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. 2020 Feb 3;72(2):247–258. doi: 10.1007/s10616-020-00374-y

Photobiomodulation effects on osteogenic differentiation of adipose-derived stem cells

Gamze Bölükbaşı Ateş 1,✉, Ayşe Ak 2, Bora Garipcan 1, Murat Gülsoy 1
PMCID: PMC7192995  PMID: 32016710

Abstract

Increasing interest has been observed in the use of photobiomodulation (PBM) to enhance the proliferation of stem cells and induce their differentiation. The effects of PBM at two different wavelengths (635 and 809 nm) with three different energy densities (0.5, 1 and 2 J/cm2) on the osteogenic differentiation of adipose-derived stem cells (ADSC) were investigated. Cell viability and proliferation were evaluated by MTT and Alamar Blue assays. Osteoblast differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red staining and reverse-transcription polymerase chain reaction (RT-PCR) for the expression of collagen type I (COL1A), ALP and osteocalcin. 635 nm and 809 nm laser irradiation had no effect on the cell viability on days 7 and 14, except for 0.5 J/cm2 group at 14th day after 635 nm irradiation (p < 0.05). Cell proliferation was not changed significantly. Mineralization was increased significantly in 809 nm laser groups but no enhancement was detected in the osteogenic differentiation by ALP activity and gene expression results. In 0.5 and 1 J/cm2 groups, ALP and COL1A expressions were down regulated at day 7 after 809 nm laser exposure. These results suggest that PBM may alter osteogenic differentiation of ADSC and increase mineralization but further investigation is needed to define adequate parameters.

Keywords: Photobiomodulation (PBM), Adipose derived stem cells (ADSC), Low-level laser therapy (LLLT), 635 nm, 809 nm

Introduction

Stem cells are currently under investigation especially in fields of tissue engineering and regenerative medicine. Stem cells can renew themselves, expand in an undifferentiated stage and have the ability to differentiate into specific cell types. They are promising for therapeutic interventions but there are some important problems. First it is practically hard to produce enough number of cells; secondly, immune reactions may occur at the acceptor site; thirdly there are complicated ethical concerns. Among stem cells, adipose derived stem cells (ADSC) may be preferred due to their easy isolation, greater abundance and considerably less morbidity to donors (Abrahamse 2011). They have been shown to be very similar to bone marrow stem cells (de Villiers et al. 2011; Wang et al. 2016b). In vitro and in vivo studies have shown that adipose derived cells (ADSC) can differentiate into multiple mature cell phenotypes including adipocytes, osteoblasts and chondrocytes when treated with specialized induction media in vitro (Rodríguez et al. 2006; Watt and Driskell 2010).

Bone tissue engineering (BTE) aims to find out alternative methods for bone repair and regeneration since clinically used treatments have present problems such as donor site injury and morbidity, immune reactions and operative difficulties (Amini et al. 2012). Use of stem cells combined with biomaterial scaffolds is one of the cellular approaches of BTE. The osteogenic differentiation potential of ADSC makes them suitable for this area and increasing proliferation and differentiation is a challenge that needs further in vitro and in vivo studies (Halvorsen et al. 2001; Grottkau and Lin 2013; Zuk 2013). Differentiation can be induced by growth factors and physical factors (Engler et al. 2006; Green et al. 2009; Liu et al. 2009; Peng et al. 2012) or by using a scaffold with the appropriate characteristics (three-dimensional structure, composition and addition of exogenous growth factors) (Choi et al. 2013; Polo-Corrales et al. 2014; Wang et al. 2016b). An ideal scaffold is biocompatible and biodegradable with a controlled degradation rate structurally and functionally mimic the target tissue (Caetano et al. 2015). ADSC are differentiated into osteoblasts when cultured with DMEM containing dexamethasone, ascorbic acid and B-glycerophosphate (Kim et al. 2009; Trentz et al. 2010).

