Effect of Low-Level Diode Laser and Red Light-Emitting Diode on Survival and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Background: This investigation set out to compare the impacts of low-level diode laser (LLDL) and red light-emitting diode (LED) on the survival of human dental pulp stem cells (hDPSCs) and osteogenic/odontogenic differentiation. Methods and materials: In this ex vivo experimental study, the experimental groups underwent the irradiation of LLDL (4 J/cm2 energy density) and red LED in the osteogenic medium. Survival of hDPSCs was assessed after 24 and 48 h (n = 9) using the methyl thiazolyl tetrazolium (MTT) assay. The assessment of osteogenic/odontogenic differentiation was conducted using alizarin red staining (ARS; three repetitions). The investigation of osteogenic and odontogenic gene expression was performed at two time points, specifically 24 and 48 h (n = 12). This analysis was performed utilizing real-time reverse-transcription polymerase chain reaction (RT-PCR). The groups were compared at each time point using SPSS version 24. To analyze the data, the Mann-Whitney U test, analysis of variance, Tukey's test, and t-test were utilized. Results: The MTT assay showed that LLDL significantly decreased the survival of hDPSCs after 48 h, compared with other groups (p < 0.05). The qualitative results of ARS revealed that LLDL and red LED increased the osteogenic differentiation of hDPSCs. LLDL and red LED both upregulated the expression of osteogenic/odontogenic genes, including bone sialoprotein (BSP), alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP), in hDPSCs. The LLDL group exhibited a higher level of gene upregulation (p < 0.0001). Conclusions: The cell survival of hDPSCs was reduced, despite an increase in osteogenic/odontogenic activity. Clinical relevance: Introduction of noninvasive methods in regenerative endodontic treatments.

NIR irradiation of human buccal fat pad adipose stem cells and its effect on TRP ion channels.

The effect of near infrared (NIR) laser irradiation on proliferation and osteogenic differentiation of buccal fat pad-derived stem cells and the role of transient receptor potential (TRP) channels was investigated in the current research. After stem cell isolation, a 940 nm laser with 0.1 W, 3 J/cm2 was used in pulsed and continuous mode for irradiation in 3 sessions once every 48 h. The cells were cultured in the following groups: non-osteogenic differentiation medium/primary medium (PM) and osteogenic medium (OM) groups with laser-irradiated (L +), without irradiation (L -), laser treated + Capsazepine inhibitor (L + Cap), and laser treated + Skf96365 inhibitor (L + Skf). Alizarin Red staining and RT-PCR were used to assess osteogenic differentiation and evaluate RUNX2, Osterix, and ALP gene expression levels. The pulsed setting showed the best viability results (P < 0.05) and was used for osteogenic differentiation evaluations. The results of Alizarin red staining were not statistically different between the four groups. Osterix and ALP expression increased in the (L +) group. This upregulation abrogated in the presence of Capsazepine, TRPV1 inhibitor (L + Cap); however, no significant effect was observed with Skf96365 (L + Skf).

Low-level laser therapy with different irradiation methods modulated the response of bone marrow mesenchymal stem cells in vitro.

Low-level laser therapy (LLLT) also known as photobiomodulation is a treatment to change cellular biological activity. The exact effects of LLLT remain unclear due to the different irradiation protocols. The purpose of this study was to investigate the effects of LLLT by three different irradiation methods on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs were inoculated in 24-well plates and then irradiated or not (control) with a laser using three different irradiation methods. The irradiation methods were spot irradiation, covering irradiation, and scanning irradiation according to different spot areas (0.07 cm2 or 1.96 cm2) and irradiation areas (0.35 cm2 or 1.96 cm2), respectively. The laser was applied three times at energy densities of 4 J/cm2. The cell proliferation by CCK-8. ALP activity assay, alizarin red, and quantitative real-time polymerase chain reaction (RT-PCR) were performed to assess osteogenic differentiation and mineralization. Increases in cell proliferation was obvious following irradiation, especially for covering irradiation. The ALP activity was significantly increased in irradiated groups compared with non-irradiated control. The level of mineralization was obviously improved following irradiation, particularly for covering irradiation. RT-PCR detected significantly higher expression of ALP, OPN, OCN, and RUNX-2 in the group covering than in the others, and control is the lowest. The presented results indicate that the biostimulative effects of LLLT on BMSCs was influenced by t he irradiation method, and the covering irradiation is more favorable method to promote the proliferation and osteogenic differentiation of BMSCs.

