Effectiveness of 780 nm photobiomodulation as adjunct treatment for bone exposed fractures: A pilot study on radiograph, pain, and cytokines analysis.

The aim of this study was to evaluate the effectiveness of photobiomodulation with a 780 nm laser as an adjunct to surgical treatment in the regeneration of bone fractures. Twenty patients diagnosed with open fractures in the lower limbs were selected and randomly divided into two groups: control and LLLT. LLLT parameter: 780 nm, 0.04 cm2 of light beam diameter, 40 mW of power, 10 s per point, 0.4 J of energy, fluence of 10 J/cm2 and irradiance of 1 W/cm2. The evaluated data were: pain, using McGill scale, use of analgesics and anti-inflammatories, levels of cytokines TNF-α, IFN-γ, IL-1ß, IL-10, and IL-17, and bone level regeneration. Data were analyzed using Wilcoxon and Mann-Whitney tests (5%). We can conclude that LLLT was effective as an adjuvant in the bone fracture regeneration process, altered IL-1ß levels, reduced the use of analgesics and anti-inflammatories, reducing the pain pattern throughout the sessions.

Low-intensity pulsed ultrasound enhances bone marrow-derived stem cells-based periodontal regenerative therapies.

BACKGROUND: Alveolar bone loss is one of the most common consequence for periodontitis, which is a major obstacle in periodontal regeneration. Bone marrow stromal cells (BMSCs) have shown significant promise in the treatment of various disease, which also contribute to the natural bone repair process. Low-intensity pulsed ultrasound (LIPUS) is a therapeutic ultrasound used in our previous studies to promotes alveolar bone regeneration. In addition, LIPUS was found to be a promising method to enhance mesenchymal stromal cell-based therapies. In the current study, we have investigated the effects of LIPUS combined with BMSCs therapies on BMSCs homing and its potential to promote alveolar bone regeneration. METHODS: BMSCs were isolated from rat and characterized by multilineages differentiation assay. Then these cells were labeled with luciferase and green fluorescent protein (GFP) by lentivirus in vitro. Periodontal bone defect was made on the mesial area of the maxillary first molar in rats. A total of 1 × 106 Luc-GFP labeled BMSCs were injected into rat tail vein. Bioluminescence imaging was utilized to track BMSCs in vivo. The rats were sacrificed eight weeks after surgery and the samples were harvested. Micro-computed tomography (Micro-CT) was performed to evaluate alveolar bone regeneration. Paraffin sections were made and subject to hematoxylin-eosin staining, masson staining and immunohistochemistry staining. RESULTS: BMSCs display a fibroblast-like morphology and can differentiate into adipocytes or osteoblasts under appropriate condition. The transfected BMSCs are strongly positive for GFP express. Bioluminescence imaging showed that most of BMSCs were trapped in the lung. A small portion BMSCs were homed to the alveolar bone defect area in BMSCs group, while more cells were observed in BMSCs/LIPUS group compare to other groups on day 3 and 7. Micro-CT results showed that BMSCs/LIPUS group resulted in more new bone formation than other groups. Immunohistochemical results showed higher expression of COL-I and osteopontin in BMSCs/LIPUS group compared with the other groups. CONCLUSIONS: These results suggested that LIPUS can enhance BMSCs-based periodontal alveolar bone regeneration. This study provides new insights into how LIPUS might provide therapeutic benefits by promoting BMSCs homing.

Bone substitutes and photobiomodulation in bone regeneration: A systematic review in animal experimental studies.

