Abstract
In stem cell-based chondrogenesis for articular cartilage regeneration, TGF-β3 is dosed to the stem cells to drive differentiation into chondrocytic cells. Meanwhile, type I collagen, which is endogenously expressed in some stem cells (e.g., synovium-derived mesenchymal stem cells) and upregulated by TGF-β3, poses a threat to chondrogenesis, as type I collagen may alter the components and stiffness of articular cartilage. Therefore, a wiser strategy would be to feed the cells with TGF-β3 while at the same time silencing the expression of type I collagen. In this chapter, methods for construction of adenoviral vectors and lentiviral vectors having both of the above functions are given. Their transduction into synovium-derived mesenchymal stem cells for articular cartilage engineering and following characterizations are also described.
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Flugge LA, Miller-Deist LA, Petillo PA (1999) Towards a molecular understanding of arthritis. Chem Biol 6:R157–R166
Bader RMSA (2007) Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes. Eur Biophys J 36:539–568
Chancellor JV, Hunsche E, de Cruz E, Sarasin FP (2001) Economic evaluation of celecoxib, a new cyclo-oxygenase 2 specific inhibitor, in Switzerland. Pharmacoeconomics 19:59–75
Darling EM, Hu JC, Athanasiou KA (2004) Zonal and topographical differences in articular cartilage gene expression. J Orthop Res 22:1182–1187
Hidaka C, Cheng C, Alexandre D, Bhargava M, Torzilli PA (2006) Maturational differences in superficial and deep zone articular chondrocytes. Cell Tissue Res 323:127–135
Goessler UR, Bugert P, Bieback K, Baisch A, Sadick H, Verse T, Klüter H, Hörmann K, Riedel F (2004) Expression of collagen and fiber-associated proteins in human septal cartilage during in vitro dedifferentiation. Int J Mol Med 14:1015–1022
Goessler UR, Bugert P, Bieback K, Sadick H, Baisch A, Hormann K, Riedel F (2006) In vitro analysis of differential expression of collagens, integrins, and growth factors in cultured human chondrocytes. Otolaryngol Head Neck Surg 134:510–515
Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I (2007) Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 327:449–642
Na K, Kim S, Woo DG, Sun BK, Yang HN, Chung HM, Park KH (2007) Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test. J Biotechnol 128:412–422
Yun K, Moon HT (2008) Inducing chondrogenic differentiation in injectable hydrogels embedded with rabbit chondrocytes and growth factor for neocartilage formation. J Biosci Bioeng 105:122–126
Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F (2004) Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials 25:3211–3222
Steinert A, Weber M, Dimmler A, Julius C, Schütze N, Nöth U, Cramer H, Eulert J, Zimmermann U, Hendrich C (2003) Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate. J Orthop Res 21:1090–1097
Park H, Kang SW, Kim BS, Mooney DJ, Lee KY (2009) Shear-reversibly crosslinked alginate hydrogels for tissue engineering. Macromol Biosci 9:895–901
Yao Y, Zhang F, Zhou RJ, Li M, Wang DA (2010) Continuous supply of TGFbeta3 via adenoviral vector promotes type I collagen and viability of fibroblasts in alginate hydrogel. J Tissue Eng Regen Med 4:497–504
Zhang F, Yao Y, Hao J, Zhou R, Liu C, Gong Y, Wang DA (2010) A dual-functioning adenoviral vector encoding both transforming growth factor-beta3 and shRNA silencing type I collagen: construction and controlled release for chondrogenesis. J Control Release 142:70–77
Oberholzer A, John T, Kohl B, Gust T, Müller RD, La Face D, Hutchins B, Zreiqat H, Ertel W, Schulze-Tanzil G (2007) Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes. Cell Tissue Res 328:383–390
Zhang F, Yao YC, Zhou RJ, Su K, Citra F, Wang DA (2011) Optimal construction and delivery of dual-functioning lentiviral vectors for type I collagen-suppressed chondrogenesis in synovium-derived mesenchymal stem cells. Pharm Res 28:1338–1348
Wang CM, Hao JH, Zhang F, Su K, Wang DA (2008) RNA extraction from polysaccharide-based cell-laden hydrogel scaffolds. Anal Biochem 380:333–334
Wang CM, Li X, Yao YC, Wang DA (2009) Concurrent extraction of proteins and RNA from cell-laden hydrogel scaffold free of polysaccharide interference. J Chromatogr B 877:3762–3766
Acknowledgements
This work was supported by AcRF Tier 1 RG 36/12 and AcRF Tier 2 ARC 1/13, Ministry of Education, Singapore.
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Zhang, F., Wang, DA. (2015). Gene Transfer and Gene Silencing in Stem Cells to Promote Chondrogenesis. In: Doran, P. (eds) Cartilage Tissue Engineering. Methods in Molecular Biology, vol 1340. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2938-2_7
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DOI: https://doi.org/10.1007/978-1-4939-2938-2_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2937-5
Online ISBN: 978-1-4939-2938-2
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