In vitro and in vivo biological performance of hydroxyapatite from fish waste.

The aim of this study was to evaluate biocompatibility of hydroxyapatite (HAP) from fish waste using in vitro and in vivo assays. Fish samples (whitemouth croaker - Micropogonias furnieri) from the biowaste was used as HAP source. Pre-osteoblastic MC3T3-E1 cells were used in vitro study. In addition, bone defects were artificially created in rat calvaria and filled with HAP in vivo. The results demonstrated that HAP reduced cytotoxicity in pre-osteoblast cells after 3 and 6 days following HAP exposure. DNA concentration was lower in the HAP group after 6 days. Quantitative RT-PCR did not show any significant differences (p > 0.05) between groups. In vivo study revealed that bone defects filled with HAP pointed out moderate chronic inflammatory cells with slight proliferation of blood vessels after 7 and 15 days. Chronic inflammatory infiltrate was absent after 30 days of HAP exposure. There was also a decrease in the amount of biomaterial, being followed by newly formed bone tissue. All experimental groups also demonstrated strong RUNX-2 immoexpression in the granulation tissue as well as in cells in close contact with biomaterial. The number of osteoblasts inside the defect area was lower in the HAP group when compared to control group after 7 days post-implantation. Similarly, the osteoblast surface as well as the percentage of bone surface was higher in control group when compared with HAP group after 7 days post-implantation. Taken together, HAP from fish waste is a promising possibility that should be explored more carefully by tissue-engineering or biotechnology.

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.

Photobiomodulation and bone healing in diabetic rats: evaluation of bone response using a tibial defect experimental model.

Diabetes mellitus (DM) leads to a delay in bone healing. Thus, some therapeutic approaches have been used to accelerate the process of bone repair such as photobiomodulation (PBM). Therefore, the present study aimed to evaluate the effects of PBM, in different fluences, in bone repair in an experimental model of tibial bone defects in diabetic rats. Sixty-four Wistar rats were submitted to a surgical procedure to perform bone defect and distributed in four groups: diabetic control group (DCG), diabetic laser group 30 J/cm(2) (L30), diabetic laser group 60 J/cm(2) (L60), and diabetic laser group 120 J/cm(2) (L120). A 808 nm Ga-Al-As (DMC Equipment, São Carlos, SP, Brazil) laser, 100 mW; 0.028 cm(2); 3.57 W/cm(2); 30, 60, and 120 J/cm(2); 0.84, 1.68, and 3.36 J; 8, 16, and 33 s was used. Animals were euthanized 15 and 30 days after the surgery. Histological, morphometric, immunohistochemistry, and biomechanical analyses were performed. In the histological and morphometric evaluation, all laser-treated groups showed a better histological pattern and a higher amount of newly formed bone compared to DCG. An intense RUNX2 immunoexpression was observed in the laser-treated groups, 15 days after the surgery. Receptor activator of nuclear factor κ-ß ligand (RANK-L) immunohistochemistry analysis showed a significant decrease in the immunoreactivity for L30 and L120, 30 days after surgery. There was no statistical difference in the biomechanical analysis among the groups. In conclusion, PBM, in all fluences used, showed an osteogenic potential in bone healing of diabetic rats.

Anti-inflammatory Effects of Compounds Extracted from Marine Sponge s: A Systematic Review.

BACKGROUND: Previous studies have experimentally validated and reported that chemical constituents of marine sponges are a source of natural anti-inflammatory substances with the biotechnological potential to develop novel drugs. AIMS: Therefore, the aim of this study was to perform a systematic review to provide an overview of the anti-inflammatory substances isolated from marine sponges with therapeutic potential. METHODS: This systematic review was performed on the Embase, PubMed, Scopus and Web of Science electronic databases. In total, 613 were found, but 340 duplicate studies were excluded, only 100 manuscripts were eligible, and 83 were included. RESULTS: The results were based on in vivo and in vitro assays, and the anti-inflammatory effects of 251 bioactive compounds extracted from marine sponges were investigated. Their anti-inflammatory activities include inhibition of pro-inflammatory mediators, such as tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), nitrite or nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 1ß (IL-1ß), prostaglandin E2 (PGE2), phospholipase A2 (PLA2), nuclear transcription factor-kappa B (NF-κB), leukotriene B4 (LTB4), cyclooxygenase- 1 (COX-1), and superoxide radicals. CONCLUSION: In conclusion, data suggest (approximately 98% of articles) that substances obtained from marine sponges may be promising for the development of novel anti-inflammatory drugs for the treatment of different pathological conditions.

