Peri-implantitis is an infectious disease that occurs in the tissue around dental implants. It is characterized by the inflammation of tissue in the vicinity of an implant and the progressive loss of supporting bone .Both Er:YAG and Nd:YAG lasers apply to the treatment of peri-implantitis because of their anti-inflammatory and sterilizing effects, and their use can also promote bone healing .The Er:YAG laser can make the water molecules in the irradiation area absorb the laser energy and instantly evaporate, after which soft and hard tissue are melted by microblasting. The laser’s low penetrability and water misting effect reduce thermal injury to the tissue, which is beneficial in terms of supporting better bone healing.
The immediate high energy produced by an Nd:YAG laser causes bacterial solution vaporization, leading to cell-wall disintegration and, accordingly, sterilization and disinfection . In addition, it has a biostimulation effect on cell activity and supports the proliferation of osteoblasts .
Some researchers have combined the two above-noted laser methods in the treatment of peri-implantitis. Compared with a traditional treatment method, Twinlight laser therapy can effectively reduce the sulcus bleeding index and the probing depth, thereby achieving better clinical efficacy .Meanwhile, Twinlight laser treatment contributes to improving cutting efficacy, shortens the operation duration, and promotes the improvement of patients' intraoperative comfort level and postoperative satisfaction, giving it high clinical application and promotional value. However, there are currently few reports on the mechanism of action involved in Twinlight laser treatment.
The acquisition of MSCs and establishing the peri-implantitis model
MSCs derive from oral alveolar bone and have multiple differentiation potentials. They can be differentiated into, e.g., osteoblasts, adipocytes, and endothelial cells, and contribute to repairing hard and soft tissue around the implant .Bone marrow mesenchymal stem cells are an important source of osteoblasts and are often used as core cells for repairing local bone, gristle, and myelogenous adipose tissue. These cells can be adhered to a titanium surface and activated in the osteogenesis and osseointegration phases . There are two ways in which to obtain inflammatory MSCs, i.e., the in-vitro induction of an inflammatory microenvironment and culturing in the periodontia of patients with peri-implantitis whose implants must be removed. The major pathogenic bacterium of peri-implantitis is a gram-negative anaerobic bacterium. Liposaccharides, as one of the strongest virulence factors, can activate a series of host cells to release proinflammatory cytokines and inflammatory mediators, thereby directly or indirectly causing inflammation. Through PCR tests, Lu et al.  found that LPSs could up-regulate the expression of inflammatory cytokines (IL-6 and IL-1β) and transforming growth factor beta in BMSCs. Yuli Wang et al.  used 150-μg/L LPS osteogenesis to establish a BMSC inflammation model. By conducting a western blot analysis, the authors found that LPSs could lower the content or relative expression level of both bone morphogenetic protein-2 and the ALP gene.
The above studies indicate the feasibility of establishing an in-vitro inflammation model. Compared with other modeling methods, this approach lacks a bacterial effect; however, LPSs can rapidly induce the host’s immune response in a manner similar to the endotoxins of some pathogenic bacteria present in peri-implantitis .In the current study, a concentration of 1-μg/ml LPSs was used. By referring to domestic and foreign literature and combining existing preliminary experiments with the results of the current study, the authors found that this concentration was perfectly suitable and could cause inflammatory injury in MSCs without causing significant cell death. Hence, it was concluded as a reasonable induction dose. In this study, the significant elevation in the expression level of IL-6 and IL-8 mRNA in group L verified this.
Because of its excellent mechanical properties and biocompatibility, titanium has become a mainstay material for use in dental implants. Many studies have shown that treating the surface of titanium with an Er:YAG laser can effectively enhance osseointegration between titanium disks and cells, without damaging the surface .Hauser et al.  compared the surface of a titanium implant treated with an Er:YAG laser (63.69 J/cm2) with the same surface in a control group without any treatment and found that laser treatment was beneficial to the colonization and proliferation of osteoblasts. Hani et al.  compared the removal effect of an Er:YAG laser, titanium brush, and a carbon fiber tip on the biofilm of implants with a titanium surface. The researchers created a titanium plate for 8 individuals who wore the device for 72 h to form biofilms. The titanium plate was then removed. The 8 individuals were then randomly divided into 4 treatment groups. The residual biofilms were observed under a fluorescence microscope, and image analysis software was used for the quantitative analysis of the residual biofilms. It was concluded that for titanium surfaces without threads, Er:YAG laser therapy was an effective approach for reducing bacterial biofilms on the titanium plate. Guo Zehong et al.  inoculated osteoblasts onto laser-etched and smooth titanium disks and observed cell adhesion microscopically. After inoculation, immunofluorescence staining was performed, and a laser scanning confocal microscope was used to observe the cytoskeleton and morphology. It was concluded that, following laser etching treatment of the titanium surface, the surface morphology was controllable and the microstructure of the titanium surface could be increased without causing cytotoxicity, thereby promoting the early proliferation of human osteosarcoma (MG63) cells.