Photobiomodulation therapy upregulates the growth kinetics and multilineage differentiation potential of human dental pulp stem cells-an in vitro Study.

This study aims to evaluate the impact of red LED irradiation on the viability, proliferation, colonogenic potential, markers expression along with osteogenic and chondrogenic differentiation of dental pulp stem cells. DPSCs were isolated from sound human permanent teeth using enzymatic digestion method and seeded with regular culture media. Cells at P4 were irradiated using red LED Light (627 nm, 2 J/cm2) and examined for growth kinetics, and multilineage differentiation using the appropriate differentiation media. The irradiated groups showed an increase in cellular growth rates, cell viability, clonogenic potential, and decrease in population doubling time compared to the control group. Cells of the irradiated groups showed enhanced differentiation towards osteogenic and chondrogenic lineages as revealed by histochemical staining using alizarin red and alcian blue stains. Photobiomodulation is an emerging promising element of tissue engineering triad besides stem cells, scaffolds, and growth factors.

The effect of 805 nm near-infrared photobiomodulation on proliferation and differentiation of bone marrow stem cells in murine rats.

OBJECTIVE: To evaluate the effect of near infra-red gallium-aluminium-arsenide (GaAlAs) diode laser (805 nm) irradiation on proliferation and differentiation of rat femoral bone marrow-derived mesenchymal stem cells (BMSCs) cultured in osteogenic medium. MATERIALS AND METHODS: BMSCs were obtained from femurs of 60 Sprague Dawley rats (200 gm). The control group comprised isolated BMSCs supplemented with an osteogenic differentiation medium. On the other hand, in the experimental group, the BMSCs were irradiated with a near-infrared laser in addition to an osteogenic differentiation medium. The experimental group was irradiated with a soft tissue laser comprising of allium-aluminium-arsenic (Ga-Al-Ar) Diode at a near-infrared wavelength of 805 nm in continuous mode. The different output powers applied were 0.5 W, 1.0 W, 1.5 W and 2.0 W respectively. Various energy levels of 1, 4, 7 and 10 J were used for irradiation. Alkaline phosphatase (ALP) assay and Alizarin staining were performed to confirm osteogenic differentiation. Statistical analysis was done using a one-way ANOVA and a p-value of <0.05 was considered significant. RESULTS: According to our findings, 1.27 J/cm2 was the optimal energy density value that significantly increased the BMSC proliferation at the output of 1.5 W with the power density of 1.27 W/cm2. On 1.27 J/cm2, there was a significant difference compared to the control group on the first day, and the osteogenic differentiation increased significantly on the 4th day compared to the 1st day. CONCLUSIONS: According to our findings, 1.27 J/cm2 was the optimal energy density value that significantly increased the BMSC proliferation at the output of 1.5 W with the power density of 1.27 W/cm2.

Electromagnetic stimulation increases mitochondrial function in osteogenic cells and promotes bone fracture repair.

Bone fracture is a growing public health burden and there is a clinical need for non-invasive therapies to aid in the fracture healing process. Previous studies have demonstrated the utility of electromagnetic (EM) fields in promoting bone repair; however, its underlying mechanism of action is unclear. Interestingly, there is a growing body of literature describing positive effects of an EM field on mitochondria. In our own work, we have previously demonstrated that differentiation of osteoprogenitors into osteoblasts involves activation of mitochondrial oxidative phosphorylation (OxPhos). Therefore, it was reasonable to propose that EM field therapy exerts bone anabolic effects via stimulation of mitochondrial OxPhos. In this study, we show that application of a low intensity constant EM field source on osteogenic cells in vitro resulted in increased mitochondrial membrane potential and respiratory complex I activity and induced osteogenic differentiation. In the presence of mitochondrial inhibitor antimycin A, the osteoinductive effect was reversed, confirming that this effect was mediated via increased OxPhos activity. Using a mouse tibial bone fracture model in vivo, we show that application of a low intensity constant EM field source enhanced fracture repair via improved biomechanical properties and increased callus bone mineralization. Overall, this study provides supporting evidence that EM field therapy promotes bone fracture repair through mitochondrial OxPhos activation.

Pulsed Electromagnetic Fields in Bone Healing: Molecular Pathways and Clinical Applications.

