Kitosan Bazlı Yüzeylerin Mezenşimal Kök Hücrelerin Nöronal ve Glial Yönde Farklılaşmalarına Etkileri

Öz

Doku mühendisliği, sinir sisteminin rejenerasyonu konusunda ümit vadeden yeni tekniklerden biridir.  Biyouyumlu ve biyobozunur doku iskelelerinin kök hücreler için taşıyıcı olarak kullanıldığı bu yöntemde, seçilen doku iskelesinin kimyasal ve fiziksel özellikleri, üzerine ekilen hücrelerin davranışlarını yönlendirerek tedavi sürecine katkıda bulunabilmektedir.

Bu çalışmada biyouyumluluğu çok farklı dokular için gösterilmiş bir biyopolimer olan kitosan ile sülfat ve fosfat gruplarıyla fonksiyonelleştirilmiş türevlerinin sinir doku mühendisliği çalışmalarında kullanılma amacıyla sıçan kemik iliği kökenli mezenşimal kök hücrelerin (MKH) nöronal ve glial yönde farklılaşmalarına etkileri incelenmiştir.

Hücre kültürü çalışmaları için sıçan kemik iliğinden MKH'ler izole edilmiş, osteojenik ve kondrojenik hücrelere farklılaşmaları sağlanarak hücrelerin farklılaşma potansiyelleri kalitatif yöntemlerle gösterilmiştir. Yüzey kimyasına hassas olduğu bilinen MKH’lerin kitosan, kitosan fosfat ve kitosan sülfat yüzeyler üzerinde tutunma ve proliferasyon özellikleri incelenmiştir. Kitosan ve kitosan fosfat yüzeylerde hücre tutunması düşükken, sülfat fonksiyonel grubunun eklenmesinin hücre tutunmasını anlamlı şekilde arttırdığı belirlenmiştir. MKH’ler kitosan sülfat yüzeyler üzerinde hem nöronal hem de glial yönde farklılaştırılabilmişlerdir. Bu çalışma, sinir doku rejenerasyonu ve doku mühendisliği amacıyla kitosan sülfat yüzey kimyasının kullanılabileceğini göstermektedir.

Anahtar Kelimeler

• Mezenşimal Kök Hücre,Kitosan,Kitosan SüLfat,Sinir Doku Mühendisliği

Kaynakça

1. Schmidt, C.E., and Leach, J.B. Neural Tissue Engineering: Strategies for Repair and Regeneratio. Annual Review of Biomedical Engineering, 2003;5:293-347.
2. Keirstead, H.S. Stem cell transplantation into the central nervous system and the control of differentiation. Journal of Neuroscience Research. 2001;63:233-236.
3. Caplan, A. I. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. Journal of Cellular Physiology. 2007 Volume: 213 Issue: 2 Pages: 341-347.
4. Pountos, I., Corscadden, D., Emery, P., and Giannoudis, P.V. Mesenchymal stem cell tissue engineering: Techniques for isolation, expansion and application. Injury-Int. J. Care Inj. 2007;38:S23-S33.
5. Barry, F.P., and Murphy, J.M., Mesenchymal stem cells: clinical applications and biological characterization. The International Journal of Biochemistry & Cell Biology. 2004;36:568-584.
6. Uccelli, A., Moretta, L., Pistoia, V. Mesenchymal stem cells in health and disease. Nature Reviews Immunology. 2008;Volume: 8 Issue: 9 Pages: 726-736.
7. Kivanc, M., Ozturk, S., Gokalp, S., Ozdemir, I., Tuglu, I., Adipose-Derived Stem Cells and Application Areas. Cukurova Medical Journal. 2015;Volume: 40 Issue: 3 Pages: 399-408.
8. Sendemir-Urkmez, A. and Jamison, R. D. The addition of biphasic calcium phosphate to porous chitosan scaffolds enhances bone tissue development in vitro. Journal of Biomedical Materials Research. 2007; Part A, 81A (3): p. 624-633.

