Influence of photobiomodulation therapy on the treatment of pulmonary inflammatory conditions and its impact on COVID-19.

We are currently facing a pandemic that continuously causes high death rates and has negative economic and psychosocial impacts. Therefore, this period requires a quick search for viable procedures that can allow us to use safe and non-invasive clinical tools as prophylactic or even adjuvant methods in the treatment of COVID-19. Some evidence shows that photobiomodulation therapy (PBMT) can attenuate the inflammatory response and reduce respiratory disorders similar to acute lung injury (ALI), complications associated with infections, such as the one caused by the new Coronavirus (SARS-CoV-2). Hence, the aim of the present study was to evaluate the influence of PBMT (infrared low-level laser therapy) on the treatment of ALI, one of the main critical complications of COVID-19 infection, in an experimental model in rats. Twenty-four male Wistar rats were randomly allocated to three experimental groups (n = 8): control group (CG), controlled ALI (ALI), and acute lung injury and PBM (ALIP). For treatment, a laser equipment was used (808 nm; 30 mw; 1.68 J) applied at three sites (anterior region of the trachea and in the ventral regions of the thorax, bilaterally) in the period of 1 and 24 h after induction of ALI. For treatment evaluation, descriptive histopathological analysis, lung injury score, analysis of the number of inflammatory cells, and expression of interleukin 1 ß (IL-1ß) were performed. In the results, it was possible to observe that the treatment with PBMT reduced inflammatory infiltrates, thickening of the alveolar septum, and lung injury score when compared to the ALI group. In addition, PBMT showed lower immunoexpression of IL-1ß. Therefore, based on the results observed in the present study, it can be concluded that treatment with PBMT (infrared low-level laser therapy) was able to induce an adequate tissue response capable of modulating the signs of inflammatory process in ALI, one of the main complications of COVID-19.

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.

Incorporation of collagen and PLGA in bioactive glass: in vivo biological evaluation.

Bioactive glasses (BG) are known for their unique ability to bond to bone tissue. However, in critical situations, even the osteogenic properties of BG may be not sufficient to produce bone consolidation. The use of composite materials may constitute an optimized therapeutical intervention for bone stimulation. The aim of this study was to characterize BG/collagen/poly (d,l-lactic-co-glycolic) acid (BG/COL/PLGA) composites, in vitro biocompatibility and in vivo biological properties. MC3T3-E1 cells were evaluated by cell proliferation, ALP activity, cell adhesion and morphology. Qualitative histology and immunohistochemistry were performed in a calvarial bone defect model in rats. The in vitro study demonstrated, after 3 and 6 days of culture, a significant increase of proliferation was observed for BG/PLGA compared to BG/COL and BG/COL/PLGA. BG/COL/PLGA presented a higher value for ALP activity after 3 days of culture compared to BG/PLGA. For in vivo analysis, 6 weeks post-surgery, BG/PLGA showed a more mature neoformed bone tissue. As a conclusion, the in vitro and in vivo studies pointed out that BG/PLGA samples improved biological properties in calvarial bone defects, highlighting the potential of BG/PLGA composites to be used as a bone graft for bone regeneration applications.

Vacuumed collagen-impregnated bioglass scaffolds: Characterization and influence on proliferation and differentiation of bone marrow stromal cells.

This study evaluated physical-chemical characteristics of a vacuumed collagen-impregnated bioglass (BG) scaffolds and bone marrow stromal cells (BMSCs) behavior on those composites. scanning electron microscope and energy dispersive x-ray spectroscope demonstrated collagen (Col) was successfully introduced into BG. Vacuum impregnation system has showed efficiency for Col impregnation in BG scaffolds (approximately 20 wt %). Furthermore, mass weight decreasing and more stabilized pH were observed over time for BG/Col upon incubation in phosphate buffered saline compared to plain BG under same conditions. Calcium evaluation (Ca assay) demonstrated higher calcium uptake for BG/Col samples compared to BG. In addition, BG samples presented hydroxyapatite crystals formation on its surface after 14 days in simulated body fluid solution, and signs of initial degradation were observed for BG and BG/Col after 21 days. Fourier transform infrared spectroscopy spectra for both groups indicated peaks for hydroxyapatite formation. Finally, a significant increase of BMSCs viability for both composites was observed compared to control group, but no increase of osteogenic differentiation-related gene expressions were found. In summary, BG/Col scaffolds have improved degradation, pH equilibrium and Ca mineralization over time, accompanied by hydroxyapatite formation. Moreover, both BG and BG/Col scaffolds were biocompatible and noncytotoxic, promoting a higher cell viability compared to control. Future investigations should focus on additional molecular and in vivo studies in order to evaluate biomaterial performance for bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 211-222, 2019.

