BPs are the most widely used anti-resorptive drugs for metabolic bone diseases . BPs are pyrophosphate-like structures with two variable regions (R1 and R2) on the carbon atom of the BP molecule attached to the basic P-C-P structure. This allows for variations in the molecular structure and a range of corresponding potencies. BPs are classified according to the chemical group added to the base pyrophosphoric nucleus at the R2 side chain. Alkyl derivatives are the first generation of drugs (e.g., etidronate). The second generation includes amino-bisphosphonates with a terminal amino group (e.g., alendronate and pamidronate), while the third generation is characterized by a cyclic side chain (e.g., zoledronate). Depending on whether or not nitrogen is attached to the R2 side chain, BPs are classified as nitrogen-containing BPs (NBPs) or non-nitrogen-containing BPs (NNBPs). NBPs (e.g., pamidronate, alendronate, risedronate, ibandronate, and zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), which is an enzyme in the mevalonate pathway, and block the prenylation of small GTPase-signaling proteins. This results in the accumulation of active unprenylated GTPases in the cytoplasm of the osteoclast, which causes an inappropriate activation of the downstream signaling pathways, thereby leading to the disruption of normal osteoclast function and survival. As a result, NBPs suppress bone resorption through a direct effect on the osteoclasts and their precursors [11, 12].
In the present study, we examined the effects of alendronate on osteoblasts. Alendronate is the most widely prescribed oral BP, which is more likely to cause BRONJ. However, the precise mechanism of alendronate in BRONJ remains elusive .
The effects of BPs on osteoclasts are well understood, and their toxicity effects on osteoclast are thought to influence the onset of BRONJ. Besides the inhibition of osteoclasts, many complicated events may be related to BRONJ development, and interactions among the bone cells must be considered as a whole . Although the majority of in vitro BP studies have focused on the activities of the osteoclast lineage cells, recent studies have suggested that the presence of osteoblastic family cells is required in order for the anti-resorptive effects of BPs to occur. This effect may depend upon soluble factors that are secreted by the osteoblasts, which inhibit the formation and activity of the osteoclasts . RANKL, OPG, and M-CSF are essential factors that are produced by the osteoblast/stromal cells for osteoclast-osteoblast interactions. RANKL is an important factor in protecting bone resorption, extending the life of osteoclasts, and promoting differentiation. OPG is known as a decoy receptor protein that prohibits osteoclast activation by protecting the function of the receptor activator nuclear factor-kB (RANK), which is involved in osteoclast differentiation by combining with RANKL . The role of OPG is largely associated with an initiation phase, in which OPG counteracts the osteoclastogenic activity of RANKL. During bone formation, osteoclast differentiation is suppressed through the OPG that is produced by the osteoblast . Osteoblasts produce M-CSF, which is required for cell survival in the macrophage-osteoclast lineage, and the control of cell migration and reorganization . However, studies on the effects of BPs on osteoblasts are the subject of debate. In addition, the effects of BPs on osteoblastic activities have been sparsely investigated in terms of BRONJ development [19–21]. Knowledge regarding the effects of alendronate on the hFOB cells, particularly in the OPG/RANKL system, is lacking.
In order to address this data gap, we investigated the expressions of RANKL, OPG, and M-CSF in BP-treated hFOB cells. In the cells treated with alendronate at concentrations of 50 μM and higher, the cell survivability significantly decreased after 48 h, and there was a strongly negative dose-dependent influence on the viability of the osteoblasts and induced toxic effects in the hFOB cells. Enjuanes et al.  reported that high concentrations of alendronate inhibited osteoblast proliferation in the primary hFOB cells and indicated that the drug did not significantly inhibit proliferative effects, as compared to controls at lower concentrations (≤10–5 M). Naidu et al.  reported that high concentrations of alendronate and zoledronate were cytotoxic and decreased the cell survivability at 72 h, and cytotoxicity leading to cell death was likely to result in osteonecrosis. Our results are similar to these studies and suggest that alendronate at higher concentrations, more than 50 μM will affect hFOB cell proliferation and viability significantly after 48 h. We observed that 50 μM alendronate appeared to suppress M-CSF and RANKL expressions, and decreased the OPG expression, as compared to the control group. The RANKL expression appeared to be more suppressed than the OPG expression. RANKL expression level was more decreased than OPG or M-CSF in RT-PCR and ELISA result. Appeared by Western blot analysis, OPG, RNAKL, and M-CSF proteins were decreased in alendronate-treated cells.
