اثر تمایزی اگزوزوم های مشتق شده از مونوسیت ها و ماکروفاژها در داربست های قطره ای کیتوزان آلژینات

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه زیست شناسی، واحد مشهد، دانشگاه آزاداسلامی، مشهد، ایران

2 گروه زیست شناسی و مرکز تحقیقات بیولوژی کاربردی تکوین جانوری، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران

چکیده

آسیب­های استخوانی یکی از چالش­های علم پزشکی محسوب می­شود که هر ساله هزینه­ی زیادی را در دنیا برای درمان به خود اختصاص می­دهد. اگزوزوم­ها، نانووزیکول­هایی هستند که پروتئین­ها و ماده­ی ژنتیک به سلول هدف وارد می­کنند و به دنبال آن، تکثیر، بقای سلولی و تمایز را در سلول گیرنده القا می­نمایند. این ویژگی­ها، می­تواند اگزوزوم­ها را به عامل تمایزی مناسب تبدیل کند. هدف از انجام این مطالعه، بررسی اثر تمایزی اگزوزوم­های مشتق شده از مونوسیت­ها و ماکروفاژها در داربست­های قطره­ای کیتوزان آلژینات. داربست­های کیتوزانی- آلژیناتی به روش قطره­ای به همراه سلول­های بنیادی چربی ساخته شد. سپس سلول­های مونوسیتی کشت و مایع رویی جمع­آوری شد و اگزوزوم­ها به روش اولتراسانتریفوژ جمع شدند و توسط روش DLS  وSEM  شناسایی شدند. نمونه­ها تیمار و سپس تستMTT  و  DAPI و برای بررسی فرآیند تمایز تست آلکالین فسفاتاز و Real-time PCR انجام شد. بررسی یافته­های آزمون MTT  نشان داد میزان بقای سلول­های تمایزیافته با اگزوزوم­ها در روزهای 7، 14 و 21 نسبت به کنترل تفاوت معناداری داشت. نتایج DAPI نشان­دهنده عدم مرگ سلولی در گروهای تیماری بود و نتایج تست آلکالین فسفاتاز تفاوت معناداری با گروه کنترل نشان داد و همچنین نتایج Real-time PCR افزایش بیان ژن­هایBMP 2/6، SMAD4، ژن­های تمایزی استئوکلسین (Osc) و استئوپونتین(Opn) نسبت به نمونه کنترل را به همراه داشت. این مطالعه، نشان داد اگزوزوم مشتق از سلول­های مونوسیتی و ماکروفاژی، می­توانند سبب بقا و تمایز استئوژنیک سلول­های مزانشیمی چربی در درابست­های کیتوزان آلژینات شوند.

کلیدواژه‌ها

عنوان مقاله [English]

Differentiation Effect of Exosomes Derived from Monocytes and Macrophages on Chitosan Alginate Drip Scaffolds

نویسندگان [English]

  • Nasrin Hosseini 1
  • Javad Baharara 2
  • Khadijeh Nejad Shahrokhabadi 1
  • Saeedeh Zafar Balanezhad 1
1 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran
2 Department of Biology & Research Center for Animal Development Applied Biology, Mashhad
چکیده [English]

Bone injuries are one of the challenges of medical science which costs a lot of money in the world for the treatment every year. Exosomes are nanovesicles which carry the proteins and genetic material into the target cell, which in turn, induces the proliferation, cell survival, and differentiation in the recipient cell. These features make exosomes a proper differentiation factor. This study aimed to investigate the differentiating effect of exosomes derived from the monocytes and macrophages on chitosan alginate drip scaffolds. Chitosan-alginate scaffolds were made by drip method with fat stem cells. Monocytes were cultured, and the supernatant was collected, and the exosomes were collected by the ultracentrifugation. Exosomes were identified by DLS and SEM methods. The samples were treated, and then MTT and DAPI tests were performed. The osteogenic differentiation was examined by alkaline phosphatase and real-time PCR. MTT results showed that the survival rate of differentiated cells with exosomes on days 7, 14, and 21 was increased compared to control group. Moreover, DAPI results showed no cell death in the treatment groups, and the results of the alkaline phosphatase test showed an increase with the control group, and also the results of real-time PCR increased the expression of genes BMP2/6, SMAD4 and differentiation genes Osc and Opn in the treatment group. This study showed that the exosomes derived from the monocytes and macrophages could cause survival and osteogenic differentiation of the fat mesenchymal cells in the chitosan alginate scaffold.