Photobiomodulation guided healing in a sub-critical bone defect in calvarias of rats.

BACKGROUND: Photobiomodulation presents stimulatory effects on tissue metabolism, constituting a promising strategy to produce bone tissue healing. OBJECTIVE: the aim of the present study was to investigate the in vivo performance of PBM using an experimental model of cranial bone defect in rats. MATERIAL AND METHODS: rats were distributed in 2 different groups (control group and PBM group). After the surgical procedure to induce cranial bone defects, PBM treatment initiated using a 808 nm laser (100 mW, 30 J/cm2, 3 times/week). After 2 and 6 weeks, animals were euthanized and the samples were retrieved for the histopathological, histomorphometric, picrosirius red staining and immunohistochemistry analysis. RESULTS: Histology analysis demonstrated that for PBM most of the bone defect was filled with newly formed bone (with a more mature aspect when compared to CG). Histomorphomeric analysis also demonstrated a higher amount of newly formed bone deposition in the irradiated animals, 2 weeks post-surgery. Furthermore, there was a more intense deposition of collagen for PBM, with ticker fibers. Results from Runx-2 immunohistochemistry demonstrated that a higher immunostaining for CG 2 week's post-surgery and no other difference was observed for Rank-L immunostaining. CONCLUSION: This current study concluded that the use of PBM was effective in stimulating newly formed bone and collagen fiber deposition in the sub-critical bone defect, being a promising strategy for bone tissue engineering.

Scaffolds of bioactive glass-ceramic (Biosilicate®) and bone healing: A biological evaluation in an experimental model of tibial bone defect in rats.

This study aimed to investigate the in vivo tissue response of the Biosilicate® scaffolds in a model of tibial bone defect. Sixty male Wistar rats were distributed into bone defect control group (CG) and Biosilicate® scaffold group (BG).  Animals were euthanized 15, 30 and 45 days post-surgery. Stereomicroscopy, scanning electron microscopy, histopathological, immunohistochemistry and biomechanical analysis were used. Scaffolds had a total porosity of 44%, macroporosity of 15% with pore diameter of 230 µm. Higher amount of newly formed bone was observed on days 30 and 45 in BG. Immunohistochemistry analysis showed that the COX-2 expression was significantly higher on days 15 and 30 in BG compared with the CG. RUNX-2 immunoexpression was significantly higher in BG on days 15 and 45. No statistically significant difference was observed in RANKL immunoexpression in all experimental groups. BMP-9 immunoexpression was significantly upregulated in the BG on day 45. Biomechanical analysis showed a decrease in the biomechanical properties of the bone callus on days 30 and 45. The implantation of the Biosilicate® scaffolds was effective in stimulating newly bone formation and produced an increased immunoexpression of markers related to the bone repair.

Photobiomodulation and bacterial cellulose membrane in the treatment of third-degree burns in rats.

Burns are injuries caused mainly by thermal trauma, which can progress to unsatisfactory results healing. This study aimed to evaluate the biomaterial (bacterial cellulose membrane) and photobiomodulation, exclusively and associated, in the treatment of third degree burns in rats. Forty male Wistar rats (±280 g) were randomly divided into four groups, with 10 animals each: control group (CG); bacterial cellulose membrane group (MG); laser group (LG) and bacterial cellulose membrane and laser group (MG + L). The burn was caused with a 1 cm2 aluminum plate heated to 150 °C and pressed on the animal's back for 10 s. The treatments were started immediately after induction of injury. For to laser irradiation (660 nm, 100 mW, 25 J/cm2 and energy of 1 J) on five distinct application points were used, on alternate days, a total of five sessions. After ten days of treatment the animals were euthanized for collected samples. One-way ANOVA and Tukey's tests (P < 0.05) were used. Histological analysis revealed differences regarding the healing process phase in each experimental group. MG showed the proliferative phase. The LG demonstrated greater amount of blood vessels and immune expression of VEGF. However, when the treatments were combined, the number of vessels and the immune expression of VEGF factor was lower than LG. Thus, it was concluded that both treatments proposed (biomaterial and LLLT) are good alternatives for third degree burns when applied isolated because they stimulate the healing process by acting on the modulation of the inflammatory phase and promote stimulation of angiogenesis.

