Carla Roberta Tim

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.

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.

Aerobic exercise training and low-level laser therapy modulate inflammatory response and degenerative process in an experimental model of knee osteoarthritis in rats

OBJECTIVE The aim of this study was to evaluate the effects of an aerobic exercise training and low-level laser therapy (LLLT) (associated or not) on degenerative modifications and inflammatory mediators on the articular cartilage using an experimental model of knee OA. MATERIAL AND METHODS Fifty male Wistar rats were randomly divided into five groups: control group (CG); knee OA control group (OAC); OA plus exercise training group (OAT); OA plus LLLT group (OAL); OA plus exercise training associated with LLLT group (OATL). The exercise training (treadmill; 16 m/min; 50 min/day) and the laser irradiation (two points-medial and lateral side of the left joint; 24 sessions) started 4 weeks after the surgery, 3 days/week for 8 weeks. RESULTS The results showed that all treated groups showed (irradiated or not) a better pattern of tissue organization, with less fibrillation and irregularities along the articular surface and chondrocytes organization, a lower degenerative process measured by OARSI score and higher thickness values. Additionally, all treated group showed a reduced expression in IL-1β, caspase-3 and MMP-13 compared to OAC. Moreover, a lower caspase-3 expression was observed in OATL compared to OAL and OAT. CONCLUSION These results suggest that exercise training and LLLT were effective in preventing cartilage degeneration and modulating inflammatory process induced by knee OA.

Comparison of the effects of electrical field stimulation and low-level laser therapy on bone loss in spinal cord-injured rats.

OBJECTIVE This study investigated the effects of low-level laser therapy (LLLT) and electrical stimulation (ES) on bone loss in spinal cord-injured rats. MATERIALS AND METHODS Thirty-seven male Wistar rats were divided into four groups: standard control group (CG); spinal cord-injured control (SC); spinal cord-injured treated with laser (SCL; GaAlAs, 830 nm, CW, 30 mW/cm, 250 J/cm(2)); and spinal cord-injured treated with electrical field stimulation (SCE; 1.5 MHz, 1:4 duty cycles, 30 mW, 20 min). Biomechanical, densitometric, and morphometric analyses were performed. RESULTS SC rats showed a significant decrease in bone mass, biomechanical properties, and morphometric parameters (versus CG). SCE rats showed significantly higher values of inner diameter and internal and external areas of tibia diaphyses; and the SCL group showed a trend toward the same result (versus SC). No increase was found in either mechanical or densitometric parameters. CONCLUSION We conclude that the mentioned treatments were able to initiate a positive bone-tissue response, maybe through stimulation of osteoblasts, which was able to determine the observed morphometric modifications. However, the evoked tissue response could not determine either biomechanical or densitometric modifications.

Effects of biosilicate(®) scaffolds and low-level laser therapy on the process of bone healing.

OBJECTIVE This study aimed to investigate the in vivo tissue performance of the association of Biosilicate(®) scaffolds and low-level laser therapy (LLLT) in a tibial bone defects model in rats. BACKGROUND DATA Many studies have been demonstrating the osteogenic potential of Biosilicate and LLLT. However, there is a need to investigate the effects of both treatments for bone consolidation. METHODS The animals were divided into control group (CG), Biosilicate scaffold group (BG), and Biosilicate scaffolds plus LLLT group (BLG). Animals were euthanized after 15, 30, and 45 days post-injury. RESULTS The histological analysis revealed that all the experimental groups showed inflammatory infiltrate and granulation tissue, at the area of the defect at day 15. After 30 days, CG still showed granulation tissue and bone ingrowth. Both Biosilicate groups presented newly formed bone and interconected trabeculae. At 45 days, CG showed immature newly formed bone. A more mature newly formed bone was observed in BG and BLG. On day 15, BG demonstrated a statistically higher expression of cyclooxygenase (COX)-2 compared with CG and BLG. No statistically significant difference was observed in COX-2 immunoexpression among the groups at 30 and 45 days. Similar expression of bone morphogenetic protein (BMP)-9 was demonstrated for all experimental groups at 15 and 30 days. At 45 days, the BMP-9 immunoexpression was statistically upregulated in the BLG compared with the CG and BG. No statistically significant difference was observed in the receptor activator of nuclear factor kappa-B ligand (RANKL) immunoexpression among the groups in all periods evaluated. Biosilicate groups presented a decrease in biomechanical properties compared with CG at 30 and 45 days post-surgery. CONCLUSIONS Our findings suggest that Biosilicate presented osteogenic activity, accelerating bone repair. However, laser therapy was not able to enhance the bioactive properties of the Biosilicate.

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.

Effects of phototherapy on cartilage structure and inflammatory markers in an experimental model of osteoarthritis.