کلیدواژه‌ها [English]

  • Exosome
  • Fat Stem Cell
  • cell differentiation
  • Monocyte
  • Chitosan
  • Alginate Scaffold

مراجع

1. Ahmed G.M., Abouauf E.A., Abubakr N., Dörfer C.E., El-sayed K.F. 2020. Review Article Tissue Engineering Approaches for Enamel , Dentin , and Pulp Regeneration : An Update. stem cell International, 2020(2): 1-15.
2. Bendtsen S.T. 2017. Alginate Hydrogels for Bone Tissue Regeneration. Uconn, 4(7): 1-149.
3. Ciuffi S., Zonefrati R., Brandi ML. 2017, Adipose stem cells for bone tissue repair. Clinical Cases of Mineral Bone Metabolism, 14(4): 217.
4. De C., Gomez T., Goreham R.V., Serra JJB., Nann T., Kussmann M. 2018, Exosomics A Review of Biophysics ,Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk. Front Genetics, 9(5): 1-11.
5. Do A.V., Khorsand B., Geary S.M., Salem A.K. 2015. 3D Printing of Scaffolds for Tissue Regeneration Applications. Advacces in Healthcare Materials, 4(6): 1742-1762.
 6. Doustgani A., Vasheghani-Farahani E., Soleimani M., Hashemi-Najafabadi S. 2011. Preparation and characterization of aligned and random nanofibrous nanocomposite scaffolds of poly (vinyl alcohol), poly (e-Caprolactone) and nanohydroxyapatite. International Journal of Nanosciece a Nanotechnology, 7(7): 127-132.
7. Ekström K., Omar O., Granéli C., Wang X., Vazirisani ndF., Thomsen P. 2013. Monocyte Exosomes Stimulate the Osteogenic Gene Expression of Mesenchymal Stem Cells. PLoS One, 8(9): 1-14.
8. Ghosh M., Halperin-sternfeld M., Grinberg I., Adler-abramovich L. 2019. Injectable Alginate-Peptide Composite Hydrogel as a Scaffold for Bone Tissue Regeneration. Nanomaterials, 9(3):14.
9. Guihard P., Danger Y., Brounais B., David E., Brion R., Delecrin J., Richards CD., Chevalier S., Rédini F., Heymann D., Gascan H., Blanchard F . 2012. Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling. Stem Cells, 30: 762-772.
10. Hu L., Wang J., Zhou X., Xiong Z., Zhao J., Yu R., Huang F., Zhang H., Chen L. 2016. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Scientific Reports, 6: 32993
11. Kapellos T.S., Bonaguro L., Gemünd I., Reusch N., Saglam A., Hinkley ER., Schultze J.L. 2019. Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases. Immunology, 10:1-13.
12. Kumbhar S.G., Pawar S.H. 2017 Self-Functionalized, Oppositely Charged Chitosan-Alginate Scaffolds for Biomedical Applications. Biotechnology Indian Journal, 13(2): 130
13. Liu X., Yang Y., Li Y., Niu X., Zhao B., Wang Y., Bao C., Xie Z., Lin Q., Zhu L. 2017. Integration of stem cell-derived exosomes with in situ hydrogel glue as a promising tissue patch for articular cartilage regeneration. Nanoscale,9(7): 4430-4438.
14. Maji K., Dasgupta S., Pramanik K., Bissoyi A. 2016. Preparation and Evaluation of Gelatin-Chitosan-Nanobioglass 3D Porous Scaffold for Bone Tissue Engineering. International Journal of Biomaterials,2016: 1-14.
15. Malda J., Boere J., van de Lest C.H.A., van Weeren P.R., Wauben M.H.M. 2016. Extracellular vesicles - new tool for joint repair and regeneration. Natural Review of Rheumatology, 12(3):243-249.
16. Markwald RR., Mironov V. 2015. Organ printing: from bioprinter to organ biofabrication line. Expert Opin Biological Therapy, 10: 409-420.
