نوع مقاله : مقاله کامل پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد، دانشکده علوم و فنون نوین، دانشگاه تهران، تهران، تهران، ایران

2 دانشیار، دانشکده علوم و فنون نوین، دانشگاه تهران، تهران، تهران، ایران

چکیده

هدف از این پژوهش، ساخت و بررسی خواص داربست فیبروئینی حاوی نانوذرات کیتوسان بارگذاری شده با آسکوربیک اسید بود. برای این منظور، نانوذرات آسکوربیک اسید-کیتوسان به روش ژل‌شدن یونی ساخته شدند. تصاویر میکروسکوپ الکترونی روبشی (SEM) و نتایج پراکندگی نور دینامیکی (DLS) نشان دادند که نانوذرات کروی‌شکل دارای ذراتی با اندازه میانگین 200 نانومتر هستند. سپس، مقادیر مختلفی از نانوذرات در داخل محلول فیبروئین ابریشم قرار گرفت و داربست‌های مورد‌نظر به روش خشکایش انجمادی تهیه شدند. تأثیر غلظت‌های مختلف نانوذرات بر ویژگی‌های ریخت‌شناسی، تغییرات ساختاری، جذب آب، رهایش دارو، سمّیت، چسبندگی و فعالیت آلکالین فسفاتاز سلول‌های استئوسارکومای رد‌ه MG63، مطالعه و بررسی شد. نتایج به‌دست‌آمده از طیف‌سنجی تبدیل فوریه فروسرخ (FTIR)، وجود نانوذرات در داربست را تأیید کرد. بررسی‌ ریخت‌شناسی سطح مقطع داربست‌ها نشان داد که همه داربست‌ها دارای ساختاری متخلخل با حفره‌های به‌هم‌متصل هستند. افزایش نانوذرات سبب کاهش اندازه قطر حفره‌ها و درصد تخلخل شد. رهایش آسکوربیک اسید در همه نمونه‌ها، با رهایش انفجاری در ۲۴ ساعت اولیه، شروع شد و سپس به صورت کنترل شده تا ۱۴ روز ادامه یافت. مقدار رهایش آسکوربیک اسید  با افزایش درصد نانو ذرات در داربست افزایش یافت. بررسی‌های سلولی نشان داد که داربست، دارای سمّیت نیست و سلول‌های MG63 به‌خوبی به سطح داربست‌ها و دیواره حفره‌ها می‌چسبند. همچنین تکثیر و فعالیت آلکالین فسفاتاز سلول‌های MG63، با افزایش مقدار آسکوربیک اسید، افزایش می‌یابد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Fabrication and Characterization of Properties of Silk Fibroin Scaffold Containing Ascorbic Acid-Loaded Chitosan Nanoparticles for Bone Regeneration Applications

نویسندگان [English]

  • Pegah Sanjarnia 1
  • Jhamak Nourmohammadi 2
  • Ali Hossein Rezayan 2
  • Mehrnaz Moaddab 1

1 M. Sc. Student, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Tehran, Iran

2 Associate Professor, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Tehran, Iran

چکیده [English]

The aim of this study was to fabricate and characterize silk fibroin scaffold containing chitosan nanoparticle loaded with ascorbic acid. Therefore, ascorbic acid- chitosan nanoparticles were fabricated using the ionic gelation method. Scanning Electron Microscopy (SEM) images and Dynamic Light Scattering (DLS) results showed that the nanoparticles are spherical with the average size of 200 nm. Then, different amounts of nanoparticles were placed in the silk fibroin solution, and finally, the scaffolds were prepared by the freeze-drying method. The effect of nanoparticle concentrations on various properties such as morphology, structural changes, water absorption, drug release, toxicity, adhesion, and alkaline phosphatase activity of MG63 cells were studied. The results of Fourier Transform Infrared Spectroscopy (FTIR) confirmed the presence of nanoparticles in the scaffold. Morphological examinations of the cross-section of the scaffold showed that all scaffolds have a porous structure with interconnected pores. The mean size of the pores and porosity percentages reduced as the nanoparticle content rose. The release of ascorbic acid in all samples started with the burst release in the first 24 hours and then continued with a controlled release for up to 14 days. Higher amounts of ascorbic acid were released from the scaffold containing more nanoparticles. Cellular studies showed that the scaffold was non-toxic and that MG63 cells adhered well onto the surface of the scaffold and the pores’ wall. Also, the proliferation and alkaline phosphatase activity of MG63 cells increased with increasing ascorbic acid amounts.