9. Singh, S., Jones, B.J., Crawford, R., and Xiao, Y. Characterization of a Mesenchymal-Like Stem Cell Population from Osteophyte Tissue. Stem Cells Dev. 2008;17:245-254.
10. Danoviz, M.E., Bassaneze, V., Nakamuta, J.S., dos Santos-Junior, G.R., Saint-Clair, D., Bajgelman, M.C. Adipose Tissue–Derived Stem Cells from Humans and Mice Differ in Proliferative Capacity and Genome Stability in Long-Term Cultures. Stem Cells Dev. 2011;20:661-670.
11. Yang, L., Wang, N.-L., and Cai, G.-P. Maohuoside A promotes osteogenesis of rat mesenchymal stem cells via BMP and MAPK signaling pathways. Molecular and cellular biochemistry. 2011;1-8.
12. Gimble, J.M., Guilak, F., Nuttall, M.E., Sathishkumar, S., Vidal, M., and Bunnell, B.A. In vitro Differentiation Potential of Mesenchymal Stem Cells. Transfusion Medicine and Hemotherapy. 2008;35:228-238.
13. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science. 1999;284:143-147.
14. Ninomiya, Y., Sugahara-Yamashita, Y., Nakachi, Y., Tokuzawa, Y., Okazaki, Y., and Nishiyama, M. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells. Biochem. Biophys. Res. Commun. 2010;394:303-308.
15. Chen, Y., Teng, F. Y. H. and Tang, B. L. Coaxing bone marrow stromal mesenchymal stem cells towards neuronal differentiation: progress and uncertainties. Cell. Mol. Life Sci. 2006:63:1649–1657.
16. Neuhuber, B., Gallo, G., Howard, L., Kostura, L., Mackay, A. and Fischer I. Revaluation of In Vitro Differentiation Protocols for Bone Marrow Stromal Cells: Disruption of Actin Cytoskeleton Induces Rapid Morphological Changes and Mimics Neuronal Phenotype. Journal of Neuroscience Research. 2004;77:192–204.
17. Zhang, H., Liu, Z., Yao, X., Yang, Z. and Xu, R. Neural Differentiation Ability of Mesenchymal Stromal Cells from Bone Marrow And Adipose Tissue: A Comparative Study. Cytotherapy. 2012;14: 1203–1214.
18. Montzka, K., Lassonczyk, N., Tschoke, B., Neuss, S., Fuhrmann, T., Franzen, R. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression. BMC Neurosci. 2009;10:1- 12.
19. Bossolasco, P., Cova, L., Calzarossa, C., Rimoldi, S.G., Borsotti, C., Lambertenghi-Deliliers. Neuro-glial differentiation of human bone marrow stem cells in vitro, Experimental Neurology. 2005;193: 312–325.
20. Woodbury, D., Schwarz, E.J., Prockop, D.J., and Black, I.B. Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of Neuroscience Research. 2000;61:364-370.
21. Deng, W., Obrocka, M., Fischer, I., and Prockop, D.J. In Vitro Differentiation of Human Marrow Stromal Cells into Early Progenitors of Neural Cells by Conditions That Increase Intracellular Cyclic AMP. Biochem. Biophys. Res. Commun. 2001;282:148-152.
22. Zhang, J., Lu, X., Feng, G., Gu, Z., Sun, Y., Bao, G. Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy. Cell Tissue Res, 2016;366:129–142.
23. Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., Hazzi, C., Stedeford, T., Willing, A. Adult Bone Marrow Stromal Cells Differentiate into Neural Cells In Vitro. Experimental Neurology. 2000;164:247-256.
24. Pillai, C.K.S., Paul, W., and Sharma, C.P. Chitin and chitosan polymers: Chemistry, solubility and fiber formation. Prog. Polym. Sci. 2009;34:641-678.
25. Kim, I.Y., Seo, S.J., Moon, H.S., Yoo, M.K., Park, I.Y., Kim, B.C. Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 2008;26:1-21.
26. Lim, S. M., Song, D.K., Oh, S. H., Lee-Yoon, D. S. Bae, E. H., Lee, J. H. In vitro and in vivo degradation behavior of acetylated chitosan porous beads. Journal of Biomaterials Science-Polymer Edition. 2008;Volume: 19 Issue: 4 Pages: 453-466.
27. Kumar, M., Muzzarelli, R.A.A., Muzzarelli, C., Sashiwa, H., and Domb, A.J. Chitosan chemistry and pharmaceutical perspectives. Chemical Reviews. 2004;104:6017-6084.
