Fibronectin within Sodium Alginate Microcapsules Improved Osteogenic Differentiation of BMMSCs in Dose Dependent Manner by Targeting SP7, OCN, CDK1, ZBTB16, and Twist1 Expression.

Karim Shamsasenjan,Nesrin Gareayaghi,Mahsa Mahmoodi,Younes Beygi Khosrowshahi,Babak Nejati,Parvin Akbarzadehlaleh

doi:10.34172/apb.2022.012

Abstract

Purpose: Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs). However, there is limited information about the effects of fibronectin various concentrations on bone marrow-derived MSCs (BMMSCs) function and differentiation. Methods: In this experimental study, using a gel injection device, BMMSCs were encapsulated in sodium alginate microcapsules containing 1.25% alginate, 1% gelatin, and fibronectin (0.01, 0.05, 0.1, and 0.2 μg/ml). MTT assay was used to examine the proliferation of BMMSCs. Also, BMMSCs apoptosis were analyzed using Annexin-V/PI staining and fluorescence activated cell sorting (FACS). Alkaline phosphatase (ALP) test was conducted to assess BMMSCs osteogenic differentiation potential. Finally, mRNA expression levels of the SP7, osteocalcin (OCN), Twist Family BHLH Transcription Factor 1 (Twist1), Peroxisome proliferator‐activated receptor γ2 (PPARγ2), Cyclin-dependent kinase 1 (CDK1), and Zinc Finger and BTB Domain Containing 16 (ZBTB16), following exposure with fibronectin 0.1 μg/ml. Results: According to results, fibronectin had the potential to promote proliferation rates of the BMMSCs, in particular at 0.1 and 0.2 μg/ml concentrations. we showed that the fibronectin was not able to modify apoptosis rates of the BMMSCs. ALP test results approved the notable potential of the fibronectin, to trigger osteogenic differentiation of the BMMSCs. Also, RT-PCR results indicated that fibronectin 0.1 μg/ml could augment osteogenic differentiation of cultured BMMSCs through targeting of OCN, SP7, Twist1, CDK1, and ZBTB16, strongly or slightly. Conclusion: Results showed that fibronectin can improve proliferation and osteogenic differentiation of BMMSCs without any effect on these cells' survival.

Highlights

Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs)
3.1 Confirming of the bone marrowderived MSCs (BMMSCs) osteoblastic differentiation by Alizarin‐red staining c The osteoblastic differentiation of the BMMSCs was approved by absorption of the Alizarin‐red stain in ECM of the BMMSCs because of the occurring of the calcium mineralization on [10, 15] and 21days s of cell cultures (Figure 1)
3.4 Fibronectin had not any effect on BMMSCs apoptosis te There was no significant shift in apoptosis rates of BMMSCs-exposed with fibronectin 0.01, 0.05, 0.1 and 0.2 μg/ml in comparison to control cells at 48 hours of exposure measured by Annexin-V/propidium iodide (PI) staining and FACS analysis (P

Summary

Introduction

Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs). P there is limited information about the effects of fibronectin various concentrations on bone marrowderived MSCs (BMMSCs) function and differentiation. According to the wide range of studies, the cells should make contact with other cells and extracellular c matrix (ECM) to become biologically functional 1. The ECM is a three-dimensional (3D) network that plays key roles in supporting the physical and biological structure and function of the living cells s 2. As cells in the body perform their function within a complex 3D environment that cannot be formed by two-dimensional (2D) medium and animal models, 3D culture media have been used widely in u medical science and tissue engineering.

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Conclusion