Continuous characteristics of chondrogenic differentiation of mesenchymal stem cells from human bone marrow in certain culture media

Hang YIN; Ya ZHANG; Xiaodong WANG; Xiqing ZHANG

ABSTRACT

BACKGROUND:

The mesenchymal stem calls (MSCs) have multiple-direction differentiation potential.How to induce human mesenchymal stem cells (hMSCs) into chondrogenic in vitro is an advanced researching direction.

OBJECTIVE:

Experimental study on chondrogenic differentiation of mesenchymal stem cells from human bone marrow in study culture medium so as to observe the characteristic of the cells in the continuous progressive process,such as culture of the primary generation and passage culture.

DESIGN:

A single sample and open study.

SETTING:

Department of Orthopaedics, Children's Hospital Affiliated to Soochow University.MATERIALSA total of 5 children with single bone cyst were selected from Children's Hospital Affiliated to Soochow University from August 2002 to September 2003,including 3 boys and 2 girls aged from 3 to 12 years.All of them donated 5 Ml bone marrow, which was added with 1 Ml (20 U/Ml) heparinization, from which MSCs was obtained. Main reagents were detailed as follows F-12 (Gibco, USA), fetal calf serum (Sijiqing Bioengineering Company, Hangzhou), trypsin (Sigma, USA), transforming growth factor-β1 (TGF-β1) (Sigma, USA) and vitamin C (the Third Medical Company,Nanjing; batch number 021017).

METHODS:

① Standard culture medium0.1 volume fraction of fetal calf serum,100 U/Ml penicillin,100 U/Ml streptomycin, 5.8 g/L Hepes, 2 g/L NaHCO3 and 0.3 g/L glutamine were added into F-12 culture medium (Ph 7.2-7.4). ②Study culture medium 50 μg/L vitamin C and 1 μg/L TGF-β1 were added into standard culture medium (Ph 7.2-7.4). ③Cell culture of the primary generation 12 Ml Hanks which contained 1 Ml of heparinization (20 μ/Ml) and 5 Ml bone marrow was centrifugated. Then the cells which had nucleolus were rinsed. Then cells were cultured in a 50 Ml plastic culture bottle at the density of 3×109 L-1 and were cultured in 6-well culture plate at the density of 1×109 L-1. One bottle and 3 well was used study culture medium,the other bottle and 3 well was used standard culture medium.The cells were cultured in incubator containing 0.05 volume fraction of CO2 and saturated tumidity at 37℃. The medium was changed every 48 hours. The cells were observed under inverted microscope. ④ Passage culture When the primary generation cells had been cultured with standard culture medium for 10-14 days, we would find that the cells covered 80％ of the bottle bottom. Then 2.5 g/L of trypsin containing 0.2 g/L of EDTA was used to digestion and passage. The third generation cells were separated at the ratio of 11, and were cultured in two bottles. One bottle used standard culture medium, the other used study culture medium. At the same time, the cells in 6-well plate which contains coverslip were deal with in the same way. 3-wells used standard culture medium, and the others used study culture medium. Culture time was7 days.The cells were observed under inverted microscope.⑤ Index detection Alcian blue staining was used to show the expression of the glycosaminoglycan of the primary generation and the third generation. Type- Ⅱ collagen immunohistochemical staining was used to show the express of the type- Ⅱ collagen of the primary generation and thethird generation.MAIN OUTCOME MEASUREMENTS① Morphological observation; ② secretion of glycosaminoglycan; ③ expression of type- Ⅱ collagen.

RESULTS:

① Observation under inverted microscope Most of the hMSCs in primary culture were spindle; a few of them were wide, flat and polygon. After passage, the cells were found in a uniform spindle shape. The cells, which were induced in TGF-β1 and vitamin C, became round or oval shape. ② The positive rate of primary generation was 82.4％, the 3rd generation which were cultured in study culture medium was 76.3％ (x2 =1.14,P＞ 0.05), and the 6th generation was 68.5％(x2 =5.22, P ＜ 0.05). The cells, which were culturedinstandardculturemedium, were negative. ③ The 3rd generation and 6th generation cells which were cultured in study culture medium, were found that brown-yellow granules distributed in cytoplast. It was the positive reaction. The cells, which were cultured in standard culture medium, were negative.

CONCLUSION:

MSCs derived from human bone marrow may be inducedinto hondrocytes under study culture medium which contained TGF-β1 and vitamin C in vitro.The express of the glycosaminoglycan and type Ⅱ collagen show the characteristic of chondrocyte.