Effect of indianhedgehog gene transfection into rabbit bone marrow mesenchymal stem cells in promoting chondrogenic differentiation and inhibiting cartilage aging in rotary cell culture system_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIUPengcheng , LIUKuan , LIUJunfeng , XIAKuo , CHENLiyang ,  WUXing

Department of Orthopaedics, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai, 200072, P. R. China;

Corresponding author：

WUXing, Email: orthopedics_dsyy@126.com

Keywords：

Bone marrow mesenchymal stem cells; Hedgehog signaling pathway; Indianhedgehog; Rotary cell culture system; Cartilage tissue engineering; Gene transfection

DOI：

10.7507/1002-1892.20160180

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Objective To investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. Methods The 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. Results After transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. Conclusion Under the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.

Citation： LIUPengcheng, LIUKuan, LIUJunfeng, XIAKuo, CHENLiyang, WUXing. Effect of indianhedgehog gene transfection into rabbit bone marrow mesenchymal stem cells in promoting chondrogenic differentiation and inhibiting cartilage aging in rotary cell culture system. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(7): 892-902. doi: 10.7507/1002-1892.20160180 Copy

1.	Moran CJ, Pascual-Garrido C, Chubinskaya S, et al. Restoration of articular cartilage. J Bone Joint Surg (Am), 2014, 96(4): 336-344.
2.	Camp CL, Stuart MJ, Krych AJ. Current concepts of articular cartilage restoration techniques in the knee. Sports Health, 2014, 6(3): 265-273.
3.	Gopal K, Amirhamed HA, Kamarul T. Advances of human bone marrow-derived mesenchymal stem cells in the treatment of cartilage defects: a systematic review. Exp Biol Med (Maywood), 2014, 239(6): 663-669.
4.	Bagaria V, Patil N, Sapre V, et al. Stem cells in orthopedics: current concepts and possible future applications. Indian J Med Sci, 2006, 60(4): 162-169.
5.	Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
6.	St-Jacques B, Hammerschmidt M, Mcmahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 1999, 13(16): 2072-2086.
7.	Mak KK, Kronenberg HM, Chuang PT, et al. Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy. Development, 2008, 135(11): 1947-1956.
8.	Steinert AF, Weissenberger M, Kunz M, et al. Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells. Arthritis Res Ther, 2012, 14(4): R168.
9.	Sewing J, Klinger M, Notbohm H. Jellyfish collagen matrices conserve the chondrogenic phenotype in two-and three-dimensional collagen matrices. J Tissue Eng Regen Med, 2015. [Epub ahead of print].
10.	Yu B, Yu D, Cao L, et al. Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway. Biochem Biophys Res Commun, 2011, 414(2): 412-418.
11.	Wu X, Li SH, Lou LM, et al. The effect of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells. Mol Biotechnol, 2013, 54(2): 331-336.
12.	Lei X, Deng Z, Zhang H, et al. Rotary suspension culture enhances mesendoderm differentiation of embryonic stem cells through modulation of Wnt/beta-catenin pathway. Stem Cell Rev, 2014, 10(4): 526-538.
13.	Wu X, Cai ZD, Lou LM, et al. The effects of inhibiting hedgehog signaling pathways by using specific antagonist cyclopamine on the chondrogenic differentiation of mesenchymal stem cells. Int J Mol Sci, 2013, 14(3): 5966-5977.
14.	Chung R, Wong D, Macsai C, et al. Roles of Wnt/beta-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats. Bone, 2013, 52(2): 651-658.
15.	Kronenberg HM. Developmental regulation of the growth plate. Nature, 2003, 423(6937): 332-336.
16.	Xu F, Xu L, Wang Q, et al. 3D dynamic culture of rabbit articular chondrocytes encapsulated in alginate gel beads using spinner flasks for cartilage tissue regeneration. Biomed Res Int, 2014, 2014: 539789.
17.	Fischer J, Aulmann A, Dexheimer V, et al. Intermittent PTHrP (1-34) exposure augments chondrogenesis and reduces hypertrophy of mesenchymal stromal cells. Stem Cells Dev, 2014, 23(20): 2513-2523.
18.	Sambandam Y, Townsend MT, Pierce JJ, et al. Microgravity control of autophagy modulates osteoclastogenesis. Bone, 2014, 61: 125-131.
19.	Morabito C, Steimberg N, Mazzoleni G, et al. RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures. Biomed Res Int, 2015, 2015: 754283.
20.	Yanagisawa M, Suzuki N, Mitsui N, et al. Effects of compressive force on the differentiation of pluripotent mesenchymal cells. Life Sci, 2007, 81(5): 405-412.
21.	Meyers VE, Zayzafoon M, Douglas JT, et al. RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity. J Bone Miner Res, 2005, 20(10): 1858-1866.
22.	Huang Y, Dai ZQ, Ling SK, et al. Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells. J Biomed Sci, 2009, 16: 87.
23.	Dai ZQ, Wang R, Ling SK, et al. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Prolif, 2007, 40(5): 671-684.