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.

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.

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.

Low-level laser therapy modulates demyelination in mice.

There are no effective therapies for remyelination. Low-level laser therapy (LLLT) has been found advantageous in neurogenesis promotion, cell death prevention, and modulation of inflammation in central and peripheral nervous system models. The purpose of this study was to analyse LLLT effects on cuprizone-induced demyelination. Mice were randomly distributed into three groups: Control Laser (CTL), Cuprizone (CPZ), and Cuprizone Laser (CPZL). Mice from CPZ and CPZL groups were exposed to a 0.2% cuprizone oral diet for four complete weeks. Six sessions of transcranial laser irradiation were applied on three consecutive days, during the third and fourth weeks, with parameters of 36 J/cm2, 50 mW, 0.028 cm2 spot area, continuous wave, 1 J, 20 s, 1.78 W/cm2 in a single point equidistant between the eyes and ears of CTL and CPZL mice. Motor coordination was assessed by the rotarod test. Twenty-four hours after the last laser session, all animals were euthanized, and brains were extracted. Serum was obtained for lactate dehydrogenase toxicity testing. Histomorphological analyses consisted of Luxol Fast Blue staining and immunohistochemistry. The results showed that laser-treated animals presented motor performance improvement, attenuation of demyelination, increased number of oligodendrocyte precursor cells, modulated microglial and astrocytes activation, and a milder toxicity by cuprizone. Although further studies are required, it is suggested that LLLT represents a feasible therapy for demyelinating diseases.

Low level laser therapy accelerates bone healing in spinal cord injured rats.

Bone loss occurs rapidly and consistently after the occurrence of a spinal cord injury (SCI), leading to a decrease in bone mineral density (BMD) and a higher risk of fractures. In this context, the stimulatory effects of low level laser therapy (LLLT) also known as photobiomodulation (PBM) have been highlighted, mainly due to its osteogenic potential. The aim of the present study was to evaluate the effects of LLLT on bone healing using an experimental model of tibial bone defect in SCI rats. Twenty-four female Wistar rats were randomly divided into 3 groups: Sham group (SG), SCI control group (SC) and SCI laser treated group (SL). Two weeks after the induction of the SCI, animals were submitted to surgery to induce a tibial bone defect. Treatment was performed 3days a week, for 2weeks, at a single point over the area of the injury, using an 808nm laser (30mW, 100J/cm(2); 0.028cm(2), 1.7W/cm², 2.8J). The results of the histological and morphometric evaluation demonstrated that the SL group showed a larger amount of newly formed bone compared to the SC group. Moreover, a significant immunoexpression of runt-related transcription factor 2 (RUNX2) was observed in the SL group. There was no statistical difference in the biomechanical evaluation. In conclusion, the results suggest that LLLT accelerated the process of bone repair in rats with complete SCI.

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.

Low-level laser therapy associated to a resistance training protocol on bone tissue in diabetic rats.

OBJECTIVE: The present study aimed to evaluate the in vivo response of a resistance training and low-level laser therapy (LLLT) on tibias and femurs of rats with diabetes mellitus (DM). MATERIALS AND METHODS: Forty male Wistar rats were randomly distributed into four experimental groups: control group (CG), diabetic group (DG), diabetic trained group (TG) and diabetic trained and laser irradiated group (TLG). DM was induced by streptozotocin (STZ) and after two weeks laser and resistance training started, performed for 24 sessions, during eight weeks. At the end of the experiment, animals were euthanized and tibias and femurs were removed for analysis. Histological, histomorphometrical, immunohistochemistry and mechanical analyses were performed. RESULTS: Trained groups, with or without laser irradiation, showed increased cortical area, bone density and biomechanical properties. The immunohistochemical analysis revealed that TG and TLG demonstrated an increased RUNX2 expression. RANK-L immunoexpression was similar for all experimental groups. CONCLUSION: In conclusion, it can be suggested that the resistance exercise program stimulated bone metabolism, culminating in increased cortical tibial area, bone mineral content, bone mineral density and biomechanical properties. Furthermore, the association of physical exercises and LLLT produced higher values for bone mineral content and stiffness. Consequently, these data highlight the potential of physical exercise in the management of bone loss due to DM and the possible extra osteogenic stimulus offered by lasertherapy. Further long-term studies should be carried out to provide additional information.