In this article, we provide an extensive review of the recent literature of the signaling pathways modulated by Pulsed Electromagnetic Fields (PEMFs) and PEMFs clinical application. A review of the literature was performed on two medical electronic databases (PubMed and Embase) from 3 to 5 March 2021. Three authors performed the evaluation of the studies and the data extraction. All studies for this review were selected following these inclusion criteria: studies written in English, studies available in full text and studies published in peer-reviewed journal. Molecular biology, identifying cell membrane receptors and pathways involved in bone healing, and studying PEMFs target of action are giving a solid basis for clinical applications of PEMFs. However, further biology studies and clinical trials with clear and standardized parameters (intensity, frequency, dose, duration, type of coil) are required to clarify the precise dose-response relationship and to understand the real applications in clinical practice of PEMFs.

Amelioration of bone fragility by pulsed electromagnetic fields in type 2 diabetic KK-Ay mice involving Wnt/ß-catenin signaling.

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMFs) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMFs resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 wk remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate, and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number, or osteoclast-specific gene expression (TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a, and Wnt10b), but not noncanonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3ß and ß-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength, and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/ß-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration.NEW & NOTEWORTHY PEMF improved trabecular bone microarchitecture and suppressed cortical bone porosity in T2DM KK-Ay mice. It attenuated T2DM-induced detrimental consequence on trabecular and cortical bone material properties. PEMF resisted bone deterioration in KK-Ay mice by enhancing osteoblast-mediated bone formation. PEMF also significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. The therapeutic potential of PEMF on T2DM-induced bone deterioration was associated with the activation of Wnt/ß-catenin signaling.

Evaluation of photobiomodulation therapy to accelerate bone formation in the mid palatal suture after rapid palatal expansion: a randomized clinical trial.

To evaluate the efficiency of photobiomodulation therapy (PBMT) in the midpalatal suture (MPS) and pain sensation in patients undergoing rapid palatal expansion (RPE). Thirty-four individuals with the diagnosis of skeletal maxillary hypoplasia were divided in two groups: laser (n = 18) and control (n = 16). Treatment plan consisted of the use of the Hyrax expander in all patients. Subjects in the laser group were irradiated with diode laser (980 nm, 0.3 W) in six spots bilaterally distributed along the MPS for 10 s during the active phase of treatment and after overcorrection (passive phase of RPE). Control group received sham irradiations with the laser in standby mode to characterize the placebo effect. Digital occlusal radiographs were performed at different time-points for bone formation evaluation in both groups. The effects of laser irradiation on pain were assessed by the visual analog scale (Wong-Baker Faces Pain Scale). Bone formation between groups was not significantly different (p = 0.2273). At 3 months, bone formation was not yet complete in both groups. Pain sensation was similar between groups (p = 0.3940). However, pain was significantly higher for the first 7 days of treatment compared with the 14th day. PBMT did not accelerate bone regeneration in the MPS and pain sensation was similar.

Laser and LED photobiomodulation effects in osteogenic or regular medium on rat calvaria osteoblasts obtained by newly forming bone technique.

The purposes of this study are to evaluate the effects of photobiomodulation (PBM) with laser and LED on rat calvaria osteoblasts (rGO lineage), cultured in osteogenic (OST) or regular (REG) medium, after induction of a quiescent state and to test if PBM is capable of osteogenic induction and if there is a sum of effects when combining OST medium with PBM. Before irradiation, the cells were put in a quiescent state (1% FBS) 24 h, when red (AlGaInP-660 nm) and infrared laser (GaAlAs-808 nm) and LED (637 ± 15 nm) were applied. The groups were as follows: red laser (RL3-5 J/cm2, 3 s and RL5-8.3 J/cm2, 5 s, 1.66 W/cm2); infrared laser (IrL3-5 J/cm2, 3 s and IrL5-8.3 J/cm2, 5 s); LED (LED3-3 s and LED5-5 s, 0.02 J/cm2, 0.885 W/cm2); positive (C+, 10% FBS) and negative control (C-, 1% FBS). For alkaline phosphatase (ALP) and mineralization assays, the cells were cultured in REG (DMEM 10% FBS) and OST medium (DMEM 10% FBS, 50 µg/mL ascorbic acid, 10 mM ß-glycerophosphate). Statistical analysis was performed using ANOVA and Tukey's tests (p < 0.05). RL5 and LED5 increased proliferation, in vitro wound closure, ALP, and mineralization in rGO cells (p < 0.05). PBM with red laser and LED induced mineralization by itself, without osteogenic medium, not observed for infrared laser (p < 0.05). A sum of effects was observed in osteogenic medium and PBM by infrared, red laser, and LED (5 s). Red laser and LED increased proliferation, migration, and secretory phases in rGO cells in a dose-dependent manner. PBM with red laser and LED promotes osteogenic induction by itself. PBM with infrared laser and osteogenic medium potentializes mineralization.

