Ana Claudia Muniz Renno

Effect of 830 nm Laser Phototherapy on Osteoblasts Grown In Vitro on Biosilicate® Scaffolds

OBJECTIVE The purpose of this study was (i) to develop a method for successfully seeding osteoblasts onto a glass-ceramic scaffold designed for use in clinical settings, and (ii) to determine whether the application of laser phototherapy at 830 nm would result in osteoblast proliferation on the glass-ceramic scaffold. BACKGROUND The use of bioscaffolds is considered a promising strategy for a number of clinical applications where tissue healing is sub-optimal. As in vitro osteoblast growth is a slow process, laser phototherapy could be used to stimulate osteoblast proliferation on bioscaffolds. METHODS A methodology was developed to seed an osteoblastic (MC3T3) cell line onto a novel glass-ceramic scaffold. Seeded scaffolds were irradiated with a single exposure of 830 nm laser at 10 J/cm(2) (at diode). Non-irradiated seeded scaffolds acted as negative controls. Cell proliferation was assessed seven days after irradiation. RESULTS Osteoblastic MC3T3 cells were successfully grown on discs composed of a glass-ceramic composite. Laser irradiation produced a 13% decrease in MC3T3 cell proliferation on glass-ceramic discs (mean +/- SD = 0.192 +/- 0.002) compared with control (non-irradiated) discs (mean +/-SD = 0.22 +/- 0.002). CONCLUSIONS Despite successful seeding of bioscaffolds with osteoblasts, laser phototherapy resulted in a reduction in cell growth compared to non-irradiated controls. Future research combining laser phototherapy and glass-ceramic scaffolds should take into account possible interactions of the laser with matrix compounds.

Low-Level Laser Therapy Induces Differential Expression of Osteogenic Genes During Bone Repair in Rats

OBJECTIVES The aim of this study was to measure the temporal pattern of the expression of osteogenic genes after low-level laser therapy during the process of bone healing. We used quantitative real-time polymerase chain reaction (qPCR) along with histology to assess gene expression following laser irradiation on created bone defects in tibias of rats. MATERIAL AND METHODS The animals were randomly distributed into two groups: control or laser-irradiated group. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation started 24 h post-surgery and was performed for 3, 6, and 12 sessions, with an interval of 48 h. A 830 nm laser, 50 J/cm(2), 30 mW, was used. On days 7, 13, and 25 post-injury, rats were sacrificed individually by carbon dioxide asphyxia. The tibias were removed for analysis. RESULTS The histological results revealed intense new bone formation surrounded by highly vascularized connective tissue presenting slight osteogenic activity, with primary bone deposition in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). The quantitative real-time PCR showed that laser irradiation produced an upregulation of BMP-4 at day 13 post-surgery and an upregulation of BMP4, ALP, and Runx 2 at day 25 after surgery. CONCLUSION Our results indicate that laser therapy improves bone repair in rats as depicted by differential histopathological and osteogenic genes expression, mainly at the late stages of recovery.

Low level laser therapy (830 nm) improves bone repair in osteoporotic rats: Similar outcomes at two different dosages

BACKGROUND AND OBJECTIVE The goal of this study was to investigate the effects of low level laser therapy (LLLT) in osteoporotic rats by means of subjective histopathological analysis, deposition of collagen at the site of fracture, biomechanical properties and immunohistochemistry for COX-2, Cbfa-1 and VEGF. MATERIAL AND METHODS A total of 30 female Wistar rats (12weeks-old, ±250g) were submitted to ovariectomy (OVX). Eight weeks after the OVX, a tibial bone defect was created in all animals and they were randomly divided into 3 groups (n=10): control bone defect group (CG): bone defects without any treatment; laser 60J/cm(2) group (L60): animals irradiated with LLLT, at 60J/cm(2) and laser 120J/cm(2) group (L120): animals irradiated with LLLT, at 120J/cm(2). RESULTS In the laser treated groups, at both fluences, a higher amount of newly formed bone was evidenced as well as granulation tissue compared to control. Picrosirius analysis demonstrated that irradiated animals presented a higher deposition of collagen fibers and a better organization of these fibers when compared to other groups, mainly at 120J/cm(2). COX-2, Cbfa-1 or VEGF immunoreactivity was detected in a similar manner either 60J/cm(2) or 120J/cm(2) fluences. However, no differences were shown in the biomechanical analysis. CONCLUSION Taken together, our results support the notion that LLLT improves bone repair in the tibia of osteoporotic rats as a result of stimulation of the newly formed bone, fibrovascularization and angiogenesis.