1. Moran CJ, Pascual-Garrido C, Chubinskaya S, et al. Restoration of articular cartilage. J Bone Joint Surg (Am), 2014, 96(4): 336-344.
2. Camp CL, Stuart MJ, Krych AJ. Current concepts of articular cartilage restoration techniques in the knee. Sports Health, 2014, 6(3): 265-273.
3. Gopal K, Amirhamed HA, Kamarul T. Advances of human bone marrow-derived mesenchymal stem cells in the treatment of cartilage defects: a systematic review. Exp Biol Med (Maywood), 2014, 239(6): 663-669.
4. Bagaria V, Patil N, Sapre V, et al. Stem cells in orthopedics: current concepts and possible future applications. Indian J Med Sci, 2006, 60(4): 162-169.
5. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv, 2008, 26(1): 1-21.
6. St-Jacques B, Hammerschmidt M, Mcmahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 1999, 13(16): 2072-2086.
7. Mak KK, Kronenberg HM, Chuang PT, et al. Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy. Development, 2008, 135(11): 1947-1956.
8. Steinert AF, Weissenberger M, Kunz M, et al. Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells. Arthritis Res Ther, 2012, 14(4): R168.
9. Sewing J, Klinger M, Notbohm H. Jellyfish collagen matrices conserve the chondrogenic phenotype in two-and three-dimensional collagen matrices. J Tissue Eng Regen Med, 2015. [Epub ahead of print].
10. Yu B, Yu D, Cao L, et al. Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway. Biochem Biophys Res Commun, 2011, 414(2): 412-418.
11. Wu X, Li SH, Lou LM, et al. The effect of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells. Mol Biotechnol, 2013, 54(2): 331-336.
12. Lei X, Deng Z, Zhang H, et al. Rotary suspension culture enhances mesendoderm differentiation of embryonic stem cells through modulation of Wnt/beta-catenin pathway. Stem Cell Rev, 2014, 10(4): 526-538.
13. Wu X, Cai ZD, Lou LM, et al. The effects of inhibiting hedgehog signaling pathways by using specific antagonist cyclopamine on the chondrogenic differentiation of mesenchymal stem cells. Int J Mol Sci, 2013, 14(3): 5966-5977.
14. Chung R, Wong D, Macsai C, et al. Roles of Wnt/beta-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats. Bone, 2013, 52(2): 651-658.
15. Kronenberg HM. Developmental regulation of the growth plate. Nature, 2003, 423(6937): 332-336.
16. Xu F, Xu L, Wang Q, et al. 3D dynamic culture of rabbit articular chondrocytes encapsulated in alginate gel beads using spinner flasks for cartilage tissue regeneration. Biomed Res Int, 2014, 2014: 539789.
17. Fischer J, Aulmann A, Dexheimer V, et al. Intermittent PTHrP (1-34) exposure augments chondrogenesis and reduces hypertrophy of mesenchymal stromal cells. Stem Cells Dev, 2014, 23(20): 2513-2523.
18. Sambandam Y, Townsend MT, Pierce JJ, et al. Microgravity control of autophagy modulates osteoclastogenesis. Bone, 2014, 61: 125-131.
19. Morabito C, Steimberg N, Mazzoleni G, et al. RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures. Biomed Res Int, 2015, 2015: 754283.
20. Yanagisawa M, Suzuki N, Mitsui N, et al. Effects of compressive force on the differentiation of pluripotent mesenchymal cells. Life Sci, 2007, 81(5): 405-412.
21. Meyers VE, Zayzafoon M, Douglas JT, et al. RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity. J Bone Miner Res, 2005, 20(10): 1858-1866.
22. Huang Y, Dai ZQ, Ling SK, et al. Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells. J Biomed Sci, 2009, 16: 87.
23. Dai ZQ, Wang R, Ling SK, et al. Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells. Cell Prolif, 2007, 40(5): 671-684.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of indianhedgehog gene transfection into rabbit bone marrow mesenchymal stem cells in promoting chondrogenic differentiation and inhibiting cartilage aging in rotary cell culture system

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