Analysis of the effect of low level laser phototherapy on stem cells derived from human deciduous dental pulp (SHED-LacZ): In vitro and in vivo study of dentin matrix protein production

Abstract

The endodontic treatment aims to keep any teeth with pulp necrosis or irreversible pulpitis in function. Such pathological conditions are common responses to severe dental trauma or deep caries lesions. When it occurs in young permanent teeth, the loss of vascularization and pulp necrosis leads the cessation of the to dental root formation process. The cellular therapy with the purpose of creating a new pulp tissue in these teeth could allow the maintenance of root formation in order to finalize this process. Studies on tissue engineering to understand the mechanisms of stem cells proliferation and differentiation are very important for a future use on cellular therapy. The dental pulp, a soft connective tissue composed by cells and extracellular matrix (ECM), is responsible for the formation, protection and nutrition of dental structure. Several studies have shown that undifferentiated cells from this tissue can act as stem cells, capable of differentiating in other cell types. There is a huge potential of these dental pulp stem cells (DPSC) for the future treatment of pulpless immature permanent teeth with incomplete root formation. This explains the high interest in isolating and studying these cells. Another therapy that could be joined to cellular therapy is the phototherapy with low intensity lasers (LPT). In fact, LPT studies have been demonstrated its capability of modulating cell growth, viability and differentiation, as well as the protein synthesis and secretion processes of several types of cells, such as fibroblasts, osteoblasts and endothelial cells. It is important to state that the increase of cell viability becomes a major issue for stem cells, mostly when the objective of their culture is the implantation on human organs during cellular therapies. Some in vitro studies already explored the LPT effects on mesenquimal stem cells (derived from bone marrow and fat tissue). However, there is little knowledge about LPT effects on DPSC biology. The comprehension of how to stimulate the proliferation and differentiation of these stem cells, as well as the synthesis and secretion of ECM proteins could allow future use on dental tissues regeneration. Therefore, the objective of this study is to evaluate the LPT influence on growing and production of dentin matrix proteins of a dental pulp stem cells lineage originated from deciduous teeth (SHED-LacZ cell line). More specifically, we intend to study in vitro the proliferation and expression of SHED-LacZ ECM proteins related to dentinogenesis submitted or not to LPT. For this study, it will be used cell viability analysis techniques (MTT reduction), immunofluorescence, Western-blot and flow cytometry. Additionally, we will study, in vivo, the morphologic and ultrastructural characteristics of the formed tissue inside dental slices containing SHED-LacZ cells sieved in biodegradable scaffolds and implanted immunosupressed mice. For this study histological techniques will be used (HE and ²-galactosidase stainings), immunohistochemistry and transmission electron microscopy. Finally, the influence of LPT before and after the implantation will be investigated on the morphological characteristics of this tissue. Numeric data will be statistically compared with a proper test, selected after data normality analysis. Morphologic and ultrastructure data will be presented in a qualitative and/or illustrative manner.