28. Alves, N.M., and Mano, J.F. Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications. Int. J. Biol. Macromol. 2008;43:401-414.
29. Galed, G., Miralles, B., Paños, I., Santiago, A., and Heras, Á., N- Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin. Carbohydr. Polym. 2005;62:316-320.
30. Sjoholm, K.H., Cooney, M., and Minteer, S.D., Effects of degree of deacetylation on enzyme immobilization in hydrophobically modified chitosan. Carbohydr. Polym. 2009;77:420-424.
31. Gamzazade, A., Sklyar, A., Nasibov, S., Sushkov, I., Shashkov, A., and Knirel, Y. Structural features of sulfated chitosans, Carbohydr. Polym. 1997;34:113-116.
32. Pramanik, N., Mishra, D., and Banerjee, I. Chemical Synthesis, Characterization, and Biocompatibility Study of Hydroxyapatite/Chitosan Phosphate, International Journal of Biomaterials, 2009:1-8.
33. Li, B., Huang, L., Wang, X., Ma, J., and Xie, F. Biodegradation and compressive strength of phosphorylated chitosan/chitosan/hydroxyapatite bio-composites. Materials & Design. 2011;32:4543-4547.
34. Zhang, X., Goncalves, R., and Mosser, D.M. The Isolation and Characterization of Murine Macrophages, Current Protocols in Immunology. John Wiley & Sons, Inc., Chapter. 2008;14:Unit 14.1.
35. Davies, J.Q., and Gordon, S. Isolation and Culture of Murine Macrophages, Methods Mol Biol. 2005;290:91-103.
36. Gregory, C.A., Grady Gunn, W., Peister, A., and Prockop, D.J. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction, Analytical Biochemistry. 2004;329:77-84.
37. Şenses, Y. M. Mezenşimal Kök Hücrelerin Kitosan Bazlı Yüzeylerde Nöronal ve Glial Yönde Farklılaşmalarının İncelenmesi, Ege Üniversitesi Fen Bilimleri Enstitüsü, Bornova-İzmir (Yüksek Lisans Tezi). 2011.
38. Mareschi, K., Novara, M., Rustichelli, D., Ferrero, I., Guido, D., Carbone, E., Medico, E., Madon, E., Vercelli, A., and Fagioli, F. Neural differentiation of human mesenchymal stem cells: evidence for expression of neural markers and eag K+ channel types, Exp. Hematol. 2006;34:1563- 1572.
39. Marcoli, M., Candiani, S., Tonachini, L., Monticone, M., Mastrogiacomo, M., Ottonello, A. In vitro modulation of gamma amino butyric acid (GABA) receptor expression by bone marrow stromal cells. Pharmacol. Res. 2008;57:374-382.
40. Woodbury, D., Reynolds, K., and Black, I.B. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis, Journal of Neuroscience Research, 2002;69:908- 917.
41. Jori, F.P., Melone, M.A.B., Napolitano, M.A., Cipollaro, M., Cascino, A., Giordano, A., and Galderisi, U. RB and RB2//p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells, Cell Death Differ. 2004;12:65-77.
42. Bi, Y., Gong, M., Zhang, X., Zhang, X., Jiang, W., Zhang, Y., Chen, J., Liu, Y., He, T.-C., and Li, T. Pre-activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells, Development, Growth & Differentiation. 2010;52:419-431.
43. Tığlı, S. R., Karakeçili, A., Gümüşderelioğlu, M., In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree, J Mater Sci Mater Med. 2007;18(9):1665-74.
44. Lanniel, M., Huq, E., Allen, S., Buttery, L., Williams, P.M., Alexander, M.R., Substrate induced differentiation of human mesenchymal stem cells on hydrogels with modified surface chemistry and controlled modulus, Soft Matter. 2011;Volume: 7 Issue: 14 Pages: 6501-6514.
45. Dulgar-Tulloch, A. J., Bizios, R., Siegel, R.W. Human mesenchymal stem cell adhesion and proliferation in response to ceramic chemistry and nanoscale topography, Journal of Biomedical Materials Research Part A. 2009;Volume: 90A Issue: 2 Pages: 586-594.
