RESUMO
The purposes of this study are to evaluate the effects of photobiomodulation (PBM) with laser and LED on rat calvaria osteoblasts (rGO lineage), cultured in osteogenic (OST) or regular (REG) medium, after induction of a quiescent state and to test if PBM is capable of osteogenic induction and if there is a sum of effects when combining OST medium with PBM. Before irradiation, the cells were put in a quiescent state (1% FBS) 24 h, when red (AlGaInP-660 nm) and infrared laser (GaAlAs-808 nm) and LED (637 ± 15 nm) were applied. The groups were as follows: red laser (RL3-5 J/cm2, 3 s and RL5-8.3 J/cm2, 5 s, 1.66 W/cm2); infrared laser (IrL3-5 J/cm2, 3 s and IrL5-8.3 J/cm2, 5 s); LED (LED3-3 s and LED5-5 s, 0.02 J/cm2, 0.885 W/cm2); positive (C+, 10% FBS) and negative control (C-, 1% FBS). For alkaline phosphatase (ALP) and mineralization assays, the cells were cultured in REG (DMEM 10% FBS) and OST medium (DMEM 10% FBS, 50 µg/mL ascorbic acid, 10 mM ß-glycerophosphate). Statistical analysis was performed using ANOVA and Tukey's tests (p < 0.05). RL5 and LED5 increased proliferation, in vitro wound closure, ALP, and mineralization in rGO cells (p < 0.05). PBM with red laser and LED induced mineralization by itself, without osteogenic medium, not observed for infrared laser (p < 0.05). A sum of effects was observed in osteogenic medium and PBM by infrared, red laser, and LED (5 s). Red laser and LED increased proliferation, migration, and secretory phases in rGO cells in a dose-dependent manner. PBM with red laser and LED promotes osteogenic induction by itself. PBM with infrared laser and osteogenic medium potentializes mineralization.
Assuntos
Lasers , Terapia com Luz de Baixa Intensidade , Osteoblastos/efeitos da radiação , Osteogênese/efeitos da radiação , Crânio/efeitos da radiação , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , RatosRESUMO
BACKGROUND: Calvaria skin has a reduced thickness, and its initial damage produced by irradiation was scarcely reported. We aimed to identify the initial effects of x-ray irradiation in the rat calvaria skin. METHODS: After approval by the Animal Ethical Committee, calvaria skin sections of five Wistar rats per time point were evaluated on days 4, 9, 14, and 25 following a single 15-Gy x-ray irradiation of the head. The control group was composed of five rats and evaluated on day 4. Sections were assessed using hematoxylin-eosin and Masson's trichrome staining for morphology, inflammation, and fibrosis. Fibrosis was also evaluated by the collagen maturation index from Picrosirius red staining and by cell proliferation using the immunohistochemistry, after 5-bromo-2-deoxyuridine intraperitoneal injection. RESULTS: In irradiated rats, we observed a reduction in epithelial cell proliferation (p = 0.004) and in matrix metalloproteinase-9 expression (p < 0.001), an increase in the maturation index, and with a predominance in the type I collagen fibers, on days 9 and 14 (1.19 and 1.17, respectively). A progressive disorganization in the morphology of the collagen fibers at all time points and changes in morphology of the sebaceous gland cells and hair follicle were present until day 14. CONCLUSIONS: The initial damage produced by a single 15-Gy x-ray irradiation to the rat calvaria skin was a change in the normal morphology of collagen fibers to an amorphous aspect, a temporary absence of the sebaceous gland and hair follicles, and without a visible inflammatory process, cell proliferation, or fibrosis process in the dermis.
Assuntos
Lesões por Radiação/patologia , Pele/efeitos da radiação , Animais , Proliferação de Células , Masculino , Ratos , Ratos Wistar , Crânio/efeitos da radiação , Coloração e Rotulagem , Raios XRESUMO
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.
Assuntos
Substitutos Ósseos/uso terapêutico , Cerâmica/uso terapêutico , Fraturas Ósseas/terapia , Luz , Ácido Poliglicólico/uso terapêutico , Crânio/lesões , Cicatrização/efeitos dos fármacos , Animais , Substitutos Ósseos/química , Substitutos Ósseos/efeitos da radiação , Transplante Ósseo/métodos , Cimentação/métodos , Cerâmica/química , Terapia Combinada , Masculino , Teste de Materiais , Osteogênese/efeitos dos fármacos , Osteogênese/efeitos da radiação , Fototerapia/métodos , Ácido Poliglicólico/química , Ratos , Ratos Wistar , Crânio/efeitos dos fármacos , Crânio/efeitos da radiação , Engenharia TecidualRESUMO
Objective: To evaluate the effect of the Er,Cr:YSGG laser on healing of critical-sized calvarial defects (CSDs) in rats submitted to inhalation of cigarette smoke. Background: Smoking has been implicated with the delay in the bone healing after osteotomy procedures, then the use of the Er,Cr:YSGG laser for osteotomy in smokers could be an alternative to the conventional drills. Methods: One hundred animals were randomly allocated into four groups: trephine-the CSDs were made with a trephine drill in healthy rats; Er,Cr:YSGG-the CSDs were made with the Er,Cr:YSGG laser in healthy rats; Trephine-S-the CSDs were made with a trephine drill in rats exposed to cigarette smoke; and Er,Cr:YSGG-S-the CSDs were made with the Er,Cr:YSGG laser in rats exposed to cigarette smoke. The inhalation of cigarette smoke started 7 days before the surgical procedure until euthanasia (immediately, 7, 15, 30, or 60 days after the surgical procedure). A histometric analysis and a histological description were performed to evaluate (1) the residual linear lengths and bone formation in the CSDs; (2) the quality of bone healing. Results: The use of Er,Cr:YSGG laser induces more bone formation compared with the trephine in smokers; however, the closure of the CSD was only superior in the Er,Cr:YSGG-S group compared to the Trephine-S group at the 60-day period. Conclusions: The use of the Er,Cr:YSGG laser stimulated the bone repair process after osteotomy procedures in animals submitted to exposure of inhalation of cigarette smoke.
