P R Garrido1, A C F Pedroni2, D P Cury3, M S Moreira4, F Rosin5, G Sarra6, M M Marques7. 1. Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil; School of Dentistry, Central University of Ecuador, Quito, Ecuador. Electronic address: pgarrido@uce.edu.ec. 2. Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil. Electronic address: anapedroni@usp.br. 3. Biomedical Sciences Institute III, University of Sao Paulo, SP, Brazil. Electronic address: diegocury@usp.br. 4. Post Graduation Program, School of Dentistry, Ibirapuera University, Sao Paulo, Brazil. Electronic address: stellam@usp.br. 5. Experimental Pathology Laboratory, School of Dentistry, University of Sao Paulo, Brazil. Electronic address: flarosin@usp.br. 6. Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil. Electronic address: gisarra@usp.br. 7. Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo, Brazil. Electronic address: mmmarques@usp.br.
Abstract
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). METHODS: 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, 0.028cm2, 0.71 W/cm2) in punctual and contact mode. The CSs were allocated in 3 experimental groups: Control: no further treatment; PBMT1 [4 s, 3 J/cm2 (lower energy density), 0.08 J/point] and PBMT2 [7 s, 5 J/cm2 (higher energy density), 0.14 J/point]. Statistical comparisons were performed (p ≤ .05). RESULTS: 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. CONCLUSION: 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.
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). METHODS: 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, 0.028cm2, 0.71 W/cm2) in punctual and contact mode. The CSs were allocated in 3 experimental groups: Control: no further treatment; PBMT1 [4 s, 3 J/cm2 (lower energy density), 0.08 J/point] and PBMT2 [7 s, 5 J/cm2 (higher energy density), 0.14 J/point]. Statistical comparisons were performed (p ≤ .05). RESULTS: 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. CONCLUSION: 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.
Authors: Polina Y Bikmulina; Nastasia V Kosheleva; Anastasia I Shpichka; Yuri M Efremov; Vladimir I Yusupov; Peter S Timashev; Yury A Rochev Journal: J Biomed Opt Date: 2020-04 Impact factor: 3.170
Authors: Harim T Dos Santos; Kyungsook Kim; Teruo Okano; Jean M Camden; Gary A Weisman; Olga J Baker; Kihoon Nam Journal: Cells Date: 2020-12-09 Impact factor: 6.600