Photobiomodulation (PBM) is the application of light in the visible and near infrared range via coherent or incoherent light sources at low powers. Light within those ranges is a nonionizing electromagnetic radiation but can cause photophysical and photochemical responses when absorbed by endogenous chromophores (De Freitas and Hamblin 2016; Anders et al. 2019). The photobiomodulation therapy is investigated for therapeutic purposes for a long time but research studies gained speed after the invention of lasers in 1960s and development of new generation light emitting diodes with a variety of wavelengths in 1990s. Until 2015, those studies were known as low level laser therapy (LLLT), but at that time the term LLLT was replaced by photobiomodulation with several reasons; first, the studies were not limited with lasers anymore and also the word “therapy” could not describe the diversity of the effects investigated (Anders et al. 2015). PBM has been demonstrated to influence the behavior of various cell types, including stem cells. To establish a method to increase the proliferation or accelerate the differentiation process of the stem cells have been the immediate goals of PBM studies. Results of research studies suggested that photochemical processes were responsible for changes in cellular mechanisms, but the quest for optimum application protocols (choice of wavelength and energy doses) is still ongoing (Wang et al. 2016b; Deana et al. 2018; de Andrade et al. 2019).

Previously, we have investigated the effects of PBM with 635 and 809 nm on osteoblasts (Bölükbaşı Ateş et al. 2017). In the present study, the possible effects of a visible (635-nm) and an infrared (809-nm) laser irradiation on the proliferation and differentiation of ADSC into osteoblasts were examined.

Materials and methods

Cell culture

Human ADSC used in this research was purchased (Invitrogen R7788115) and were maintained at 37 °C in low glucose Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY, USA), supplemented by 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution (Sigma, st. Louis, MO, USA) in humidified atmosphere of 5% CO2 and 95% air. The medium was refreshed every 3 or 4 days. 0.25% trypsin–EDTA solution (Sigma, St. Louis, Mo, USA) was used to passage the cells. The cells used in the experiments were at 2–4 passages. To induce osteogenesis, 1 nm dexamethasone, 2 mm β-glycerolphosphate and 50 μm ascorbate-2-phosphate (ODM) were added to the culture medium (Bunnell et al. 2008). Immediately after irradiation ODM were added to plates.

Laser irradiation

Two lasers at 635 and 809 nm wavelengths were used to irradiate ADSC (Fig. 1). Red light irradiation was executed with a continuous diode laser at 635 nm (635 nm-VA-I- 400-635, Optotronics, CO, USA) with 400 mW maximum output power. The light was delivered via 600 μm fiber and a collimator was used for homogeneous illumination of the target cells.

Fig. 1.

Fig. 1

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Lasers used in the experiments. On the left photograph, 809-nm Infrared Diode Laser System (developed in our Laboratory) and on the right 635 nm (Optotronics)

A computer controlled high power 809 nm diode laser system was developed in Bogazici University, Institute of Biomedical Engineering, Biophotonics Laboratory. The laser had a maximum 10 W output power at 35 A applied current. A 400 μm fiber was used to transfer laser energy to the target and a collimator was used for homogeneous irradiation (Ateş et al. 2018).

Four wells of 96 well-plate were irradiated at a time. Only the center of the Gaussian distribution profile was used. The irradiance was adjusted by changing the distance between the laser and the cell culture dish and measured by an optical power meter (1918-R, Newport Corp., CA, USA) with a low-power sensor (918D-SL-OD3) at the cell surface as 50 mW/cm2. A blackout foil (Thorlabs Inc., NJ, USA) was used to avoid remaining wells from scattered photons (Table 1).

Table 1.

