Abstract
Immune reactions play an important role in determining the biostimulation of bone formation, either in new bone formation or inflammatory fibrous tissue encapsulation. Macrophage cell, the important effector cells in the immune reaction, which are indispensable for osteogenesis and their heterogeneity and plasticity, render macrophages a primer target for immune system modulation. However, there are very few studies about the effects of macrophage cells on laser treatment-regulated osteogenesis. In this study, we used CO2 laser as a model biostimulation to investigate the role of macrophage cells on the CO2 laser stimulated osteogenesis. Bone morphogenetic protein 2 (BMP2) was also significantly up regulated by the CO2 laser stimulation, indicating that macrophage may participate in the CO2 laser stimulated osteogenesis. Interestingly, when laser treatment macrophage-conditioned medium were applied to human periodontal ligament cells (hPDLs), the osteogenesis differentiation of hPDLs was significantly enhanced, indicating the important role of macrophages in CO2 laser-induced osteogenesis. These findings provided valuable insights into the mechanism of CO2 laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment.
Keywords: CO2 laser, macrophage, periodontal ligament cell, bone morphogenetic protein 2, osteogenesis.
1. Introduction
Laser is classified as light amplification by stimulated involves the modification of the environment to stimulate existing bacteria capable of bioremediation [1]. The application of laser light was in many fields including medical, industrial, and the military. In the clinical, there are several commercial lasers, including CO2, diode, and erbium (Er):yttrium aluminum garnet (YAG) lasers[2]. The advantage of laser light over conventional treated with chemical agents was its ability to partially travel through the biofilm [3]. Some studies proved that the antibacterial effect of CO2 laser irradiation on bacteria was high efficiency when bacteria were embedded in biofilm, due to a photo-thermal mechanism [1-4]. However, some researches shown that lasers affect fibroblast proliferation and collagen synthesis and reduce inflammation [5]. In addition, the laser light can promote periodontal cell differentiation and it has potentially be used to enhance periodontal tissue regeneration [2,6].
The interaction between immune cells and bone cells has been studied more thoroughly in osteoimmunology [7]. The skeletal and immune systems are closely related, haring several receptors, cytokines, signalling molecules and transcription factors. Among the immune cells, the macrophage is one of the most important cells in the laser treatment-induced immune response [8,9]. The macrophages can change their physiology and phenotype through the environmental signals [10]. In addition, the inflammation macrophages are also known to affect bone pathology and physiology [11,12]. In addition, it is well known that bone remodeling is regulated by the replacement of old bone with new bone through sequential osteoblastic bone formation and osteoclastic resorption [13]. Several inflammatory cytokines, growth factors, and
hormones result in an imbalance between osteoblast and osteoclast activities and can result in skeletal abnormalities, such as osteopetrosis and osteoporosis [14]. Macrophages contribute to osteogenic through the secretion of a wide range of regulatory biomolecules, such as transforming growth factor β (TGF-β) and BMP2 [15]. Thus, macrophages are needed for efficient osteoblast differentiation and the attrition of macrophages can reduce osteoblast bone formation ability [11]. Given the important roles of macrophages in the bone formation, some studies have analyzed the interactions between laser treatment and macrophages. However, these studies are focused on either the inflammatory contributions or the differentiation into osteoclasts under various laser treatment [2,16]. Very few reporters have been made on the effects of macrophages in regulating laser-treatment stimulated osteogenesis.
The CO2 laser treatment is well recognized as the osteoconductive stimulation and has been widely used for clinical dentistry regeneration application. Although there are many studies about the in vitro and in vivo osteogenesis of CO2 laser, the effect of CO2 laser treatment on the cellular behavior of macrophages is unclear, and the effect of macrophages on laser-coordinated osteogenesis of hPDLs has not been investigated. Therefore, the aim of this study, the CO2 laser is used as a model to investigate the interactions between laser-treatment and macrophages, and to further reveal whether macrophages participate in the laser-stimulated osteogenesis.