In this study, an Er:YAG laser (MSP, 60 mJ, 20 Hz, 1.2 W; water, 4; gas, 4) was used to treat a titanium disk surface to simulate debridement in the clinical treatment of peri-implantitis. Cells treated using different methods were inoculated onto the titanium disk surface, and SEM showed that inflammatory MSCs could grow and extend on the titanium disk’s surface. Additionally, qRT-PCR results showed that compared with group C, the mRNA expression level of inflammatory cytokines (IL-6 and IL-8) rose significantly in group L (P < 0.01), indicating the successful establishment of a peri-implantitis model.
The effect of Twinlight laser treatment on titanium surface proliferation and the morphology of inflammatory MSCs
Scanning electron microscopy indicated that cells treated with a variety of methods grew and adhered well to the titanium disks. Compared with the LPS induction group, the cells in group C were the densest. Compared with the L + E and L + N groups, the MSCs in the L + E + N group had richer filopodia; additionally, the slender filopodia of MSCs could potentially extend continuously to seek more suitable adhesion sites. The proliferation capacity of MSCs on the titanium disk’s surface was tested by CCK-8, and the results showed that the absorbance proliferation trend of MSCs in the five groups on the titanium disk’s surface was consistent with the growth trend of the cells in the 6-well plate, presenting a trend showing a slow decline after an initial rise. Days 1–3 represented a rapid growth period and days 5–7 a gentle growth period; the reason for this may have been because cell growth space had been reduced. Hou et al.  stimulated the BMSCs of rats with a 5.0 J/cm2 diode laser; MTT test results showed that, compared with the C group, the diode laser significantly promoted BMSCs’ proliferation, growth factor secretion, and differentiation. On days 5–7, the absorbance value of the L + N, L + E, and L + E + N groups in the present study that had been laser-treated was higher compared with group L, verifying that laser treatment could promote inflammatory cell proliferation.
Concerning absorbance value, on days 3–7, in the L + E + N group, this value was higher compared with the L + N and L + E groups, while the value in the L + E group was higher compared with that of the L + N group. It was thus concluded that Er:YAG laser treatment had a better effect compared with Nd:YAG laser treatment in terms of promoting the proliferation capacity of inflammatory MSCs. Twinlight laser treatment had a better effect compared with using an Er:YAG or Nd:YAG laser on their own in terms of promoting the proliferation capacity of inflammatory MSCs.
The effect of Twinlight laser treatment on the inflammatory response of inflammatory MSCs
Peri-implantitis is typically caused by the overexpression of proinflammatory cytokines and inflammatory mediators in a local microenvironment. According to the pathogenesis of peri-implantitis, reducing the production of proinflammatory cytokines, inhibiting alveolar resorption, and promoting new bone formation may be effective ways of treating this condition. Interleukin-6 is a cytokine produced by activated T-cells and fibroblasts, which are involved in the development of inflammation and influence the growth and differentiation of various cells through their pleiotropy. Interleukin-8 is an inflammatory chemokine that promotes an inflammatory response, thus stimulating angiogenesis and facilitating mitosis, which is closely linked to the occurrence and development of various diseases . Chu Tienan et al.  studied patients receiving implant repair therapy and found that inflammatory cytokines, such as IL-6, IL-8, and tumor necrosis factor alpha (TNF-α), could be detected in gingival crevicular fluid with inflammation manifestation in gingival implant tissue. Jin Xiaolan et al.  detected TNF-α, IL-8, IL-1β, and IL-6 in the supernatant of inflammatory and normal cells using an ELISA method, and concluded that low-level laser irradiation could protect against the inflammatory damage of LPS-induced human periodontal ligament fibroblasts. Huang et al.  found that the diode laser reduced the expression of inflammatory markers IL-6 and IL-1 in MG63 induced by LPSs and improved cell proliferation capacity.