کلیدواژه‌ها [English]

  • Ascorbic acid
  • Bone scaffold
  • Silk fibroin
  • Chitosan Nanoparticles
  1. Chocholata, P., Kulda, V., Babuska, V., "Fabrication of scaffolds for bone-tissue regeneration", Materials, Vol. 12, No. 4, (2019), 568. https://doi.org/10.3390/ma12040568h
  2. Sommer, M. R., Vetsch, J. R., Leemann, J., Müller, R., Studart, A. R., Hofmann, S., "Silk fibroin scaffolds with inverse opal structure for bone tissue engineering", Journal of Biomedical Materials Research, Part B: Applied Biomaterials, Vol. 105, No. 7, (2017), 2074-2084. https://doi.org/10.1002/jbm.b.33737
  3. Montazeri, M., Karbasi, S., Monshi, A., Ebrahimi Kahrizsangi, R., "Evaluation of bioactivity poly-3-hydroxybutyrate coated nano-bioglass 45S5 composite scaffolds for bone tissue engineering", Journal of Advanced Materials and Technologies (JAMT), Vol. 2, No. 4, (2014), 10-18. https://dx.doi.org/10.30501/jamt.2010.70229
  4. Naskar, D., Ghosh, A. K., Mandal, M., Das, P., Nandi, S. K., Kundu, S. C., "Dual growth factor loaded nonmulberry silk fibroin/carbon nanofiber composite 3D scaffolds for in vitro and in vivo bone regeneration", Biomaterials, Vol. 136, (2017), 67-85. https://doi.org/10.1016/j.biomaterials.2017.05.014
  5. Dash, M., Chiellini, F., Ottenbrite, R. M., Chiellini, E., "Chitosan—A versatile semi-synthetic polymer in biomedical applications", Progress in Polymer Science, Vol. 36, No. 8, (2011), 981-1014. https://doi.org/10.1016/j.progpolymsci.2011.02.001
  6. Inamdar, N. N., Mourya, V., "Chitosan and low molecular weight chitosan: Biological and biomedical applications", Advanced Biomaterials and Biodevices, 1st, Tiwari, A., Nordin, A. Edition, Elsevier, Wiley Blackwell, (2014), 1-554. https://doi.org/10.1002/9781118774052.ch6
  7. Leena, R. S., Vairamani, M., Selvamurugan, N., "Alginate/gelatin scaffolds incorporated with silibinin-loaded chitosan nanoparticles for bone formation in vitro", Colloids and Surfaces, B: Biointerfaces, Vol. 158, (2017), 308-318. https://doi.org/10.1016/j.colsurfb.2017.06.048
  8. Berger, J., Reist, M., Mayer, J. M., Felt, O., Peppas, N. A., Gurny, R., "Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications", European Journal of Pharmaceutics and Biopharmaceutics, Vol. 57, No. 1, (2004), 19-34. https://doi.org/10.1016/s0939-6411(03)00161-9
  9. Zhou, X., Liu, P., Nie, W., Peng, C., Li, T., Qiang, L., He, C., Wang, J., "Incorporation of dexamethasone-loaded mesoporous silica nanoparticles into mineralized porous biocomposite scaffolds for improving osteogenic activity", International Journal of Biological Macromolecules, Vol. 149, (2020), 116-126. https://doi.org/10.1016/j.ijbiomac.2020.01.237
  10. Jang, K. I., Lee, H. G., "Stability of chitosan nanoparticles for l-ascorbic acid during heat treatment in aqueous solution", Journal of Agricultural and Food Chemistry, Vol. 56, No. 6, (2008), 1936-1941. https://doi.org/10.1021/jf073385e
  11. Bazelli, F., Attar, H., Amoabedini, G., Iman, , "Encapsulated of ascorbic acid in the nanomicelle", New Cellular and Molecular Biotechnology Journal, Vol. 5, No. 20, (2015), 89-96. http://ncmbjpiau.ir/article-1-703-en.html
  12. Moaddab, M., Nourmohammadi, J., Rezayan, A. H., "Bioactive composite scaffolds of carboxymethyl chitosan-silk fibroin containing chitosan nanoparticles for sustained release of ascorbic acid", European Polymer Journal, Vol. 103, (2018), 40-50. https://doi.org/10.1016/j.eurpolymj.2018.03.032
  13. Melke, J., Midha, S., Ghosh, S., Ito, K., Hofmann, S., "Silk fibroin as biomaterial for bone tissue engineering", Acta Biomaterialia, Vol. 31, (2016), 1-16. https://doi.org/10.1016/j.actbio.2015.09.005
  14. Sultana, N., Hassan, M. I., Ridzuan, N., Ibrahim, Z., Soon, C. F., "Fabrication of gelatin scaffolds using thermally induced phase separation technique", International Journal of Engineering, Vol. 31, No. 8, (2018), 1302-1307. http://dx.doi.org/10.5829/ije.2018.31.08b.19
  15. Ghalei, S., Nourmohammadi, J., Solouk, A., Mirzadeh, H., "Enhanced cellular response elicited by addition of amniotic fluid to alginate hydrogel-electrospun silk fibroin fibers for potential wound dressing application", Colloids and Surfaces, B: Biointerfaces, Vol. 172, (2018), 82-89. https://doi.org/10.1016/j.colsurfb.2018.08.028
  16. Mirhadi, S. M., Hassanzadeh Nemati, N., "Synthesis and characterization of highly porous TiO2 scaffolds for bone defects", International Journal of Engineering, Vol. 33, No. 2, (2020), 134-140. https://dx.doi.org/10.5829/ije.2020.33.01a.15
