Differentiation of bone marrow mesenchymal stem cells into chondrocytes in transforming growth factor beta-1 culture medium in two-dimensional culture in vitro☆

Peng Wu-xun1, Wang Lei2, Deng Jin1, Li Peng1

1Department of Emergency and Trauma Surgery, the Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, China; 2Guizhou Provincial Disease Prevention and Control Center, Guiyang 550008, Guizhou Province, China

Peng Wu-xun☆ Doctor, Associate chief physician, Department of Emergency and Trauma Surgery, the Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, China
pengwuxun@ sina.com

Correspondence to: Wang Lei, Master, Guizhou Provincial Disease Prevention and Control Center, Guiyang 550008, Guizhou Province, China

Received: 2008-02-15
Accepted:2008-02-17
(08-50-1-798/GW)

Peng WX, Wang L, Deng J, Li P.Differentiation of bone marrow mesenchymal stem cells into chondrocytes in transforming growth factor beta-1 culture medium in two-dimensional culture in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):
5759-5762(China)

[www.zglckf.com/ zglckf/ ejournal/ upfiles/08-29/ 29k-5759(ps).pdf]

Abstract
BACKGROUND: Bone marrow mesenchymal stem cells may directionally differentiate into chondrocytes.
OBJECTIVE: To establish an inducing system for differentiation of bone marrow mesenchymal stem cells into chondrocytes in two-dimensional culture in vivo, to analyze the best concentration of transforming growth factor beta-1 (TGF-β1) to induce the differentiation and the correlated influence factors for the directional differentiation, and to observe the changes of cell form and phenotype.
DESIGN: Randomized controlled animal study.
SETTING: Animal Experimental Center, Guiyang Medical College.
MATERIALS: This study was performed at Animal Experimental Center, Guiyang Medical College from September 2005 to November 2006. Twenty-four 4-week-old male SD rats weighing (98.23±7.97) g were provided by Animal Center of Guiyang Medical College. The animal experiment received informed consent from the local ethic committee.
METHODS: Bone marrow mesenchymal stem cells were separated and purified from femur and tibia using attachment culture method. Surface antigen of the fourth-generated bone marrow mesenchymal stem cells was determined by using flow cytometry. Subsequently, the fourth-generated bone marrow mesenchymal stem cells were induced with TGF-β1 culture medium (1, 5, 10, 15, and 20 μg/L) in two-dimensional culture. On the other hand, bone marrow mesenchymal stem cells in the control group were induced with negative culture medium. Collagen type Ⅱ was qualitatively determined by using immunohistochemical method after two weeks, and glycosaminoglycan expression of extracellular matrix was quantitatively detected by using dimethyl-methylene-blue colorimetric method.
MAIN OUTCOME MEASURES: Surface antigen, collagen type Ⅱ, and glycosaminoglycan expression of extracellular matrix.
RESULTS: Bone marrow mesenchymal stem cells were separated and purified by using attachment culture method. The fourth-generated bone marrow mesenchymal stem cells were positive for surface antigen CD44, but negative for surface antigen CD34 and CD45. Cell form was irregular two weeks after induction. Immunohistochemical staining on collagen type Ⅱ indicated that positive cells were observed in TGF-β1 (1, 5, 10, 15, and 20 μg/L) groups. On the other hand, dimethyl-methylene-blue colorimetric method demonstrated that glycosaminoglycan expression of extracellular matrix in the TGF-β1 groups were significantly higher than in the control group (P < 0.01). In particular, glycosaminoglycan expression in the 10 μg/L TGF-β1 group was significantly higher than in other concentration groups (P < 0.01), and the capability of excreting glycosaminoglycan of extracellular matrix was positively correlated with cell densities (r=0.822, P < 0.01).
CONCLUSION: A high cell density is beneficial to differentiation of bone marrow mesenchymal stem cells into chondrocytes induced by TGF-β1 (10 μg/L) in two-dimensional culture.

INTRODUCTION

Bone marrow mesenchymal stem cells have been paid more and more attention due to the advantages of convenient collection, strong proliferation capability in vitro, differentiation into bone, cartilage, fat, and muscle tissues, and weak immunogenicity[1]. In particular, the differentiation of bone marrow mesenchymal stem cells into chondrocytes is a hot topic in tissue engineering research[2]. This study was designed for separating and purifying bone marrow mesenchymal stem cells in SD rats to establish an inducing system for the differentiation of bone marrow mesenchymal stem cells into chondrocytes in two-dimensional culture in vivo, to analyze the best concentration of transforming growth factor beta-1 (TGF-β1), to induce the differentiation and the correlated influence factors for the directional differentiation, and to observe the changes of cell form and phenotype.