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

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/cm(2) (L10), and injured laser treated group at 50 J/cm(2) (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/cm(2), 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 IL1ß, tumor necrosis factor-α, and metalloprotein-13. Furthermore, laser irradiated animals, at 50 J/cm(2) showed a lower amount of collagen type 1.

Low-level laser therapy associated to a resistance training protocol on bone tissue in diabetic rats

ABSTRACT Objective The present study aimed to evaluate the in vivo response of a resistance training and low-level laser therapy (LLLT) on tibias and femurs of rats with diabetes mellitus (DM). Materials and methods Forty male Wistar rats were randomly distributed into four experimental groups: control group (CG), diabetic group (DG), diabetic trained group (TG) and diabetic trained and laser irradiated group (TLG). DM was induced by streptozotocin (STZ) and after two weeks laser and resistance training started, performed for 24 sessions, during eight weeks. At the end of the experiment, animals were euthanized and tibias and femurs were removed for analysis. Histological, histomorphometrical, immunohistochemistry and mechanical analyses were performed. Results Trained groups, with or without laser irradiation, showed increased cortical area, bone density and biomechanical properties. The immunohistochemical analysis revealed that TG and TLG demonstrated an increased RUNX2 expression. RANK-L immunoexpression was similar for all experimental groups. Conclusion In conclusion, it can be suggested that the resistance exercise program stimulated bone metabolism, culminating in increased cortical tibial area, bone mineral content, bone mineral density and biomechanical properties. Furthermore, the association of physical exercises and LLLT produced higher values for bone mineral content and stiffness. Consequently, these data highlight the potential of physical exercise in the management of bone loss due to DM and the possible extra osteogenic stimulus offered by lasertherapy. Further long-term studies should be carried out to provide additional information.

Avaliação funcional da disfunção temporomandibular após bioestimulação associado à cinesioterapia / Functional evaluation of temporomandibular joint disorders after bioestimulation combined with kinesiotherapy

A disfunção temporomandibular (DTM) é altamente debilitantee causa alteração na realização de algumas funções importantescomo mastigar alimentos ou falar adequadamente. Esta disfunçãogeralmente segue uma trajetória de eventos progressivos, podendodesencadear uma condição articular degenerativa. Diante disso, otratamento fisioterapêutico se destaca, por ser um tratamento nãomedicamentoso e não invasivo. Há uma escassez de artigos na literaturasobre a utilização de técnicas cinesioterapêuticas associadascom aplicação de laser de baixa intensidade. Diante deste contexto,o presente estudo teve como objetivo verificar o quadro clínico depacientes portadores de DTM, após intervenção fisioterapêutica.Cinco pacientes receberam tratamento fisioterapêutico, associandotécnicas cinesioterapêuticas e aplicação de laser de baixa intensidade.Foram realizadas três sessões semanalmente, durante dois meses. Paraverificar o quadro clínico, foi realizada avaliação da amplitude demovimento e aplicação dos questionários: questionário de Fonseca,índice de disfunção clínica craniomandibular, índice temporomandibulare o SF36. Através dos resultados deste estudo, a associação dasduas técnicas de tratamento demonstrou um aumento da amplitudede movimento articular e melhora nos escores de funcionalidade,correlacionados com a melhora importante dos aspectos sociais eemocionais. Assim, este é um importante indicador para o surgimentode novos estudos que enfatizem esta associação de técnicas.

The temporomandibular disorder (TMD) is a debilitating diseasethat causes changes in some important functions such as to chewfood or speak properly. This disorder usually develops a progressivetrajectory of events, which can lead to a degenerative joint disease.Therefore, physical therapy stands out for being a non-medicationand noninvasive treatment. There is little articles in the literatureconcerning the use of kinesiotherapy techniques associated withlow level laser. This study aimed to verify the clinical picture of patientswith TMD, after physical therapy intervention. Five patientsunderwent physical therapy combining kinesiotherapy and lowlevel laser application. Three sessions were held weekly during twomonths. In order to verify clinical current status, we carried out anevaluation of range of motion and used the following questionnaires:Fonseca questionnaire, Clinical Craniomandibular dysfunctionIndex, Temporomandibular Index and SF36. From the results ofthis study we observed that the combination of the two treatmenttechniques showed an increase in range of motion and improvementin functional scores, correlated with significant improvement ofsocial and emotional aspects. Thus, new studies emphasizing thecombination of the two techniques should be conducted.