In general, bone fractures are able of healing by itself. However, in critical situations such as large bone defects, poor blood supply or even infections, the biological capacity of repair can be impaired, resulting in a delay of the consolidation process or even in non-union fractures. Thus, technologies able of improving the process of bone regeneration are of high demand. In this context, ceramic biomaterials-based bone substitutes and photobiomodulation (PBM) have been emerging as promising alternatives. Thus, the present study performed a systematic review targeting to analyze studies in the literature which investigated the effects of the association of ceramic based bone substitutes and PBM in the process of bone healing using animal models of bone defects. The search was conducted from March and April of 2019 in PubMed, Web of Science and Scopus databases. After the eligibility analyses, 16 studies were included in this review. The results showed that the most common material used was hydroxyapatite (HA) followed by Biosilicate associated with infrared PBM. Furthermore, 75% of the studies demonstrated positive effects to stimulate bone regeneration from association of ceramic biomaterials and PBM. All studies used low-level laser therapy (LLLT) device and the most studies used LLLT infrared. The evidence synthesis was moderate for all experimental studies for the variable histological analysis demonstrating the efficacy of techniques on the process of bone repair stimulation. In conclusion, this review demonstrates that the association of ceramic biomaterials and PBM presented positive effects for bone repair in experimental models of bone defects.

Bone marrow coagulated and low-level laser therapy accelerate bone healing by enhancing angiogenesis, cell proliferation, osteoblast differentiation, and mineralization.

The present study evaluated bone marrow aspirate (BMA) and low-level laser therapy (LLLT) on bone healing. It was created critical-size defects (CSD) of 5 mm diameter in rat calvaria of 64 rats. Animals were randomly divided into four groups: Control (blood clot), BMA (coagulated BMA), LLLT (laser irradiation and blood clot), and BMA/LLLT (laser irradiation and coagulated BMA). Euthanasia was performed at 15 or 30 days postoperative. Immunohistochemical reactions were performed to identify vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The markers were quantified, and data were statistically analyzed. Groups BMA/LLLT and LLLT presented significantly higher VEGF expression than group control. Group BMA/LLLT presented a significantly higher expression of PCNA than all experimental groups. Groups BMA and BMA/LLLT presented significantly higher expression of BMP-2 than all experimental groups. Groups LLLT and BMA/LLLT presented significantly higher expression of OPN than groups control and BMA. Groups LLLT, BMA, and BMA/LLLT presented a significantly higher expression of OCN than group control. It can be concluded that the association of BMA and LLLT enhanced bone healing by improving expression of VEGF, PCNA, Runx2, BMP-2, OPN, and OCN.

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.

Growth factors regional patterned and photoimmobilized scaffold applied to bone tissue regeneration.

Bone diseases such as osteomalacia, osteoporosis, and osteomyelitis are major illnesses that threaten the health of human. This study aimed to provide an idea at the molecular level of material properties determined with UV specific surface approaches. The tert-butyl hydroperoxide (t-BHP) exposure aging model bone mesenchymal stem cells (BMSCs) were reverted by using a poly-hybrid scaffold (PS), which is a carbon nanotube (CNT) coated polycaprolactone (PCL) and polylactic acid (PLA) scaffold, combined with insulin-like growth factor-1 (IGF). Then, the region-specific PS photo-immobilized with different growth factors (GFs) was obtained by interference and diffraction of ultraviolet (UV) light. Additionally, the reverted BMSCs were regionally pattern differentiated into three kinds of cells on the GF immobilized PS (GFs/PS). In vivo, the GFs/PS accelerate bone healing in injured Sprague-Dawley (SD) rats. The data showed that GFs/PS effectively promoted the differentiation of reverted BMSCs in the designated area on 21st day. These results suggest region-specific interface immobilization of GFs concurrently differentiating reverted BMSCs into three different cells in the same scaffold. This method might be considered as a short-time, low cost, and simple operational approach to scaffold modification for tissue regeneration in the future.

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.

NIR light-assisted phototherapies for bone-related diseases and bone tissue regeneration: A systematic review.

Recently, the rapid development of biomaterials has induced great interest in the precisely targeted treatment of bone-related diseases, including bone cancers, infections, and inflammation. Realizing noninvasive therapeutic effects, as well as improving bone tissue regeneration, is essential for the success of bone­related disease therapies. In recent years, researchers have focused on the development of stimuli-responsive strategies to treat bone-related diseases and to realize bone regeneration. Among the various external stimuli for targeted therapy, near infrared (NIR) light has attracted considerable interests due to its high tissue penetration capacity, minimal damage toward normal tissues, and easy remote control properties. The main objective of this systematic review was to reveal the current applications of NIR light-assisted phototherapy for bone-related disease treatment and bone tissue regeneration. Database collection was completed by June 1, 2020, and a total of 81 relevant studies were finally included. We outlined the various therapeutic applications of photothermal, photodynamic and photobiomodulation effects under NIR light irradiation for bone­related disease treatment and bone regeneration, based on the retrieved literatures. In addition, the advantages and promising applications of NIR light-responsive drug delivery systems for spatiotemporal-controlled therapy were summarized. These findings have revealed that NIR light-assisted phototherapy plays an important role in bone-related disease treatment and bone tissue regeneration, with significant promise for further biomedical and clinical applications.

