IDENTIFYING THE POTENTIAL OF TRANSCRIPTION FACTOR SOX9 GENE TRANSFER IN CHONDROCYTES DIFFERENTIATION AND ARTICULAR CARTILAGE FORMATION IN VITRO
Keywords:Sox9 gene transfer, articular cartilage, transient transfection, chondrocytes, PLGA, fibrin
AbstractSox9 plays an important role as transcription factor for chondrogenesis; the formation of cartilage. This study aimed to identify the potential of the transiently overexpressed Sox9 gene in human chondrocytes differentiation and tissue engineered cartilage (TEC) formation in vitro. Articular cartilage samples were obtained from osteoarthritic patients who underwent joint replacement surgery. The isolated chondrocytes were cultured and transfected with pcDNA3-Sox9 using lipofection technique. The TEC constructs were formed by incorporating the transfected and the non-transfected cells onto poly(lactic-co-glycolic acid) (PLGA) scaffold with or without fibrin. This approach allows a comparison between four groups i.e. (1) transfected chondrocytes seeded on PLGA/fibrin [PFTC], (2) non-transfected chondrocytes on PLGA/fibrin [PFC], (3) transfected chondrocytes on PLGA [PTC] and (4) non-transfected chondrocytes on PLGA [PC]. All TEC constructs were cultured and evaluated at each time point of 1, 2 and 3 weeks in vitro. All TEC constructs were analysed for gross observation, histology, immunohistochemistry, cell proliferation activity, gene expression and sulphated glycosaminoglycan (sGAG) production assay. After 3 weeks, all PFTC and PFC showed higher cell viability, higher sGAG content, better histological features and distribution of extracellular matrix in concert with positive glycosaminoglycan (GAG) accumulation when compared to the PTC and PC. However, at week 3, the PFC and PC exhibited significantly higher sGAG production than PFTC and PTC. Chondrogenic properties of the constructs were evidenced by the expression of cartilage-specific markers; collagen II, collagen XI and aggrecan core protein. In this study, due to the nature of a new cartilage formation, the co-expression of collagen I in all constructs can be an indication of early cartilage development. Based on the outcomes, it is hoped that this study will provide a good ground for future tissue engineering application.
Buckwalter, J., Mankin, H., &Grodzinsky, A. 2005. Articular Cartilage and Osteoarthritis. Instructional Course Lectures. 54: 465-480.
Hunziker, E. B. 2002. Articular Cartilage Repair: Basic Science and Clinical Progress. A Review of the Current Status and Prospects. Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society. 10(6): 432-63.
Wright, E., Hargrave, M. R., Christiansen, J., Cooper, L., Kun, J., Evans, T., ...& Koopman, P. 1995. The Sry-related Gene Sox9 is Expressed During Chondrogenesis in Mouse Embryos. Nature Genetics. 9(1): 15-20.
Kulyk, W. M., Franklin, J. L., & Hoffman, L. M. 2000. Sox9 Expression During Chondrogenesis in Micromass Cultures of Embryonic Limb Mesenchyme. Experimental Cell Research. 255(2): 327-332.
Lefebvre, V., Huang, W., Harley, V. R., Goodfellow, P. N., & de Crombrugghe, B. 1997. SOX9 is A Potent Activator of the Chondrocyte-Specific Enhancer of the Pro Alpha1(II) Collagen Gene. Molecular and Cellular Biology. 17(4): 2336-46.
Sekiya, I., Tsuji, K., Koopman, P., Watanabe, H., Yamada, Y., Shinomiya, K., â€¦ Noda, M. 2000. SOX9 Enhances Aggrecan Gene Promoter/Enhancer Activity and Is Up-regulated by Retinoic Acid in a Cartilage-derived Cell Line, TC6. Journal of Biological Chemistry. 275(15): 10738-10744.
Zhang, P., Jimenez, S. a, & Stokes, D. G. 2003. Regulation of Human COL9A1 Gene Expression. Activation of the Proximal Promoter Region by SOX9. Journal of Biological Chemistry. 278(1): 117-23.
Bridgewater, L. C., Lefebvre, V., & de Crombrugghe, B. 1998. Chondrocyte-specific Enhancer Elements in the Col11a2Gene Resemble the Col2a1 Tissue-specific Enhancer. Journal of Biological Chemistry. 273(24): 14998-15006.
Wang, W., Li, B., Li, Y., Jiang, Y., Ouyang, H., & Gao, C. 2010. In vivo Restoration of Full-Thickness Cartilage Defects By Poly(lactide-co-glycolide) Sponges Filled With Fibrin Gel, Bone Marrow Mesenchymal Stem Cells and DNA Complexes. Biomaterials. 31(23): 5953-65.
Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., â€¦Lun, K. 2004. New Non-Viral Method for Gene Transfer Into Primary Cells. Methods (San Diego, California). 33(2): 151-63.
Shaâ€™ban, M., Kim, S. H., Idrus, R. B., &Khang, G. 2008. Fibrin and Poly(lactic-co-glycolic acid) Hybrid Scaffold Promotes Early Chondrogenesis of Articular Chondrocytes: An in Vitro Study. Journal of Orthopaedic Surgery and Research. 3: 17.