Comparative effects of low-intensity pulsed ultrasound and low-level laser therapy on injured skeletal muscle.

OBJECTIVE The main purpose of this study was to compare the effects of low-intensity pulsed ultrasound (US) and low-level laser therapy (LLLT) on injured skeletal muscle after cryolesion by means of histopathological analysis and immunohistochemistry for cyclo-oxygenase-2 (COX-2). BACKGROUND AND METHODS Thirty-five male Wistar rats were randomly distributed into four groups: intact control group with uninjured and untreated animals; injured control group with muscle injury and no treatment; LLLT-treated group with muscle injury treated with 830-nm laser; and US-treated group with muscle injury treated with US. Treatments started 24 h postsurgery and were performed during six sessions. RESULTS LLLT-treated animals presented minor degenerative changes of muscle tissue. Exposure to US reduced tissue injuries induced by cryolesion, but less effectively than LLLT. A large number of COX-2 positive cells were found in untreated injured rats, whereas COX-2 immunoexpression was lower in both LLLT- and US-treated groups. CONCLUSION This study revealed that both LLLT and US therapies have positive effects on muscle metabolism after an injury in rats, but LLLT seems to produce a better response.

In vivo biological performance of a novel highly bioactive glass-ceramic (Biosilicate®): A biomechanical and histomorphometric study in rat tibial defects

This study aimed to investigate bone responses to a novel bioactive fully crystallized glass-ceramic of the quaternary system P(2)O(5)-Na(2)O-CaO-SiO(2) (Biosilicate®). Although a previous study demonstrated positive effects of Biosilicate® on in vitro bone-like matrix formation, its in vivo effect was not studied yet. Male Wistar rats (n = 40) with tibial defects were used. Four experimental groups were designed to compare this novel biomaterial with a gold standard bioactive material (Bioglass® 45S5), unfilled defects and intact controls. A three-point bending test was performed 20 days after the surgical procedure, as well as the histomorphometric analysis in two regions of interest: cortical bone and medullary canal where the particulate biomaterial was implanted. The biomechanical test revealed a significant increase in the maximum load at failure and stiffness in the Biosilicate® group (vs. control defects), whose values were similar to uninjured bones. There were no differences in the cortical bone parameters in groups with bone defects, but a great deal of woven bone was present surrounding Biosilicate® and Bioglass® 45S5 particulate. Although both bioactive materials supported significant higher bone formation; Biosilicate® was superior to Bioglass® 45S5 in some histomorphometric parameters (bone volume and number of osteoblasts). Regarding bone resorption, Biosilicate® group showed significant higher number of osteoclasts per unit of tissue area than defect and intact controls, despite of the non-significant difference in the osteoclastic surface as percentage of bone surface. This study reveals that the fully crystallized Biosilicate® has good bone-forming and bone-bonding properties.

Biosilicate® and low-level laser therapy improve bone repair in osteoporotic rats

The aim of this study was to investigate the effects of a novel bioactive material (Biosilicate®) and low‐level laser therapy (LLLT) on bone fracture consolidation in osteoporotic rats. Forty female Wistar rats were submitted to ovariectomy (OVX) to induce osteopenia. Eight weeks after surgery, the animals were randomly divided into four groups of 10 animals each: a bone defect control group (CG); a bone defect filled with Biosilicate group (BG); a bone defect filled with Biosilicate and irradiated with LLLT at 60 J/cm2 group (BG60); and a bone defect filled with Biosilicate and irradiated with LLLT at 120 J/cm2 group (BG120). Bone defects were surgically performed on both tibias. The size of particle used for Biosilicate was 180–212 µm. Histopathological analysis showed that bone defects were predominantly filled with the biomaterial in specimens treated with Biosilicate. LLLT with either 60 or 120 J/cm2 was able to increase collagen, Cbfa‐1, VGEF and COX‐2 expression in the circumjacent cells of the biomaterial. A morphometric analysis revealed that the Biosilicate + laser groups showed a higher amount of newly formed bone. Our results indicate that laser therapy improves bone repair process in contact with Biosilicate as a result of increasing bone formation, as well as COX‐2 and Cbfa‐1 immunoexpression, angiogenesis and collagen deposition in osteoporotic rats. Copyright