Development of a Rat Model of Mandibular Irradiation Sequelae for Preclinical Studies of Bone Repair.

Repairing mandibular bone defects after radiotherapy of the upper aerodigestive tract is clinically challenging. Although bone tissue engineering has recently generated a number of innovative treatment approaches for osteoradionecrosis (ORN), these modalities must be evaluated preclinically in a relevant, reproducible, animal model. The objective of this study was to evaluate a novel rat model of mandibular irradiation sequelae, with a focus on the adverse effects of radiotherapy on bone structure, intraosseous vascularization, and bone regeneration. Rats were irradiated with a single 80 Gy dose to the jaws. Three weeks after irradiation, mandibular bone defects of different sizes (0, 1, 3, or 5 mm) were produced in each hemimandible. Five weeks after the surgical procedure, the animals were euthanized. Explanted mandibular samples were qualitatively and quantitatively assessed for bone formation, bone structure, and intraosseous vascular volume by using micro-computed tomography, scanning electron microscopy, and histology. Twenty irradiated hemimandibles and 20 nonirradiated hemimandibles were included in the study. The bone and vessel volumes were significantly lower in the irradiated group. The extent of bone remodeling was inversely related to the defect size. In the irradiated group, scanning electron microscopy revealed a large number of polycyclic gaps consistent with periosteocytic lysis (described as being pathognomonic for ORN). This feature was correlated with elevated osteoclastic activity in a histological assessment. In the irradiated areas, the critical-sized defect was 3 mm. Hence, our rat model of mandibular irradiation sequelae showed hypovascularization and osteopenia. Impact statement Repairing mandibular bone defects after radiotherapy of the upper aerodigestive tract is clinically challenging. Novel tissue engineering approaches for healing irradiated bone must first be assessed in animal models. The current rat model of mandibular irradiation sequelae is based on tooth extraction after radiotherapy. However, the mucosal sequelae of radiotherapy often prevent the retention of tissue-engineered biomaterials within the bone defect. We used a submandibular approach to create a new rat model of mandibular irradiation sequelae, which enables the stable retention of biomaterials within the bone defect and should thus facilitate the assessment of bone regeneration.

Low-level laser therapy (LLLT) in sites grafted with osteoconductive bone substitutes improves osseointegration.

The aim of this study was to evaluate the osseointegration of implants placed in areas grafted with different osteoconductive bone substitutes irradiated with infrared low-level laser therapy (LLLT). Fifty-six rats were randomly allocated into 4 groups: DBB, bone defects filled with deproteinized bovine bone graft (DBB); HA/TCP, bone defects filled with biphasic ceramic made of hydroxyapatite and ß-tricalcium phosphate (HA/TCP); DBB-L, bone defects filled with DBB and treated by LLLT; HA/TCP-L, bone defects filled with HA/TCP and treated by LLLT. Bone defects were performed in the tibia of each animal and filled with the different biomaterials. The grafted areas were treated with LLLT (λ 808 nm, 100 mW, ϕ ∼ 0.60 mm) in 7 sessions with 48 h between the irradiations. After the 60-day period, the implants were placed, and the animals were euthanized after 15 and 45 days. The osseointegration and bone repair in the grafted area were evaluated by biomechanical, microtomographic and histometric analyses, and the expression of some bone biomarkers was evaluated by immunohistochemistry analysis. LLLT induced higher degree of osseointegration, which was associated with the greater expression of BMP2 and OCN. LLLT performed in areas grafted with osteoconductive bone substitutes prior to implant placement improves osseointegration.