  17. Abbasizadeh, N., Rezayan, A. H., Nourmohammadi, J., Kazemzadeh-Narbat, M., "HHC-36 antimicrobial peptide loading on silk fibroin (SF)/hydroxyapatite (HA) nanofibrous-coated titanium for the enhancement of osteoblast and bactericidal functions", International Journal of Polymeric Materials and Polymeric Biomaterials, Vol. 69, No. 10, (2020), 629-639. https://doi.org/10.1080/00914037.2019.1596913
  18. Abul Kalam, M., Khan, A. A., Khan, S., Almalik, A., Alshamsan, A., "Optimizing indomethacin-loaded chitosan nanoparticle size, encapsulation, and release using Box-Behnken experimental design", International Journal of Biological Macromolecules, Vol. 87, (2016), 329-340. https://doi.org/10.1016/j.ijbiomac.2016.02.033
  19. Ghaee, A., Nourmohammadi, J., Danesh, P., "Novel chitosan-sulfonated chitosan-polycaprolactone-calcium phosphate nanocomposite scaffold", Carbohydrate Polymers, Vol. 157, (2017), 695-703. https://doi.org/10.1016/j.carbpol.2016.10.023
  20. Ryu, S., Kim, H. H., Park, Y. H., Lin, C. C., Um, I. C., Ki, C. S., "Dual mode gelation behavior of silk fibroin microgel embedded poly (ethylene glycol) hydrogels", Journal of Materials Chemistry B, Vol. 4, No. 26, (2016), 4574-4584. https://doi.org/10.1039/C6TB00896H
  21. Nourmohammadi, J., Roshanfar, F., Farokhi, M., Haghbin Nazarpak, M., "Silk fibroin/kappa-carrageenan composite scaffolds with enhanced biomimetic mineralization for bone regeneration applications", Materials Science and Engineering: C, Vol. 76, (2017), 951-958. https://doi.org/10.1016/j.msec.2017.03.166
  22. Shao, J., Zheng, J., Liu, J., Carr, C. M., "Fourier transform raman and fourier transform infrared spectroscopy studies of silk fibroin", Journal of Applied Polymer Science, Vol. 96, No. 6, (2005), 1999-2004. https://doi.org/10.1002/app.21346
  23. Budiraharjo, R., Neoh, K. G., Kang, E. T., "Hydroxyapatite-coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation", Journal of Colloid and Interface Science, Vol. 366, No. 1, (2012), 224-232. https://doi.org/10.1016/j.jcis.2011.09.072
  24. Bunaciu, A. A., Bacalum, E., Aboul-Enein, H. Y., Elena Udristioiu, G., Fleschin, Ş., "FT-IR spectrophotometric analysis of ascorbic acid and biotin and their pharmaceutical formulations", Analytical Letters, Vol. 42, No. 10, (2009), 1321-1327. https://doi.org/10.1080/00032710902954490
  25. Zadegan, S., Nourmohammadi, J., Vahidi, B., Haghighipour, N., "An investigation into osteogenic differentiation effects of silk fibroin-nettle (Urtica dioica) nanofibers", International Journal of Biological Macromolecules, Vol. 133, (2019), 795-803. https://doi.org/10.1016/j.ijbiomac.2019.04.165
  26. Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R. I., "Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells", Cell Biology International, Vol. 28, No. 4, (2004), 255-265. https://doi.org/10.1016/j.cellbi.2004.01.010
  27. Santo, V. E., Duarte, A. R. C., Popa, E. G., Gomes, M. E., Mano, J. F., Reis, R. L., "Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming", Journal of Controlled Release : Official Journal of the Controlled Release Society, Vol. 162, No. 1, (2012), 19-27. https://doi.org/10.1016/j.jconrel.2012.06.001
  28. Choi, H. K., Kim, G. J., Yoo, H. S., Song, D. H., Chung, K. H., Lee, K. J., Koo, Y., An, J. H., "Vitamin C activates osteoblastogenesis and inhibits osteoclastogenesis via Wnt/β-catenin/ATF4 signaling pathways", Nutrients, Vol. 11, No. 3, (2019), 506. https://doi.org/10.3390/nu11030506
  29. Lin, H. Y., Lai, R. H., Lin, S. T., Lin, R. C., Wang, M. J., Lin, C. C., Lee, H., Wang, F., Chen, J. Y., "Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission via regulating DRP1 translocation", Cell Death and Differentiation, Vol. 20, No. 1, (2013), 139-153. https://doi.org/10.1038/cdd.2012.106
  30. Fujisawa, K., Hara, K., Takami, T., Okada, S., Matsumoto, T., Yamamoto, N., Sakaida, I., "Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells", Stem Cell Research & Therapy, Vol. 9, No. 1, (2018), 93. https://doi.org/10.1186/s13287-018-0825-1
  31. Ishikawa, S., Iwasaki, K., Komaki, M., Ishikawa, I., "Role of ascorbic acid in periodontal ligament cell differentiation", Journal of Periodontology, Vol. 75, No. 5, (2004), 709-716. https://doi.org/10.1902/jop.2004.75.5.709