Low-level laser therapy (LLLT) (660 nm) alters gene expression during muscle healing in rats

INTRODUCTION The effects of LLLT were studied during muscle regeneration through gene expression. METHODS It was evaluated 10 and 50J/cm(2) doses during 7, 14 and 21days post cryoinjury, through histopathological analysis and mRNA MyoD, Myogenin, Vegf and Cox-2 expression. RESULTS Irradiated groups presented less inflammatory process than control group after 14 and 21days. Cox-2 levels were downregulated in all irradiated groups after 7, 14 and 21days. On day 7, both treated groups had a downregulation of Vegf levels, and an upregulation after 14 and 21days, mainly with 50J/cm(2). The MyoD levels were upregulated with high dose in all periods and with low dose after 21days. Myogenin expression was downregulated in both treated groups after 7days, and was upregulated with 10J/cm(2) after 21days. CONCLUSION These responses suggest that LLLT can improve the skeletal muscle regeneration through the gene expression stimulation.

Effects of low-level laser therapy on the expression of osteogenic genes related in the initial stages of bone defects in rats

Abstract. We evaluate the effects of low-level laser therapy (LLLT) on the histological modifications and temporal osteogenic genes expression during the initial phase of bone healing in a model of bone defect in rats. Sixty-four Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 0.028  cm2, 1.071  W/cm2, 1 min and 34 s, 2.8 Joules, 100  J/cm2) was performed for 1, 2, 3, and 5 sessions. Histopathology revealed that treated animals presented higher inflammatory cells recruitment, especially 12 and 36 h postsurgery. Also, a better tissue organization at the site of the injury, with the presence of granulation tissue and new bone formation was observed on days three and five postsurgery in the treated animals. The quantitative real time polymerase chain reaction showed that LLLT produced a significantly increase in mRNA expression of Runx-2, 12 h and three days post-surgery, a significant upregulation of alkaline phosphatase mRNA expression after 36 h and three days post-surgery and a significant increase of osteocalcin mRNA expression after three and five days. We concluded that LLLT modulated the inflammatory process and accelerated bone repair, and this advanced repair pattern in the laser-treated groups may be related to the higher mRNA expression of genes presented by these animals.

Effect of low-level laser therapy (660 nm) on the healing of second-degree skin burns in rats

The aim of this study was to investigate the effects of 660 nm laser on the healing of burn wounds made on the backs of rats. Thirty-two Wistar male rats were used. The animals were randomly distributed into 2 groups of 16 animals each: control group (burned rats without treatment) and laser-treated group (burned rats treated with laser therapy). Each group was divided into two different subgroups, euthanized in different periods (subgroup A: 7 days post-surgery and subgroup B: 14 days post-surgery). Histopathological analysis revealed a significant decrease in the necrotic area in the laser-treated group compared to the controls at days 7 and 14 post-injury. COX-2 positive cells were found in a strong pattern in the group submitted to laser therapy after 7 days. Regarding VEGF immunomarker, a significant VEGF immunoexpression was detected in the laser-exposed group after 14 days when compared to the negative control group. Taken together, our results demonstrate that laser therapy is able to promote skin repair of burned rats as a result of decreasing necrotic area and an up-regulation of COX-2 and VEGF immunoexpression.

Characterization and In Vivo Biological Performance of Biosilicate

After an introduction showing the growing interest in glasses and glass-ceramics as biomaterials used for bone healing, we describe a new biomaterial named Biosilicate. Biosilicate is the designation of a group of fully crystallized glass-ceramics of the Na2O-CaO-SiO2-P2O5 system. Several in vitro tests have shown that Biosilicate is a very active biomaterial and that the HCA layer is formed in less than 24 hours of exposure to “simulated body fluid” (SBF) solution. Also, in vitro studies with osteoblastic cells have shown that Biosilicate disks supported significantly larger areas of calcified matrix compared to 45S5 Bioglass, indicating that this bioactive glass-ceramic may promote enhancement of in vitro bone-like tissue formation in osteogenic cell cultures. Finally, due to its special characteristics, Biosilicate has also been successfully tested in several in vivo studies. These studies revealed that the material is biocompatible, presents excellent bioactive properties, and is effective to stimulate the deposition of newly formed bone in animal models. All these data highlight the huge potential of Biosilicate to be used in bone regeneration applications.