Glycosaminoglycans Content and Type II Collagen Localization in Chondrogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Induced by L-Ascorbic Acid 2-Phosphate
Keywords:chondrocyte, chondrogenesis, collagen II, glycosaminoglycan, L-ascorbic acid 2-phosphate
AbstractL-ascorbic acid 2-phosphate (LAA) is known to induce chondrocyte differentiation. The objective of this study was to analyze the potency of LAA in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSC) by analyzing the glycosaminoglycans (GAG) content and type II collagen (Coll2) localization. ADSC was characterized using flow cytometry and cultured in media containing various concentrations of LAA (0, 25, 50, 100 μg/mL) for 2, 3 and 4 weeks. Coll2 localization was analyzed by immunocytochemistry (ICC) using a confocal microscope. The quantification of GAG was performed by Alcian Blue staining and calcium deposition by Alizarin Red S staining. The results showed that ADSC was positive for mesenchymal stem cell (MSC) markers. Coll2 was localized in the cytoplasm and showed increasing abundance along with the increase of the LAA concentration. The highest intensity of Coll2 localization was shown in LAA 100 μg/mL. ADSC in LAA induction medium showed higher GAG content compared to the control group (LAA 0 μg/mL) (p<0.05). The highest calcium deposit was shown by LAA 25 μg/mL after 4 weeks of culture (p<0.05) and it decreased at higher concentrations. In conclusion, LAA 100 μg/mL is considered the optimum LAA concentration for chondrogenic differentiation.
Sophia Fox, A.J., Bedi, A. & Rodeo, S.A., The Basic Science of Articular Cartilage: Structure, Composition, and Function Sport. Heal. A Multidiscip. Approach, 1(6), pp. 461-468, 2009.
Phull, A.R., Eo, S.H., Abbas, Q., Ahmed, M. & Kim, S.J., Applications of Chondrocyte-Based Cartilage Engineering: An Overview, Biomed Res. Int., 2016, pp. 1-17, 2016.
Gelse, K., Collagens - Structure, Function, and Biosynthesis, Adv. Drug Deliv. Rev., 55(12), pp. 1531-1546, 2003.
Hoshi, K., Fujihara, Y., Asawa, Y., Nishizawa, S., Kanazawa, S., Sakamoto, T., Watanabe, M., Ogasawara, T., Saijo, H., Mori, Y. & Takato, T., Recent Trends in Cartilage Regenerative Medicine and its Application to Oral and Maxillofacial Surgery, Oral Sci. Int., 10(1), pp. 15-19, 2013.
Strioga, M., Viswanathan, S., Darinskas, A., Slaby, O. & Michalek, J., Same or Not the Same? Comparison of Adipose Tissue-Derived Versus Bone Marrow-Derived Mesenchymal Stem and Stromal Cells, Stem Cells Dev., 21(14), pp. 2724-2752, 2012.
Choi, K.M., Seo, Y.K., Yoon, H.H., Song, K.Y., Kwon, S.Y., Lee, H.S. & Park, J.K., Effect of Ascorbic Acid on Bone Marrow-Derived Mesenchymal Stem Cell Proliferation and Differentiation, J. Biosci. Bioeng., 105(6), pp. 586-594, 2008.
Kim, J.H., Kim, W.K., Sung, Y.K, Kwack, M.H., Song, S.Y., Choi, J.S., Park, S.G., Yi, T., Lee, H.J., Kim, D.D., Seo, H.M., Song, S.U. & Sung. J.H., The Molecular Mechanism Underlying the Proliferating and Preconditioning Effect of Vitamin C on Adipose-Derived Stem Cells, Stem Cells Dev., 23(12), pp. 1364-1376, 2014.
Yamashita, A., Nishikawa, S. & Rancourt, D.E., Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells, PLoS One, 5(6), pp. e10998, 2010.
Barry, F., Boynton, R.E., Liu, B. & Murphy, J.M., Chondrogenic Differentiation of Mesenchymal Stem Cells from Bone Marrow: Differentiation-Dependent Gene Expression of Matrix Components, Exp. Cell Res., 268(2), pp. 189-200, 2001.
Freshney, R.I., Culture of Animal Cells. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010.
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W., NIH Image to ImageJ: 25 Years of Image Analysis., Nat. Methods, 9(7), pp. 671-5, Jul. 2012.
Trujillo, N. & Popat, K., Increased Adipogenic and Decreased Chondrogenic Differentiation of Adipose Derived Stem Cells on Nanowire Surfaces, Materials (Basel)., 7(4), pp. 2605-2630, 2014.
Tavakolinejad, S., Khosravi, M., Mashkani, B., Ebrahimzadeh Bideskan, A., Sanjar Mossavi, N., Parizadeh, M.R. & Hamidi Alamdari, D., The Effect of Human Platelet-Rich Plasma on Adipose-Derived Stem Cell Proliferation and Osteogenic Differentiation, Iran. Biomed. J., 18(3), pp. 151-157, 2014.
Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach., I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, Dj. & Horwitz, E., Minimal Criteria for Defining Multipotent Mesenchymal Stromal Cells, The International Society for Cellular Therapy Position Statement, Cytotherapy, 8(4), pp. 315-7, 2006.
Ryu, Y.J., Cho, T.J., Lee, D.S., Choi, J.Y. & Cho, J., Phenotypic Characterization and in Vivo Localization of Human Adipose-Derived Mesenchymal Stem Cells, Mol. Cells, 35(6), pp. 557-564, 2013.
Ullah, I., Subbarao, R.B. & Rho, G.J., Human Mesenchymal Stem Cells - Current Trends and Future Prospective Bioscience Reports, Biosci. Rep., 35, 2015.
Jiang, T., Liu, W, Lv, X., Sun, H., Zhang, L., Liu, Y., Zhang, W.J., Cao, Y. & Zhou, G., Potent in Vitro Chondrogenesis of CD105 Enriched Human Adipose-Derived Stem Cells, Biomaterials, 31(13), pp. 3564-3571, May 2010.
Baer, P.C., Griesche, N., Luttmann, W., Schubert, R., Luttmann, A. & Geiger, H., Human Adipose-Derived Mesenchymal Stem Cells in Vitro: Evaluation of an Optimal Expansion Medium Preserving Stemness, Cytotherapy, 12(1), pp. 96-106, 2010.
Bourin, P., Bunnell, B.A., Casteilla, L., Dominici, M., Katz, A.J, March, K.L, Redl, H., Rubin, J.P., Yoshimura, K. & Gimble, J.M., Stromal Cells From the Adipose Tissue-Derived Stromal Vascular Fraction and Culture Expanded Adipose Tissue-Derived Stromal/Stem Cells: A Joint Statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International So, Cytotherapy, 15(6), pp. 641-648, 2013.
Barlian, A., Judawisastra, H., Alfarafisa, N.M., Wibowo, U.A. & Rosadi, I., Chondrogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Induced by L-Ascorbic Acid and Platelet Rich Plasma on Silk Fibroin Scaffold, PeerJ, 6:e5809, 2018. DOI: 10.7717/peerj.5809
Sorushanova, A., Delgado, L.M., Wu, Z., Shologu, N., Kshirsagar, A., Raghunath, R., Mullen, A.M., Bayon, Y., Pandit, A., Raghunath, M. & Zeugolis, D.I., The Collagen Suprafamily: from Biosynthesis to Advanced Biomaterial Development, Adv. Mater., 31(1), pp. 1801651, 2019.
Qiao, H., Bell, J., Juliao, S., Li, L. & May, J.M., Ascorbic Acid Uptake and Regulation of Type I Collagen Synthesis in Cultured Vascular Smooth Muscle Cells, J. Vasc. Res., 46(1), pp. 15-24, 2009.
Akbari, A., Jelodar, G., Nazifi, S. & Sajedianfard, J., An Overview of the Characteristics and Function of Vitamin C in Various Tissues: Relying on its Antioxidant Function, Zahedan J. Res. Med. Sci., 18(11):e4037, 2016. DOI:10.17795/zjrms-4037
Leboy, P.S., Vaias, L., Uschmann, B., Golub, E., Adams, S.L. & Pacifici, M., Ascorbic Acid Induces Alkaline Phosphatase, Type X Collagen, and Calcium Deposition in Cultured Chick Chondrocytes., J. Biol. Chem., 264(29), pp. 17281-6, 1989.
Hu, D. & Shan, X., Effects of Different Concentrations of Type-I Collagen Hydrogel on the Growth and Differentiation of Chondrocytes, Exp. Ther. Med., 14(6), pp. 5411-5416, 2017.
Erden Tayhan, S., TaAYdemir, A?., DeliloAYlu G1/4rhan, S.A. & Mir, E., Comparison of the Osteogenic Differentiation Capacity of Adipose Tissuederived Mesenchymal Stem Cells From Humans and Rats, Turkish J. Biol., 40, pp. 1090-1095, 2016.
Jaiswal, N., Haynesworth, S.E., Caplan, A.I. & Bruder, S.P., Osteogenic Differentiation of Purified, Culture-Expanded Human Mesenchymal Stem Cells In Vitro., J. Cell. Biochem., 64(2), pp. 295-312, Feb. 1997.
Aghajanian, P., Hall, S., Wongworawat, M.D. & Mohan , S., The Roles and Mechanisms of Actions of Vitamin C in Bone: New Developments, J. Bone Miner. Res., 30(11), pp. 1945-1955, Nov. 2015.