MATERIALS AND METHODS

Materials
This study was performed at Animal Experimental Center, Guiyang Medical College from September 2005 to November 2006. Twenty-four 4-week-old male SD rats weighing (98.23±7.97) g were provided by Animal Center of Guiyang Medical College (certification: gqzz200501001). The animal experiment received informed consent from the local ethic committee. Fetal bovine serum was provided by Sijiqing Company; DMEM-LG and DMEM-HG culture media, 1,9-dimethyl- methylene-
blue, and 4-chondroitin sulfate standard samples were provided by Gibco Company; TGF-β1 was provided by Peprotech Company; collagen type Ⅱ immunohistochemical kit, SABC kit, and DAB coloring kit were provided by Wuhan Boster Company.

Methods
Separation, purification, and evaluation of bone marrow mesenchymal stem cells
Rats were sacrificed by luxation of the neck and maintained in 0.75 volume fraction of ethanol for 5 minutes. And then, bilateral femurs were exposed under sterility condition to shear bone matrix at both ends. Subsequently, bone marrow was put in serum-free DMEM-LG culture media, and centrifuged

With 10 mL at 1 000 r/min for 5 minutes. In addition, cells were re-suspended and deposited in 5-mL complete culture medium containing 0.1 volume serum. The obtained cells were inoculated in culture bottle with the density of 2×106 and cultured in incubator containing 0.05 volume CO2 at 37 ℃. The liquid was changed firstly 24 hours later, subsequently once every 2 days. About 80% cells over the bottom were digested and generated with 0.25% pancreatin. The fourth-generated bone marrow mesenchymal stem cells during log growth period were collected to detect surface antigen CD34, CD44, and CD45 expressions by using flow cytometry.

Differentiation of bone marrow mesenchymal stem cells into chondrocytes
80% confluence in the third generation, well-grown bone marrow mesenchymal stem cells were divided into 6 groups with 4 bottles in each group. One group was randomly drawn as a control group, and other five groups were regarded as experimental group and randomly named as groups A, B, C, D, and E. After digestion, cells in all groups were made into single cell suspension separately. The density was adjusted to 2×108 L-1. And then, the cells were inoculated on 6-well plates with 2 mL in each well. Under 80% confluence, inducing culture media containing 1, 5, 10, 15, and 20 μg/L TGF-β1 were respectively added in groups A, B, C, D, and E. On the other hand, negative culture medium was added in the control group. The liquid was changes firstly 48 hours later, subsequently once every 3 days. Finally, the samples were observed under inverted microscope.

Detection of collagen type Ⅱ using immunohistochemical method
Collagen type Ⅱ was qualitatively determined by using NYI immunohistochemical method (SABC method based on kit introduction) two weeks after induction, and glycosaminoglycan expression of extracellular matrix was quantitatively detected by using dimethyl-methylene-blue (DMB) colorimetric method.

Statistical analysis
Statistical processing was finished by Wang Lei who came from Guizhou Provincial Disease Prevention and Controlling Center. Measurement data were expressed as Mean ± SD. SPSS 11.5 software was used in this study. Data were processed with analysis of variance and correlation analysis, and α=0.05 was regarded as the significant difference.

RESULTS

Separation and culture of bone marrow mesenchymal stem cells
Inverted phase contrast microscope showed cell adherence in round or oval shape and in various sizes that attached at 30 minutes after inoculation. After the first liquid changing, most colony cells grew rapidly and gradually expanded outward. Central cells were like round shape, and peripheral cells were fusiform shape. A lot of cells in division phase were also observed. A few of colony cells gradually shrank into round shape, and the light refractivity strengthened. Finally, the colony cells gradually dropped off and disappeared. The growth of scattered solitary fusiform adhered cells was slower than colony cells, and a lot of cells dropped off and disappeared. However, growth was increased during division phase. Liquid changing for three times, both hemocytes and hematopoietic cells were removed in primary-cultured cells. Cell forms were coincident, i.e., big nucleus, clear nucleolus, and fusiform shape. Light penetration of plasma was uniform. Five to seven days after culture, about 80%-90% cells in the primary generation were confluent. Cells were distributed uniformly after generation (the ratio of 1∶2), and then the cells rapidly grew following a transitory quiescent period and counted for 80%-90% after 4 days. After subculturing, bone marrow mesenchymal stem cells still grew as the same as colony cells, and the cell form was deeply coincident.

