Efeitos do laser de baixa intensidade em osteoblastos cultivados em arcabouços nanofibrosos de ácido polilático / Effects of low-intensity laser on osteoblasts cultured on polylactic acid nanofibrous scaffolds

A engenharia de tecidos ósseos é um ramo importante da medicina regenerativa e envolve o desenvolvimento de arcabouços com composição e arquitetura favoráveis à integração celular, além do estudo de fatores capazes de promover a adesão e proliferação celular, incluindo estímulos químicos e biofísicos. O objetivo do estudo foi avaliar a utilização do laser de baixa intensidade (LBI) como uma ferramenta para promover a bioestimulação in vitro de células osteoblásticas cultivadas em arcabouços nanofibrosos de ácido polilático (PLA). Os arcabouços foram produzidos pela técnica de eletrofiação e caracterizados quanto à molhabilidade, composição pela espectroscopia no infravermelho por transformada de Fourier (FTIR), morfologia da superfície por microscópica eletrônica de varredura (MEV), caracterização termogravimétrica (TGA), calorimetria diferencial exploratória (DSC) e cristalinidade por difração de raios-X (DRX). Os ensaios biológicos foram conduzidos com osteoblastos da linhagem OFCOL II cultivados na superfície dos arcabouços e submetidos ou não (grupo controle) a irradiação com laser diodo InGaAIP na potência de 30 mW, nas doses de 1, 4 e 6 J/cm² e nos comprimentos de onda de 660 nm (grupos V1, V4, V6, respectivo as doses) e 780 nm (grupos I1, I4 e I6, respectivo as doses). Os efeitos do LBI na proliferação dos osteoblastos foram avaliados através do método bioquímico Alamar Blue, nos intervalos de 24, 48 e 72h, enquanto a viabilidade e a morfologia celular foram analisadas no intervalo de 72h, através do ensaio Live/Dead e da microscopia eletrônica de varredura (MEV), respectivamente. Os dados do ensaio bioquímico de Alamar Blue mostraram uma maior proliferação celular nos grupos V6 em todos os intervalos analíticos em comparação ao grupo controle (p<0,05). Outras diferenças entre o grupo controle e irradiados foram encontradas apenas nos intervalos de 48h e 72h para V1, e para o grupo IV6 em 72h. O ensaio Live/Dead revelou um aumento na viabilidade celular nos grupos trados com LBI, sendo significativamente maior no grupo V1 quando comparado ao grupo controle. A análise por MEV mostrou adequada interação dos osteoblastos aos arcabouços, com o corpo celular se espalhando ao longo do eixo da nanofibra e a presença de contatos físicos mais evidentes, através da formação de ligação por meio de filopódios e lamelipódios, nos grupos V1, V6 e I6. Em conjunto, os dados do presente trabalho mostraram que o LBI promove a bioestimulação de osteoblastos cultivados sobre nanofibras de PLA, o que aponta para o seu uso potencial nas técnicas de engenharia tecidual óssea, sobretudo no que se refere ao uso do comprimento de onda de 660 nm, a qual apresentou grupos com mais resultados significativos (AU).

Bone tissue engineering is a relevant branch of regenerative medicine and involves the development of scaffolds with composition and architecture favorable to cell integration, in addition to studying factors capable of promoting cell adhesion and proliferation, including chemical and biophysical stimuli. The study aimed to evaluate the use of low-level laser irradiation (LLLI) to promote in vitro biostimulation of osteoblastic cells cultured on polylactic acid (PLA) nanofibrous scaffolds. The scaffolds were produced by the electrospinning technique and characterized in terms of wettability, composition by Fourier transform infrared spectroscopy (FTIR), surface morphology by scanning electron microscopy (SEM), thermogravimetric characterization (TGA), differential scanning calorimetry (DSC) and crystallinity by Xray diffraction (XRD). The biological assays were conducted with osteoblasts of the OFCOL II lineage cultured on the surface of the scaffolds and submitted or not (control group) to irradiation with InGaAIP diode laser, power of 30 mW, with doses of 1, 4 and 6 J/cm² and wavelengths of 660 nm (groups V1, V4, V6, respectively doses) and 780 nm (groups I1, I4 and I6, respectively doses). The effects of LLLT from the perspective of osteoblasts were evaluated using the biochemical method Alamar Blue assay, at intervals of 24, 48 and 72h, while cell viability and morphology were observed at 72h, using the Live/Dead assay and electron microscopy. scan (SEM), respectively. The Alamar Blue assay data showed more significant cell proliferation in groups in the V6 groups at all analytical intervals compared to the control group (p<0.05). Other differences between the control and irradiated groups were found only at intervals of 48h and 72h for V1, and for group IV6 at 72h. The Live/Dead assay revealed an increase in cell viability in the groups treated with LLLT, being significantly higher in the V1 group when compared to the control group. SEM analysis showed adequate interaction between osteoblasts and scaffolds, with the cell body spreading along the nanofiber axis and the presence of more evident physical contacts, through the formation of bonds through filopodia and lamellipodia, in groups V1, V6 and I6. Together, the data from the present study observed that LLLT promotes the biostimulation of osteoblasts cultured on PLA nanofibers, which pointed to its potential use in bone tissue engineering techniques, especially with regard to the use of the wavelength of 660 nm, which presented groups with more significant results (AU).