2. Materials and Methods 2.1 Cell culture
LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway) was used to isolate monocytes from healthy donors. The patient gave informed consent, and approval from the Ethics Committee of the Chung Shan Medicine University Hospital was obtained (CSMUH No. CS11187). The blood was diluted by the addition of an equal volume of 0.9% sodium chloride. The dilute blood (8 mL) was mixed with LymphoprepTM (4 mL) and centrifuged at 800 g for 20 minutes at room temperature. The interphase band containing peripheral blood mononuclear cells was then removed and washed in PBS, after which the monocytes were resuspended in RPMI 1640 medium (Caisson Laboratories, North Logan, UT) with 2 mM of L-glutamine (Caisson Laboratories), 100 U/mL penicillin, and 100 μg/mL streptomycin (PS, Caisson Laboratories), and supplemented with 10% fetal bovine serum (FBS, GeneDireX, Taiwan).
Human PDL tissues were freshly derived from the third premolar teeth was extracted for orthodontic reasons. The patient gave informed consent. The extracted tooth with PDL tissue was rinsed three times in phosphate-buffered saline (PBS; Caisson Laboratories, North Logan, UT) solution and digestion in 0.1% collagenase type I (Sigma-Aldrich, St Louis, MO) for 30 min. After transferred into a new plate, the cells suspension were cultured in Dulbecco’s modified Eagle medium (DMEM; Caisson), supplemented with 10% FBS, 10 units/ml penicillin G solution, and 10 mg/ml streptomycin (PS, Caisson) in a humidified atmosphere of 5 % CO2 at 37oC. PDL cells were subculture by successive passaging at a 1:3 ratio until they were used for experiments (passages 3–8).
2.2 Macrophage cell viability
To differentiate monocytes into monocyte-derived macrophages, the monocytes were resuspended in 96 well (GeneDireX) with a medium containing 100 nM phorbol 12-myristate 13-acetate (PMA, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 day, than treatment with carbon dioxide (CO2) laser (Yoshida Dental Laser, Tokyo, Japan) for 1 w, 0.002s. After different culture times, cell viability was evaluated by the PrestoBlue® assay (Invitrogen, Grand Island, NY). Briefly, at the end of the culture period, the medium was discarded and the wells were washed with cold PBS twice. Each well was filled with medium with a 1:9 ratio of PrestoBlue® in fresh DMEM and incubated at 37°C for 30 min. The solution in each well was transferred to a new 96-well plate. Plates were read in a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test.
2.3 The inflammatory protein of macrophage cells
Macrophage cells were seeded in a 96-well plate at a density of 5 x 104 cells per well. After 24 h of incubation, the culture medium was removed and treatment with CO2-laser (1 w, 0.002s, 2 times). Culture medium without treatment with CO2-laser was used as a control. After cultured for different time-points, the cell lysates of all groups was analysis by ELISA kit.
Macrophage cells were seeded in a 96-well plate at a density of 5 x 104 cells per well. After 24 h of incubation, the culture medium was removed and treatment with CO2 laser (1 w, 0.002s, 2 times). Culture medium without treatment with laser was used as a control. After treatment for 3 days, BMP2, TGF-1, and VEGF protein released from cells were determined by enzyme-linked immunosorbent assay kit (ELISA, Invitrogen) following the manufacturer’s instruction. The protein concentration was measured by correlation with a standard curve. All experiments were done in triplicate.
2.5 Effects of macrophage cells-conditioned medium on the osteogenic differentiation of PDL cells
Macrophage cells were seeded in a 96-well plate at a density of 5 x 103 cells per well. After 24 h of incubation, the culture medium was removed and treatment with CO2-laser for 1 or 2 times. Culture medium with 5% FBS was used as a negative control. After 3 days, media from cells without/with laser treatment were collected, and centrifuged at 1500 rpm to gain the supernatants for further conditioned media experiments. To investigate whether macrophage cells could regulate osteogenesis of PDL cells in response to laser treatment, the PDL cells were cultured with the collected macrophage cells supernatants conditioned media.
For the detection of bone-related gene [alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC)] and protein expression (ALP, OC) of PDL cells, which were cultured at a density of 104 cells per well in separate 6-well plates. After 24 h of incubation, the culture medium was removed and replaced by conditioned medium or control medium. Total RNA of all four groups was extracted using TRIzol reagent
(Invitrogen) after 3 days and analyzed by RT- qPCR. Total RNA (500 ng) was used for the synthesis of complementary DNA using cDNA Synthesis Kit (GenedireX) following the manufacturer’s instructions. RT-qPCR primers (Table 1) were designed based on cDNA sequences from the NCBI Sequence database. SYBR Green qPCR Master Mix (Invitrogen) was used for detection and the target mRNA expressions were assayed on the ABI Step One Plus real-time PCR system (Applied Biosystems, Foster City, California, USA). Each sample was performed in triplicate.