Abstract. The aim of this study was to evaluate the effects of laser phototherapy on the degenerative modifications on the articular cartilage after the anterior cruciate ligament transection (ACLT) in the knee of rats. Eighty male rats (Wistar) were distributed into four groups: intact control group (IG), injured control group (CG), injured laser treated group at 10  J/cm2 (L10), and injured laser treated group at 50  J/cm2 (L50). Animals were distributed into two subgroups, sacrificed in 5 and 8 weeks postsurgery. The ACLT was used to induce knee osteoarthritis in rats. After 2 weeks postsurgery, laser phototherapy initiated and it was performed for 15 and 30 sessions. The histological findings revealed that laser irradiation, especially at 10  J/cm2, modulated the progression of the degenerative process, showing a better cartilage structure and lower number of condrocytes compared to the other groups. Laser phototherapy was not able to decrease the degenerative process measured by Mankin score and prevent the increase of cartilage thickness related to the degenerative process. Moreover, it did not have any effect in the biomodulation of the expression of markers IL1β, tumor necrosis factor-α, and metalloprotein-13. Furthermore, laser irradiated animals, at 50  J/cm2 showed a lower amount of collagen type 1.

Characterization and biocompatibility of a fibrous glassy scaffold

Bioactive glasses (BGs) are known for their ability to bond to living bone and cartilage. In general, they are readily available in powder and monolithic forms, which are not ideal for the optimal filling of bone defects with irregular shapes. In this context, the development of BG‐based scaffolds containing flexible fibres is a relevant approach to improve the performance of BGs. This study is aimed at characterizing a new, highly porous, fibrous glassy scaffold and evaluating its in vitro and in vivo biocompatibility. The developed scaffolds were characterized in terms of porosity, mineralization and morphological features. Additionally, fibroblast and osteoblast cells were seeded in contact with extracts of the scaffolds to assess cell proliferation and genotoxicity after 24, 72 and 144 h. Finally, scaffolds were placed subcutaneously in rats for 15, 30 and 60 days. The scaffolds presented interconnected porous structures, and the precursor bioglass could mineralize a hydroxyapatite (HCA) layer in simulated body fluid (SBF) after only 12 h. The biomaterial elicited increased fibroblast and osteoblast cell proliferation, and no DNA damage was observed. The in vivo experiment showed degradation of the biomaterial over time, with soft tissue ingrowth into the degraded area and the presence of multinucleated giant cells around the implant. At day 60, the scaffolds were almost completely degraded and an organized granulation tissue filled the area. The results highlight the potential of this fibrous, glassy material for bone regeneration, due to its bioactive properties, non‐cytotoxicity and biocompatibility. Future investigations should focus on translating these findings to orthotopic applications. Copyright

Effects of low level laser therapy on inflammatory and angiogenic gene expression during the process of bone healing: A microarray analysis

The process of bone healing as well as the expression of inflammatory and angiogenic genes after low level laser therapy (LLLT) were investigated in an experimental model of bone defects. Sixty Wistar rats were distributed into control group and laser group (830nm, 30mW, 2,8J, 94seg). Histopathological analysis showed that LLLT was able to modulate the inflammatory process in the area of the bone defect and also to produce an earlier deposition of granulation tissue and newly formed bone tissue. Microarray analysis demonstrated that LLLT produced an up-regulation of the genes related to the inflammatory process (MMD, PTGIR, PTGS2, Ptger2, IL1, 1IL6, IL8, IL18) and the angiogenic genes (FGF14, FGF2, ANGPT2, ANGPT4 and PDGFD) at 36h and 3days, followed by the decrease of the gene expression on day 7. Immunohistochemical analysis revealed that the subjects that were treated presented a higher expression of COX-2 at 36h after surgery and an increased VEGF expression on days 3 and 7 after surgery. Our findings indicate that LLLT was efficient on accelerating the development of newly formed bone probably by modulating the inflammatory and angiogenic gene expression as well as COX2 and VEGF immunoexpression during the initial phase of bone healing.

Bone regeneration and gene expression in bone defects under healthy and osteoporotic bone conditions using two commercially available bone graft substitutes.

Biosilicate(®) and Bio-Oss(®) are two commercially available bone substitutes, however, little is known regarding their efficacy in osteoporotic conditions. The purpose of this study was to evaluate the osteogenic properties of both materials, at tissue and molecular level. Thirty-six Wistar rats were submitted to ovariectomy (OVX) for inducing osteoporotic conditions and sham surgery (SHAM) as a control. Bone defects were created in both femurs, which were filled with Biosilicate(®) or Bio-Oss(®), and empty defects were used as control. For the healthy condition both Biosilicate(®) and Bio-Oss(®) did not improve bone formation after 4 weeks. Histomorphometric evaluation of osteoporotic bone defects with bone substitutes showed more bone formation, significant for Bio-Oss(®). Molecular biological evaluation was performed by gene-expression analysis (Runx-2, ALP, OC, OPG, RANKL). The relative gene expression was increased with Biosilicate(®) for all genes in OVX rats and for Runx-2, ALP, OC and RANKL in SHAM rats. In contrast, with Bio-Oss(®), the relative gene expression of OVX rats was similar for all three groups. For SHAM rats it was increased for Runx-2, ALP, OC and RANKL. Since both materials improved bone regeneration in osteoporotic conditions, our results suggest that bone defects in osteoporotic conditions can be efficiently treated with these two bone substitutes.