190 | Surface engineering of titanium implants with vanillin-chitosan and cerium doped-nanoparticle coatings for improved osteoimmune response

Ioannis Tsamesidis¹, Styliani Axypolitou¹, Georgia K. Pouroutzidou¹, Maria Bousnaki¹, Dimitrios Bikiaris¹, Evi Christodoulou¹, Antonios D. Anastasiou², Eleana Kontonasaki¹ | ¹Aristotle University of Thessaloniki, Greece ²University of Manchester, UK.

Approximately 10–15 million dental implants are placed annually worldwide, with titanium and its alloys remaining the materials of choice due to their favorable mechanical properties and biocompatibility. However, the bioinert nature of conventional titanium surfaces limits their ability to actively promote bone regeneration and modulate inflammatory responses, driving interest in advanced surface modification strategies. In this study, smart implant coatings based on vanillin (VA)-crosslinked chitosan (CHS) with 5% incorporated cerium (Ce) silica mesoporous nanoparticles (Ce-MSNs) were developed to enhance implant–tissue interactions. The coatings abbreviated as CHS-VA-Ce blend, were synthesized via Schiff base formation between vanillin aldehyde groups and chitosan amino groups, with additional stabilization through hydrogen bonding and the addition of the Ce-MSNs at the final stage of the synthetic route. The biological performance of the coated implants was evaluated under both static and dynamic conditions with human periodontal ligament cells (hPDLCs) and their immunomodulatory capacity through RAW-264.7 macrophages. In details, the tested materials were disinfected by UV irradiation for 30 minutes before being used for further experiments. CHS and CHS-VA-Ce blend tested at three different concentrations (C1:1, C2:0.75, and C3:0.5 mg/mL). CHS and CHS- VA blend samples for the in vitro experiments were added in a 96-well plate and subsequently hPDLCs with 104 cells per well were seeded to each well and incubated at 37°C for 1, 3, and 5 days for the MTT assay and up to 28 days for osteogenic differentiation evaluation. RT-PCR was used to evaluate the expression of various osteogenesis genes such as BMP-2, RUNX-2, OCN, ALP and genes related to immunomodulation such as TNF-alpha and Araginase-1. Biomineralization was evaluated with ALP activity at days 7 and 14 and Alizarin red staining (ARS) at days 21 and 28. To assess the biological behaviour of the polymers and its interaction with cells in simulated microenvironment, experiments in microfluidics were also conducted. The CHS–VA-Ce blend coatings demonstrated favorable cytocompatibility and osteogenic potential under both static and dynamic conditions. MTT assays revealed sustained high viability of hPDLCs cultured on CHS and CHS–VA blend surfaces over 1, 3, and 5 days, with no cytotoxic effects observed. Gene expression analysis by RT-PCR showed upregulation of key osteogenic markers, including BMP-2, RUNX-2, ALP, and OCN, indicating enhanced osteogenic differentiation in cells exposed to the vanillin-crosslinked coatings. Biomineralization studies further supported these findings, as increased ALP activity was detected at day 7 and decreased at day 14, along with pronounced calcium deposition confirmed by Alizarin Red S staining at days 21 and 28. In microfluidic experiments simulating a dynamic microenvironment, the VA-Ce blend coatings maintained excellent cell viability across all tested concentrations the experimental period, highlighting their stability and biofunctionality under flow conditions. Overall, these results suggest that vanillin-crosslinked chitosan incorporated Ce-MSNs coatings represent a promising smart surface modification strategy for titanium dental implants, capable of supporting cell viability, promoting osteogenic differentiation, and performing effectively under physiologically relevant dynamic conditions.