Photobiomodulation as adjunctive therapy for guided bone regeneration. A microCT study in osteoporotic rat model.

Regeneration of diseased bone is challenging. Guided bone regeneration (GBR) has been applied to favor the bone repair. Photobiomodulation (PBM) is also a recognized therapy able to improve bone repair in healthy and diseased individuals. Thus, with the hypothesis that PBM therapy could improve the GBR of diseased bone, this study evaluated the effect of PBM as adjunctive therapy to GBR in osteoporotic rats. Osteoporosis was induced in rats using the oophorectomy model. Then, 5-mm calvaria bone defects were created and treated according to the experimental groups, as follows: with no further treatment (Control); conventional GBR (Membrane), GBR and PBM applied with 3 s, 4 J/cm2 and 0.12 J per point (PBM-1) and GBR and PBM applied with 10s, 14 J/cm2, 0.4 J per point (PBM-2). PBM therapy (808 nm, 40 mW, 1.42 W/cm2) was applied immediately, 48 and 96 h postoperatively. Four and eight weeks later, the samples were harvested and processed for micro-computerized tomography (Micro CT). Data were statistically compared (p < 0.05). From 4 to 8 weeks mostly significant changes were observed in the PBM groups. The bone volume fraction and number of trabeculae of the PBM groups, especially the PBM-1, were significantly higher than those of Control (p < 0.0001). The values of thickness and separation of the trabeculae and structural model index of the PBM groups were significantly smaller than Control (p < 0.0001). The connectivity density was significantly higher on Membrane and PBM groups than Control (p < 0.0004). The application of PBM as adjunctive therapy to GBR results in enhanced bone formation and maturation in comparison to the conventional GBR in the regeneration of lesions of osteoporotic bone in rats. Overviewing the challenges that face bone regeneration in patients with osteoporosis, our findings open new perspectives on the treatment of bone defects under osteoporotic conditions.

The effect of hyperbaric oxygen therapy on bone macroscopy, composition and biomechanical properties after ionizing radiation injury.

BACKGROUND: Radiotherapy used in tumor treatment compromises vascularization of bone tissue. Hyperbaric oxygenation (HBO) increases oxygen availability and improves vascularization, minimizing the deleterious effects of ionizing radiation (IR). Therefore, the aim of this study was to evaluate HBO therapy effect on bone macroscopy, composition and biomechanical properties after IR damage. METHODS: Twenty male Wistar rats weighing 300 ± 20 g (10 weeks of age) were submitted to IR (30 Gy) to the left leg, where the right leg was not irradiated. After 30 days, ten animals were submitted to HBO therapy, which was performed daily for 1 week at 250 kPa for 90-min sessions. All animals were euthanized 37 days after irradiation and the tibia were separated into four groups (n = 10): from animals without HBO - right tibia Non-irradiated (noIRnoHBO) and left tibia Irradiated (IRnoHBO); and from animals with HBO - right tibiae Non-irradiated (noIRHBO) and left tibia Irradiated (IRHBO). The length (proximal-distal) and thickness (anteroposterior and mediolateral) of the tibiae were measured. Biomechanical analysis evaluated flexural strength and stiffness. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to calculate the amide I ratio, crystallinity index, and matrix to mineral ratios. RESULTS: In the macroscopic and ATR-FTIR analysis, the IRnoHBO showed lower values of length, thickness and amide I ratio, crystallinity index and matrix to mineral ratios compared to noIRnoHBO (p < 0.03). IRnoHBO showed no statistical difference compared to IRHBO for these analyses (p > 0.05). Biomechanics analysis showed that the IRnoHBO group had lower values of flexural strength and stiffness compared to noIRnoHBO and IRHBO groups (p < 0.04). In addition, the noIRHBO group showed higher value of flexural strength when compared to noIRnoHBO and IRHBO groups (p < 0.02). CONCLUSIONS: The present study concluded that IR arrests bone development, decreases the collagen maturation and mineral deposition process, thus reducing the flexural strength and stiffness bone mechanical parameters. Moreover, HBO therapy minimizes deleterious effects of irradiation on flexural strength and the bone stiffness analysis.