Effects of the combination of low-level laser therapy and anionic polymer membranes on bone repair.

In view of the limitations of bone reconstruction surgeries using autologous grafts as a gold standard, tissue engineering is emerging as an alternative, which permits the fabrication and improvement of scaffolds to stimulate osteogenesis and angiogenesis, processes that are essential for bone repair. Polymers are used to mimic the extracellular bone matrix and support cell growth. In addition, bone neoformation can be induced by external factors such as laser irradiation, which stimulates bone metabolism. The objective of this study was to evaluate the regeneration of bone defects using collagen and elastin membranes derived from intestinal serosa and bovine auricular cartilage combined with low-level laser application. Thirty-six Wistar rats were operated to create a 3-mm defect in the distal metaphysis of the left femur and divided into six groups: G1 (control, no treatment); G2 (laser); G3 (elastin graft), G4 (elastin+laser); G5 (collagen graft); G6 (collagen+laser). The animals were sacrificed 6 weeks after surgery and the femurs were removed for analysis of bone repair. Macroscopic and radiological results showed the absence of an infectious process in the surgical area. This was confirmed by histological analysis, which revealed no inflammatory infiltrate. Histomorphometry showed that the formation of new bone started from the margins of the bone defect and its volume was greater in elastin+laser and collagen+laser. We conclude that newly formed bone in the graft area was higher in the groups that received the biomaterials and laser. The collagen and elastin matrices showed biocompatibility.

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.

Low-intensity pulsed ultrasound promotes tissue regeneration in rat dental follicle cells in a porous ceramic scaffold.

The aim of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of dental follicle cells (DFCs) in vitro and on the regenerative effects of DFC-OsteoBoneTM complexes in vivo. DFCs were isolated and characterized. In the in vitro study, DFCs were cultured in an osteogenic medium in the presence or absence of LIPUS. The expression levels of ALP, Runx2, OSX, and COL-I mRNA were analyzed using real-time polymerase chain reaction (RT-PCR) on day 7. Alizarin red staining was performed on day 21. The state of the growth of the DFCs that were seeded on the scaffold at 3, 5, 7, and 9 days was detected by using a scanning electron microscope. In our in vivo study, 9 healthy nude mice randomly underwent subcutaneous transplantation surgery in one of three groups: group A, empty scaffold; group B, DFCs + scaffold; and group C, DFCs + scaffold + LIPUS. After 8 weeks of implantation, a histological analysis was performed by HE and Mason staining. Our results indicate that LIPUS promotes the osteogenic differentiation of DFCs by increasing the expression of the ALP, Runx2, OSX, and COL-I genes and the formation of mineralized nodules. The cells can adhere and grow on the scaffolds and grow best at 9 days. The HE and Mason staining results showed that more cells, fibrous tissue and blood vessels could be observed in the DFCs + scaffold + LIPUS group than in the other groups. LIPUS could promote the osteogenic differentiation of DFCs in vitro and promote tissue regeneration in a DFCs-scaffold complex in vivo. Further studies should be conducted to explore the underlying mechanisms of LIPUS.

Effects of an Er,Cr:YSGG Laser on Bone Regeneration in Critical-Sized Calvarial Defects of Rats Exposed to Inhalation of Cigarette Smoke.

Objective: To evaluate the effect of the Er,Cr:YSGG laser on healing of critical-sized calvarial defects (CSDs) in rats submitted to inhalation of cigarette smoke. Background: Smoking has been implicated with the delay in the bone healing after osteotomy procedures, then the use of the Er,Cr:YSGG laser for osteotomy in smokers could be an alternative to the conventional drills. Methods: One hundred animals were randomly allocated into four groups: trephine-the CSDs were made with a trephine drill in healthy rats; Er,Cr:YSGG-the CSDs were made with the Er,Cr:YSGG laser in healthy rats; Trephine-S-the CSDs were made with a trephine drill in rats exposed to cigarette smoke; and Er,Cr:YSGG-S-the CSDs were made with the Er,Cr:YSGG laser in rats exposed to cigarette smoke. The inhalation of cigarette smoke started 7 days before the surgical procedure until euthanasia (immediately, 7, 15, 30, or 60 days after the surgical procedure). A histometric analysis and a histological description were performed to evaluate (1) the residual linear lengths and bone formation in the CSDs; (2) the quality of bone healing. Results: The use of Er,Cr:YSGG laser induces more bone formation compared with the trephine in smokers; however, the closure of the CSD was only superior in the Er,Cr:YSGG-S group compared to the Trephine-S group at the 60-day period. Conclusions: The use of the Er,Cr:YSGG laser stimulated the bone repair process after osteotomy procedures in animals submitted to exposure of inhalation of cigarette smoke.