Detection of surface markers of bone marrow mesenchymal stem cells
Flow cytometer showed that positive rates of surface markers of bone marrow mesenchymal stem cells were 95.5% of CD44, 6.4% of CD45, and 4.7% of CD34. Primarily, the separated and cultured cells in this study were bone marrow mesenchymal stem cells, which were purified after subculturing.

Detection of differentiation of bone marrow mesenchymal stem cells into chondrocytes
Morphological changes of cells after induction
Three days before induction, cell form did not change apparently. From days 4 and 5, optic microscope showed that cells shrank gradually, stereoscopic impression was powerful, intercellular space gradually enlarged, and cell shapes were irregular.

Detection of collagen type Ⅱ after induction using immunohistochemistry
Brown-yellow granules were distributed in plasma of positive cells, and circumference region was light yellow. Cells were positively stained in 10 μg/L TGF-β1 group, and yellow granules were dense in plasma. On the other hand, cells were weakly positively stained in 5, 15, and 20 μg/L TGF-β1 groups and negatively stained in 1 μg/L TGF-β1 group and the control group.

Detection and statistical analysis of glycosaminoglycan content of extracellular matrix after induction
Random analysis of variance showed that glycosaminoglycan content of extracellular matrix in the experimental groups was significantly higher than in the control group (P < 0.01). In particular, glycosaminoglycan content in the 10 μg/L TGF-β1 group was significantly higher than in other concentration groups (P < 0.01), but there were no significant differences between 5 μg/L and 15 μg/L TGF-β1 groups (P > 0.05). Glycosaminoglycan contents in the 5 μg/L and 15 μg/L TGF-β1 groups were significantly higher than in the 1 μg/L and 20 μg/L TGF-β1 groups (P < 0.01). Glycosaminoglycan content in the 20 μg/L TGF-β1 group was significantly higher than in the 1 μg/L TGF-β1 group (P < 0.01). Analysis of variance for cell density showed that there was no significant difference in the density between experimental groups and control group (P > 0.05, Table 1). Correlation analysis demonstrated that cell density in the 10 μg/L TGF-β1 group was positive correlation with glycosaminoglycan content (r=0.822, P < 0.01), but there were no significant differences in the correlation between cell density and glycosaminoglycan content in other experimental groups (P > 0.05).