Osteogenic Potential of Bovine Bone Graft in Combination with Laser Photobiomodulation: An Ex Vivo Demonstrative Study in Wistar Rats by Cross-Linked Studies Based on Synchrotron Microtomography and Histology.

BACKGROUND: Alveolar bone defects are usually the main concern when planning implant treatments for the appropriate oral rehabilitation of patients. To improve local conditions and achieve implant treatments, there are several methods used for increasing bone volume, among which one of the most successful, versatile, and effective is considered to be guided bone regeneration. The aim of this demonstrative study was to propose an innovative analysis protocol for the evaluation of the effect of photobiomodulation on the bone regeneration process, using rat calvarial defects of 5 mm in diameter, filled with xenograft, covered with collagen membrane, and then exposed to laser radiation. METHODS: The animals were sacrificed at different points in time (i.e., after 14, 21, and 30 days). Samples of identical dimensions were harvested in order to compare the results obtained after different periods of healing. The analysis was performed by cross-linking the information obtained using histology and high-resolution synchrotron-based tomography on the same samples. A comparison was made with both the negative control (NC) group (with a bone defect which was left for spontaneous healing), and the positive control (PC) group (in which the bone defects were filled with xenografts and collagen membrane without receiving laser treatment). RESULTS: We demonstrated that using photobiomodulation provides a better healing effect than when receiving only the support of the biomaterial. This effect has been evident for short times treatments, i.e., during the first 14 days after surgery. CONCLUSION: The proposed analysis protocol was effective in detecting the presence of higher quantities of bone volumes under remodeling after photobiomodulation with respect to the exclusive bone regeneration guided by the xenograft.

Utility of High-Intensity Laser Therapy Combined with Photobiomodulation Therapy for Socket Preservation After Tooth Extraction.

Objective: High-intensity laser therapy (HILT) combined with photobiomodulation therapy (PBMT) using a diode or CO2 laser was administered after extraction of the left first molar in rats. Effects on socket preservation (preservation of the alveolar bone and healing time after extraction) were evaluated histopathologically. Background: Irradiation using a diode or CO2 laser has been shown to hasten wound healing, but the effects remain controversial. Methods: Five-week-old male Wistar rats that underwent extraction of the left maxillary first molar were divided into three groups: diode laser irradiation (diode group), CO2 laser irradiation (CO2 group), and no laser irradiation (control group). HILT (27 J) was performed immediately after tooth extraction to enhance blood coagulation, followed by PBMT (0.7 J) 1 day later to enhance healing. Tissues, including the extraction socket, were removed en bloc 3, 5, 7, 10, and 21 days postextraction to determine the morphological characteristics of wound healing and the distribution of myofibroblasts involved in scar formation. Results: In the diode and CO2 groups, new bone formation and cancellous bone maturation were observed at an early stage of wound healing. The number of myofibroblasts was significantly lower in the laser treatment groups than the control (p < 0.001), and both treatment groups had a significantly higher alveolar crest height (p < 0.01), with almost no concavity in the mucosa of the extraction wound. Conclusions: Combined HILT and PBMT following tooth extraction hastened wound healing and preserved alveolar crest height, suggesting a role in socket preservation.

The Effects of Photobiomodulation on MC3T3-E1 Cells via 630 nm and 810 nm Light-Emitting Diode.