In addition, the ALP activity was determined after cell cultured for 7 days. The process was as follows: the cells were lysed from dish using 0.2 % NP-40, and centrifuged for 10 min at 2000 rpm after washing with PBS. ALP activity was determined using p-nitrophenyl phosphate (pNPP, Sigma) as the substrate. Each sample was mixed with pNPP in 1 M diethanolamine buffer for 15 min, after which the reaction was stopped by the addition of 5 N NaOH and quantified by absorbance at 405 nm. All experiments were done in triplicate. The OC protein released from cells were determined by enzyme-linked immunosorbent assay kit (ELISA, Invitrogen) following the manufacturer’s instruction. The protein concentration was measured by correlation with a standard curve.
2.6 The mineralization of PDL cells
The accumulated calcium deposition after 14 days was analyzed using Alizarin Red S staining, as in a previous study [17,18]. In brief, the cells were fixed with 4% paraformadedyde (Sigma-Aldrich) for 15 min and then incubated in 0.5% Alizarin Red S (Sigma-Aldrich) at pH 4.0 for 15 min at room temperature in an orbital shaker (25 rpm).
After the cells were washed with PBS, photographs were observed using an optical microscope (BH2-UMA, Olympus, Tokyo, Japan) equipped with a digital camera (Nikon, Tokyo, Japan) at 200x magnification. To quantify the stained calcified nodules after staining, samples were immersed with 1.5 mL of 5% SDS in 0.5N HCl for 30 min at room temperature. After that, the tubes were centrifuged to 5,000 rpm for 10 min and the supernatant was transferred to the new 96-well plate (GeneDireX); absorbance was measured at 405 nm (Hitachi).
2.7 BMP2-related signaling pathway of PDL cells
To investigate the activation of the BMP2 signaling pathway in hPDLs, BMP2 signaling pathway related genes (SMAD5), bone morphogenetic protein receptor type IA (BMPR1) and bone morphogenetic protein receptor type II (BMPR2) were analyzed by RT-qPCR as described in section 2.5.
2.8 Statistical Analysis
A one-way variance statistical analysis was used to evaluate the significance of the differences between the groups in each experiment. Scheffe’s multiple comparison test was used to determine the significance of the deviations in the data for each specimen. In all cases, the results were considered statistically significant with a p value < 0.05.
3. Results
3.1 Macrophage cell viability
At 1, 2, and 3 days post-irradiation, the macrophage cell viability was similar (p > 0.05) between the CO2 laser-treatment for 1 and 2 times groups and Ctl (Fig 1). PrestoBlue® analysis showed that the overall metabolic activity of most groups with laser-treatment increased in a time-dependent manner. However, the viability of macrophage cells in the presence of laser-treatment for 3 times significantly decreased than other groups (p < 0.05).
3.2 Inflammatory protein expression of macrophage cells
The inflammatory protein of TNF-α (Fig 2A) was showed no significant change after CO2 laser stimulation. On the contrary, IL-1 (Fig 2B) was down regulated significantly with the treatment with CO2 laser 2 times (p < 0.05). Anti-inflammatory protein IL-10 expression was showed no significant change after stimulation (Fig. 2C).
3.3 Osteogenic protein secretion from RAW 264.7 cells
BMP2 protein secretion was up regulated by the stimulation of CO2 laser (P < 0.05) (Fig 3A). The BMP2 secretions from cells with laser-treatment for 1- and 2-times were increased 57% and 95% (p < 0.05) than on the macrophage cell alone, respectively. However, TGF-β1 (Fig 3B) and VEGF (Fig 3C) protein secretion showed no significant change with the all treatment.
Fig. 4 showed the expressions of three-measured osteogenic gene (ALP, OPN, and OC) by PDL cells stimulated by macrophage cells-conditioned media with laser-treatment were significantly higher than DMEM (p < 0.05). However, the macrophage cell conditioned medium alone without laser-treatment showed no significant changes in expression of ALP in comparison with DMEM (P > 0.05) (Fig. 4A).
Protein expression of ALP and OC showed a similar trend to the RT-qPCR results. Both measured proteins were significantly promoted by PDL cells stimulated by macrophage cell with laser-treatment relative to cell alone (p < 0.05) (Fig. 5).
