بررسی چسبندگی و نفوذ سلول های مزانشیم به داربست متخلخل از کامپوزیت هیدروکسی آپاتیت/ PLGA با روکش پلیمر سه قطعه ای

نوع مقاله: مقاله کامل پژوهشی

نویسندگان

1 دکترای بیومواد، عضو هیئت علمی پژوهشکده فناور یهای نو، دانشگاه صنعتی امیرکبیر

2 دکترای بیوشیمی، عضو هیئت علمی شرکت فنآوری بن یاخته

3 دکترای پلیمر، عضو هیئت علمی مرکز پژوهشی زیس تمواد، دانشگاه تهران

10.22041/ijbme.2010.13340

چکیده

داربست های مورد استفاده در مهندسی بافت باید علاوه بر عملکرد مناسب، متخلخل، زیست سازگار و زیست تخریب پذیر باشند. در این تحقیق، داربست های متخلخل کامپوزیتی PLGA/HA به روش تعویض حلال ساخته شده و با پلیمر سه قطعه ای روکش دهی و با نور UV استریل شدند. مشاهدات حاصل از میکروسکوپ الکترونی روبشی حاکی از تشکیل ریزساختار متخلخل با اندازه حفرات حدود50 mm  و حفرات به هم پیوسته است. سلول های بنیادی مزانشیم انسانی بر روی داربست ها بذرافشانی شدند و سلول ها در داخل این ساختار به طور مطلوب چسبیدند. رنگ آمیزی فلورسانس باDAPI نشان دهنده چسبندگی سلول های مزانشیم به نمونه های دارای روکش و نفوذ سلول ها به داخل حفرات بود. همچنین، به منظور بررسی میزان تکثیر سلول ها روی داربست ها، آزمایش MTT روی آنها انجام شد و نشان داد که تعداد سلول های کشت شده روی داربست ها در مقایسه با نمونه های کنترل تفاوت معناداری ندارد. از نتایج به دست آمده استنباط می شود که داربست های روکش دار شده با پلیمر سه قطعه ای بستر مناسبی برای سلول های مزانشیم و روش به کار رفته روشی کارامد در ساخت داربست مهندسی بافت استخوان است.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Investigation of Human Mesenchymal Stem Cells Adhesion and Diffusion into Poly (lactic-co-glycolic acid)/Hydroxyapatite Porous Scaffold Coated with a Biodegradable Triblock Copolymer

نویسندگان [English]

  • Masoume Haghbin Nazarpak 1
  • Farzane Pourasgari 2
  • Mohammad Nabi Sarbolouki 3
1 PhD, New Technologies Research Center (NTRC), Amirkabir University of Technology
2 PhD, Stem Cells Technology Research Center
3 PhD, Biomaterials Research Center (BRC), University of Tehran
چکیده [English]

The scaffolds for bone tissue engineering should consider the functional requirements: porosity, biocompatibility, and biodegradability. In this study, porous Poly (lactic-co-glycolic acid)/Hydroxyapatite composites were prepared with different weight ratios. Porous samples were fabricated by freeze-extraction method, coated with triblock copolymer and sterilized by UV. Then, human mesenchymal stem cells were cultured on scaffolds. Microstructural studies with SEM suggest the formation of about 50 micrometer size porous structure and interconnected porosity so that cells adhesion within the structure is well in depth in coated samples. DAPI fluorescence microscopy showed cells adhesion to the coated scaffolds and cells diffusion into the pores. Also, direct assay of cell proliferation performed with MTT test showed that, cells grew on the scaffold similar to or more than control samples result. Therefore, these findings suggest that the triblock-coated Poly (lactic-coglycolic acid)/ Hydroxyapatite porous composite scaffolds could provide cells adhesion and proliferation and are appropriate matrices for bone tissue engineering.

کلیدواژه‌ها [English]

  • Scaffold
  • Bone tissue engineering
  • Freeze-extraction
  • Mesenchymal stem cells
  • Cell adhesion
  • Porosity
  • PLGA
  • Composite
[1]     Vindigni V., Cortivo R., Iacobellis L., Abatangelo G. and Zavan B., Hyaluronan benzyl ester as a scaffold for tissue engineering, Int. J. Mol. Sci., 2009; 10: 2972-2985.