BACKGROUND Photobiomodulation (PBM) has been explored as a promising therapeutic strategy to regulate bone cell growth; however, the effects of PBM on osteoblast cell lines remains poorly understood. In addition, as a light source of PBM, the light uniformity of light-emitting diode (LED) devices has not been given enough attention. MATERIAL AND METHODS Here, we sought to investigate the effects of PBM on MC3T3-E1 cells via 630 nm and 810 nm light from a newly designed LED with high uniformity of light. Cell proliferation, flow cytometric analysis, alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red S staining, and quantitative real-time polymerase chain reaction (qRT-PCR) were carried out to assess treatment response. MC3T3-E1 cells were irradiated with LED devices (630±5 nm and 810±10 nm, continuous wave) for 200 seconds at a power density of 5 mW/cm² once daily. RESULTS Increases in cell proliferation and decreases in cell apoptosis were evident following irradiation. ALP staining intensity and activity were also significantly increased following irradiation. Level of mineralization was obviously enhanced in irradiated groups compared with non-irradiated controls. qRT-PCR also showed significant increases in mRNA expression of osteocalcin (OCN) and osteoprotegerin (OPG) in the irradiated groups. CONCLUSIONS Our results showed that LED PBM could promote the proliferation, ALP staining intensity and activity, level of mineralization, gene expression of OCN and OPG of MC3T3-E1 cells, with no significant difference between the 630 nm- and 810 nm-irradiated groups.

Photobiomodulation therapy (PBMT) in bone repair: A systematic review.

BACKGROUND: Photobiomodulation therapy (PBMT) using low-level laser influences the release of several growth factors involved in the formation of epithelial cells, fibroblasts, collagen and vascular proliferation, besides accelerating the synthesis of bone matrix due to the increased vascularization and lower inflammatory response, with significant increase of osteocytes in the irradiated bone. Considering its properties, beneficial effects and clinical relevance, the aim of this review was to analyze the scientific literature regarding the use of PBMT in the process of bone defect repair. METHODS: Electronic search was carried out in PubMed/MEDLINEⓇ and Web of Science databases with combination of the descriptors low-level laser therapy AND bone repair, considering the period of publication until the year 2018. RESULTS: The literature search identified 254 references in PubMed/MEDLINE and 204 in Web of Science, of which 33 and 4 were selected, respectively, in accordance with the eligibility requirements. The analysis of researches showed articles using PBMT in several places of experimentation in the subjects, different types of associated biomaterials, stimulatory effects on cell proliferation, besides variations in the parameters of use of laser therapy, mainly in relation to the wavelength and density of energy. Only four articles reported that the laser did not improve the osteogenic properties of a biomaterial. CONCLUSIONS: Many studies have shown that PBMT has positive photobiostimulatory effects on bone regeneration, accelerating its process regardless of parameters and the use of biomaterials. However, standardization of its use is still imperfect and should be better studied to allow correct application concerning the utilization protocols.

Bioglass/PLGA associated to photobiomodulation: effects on the healing process in an experimental model of calvarial bone defect.

Bioactive glasses (BG) are known for their ability to bond to bone tissue. However, in critical situations, even the osteogenic properties of BG may be not enough to induce bone consolidation. Thus, the enrichment of BG with polymers such as Poly (D, L-lactic-co-glycolic) acid (PLGA) and associated to photobiomodulation (PBM) may be a promising strategy to promote bone tissue healing. The aim of the present study was to investigate the in vivo performance of PLGA supplemented BG, associated to PBM therapy, using an experimental model of cranial bone defect in rats. Rats were distributed in 4 different groups (Bioglass, Bioglass/PBM, Bioglas/PLGA and BG/PLGA/PBM). After the surgical procedure to induce cranial bone defects, the pre-set samples were implanted and PBM treatment (low-level laser therapy) started (808 nm, 100 mW, 30 J/cm2). After 2 and 6 weeks, animals were euthanized, and the samples were retrieved for the histopathological, histomorphometric, picrosirius red staining and immunohistochemistry analysis. At 2 weeks post-surgery, it was observed granulation tissue and areas of newly formed bone in all experimental groups. At 6 weeks post-surgery, BG/PLGA (with or without PBM) more mature tissue around the biomaterial particles. Furthermore, there was a higher deposition of collagen for BG/PLGA in comparison with BG/PLGA/PBM, at second time-point. Histomorphometric analysis demonstrated higher values of BM.V/TV for BG compared to BG/PLGA (2 weeks post-surgery) and N.Ob/T.Ar for BG/PLGA compared to BG and BG/PBM (6 weeks post-surgery). This current study concluded that the use of BG/PLGA composites, associated or not to PBM, is a promising strategy for bone tissue engineering.

Effect of low-level laser therapy and zoledronic acid on bone repair process.

