Within dentistry, tissue regenerative concepts have been developed to be applied in the clinical areas of periodontology and implantology (Izumi et al., 2003; Egusa et al., 2012). Oro-maxillofacial reconstruction procedures can assist in facial tissue repair and regeneration, such as may be the result of trauma or neoplasm. In Europe, about 1.5 million patients undergo craniofacial reconstruction procedures annually. However, ~20% of them continue to experience functional deficiencies despite the surgical interventions. Furthermore, about 30,000 patients per year would develop donor-site morbidity following oral and maxillofacial reconstruction (Czerwinski et al., 2010; Rodriguez y Baena et al., 2016).

The research area of utilizing stem cells toward restoring the structure and function of damaged tissues or organs in medicine and dentistry (Huang et al., 2009; Meirelles Lda and Nardi, 2009) is moving fast and attracting the interest of researchers. Many studies have shown that the use of mesenchymal stromal cells (MSCs) for bone regeneration is one of the most important applications in oro-maxillofacial reconstruction (Egusa et al., 2012; Zigdon-Giladi et al., 2014), periodontology and implantology (Izumi et al., 2003; Egusa et al., 2012). Despite the positive results that were achieved in bone regeneration through utilizing the MSCs, the destiny of the transplanted cells and their effect on the clinical outcome remains uncertain. This could be related to the fact that the transplanted cells die very quickly or migrate out of the transplantation site according to several animal studies (Meijer et al., 2008; Zimmermann et al., 2011; Egusa et al., 2012). Therefore, the challenge of stem cell research is related to the identification of supporting therapies, which can improve and enhance the effectiveness of various tissues repair and regeneration.

Utilization of a low-energy light intensity within the visible red and near infrared portion of the electromagnetic spectrum has been shown to stimulate irradiated cellular activity (Avci et al., 2013). This phenomenon has been referred to as photobiomodulation (PBM) (Avci et al., 2013; Amaroli et al., 2015a). Many cellular responses have been observed in in vitro models after irradiation with light sources of different wavelengths at specific energy density, which has induced PBM effects. These cellular responses resulted in: an increase of mitochondrial respiration and ATP production (Karu, 2010; Amaroli et al., 2015b,c, 2016c), the synthesis of proteins (Avci et al., 2013; Lipovsky et al., 2013) and in cells migration and proliferation (Avci et al., 2013; Amaroli et al., 2015a). Furthermore, reduction in the inflammation, acceleration of wound healing (Avci et al., 2013; Amaroli et al., 2018) and bone formation (Santinoni et al., 2017) were observed. Few studies have investigated the use of PBM on MSCs' stimulation (Lipovsky et al., 2013; Kushibiki et al., 2015). These studies have pointed out that the effectiveness of PBM on MSCs was related to enhanced angiogenic effects of adipose-derived stromal cells (Park et al., 2014) and to the modulation of gene expression, proliferation, intracellular cAMP levels, and osteogenic differentiation (Kushibiki et al., 2015). However, despite in vivo studies (animal models and randomized controlled clinical trials) with a positive PBM outcome, it remains a controversial subject as a consequence of conflicting effects produced by various operating parameters such as wavelength, fluence, laser power output, exposure time, number of applications (Jenkins and Carroll, 2011), and the beam profile (Amaroli et al., 2016b).

Recently, the AB2799 flat-top hand-piece has been developed and marketed. It can generate a more homogenous irradiation at ~1 cm² (Spot size) from contact to 105 cm distance from the target tissue (Amaroli et al., 2016b; Selting, 2016). This probe allows the use of relatively higher power densities and fluences with lower risk of causing collateral thermal damage to adjacent tissues, in comparison to standard (Gaussian profile) hand-pieces (Amaroli et al., 2015a,b; Selting, 2016). Previous studies by Amaroli and co-workers have shown that, the use of the 808 nm diode laser with the flat-top hand-piece at a higher-fluence of 64 J/cm² and power output of 1 Watt (W) in continuous-wave (CW) emission, resulted in increased mitochondrial activity (Amaroli et al., 2016b), in stimulation of oxygen consumption (Amaroli et al., 2015b) and in ATP production (Amaroli et al., 2015c, 2016c) in the unicellular organism Paramecium primaurelia. In the same unicellular model, modulation of calcium fluxes and intracellular calcium concentration (Amaroli et al., 2015c, 2016a) was also observed, as well as an increment of fission rate rhythm (Amaroli et al., 2015a). In addition, it was evidenced that this higher-fluence did not generate genotoxic damage or adverse effects (Amaroli et al., 2017) while it was able to induce anti-inflammatory effects that promoted the wound healing in an animal model (Cuteri et al., 2016; Amaroli et al., 2018).

