Chondrogenic Differentiation of Human Umbilical Cord Blood-Derived Unrestricted Somatic Stem Cells on A 3D Beta-Tricalcium Phosphate-Alginate-Gelatin Scaffold

Abstract

Objective: Finding cell sources for cartilage tissue engineering is a critical procedure. The purpose of the present experimental study was to test the in vitro efficacy of the beta-tricalcium phosphate-alginate-gelatin (BTAG) scaffold to induce chondrogenic differentiation of human umbilical cord blood-derived unrestricted somatic stem cells (USSCs).

Materials and methods: In this experimental study, USSCs were encapsulated in BTAG scaffold and cultured for 3 weeks in chondrogenic medium as chondrogenic group and in Dulbecco's Modified Eagle's Medium (DMEM) as control group. Chondrogenic differentiation was evaluated by histology, immunofluorescence and RNA analyses for the expression of cartilage extracellular matrix components. The obtain data were analyzed using SPSS version 15.

Results: Histological and immunohistochemical staining revealed that collagen II was markedly expressed in the extracellular matrix of the seeded cells on scaffold in presence of chondrogenic media after 21 days. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significant increase in expression levels of genes encoded the cartilage-specific markers, aggrecan, type I and II collagen, and bone morphogenetic protein (BMP)-6 in chondrogenic group.

Conclusion: This study demonstrates that BTAG can be considered as a suitable scaffold for encapsulation and chondrogenesis of USSCs.

Keywords: Chondrogenesis; Mesenchymal Stem Cells; Scaffold.

Fig 1

Characterization of the different surface markers. A. CD29, B. CD166, C. FLK-1 and D. CD49. Red and white histograms showed control and cell surface markers, respectively. The bars on the peak levels of histograms are a measure of positive expression of surface markers.

Fig 2

Scanning electron microscopy images of both unseeded and seeded scaffolds. A. Unseeded scaffold, B. Control group and C. Chondrogenic group.

Fig 3

MTT results showing the metabolic activities of seeded cells on scaffold in chondrogenic medium as chondrogenic group and in DMEM as control group. P>0.05.

Fig 4

Alcian blue staining of USSCs cultured on control and BTAG scaffold.

Fig 5

Immunofluorescence staining of collagen II (Red) with nuclear counterstain (Blue) of USSCs on BTAG scaffold. A. control group, only nucleous was stained . B. chondrogenic group, extra cellular matrix and nucleous were stained.

Fig 6

The expression of genes encoded the cartilage-specific markers in control (A) and chondrogenic (B) groups. 1; B2M, 2; aggrecan, 3; Collegen I, 4; Collagen II, 5; BMP-6.