2.1.

Cell Culture

The human fetal osteoblast cell line (hFOB) was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). The cells were maintained in a growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco, California) with F12 containing 10% ($\mathrm{v}/\mathrm{v}$) fetal bovine serum (FBS, Lonza, Maryland) and 1% ($\mathrm{v}/\mathrm{v}$) penicillin–streptomycin solution (50-U/ml penicillin and $50-\mathrm{mg}/\mathrm{ml}$ streptomycin; Sigma Chemical, St. Louis, Missouri) at 37°C in a humidified atmosphere of 95% air/5% ${\mathrm{CO}}_2$; the medium was changed twice weekly.

2.2.

Procedure of LLLT

The hFOB cells were plated in 96-well plates at a density of $2\times {10}^4\text{cells}/{\mathrm{cm}}^2$. After a 24-h incubation for adhesion, the cells were irradiated using a Ga–Al–As diode laser (ezlase, Biolase, California) with a wavelength of 940 nm in a continuous wave mode of operation. The laser beam was delivered by a 0.4-mm-diameter optical fiber defocused at the tip by a concave lens to provide a uniform circle of irradiation that was 7 mm in diameter at the cell layer level; irradiation was performed from 14 mm above the cell layer, as recommended by Aihara et al.¹⁸ The power density of the laser beam was measured with a laser power meter (Thorlabs, New Jersey). The 96-well plates were uniformly irradiated for a period of up to 7 days. The plates were grouped into 3 main experimental groups according to the power delivered: 100, 200, and 300 mW; these laser powers were obtained from a previous preliminary study. Then, each main group was further subdivided to subgroups according to periods of exposure, which either were 3 or 6 min/day for each laser power. The energy densities each subgroup received at 3 or 6 min exposure times, respectively, were as follows: the first group that was exposed to 100 mW received 22.92 or $45.85\mathrm{J}/{\mathrm{cm}}^2$; the second group that was exposed to 200 mW received 45.85 or $91.79\mathrm{J}/{\mathrm{cm}}^2$; and the third group of 300 mW exposure received 68.78 or $137.57\mathrm{J}/{\mathrm{cm}}^2$. When not receiving laser irradiation, the cells were kept in a 5% ${\mathrm{CO}}_2$ incubator at 37°C. All experimental groups were compared with a control group of cells that did not receive any laser treatment.

2.3.

MTT Assay

The (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) (MTT) is transformed by mitochondrial dehydrogenases of active cells, providing a measurement of cell proliferation and viability.³⁰ All experimental groups were measured by this test at days 1, 3, and 7. The MTT (Sigma, Missouri M2128) was dissolved at a concentration of $5\mathrm{mg}/\mathrm{ml}$ in sterile phosphate buffered saline, filtered through a 0.22-pm filter to remove any formazan crystals, and stored at 4°C in the dark; The MTT was added to osteoblast cultures in the 96-well plates at a 1:l0 ratio. Following incubation at 37°C for 4 h in 5% ${\mathrm{CO}}_2/95\%$ air humidified atmosphere, the supernatants in the wells were removed and replaced with 100 μl of dimethyl sulphoxide per well. The absorbance was measured at the 570-nm wavelength using a microplate reader (Sunrise, TECAN, Switzerland). These values were expressed as the percent viability of the samples versus control cells, which were set equal to 100%.

2.4.

Alkaline Phosphatase Activity Assay

The Alkaline phosphatase (ALP) is expressed at high levels in osteoblasts; the level of ALP in serum is a systemic indictor of bone formation.³¹ The hFOB cells were measured for the ALP activity at days 1, 3, and 7. The procedure was conducted by following the ALP Activity Colorimetric Assay Kit (Biovision, California) manufacturer’s protocol. Briefly, the samples were added to the wells of 96-well plates and the volume was brought to 80 μl. Because the colored samples may interfere with optical density (O.D.) readings, sample background controls were maintained by adding the same amount of the sample to separate wells, also bringing the volume to 80 μl. To terminate the ALP activity, 20 μl of stop solution was added to the background sample. Then, 50 μl of 5 mM $p$-nitrophenyl phosphate solution was added to the wells containing the test, background, and control samples. The ALP reaction incubated for 60 min at 25°C in the dark. Twenty microliters of the stop solution was added to each standard and sample reaction, except the background reaction, and after gentle shaking of the plates, a microplate reader (Sunrise, TECAN, Switzerland) was used to measure the O.D. at 405 nm. A standard curve was generated with the assay. The ALP activity was expressed as a relative sample percentage compared with the control, which was set equal to 100%.

2.5.