Assuntos
Regeneração Óssea/efeitos da radiação , Lasers de Estado Sólido , Crânio/efeitos da radiação , Fumar , Cicatrização/efeitos da radiação , Animais , Exposição por Inalação , Ratos , Crânio/cirurgiaRESUMO
OBJECTIVE: Considering the global public health problem of smoking, which can negatively influence bone tissue repair, the aim of this study is to analyze the influence of photobiomodulation therapy (PBM) on calvaria defects created surgically in specimens under the effect of cigarette smoke and analyzed with use of histomorphometric and immunohistochemistry techniques. METHODOLOGY: Calvaria defects 4.1 mm in diameter were surgically created in the calvaria of 90-day-old rats (n=60) that were randomly divided into 4 experimental groups containing 15 animals each: control group (C), smoking group (S), laser group (L), and smoke associated with laser group (S+L). The animals were subjected to surgery for calvaria defects and underwent PBM, being evaluated at 21, 45, and 60 days post-surgery. The specimens were then processed for histomorphometric and immunohistochemistry analyses. The area of bone neoformation (ABN), percentage of bone neoformation (PBNF), and the remaining distance between the edges of the defects (D) were analyzed histometrically. Quantitative analysis of the TRAP immunolabeled cells was also performed. The data were subjected to analysis of variance (ANOVA) in conjunction with Tukey's test to verify the statistical differences between groups (p<0.05). RESULTS: The smoking group showed less ABN compared to the other experimental groups in all periods, and it also showed more D at 21 days compared to the remaining groups and at 45 days compared to the laser group. The smoking group showed a lower PNBF compared to the laser group in all experimental periods and compared to smoking combined with LLLT group at 21 days. CONCLUSIONS: PBM acted on bone biomodulation, thus stimulating new bone formation and compensating for the negative factor of smoking, which can be used as a supportive therapy during bone repair processes.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Fumar Cigarros/fisiopatologia , Terapia com Luz de Baixa Intensidade/métodos , Crânio/efeitos da radiação , Cicatrização/efeitos da radiação , Animais , Fumar Cigarros/efeitos adversos , Feminino , Imuno-Histoquímica , Osteogênese/efeitos dos fármacos , Distribuição Aleatória , Ratos Wistar , Reprodutibilidade dos Testes , Crânio/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Abstract Objective Considering the global public health problem of smoking, which can negatively influence bone tissue repair, the aim of this study is to analyze the influence of photobiomodulation therapy (PBM) on calvaria defects created surgically in specimens under the effect of cigarette smoke and analyzed with use of histomorphometric and immunohistochemistry techniques. Methodology Calvaria defects 4.1 mm in diameter were surgically created in the calvaria of 90-day-old rats (n=60) that were randomly divided into 4 experimental groups containing 15 animals each: control group (C), smoking group (S), laser group (L), and smoke associated with laser group (S+L). The animals were subjected to surgery for calvaria defects and underwent PBM, being evaluated at 21, 45, and 60 days post-surgery. The specimens were then processed for histomorphometric and immunohistochemistry analyses. The area of bone neoformation (ABN), percentage of bone neoformation (PBNF), and the remaining distance between the edges of the defects (D) were analyzed histometrically. Quantitative analysis of the TRAP immunolabeled cells was also performed. The data were subjected to analysis of variance (ANOVA) in conjunction with Tukey's test to verify the statistical differences between groups (p<0.05). Results The smoking group showed less ABN compared to the other experimental groups in all periods, and it also showed more D at 21 days compared to the remaining groups and at 45 days compared to the laser group. The smoking group showed a lower PNBF compared to the laser group in all experimental periods and compared to smoking combined with LLLT group at 21 days. Conclusions PBM acted on bone biomodulation, thus stimulating new bone formation and compensating for the negative factor of smoking, which can be used as a supportive therapy during bone repair processes.
