Chitosan Coating of TiO2 Nanotube Arrays for Improved Metformin Release and Osteoblast Differentiation

Abstract

Background: Ineffective integration has been recognized as one of the major causes of early orthopedic failure of titanium-based implants. One strategy to address this problem is to develop modified titanium surfaces that promote osteoblast differentiation. This study explored titanium surfaces modified with TiO2 nanotubes (TiO2 NTs) capable of localized drug delivery into bone and enhanced osteoblast cell differentiation.

Materials and methods: Briefly, TiO2 NTs were subjected to anodic oxidation and loaded with Metformin, a widely used diabetes drug. To create surfaces with sustainable drug-eluting characteristics, TiO2 NTs were spin coated with a thin layer of chitosan. The surfaces were characterized via scanning electron microscopy, atomic force microscopy, and contact angle measurements. The surfaces were then exposed to mesenchymal bone marrow stem cells (MSCs) to evaluate cell adhesion, growth, differentiation, and morphology on the modified surfaces.

Results: A noticeable increase in drug release time (3 days vs 20 days) and a decrease in burst release characteristics (85% to 7%) was observed in coated samples as compared to uncoated samples, respectively. Chitosan-coated TiO2 NTs exhibited a considerable enhancement in cell adhesion, proliferation, and genetic expression of type I collagen, and alkaline phosphatase activity as compared to uncoated TiO2 NTs.

Conclusion: TiO2 NT surfaces with a chitosan coating are capable of delivering Metformin to a bone site over a sustained period of time with the potential to enhance MSCs cell attachment, proliferation, and differentiation.

Keywords: MSCs; anodization; mesenchymal bone marrow stem cells; osteogenic differentiation; titania nanotubes; titanium.

Figure 1

Series of SEM images of the pore surface of TiO2 nanotube film using different anodizing times and voltages of (A) 30 V, 1.5 h; (B) 30 V, 3 h; (C) 60 V, 1.5 h; (D) 60 V, 3 h; (E) 85 V, 1.5 h; (F) 85 V, 3 h; (G) 95 V, 1.5 h; (H) 95 V, 3 h; (I) 105 V, 1.5 h; (J) 105 V, 3 h. SEM images of TiO2 nanotube structures fabricated by electrochemical anodization in NH4F/ethylene glycol electrolyte. (K) Typical cross-sectional image of self-supporting TiO2 nanotube layer and the entire structure (nanotube film) on the Ti substrate. (L) The bottom surface showing the nanotube structures detached from underlying Ti substrate.

Figure 2

FTIR spectra of the TAmc sample.

Figure 3

Water contact angle measurements of samples T (A), TA (B), and TAmc (C). AFM images of T (D), TA (E), and TAmc (F). The samples were measured in the dry state.

Figure 4

SEM images of TiO2 NTs after drug loading (Metformin) and spin coating of Chitosan, showing the cross-section of the three (A), five (B), seven (C), ten (D), and fifteen (E) layers of Chitosan. Comparative drug release graph of Metformin from different layers of Chitosan (F).

Figure 5

Flow cytometric analysis of rat bone marrow mesenchymal stem cells, the analysis revealed that their expression of surface antigens CD44 (Passage 3) (A) was strongly positive; while CD45 (B) was negative.

Figure 6

Scanning electron microscope (SEM) images of MSCs cultured on Ti samples after 3 days of incubation, (A) T, (B) TA, and (C) TAmc, (D) MSCs adhesion on titanium surfaces after 2, 4, and 6 h. Data are presented as mean ± SD; n = 3. *p<0.05 compared to control, **p<0.05 compared to control and TA, (E) Cell proliferation of MSCs cells after 1, 3 and 7 days on titanium surfaces was measured with MTT assay. Each bar represents the mean of cell proliferation ± SD (n=3). *p<0.05 compared to control, and TA.

Figure 7

Alkaline phosphatase (ALP) activity (normalized to total protein contents) (A) and Type 1 Collagen expression (B) of MSCs on different titanium samples after 7, and14 days. *p<0.05 compared to control, and TA, **p<0.05 compared to control and TA.

Figure 8

PCR amplification for ALP (TAmc, TA, T) after 14 days and Collagens (TAmc₁, TA_1, T₁) after 21 days of cell culture.