05, **P < 0.01, ***P < 0.001).

6

TAMA implants accelerate osseointegration by rapid activation of Wnt/β-catenin pathway. (a) and (b) Representative images of immunofluorescence costaining RUNX2 (red) and OSX (green) of the bone tissue in each group of implants after 8 weeks and the quantitative analysis of fluorescence intensity. (c and d) Representative images of immunofluorescence costaining β-catenin (red), ALP (green) and AXIN2 (pink) of the bone tissue in each group of implants after 8 weeks and the quantitative analysis of fluorescence intensity. (e and f) Representative images of immunofluorescence costaining Collagen-I (red), OPN (green) and OCN (pink) of the bone tissue in each group of implants after 8 weeks and the quantitative analysis of fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001.