The purpose of this study was to assess the in vitro ability of 808 nm diode laser therapy with flat-top hand-piece delivery system at a higher-fluence and -power (64 J/cm²; 1 W, 1 W/cm²) on inducing osteoblast maturation and on modulating the bone marrow stromal cells (BMSCs) secretion of important inflammatory mediators.

Latterly, increasing findings point to the properties of BMSCs; a cell population isolated from the bone marrow stroma and constituted by multiple cell types including multipotent progenitors for skeletal lineages (Bianco and Robey, 2015). In addition to their ability to differentiate into skeletal tissue cells, the BMSC possess immunomodulatory characteristics (Kuroda et al., 2017). The current research is increasingly demonstrating the importance of the interaction of the bone marrow stem/progenitor and the mature cell in their role in bone metabolism, turnover, and regeneration (Agas et al., 2015; Kassem and Bianco, 2015). Within this investigation, the BMSCs of 3 month old female Balb-c mice were analyzed through histochemical staining (alkaline phosphatase and alizarin red S assays), immunoassay (cytokines and chemokines assays) and western blotting (expression of runt-related transcription factor 2, osterix, peroxisome proliferator-activated receptor gamma, transforming growth factor β1, tumor necrosis factor α).

Stem cell research is related to identifying optimal therapies that can be utilized in promoting tissue healing. Photobiomodulation could be a useful adjunctive, as it plays a role in acceleration of tissue regeneration and repair (Avci et al., 2013). Such application on stem cells may be therefore beneficial in many areas of biomedical application and ultimately, this would augment the success of regenerative medicine (Abrahamse, 2012). Our data appears to support this line of thought. In fact, it was found that the 808 nm laser irradiation at a fluence of 64 J/cm² increased the BMSCs' Runx2, which is one of the early cell markers that promotes MSCs differentiation into immature osteoblasts and inhibits lineage development into adipocytes (Komori, 2010). Wu et al. (2012) study has shown that irradiation with gallium-aluminum-arsenide (GaAlAs) red laser (wavelength 660 nm) at different fluences of 1 J/cm², 2 J/cm², or 4 J/cm² with power density 10 mW/cm² can induce the generation of insulin-like growth factors 1 (IGF-1), to promote both the proliferation and the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (D1 cells), whereas it may induce bone morphogenetic protein 2 (BMP2) expression primarily to enhance osteogenic differentiation. It is known that IGF-1 can regulate Runx2 DNA binding (Qiao et al., 2004; Guntur and Rosen, 2013) and BMP2 is able to induce osteoblast differentiation through Runx2-dependent activating transcription factor 6 (ATF6) expression (Jang et al., 2012). Therefore, our protocol of utilizing the 808 nm laser therapy at a higher-fluence can photobiomodulate the BMSCs in a way which is similar to what has been observed in Wu et al. (2013) results, when 660 nm was utilized to irradiate the cells at low-fluence. However, previous results by Bouvet-Gerbettaz et al. (2009) have failed to induce osteoblast differentiation with 808 nm wavelength at low-fluence (4 J/cm²). In contrast, Soleimani et al. (2012) has observed that 810 nm GaAlAs with range of fluences of 3–6 J/cm² had a positive effect on the BMSCs differentiation. However, the author did not provide an explanation of the mechanism of action of the laser therapy.

In our study an induction of MSC to osteoblast precursors and an inhibition of their commitment to adipocytes lineage was demonstrated by the increase of Runx2 and the decrease of PPARγ (Komori, 2010) in culture exposed to irradiation for 5 (T1) and 10 days (T2). In addition, the differentiation of osteoblasts precursors into mature osteoblasts was demonstrated in laser-irradiated cells by the increase of Osx (Zou et al., 2006) overall, evident after 15 days (T3) of irradiation. At the same time point (T3) an enhanced ALP positivity and matrix mineralization (revealed by Alizarin red staining analysis) was found in irradiated cultures, compared to the untreated control cells. As anticipated, for the evaluation of matrix deposition both untreated (control) and treated cultures were grown on osteogenic medium to accelerate and increase the mineralization process, since cells growth in RPMI culture medium showed slow matrix deposition after laser treatment. In addition, the untreated cultures did not show any mineralization, in line with the fact that our experiments were conducted on a cell population involving overall undifferentiated cells.

