EFFECT OF BASIC FIBROBLAST GROWTH FACTOR AND PARATHYROID HORMONE-RELATED PROTEIN ON EARLY AND LATE CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β1_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Yin , HE Liping , TIAN Jing.

Department of Orthopedics, Zhujiang Hospital of Southern Medical University, Guangzhou Guangdong, 510282, P.R.China. Corresponding author: TIAN Jing, E-mail: tian_jing672@yahoo.com.cn;

Corresponding author：

Keywords：

Cartilage tissue engineering; Bone marrow mesenchymal stem cells; Basic fibroblast growth factor; Parathyroid hormone-related protein; Chondrogenic differentiation; Rabbit

DOI：

10.7507/1002-1892.20130044

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Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.

Citation： LIU Yin,HE Liping,TIAN Jing.. EFFECT OF BASIC FIBROBLAST GROWTH FACTOR AND PARATHYROID HORMONE-RELATED PROTEIN ON EARLY AND LATE CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β1. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(2): 199-206. doi: 10.7507/1002-1892.20130044 Copy

1.	Zheng YH, Su K, Jian YT, et al. Basic fibroblast growth factor enhances osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells in coral scaffold constructs. J Tissue Eng Regen Med, 2011, 5(7): 540-550.
2.	Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8): 991-1002.
3.	Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11): 3401-3415.
4.	Park KH, Na K. Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels. J Biosci Bioeng, 2008, 106(1): 74-79.
5.	Yang KG, Saris DB, Geuze RE, et al. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng, 2006, 12(9): 2435-2447.
6.	Bosnakovski D, Mizuno M, Kim G, et al. Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system. Jpn J Vet Res, 2006, 53(3-4): 127-139.
7.	Mizuta H, Kudo S, Nakamura E, et al. Expression of the PTH/PTHrP receptor in chondrogenic cells during the repair of full-thickness defects of articular cartilage. Osteoarthritis Cartilage, 2006, 14(9): 944-952.
8.	Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 2008, 112(2): 295-307.
9.	Ito T, Sawada R, Fujiwara Y, et al. FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling. Cytotechnology, 2008, 56(1): 1-7.
10.	Harrington EK, Roddy GW, West R, et al. Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation. Anat Rec (Hoboken), 2007, 290(2): 155-167.
11.	Amizuka N, Davidson D, Liu H, et al. Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004, 34(1): 13-25.
12.	Hankemeier S, Keus M, Zeichen J, et al. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng, 2005, 11(1-2): 41-49.
13.	Dickhut A, Gottwald E, Steck E, et al. Chondrogenesis of mesenchymal stem cells in gel-like biomaterials in vitro and in vivo. Front Biosci, 2008, 13: 4517-4528.
14.	Varas L, Ohlsson LB, Honeth G, et al. Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential. Stem Cells Dev, 2007, 16(6): 965-978.
15.	Iwamoto M, Jikko A, Murakami H, et al. Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation. J Biol Chem, 1994, 269(25): 17245-17251.
16.	Kafienah W, Mistry S, Dickinson SC, et al. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum, 2007, 56(1): 177-187.
17.	Kim YJ, Kim HJ, Im GI. PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs. Biochem Biophys Res Commun, 2008, 373(1): 104-108.
18.	晏杰, 刘玲蓉, 张其清. 诱导骨髓间充质干细胞向软骨细胞分化的体外研究. 中国修复重建外科杂志, 2006, 20(11): 1114-1118.

1. Zheng YH, Su K, Jian YT, et al. Basic fibroblast growth factor enhances osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells in coral scaffold constructs. J Tissue Eng Regen Med, 2011, 5(7): 540-550.
2. Kim JH, Lee MC, Seong SC, et al. Enhanced proliferation and chondrogenic differentiation of human synovium-derived stem cells expanded with basic fibroblast growth factor. Tissue Eng Part A, 2011, 17(7-8): 991-1002.
3. Lee S, Kim JH, Jo CH, et al. Effect of serum and growth factors on chondrogenic differentiation of synovium-derived stromal cells. Tissue Eng Part A, 2009, 15(11): 3401-3415.
4. Park KH, Na K. Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels. J Biosci Bioeng, 2008, 106(1): 74-79.
5. Yang KG, Saris DB, Geuze RE, et al. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng, 2006, 12(9): 2435-2447.
6. Bosnakovski D, Mizuno M, Kim G, et al. Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system. Jpn J Vet Res, 2006, 53(3-4): 127-139.
7. Mizuta H, Kudo S, Nakamura E, et al. Expression of the PTH/PTHrP receptor in chondrogenic cells during the repair of full-thickness defects of articular cartilage. Osteoarthritis Cartilage, 2006, 14(9): 944-952.
8. Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 2008, 112(2): 295-307.
9. Ito T, Sawada R, Fujiwara Y, et al. FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling. Cytotechnology, 2008, 56(1): 1-7.
10. Harrington EK, Roddy GW, West R, et al. Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation. Anat Rec (Hoboken), 2007, 290(2): 155-167.
11. Amizuka N, Davidson D, Liu H, et al. Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone, 2004, 34(1): 13-25.
12. Hankemeier S, Keus M, Zeichen J, et al. Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng, 2005, 11(1-2): 41-49.
13. Dickhut A, Gottwald E, Steck E, et al. Chondrogenesis of mesenchymal stem cells in gel-like biomaterials in vitro and in vivo. Front Biosci, 2008, 13: 4517-4528.
14. Varas L, Ohlsson LB, Honeth G, et al. Alpha10 integrin expression is up-regulated on fibroblast growth factor-2-treated mesenchymal stem cells with improved chondrogenic differentiation potential. Stem Cells Dev, 2007, 16(6): 965-978.
15. Iwamoto M, Jikko A, Murakami H, et al. Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation. J Biol Chem, 1994, 269(25): 17245-17251.
16. Kafienah W, Mistry S, Dickinson SC, et al. Three-dimensional cartilage tissue engineering using adult stem cells from osteoarthritis patients. Arthritis Rheum, 2007, 56(1): 177-187.
17. Kim YJ, Kim HJ, Im GI. PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs. Biochem Biophys Res Commun, 2008, 373(1): 104-108.
18. 晏杰, 刘玲蓉, 张其清. 诱导骨髓间充质干细胞向软骨细胞分化的体外研究. 中国修复重建外科杂志, 2006, 20(11): 1114-1118.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF BASIC FIBROBLAST GROWTH FACTOR AND PARATHYROID HORMONE-RELATED PROTEIN ON EARLY AND LATE CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β1

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