46. Ozdal-Kurt, F., Tuglu, I., Vatansever, H. S., Tong, S., Sen, B. H., Deliloglu-Gurhan, S. I., 2016, The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts, Biotechnic & Histochemistry, Volume: 91 Issue: 6, Pages: 412-422.
47. English, K., French, A., and Wood, K.J. Mesenchymal Stromal Cells: Facilitators of Successful Transplantation?. Cell stem cell. 2010;7:431-442.
48. Si, Y.L., Zhao, Y.L., Hao, H.J., Fu, X.B., and Han, W.D. MSCs: Biological characteristics, clinical applications and their outstanding concerns, Ageing Res. Rev. 2011;10:93-103.
49. Kolf, C.M., Cho, E., and Tuan, R.S., Mesenchymal stromal cells - Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation, Arthritis Res. Ther. 2007;9(1):204.
50. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., and Horwitz, E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy. 2006;8:315-317.
51. Peng, L., Jia, Z., Yin, X., Zhang, X., Liu, Y., Chen, P., Ma, K., and Zhou, C., Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Cartilage, and Adipose Tissue, Stem Cells Dev. 2008;17:761-774.
52. Karaoz, E., Aksoy, A., Ayhan, S., Sarıboyacı, A., Kaymaz, F., and Kasap, M., Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers, Histochemistry and Cell Biology. 2009;132:533-546.
53. Khatri, M., O’Brien, T.D., and Sharma, J.M. Isolation and Differentiation of Chicken Mesenchymal Stem Cells From Bone Marrow, Stem Cells Dev., 2009;18:1485-1492.
54. Solchaga, L.A., Penick, K.J., and Welter, J.F., Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells: Tips and Tricks, Mesenchymal Stem Cell Assays and Applications, Humana Press, 2011;698:253-278.
55. Donzelli, E., Salvadè, A., Mimo, P., Viganò, M., Morrone, M., Papagna, R., Carini, F., Zaopo, A., Miloso, M., Baldoni, M., and Tredici, G. Mesenchymal stem cells cultured on a collagen scaffold: In vitro osteogenic differentiation, Archives of Oral Biology, 2007;52:64-73.
56. Koch, T., Heerkens, T., Thomsen, P., and Betts, D. Isolation of mesenchymal stem cells from equine umbilical cord blood, BMC Biotechnology. 2007;7:26.
57. Hong, D., Chen, H. X., Xue, Y., Li, D.-M., Wan, X. C., Ge, R., and Li, J. C. Osteoblastogenic effects of dexamethasone through upregulation of TAZ expression in rat mesenchymal stem cells, The Journal of Steroid Biochemistry and Molecular Biology. 2009;116:86-92.
58. Zhang, L., Su, P., Xu, C., Yang, J., Yu, W., and Huang, D. Chondrogenic differentiation of human mesenchymal stem cells: a comparison between micromass and pellet culture systems, Biotechnology Letters. 2010;32:1339-1346.
59. Muzzarelli, R., and Muzzarelli, C. Chitosan Chemistry: Relevance to the Biomedical Sciences, Polysaccharides I, Springer Berlin / Heidelberg. 2005;186:151-209.
60. Pavinatto, F.J., Caseli, L., and Oliveira, O.N. Chitosan in Nanostructured Thin Films, Biomacromolecules, 2010;11:1897-1908.
61. Ozdemir E., Sendemir-Urkmez A., Yesil Celiktas O. Supercritical CO2 processing of a chitosan-based scaffold: Can implantation of osteoblastic cells be enhanced?, The Journal of Supercritical Fluids. 2013;27: 120 – 127.
62. Mi, F. L., Tan, Y. C., Liang, H. F., and Sung, H. W. In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant, Biomaterials. 2002;23:181-191.
63. VandeVord, P.J., Matthew, H.W.T., DeSilva, S.P., Mayton, L., Wu, B., and Wooley, P.H. Evaluation of the biocompatibility of a chitosan scaffold in mice, Journal of Biomedical Materials Research. 2002;59:585-590.
64. Yang, B., Li, X.Y., Shi, S.A., Kong, X.Y., Guo, G., Huang, M.J. Preparation and characterization of a novel chitosan scaffold, Carbohydr. Polym. 2010;80:860-865.
65. Jayakumar, R., Selvamurugan, N., Nair, S.V., Tokura, S., and Tamura, H. Preparative methods of phosphorylated chitin and chitosan—An overview, Int. J. Biol. Macromol. 2008;43:221-225.