Alizarin Red S staining showed evidence of calcium deposition and nodule formation. Mineralized nodules formation was observed in all groups with osteogenic medium (Fig. 6A). More distinct nodules were observed in PDL cells stimulated by macrophage cells with laser-treatment relative to cell alone (p < 0.05) (Fig. 6B).
3.5 BMP2 signaling pathway-related gene expression
To further explore the mechanisms of enhanced osteogenic, we considered the gene expressions of BMP2 signaling pathways (Fig. 7). Macrophage cells conditioned medium alone could not up-regulate the BMP2 signaling pathways related genes (BMPR1, BMPR2 and SMAD5), compared with the normal culture medium. However, The expressions of BMPR2, and SMAD5 cultured with laser treated-macrophage cells extracts showed significantly up-regulation compared to those cultured with DMEM (p < 0.05). Moreover, the BMPR2 and SMAD5 gene expression of PDL cells cultured with
the conditioned medium of macrophage cells under laser-treatment-2 times stimulated were higher than laser-treatment-1 time.
4. Discussion
Macrophage cells play an important role in the interaction between bone modeling and remodeling. Such effects are commonly attributed to an inflammatory response and laser treatment. While these effects are undoubtedly important, our study further revealed that in response to the CO2-laser treatment, leading to the release of osteoinductive molecules and anti-inflammatory cytokines from macrophage cells, which enhanced the osteogenesis of hPDLs through the BMP2 pathway. These findings demonstrate that macrophage participated and made important contributions to the CO2-laser stimulated osteogenesis.
Although macrophages have been confirmed to be involved in the osteogenesis, there is still no consensus on which phenotype is more useful for the osteogenic differentiation. The classically activated inflammatory M1 macrophages cells were lead osteogenesis in hMSCs [19]. However, it was also verified that culture medium from lipopolysaccharide induced macrophages lead osteoblast cells to differentiate towards fibroblasts even with osteogenic medium, which might be related to the up-regulation of TNF-α, IL-1β and IL-6 [20]. In the current study, immune response was evaluated by measuring the expression of TNF-α, 1 and 10, and macrophage cells decreased IL-1 expression when stimulated with CO2-laser as compared to those in the control environment. IL-1 is the early “immune” cytokine identified to be involved in the control of bone formation and affected pre-osteoclast cell behavior. It is also a typical example of multifunctional cytokines that are activated by environmental stress [21]. iNOS is only expressed in responses to inflammatory stimuli [22] and IL-1 activation of the iNOS pathway in cells [23]. It was found that both IL-1 and iNOS expression of several type
cells after treatment with laser with increasing culture time [2,6]. Similar studies have shown that inflammatory responses to laser irradiation was shown to inhibit prostaglandin E2 and IL-1β production and gene expression; the inhibition of the prostaglandin E2 and IL-1β might be of therapeutic value [24].
During orthodontic treatment, the macrophage cells participate in both ends, via secreting relating cytokines. They may contribute osteoinductive and osteogenic cytokines (BMP2, VEGF) to promote osteogenesis, but also assist inflammatory and fibrous agents (TNF-α, VEGF, TGF-β) to promote pathological fibrosis [24]. In addition, it was indicated that excess switch of M2 phenotype may result in scar tissue or a delay in wound healing. Therefore, the conclusion still could not be made that the macrophage cells played an important role in regulating osteogenesis. An effective and adequate switch in macrophage might be of great importance for enhancing osteogenesis. Possible effects of macrophages in the process of CO2-laser inducing osteogenesis are summarized in Fig. 8. The CO2-laser stimulated macrophages were result in the down-regulation inflammatory cytokines 1), and the up-regulation of anti-inflammatory cytokines (IL-10). The stimulated macrophages also up-regulate BMP2 gene expression, which activates the BMP2 signalling pathway in PDL cells, leading to osteogenic differentiation. For evaluating the osteogenic capacity of laser-treatment, PDL cells are typically used [2,6,16]. However, the results of biological responses to laser stimulation are not always consistent, due to the lack of immune cells in vitro. CO2 laser-treatment itself was not sufficient to enhance the osteogenic effect of hPDLs, however, laser stimulated macrophages conditioned medium could release osteogenic cytokines, thereby promoting the osteogenesis effect of hPDLs, which was more consistent with in vivo
results. The confirmation that macrophage cells behave actively during the osteogenesis differentiation caused by CO2 laser is meaningful. Inadequately activated macrophage cells will affect failure of bone regeneration and formation, leading to undesired inflammatory fibrosis. The macrophage cells should be taken into consideration for evaluating the in vitro osteogenic capacity of CO2 laser-treatment therapy.
