Paulo Sérgio Bossini

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm(2) on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm(2) and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm(2). The random skin flap was performed measuring 10×4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner.

Effect of low level laser therapy (830 nm) with different therapy regimes on the process of tissue repair in partial lesion calcaneous tendon

Calcaneous tendon is one of the most damaged tendons, and its healing may last from weeks to months to be completed. In the search after speeding tendon repair, low intensity laser therapy has shown favorable effect. To assess the effect of low intensity laser therapy on the process of tissue repair in calcaneous tendon after undergoing a partial lesion.

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

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.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery.

The effects of 660 nm and 780 nm laser irradiation on viability of random skin flap in rats.

BACKGROUND AND OBJECTIVE Some studies have shown that laser phototherapy is able to increase skin flap viability by decreasing the necrotic area and increasing neoangiogenesis. However, the mechanism by which laser acts on cells is not fully understood. The present study investigated the effects of two different laser wavelengths at 30 and 40 J/cm(2) on the viability of skin flap in rats. MATERIAL AND METHODS Sixty male animals were used in this study. They were distributed into the following groups (n = 12 each group): control group, group irradiated with 660 nm at 30 J/cm(2); group irradiated with 780 nm, at 30 J/cm(2), group irradiated with 660 nm at 40 J/cm(2); and group irradiated with 780 nm at 40 J/cm(2). The skin flap was performed on the back of all animals studied, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was done immediately after the surgery and on days 1, 2, 3, and 4 post-surgery. The percentage of the necrotic area of the flap was calculated at day 7 post-surgery. RESULTS Control group showed a necrotic area of 62.83%. Interestingly, no statistically significant differences were found among the treated groups and the control group. CONCLUSION This present study showed that 660 nm and 780 nm lasers at doses of 30 and 40 J/cm(2) were not effective for decreasing the necrotic area of the skin flaps in rats.