[2]     Mistry A.S., Mikos A.G., Tissue engineering Strategies for bone regeneration, Adv Biochem Engin/ Biotechnol, 2005; 94: 1 22.

[3]     Tan Q., Steiner R., Hoerstrup S.P., Weder W., Tissueengineered trachea: History, problems and the future, European Journal of Cardio-thoracic Surgery, 2006; 30: 782-786.

[4]     Liu X. and Ma P.X., Polymeric scaffolds for bone tissue engineering, Annals of Biomedical Engineering, 2004; 32 (3): 477 486.

[5]     Tabata Y., Recent progress in tissue engineering, DDT, 2001; 6 (1): 483-487.

[6]     Mikos A.G., Temenoff J.S., Formation of highly porous biodegradable scaffolds for tissue engineering, EJB Electronic Journal of Biotechnology, 2000; 3 (2). http://www.scielo.cl/pdf/ejb/v3n2/art03.pdf

[7]     Chen G., Ushida T., Tateishi T., Development of biodegradable porous scaffolds for tissue engineering, Materials Science and Engineering C, 2001; 17: 63–69.

[8]     Hutmacher D. W., Garcia A. J., Scaffold-based bone engineering by using genetically modified cells, Gene, 2005; 347: 1–10.

[9]     Zhu X., Cui W., Li X. and Jin Y., Electrospun fibrous mats with high porosity as potential scaffolds for skin tissue engineering, Biomacromolecules, 2008; 9: 1795–1801.

[10] Ma P. X., Biomimetic materials for tissue engineering, Adv Drug Deliv Rev, 2008; 60 (2): 184–198.

[11] Budyanto L., Ooi C.P. and Goh Y.Q., Fabrication and characterization of porous poly (L-lactide) PLLA scaffolds using liquid-liquid phase separation, IFMBE Proceedings, 2008; 21: 322-325.

[12] Gunatillake P.A. and Adhikari R., Biodegradable synthetic polymers for tissue engineering, European Cells and Materials, 2003; 5: 1-16.

[13] Yang S., Leong K.F., Du Z., Chua C.K., The design of scaffolds for use in tissue engineering. Part І. Traditional Factors, Tissue Engineering, 2001; 7 (6): 679-689.

[14] Jones A. C., Milthorpe B., Averdunka H., Limaye A., Senden T. J., Sakellariou A., Sheppard A. P., Soka R. M., Knackstedt M. A., Brandwood A., Rohner D., Hutmacher D. W., Analysis of 3D bone ingrowth into polymer scaffolds via micro computedtomography imaging, Biomaterials, 2004; 25: 4947–4954.

[15] Wei G., Ma P. X., Macroporous and nanofibrous polymer scaffolds and polymer/bone-like apatite composite scaffolds generated by sugar spheres, J Biomed Mater Res, 2006; 78A: 306–315.

[16] Ma P. X., Zhang R., Xiao G., Franceschi R., Engineering new bone tissue in vitro on highly porous poly (α- hydroxyl acids)/hydroxyapatite composite scaffolds, J Biomed Mater Res, 2001; 54: 284-293.

[17] Papenburg B.J., Vogelaar L., Bolhuis-Versteeg L.A.M., Lammertink R.G.H., Tamatialis D. S., Wessling M., One-step fabrication of porous micropatterned scaffolds to control cell behavior, Biomaterials, 2007; 28: 1998–2009.

[18] Zhang R., Ma P. X., Porous poly (L- lactic acid)/apatite composites created by biomimetic process, J Biomed Mater Res, 1999; 45: 258-293.

[19] Chua C. K., Leong K. F., Cheah C. M. and Chua S.W., Development of a tissue engineering scaffold Structure library for rapid prototyping. Part 2: Parametric library and assembly program, Int J Adv Manuf Technol, 2003; 21:302–312.

[20] Horch R. A., Shahid N., Mistry A. S., Timmer M. D., Nanoreinforcement of poly (propylene fumarate)- based networks with surface modified alumoxane nanoparticles for bone tissue engineering, Biomacromolecules, 2004; 5 (5): 1990–1998.