Mitochondrial dynamics (fission and fusion) and collagen production in a rat model of diabetic wound healing treated by photobiomodulation: comparison of 904 nm laser and 850 nm light-emitting diode (LED).

OBJECTIVE: Mitochondrial dysfunction has been associated with the development of diabetes mellitus which is characterized by disorders of collagen production and impaired wound healing. This study analyzed the effects of photobiomodulation (PBM) mediated by laser and light-emitting diode (LED) on the production and organization of collagen fibers in an excisional wound in an animal model of diabetes, and the correlation with inflammation and mitochondrial dynamics. METHODS: Twenty Wistar rats were randomized into 4 groups of 5 animals. Groups: (SHAM) a control non-diabetic wounded group with no treatment; (DC) a diabetic wounded group with no treatment; (DLASER) a diabetic wounded group irradiated by 904 nm pulsed laser (40 mW, 9500 Hz, 1 min, 2.4 J); (DLED) a diabetic wounded group irradiated by continuous wave LED 850 nm (48 mW, 22 s, 1.0 J). Diabetes was induced by injection with streptozotocin (70 mg/kg). PBM was carried out daily for 5 days followed by sacrifice and tissue removal. RESULTS: Collagen fibers in diabetic wounded skin were increased by DLASER but not by DLED. Both groups showed increased blood vessels by atomic force microscopy. Vascular endothelial growth factor (VEGF) was higher and cyclooxygenase (COX2) was lower in the DLED group. Mitochondrial fusion was higher and mitochondrial fusion was lower in DLED compared to DLASER. CONCLUSION: Differences observed between DLASER and DLED may be due to the pulsed laser and CW LED, and to the higher dose of laser. Regulation of mitochondrial homeostasis may be an important mechanism for PBM effects in diabetes.

Bacterial cellulose membrane used as biological dressings on third-degree burns in rats.

INTRODUCTION: Burn injuries represent a high risk of morbidity and mortality. The wound healing process is complex and requires the participation of different types of cells. Therefore, new biomaterials, which innovate the wound healing process, are being investigated. OBJECTIVE: The aim of this study was to investigate the use of bacterial cellulose both in its pure state and enriched with lidocaine in full-thickness burns in rats. METHODS: Thirty rats (Wistar) (260 ± 20 gramas) divided into control group (CG), bacterial cellulose membrane group (MG) and bacterial cellulose membrane enriched with lidocaine group (MLG) were used. The burns were induced using a 150°C heated soldering iron, held on the animal neck for 10 seconds. The biomaterial was applied immediately after injury and skin samples were collected on the tenth day of the treatment. The level of significance of p⩽0.05 was used for the conclusion of the statistical analysis. RESULTS: The groups treated with the biomaterials, a histological pattern compatible with a more advanced repair stage showing skin appendages, mild inflammatory infiltrate, better collagen fiber organization and mild immunostaining COX-2 and MMP-9 was observed, when compared to the control group that did not receive any type of treatment. CONCLUSION: Thus, was concluded that the bacterial cellulose-based biomaterial both in its pure state and enriched with lidocaine optimizing the full-thickness burn wound healing in rats.

Low-level laser therapy induces an upregulation of collagen gene expression during the initial process of bone healing: a microarray analysis.