Device information and irradiation parameters

Technical specifications and dosimetry
Type Diode laser Diode laser
Wavelength (nm) 635 809
Manufacture Optotronics, CO, USA In-house developed
Model VA-I-400-635
Delivery 600 μm fiber 400 μm fiber
Treatment mode Non contact Non contact
Irradiation mode Continuous Continuous
Exposure time (s) 10/20/40 10/20/40
Irradiance at target area (mW/cm2) 50 50
Radiant exposure (J/cm2) 0.5/1/2 0.5/1/2
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The study had one control and three experimental groups for both lasers. Control group did not receive any illumination but the plates were kept outside the incubator for the same period of time as irradiated cells. Laser group 1 (L1) was irradiated for 10 s, at 0.5 J/cm2 radiant exposure. Laser group 2 (L2) was irradiated for 20 s, at 1 J/cm2 radiant exposure. Finally, the laser group 3 (L3) had 2 J/cm2 radiant exposure by an irradiation of 40 s.

Before the laser treatment, the cell culture medium was replaced with PBS to avoid any possible serum-light interaction. Immediately after irradiation, cells were grown in ODM and the medium was changed every 3 days. All the experiments were performed in triplicate.

Cell viability

Cell viability was evaluated by [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) assay at 7th and 14th days post-irradiation. The yellow MTT substance can be transformed into the purple formazan by living cells, which can be dissolved in DMSO. This colorimetric assay measures cell viability with a spectrophotometer (Mosmann 1983; Gerlier and Thomasset 1986). 5 × 103 cells/well were seeded on a 96 well-plate. After 24 h incubation for adhesion, irradiation was applied. After irradiation, the ODM was added to the wells. At 7th and 14th day, 10 μl of MTT solution (5 mg/ml) dissolved in 100 μl of medium was added to each well and the plates were kept in the incubator for 4 h at 37 °C. At the end of 4 h, to solubilize formazan crystals 150 μl/well of DMSO were added and the plates were agitated on a plate shaker for 15 min in dark. The optical absorbance was measured by a microplate reader (iMark, Bio-Rad Lab., USA) using a 570 nm filter. Normalized absorbance values were used to plot viability graphs.

Cell proliferation

Alamar Blue (AB; Invitrogen, CA, USA) assay was used to determine proliferation activity. It is a water-soluble, non-toxic dye, which changes color in response to chemical reduction from blue to red (Al-Nasiry et al. 2007). 5 × 103 cells/well were seeded on a 96 well-plate. After 24 h incubation for adhesion, irradiation was applied. After irradiation, the ODM was added to the wells. At 7th and 14th day, the cells were incubated for 4 h at 37 °C with 100 μl of ODM containing 10 μl of AB solution. The optical absorbance was measured with a filter at 570 nm by the microplate reader (iMark, Bio-Rad). The relative cell proliferation was normalized to the untreated control.

Alkaline phosphatase activity

Cells were plated at a density of 1 × 105 cells/cm2 in 24-well culture plates and cultured in ODM after laser irradiation. At days 7 and 14, a commercial colorimetric kit (Biovision, Research Products, CA, USA) was used to measure Alkaline Phosphatase (ALP) activity by following manufacturer’s protocol. Similarly, Bradford’s assay was performed to determine the total protein amount according to the protocol.

Alizarin red staining for mineralization

In the presence of osteoblast differentiation medium, ADSC differentiate into osteoblasts and the matrix starts to mineralize. ADSC differentiation into osteoblasts can be measured by Alizarin Red staining, which stains the precipitated calcium in the matrix. The staining was performed at 14th day. Another control group cultured in DMEM (without osteogenic supplements) was used in Alizarin Red staining experiments. Briefly, cells were rinsed with PBS and fixed in 10% formalin for 30 min at room temperature, washed twice with deionized H2O, stained with 2% Alizarin Red S (Sigma, St. Louis, MO, USA) solution, pH 4.2, for 10 min. After staining, the cells were rinsed with PBS again to remove excess solution and were photographed. Calcification deposits are seen as bright red/orange dots under an optical microscopy (DMIL, Leica, Germany). For quantitative analysis, a solution obtained by mixing 15% acetic acid and 20% methanol was added to the plates to solubilize the precipitate. The plates were kept in dark for 45 min and the optical density of the solution was read at 405 nm (Gregory et al. 2004).