Osteoblast differentiation is generally accompanied by the production of ALP, OPN, OC, and in vitro mineralization. ALP played a major role in the initiation of osteogenic differentiation, and ALP expression was up regulated after the start of differentiation, as shown in cells cultured with laser treatment-macrophage medium. Our results were consistent with previous reports [2]. As an example, ALP protein expression of hPDLs was similar to gene expression. [2]. The explanation for the low number of increments seen in ALP activity with culture time may be an indicator of the cell progressing into mineralization. OC is the most abundant non-collagenous bone-matrix protein and is secreted at the late stage in osteoblast differentiation and mineralization [25]. hPDLs cultured with laser treatment-macrophage medium showed significantly increased OPN and OC expression levels with increased treatment time compared to those in the normal DMEM and macrophage-medium. Likewise, similar results have been demonstrated after microarray analysis for the osteogenic biomarker [26]. The results of the current study consistently indicated that the presence of laser-treatment macrophage was effective in supporting the differentiation of hPDLs and strongly promoted a biological response in cells through the production of protein specific to bone formation. Therefore, initiation of the osteogenesis effect could be observed after cellular
inflammation lessened. It is reasonable to assume that the alveolar bone had good osteogenic abilities on the laser treatment.
At very low-power densities, the energy fluence range from 1 to 10 Jcm−2, and frequently, the photochemical interactions can cause the biostimulation of several tissues. In contemporary dental practice, the orthodontist treatment is an increasingly daily occurrence. From our present study, the CO2 laser can be considered a non-invasive, painless and a thermal therapy that restores tissue functionality through its bio-stimulation, regenerative and anti-inflammatory effects. In addition, we demonstrate that although most CO2 laser induces the secretion from macrophage cells to affect behavior of hPDLs. Consequently, CO2 laser lead to cell signal transduction and outcomes through similar mechanoreceptors and signaling effectors in cells and tissues, and it is therefore suggested that common signaling mechanisms are involved in laser-transduction pathways.
5. Conclusion
Macrophages played important roles in CO2 laser treatment-induced osteogenesis. CO2 laser treatment macrophage condition medium inhibited the inflammation and enhance the osteogenic differentiation of hPDLs. Adequately activated macrophages are vital for the success of laser-stimulated bone regeneration. The interaction with immune cells, especially macrophages, should be elucidated when evaluating the in vitro osteogenesis capacity of laser treatment in orthodontic.
Acknowledgements
The authors acknowledge receipt of a grant from the National Science Council grants (NSC 102-2314-B-040-007-MY3) of Taiwan.
Author disclosure statement
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Figure Legends
Figure 1. The PrestoBlue® assay performed for viability macrophage cells treated with CO2 laser for different times. *p < 0.05, compared with Ctl.
Figure 2. (A) TNF-α, (B) IL-1, and (C) IL-10 secreted form macrophage cells after treatment with CO2 laser for different times. *p < 0.05, compared with Ctl.
Figure 3. (A) BMP2, (B) TGF-β1, and (C) VEGF secreted form macrophage cells after treatment with CO2 laser for different times. *p < 0.05, compared with Ctl.
Figure 4. Relative mRNA expressions of (A) ALP, (B) OPN, and (C) OC by comparing hPDLs cultured in complete culture medium or CO2 laser treatment-macrophage cell conditioned media. *p < 0.05, compared with DMEM.
Figure 5. (A) ALP and (B) OC secretion from hPDLs cultured in complete culture medium or CO2 laser treatment-macrophage cell conditioned media. *p < 0.05, compared with DMEM.
Figure 6. (A) Photograph and (B) quantification of calcium mineral deposits by Alizarin Red S assay of hPDLs cultured in complete culture medium or CO2 laser treatment-macrophage cell conditioned media for 14 days. *p < 0.05, compared with DMEM. Figure 7. Relative mRNA expressions of (A) BMPR1, (B) BMPR2, and (C) SMAD5 of BMP2 signalling pathway genes in hPDLs cultured with complete culture medium or CO2 laser treatment-macrophage cell conditioned media. *p < 0.05, compared with DMEM.
Figure 8. Summary of possible effects of macrophages in the process of the CO2 laser treatment inducing osteogenesis.