Assuntos
Animais , Feminino , Ratos , Crânio/efeitos da radiação , Cicatrização/efeitos da radiação , Regeneração Óssea/efeitos dos fármacos , Terapia com Luz de Baixa Intensidade/métodos , Fumar Cigarros/fisiopatologia , Osteogênese/efeitos dos fármacos , Crânio/patologia , Fatores de Tempo , Imuno-Histoquímica , Distribuição Aleatória , Reprodutibilidade dos Testes , Resultado do Tratamento , Ratos Wistar , Fumar Cigarros/efeitos adversosRESUMO
Biocompatible membranes are widely used in medicine to stimulate bone repair. Several studies have demonstrated that laser photobiomodulation (PBM) also stimulates osteoblast proliferation and osteogenesis at the fracture site, leading to a greater deposition of bone mass and accelerating the process of bone consolidation. This work assessed the therapeutic effect of 780-nm laser PBM and a polystyrene membrane coated with norbixin and collagen (PSNC) on bone healing in rats with calvarial bone defect. Histological staining, Raman spectroscopy, and scanning electron microscopy (SEM) were used to evaluate the bone repair process. Four experimental treatment groups were compared: C, control; M, membrane only; L, laser PBM only; and ML, membrane + laser PBM. A bone defect was created in the calvaria of each animal, with each group subdivided into two subgroups that underwent euthanasia after 15 and 30 days treatment. The L and ML groups were irradiated (λ = 780 nm, ED = 6 J/cm2, P = 60 mW, t = 4 s) postoperatively on alternate days until they were euthanized. The bone concentration of hydroxyapatite (CHA) showed a clear gradation with increasing phosphate area in the order B (normal cortical bone) > L > M > ML > C for both periods. The PSNC membrane was effective in reducing the inflammatory process and served as a scaffold for bone repair. The laser PBM also showed positive effects on the bone repair process with increased deposition and organization of the newly formed bone. However, laser PBM failed to improve the bioactive properties of the membrane scaffold.
Assuntos
Osso e Ossos/patologia , Osso e Ossos/efeitos da radiação , Carotenoides/farmacologia , Terapia com Luz de Baixa Intensidade/métodos , Poliestirenos/química , Cicatrização/efeitos dos fármacos , Cicatrização/efeitos da radiação , Animais , Osso e Ossos/efeitos dos fármacos , Colágeno/farmacologia , Durapatita , Masculino , Microscopia Eletrônica de Varredura , Ratos Wistar , Crânio/efeitos dos fármacos , Crânio/patologia , Crânio/efeitos da radiação , Análise Espectral RamanRESUMO
The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.
Assuntos
Regeneração Óssea/efeitos da radiação , Regeneração Tecidual Guiada/métodos , Terapia com Luz de Baixa Intensidade/métodos , Animais , Autoenxertos , Regeneração Óssea/fisiologia , Colágeno/análise , Masculino , Osteogênese/fisiologia , Osteogênese/efeitos da radiação , Distribuição Aleatória , Ratos Wistar , Valores de Referência , Reprodutibilidade dos Testes , Crânio/fisiologia , Crânio/efeitos da radiação , Crânio/cirurgia , Resultado do Tratamento , Cicatrização/fisiologia , Cicatrização/efeitos da radiaçãoRESUMO
Abstract The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.
Assuntos
Animais , Masculino , Regeneração Óssea/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , Regeneração Tecidual Guiada/métodos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Valores de Referência , Crânio/cirurgia , Crânio/efeitos da radiação , Crânio/fisiologia , Cicatrização/efeitos da radiação , Cicatrização/fisiologia , Regeneração Óssea/fisiologia , Distribuição Aleatória , Reprodutibilidade dos Testes , Colágeno/análise , Resultado do Tratamento , Ratos Wistar , AutoenxertosRESUMO
The aim of this study was to evaluate the effect of collagen sponge scaffold (CSS) implantation associated with low-level laser therapy (LLLT) on repairing bone defects. A single 5-mm cranial defect was surgically created in forty Wistar rats, which then received one of the following four interventions (n = 10 per group): no treatment (G0); bone defect implanted with collagen sponge scaffold (CSS) alone (G1); defect treated with low-level laser therapy (LLLT) (wavelength 780 nm; total energy density 120 J/cm2 ; power 50 mW) alone (G2); and CSS associated with LLLT treatment (G3). After surgery, animals in each group were euthanized at 21 days and 30 days (n = 5 per euthanasia time group). Bone formation was monitored by X-ray imaging analysis. Biopsies were collected and processed for histological analysis and immunohistochemical evaluation of transforming growth factor-beta (TGF-ß), fibroblast growth factor-2 (FGF-2), osteoprotegerin (OPG) and receptor activator of nuclear factor Æ (RANK). Osteocalcin (OCN) was detected by immunofluorescence analysis. Compared to the G0 group, defects in the 30-day G3 group exhibited increased bone formation, both by increase in radiopaque areas (P < 0.01) and by histomorphometric analysis (P < 0.001). The histopathological analysis showed a decreased number of inflammatory cells (P < 0.001). The combined CCS + LLLT (G3) treatment also resulted in the most intense immunostaining for OPG, RANK, FGF-2 and TGF-ß, and the most intense and diffuse OCN immunofluorescent labelling at 30 days postsurgery (G3 vs. G0 group, P < 0.05). Therefore, the use of CCS associated with LLLT could offer a synergistic advantage in improving the healing of bone fractures.