Gene expressions by RT-PCR analysis

In order to investigate the effects of PBM on osteogenic markers, RT-PCR was performed at 7th and 14th day. Collagen type I (COL1), Alkaline phosphatase (ALP) and bone GLA protein bone gamma-carboxyglutamate protein (BGLAP), also known as osteocalcin) expressions were analyzed by using High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The details of the protocol were described in previous studies (Tabatabaei et al. 2015; Ateş et al. 2017; Bölükbaşı Ateş et al. 2017; Ateş et al. 2018).

Statistical analysis

The results were presented as the mean value ± standard deviation of the mean. To assess the data normality Saphiro Wilk test was performed and it was found that data were sampled from a normally distributed population. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) supplemented with Tukey’s test with the use of Statistical Package for the Social Sciences (SPSS) v 22.0 software (SPSS Inc., Chicago, IL). A p-value of < 0.05 was considered to define statistically significant differences.

Results

Cell viability

MTT assays were performed on day 7 and 14 to examine the effects of PBM on cell viability. No significant differences between irradiated at 635 nm and control groups were found after analysis of normalized optical absorbance values with the exception of the group that received 0.5 J/cm2 at 14th day (p < 0.05) (Fig. 2). Similarly, cell viability at 7th and 14th day did not seem to be effected by irradiation at 809 nm (Fig. 3).

Fig. 2.

Fig. 2

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Normalized absorbance for MTT assay performed after 635 nm laser irradiation at day 7 and day 14. 0.5 J/cm2 group of 635 nm at 14th day had significantly increased cell viability. * indicates statistically significant difference (p < 0.05) from the control group

Fig. 3.

Fig. 3

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Normalized absorbance for MTT assay performed after 809 nm laser irradiation at day 7 and day 14. No statistical differences were observed between 809 nm laser groups

Cell proliferation

The proliferation rate of ADSC cultured in ODM was assessed by Alamar Blue assay. The results showed that irradiation at both wavelengths did not cause any significant change in the proliferation (Figs. 4 and 5).

Fig. 4.

Fig. 4

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Normalized absorbance of Alamar Blue assay at days 7 and 14 after 635 nm laser irradiation. There were no significant differences between groups

Fig. 5.

Fig. 5

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Normalized absorbance of Alamar Blue assay at days 7 and 14 after 809 nm laser irradiation. There were no significant differences between groups

Alkaline phosphatase activity

The ALP activity of the cells irradiated with 635 nm did not show any differences from the control group at day 7 and 14. Although there was a slight increase in ALP activity of cells irradiated with 809 nm (1 J/cm2 and 2 J/cm2), no statistical differences were observed between control and laser groups at both time intervals (Figs. 6 and 7).

Fig. 6.

Fig. 6

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Normalized ALP activity at 7th and 14th day after irradiation at 635 nm wavelength. No significant differences were observed between groups

Fig. 7.

Fig. 7

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Normalized ALP activity at 7th and 14th day after irradiation at 809 nm. Although there were slight increases for 1 and 2 J/cm2 groups, the differences were not statistically significant

Alizarin red staining for mineralization

Microscopy analysis of cells cultured in ODM stained with Alizarin Red at 14th day has demonstrated many nodules of calcium in the mineralized extracellular matrix whereas cells cultured in DMEM were not stained with Alizarin Red showing that they were not differentiated into osteoblasts (Fig. 8a, b). Measurement of normalized optical absorbance of irradiated groups is given in Fig. 9. Cells subjected to 809 nm irradiation at all energy densities showed statistically increased mineralization, 1 J/cm2 group being the least. On the other hand, 635 nm irradiation results were not different than the control group except for the 2 J/cm2 group which had statistically increased mineralization (p < 0.05).

Fig. 8.