DISCUSSION

Recently, only early attachment separation method can be used to obtain bone marrow mesenchymal stem cells in various densities[3]. The acquired cells are characteristics by strong proliferation and powerful multi-differentiation. A lot of bone marrow mesenchymal stem cells can be derived from only a few of bone marrow tissues in a short period. After subculturing, the acquired bone marrow mesenchymal stem cells are coincident in form and pure[4-5]. 10% fetal bovine serum is the best beneficial to culture of bone marrow mesenchymal stem cells[6]. In this study, bone marrow mesenchymal stem cells were separated by using early attachment separation method and purified with DMEM-LG culture medium containing 10% fetal bovine serum. In primary culture, most adhered cells were like fibroblast and epithelioid cells. The latency of primary culture was 2-4 days. During this time, granule-shaped substances with high refraction were mostly observed in plasma. Cell colony formed, but grew slowly. Cells in division phase were not observed at the colony center. The latency was mainly beneficial to adaptively adhered growth of bone marrow mesenchymal stem cells. From the fourth day, phase contrast microscope showed that cell colony grew rapidly and expanded outward, and cells in division phase were increased. This suggested that cells were in log growth phase. Seven or eight days later, cell clone was further enlarged, and more and more cell clones connected each other. About 80% confluence, cells were in growth balance phase due to contact inhibition. After subculturing, cell form was generally coincident, and cell extension was sufficient. Cells in the low density were large and flat, and characteristics of them were similar to fibroblasts. On the other hand, cells in the high density were fusiform and grew like colony. This study demonstrated that although bone marrow mesenchymal stem cells were not abundant in bone marrow, its proliferation was very active and powerful. Therefore, it is feasibility for deficient bone marrow mesenchymal stem cells to proliferate in vivo.
Up to now, specific mark of bone marrow mesenchymal stem cells is still unclear[7]. Generally speaking, integrin family CD29, adhesion molecule CD44, and CD105 are regarded as important marks[8]. Flow cytometry was used in this study to detect surface marker of the fourth-generated bone marrow mesenchymal stem cells. The results indicated that positive rate of CD44 was 95.5%, positive rate of CD45 was 6.4%, and positive rate of CD34 was 4.7%. This was coincident with the reference reports[6,9] and suggested that the separated and cultured cells were bone marrow mesenchymal stem cells but not haemopoietic stem cells or fibroblasts. In addition, purer bone marrow mesenchymal stem cells were cultured in vitro by using attachment culture method.
At present, cell factors promoting differentiation in vitro of bone marrow mesenchymal stem cells into chondrocytes mainly contain transforming growth factor β superfamily, insuliu-like growth factor (IGF), epidermal growth factor (EGF), etc[10]. It is necessary for TGF-β1 to induce the differentiation of bone marrow mesenchymal stem cells into chondrocytes, and the promotion of TGF-β1 is dose-dependent[11-12]. Synthesis and excretion of extracellular matrix are regarded as a main indicator to evaluate function of chondrocytes[13]. Extra matrix excreted from chondrocytes mainly contains collagen (collagen type Ⅱ) and proteoglycan (glycosaminoglycan)[14]. Therefore, glycosaminoglycan content of extracellular matrix is a reliable evidence to determine excretion function of chondrocytes; while, DMB chromatometry is used to quantitatively measure glycosaminoglycan content. Results in this study demonstrated that glycosaminoglycan content in the experimental groups were higher than in the control group, and this suggested that TGF-β1 in different dosages had different inducing effects on bone marrow mesenchymal stem cells. In particular, 10 μg/L was the best concentration of TGF-β1 to induce the differentiation of bone marrow mesenchymal stem cells into chondrocytes. After induction, bone marrow mesenchymal stem cells shaped from fusiform to polygon. Fourteen days after induction, most cells were flat and polygon, and multangular neurites or multangular spindle were observed at the same time. Culture media was glutinous, and this suggested that cell secretion was more active as compared before induction. Collagen type Ⅱ immunohistochemistry showed that partial positive cells were observed in 5, 10, 15, and 20 μg/L TGF-β1 groups at two weeks after induction, and brown-yellow granules were distributed in plasma. In particular, collagen type Ⅱ expression was the most in the 10 μg/L TGF-β1 group, but cells in the 1 μg/L TGF-β1 group and the control group were negative. Comparisons among groups indicated that glycosaminoglycan content in the 10 μg/L TGF-β1 group was higher than in other experimental groups, and this suggested that inducing system designed in this study could induce the differentiation of bone marrow mesenchymal stem cells into chondrocytes. Collagen type Ⅱ expression in the high concentration group was inhibited, and synthesis and excretion of glycosaminoglycan were decreased, and this suggested that TGF-β1 in high concentration could decrease the differentiation and was not beneficial to chondrification. The above-mentioned conclusion was similar to researching results of Worster et al[15]. In this study, 1 μg/L TGF-β1 could not induce chondrification, and this might be related that low concentration of TGF-β1 could not cause endogenous bone morphogenetic protein expression[16-17]. Correlation analysis in the 10 μg/L TGF-β1 group demonstrated that cell density was positively correlated to glycosaminoglycan content (r=0.822, P < 0.01), and this suggested that a high cell density was beneficial to differentiation of bone marrow mesenchymal stem cells into chondrocytes induced by TGF-β1 (10 μg/L) in two- dimensional culture. This might be related to high-density culture which could enhance communication between two cells to promote differentiation of bone marrow mesenchymal stem cells into chondrocytes[18-20].
The fourth-generated cells derived from 5 samples were induced in this study. Specific expression of collagen type Ⅱ was found in all groups, and this suggested that bone marrow mesenchymal stem cells had a potency to differentiate into chondrocytes under our culture condition. However, whether the induced chondrocytes might form a normal cartilage tissue after transplantation, whether chondrocytes might differentiate into hypertrophic chondrocyte, and whether chondrocytes might attack phosphoric acid mineral matter deposition and internal calcification need further studies.

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