Odonto/osteogenic differentiation of photomodulated mesenchymal stem cells in BMP4-loaded hydrogel

Author(s)

Ivana Márcia Alves Diniz

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia (FO/SDO)
• Defense date: 2015-08-06

Examining board members

Marcia Martins Marques; Carlos José Soares; Mari Cleide Sogayar

• Advisor: Marcia Martins Marques

Abstract

This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied. (AU)

• FAPESP's process: 12/16552-0 - Effects of low level laser therapy associated to a BMP4 incorporated scaffold on dental pulp stem-cells differentiation and migration. An in vitro study.
• Grantee: Ivana Marcia Alves Diniz
• Support Opportunities: Scholarships in Brazil - Doctorate