Int J Stem Cells.  2015 Nov;8(2):219-227. 10.15283/ijsc.2015.8.2.219.

Fibrin Scaffolds Designing in order to Human Adipose-derived Mesenchymal Stem Cells Differentiation to Chondrocytes in the Presence of TGF-beta3

• Sheykhhasan M
• Qomi RT
• Ghiasi M

• KMID: 2380816
• DOI: http://doi.org/10.15283/ijsc.2015.8.2.219

Abstract

BACKGROUND AND OBJECTIVES
One of the most cellular source used for cartilage tissue engineering are mesenchymal stem cells (MSCs). In present study, human MSCs were used as cellular source. Since scaffold plays an important role in tissue engineering the aim of this study is to assess fibrin scaffold ability in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs).
METHODS
ADMSCs were isolated and cultured in DMEM medium supplemented with 10% FBS. Also ADMSCs expanded and characterised by flow cytometry. ADMSCs expressed CD44, CD90, CD105 but not CD34. After trypsinization, cells were entered within the fibrin scaffold. Then, chondrogenic medium was added to the scaffold. Seven days after cell culture, cell viability and proliferation were assessed by MTT test. Finally, 14 days after the ending of chondrogenic differentiation, analysis of chondrogenic genes expression was evaluated by RT-PCR and Real time PCR. Also, formation and development of chondrocyte cells was analysed by histological and immunohistochemistry evaluations.
RESULTS
Viability and proliferation as well as chondrogenic genes expression within fibrin scaffold increased significantly compared with control group (cells free scaffold). Also, histological and immunohistochemistry evaluation showed that chondrocyte cells and collagen type II are formed on fibrin scaffold.
CONCLUSIONS
Fibrin is a suitable scaffold for chondrogenic differentiation of ADMSCs.

Keyword

Tissue Engineering; Adipose-Derived Mesenchymal Stem Cells (ADMSCs); Chondrogenic Differentiation; Fibrin

MeSH Terms

Cartilage
Cell Culture Techniques
Cell Survival
Chondrocytes*
Collagen Type II
Fibrin*
Flow Cytometry
Humans*
Immunohistochemistry
Mesenchymal Stromal Cells*
Real-Time Polymerase Chain Reaction
Tissue Engineering
Transforming Growth Factor beta3*
Trypsin
Collagen Type II
Fibrin
Transforming Growth Factor beta3
Trypsin

Figure

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  Fig. 1 Flow cytometeric analysis for the determination of the surface markers of MSCs expression. (A) CD44, (B) CD90, (C) CD105 and (D) CD34.

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  Fig. 2 The comparison of cell viability between MSCs embedded in fibrin scaffold and control group.

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  Fig. 3 Image produced by invert microscope of living mesenchymal stem cells isolated from human adipose tissue which spindle cells in the third passage are visible. ×40.

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  Fig. 4 The analysis of collagen Type II, Aggrecan and SOX9 genes expression in cell/fibrin using RT-PCR method.

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  Fig. 5 The expression of collagen I, Aggrecan, SOX9 and Collagen II in cell/fibrin vs. MSCs.

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  Fig. 6 Histological examination of the ADMSCs using haematoxylin and eosin staining. After 4 weeks there was significant cartilage formation with high cell density in ADMSCs seeded on the Fibrin glue scaffold (original magnification ×10) (A). Staining of native cartilage used as control with hematoxylin/eosin is demonstrated in (B) (×50 magnification).

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  Fig. 7 The analysis of collagen type II antibody (arrowheads) in cell/fibrin (A) and control group (B) using immunohistochemistry evaluation.

Reference

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