مطالعه اثر داربست پلی-ال-لاکتیک-‌‌اسید در تمایز استخوانی سلول‌های بنیادی مزانشیمی جدا شده از بافت چربی اسب

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه علوم دامی، دانشکده علوم کشاورزی و منابع طبیعی، دانشگاه گنبد کاووس،گنبد کاووس، گلستان، ایران

2 گروه علوم دام و طیور، پردیس ابوریحان، دانشگاه تهران، ورامین، ایران

3 گروه زیست فناوری، پردیس علوم، دانشگاه تهران، تهران، ایران

چکیده

هدف از این مطالعه بررسی امکان جداسازی سلول‌های بنیادی مزانشیمی از بافت چربی اسب و مطالعه بازده تمایز استخوانی این سلول‌ها در شرایط کشت تک بعدی (در ظرف کشت بافت پلاستیکی) و سه بعدی (روی داربست های پلی-ال-لاکتیک-اسید) بود. بافت چربی به روش بیوپسی از ناحیه قاعده دم اسب تهیه  و سلول‌های بنیادی مزانشیمی با کمک هضم مکانیکی و آنزیمی از بافت چربی جدا شد. سلول‌ها بنیادی جداشده، در دو شرایط جداگانه شامل شرایط ظرف کشت بافت پلاستیکی (گروه شاهد) و شرایط داربست پلی-ال-لاکتیک-اسید با سه تکرار، به رده استخوان تمایز داده شدند. در طول 21 روز تمایز آزمونهای آلیزارین رد، اندازه‌گیری آنزیم آلکالین‏فسفاتاز و اندازه‌گیری میزان کلسیم رسوبی برای ارزیابی راندمان هر یک از این شرایط در تمایز سلول‌ها به رده استخوان مورد استفاده قرار‌گرفت. دادهای حاصل در قالب طرح کاملاً تصادفی تجزیه شدند. رنگ‌آمیزی آلیزارین رد بهعنوان یک آزمون کیفی نشان داد در هر دو شرایط سلول‏های بنیادی مزانشیمی مشتقشده از بافت چربی اسب می‌توانند به رده استخوان تمایز یابند. با این حال سلول‌های بنیادی مزانشیمی کشت داده شده روی داربست پلی-ال-لاکتیک-اسید در مقایسه با سلول‌های کشت داده شده در شرایط ظرف کشت بافت پلاستیکی دارای فعالیت آنزیم آلکالین‏فسفاتاز و مقدار کلسیم رسوبی بیشتری بودند. یافتههای این مطالعه نشان داد استفاده از داربستهای پلی-ال-لاکتیک-اسید امکان رشد و تمایز بهینه سلول‌های بنیادی مزانشیمی مشتقشده از بافت چربی اسب را به رده استخوانی فراهم می‌سازد.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of poly (L-lactide) nanofiber scaffolds on osteogenic differentiation of equine adipose tissue-derived mesenchymal stem cells

نویسندگان [English]

  • behnaz bageshlooyafshar 1
  • Reza Rahchamani 1
  • Abdollah Mohammadi-Sangcheshmeh 2
  • Ehsan Seyedjafari 3
  • Yussof Mostafaloo 1
1 2. Department of Animal Sciences, College of Agriculture and Natural Resources, University of Gonbad-e Qabus ,golestan,iran
2 Department of Livestock and Poultry Sciences, ABOURYHAN, University of Tehran,varamin,iran
3 Department of Biotechnology, College of Science, University of Tehran ,Tehran, Iran
چکیده [English]

This study was conducted to investigate the differentiation potential of equine adipose-derived mesenchymal stem cell into bone in single-dimensional culture system (in plastic tissue culture) and in three-dimensional system (on poly-l-lactic acid scaffolds; PLLA). A porous structure that allows use of three-dimensional distribution and provides optimal growth of cells is of great clinical significance in the field of tissue engineering. In current study using equine adipose-derived stem cells (ASCs), we intended to compare the osteogenic differentiation potential of PLLA nanofibrous scaffold with tissue culture plastic (TCP). Adipose tissues were collected from 3 adult horses, and ASCswere isolated by enzymatic digestion. PLLA nanofibrous scaffold was successfully prepared using a phase separation method. Viability and growth characteristics of ASCs on TCP and scaffold were investigated by tetrazolium (MTT) based colorimetric assay. Alizarin Red staining was performed for determination of calcium deposition following osteogenic differentiation. Furthermore, other common osteogenic markers such as alkaline phosphatase (ALP) activity, and calcium content were also analyzed. Our data showed that the PLLA scaffold had no detrimental effect on the cell growth rate as evaluated by MTT assay. However, ASCs that differentiated on PLLA nanofibrous scaffolds indicated higher ALP activity and more calcium content than that on TCP. Adequate proliferation rate and higher expression of osteogenic markers of stem cells cultured on PLLA nanofibrous scaffolds provide this scaffold as a suitable substrate to support proliferation and differentiation of ASCs in equine.