Fig. 8

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Microscopic images (×10) of two different controls (scale bar: 50 μm). Cells cultured in DMEM (a), cells cultured in ODM (b). Small orange dots are calcium deposits. Cells cultured in DMEM were not stained with Alizarin Red showing that they were not differentiated into osteoblasts

Fig. 9.

Fig. 9

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Normalized absorbance at 405 nm for mineralization. 809 nm irradiation at all energy densities and 2 J/cm2 group of 635 nm showed statistically increased mineralization (p < 0.05)

Gene expressions by RT-PCR analysis

The effect of PBM on the expression of three osteoblast markers, namely BGLAP, ALP and COL1A were analyzed by RT-PCR. Figure 10 illustrates the effect of 635 nm laser irradiation on the relative gene expressions at 7 and 14 days. At 7th day, similar expression patterns of BGLAP and ALP were observed for the laser and control groups. 1 J/cm2 group showed statistically decreased COL1A expression at 7th day. Cells demonstrated slightly increased ALP expression at 14th day in all groups although no statistically significance was detected. All groups expressed BGLAP slightly low, but statistically significant difference was recorded for only 0.5 J/cm2 group. Similar COL1A expressions were obtained between laser and control groups at 14th day.

Fig. 10.

Fig. 10

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The relative gene expressions normalized to GAPDH at 7th and 14th days after 635 nm laser irradiation.*Statistically significant compared to the control group. No statistical differences were observed between groups. (p < 0.05)

The relative gene expressions after 809 nm laser irradiation are shown in Fig. 11. In 0.5 and 1 J/cm2 groups, ALP and COL1A expressions were significantly decreased at day 7. However, ALP expressed by the cells was slightly but not significantly higher at 14th day for all irradiated groups. The expressions of BGLAP and COL1A were not altered in all groups by laser irradiation at 14th day.

Fig. 11.

Fig. 11

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The relative gene expressions normalized to GAPDH at 7th and 14th days after 809 nm laser irradiation.* Statistically significant compared to the control group, No statistical differences were observed between groups. (p < 0.05)

Discussion

The present study has focused on the possible ability of PBM to increase the proliferation and/or the differentiation of ADSC into osteoblasts. ADSC are multipotent cells and hold promise for a range of therapeutic applications such as stem cell therapy (Konno et al. 2013; Tsuji et al. 2014; Frese et al. 2016). One of the limitations of stem cell therapy is to have correct amount of cell at the injured tissue. Regarding positive results of PBM in different areas in medicine, it is a good candidate to increase the proliferation and/or the differentiation of stem cells. However, several studies suggest that PBM success is dose dependent (Huang et al. 2009; Soleimani et al. 2012) and even each cell line may have its specific dose parameters (Ferreira et al. 2009; de Oliveira et al. 2014). This is one of the reasons that may explain controversial results in PBM studies in the literature.

The MTT and Alamar Blue assay results showed that neither cell viability nor cell proliferation was significantly affected by 635 (except for 0.5 J/cm2 group at day 14) and 809 nm laser irradiation at 0.5, 1 and 2 J/cm2 after 7 and 14th day. Similarly, ALP activity assessed at same experimental days pointed out that PBM at stated parameters did not affect ALP activity. ALP mRNA expressions were slightly but not significantly higher at 14th day for all laser groups. However, mineralization at 14th day was increased after 809 nm laser application similarly as in 2 J/cm2 group of 635 nm laser which had statistically increased mineralization. BGLAP and Col1a expressions were not affected at overall by PBM with 635 or 809 nm.