Osteocalcin Activity Assay

The Osteocalcin (OST) is a noncollagenous protein found in bone that is secreted solely by osteoblasts and naturally plays a role in the body’s metabolic regulation and bone formation. The OST is also implicated in bone mineralization and calcium ion homeostasis.³² The hFOB cells were measured for OST activity at days 1, 3, and 7. The procedure was conducted by following the manufacturer’s protocol for the OST Activity Assay Kit (Invitrogen, California). Briefly, 25 μl of each kit standard, control, or sample was added to the appropriate wells. Then, 100 μl of working Anti-OST-HRP conjugate was added into all wells and incubated for 2 h at room temperature. The solution was aspirated from the wells to discard the liquid, wells were washed 3 times and 100 μl of chromogen solution was added to each well. Then, the plate was incubated for 30 min at room temperature in the dark. To stop the reaction, 100 μl of stop solution was added to each well. A standard curve was generated with the assay. The OST activity was expressed as relative sample percentage compared with the control, which was set equal to 100%.

2.6.

Statistical Analysis

All experiments were performed in triplicate. Results are expressed as the mean and standard error, and statistical analysis was performed using Kruskal–Wallis and Mann–Whitney tests. A $P$ value $\le 0.05$ was considered statistically significant.

3.1.

Cell Proliferation by MTT

The results showed that there was a significant increase in the cell proliferation rates of all groups compared with the control group in relation to time. The day 7 was the highest among all groups, as shown in Fig. 1.

Fig. 1

Effect of laser stimulation on osteoblast cell proliferation. The cells were cultured in the presence and absence of daily laser stimulation. The proliferation percentage of controls, as determined by MTT assay, for different laser powers and times: (a) 100 mW, 3 min; (b) 100 mW, 6 min; (c) 200 mW, 3 min; (d) 200 mW, 6 min; (e) 300 mW, 3 min; and (f) 300 mW, 6 min. The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups on days 1, 3, 7 and control.

When comparing all groups at day 7, the subgroups treated with the LLLT for 6 min showed a significantly higher proliferation rate than the subgroups that were treated with the LLLT for 3 min. Additionally, the cell proliferation rate significantly increased when the laser power was increased; the 300-mW laser power had the highest proliferation rate and the 100-mW laser power had the lowest proliferation rate of the groups, as shown in Fig. 2.

Fig. 2

Effect of laser stimulation on osteoblast cell proliferation at day 7. The cells were cultured in the presence and absence of daily laser stimulation. The proliferation percentage of controls, as determined by MTT assay, for laser treatments at different powers and times: (100 mW, 3 min), (100 mW, 6 min), (200 mW, 3 min), (200 mW, 6 min), (300 mW, 3 min), and (300 mW, 6 min). The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups.

3.2.

Cell Differentiation by ALP and OST Activity Assays

The results showed that there was an increase in cell differentiation (by ALP and OST assays) in all groups compared with the control group in relation to time. The day 7 was significantly increased in cell differentiation among all groups, as shown in Figs. 3 and 4.

Fig. 3

Effect of laser stimulation on alkaline phosphatase (ALP) activity of osteoblast cells. The cells were cultured in the presence and absence of daily laser stimulation. The ALP percentage of controls for different laser powers and times: (a) 100 mW, 3 min; (b) 100 mW, 6 min; (c) 200 mW, 3 min; (d) 200 mW, 6 min; (e) 300 mW, 3 min; and (f) 300 mW, 6 min. The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups on days 1, 3, 7, and control.

Fig. 4

Effect of laser stimulation on osteocalcin (OST) activity of osteoblast cells. The cells were cultured in the presence and absence of daily laser stimulation. The OST percentage of controls for different laser powers and times: (a) 100 mW, 3 min; (b) 100 mW, 6 min; (c) 200 mW, 3 min; (d) 200 mW, 6 min; (e) 300 mW, 3 min; and (f) 300 mW, 6 min. The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups on days 1, 3, 7 and control.

When comparing all groups at day 7, the subgroups treated with the LLLT for 6 min showed a significantly higher differentiation rate (by ALP and OST assays) than the subgroups that were treated with the LLLT for 3 min. Additionally, the cell differentiation rate increased when the laser power was decreased; the ALP activity rate was the more significant at the 100-mW laser power, whereas for the OST activity rate, the 200-mW laser power was the most significant, whereas the 300-mW laser group was not significant among all treatment groups for cell differentiation, as determined by both ALP and OST assays shown in Figs. 5 and 6.

Fig. 5

Effect of laser stimulation on ALP activity of osteoblast cells at day 7. The cells were cultured in the presence and absence of daily laser stimulation. The ALP percentage of controls for different laser powers and times: (100 mW, 3 min), (100 mW, 6 min), (200 mW, 3 min), (200 mW, 6 min), (300 mW, 3 min), and (300 mW, 6 min). The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups and control.

Fig. 6

Effect of laser stimulation on OST activity of osteoblast cells at day 7. The cells were cultured in the presence and absence of daily laser stimulation. The OST percentage of controls for different laser with powers and times: (100 mW, 3 min), (100 mW, 6 min), (200 mW, 3 min), (200 mW, 6 min), (300 mW, 3 min), and (300 mW, 6 min). The data are shown as the $\text{mean}\pm \mathrm{SEM}$ of three separate experiments. * $P\le 0.05$ between groups and control.