This study aimed to evaluate, through histomorphometric analysis, the bone repair process in the tibia of rats treated with zoledronic acid and submitted to 808-nm low-level laser therapy (LLLT) by using arsenide aluminum gallium laser. For this purpose, 20 rats were used and distributed according to treatment: group 1-saline administration; group 2-treated with LLLT; group 3-treated with zoledronic acid; and group 4-treated with zoledronic acid and LLLT. The zoledronic acid was administered at a dose of 0.035 mg/kg every 2 weeks for 8 weeks. Subsequently, bone defects of 2 mm were prepared in the tibias of all groups. The bone defects in groups 2 and 4 were irradiated with LLLT in the immediate post-operative period. After 14 and 28 days of application, the animals were submitted and euthanized for histomorphometric analysis. The results were submitted to statistical analysis (α = 5%), and the intragroup comparison was performed using the t test. On the other hand, for intergroup comparison, the ANOVA test was performed, and to the groups presenting statistically significant difference, the Student-Newman-Keuls test was used. In intergroup comparison, group 1 (mean ± SD= 45.2 ± 18.56%) showed a lower bone formation compared with groups 2 (64.13 ± 3.51%) (p = 0.358) and 4 (15.2 ± 78.22%) (p = 0.049), at the 14-day period. Group 3 (20.99 ± 7.42%) also presented a lower amount of neoformed bone tissue, with statistically significant difference when compared with groups 1 (p = 0.002), 2, and 4 (p ≤ 0,001). After 28 days, group 1 presented a lower amount of neoformed bone tissue compared with the other groups, with p = 0.020. Thus, it was concluded that LLLT associated with zoledronic acid is effective for stimulating bone formation in surgically created defects in rats, at the periods studied.

Synergistic effect of hyperbaric oxygen therapy with PTH [1-34] on calvarial bone graft in irradiated rat.

OBJECTIVE: To determine the synergistic effect of parathyroid hormone (PTH) [1-34] in combination with hyperbaric oxygen (HBO) on bone graft in a rat calvarial bone defect model under impaired osteogenic conditions. MATERIALS AND METHODS: Twenty-four rats were divided into three groups. Localized radiation with a single 12 Gy dose was administered to the calvaria. Four weeks after radiation, calvarial circular defects were created in the parietal bones. All defects were filled with biphasic calcium phosphate. After the bone graft, PTH [1-34] was injected subcutaneously, and HBO was administered. At 6 weeks after the bone graft, the rats were sacrificed, and specimens were harvested. RESULTS: Histomorphometric evaluation showed that the percentage of new bone area was higher in the PTH and PTH/HBO groups than in the control group. The percent residual material area was decreased in the PTH/HBO group compared with the control group. The percentage blood vessel number was highest in the PTH group. Micro-CT evaluation showed that the new bone volume was highest in the PTH/HBO group. The residual material volume was lowest in the PTH/HBO group. CONCLUSION: Within the limitations of this study, our data indicate that PTH combined with HBO may reverse radiation-induced impairment of bone healing.

Low-level laser irradiation modulates the proliferation and the osteogenic differentiation of bone marrow mesenchymal stem cells under healthy and inflammatory condition.

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under healthy and inflammatory microenvironments. Human BMSCs and BMSCs from inflammatory conditions (i-BMSCs, BMSCs treated with tumor necrosis factor α; TNF-α) were subject to LLLT (Nd:YAG;1064 nm) at different intensities. We designed one control group (without irradiation) and four testing groups (irradiation at 2, 4, 8, and 16 J/cm2) for both BMSCs and i-BMSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining and the mRNA transcript levels of genes runt-related transcription factor 2 (Runx2), ALP, and osteocalcin. Moreover, the effects of LLLT on secretion of TNF-α in BMSCs and i-BMSCs were measured by enzyme-linked immunosorbent assay. Our results demonstrated LLLT could significantly promote BMSC proliferation and osteogenesis at densities of 2 and 4 J/cm2. LLLT at density of 8 J/cm2 could promote the proliferation and osteogenesis of i-BMSCs. However, LLLT at 16 J/cm2 significantly suppressed the proliferation and osteogenesis of BMSCs both in healthy and in inflammatory microenvironment. Moreover, we also found that the expression of TNF-α was obviously inhibited by LLLT at 4, 8, and 16 J/cm2, in an inflammatory microenvironment. Considering these findings, LLLT could improve current in vitro methods of differentiating BMSCs under healthy and inflammatory microenvironments prior to transplantation.