Ginani et al. aimed to show the effects of PBM on the in vitro proliferation of mesenchymal stem cells through a systematic literature review and stated that only four studies used adipose tissue as the source of stem cells (Mvula et al. 2008, 2010; de Villiers et al. 2011; Ong et al. 2013; Ginani et al. 2015). There are studies investigating proliferation of ADSC in DMEM (Mvula et al. 2008; Abrahamse 2009; Mvula et al. 2010; Anwer et al. 2012; Wu et al. 2013; de Oliveira et al. 2014; de Andrade et al. 2019) but to our knowledge, there are not many studies using ADSC differentiating into osteoblasts to show the effect of PBM (Abramovitch-Gottlib et al. 2005; Choi et al. 2013; Wang et al. 2016a). Among them Choi et al. adipose‐derived mesenchymal stem cells seeded on an acellular dermal matrix. Abramovitch-Gottlib et al. used a three-dimensional biomatrix. Therefore, it is difficult to compare the results. Nevertheless, we have also considered studies carried out with other sort of stem cells to discuss our results since ADSCs are capable of multi-lineage differentiation and phenotypically similar to those derived from bone marrow stem cells (Caetano et al. 2015).

Soleimani et al. used bone marrow-derived mesenchymal stem cells (BMSCs) to investigate the effect of 810 nm laser application at 2 or 4 J/cm2 on their differentiation to osteoblasts (Soleimani et al. 2012). They irradiated cells at days 1, 3 and 5 after incubation of BMSCs with ODM and concluded that PBM increases BMSC proliferation and its differentiation to osteoblasts at the 7th day. Moreover, ALP activities were significantly higher at both energy densities compared to the control group. On the other hand, 808 nm laser light at an radiant exposure of 4 J/cm2 does not alter murine BMSCs proliferation and differentiation into osteoblasts according to the results of viability, specific staining assays and RT-PCR analysis for list of osteoblast markers (Bouvet-Gerbettaz et al. 2009). Another study performed by Peng et al. revealed that red light delivered every other day (LED, 620 nm, 1,2 and 4 J/cm2) suppressed proliferation of MSCs cultured in ODM but enhanced their osteogenic differentiation. The RT-PCR results showed that the Col1a1 and ALP mRNA expressions in red light irradiated cultures did not significantly differ from the control groups, but BGLAP expression levels were significantly increased on day 4 (Peng et al. 2012). Kim et al. exposed mouse MSCs to a LED light of 647 nm for 10 s, 30 s or 90 s with radiation energies of 0.093 J, 0.279 J and 0.836 J, respectively. Their results showed that cell viability was not affected by irradiation but ALP activity, mineralization, osteocalcin and collagen1 mRNA expression was increased compared to the control groups (Kim et al. 2009). Our results agree with an another study by Tani et al. who demonstrated that PBM with 635 nm on human mesenchymal stromal cell did not alter cell viability and PBM with 808 nm could positively influence osteogenic differentiation since it increased calcium deposition (Tani et al. 2018). However, they also observed an upregulation of osteogenic differentiation markers (Runx-2, ALP, OPN) as well as an increase of mineralized bone-like nodule structure formation after PBM with 635 nm. The differences in the result may arise from different laser parameters or cell sources.

The studies in which positive effect of PBM is observed, often applied irradiation multiple times during the experiment. It should be noted that number of PBM applications and timing between them are likely to play a role (Kreisler et al. 2003; Hawkins and Abrahamse 2006). This may be one of the reasons of the absence of PBM effect, in our study especially for cell viability and proliferation. A single irradiation at 635 nm with 0.5 J/cm2 radiant exposure increased cell number at 14th day after irradiation. The increase may be more prominent if the cultures were subjected to multiple applications. Interestingly, Fekrazad et al. examined the combined effect of red and infrared lasers (4 J/cm2 every other day for 3 weeks) on osteogenic differentiation of MSCs isolated from rabbit bone marrow. They concluded that the effect of combined lasers can not be predicted from the effects of each wavelength (Fekrazad et al. 2019). The biphasic response of light may also be another reason for conflicting results. Proliferation and differentiation may be promoted or suppressed depending on the dose. There will be no response if the irradiation is too low and if it is too high the response is inhibitory (Huang et al. 2009, 2011). Therefore, it is critical to determine the adequate dose in PBM studies.