Role of plasminogen activator inhibitor-1 in oral tongue squamous cell carcinoma: An immunohistochemical and in vitro analysis.

OBJECTIVE: To evaluate the influence of plasminogen activator inhibitor-1 (PAI-1) on the biological behavior and prognosis of oral tongue squamous cell carcinoma (OTSCC). METHODS: Immunoexpression of PAI-1 was analyzed in 60 OTSCC specimens and classified as low-expression (≤50% of positive cells) or high-expression (>50%). In vitro effects of recombinant human PAI-1 (rhPAI-1) were assessed through functional assays on the OTSCC-derived cell line SCC-25. Three cell groups were evaluated: G0 (control), G10 (10 nM rhPAI-1), and G20 (20 nM rhPAI-1). RESULTS: High membrane expression of PAI-1 was associated with tumor budding (p = 0.046) and high-risk cases (p = 0.043). Cytoplasmic and membrane expression of PAI-1 was not associated with patient survival. Cell viability (p = 0.020) and progression to the S-phase of the cell cycle (p = 0.024) were higher in G10 and G20 at 24 h. The percentages of apoptotic/necrotic cells were not affected by rhPAI-1. The presence of rhPAI-1 increased cell migration (p = 0.039) and invasion (p = 0.039) after 24 and 72 h, respectively. CONCLUSION: Our findings indicate the involvement of PAI-1 in the biological behavior of OTSCC, although its expression may not predict patient survival. The in vitro results suggest that PAI-1 stimulates cell proliferation, migration and invasion and may contribute to the aggressive phenotype of OTSCC.

Laser therapy increases the proliferation of preosteoblastic MC3T3-E1 cells cultured on poly(lactic acid) films.

This study aimed to verify the efficacy of low-level laser irradiation (LLLI) on the proliferation of MC3T3-E1 preosteoblasts cultured on poly(lactic acid) (PLA) films. The produced films were characterized by contact angle tests, scanning electron microscopy (SEM), atomic force microscopy, differential scanning calorimetry, and X-ray diffraction. The MC3T3-E1 cells were cultured as three different groups: Control-cultured on polystyrene plastic surfaces; PLA-cultured on PLA films; and PLA + Laser-cultured on PLA films and submitted to laser irradiation (660 nm; 30 mW; 4 J/cm2 ). Cell proliferation was analyzed by Trypan blue and Alamar blue assays at 24, 48, and 72 h after irradiation. Cell viability was assessed by Live/Dead assay, apoptosis-related events were evaluated by Annexin V/propidium iodide (PI) expression, and cell cycle events were analyzed by flow cytometry. Cell morphology on the surface of films was assessed by SEM. Cell counting and biochemical assay results indicate that the PLA + Laser group exhibited higher proliferation (p < 0.01) when compared with the Control and PLA groups. The Live/Dead and Annexin/PI assays indicate increased cell viability in the PLA + Laser group that also presented a higher percentage of cells in the proliferative cell cycle phases (S and G2/M). These findings were also confirmed by the higher cell density observed in the irradiated group through SEM images. The evidence from this study supports the idea that LLLI increases the proliferation of MC3T3-E1 cells on PLA surfaces, suggesting that it can be potentially applied in bone tissue engineering.