Effects of interactions between mesenchymal stem cells from healthy rats and rats with osteoporosis, diabetes mellitus, or arterial hypertension on osteoblast differentiation and regeneration of bone defects

Author(s)

Alann Thaffarell Portilho de Souza

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-09-30

Examining board members

Adalberto Luiz Rosa; Pedro de Sousa Gomes; Ana Clara Fagundes Pedroni; João de Jesus Viana Pinheiro

• Advisor: Adalberto Luiz Rosa; Márcio Mateus Beloti

Abstract

Bone tissue, when injured, has a great capacity for regeneration, which can be compromised by some systemic diseases such as osteoporosis, diabetes mellitus (DM), and arterial hypertension (AH). A promising alternative to treat the damage caused to the repair of bone tissue by the presence of these diseases is based on cell therapy with mesenchymal stem cells (MSC). However, several aspects need to be investigated to evaluate the effectiveness of this therapy for the regeneration of bone defects in these diseases. In this context, the aims of this study were: 1) to evaluate in vitro the effect of osteoporosis, DM, or HA on bone tissue and osteoblast differentiation of MSC; 2) to evaluate in vitro the influence of MSC from healthy rats (SD-MSC) on osteoblast differentiation of MSC from rats with osteoporosis (ORX-MSC), DM (DM-MSC) in normoglycemic or hyperglycemic medium, or HA (HA-MSC), and 3) to evaluate in vivo the effect of local injection of SD-MSC to regenerate bone defects in calvaria of rats with osteoporosis, DM or HA. For this, MSC were isolated from bone marrow to evaluate cell proliferation, gene expression of osteoblast markers runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), osteopontin (Opn) and β-catenin (Ctnnb1), gene expression of markers of BMP, WNT and ITG pathways, ALP and OPN protein expression, ALP activity and mineralized extracellular matrix formation. Then, defects were created in the calvaria of rats with osteoporosis, DM, or HA, and after 2 weeks SD-MSC or vehicle (PBS-Control) were injected. Cell permanence in bone defects was assessed by bioluminescence using cells expressing luciferase. Four weeks post-cell injection, bone tissue formation was evaluated by microcomputed tomography, histological analysis, and gene expression of osteoblast markers. Data were submitted to the t test or analysis of variance, and the significance level adopted was 5% (p≤0.05). The three diseases impaired bone tissue and osteoblast differentiation of MSC. SD-MSC promoted partial recovery of osteoblast differentiation of ORX-MSC, HA-MSC, and DM-MSC while ORX-MSC did not impair SD-MSC differentiation, DM-MSC partially impaired and HA-MSC impaired even more this differentiation. In these interactions, there was modulation of genes of bone morphogenetic proteins (BMP), Wingless/Integrated (WNT), and integrins (ITG) signaling pathways at different levels. The SD-MSC remained in the defects of all diseases and the cell therapy resulted in higher bone formation compared to the injection of PBS in the defects of animals with osteoporosis and HA, but not with DM. The results of this study are relevant for developing cell therapy aimed at regenerating bone tissue in adverse conditions such as osteoporosis, DM, or HA. (AU)

• FAPESP's process: 18/13290-0 - Cell therapy: evaluation of the effect of mesenchymal stem cells to regenerate bone tissue of rats with Osteoporosis, Diabetes Mellitus or Arterial Hypertension
• Grantee: Alann Thaffarell Portilho de Souza
• Support Opportunities: Scholarships in Brazil - Doctorate