Khang, G., Lee, S. J., Han, C. W., Rhee, J. M., & Lee, H. B. 2003. Preparation and Characterization of Natural / Synthetic Hybrid Scaffolds. In Tissue Engineering, Stem Cells and Gene Therapies. 235-245.
Berger, A. P., Bartsch, G., Deibl, M., Alber, H., Pachinger, O., Fritsche, G., â€¦Frauscher, F. 2006. Atherosclerosis as a Risk Factor for Benign Prostatic Hyperplasia. BJU international. 98(5): 1038-42.
Takebe, T., Kobayashi, S., Suzuki, H., Mizuno, M., Chang, Y., Yoshizawa, E., â€¦ Taniguchi, H. 2014. Transient Vascularization of Transplanted Human Adultâ€“Derived Progenitors Promotes Self-Organizing Cartilage. The Journal of Clinical Investigation. 124(10): 4325-4334.
Oh, C., Maity, S. N., Lu, J.-F., Zhang, J., Liang, S., Coustry, F., â€¦ Yasuda, H. 2010. Identification of SOX9 Interaction Sites in the Genome Of Chondrocytes. PloS One. 5(4): e10113.
Min, N., Bin, A., Bt, R., &Idrus, H. 2012. Non-viral Transient Transfection of hTERT Gene into hBMSCs from Elderly Patients Delays Cellular Aging in Vitro. Asian Biomedicine. 6(2): 245-254.
Esko, J. D., Kimata, K., Lindahl, U. 2009. Proteoglycans and Sulfated Glycosaminoglycans. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. Chapter 16.
Knudson, C. B., & Knudson, W. 2001. Cartilage Proteoglycans. Seminars In Cell & Developmental Biology. 12(2): 69-78.
Kiani, C. H., Chen, L. I., Wu, Y. A. O. J. I., Yee, A. L. J., & Yang, B. U. B. 2002. Structure and Function of Aggrecan. Cell Research. 12: 19-32.
Kiviranta, I., Tammi, M., Jurvelin, J., SÃ¤Ã¤mÃ¤nen, A. M., &Helminen, H. J. 1984. Fixation, Decalcification, and Tissue Processing Effects on Articular Cartilage Proteoglycans. Histochemistry. 80(6): 569-73.
Alers, J. C., Krijtenburg, P., Vissers, K. J., &Dekken, H. Van. 1999. Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and Comparative Genomic Hybridizationâ€¯: EDTA Is Highly Preferable to a Routinely Used Acid Decalcifier. The Journal of Histochemistry & Cytochemistry. 47(5): 703-709.
Camplejohn, K. L., & Allard, S. A. 1988. Limitations of Safraninâ€™ O staining in Proteoglycan-Depleted Cartilage Demonstrated with Monoclonal Antibodies. Histochemistry. 89: 185-188.
Goldring, M. B. 2012. Chondrogenesis, Chondrocyte Differentiation, and Articular Cartilage Metabolism in Health and Osteoarthritis. Therapeutic Advances in Musculoskeletal Disease. 4(4): 269-85.
Maier, T., GÃ¼ell, M., & Serrano, L. 2009. Correlation of mRNA and Protein in Complex Biological Samples. FEBS Letters. 583(24): 3966-73.
Caron, M. M. J., Emans, P. J., Coolsen, M. M. E., Voss, L., Surtel, D. a M., Cremers, a, â€¦ Welting, T. J. M. 2012. Redifferentiation of Dedifferentiated Human Articular Chondrocytes: Comparison of 2D and 3D cultures. Osteoarthritis and cartilage/OARS, Osteoarthritis Research Society. 20(10): 1170-8.
Munirah, S., Samsudin, O. C., Aminuddin, B. S., Ruszymah, B. H. I. 2010. Expansion of Human Articular Chondrocytes and Formation of Tissue-Engineered Cartilage: A Step Towards Exploring A Potential Use Of Matrix-Induced Cell Therapy. Tissue and Cell. 42(5): 282-292.
Munirah, S., Samsudin, O. C., Nor Hamdan, M. Y., Aminuddin, B. S., Ruszymah, B. H. I. 2011. Sox-9 Transient Transfection Enhances Chondrogenic Expression ofOsteoarthritic Human Articular Chondrocytes In Vitro: Preliminary Analysis. Tissue Engineering and Regenerative Medicine. 8(1): 32-41.
Hsiao, S. H., Lee, K. Der, Hsu, C. C., Tseng, M. J., Jin, V. X., Sun, WS, et al. 2010. DNA Methylation of the Trip10 Promoter Accelerates Mesenchymal Stem Cell Lineage Determination. Biochemical and Biophysical Research Communications. 400(3): 305-312.
Arnsdorf, E. J., Tummala, P., Castillo, A. B., Zhang, F., Jacobs, C. R. 2010. The Epigenetic Mechanism of Mechanically Induced Osteogenic Differentiation. Journal of Biomechanics. 43(15): 2881-2886.
How to Cite
Copyright of articles that appear in Jurnal Teknologi belongs exclusively to Penerbit Universiti Teknologi Malaysia (Penerbit UTM Press). This copyright covers the rights to reproduce the article, including reprints, electronic reproductions, or any other reproductions of similar nature.