Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chang Qing,Fan Wei-Min,Cui Wei-Ding

doi:10.1590/s0100-879x2011000400006

Chang Qing, Fan Wei-Min + Show 1 more

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https://doi.org/10.1590/s0100-879x2011000400006

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Abstract

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Highlights

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering [1,2,3,4]
Seven experimental groups were established as follows: in group 1, chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/mL penicillin G (Gibco), and 100 μg/mL streptomycin (Gibco); in groups 2-6, chondrocytes and BMSCs were co-cultured at ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 in DMEM/F12 (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin G (Gibco) and 100 μg/mL streptomycin (Gibco); in group 7, BMSCs were cultured in chondrogenic induction medium (DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin G, 100 μg/mL streptomycin, 1% ITS+ (Sigma), 10 ng/mL TGF-β1 (Invitrogen, USA), 0.1 μM dexamethasone (Sigma), and 50 μg/mL ascorbate 2-phosphate (Sigma)
After 21 days of culture, the BMSCs exposed to osteogenic medium were strongly positive to alkaline phosphatase (ALP) staining (Figure 1B)

Summary

Introduction

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering [1,2,3,4]. A satisfactory curative effect has been achieved in the clinical application of chondrocytes to the construction of tissue-engineered cartilage and the repair of defects of the articular cartilage [5,6,7]. BMSCs have been suggested as a substitute for chondrocytes in cartilage tissue engineering because of their significant proliferative and regenerative capacity [10,11]. This strategy is problematic because of the low induction efficiency of single BMSC cultures, the potential risk for tumor formation by these cells [12], and the need for growth factors and/or gene delivery systems to signal directed cell growth [13]. Novel approaches are needed before BMSCs can be used therapeutically

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