[21] Buckley C.T., O’Kelly K.U., Regular scaffold fabrication techniques for investigations in tissue engineering, Topics in Bio Mechanical Engineering, CHAPTER V, 2004:147-166.

[22] Gong S., Dong J., Xue S.T., Wang J.Y., A novel porous natural polymer scaffold for tissue Engineering, Proceedings of the IEEE Engineering in Medicine and Biology 27th Annual Conference, 2005: 4884-4887.

[23] Ho M.H., Kuo P.Y., Hsieh H.J., Hsien T.Y., Hou L.T., Lai J.Y., Wang D.M., Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods, Biomaterials, 2004; 25: 129-138.

[24] Partap S., Lyons F., O’Brien F.J., Biomaterials and tissue engineering, Chapter 5, Royal College of Surgeons in Ireland. Available at www.tara.tcd.ie/ jspui/bitstream/2262//3/5.1_ScaffoldsSurfaces_final.doc

[25] Zhang R., Ma P. X., Poly (α- hydroxyl acids) /hydroxyapatite porous composites for bone tissue engineering. І. Preparation and morphology, J Biomed Mater Res, 1999; 44: 446-445.

[26] Neumann M., Epple M., Composites of calcium phosphate and polymers as bone substitution materials, European Journal of Trauma, 2006; 32: 125-131.

[27] Marcelle Mathieua L., Muellerb T.L., Bourbana P.E., Pioletti D.P., llerb R.M., Ma°nson J.A.E., Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering, Biomaterials, 2006; 27: 905–916.

[28] Kim S., Park M.S., Jeon O., Choi C.Y., Kim B., Poly (lactide-co-glycolide)/ hydroxyapatite composite scaffolds for bone tissue engineering, Biomaterials, 2006; 27: 1399-1409.

[29] کاظم‌زاده مهدی، ساخت داربست‌های مهندسی بافت به روش گاز فومینگ، 1388، قابل مشاهده در: http://www.taksirsazan.com/tabid/250/View/866/id/4130/Default.aspx

[30] Thompson J.B., Kindt J H., Drake B., Hansma H.G., Morse D.E., Hansma P.K., Bone indentation recovery time correlates with bond reforming time, Letters to nature, Nature, 2001; 414 (13): 773-775.

[31] Mano J.F., Sousa R.A., Boesel L.F., Neves N.M., Reis R.L., Bioinert, biodegradable and injectable polymeric matrix composites for hard tissue replacement: state of the art and recent developments, Composites Science and Technology, 2004; 64: 789–817.

[32] Heo S.J., Kim S.E., Hyun Y.T., Kim D.H., Kim J. H., Lee Y.J., Kim Y.J., Shin J.W., Hwang Y.M. and Shin J.W., Fabrication of porous scaffolds for bone tissue engineering using a 3-D Robotic system: comparison with conventional scaffolds fabricated by particulate leaching, MCB, 2006; 3 (4): 179-180.

[33] Kazemzadeh Narbat M., Orang F., Solati Hashtjin M. and Goudarzi A., Fabrication of porous hydroxyapatite-gelatin composite scaffolds for bone tissue engineering, Iranian Biomedical Journal, 2006; 10 (4): 215-223.

[34] Duarte A.R.C., Mano J.F., Reis R.L., Preparation of starch-based scaffolds for tissue engineering by supercritical immersion precipitation, J. of Supercritical Fluids, 2009; 49: 279–285.

[35] Metcalfe A.D. and Ferguson M.W.J., Tissue engineering of replacement skin: the crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration, J. R. Soc. Interface, 2007; 4: 413–437.

[36] Hosseinkhani H. and Hosseinkhani M., Tissue engineered scaffolds for stem cells and regenerative medicine, Trende in Stem Cell Biology and Technology, Humana Press, 2009: 367-387.

[37] Najafi F., Sarbolouki M.N., Synthesis and characterization of block copolymers from aromatic diols, fumaric acid, sebacic acid and PEG, J Appl Polym Sci, 2003; 90: 2358–2363.

[38] Najafi F., Sarbolouki M.N., Biodegradable micelles/ polymer somes from fumaric/sebacic acids and poly (ethylene glycol), Biomaterials, 2003; 24: 1175–1182.