Effects of Er:YAG laser irradiation of different titanium surfaces on osteoblast response

Abstract

The aim of this in vitro study was to evaluate the effects of erbium-doped yttrium aluminum garnet (Er:YAG) laser irradiation on titanium surface topography and the proliferation and differentiation of osteoblasts using standard clinical treatment settings. Er:YAG laser irradiation at two levels ((1): 160 mJ, pulse at 20 Hz; (2): 80 mJ, pulse at 20 Hz) was applied to moderately rough and smooth titanium disks before MG-63 osteoblast-like cells were cultured on these surfaces. Titanium surface and cell morphology were observed by scanning electron microscopy. Cell proliferation/viability was measured by CCK-8 test. Gene expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and collagen type 1 was measured by qPCR, and OPG and OC protein production was determined by enzyme-linked immunosorbent assay. Treatment with Er:YAG laser at 160 mJ/20 Hz markedly caused heat-induced fusion of titanium and cell condensation on moderately rough surfaces, but not in smooth surfaces. MG-63 proliferation/viability decreased after 5 days in moderately rough surfaces. The expression of ALP, OC, OPG, and collagen type 1 was unaffected by laser treatment at 160 mJ/20. Laser irradiation at 80 mJ/20 Hz enhanced RANKL gene expression after 5 days in moderately rough surfaces. Study results suggest that Er:YAG laser irradiation at clinically relevant setting has no essential effect on osteogenic gene and protein expression of osteoblasts. However, surface structure, cell attachment, and proliferation are influenced by both treatment protocols, which implies that caution should be taken in the clinical treatment of peri-implant diseases when Er:YAG laser is used.

Keywords: Erbium laser; Osteoblasts; Osteogenesis; Surface decontamination; Titanium surface.

Fig. 1

Attachment of osteoblast-like cells on PT surfaces after laser irradiation. MG-63 cells were cultured on smooth or moderately rough surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (PT I and SLA I), 80 mJ/pulse at 20 Hz (PT II and SLA II) or untreated (PT and SLA) surfaces for 2 days and analyzed by scanning electron microscopy (×400). Scale bars correspond to 200 µm

Fig. 2

Proliferation/viability of MG-63 cells grown on different titanium surfaces irradiated with Er:YAG laser. MG-63 cells were cultured on smooth (A) or moderately rough (B) surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (PT I and SLA I), 80 mJ/pulse at 20 Hz (PT II and SLA II) or untreated surfaces (PT and SLA), and proliferation/viability was measured after 2 and 5 days by CCK-8 method. Cells grown on tissue culture plastic (TCP) served as control. Y-axis represents the optical density (OD) values measured at 450 nm. Data are presented as mean ± SEM of three independent experiments

Fig. 3

Expression of osteogenesis-related genes in MG-63 grown on smooth surfaces irradiated with Er:YAG laser. MG-63 cells were cultured on PT, PT I, and PT II surfaces for 2 and 5 days, and the expression levels of ALP (A), OC (B), and COL1 (C) were measured by quantitative real-time PCR. Y-axis represents n-fold expression related to MG-63 grown on tissue culture plastic calculated using 2^−ΔΔCt method, using GAPDH as housekeeping gene. Data are presented as the mean ± SEM of four independent experiments

Fig. 4

Expression of osteogenesis-related genes in MG-63 grown on moderately rough surfaces irradiated with Er:YAG laser. Following cell culture of MG-63 cells for 2 and 5 days on SLA, SLA I, and SLA II surfaces, the expression levels of ALP (A), OC (B), and COL1 (C) were measured by quantitative real-time PCR. Y-axis represents n-fold expression related to MG-63 grown on tissue culture plastic calculated using 2^−ΔΔCt method, using GAPDH as housekeeping gene. Data are presented as the mean ± SEM of four independent experiments

Fig. 5

Osteocalcin production by MG-63 cells grown on titanium disks irradiated with Er:YAG laser. MG-63 cells were cultured on smooth (A) or moderately rough (B) surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (PT I and SLA I), 80 mJ/pulse at 20 Hz (PT II and SLA II) or untreated surfaces (PT and SLA) for 2 and 5 days, and the levels of OC protein in conditioned media were measured by commercially available ELISA. Data are presented as mean ± SEM of five independent experiments

Fig. 6

Expression of bone-turnover-related genes in MG-63 grown on smooth surfaces irradiated with Er:YAG laser. MG-63 cells were cultured on smooth surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (PT II), 80 mJ/pulse at 20 Hz (PT II) or untreated (PT) surfaces for 2 and 5 days, and the expression levels of OPG (A) and RANKL (B) were measured by quantitative real-time PCR. Y-axis represents n-fold expression related to MG-63 grown on tissue culture plastic calculated using 2^−ΔΔCt method, using GAPDH as housekeeping gene. Data are presented as the mean ± SEM of four independent experiments

Fig. 7

Expression of bone-turnover-related genes in MG-63 grown on moderately rough surfaces irradiated with Er:YAG laser. MG-63 cells were cultured on moderately rough surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (SLA I), 80 mJ/pulse at 20 Hz (SLA II) or untreated (SLA) surfaces for 2 and 5 days, and the expression levels of OPG (A) and RANKL (B) were measured by quantitative real-time PCR. Y-axis represents n-fold expression related to MG-63 grown on tissue culture plastic calculated using 2^−ΔΔCt method, using GAPDH as housekeeping gene. Data are presented as the mean ± SEM of four independent experiments

Fig. 8

Osteoprotegerin production by MG-63 cells grown on titanium disks irradiated with Er:YAG laser MG-63 cells was cultured on smooth (A) or moderately rough (B) surfaces treated with Er:YAG laser with 160 mJ/pulse at 20 Hz (PT I and SLA I), 80 mJ/pulse at 20 Hz (PT II and SLA II) or untreated (PT and SLA) surfaces for 2 and 5 days, and the levels of OC protein in conditioned media were measured by commercially available ELISA. Data are presented as mean ± SEM of five independent experiments