کلیدواژه‌ها [English]

  • adipose tissue
  • Mesenchymal stem cells
  • osteogenic differentiation
  • poly-L-lactic-acid
  • scaffolds
1.     Afizah H, Yang Z, Hui JH, Ouyang HW and Lee E (2007) A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Engineering. 13(4): 659-6.
2.     Álvarez-Viejo M, Menéndez-Menéndez Y and Otero-Hernández J (2015) CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture. World journal of Stem Cells. 7(2): 470.
3.     Avril P, Le Nail L R, Brennan M A, Rosset P, De Pinieux G, Layrolle P, Heymann D, Perrot P and Trichet V (2016)  Mesenchymal stem cells increase proliferation but do not change quiescent state of osteosarcoma cells: Potential implications according to the tumor resection status. J Bone Oncol. 5(1): 5-14.
4.     Burgos-Silva M, Semedo-Kuriki P, Donizetti-Oliveira C, Costa PB, Cenedeze MA, Hiyane MI, Pacheco-Silva A and Camara NO (2015) Adipose Tissue-Derived Stem Cells Reduce Acute and Chronic Kidney Damage in Mice. PLoS One. 10(11): 142-183.
5.     Busser H, Najar M, Raicevic G, Pieters K, Velez Pombo R, Philippart P, Meuleman N, Bron D and Lagneaux L (2015) Isolation and characterization of human mesenchymal stromal cell subpopulations: comparison of bone marrow and adipose tissue. Stem cells and Development. 24(18): 2142-2157.
6.     Chi K, Fu RH, Huang YC, Chen SY, Lin SZ, Huang PC, Lin PC, Chang FK and Liu SP (2016) Therapeutic Effect of Ligustilide-Stimulated Adipose-Derived Stem Cells in a Mouse Thromboembolic Stroke Model. Cell Transplant. 25(5): 899-912.
7.     Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D and Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8(4): 315-317.
8.     Dvorak MM, Siddiqua A, Ward DT, Carter DH, Dallas SL, Nemeth EF and Riccardi D (2004) Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proceedings of the National Academy of Sciences of the United States of America. 101(14): 5140-5145.
9.     Fraser JK, Zhu M, Wulur I and Alfonso Z (2008) Adipose-derived stem cells. Mesenchymal Stem Cells: Methods and Protocols. 59-67.
10.   Friedenstein A, Piatetzky-Shapiro I and Petrakova K (1966) Osteogenesis in transplants of bone marrow cells. Development. 16(3): 381-390.
11.   Frölich K, Scherzed A, Mlynski R, Technau A, Hagen R, Kleinsasser N and Radeloff A (2011). Multipotent stromal cells for autologous cell therapy approaches in the guinea pig model. ORL. 73(1): 9-16.
12.   Guest DJ, Smith MRW and Allen WR (2008) Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: Preliminary study. Equine Veterinary Journal. 40(2): 178-181.
13.   Kim EH and Heo CY (2014) Current applications of adipose-derived stem cells and their future perspectives. World Journal Stem Cells. 6(1): 65-68.
14.   Kim JH, Choi SC, Park CY, Park JH, Choi JH, Joo HJ, Hong SJ and Lim DS (2016) Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses. PLoS One. 11(2): 147-153.
15.   Koch TG, Heerkens T, Thomsen PD and Betts DH (2007) Isolation of mesenchymal stem cells from equine umbilical cord blood. BMC Biotechnology. 7(1): 26.
16.   Koerner J, Nesic D, Romero JD, Brehm W, Mainil‐Varlet P and Grogan SP (2006) Equine peripheral blood‐derived progenitors in comparison to bone marrow‐derived mesenchymal stem cells. Stem Cells. 24(6): 1613-1619.
 17.   Lettry V, Hosoya K, Takagi S and Okumura M (2010) Coculture of equine mesenchymal stem cells and mature equine articular chondrocytes results in improved chondrogenic differentiation of the stem cells. Japanese Journal of Veterinary Research. 58(1): 5-15.
18.   Lim J-H, Boozer L, Mariani CL, Piedrahita JA and Olby NJ (2010) Generation and characterization of neurospheres from canine adipose tissue-derived stromal cells. Cellular Reprogramming (Formerly Cloning and Stem Cells). 12(4): 417-425.
19.   Marino G, Rosso F, Cafiero G, Tortora C, Moraci M, Barbarisi M and Barbarisi A (2010) β-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study. Journal of Materials Science: Materials in Medicine. 21(1): 353-363.
20.   Mohammadi-Sangcheshmeh A, Shafiee A, Seyedjafari E, Dinarvand P, Toghdory A, Bagherizadeh I, Schellander K, Cinar MU and Soleimani M (2013) Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius) In Vitro Cellular & Developmental Biology-Animal. 49(2): 147-154.
21.   Nathan S, De SD, Thambyah A, Fen C, Goh J and Lee EH (2003) Cell-based therapy in the repair of osteochondral defects: a novel use for adipose tissue. Tissue Engineering. 9(4): 733-744.
22.   Neupane M, Chang C-C, Kiupel M and Yuzbasiyan-Gurkan V (2008) Isolation and characterization of canine adipose-derived mesenchymal stem cells. Tissue Engineering Part A. 14(6): 1007-1015.
23.   Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM and Gimble JM (2007) Characterization of Equine Adipose Tissue-Derived Stromal Cells: Adipogenic and Osteogenic Capacity and Comparison with Bone Marrow-Derived Mesenchymal Stromal Cells. Veterinary Surgery. 36(7): 613-622.
24.     Xu Y, Liu L, Li Y, Zhou C, Xiong F  Liu Z, Gu R, Hou X and Zhang C (2008) Myelin-forming ability of Schwann cell-like cells induced from rat adipose-derived stem cells in vitro. Brain Research. 1239: 49-55.
25.     Yoon E, Dhar S, Chun DE, Gharibjanian NA and Evans GR (2007) In vivo osteogenic potential of human adipose-derived stem cells/poly lactide-co-glycolic acid constructs for bone regeneration in a rat critical-sized calvarial defect model. Tissue Engineering. 13(3): 619-627.