Taken together, these data point to the fact that laser irradiation not only induced the MSCs development toward immature osteoblasts but also promoted osteoblast maturation.

It has been demonstrated that within the bone marrow, the process of MSCs maturation along osteoblastic lineage is finely regulated by signals, which are released in the marrow microenvironment. For instance, TGF-β1, besides its role in promoting osteoblastic precursors or matrix-producing osteoblasts through chemotactic attraction (Park et al., 2014), also blocks the apoptosis of osteoblasts (Huang et al., 2009) and enhances the osteoblast proliferation (Horwitz et al., 2002). If it is considered that our laser irradiation of BMSCs has no effect on the anti-proliferative and pro-apoptotic TNF-α protein, we would speculate that our therapy might maintain the cell proliferation process. This assumption has been supported by our previous results which have shown that laser therapy has positive effects on the protozoa and mammalian mitochondria activities (Amaroli et al., 2015b,c, 2016b) and stimulates the cell proliferation (Amaroli et al., 2015a) without inducing cellular damage (Amaroli et al., 2017). Furthermore, the literature has supported the fact that MSCs proliferation can be induced after laser irradiation (Abramovitch et al., 2005; Hou et al., 2008; Soleimani et al., 2012; Wu et al., 2012; Giannelli et al., 2013). In addition, it has been demonstrated that elevated levels of TGF-β1 play an important role in downregulating the release of the proinflammatory cytokine such as IL-6 and IL-17, thus promoting favorable conditions for bone regeneration (Zhou et al., 2008).

In laser treated BMSCs, we have consistently observed an increase in the synthesis of TGF-β1 associated with a decrease in the secretion of the pro-inflammatory molecules Il-1β, IL-6, and IL-17. Furthermore, the production of the anti-inflammatory interleukins IL-1ra, IL-10 was increased.

Although the changes in the cytokine levels were slight, it should be considered that the BMSCs used in these experiments were derived from healthy mice with a bone marrow environment under steady-state condition. Therefore, such small fluctuations in cytokines release may be interpreted as specific responses to the treatment.

Moreover, our data has showen that utilization of a higher-fluence at a higher-power setting has confirmed the antiflogistic propriety which has coincided with the findings of the wound healing model study in muscle and epithelial tissues (Amaroli et al., 2018).

The literature has clearly demonstrated that PBM has no deleterious effects on MSC (Kushibiki et al., 2015; Marques et al., 2016). However, the current research in literature has not provided a conclusive outcome (Marques et al., 2016) but it has only pointed out the effectiveness of the red wavelengths (Kushibiki et al., 2015; Marques et al., 2016). This evidence stresses the value of the results of our current study and the possible applications of our in vivo phototherapy investigations. In fact, the results of the most in vitro studies on PBM were obtained using wavelengths in the range 600–700 nm. However, through in vivo studies, the light-energy in this range of wavelengths can quickly disperse and does not penetrate to deeper target tissue layers in order to provide therapeutic effects. On the other hand, the wavelengths ranging from 800 up to 1100 nm which have a longer optical penetration depth can target deeper tissues (Avci et al., 2013; Pandeshwar et al., 2016). Therefore, the wavelengths in the range of 390–600 nm are used to treat in vitro culture or superficial tissues while the longer wavelengths in the range of 600–1100 nm are used to treat deeper-seated tissues. Moreover, the wavelengths in the range of 700–750 nm have been found to have a limited biochemical activity and therefore, they are not often used (Avci et al., 2013; Pandeshwar et al., 2016).

In conclusion, our data prove for the first time that 808 nm diode laser irradiation, delivered by the flat-top hand-piece at the higher-fluence and -power of 64 J/cm2 and 1 W (CW) respectively, promotes BMSCs differentiation toward osteogenesis. Within the limits of our evaluation, our results suggest an additional possible laser effect based on its ability to increase TGFβ synthesis and to facilitate osteoblast differentiation by creating an anti-inflammatory effect on bone marrow stroma cells.

AA, DA, VC, MS, FL, and SB: Conceived and designed the experiments; AA, DA, VC, MS, and FL: Performed the experiments, collected data and performed the data analyses; AA, DA, VC, MS, FL, and SB: Provided analysis tools; AA, DA, VC, MS, FL, RH, and SB: Wrote the manuscript.