Mussel-inspired Ultrathin Film on Oxidized Ti-6Al-4V Surface for Enhanced Osseointegration and Antibacterial Capability

Wang, Ziyuan (Department of Mechanical Engineering, University of Manitoba)
Ojo, Olanrewaju (Department of Mechanical Engineering, University of Manitoba)
Xing, Malcolm (Department of Mechanical Engineering, University of Manitoba)

Introduction

Failure of osseointegration is the most challenging issue for bio-implants. Various modification methods have been proposed to improve titanium osseointegration capability. Among those, thermal oxidation has been demonstrated to improve in vitro and in vivo performance of titanium implants. However, the implants only passively integrate with bone, give rise to the potential of implant loosening and failure of osseointegration [1]. Meanwhile, bacterial contamination, which results in the formation of a highly resistant biofilm on implant surface, also largely comprises the practical application of thermally oxidized Ti-6Al-4V. Recently, the LBL self-assembly of polyelectrolytes has gained increasing interest. The films formed by electrostatic force attraction are ideal platforms to mimic the extracellular microenvironment [2]. Assembly of different polyelectrolytes such as chitosan/alginate, PLL/DNA and chitosan/gelatin has been carried out to regulate implant-cell interactions [3]. Meanwhile, the LBL film can also function as a carrier to load bone-related chemicals like antibacterial nano-silver, fibronectin, BMP 2, etc. [3]. Aims to improve the osseointegration and antibacterial capability of thermally oxidized Ti-6Al-4V, we herein fabricated an ultrathin alginate/chitosan surface film that contains nano-silver through poly(dopamine), which exhibits a comparable structure to the adhesive proteins secreted by mussels (Figure 1). In this study, we first monitored the alginate/chitosan film fabrication process and selected the optimal alginate/chitosan pair number to be constructed onto the thermally oxidized Ti-6Al-4V surface. Then we incorporated nano-silver into the selected alginate/chitosan film and characterized the topographical properties of different samples. Finally, the in vitro performance of the Ti-6Al-4V-based hybrid structure was evaluated through BMSC viability, morphology, proliferation and differentiation assays.

Materials and Methods

Thermal oxidation of Ti-6Al-4V was conducted in a laboratory furnace under atmospheric condition at 650°C for 8 hours. To fabricate chitosan/alginate LBL pairs, substrates were first dipped in dopamine solution overnight to get a precursor layer. The multilayer construction was conducted on spin-coater (Heady way, US) by alternatively coating chitosan (5mg/ml) and alginate (5mg/ml) at 2500rpm for 90s, followed by washing with ultrapure water twice under 1000rpm for 60s. This process was repeated until desired pairs were obtained. Nanosilver was incorporated by immerse LBL-ed substrates into dopamine(2mg/ml, 3h), then change the solution to 10mM AgNO3 for the other 3hrs. Contact angle goniometer, AFM, SEM, XPS was employed to characterize the resultant Ti-6Al-4V substrates. In vitro experiments were conducted on bone marrow stem cells by cell adhesion, MTT and rt-qPCR tests.

Results

Surface wettability and XPS demonstrated the sucessful fabrication of chitosan/alginate film onto thermally oxidized Ti-6Al-4V. AFM analysis indicates that the surface topography and roughness induced by thermal oxidation was not altered by LBL fabrication(Fig. 2). BMSC viability and morphology were significantly increased by the the (A/C)5/Ag film (Fig. 3 A and B). More importantly, the expression of ALP, type I collagen and BSP was up-regulated after BMSCs were cultured on TO/(A/C)5/Ag substrates(Fig. 3 C). Incorporation of nanosilver in the hybrid architecture was demonstrated to inhibit the growth of Escherichia coli and Staphylococcus aureus.

Discussion and Conclusion

Mussel-inspired chitosan/alginate layer-by-layer self-assembly film with nanosilver particles was fabricated on thermally oxidized sub-micron Ti-6Al-4V alloy. The chitosan-alginate film completely altered the Ti-6Al-4V surface chemistry while the sub-micron topography induced by thermal oxidation was not changed. Enhanced BMSCs adhesion, viability and demonstrated antibacterial ability was observed in vitro. ALP, Col I and BSP indicated that BMSCs differentiation was also positively stimulated by this hybrid system. Based on these results, we propose that such hybrid system has a promising application on fast osseointegration and anti-infection.

Fig. 1. Construction process of the mussel-inspired film onto thermally oxidized Ti-6Al-4V surface.

Fig. 2. (A) N 1s high resolution spectra and (B) surface topography of Ti-6Al-4V after different treatments (A: alginate; C: chitosan).

Fig. 3. (A) Viability and morphology, (B) Proliferation and (C) Differentiation of BMSCs seeded on TO and TO/(A/C)5/Ag samples, respectively.

Acknowledgements

Financial supports from NSERC, MHRC and MICH are gratefully acknowledged.

References

[1] MendonÁ§a, Gustavo, et al. "Advancing dental implant surface technology from micron-to nanotopography." Biomaterials 29.28 (2008): 3822-3835. [2]Hu, Yan, et al. "Regulation of the differentiation of mesenchymal stem cells in vitro and osteogenesis in vivo by microenvironmental modification of titanium alloy surfaces." Biomaterials 33.13 (2012): 3515-3528. [3]Gribova, et al. "Polyelectrolyte multilayer assemblies on materials surfaces: from cell adhesion to tissue engineering." Chemistry of Materials 24.5 (2011): 854-869.

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