Therefore, we proposed that the BPs interfere with osteoclastogenesis through regulating mediators (e.g., M-CSF, RANKL, and OPG) by inhibition of their expressions. However, Lin et al.  found no significant influence on osteoblast RANKL and OPG gene expressions during a 48-h experimental period when investigating alendronate and pamidronate. In contrast, Mackie et al.  reported that the RANKL gene expression was inhibited, while the OPG gene expression was not altered by stimulation with pamidronate in an osteosarcoma cell line for 6 days. Although the reasons for these differences have not been completely explained, the distinct effects of various BPs (e.g., pamidronate, zoledronate, and alendronate) and the use of different cell lines (e.g., human vs. rat and primary vs. cancer) could play a role . Additional in-depth studies will be required in order to understand the reasons for these differences.
In medicine and dentistry, diode lasers have been used predominantly in applications that are broadly termed as LLLT or biostimulation , and many studies have evaluated the therapeutic effects of LLLT on a broad range of disorders. LLLT applications, which have been promoted by some authors and manufacturers of the LLLT devices, included the acceleration of wound healing, enhancement of the remodeling and repair of bones, restoration of normal neural function following injury, pain attenuation, and modulation of the immune system . Recently, research on the use of LLLs in dentistry has proceeded gradually, and the range of clinical applications has been extended. The term LLL includes soft lasers, mid-lasers, low-energy lasers, and cold lasers. A new international definition considers LLLT to be laser therapies that do not increase tissue temperature over 36.5 °C or normal body temperature. The wavelength of such lasers is reported to be 500–1200 nm. Recent literatures regarding pre-osteoblast stimulation with red laser were reported [28–31]. We used a Ga–As–Al laser with a wavelength of 808 nm, which is within the prescribed range. It is still unclear as to which of the parameters has the greatest effect on therapeutic efficacy, even though there are information about total energy dose, energy density, and laser spectrum. In this study, the greatest biostimulatory effect was observed when a dose of 3.6 J/cm2 was used. This may be due to the use of different methodologies, such as different types of cells, experimental timings, and radiation distance.
Many previous studies have demonstrated that LLLT is optimal in tissues under a specific stress, such as hypoxia [32–34], diabetes [35–38], and nutritional deficit . Other previous studies have shown that LLLT may enhance the osteogenic potential of osteoblasts, and may promote metabolic bone activity and bone remodeling [40, 41]. LLLT has recently been used as a supportive technique in BRONJ treatment. Clinical cases that describe the application of LLLT to treat BRONJ have been reported, based on in vivo and in vitro experimental studies that demonstrated a biostimulative effect [7, 42, 43].
Vescovi et al.  reported successful results by using a combined treatment in BRONJ patients with medication plus Er:YAG laser surgery and LLLT. However, there have been no cell culture studies designed to explain the positive results obtained with the LLLT biostimulation in combination with surgery in clinical BRONJ cases. The present study investigated the effects of LLLT in hFOB cells treated with alendronate. The cell survivability significantly increased at 72 h relative to the alendronate-treated cells after LLT application. Moreover, we observed a significant increase in RANKL and M-CSF expressions, as compared to the cells treated only with alendronate. The results showed that alendronate at a concentration of 50 μM appeared to inhibit hFOB cell survivability, and suppress the M-CSF and RANKL expressions, and decreased the OPG expression, as compared to the control group. Moreover, the LLLT increased the RANKL and M-CSF expressions relative to the alendronate-treated cells.
In this study, the greatest biostimulatory effect was observed when a dose of 3.6 J/cm2 was used. It may be due to the use of different methodologies, such as different types of cells, experimental timings, and radiation distance. Recently, the choice for an appropriate laser source and standardization of radiation parameters will require further research in order to obtain an optimal result for a low-level laser study.