Department of Morphology, Federal University of Rio Grande do Norte, Natal, Brazil.
Institute of Macromolecules, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
J Tissue Eng Regen Med. 2020 Dec;14(12):1792-1803. doi: 10.1002/term.3134. Epub 2020 Oct 7.
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/cm ). 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.
本研究旨在验证低水平激光照射(LLLI)对在聚乳酸(PLA)薄膜上培养的 MC3T3-E1 前成骨细胞增殖的疗效。通过接触角试验、扫描电子显微镜(SEM)、原子力显微镜、差示扫描量热法和 X 射线衍射对所制备的薄膜进行了表征。将 MC3T3-E1 细胞培养为三个不同的组:对照-在聚苯乙烯塑料表面培养;PLA-在 PLA 薄膜上培养;PLA+激光-在 PLA 薄膜上培养并进行激光照射(660nm;30mW;4J/cm)。在照射后 24、48 和 72 小时通过台盼蓝和阿马尔蓝测定法分析细胞增殖。通过 Live/Dead 测定法评估细胞活力,通过 Annexin V/碘化丙啶(PI)表达评估细胞凋亡相关事件,并通过流式细胞术分析细胞周期事件。通过 SEM 评估细胞在薄膜表面的形态。细胞计数和生化测定结果表明,与对照和 PLA 组相比,PLA+激光组的增殖率更高(p<0.01)。Live/Dead 和 Annexin/PI 测定表明,PLA+激光组的细胞活力增加,处于增殖细胞周期(S 和 G2/M)阶段的细胞比例也更高。通过 SEM 图像观察到照射组中更高的细胞密度也证实了这一发现。本研究的证据支持 LLLI 增加 MC3T3-E1 细胞在 PLA 表面增殖的观点,提示其可能在骨组织工程中得到应用。