The current experiment found that, as time passed, the expression level of inflammatory cytokines in the cells of each group increased, and compared with group C, the expression level of IL-6 and IL-8 mRNA in group L rose significantly (P < 0.01). Compared with group L, the expression levels of IL-6 and IL-8 mRNA in the L + N, L + E, and L + E + N groups were lower; however, the decline in the L + E + N group was the most significant (P < 0.0001), indicating that Er:YAG, Nd:YAG, and Twinlight laser treatments could inhibit the expression of inflammation-related genes in inflammatory MSCs induced by LPSs on a titanium disk and reduce inflammatory injury; however, Twinlight laser treatment of inflammatory MSCs could more effectively inhibit the expression of inflammatory cytokines.
The effect of Twinlight laser treatment on the osteogenic differentiation ability of inflammatory MSCs
Among the multiple differentiation abilities of BMSCs, osteogenic differentiation is an important function for repairing tissue defects around implants. The genes related to osteogenic differentiation include the ALP and RUNX2 genes. The former is indispensable in the osteoblast mineralization process. When there is no increase in the level of inorganic phosphates in the components of bone mineral phases, the level of ALP will be high. Thus, ALP activity is considered an early indicator of osteoblast differentiation . Sun et al.  used LPSs in 1-μg/ml Escherichia coli to stimulate the BMSCs of rats to establish an inflammation environment; the researchers found that LPSs could significantly inhibit ALP expression and osteogenic differentiation in cells, thus indicating that inflammation reduced the ALP expression level of BMSCs in rats. Liu et al.  found that after adding low-concentration LPSs (100 μg/L) to an osteogenic medium, ALP gene activity in BMSCs declined. The researchers concluded that in the inflammatory response induced by LPSs, the increased expression of proinflammatory cytokines generated an inhibiting effect on the differentiation of osteoblasts.
The RUNX2 gene is an important transcription factor for osteoblast differentiation and bone formation, and also functions as a mark of osteoblast differentiation. It can activate the expression of type-I collagen, OC, and other downstream factors in the osteogenic differentiation regulatory network, promote osteogenic differentiation, accelerate extracellular matrix deposition, and form bone tissue . Luo et al.  used LPS to stimulate the BMSCs of rats; they found that LPSs inhibited the expression level of factors (ALP and RUNX2) related to osteogenic differentiation and type-I collagen. Wang et al.  assessed the effect of low-level laser therapy (LLLT) with different energy intensities on the proliferation and osteogenic differentiation of BMSCs under healthy and inflammatory microenvironments. They concluded that LLLT with a density of 8 J/cm2 could promote BMSC proliferation and osteogenesis. Bai et al. . co-cultured in vitro BMSCs and human umbilical vein endothelial cells and found that LLLT enhanced vascular bone regeneration through the coupling of angiogenesis and osteogenesis. Reactive oxygen species regulate hypoxia-inducible factor 1 alpha may be key to the formation of H-shaped blood vessels and the enhancement of osteogenic differentiation in BMSCs. This enhanced angiogenesis further promoted bone repair and regeneration and provided new perspectives on the role of LLLT in fracture healing and tissue engineering strategies. Furthermore, the effect of LLLT on cell proliferation, angiogenesis, and osteogenesis was assessed. The results of this experiment showed that, as the time of osteogenesis-induced differentiation increased, the expression of osteogenesis-related genes, i.e., ALP and RUNX2 mRNA, increased. On days 7 and 14 of osteogenesis-induced differentiation, the expression level of ALP and RUNX2 mRNA in the L, L + N, L + E, and L + E + N groups was lower than in group C, which was consistent with the findings of Bandow, indicating that LPSs could effectively inhibit the osteogenic differentiation ability of MSCs. On day 14, the expression levels of ALP and RUNX2 mRNA in the L + N, L + E, and L + E + N groups were higher than in group L, but the expression level of RUNX2 mRNA in the L + E + N group was significantly higher than in group L (P < 0.01). There was no significant difference in the expression level of ALP and RUNX2 mRNA between the L + N, L + E + N, and C groups (P > 0.05), indicating that Er:YAG, Nd:YAG, and Twinlight laser treatment could promote the expression of inflammation-related genes in inflammatory MSCs induced by LPSs on a titanium disk, and fulfill a specific osteogenic role. It is noted, however, that Twinlight laser treatment of inflammatory MSCs could more effectively promote the expression of osteogenesis-related Al-BMSC genes.
The results of the current study showed that Twinlight laser treatment could promote cell proliferation, down-regulate the mRNA expression of inflammatory cytokines, and effectively enhance the osteogenic differentiation of cells on a titanium disk surface. This study provides a theoretical basis for Twinlight laser adjuvant therapy in cases of peri-implantitis. Since this experiment was performed using an in vitro model, it is necessary to further improve animal-based experiments and clinical studies in follow-up experiments for verification purposes.