66. Varma, H.K., Yokogawa, Y., Espinosa, F.F., Kawamoto, Y., Nishizawa, K., Nagata, F. Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method, Biomaterials. 1999;20:879-884.
67. Mariappan, M.R., Alas, E.A., Williams, J.G., and Prager, M.D. Chitosan and chitosan sulfate have opposing effects on collagen–fibroblast interactions, Wound Repair and Regeneration, 1999;7:400-406.
68. Zhang, K., Helm, J., Peschel, D., Gruner, M., Groth, T., and Fischer, S. NMR and FT Raman characterisation of regioselectively sulfated chitosan regarding the distribution of sulfate groups and the degree of substitution, Polymer. 2010;51:4698-4705.
69. Xing, R.E., Liu, S., Yu, H.H., Zhang, Q.B., Li, Z., and Li, P.C. Preparation of low-molecular-weight and high-sulfate-content chitosans under microwave radiation and their potential antioxidant activity in vitro, Carbohydr. Res. 2004;339:2515-2519.
70. Zhou, H., Qian, J., Wang, J., Yao, W., Liu, C., Chen, J. Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2- N, 6-O-sulfated chitosan in vitro and in vivo, Biomaterials. 2009;30:1715-1724.
71. Amaral, I.F., Granja, P.L., Melo, L.V., Saramago, B., and Barbosa, M.A. Functionalization of chitosan membranes through phosphorylation: Atomic force microscopy, wettability, and cytotoxicity studies, Journal of Applied Polymer Science. 2006;102:276-284.
72. Wan, Y., Creber, K.A.M., Peppley, B., and Bui, V.T. Synthesis, Characterization and Ionic Conductive Properties of Phosphorylated Chitosan Membranes, Macromolecular Chemistry and Physics. 2003;204:850- 858.
73. Xiang, Y., Yang, M., Guo, Z.B., and Cui, Z. Alternatively chitosan sulfate blending membrane as methanol-blocking polymer electrolyte membrane for direct methanol fuel cell, J. Membr. Sci. 2009;337:318-323.
74. Prasitsilp, M., Jenwithisuk, R., Kongsuwan, K., Damrongchai, N., and Watts, P. Cellular responses to chitosan in vitro: The importance of deacetylation, Journal of Materials Science: Materials in Medicine. 2000;11:773-778.
75. Freier, T., Koh, H.S., Kazazian, K., and Shoichet, M.S. Controlling cell adhesion and degradation of chitosan films by N-acetylation, Biomaterials. 2005;26:5872-5878.
76. Chatelet, C., Damour, O., ve Domard, A. Influence of the degree of acetylation on some biological properties of chitosan films, Biomaterials. 2001;22:261-268.
77. Mourya, V.K., Inamdar, N. N. Chitosan-modifications and applications: Opportunities galore, Reactive & Functional Polymers. 2008;68:1013–1051.
78. Tropel, P., Platet, N., Platel, J. C., Noël, D., Albrieux, M., Benabid. Functional Neuronal Differentiation of Bone Marrow- Derived Mesenchymal Stem Cells, Stem Cells. 2006;24:2868-2876.
79. Bae, K.S., Park, J.B., Kim, H.S., Kim, D.S., Park, D.J., and Kang, S.J. Neuron-like Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells, Yonsei Med. J. 2011;52:401-412.
80. Mammadov, B., Karakas, N., and Isik, S. Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells, In Vitro Cellular & Developmental Biology – Animal. 2011;47:484-491.
81. Romero-Ramos, M., Vourc'h, P., Young, H. E., Lucas, P.A., Wu, Y., Chivatakarn, O. Neuronal differentiation of stem cells isolated from adult muscle, Journal of Neuroscience Research. 2002; 69:894-907.
82. Forsberg, M., Holmborn, K., Kundu, S., Dagalv, A., Kjellen, L. ve Forsberg-Nilsson, K. Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells. J. Biol. Chem. 2012;287: 10853−10862.
83. Ding, K., Wang, Y., Wang, H., Yuan, L., Tan, M., Shi, X. 6-O-Sulfated Chitosan Promoting the Neural Differentiation of Mouse Embryonic Stem Cells, ACS Appl. Mater. Interface. 2014;6, 20043−20050.