Morphological and Molecular Analysis of Osteoblasts Differentiated from Mesenchymal Stem Cells in Polycaprolactone/Magnesium Oxide/Graphene Oxide Scaffold

Abstract

Background: The loss or dysfunction of bone tissue that observed after bone tumor resections and severe nonunion fractures afflicts 200 million people worldwide. Bone tissue engineering is a promising approach to repair osteoporotic fractures.

Objective: In this paper, polycaprolactone (PCL)/magnesium oxide (MgO)/graphene oxide (GO) nanofibrous scaffold was fabricated by electrospining method, and its biocompatibility and osteogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) on this scaffold were evaluated and compared with pure PCL nanofibrous scaffold.

Methods: SEM analysis, DAPI staining and MTT assay were used to evaluation biocompatibility of PCL/MgO/GO composite scaffold. In addition by ALP assay and proteomic approach, osteostimulatory effect of electrospun composite scaffold was investigated and the expression level of osteogenic markers including Runt-related transcription factor cbfa1/runx2 (runx2), collagen type I (Col1a1) and osteopontin (OPN) in MSCs seeded on PCL/MgO/GO composite scaffold was determined and compared with pure PCL scaffold. Then, RT-PCR technique was used to validate the level expression of these genes.

Results: The obtained results showed that adhesion, viability and ALP activity of MSCs on PCL/MgO/GO scaffold considerably enhanced compared with pure PCL. As well as proteomic and real-time analysis illustrated the expression of osteogenic markers including runx2, Col1a1 and OPN increased (>2-fold) in cells seeded on PCL/MgO/GO composite scaffold.

Conclusion: It was concluded that MgO and GO nanoparticles could improve the biocompatibility of PCL scaffold and enhance the osteogenic differentiation of MSCs.

Keywords: Bone tissue engineering; Mesenchymal stem cells; Osteoblast; Proteomics; Scaffold.

Figure 1

Surface morphological and constructional images of nanofibers by SEM: a) PCL, b) PCL/MgO/GO. Original behaviors of MSCs on nanofibrous scaffolds; SEM images after 1 day of culture: c) PCL, d) PCL/MgO/GO. Fluorescence microscope images for DAPI staining of MSCs cultured for 1 day on nanofibrous scaffolds: e) PCL, f) PCL/MgO/GO

Figure 2

Flowcytometry analysis of MSCs labeled with a) CD90, b) CD44, and c) CD73 conjugated to FITC

Figure 3

Viability percentage of MSCs on PCL and PCL/MgO/GO scaffolds on the 1st, 4th, and 7th days (*p<0.05)

Figure 4

ALP activity of MSCs grown on PCL and PCL/MgO/GO (0.5%) nanofibrous scaffolds on the 7th and 14th days (*p<0.05)

Figure 5

2DE gel images of a) MSCs on PCL scaffold, and b) MSCs on PCL/MgO/GO scaffold

Figure 6

Images of protein spots: runx2 a) PCL; b) PCL/MgO/GO, Col1a1; c) PCL; d) PCL/MgO/GO, OPN; e) PCL; and f) PCL/MgO/GO

Figure 7

Targeted proteins have more than 2-fold up-regulation: a) runx2, b) Col1a1, c) OPN in PCL/MgO/GO scaffold compared with pure PCL

Figure 8

Various mRNA expression changes of MSCs in response to PCL and PCL/MgO/GO scaffolds on a) 7^th day and b) 14^th day (*p<0.05)