Enhanced osteogenesis of bone marrow stem cells cultured on hydroxyapatite/collagen I scaffold in the presence of low-frequency magnetic field.

As a non-invasive biophysical therapy, electromagnetic fields (EMF) have been widely used to promote the healing of fractures. In the present study, hydroxyapatite/collagen I (HAC) loaded with rabbit bone marrow mesenchymal stem cells (MSCs) were cultured in a dynamic perfusion bioreactor and exposed to EMF of 15 Hz/1mT. Osteogenic differentiation of the seeded cells was analyzed through the evaluation of ALP activity and osteogenesis-related genes expression in vitro. The in vivo osteogenesis efficacy of the cell laden HAC constructs treated with/without EMF was evaluated through a rabbit femur condyle defect model. The results showed that EMF of 15 Hz/1mT could enhance the osteogenic differentiation of the cells seeded on HAC scaffold. Furthermore, the in vivo experiments demonstrated that EMF exposure could promote bone regeneration within the defect and bone integration between the graft and host bone. Taking together, the MSCs seeded HAC scaffold combined with EMF exposure could be a promising approach for bone tissue engineering.

Bone Regeneration Effect of Hyperbaric Oxygen Therapy Duration on Calvarial Defects in Irradiated Rats.

OBJECTIVE: In this study, we evaluated changes in bone remodeling in an irradiated rat calvarial defect model according to duration of hyperbaric oxygen therapy. MATERIALS AND METHODS: The 28 rats were divided into four groups. Radiation of 12 Gy was applied to the skull, and 5-mm critical size defects were formed on both sides of the skull. Bone grafts were applied to one side of formed defects. From the day after surgery, HBO was applied for 0, 1, and 3 weeks. At 6 weeks after bone graft, experimental sites were removed and analyzed for radiography, histology, and histomorphometry. RESULTS: Micro-CT analysis showed a significant increase in new bone volume in the HBO-3 group, with or without bone graft. When bone grafting was performed, BV, BS, and BS/TV all significantly increased. Histomorphometric analysis showed significant increases in %NBA and %BVN in the HBO-1 and HBO-3 groups, regardless of bone graft. CONCLUSION: Hyperbaric oxygen therapy was effective for bone regeneration with only 1 week of treatment.

Fibrin Sealant Derived from Human Plasma as a Scaffold for Bone Grafts Associated with Photobiomodulation Therapy.

Fibrin sealants derived from human blood can be used in tissue engineering to assist in the repair of bone defects. The objective of this study was to evaluate the support system formed by a xenograft fibrin sealant associated with photobiomodulation therapy of critical defects in rat calvaria. Thirty-six rats were divided into four groups: BC (n = 8), defect filled with blood clot; FSB (n = 10), filled with fibrin sealant and xenograft; BCPBMT (n = 8), blood clot and photobiomodulation; FSBPBMT (n = 10), fibrin sealant, xenograft, and photobiomodulation. The animals were killed after 14 and 42 days. In the histological and microtomographic analysis, new bone formation was observed in all groups, limited to the defect margins, and without complete wound closure. In the FSB group, bone formation increased between periods (4.3 ± 0.46 to 6.01 ± 0.32), yet with lower volume density when compared to the FSBPBMT (5.6 ± 0.45 to 10.64 ± 0.97) group. It was concluded that the support system formed by the xenograft fibrin sealant associated with the photobiomodulation therapy protocol had a positive effect on the bone repair process.