Osteoblast differentiation may be divided into two stages. In the first stage, within 1 or 2 weeks, cells slowly proliferate, express ALP activity and bone specific genes, and produce a collagen matrix. Mineralization is a endpoint step which reflects advanced cell differentiation occurring in weeks 2 or 3 (Hoemann et al. 2009). Studies suggest that mineralization should be accompanied by increased expression or activity of osteocalcin and alkaline phosphatase (Halvorsen et al. 2001). Mineralization at 14th day was higher for all 809 nm laser groups. Our RT-PCR results for the same groups showed that ALP expressions were slightly higher at 14th day although not significant. However, osteocalcin (BGLAP) expressions did not differ from the control group. Light irradiation may result in shortening of all differentiation process. At 14th day, differentiation process has been completed and mineralization is high compared to the control group. Therefore, ALP activity may have also peaked earlier than expected that we do not observe any peak at 14th day. It should also be noted that ALP is synthesized as an inactive precursor and activated by proteolytic cleavage (Klionsky and Emr 1989; Peng et al. 2012). When ALP activity is measured, it is its active form, whereas mRNA expressions reflect inactive amounts. Light may also intervene in activation process, enhancing the proteolytic cleavage.

Our study was performed with ADSC. Their similarities to BMSCs and ease in their isolation make them good candidates for BTE. However, stem cells or cells in general have differing responses to light and other environmental conditions. The same stimulus may trigger different mechanisms in different cell types. Wang et al. hypothesized that the mechanism of action of 980 nm is based on the activation of heat gated ion channels instead activation of Cytochrome C-oxydase in mitochondria by 810 nm may be the reason (Wang et al. 2016a). Consequently, several other studies are warranted to define parameters of PBM and use it in clinical practice (Renno et al. 2007; Jawad et al. 2013; Amid et al. 2014).

The inconsistent results between studies may be attributed to differences in laser setups, wavelengths and cell culture conditions. Wang et al. compares the effect of irradiation at different wavelengths at the same dose (3 J/cm2) five times (every 2 days) on human ADSC differentiating into osteoblasts. They concluded that the 420 nm and 540 nm wavelengths were found to be more effective in stimulating osteoblast differentiation when compared to 660 nm and 810 nm (Wang et al. 2016b). In the present study we investigated two lasers of VIS and IR wavelengths and explored their PBM potentials on the same type of stem cells. Near infrared light at low energy densities seems to be more responsive since it causes more mineralization, meaning more differentiation of ADSC into osteoblasts. Interestingly, Amoroli et al. evaluated the effectiveness of PBM with 808 nm at a higher-fluence (64 J/cm2) irradiation on bone marrow stromal cells and have demonstrated that ALP and matrix mineralization is enhanced (Amaroli et al. 2018) These studies have pointed out that there is need for further studies with detailed experimental conditions to facilitate the comparison and be able to determine more convenient protocols for each cell type.

Cell culture medium may also be considered as a parameter in PBM studies. Several authors claim that cells cultured in low FBS (5% or lower), as being under nutritional deprivation, respond more evidently to light irradiation (Fujihara et al. 2006; Oliveira et al. 2010) whereas several others can not observe any differences (Ferreira et al. 2009; Tabatabaei et al. 2015).The same experiments of our study should also be performed in lower FBS concentrations to test the effect of medium and possibly enhance the light effect.

This study aimed to investigate the effect of PBM on ADSC proliferation and their differentiation potential into osteoblasts. Our findings will help to elucidate the possible effect of PBM on cells and may help to find out alternative methods for tissue repair and regeneration.

Acknowledgments

This study was supported by Grant (113Z059) of the Scientific and Research Council of Turkey (TUBITAK). The cell culture experiments were performed at Bogazici University, Institute of Biomedical Engineering, Biomaterials Research Laboratory, which was supported by Bogazici University Research Fund with the Grant number 6701.

Footnotes

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