Effects of photobiomodulation on adhesion and proliferation of stem cells from human apical papilla in chitosan scaffold with blood clot incorporation. In vitro study

Author(s)

Gabriela Laranjeira Abe

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2016-09-06

Examining board members

Marcia Martins Marques; Celso Luiz Caldeira; Flavia Gonçalves; Juliana Marchi

• Advisor: Marcia Martins Marques

Abstract

Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation. (AU)

• FAPESP's process: 14/25281-5 - Laserphototherapy effects on adhesion and proliferation of human apical papilla stem cells in chitosan sccafold incorporated with blood clot: in vitro study
• Grantee: Gabriela Laranjeira Abe
• Support Opportunities: Scholarships in Brazil - Master