This study investigates the histological modifications produced by low level laser therapy (LLLT) on the first day of bone repair, as well as evaluates the LLLT effects on collagen expression on the site of a fracture. Twenty Wistar rats were distributed into a control group (CG) and a laser group (LG). Laser irradiation of Ga-Al-As laser 830 nm, 30 mW, 94 s, 2.8 J was performed in five sessions. Animals were euthanized on day 5 postsurgery. Histopathological analysis showed that LLLT was able to increase deposition of granulation tissue and newly formed bone at the site of the injury. In addition, picrosirius analysis showed that collagen fiber organization in the LG was enhanced compared to CG. Microarray analysis demonstrated that LLLT produced an upregulation type I collagen (COL-I). Immunohistochemical analysis revealed that the subjects that were treated presented a higher immunoexpression of COL-I. Our findings indicated that LLLT improves bone healing by producing a significant increase in the expression of collagen genes.

Calcium phosphate fibers coated with collagen: In vivo evaluation of the effects on bone repair.

The aim of this study was to assess the characteristics of the CaP/Col composites, in powder and fiber form, via scanning electron microscopy (SEM), pH and calcium release evaluation after immersion in SBF and to evaluate the performance of these materials on the bone repair process in a tibial bone defect model. For this, four different formulations (CaP powder - CaPp, CaP powder with collagen - CaPp/Col, CaP fibers - CaPf and CaP fibers with collagen - CaPf/Col) were developed. SEM images indicated that both material forms were successfully coated with collagen and that CaPp and CaPf presented HCA precursor crystals on their surface. Although presenting different forms, FTIR analysis indicated that CaPp and CaPf maintained the characteristic peaks for this class of material. Additionally, the calcium assay study demonstrated a higher Ca uptake for CaPp compared to CaPf for up to 5 days. Furthermore, pH measurements revealed that the collagen coating prevented the acidification of the medium, leading to higher pH values for CaPp/Col and CaPf/Col. The histological analysis showed that CaPf/Col demonstrated a higher amount of newly formed bone in the region of the defect and a reduced presence of material. In summary, the results indicated that the fibrous CaP enriched with the organic part (collagen) glassy scaffold presented good degradability and bone-forming properties and also supported Runx2 and RANKL expression. These results show that the present CaP/Col fibrous composite may be used as a bone graft for inducing bone repair.

Comparative effects of two different doses of low-level laser therapy on wound healing third-degree burns in rats.

Burns are injuries caused by direct or indirect contact to chemical, physical, or biological agents. Low-level laser therapy (LLLT) is a promising treatment since it is low-cost, non-invasive, and induces cell proliferation. This study aimed to investigate the effects of LLLT (660 nm) at two different fluences (12.5 J/cm(2) and 25 J/cm(2) ) per point of application on third-degree burns in rats. Thirty rats (Wistar) divided into GC, GL12.5, and GL25 were used in the study, and submitted to burn injury through a soldering iron at 150°C, pressed on their back for 10 s. LLLT was applied immediately, and 2, 4, 6, and 8 days after wound induction. Histological analysis revealed a decreased inflammatory infiltrate in the group treated with 25 J/cm(2) , and intense inflammatory infiltrate in the control group and in the group treated with 12.5 J/cm(2) . The immunostaining of COX-2 was more intense in the control groups and in the group treated with 12.5 J/cm(2) than in the group treated with 25 J/cm(2) . Conversely, VEGF immunomarking was more expressive in the group treated with 25 J/cm(2) than it was in the other two groups. Therefore, our findings suggest that the use of 25 J/cm(2) and 1 J of energy was more effective in stimulating the cellular processes involved in tissue repair on third-degree burns in rats by reducing the inflammatory phase, and stimulating angiogenesis, thus restoring the local microcirculation which is essential for cell migration.

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.

Effects of low-level laser therapy on the expression of osteogenic genes during the initial stages of bone healing in rats: a microarray analysis.

This study evaluated the morphological changes produced by LLLT on the initial stages of bone healing and also studied the pathways that stimulate the expression of genes related to bone cell proliferation and differentiation. One hundred 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, 94 s, 2.8 J) was performed for 1, 2, 3, 5, and 7 sessions. Histopathology revealed that treated animals produced increased amount of newly formed bone at the site of the injury. Moreover, microarray analysis evidenced that LLLT produced a significant increase in the expression TGF-ß, BMP, FGF, and RUNX-2 that could stimulate osteoblast proliferation and differentiation, which may be related to improving the deposition of newly formed bone at the site of the injury. Thus, it is possible to conclude that LLLT improves bone healing by producing a significant increase in the expression of osteogenic genes.

