Bio-functionalization and in-vitro evaluation of titanium surface with recombinant fibronectin and elastin fragment in human mesenchymal stem cell

Abstract

Titanium is a biomaterial that meets a number of important requirements, including excellent mechanical and chemical properties, but has low bioactivity. To improve cellular response onto titanium surfaces and hence its osseointegration, the titanium surface was bio-functionalized to mimic an extracellular matrix (ECM)-like microenvironment that positively influences the behavior of stem cells. In this respect, fibronectin and elastin are important components of the ECM that regulate stem cell differentiation by supporting the biological microenvironment. However, each native ECM is unsuitable due to its high production cost and immunogenicity. To overcome these problems, a recombinant chimeric fibronectin type III9-10 and elastin-like peptide fragments (FN9-10ELP) was developed herein and applied to the bio-functionalized of the titanium surface. An evaluation of the biological activity and cellular responses with respect to bone regeneration indicated a 4-week sustainability on the FN9-10ELP functionalized titanium surface without an initial burst effect. In particular, the adhesion and proliferation of human mesenchymal stem cells (hMSCs) was significantly increased on the FN9-10ELP coated titanium compared to that observed on the non-coated titanium. The FN9-10ELP coated titanium induced osteogenic differentiation such as the alkaline phosphatase (ALP) activity and mineralization activity. In addition, expressions of osteogenesis-related genes such as a collagen type I (Col I), Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), bone sialo protein (BSP), and PDZ-binding motif (TAZ) were further increased. Thus, in vitro the FN9-10ELP functionalization titanium not only sustained bioactivity but also induced osteogenic differentiation of hMSCs to improve bone regeneration.

Fig 1. Expression of chimeric FN9-10_ELP and schematic illustration of bio-functionalization for enhanced cellular responses on titanium discs.

(A) The purity and molecular weight of chimeric FN9-10_ELP were measured by 12% SDS-PAGE and Western blotting. The molecular weight shown is approximately 38 kDa. (B) Bio-functionalization of titanium discs using chimeric FN9-10_ELP to induce osteogenic differentiation of hMSCs.

Fig 2. Protein attachment activity of FN9-10_ELP on the titanium discs.

The titanium discs were immersed overnight in various concentrations of FN9-10_ELP (0−25 μg) in 6-well plates at 4°C. The absorbance of FN9-10_ELP was quantified by ELISA using a His-tag probe and reported the mean ± SD (n = 3). p< 0.001.

Fig 3. In vitro cumulative release profiles of FN9-10_ELP from titanium discs.

The titanium discs were coated with FN9-10_ELP (10 μg·mL⁻¹) and a control substance (5% BSA) in a volume of 500 μL/disc overnight at 4°C. The amount of FN9-10_ELP absorbed from titanium discs was measured using an ELISA. The cumulative release was evaluated as the initial attachment amount (100%). The Release profiles indicate the mean ± SD (n = 3).

Fig 4. Cell adhesion activity of hMSCs according to incubation time on the FN9-10_ELP-titanium discs.

The titanium discs were immersed overnight in 10 μg·mL⁻¹ of FN9-10_ELP at 4°C, and while the non-coated titanium discs served as the control. The hMSCs were seeded at a density of 1 × 10⁵ cells/disc on the titanium discs and incubated at 37°C for up to 150 min. The hMSCs adhesion activities were evaluated by crystal violet assay and are presented as the mean ± SD (n = 3). p < 0.001.

Fig 5. Cell proliferation activity of hMSCs on the FN9-10_ELP-titanium discs for 0, 4 and 8 days.

The titanium discs were immersed in 0 or 10 μg·mL⁻¹ FN9-10_ELP overnight at 4°C then, hMSCs were seeded at a density of 1 × 10⁴ cells/disc on titanium discs and incubated for 0, 4 and 8 days at 37°C. The absorbance of formazan contained in the cells was used as a measure of cell proliferation. The cell proliferation activities are expressed as the mean ± SD (n = 3). p < 0.001.

Fig 6. ALP activity of hMSCs on the FN9-10_ELP-titanium discs for 0, 5 and 10 days.

The titanium discs were immersed in 10 μg·mL⁻¹ FN9-10_ELP overnight at 4°C, while the non-coated discs served as the control. The hMSCs were seeded at a density of 5 × 10³ cells/disc and incubated for 0, 5 and 10 days at 37°C. The ALP activities were normalized to the control and are reported as the mean ± SD (n = 3). p < 0.01.

Fig 7. Mineralization activity of hMSCs on the FN9-10_ELP-titanium discs for 0, 5 and 10 days.

The titanium discs were immersed in 10 μg·mL⁻¹ FN9-10_ELP overnight at 4°C, while the non-coated discs served as the control. The hMSCs were seeded at a density of 5 × 10³ cells/disc and incubated for 0, 5 and 10 days at 37°C. The mineralization activities are reported as the mean ± SD (n = 3). p < 0.05.

Fig 8. Osteogenic differentiation activities of hMSCs on the FN9-10_ELP-titanium discs for 20 days.

The titanium discs were immersed overnight in 10 μg·mL⁻¹ of FN9-10_ELP at 4°C, while the non-coated discs served as the control. The hMSCs were seed at a density of 5 × 10³ cells/disc and incubated for 20 days at 37°C. (A) The mRNA levels of osteogenesis-related genes (Col I, RUNX2, OPN, OCN, BSP, TAZ) were analyzed via real-time PCR, and the comparative mRNA levels of each gene were evaluated relative to that of the β-actin as an internal control. The osteogenic differentiation activities are presented as the mean ± SD (n = 3). p < 0.05 and p < 0.01. (B) The protein levels of Col I, OPN, and OCN were analyzed via western blotting. Band density values of Col I, OPN, and OCN were normalized with β-actin and these proteins were represented as an arbitrary ratio compared to control titanium.