Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil; School of Dentistry, Central University of Ecuador, Quito, Ecuador.
Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil.
J Photochem Photobiol B. 2019 May;194:149-157. doi: 10.1016/j.jphotobiol.2019.03.017. Epub 2019 Apr 1.
Photobiomodulation therapy (PBMT) and the cell sheet (CS) technology improve processes relevant to tissue regeneration. The aim of this study was to investigate the effects of different PBMT parameters on the architecture (histology), protein composition (Western blotting and immunohistochemistry) and ultrastructure [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] of the extracellular matrix (ECM) synthesized by CSs composed by human dental pulp stem cells (hDPSCs).
Thawed cells were recharacterized by the expression profile of the surface molecules of mesenchymal stem cells (MSCs) using flow cytometry. Clonogenic medium supplemented with vitamin C (20 μg/ml) was used for obtaining the CSs. PBMT was performed with continuous-wave diode laser (660 nm, 20 mW, 0.028cm, 0.71 W/cm) in punctual and contact mode. The CSs were allocated in 3 experimental groups: Control: no further treatment; PBMT1 [4 s, 3 J/cm (lower energy density), 0.08 J/point] and PBMT2 [7 s, 5 J/cm (higher energy density), 0.14 J/point]. Statistical comparisons were performed (p ≤ .05).
The cells presented the classical immunoprofile of MSCs. Type I and type III collagens and fibronectin were present in the ECM of the CSs. PBMT1 induced higher amount of fibronectin. The overall ultrastructure of the CSs in the PBMT1 was epithelial-like, whereas the PBMT2 leads to CSs with fusiform cells arranged in bundles. TEM identified a more mature ECM and signs of apoptosis and necrosis in the PBMT2 group.
PBMT influence the composition and ultrastructure of the ECM of CSs of hDPSCs. Thus, PBMT, specifically when applied in the lower energy density, could be of importance in the determination of the mechanical quality of CSs, which may favor cell therapy by improving the CS transplantation approach.
研究不同光生物调节治疗（PBMT）参数对人牙髓干细胞（hDPSC）细胞片（CS）合成细胞外基质（ECM）的结构（组织学）、蛋白质组成（Western blot 和免疫组织化学）和超微结构[扫描电子显微镜（SEM）和透射电子显微镜（TEM）]的影响。
使用流式细胞术检测间充质干细胞（MSC）表面分子的表达谱对解冻细胞进行重新鉴定。使用含有维生素 C（20μg/ml）的克隆形成培养基获得 CS。采用连续波二极管激光（660nm，20mW，0.028cm，0.71W/cm）进行 PBMT，采用点接触式和接触式。将 CS 分为 3 个实验组：对照组：无进一步治疗；PBMT1[4s，3J/cm（低能量密度），0.08J/点]和 PBMT2[7s，5J/cm（高能量密度），0.14J/点]。进行了统计学比较（p≤.05）。
细胞呈现出 MSC 的典型免疫表型。I 型和 III 型胶原和纤维连接蛋白存在于 CS 的 ECM 中。PBMT1 诱导了更多的纤维连接蛋白。PBMT1 组 CS 的整体超微结构呈上皮样，而 PBMT2 组导致 CS 中梭形细胞呈束状排列。TEM 鉴定出 PBMT2 组 ECM 更成熟，以及细胞凋亡和坏死的迹象。
PBMT 影响 hDPSC CS 的 ECM 组成和超微结构。因此，PBMT，特别是在低能量密度下应用时，可能对 CS 机械质量的确定具有重要意义，这可能通过改善 CS 移植方法来促进细胞治疗。