17. Miana V.V., González E.A.P. 2018. Adipose tissue stem cells in regenerative medicine. Ecancer, 12(2): 1-17.
18. Ms JO., Hellein Bs J., Singla Hs R., Singal PK., Faha P., Singla DK., Fiacs F. 2015. Stem cells in three-dimensional bioprinting: Future perspectives. Curr Res Cardiology, 2(5): 193-196.
19. Patel G.K., Khan M.A., Zubair H., Srivastava S.K. 2019. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield ,purity and downstream applications. scientificreports, 9(4):1-10.
20. Salehi-Nik N., Rezai Rad M., Kheiri L., Nazeman P., Nadjmi N., Khojasteh A. 2017. Buccal Fat Pad as a Potential Source of Stem Cells for Bone Regeneration: A Literature Review. Stem CellsInternational, 2017: 1-13. (In Persian).
21. Sun J., Jiao K., Niu L., Jiao Y., Song Q., Shen L., Tay F.R., Chen J. 2017. Intrafibrillar silicified collagen scaffold modulates monocyte to promote cell homing, angiogenesis and bone regeneration. Biomaterials, 113(8): 203-216.
22. Tabriz AG., Hermida MA., Leslie NR., Shu W. 2015, Three-dimensional bioprinting of complex cell laden alginate hydrogel structures. Biofabrication, 7: 45012.
 23.      Tan Y., Richards DJ., Trusk TC., Visconti RP., Yost MJ., Kindy MS., Drake CJ., Argraves WS., Markwald RR., Mei Y . 2014. 3D printing facilitated scaffold-free tissue unit fabrication. Biofabrication, 6(24): 1-11.
24. Tuğrul TD., Gülseren IM. 2017. bioprintable form of chitosan hydrogel for bone tissue engineering. iposeince, 3(2): 1-15.
25. Valido DP., Déda W., Júnior G., Andrade ME De., Rezende AA., Campos CDA., Maria A., Oliveira S., Silva B., Souza S.D. 2020. Otoliths-composed gelatin/sodium alginate scaffolds for bone regeneration. drug Delivery translational Research, 12: 14.
26. Wang G., Wang X., Huang L. 2017. Feasibility of chitosan-alginate (Chi-Alg) hydrogel used as scaffold for neural tissue engineering: a pilot study in vitro. Biotechnol Biotechnol Equipments, 31(7): 766-773.
27. Wang S., Lee J.M., Yeong WY. 2015, Smart hydrogels for 3D bioprinting. International Journal of Bioprinting,1(1): 3-14.
28. Xie Y., Chen Y., Zhang L., Ge W., Tang P . 2017, The roles of bone-derived exosomes and exosomal microRNAs in regulating bone remodelling. Journal of Cell and Molecular Medicine,21:1033-1041.
29. Yunfan H. , Derakhshanfar S., Wen Z., Li B., Lu F., Xing X.L. 2020, Nanomaterials, 2020(8):1-11.
30. Zhang Y., Liu Y., Liu H., Tang WH. 2019, Exosomes : biogenesis, biologic function and clinical potential. Cell Bioscience,9: 1-18.
 31.      Zhao J., Yan X. 2019, Regulating Preparation Of Functional Alginate-Chitosan Three-Dimensional Scaffold For Skin Tissue Engineering. International Journal of Nanomedicine, 14(7): 8891-8903.
32. Zhou S., Wang Y., Zhang K., Cao N., Yang R., Huang J., Zhao W., Rahman M., Liao H., Fu Q. 2020, The Fabrication and Evaluation of a Potential Biomaterial Produced with Stem Cell Sheet Technology for Future Regenerative Medicine. stem cell Interational, 2020(5): 1-12.
 
فصلنامه زیست شناسی جانوری
دوره 13، شماره 3
فروردین 1400
صفحه 41-53

فایل ها

اشتراک گذاری

ارجاع به این مقاله

آمار