Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells

Abstract

Background: Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials.

Methods: Laser beam irradiation at various fluences (132, 210, or 235 J/cm²) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm² was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay.

Results: The best titanium surface was that produced by laser beam irradiation at 235 J/cm² fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity.

Conclusions: The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

Keywords: Biocompatibility; Human umbilical cord; Laser beam (Yb-YAG); Mesenchymal stem cells; Osteoinduction; Surface modification; Titanium.

Fig. 1

Scanning electron microscopy images of Ti control (a), laser-processed titanium (LPT) produced using laser radiation at 132 J/cm² fluence (b), LPT produced using laser radiation at 210 J/cm² fluence (c), and LPT produced using laser radiation at 235 J/cm² fluence (d). Surfaces at × 100, ×500, ×1000, ×50,000, and × 200,000 magnification

Fig. 2

X-ray diffraction spectra of Ti control (a), laser-processed titanium (LPT) produced using laser radiation at 132 J/cm² fluence (b), LPT produced using laser radiation at 210 J/cm² fluence (c), and LPT produced using laser radiation at 235 J/cm² fluence (d). cps counts per second, Ti titanium

Fig. 3

Scanning electron microscopy micrographs of hUC-MSCs cultured after 24 h on Ti control (a) and laser-processed titanium (LPT) produced using laser radiation at 235 J/cm² fluence (b, c); surfaces at × 3000, ×5000, and × 7000 magnification. CHO-k1 cells after 24 h of culture on Ti control (d) and LPT produced using laser radiation at 235 J/cm² fluence (e, f). Surfaces at × 6000, ×5000, and × 40,000 magnification

Fig. 4

Scanning electron microscopy micrographs after culture for 7 days. hUC-MSCs on Ti control (a) and laser-processed titanium (LPT) (b, c) surfaces. CHO-k1 cells on Ti control (d) and LPT (e, f) surfaces. a x1000, b x500, c x800, d x600, e x400, f x800

Fig. 5

MTT cell metabolic activity assay. hUC-MSC and CHO-k1 cell adhesion and proliferation results on the two different types of titanium discs (laser-processed titanium (LPT) and Ti control) at different times (3 h, and 1, 3, and 7 days). ***Statistically significant differences between cell types at p < 0.001; ### represents statistically significant differences between Ti surfaces p < 0.001. Data represent means of three independent experiments and SD. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, OD optical density

Fig. 6

Alkaline phosphatase (ALP) activity. ALP activity in hUC-MSCs after culture in Dulbecco's modified Eagle’s medium on Ti control and laser-processed titanium (LPT) surfaces after 3 and 7 days (a); culture of hUC-MSCs in osteogenic medium for the same times on Ti control and LPT surfaces (b). Statistically significant differences between Ti surfaces at: ***p < 0.001, *p < 0.05. Values are mean ± SD of two independent experiments. Ti titanium

Fig. 7

Calcium deposition assay. Alizarin Red S staining of hUC-MSCs on Ti control and laser-processed titanium (LPT) after 7 and 14 days of culture in (a) basal medium and (b) osteogenic medium.***Statistically significant differences between Ti surfaces at p < 0.001. N = 3 ± SD. Ti titanium

Fig. 8

Gene expression of osteogenic markers ALPL, RUNX2, BMP2, OCN, and OPN in hUC-MSCs following culture on laser-processed titanium (LPT) for 7 days in basal medium (BM) and osteogenic medium (OM). Gene expression evaluated by ΔΔCt method normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and reported as fold changes in relation to the Ti control. Statistically significant differences between BM and OM at: **p < 0.01, *p < 0.05. Data presented as mean ± SD (n = 2). ALPL alkaline phosphatase, RUNX2 run-related transcription factor 2, BMP2 bone morphogenetic protein 2, OCN osteocalcin, OPN osteopontin