Histologic Evaluation of Early Bone Regeneration Treated with Simvastatin Associated with Low-Level Laser Therapy.

PURPOSE: This histologic study aimed at assessing bone healing after treatment with simvastatin in association with low-level laser therapy (LLLT). METHODS: Twenty-four male rats (Wistar) were submitted to surgery to create a bone defect of 5 mm in diameter in the parietal bone. These rats were randomly and equally divided into four treatment groups (n = 6): control (C), in which no treatment was performed; simvastatin (SIM), in which rats received daily subcutaneous doses of 2.5 mg/kg of simvastatin; LLLT, which was daily applied to the bone defect; and SIM-LLLT, in which both SIM and LLLT were daily applied. All laser irradiations were carried out with a 830-nm infrared diode laser (GaAlAs) with maximum output of 100 mW and a dose of 4 J, totaling 16 J per session. Rats were euthanized on the 12th postoperative day. Formalin-fixed paraffin-embedded bone samples were obtained and stained with hematoxylin-eosin (HE) and toluidine blue for optical microscope analysis. Degree of inflammation, new vascular formation, tissue repair, and osteoblastic activity were assessed. RESULTS: Categorical analysis of the histologic slides revealed newly formed bone reaching the center of the surgical wound in two animals from the SIM group, two from the LLLT group, and three from the SIM-LLLT group. Greater new bone formation and a lower degree of inflammation were observed in the animals that had bone neoformation at the center of the defect, especially in the LLLT and SIM-LLLT groups. SIM and C groups presented greater angiogenesis than LLLT and SIM-LLLT. SIMLLLT therapy showed a statistically significant reduction in the degree of inflammation when compared to the control group (P < .05). CONCLUSION: Within the limitations of this study, the present results suggest that a combination of simvastatin and low-level laser therapy may stimulate better bone formation.

Design of a biodegradable UV-irradiated gelatin-chitosan/nanocomposed membrane with osteogenic ability for application in bone regeneration.

Guided bone regeneration membranes are used in oral surgery to protect the site of a lesion exposed to connective tissue invasion which, in turn, prevents new bone formation. Although non-degradable and degradable materials have been applied in clinical treatments, biodegradable membranes have the advantage that they do not require a secondary surgical procedure to be removed. However, they have a very low mechanical strength. As biodegradable membranes, biomaterials based on gelatin-chitosan have gained importance in clinical applications due to their unique properties. Gelatin contains RGD-like sequences, promoting cell adhesion/migration, and it can be blended with chitosan, which allows the immobilization of nanoparticles. In this work, we designed a new gelatin-chitosan polymeric membrane which contains hydroxyapatite and titania nanoparticles as two very well-documented osteoconductive materials. UV radiation was used as a non-toxic cross-linking agent to improve the thermophysical/mechanical characteristics and to control the biodegradability of the nanocomposed membrane. The microstructure, thermophysical and mechanical properties of the UV-irradiated material were studied by scanning electron microscopy, differential scanning calorimetry and Young's modulus, respectively. The in vitro biocompatibility of the new nanocomposite was evaluated by cell adhesion and proliferation assays. The osteoconductive ability was determined by an alkaline phosphatase production assay using mouse embryonic fibroblast (MEF) cells. The results show a homogeneous material with an appropriate distribution of nanoparticles. Cross-linking by UV radiation improved the mechanical and biological performance of the membrane. The presence of two osteoconductive nanoparticles, such as titania and hydroxyapatite, increased the osteogenic potential of the gelatin-based material in vitro, which confers a biological function, in addition to functioning as a physical barrier. The material obtained herein represents a good alternative to current guided bone regeneration membranes, with high potential for use in oral/orthopaedic applications in patients.

Effects of Electromagnetic Field on Proliferation, Differentiation, and Mineralization of MC3T3 Cells.

IMPACT STATEMENT: We present the study about how the parameters of pulsed electromagnetic field (PEMF) stimulus affected calvarial osteoblast precursor cell in terms of growth, viability, and differentiation. This research provides insight and foundation to clinical application of noninvasive therapy using PEMF to improve bone regeneration.