Porous bioactive scaffolds: characterization and biological performance in a model of tibial bone defect in rats.

The aim of this study was to evaluate the effects of highly porous Biosilicate(®) scaffolds on bone healing in a tibial bone defect model in rats by means of histological evaluation (histopathological and immunohistochemistry analysis) of the bone callus and the systemic inflammatory response (immunoenzymatic assay). Eighty Wistar rats (12 weeks-old, weighing±300 g) were randomly divided into 2 groups (n=10 per experimental group, per time point): control group and Biosilicate® group (BG). Each group was euthanized 3, 7, 14 and 21 days post-surgery. Histological findings revealed a similar inflammatory response in both experimental groups, 3 and 7 days post-surgery. During the experimental periods (3-21 days post-surgery), it was observed that the biomaterial degradation, mainly in the periphery region, provided the development of the newly formed bone into the scaffolds. Immunohistochemistry analysis demonstrated that the Biosilicate® scaffolds stimulated cyclooxygenase-2, vascular endothelial growth factor and runt-related transcription factor 2 expression. Furthermore, in the immunoenzymatic assay, BG presented no difference in the level of tumor necrosis factor alpha in all experimental periods. Still, BG showed a higher level of interleukin 4 after 14 days post-implantation and a lower level of interleukin 10 in 21 days post-surgery. Our results demonstrated that Biosilicate® scaffolds can contribute for bone formation through a suitable architecture and by stimulating the synthesis of markers related to the bone repair.

Evaluation of the bone healing process in an experimental tibial bone defect model in ovariectomized rats.

The aim of this study was to evaluate the influence of postmenopausal bone loss (induced by ovariectomy) in the process of bone healing in a tibial bone defect model in rats by means of histological evaluation of bone defects and the analysis of the expression of genes and proteins involved in bone consolidation. Twenty female Wistar rats (12 weeks old, weighing ±250 g) were randomly divided into two groups: control group (CG) and ovariectomized group (OG). Rats of OG were submitted to ovariectomy and after 8 weeks post-surgery, all animals were submitted to the tibial bone defect model. The main histological finding analysis revealed that ovariectomized animals showed a higher amount of granulation tissue and immature newly formed bone compared to CG. Furthermore, quantitative histological analysis showed that OG presented a significant decrease in the amount of newly formed bone (p = 0.0351). RT-PCR analysis showed no difference in Runx2, ALP, RANK, RANKL and Osterix gene expression 14-day post-surgery. Interestingly, immunohistochemical evaluation showed that Runx2 was down expressed (p = 0.0001) and RANKL was up expressed (p = 0.0022) in the OG. In conclusion, these data highlight that bone loss induced by ovariectomy causes an impairment in the capacity of bone to heal mainly probably because of alterations in the imbalance of osteoblasts and osteoclasts activities.

Biocompatibility of a porous alumina ceramic scaffold coated with hydroxyapatite and bioglass.

This study aimed to evaluate the osteointegration and genotoxic potential of a bioactive scaffold, composed of alumina and coated with hydroxyapatite and bioglass, after their implantation in tibias of rats. For this purpose, Wistar rats underwent surgery to induce a tibial bone defect, which was filled with the bioactive scaffolds. Histology analysis (descriptive and morphometry) of the bone tissue and the single-cell gel assay (comet) in multiple organs (blood, liver, and kidney) were used to reach this aim after a period of 30, 60, 90, and 180 days of material implantation. The main findings showed that the incorporation of hydroxyapatite and bioglass in the alumina scaffolds produced a suitable environment for bone ingrowth in the tibial defects and did not demonstrate any genotoxicity in the organs evaluated in all experimental periods. These results clearly indicate that the bioactive scaffolds used in this study present osteogenic potential and still exhibit local and systemic biocompatibility. These findings are promising once they convey important information about the behavior of this novel biomaterial in biological system and highlight its possible clinical application.