Can TGF-β1 and rhBMP-2 act in synergy to transform bone marrow stem cells to discogenic-type cells? |
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Authors: | Sung Uk Kuh Yerun Zhu Jun Li Kai-Jow Tsai Qinming Fei William C Hutton and S Tim Yoon |
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Institution: | (1) Department of Neurosurgery, The Spine and Spinal cord Institute, Yonsei University Medical College, Seoul, Korea;(2) Department of Orthopaedics, Veterans Affairs Medical Center, Emory University School of Medicine, Atlanta, GA, USA;(3) Emory Orthopaedics and Spine Center, 59 Executive Park South, Atlanta, GA 30329, USA |
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Abstract: | Introduction The recombinant human bone morphogenic protein-2 (rhBMP-2) is known to increase the proteoglycan production and chondrogenic
gene expression in the disc cells. The transforming growth factor-beta 1 (TGF-β1) can transform the bone marrow stem cells (BMDCs) into the disc-like cells.
Materials and methods We carried out an experiment to determine if TGF-β1 and rhBMP-2 can act in synergy on BMDCs by increasing the production of sulfated-glycosaminoglycan (sGAG) and affecting the
mRNA expression of aggrecan, type I collagen, and type II collagen. The BMDCs were isolated from the iliac crest and femur
of a New Zealand white rabbit (1 year). The BMDCs were culured in monolayer and treated for 6 days with TGF-β1 10 ng/ml (group 1), rhBMP-2 200 ng/ml (group 2), and both TGF-β1 10 ng/ml and rhBMP-2 200 ng/ml (group 3: the combined group) in Dulbecco’s modified Eagle medium/F-12 with 1% fetal bovine
serum. After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining and the mRNA expression
of aggrecan, type I collagen, type II collagen, Sox-9, BMP-2, and BMP-7 were measured with the real-time PCR. The same BMDCs
were also cultured in the chamber slide at 3 × 104 cells/chamber. After 6 days treatment, the treated cells were immunofluorescence stained with aggrecan, type I collagen,
type II collagen, anti-BMP-2, anti-BMP-7 antibodies. After that, we compared the number of positive immunofluorescence stained
cells with fluorescence microscope. The sGAG production and mRNA expression for each group were normalized against the same
parameters for a non-treatment group.
Results and discussion The sGAG production was increased 1.15*, 1.34*, and 1.45* times in the TGF-β1 10 ng/ml group, the rhBMP-2 200 ng/ml group, and the combined group respectively. The mRNA expression of aggrecan was increased
1.28, 3.42*, and 5.34* times, the mRNA expression of type I collagen was increased 0.86, 1.09, 1.17 times, the mRNA expression
of type II collagen was increased 3.58*, 3.77*, and 10.78* times, the mRNA expression of Sox-9 was increased 1.29, 2.45, 2.75*
times, the mRNA expression of BMP-2 was increased 1.14, 2.07, 4.43* times, and the mRNA expression of BMP-7 was increased
1.16, 1.49, 1.97* times, respectively for each group (* indicates p < 0.05). On the immunofluorescence staining of antibodies, the average positively immunofluorescence stained cells number
for aggrecan were 4.2, 15.8*, 10*, and 22* according to the non-treatment group, TGF-β1 10 ng/ml group, rhBMP-2 200 ng/ml group, and the combined group respectively. The average positively immunofluorescence stained
cells number for type I collagen were 7, 14.2*, 9.2*, 17.4* and the average positively immunofluorescence stained cells number
for type II collagen were 8.5, 28.25*, 20.25*, 42.25* and the average positively immunofluorescence stained cells number for
anti-BMP-2 were 5, 16.75*, 8.75*, 27.25* and the average positively immunofluorescence stained cells number for anti-BMP-7
were 3.25, 7.5*, 8.75*, 15.25* (* indicates p < 0.05).
Conclusions Both TGF-β1 and rhBMP-2 alone, can increase proteoglycan production in the BMDCs. However, if they were used in combination, there is
a synergistic effect. Similarly, the mRNA expressions of both aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7 except for
type I collagen were increased significantly when TGF-β1 and rhBMP-2 were combined. The positive immunofluorescence stained cell numbers for aggrecan, type I, II collagen, BMP-2
and BMP-7 were also increased after each TGF-β1 and rhBMP-2 treatment, and also more increased significantly in the aggrecan, type I, II collagen, BMP-2, and 7 when they
were used jointly. |
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Keywords: | Proteoglycan Bone marrow derived cells TGF-β 1 rhBMP-2 |
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