Comparison of amniotic membrane (AM) transplantation and mesenchymal stem cell (MSC) transplantation in corneal alkaline burn-induced stem cell deficiency in rabbits

Abstract

The objective of this study was to compare the transformation of genus-specific rabbit bone marrow (BM)-derived MSCs into corneal epithelium with acellular AM and MSC transplantation. Alkaline burn-induced 3rd degree limbal deficiency was created in 24 female rabbit eyes (including the cornea and the limbus). Each eye was administered 1N NaOH for 60 s with the use of 12-mm ring, and was then flushed with isotonic NaCl. The 24 rabbit eyes were divided into 3 groups, as follows: Group 1 (n = 8) had placenta-derived acellular AM, group 2 (n = 8) received no additional treatment, and group 3 (n = 8) had MSC (embedded in amniotic membrane) transplanted into the cornea. Group 3 was divided into 2 subgroups. In subgroup 3a marked MSC with DAPI [4,6,diamidino-2-phenylindole]) were used, and the rabbits were euthanized 12 d after transplantation, and their corneal blocks were examined with immunofluorescence microscopy. Subgroup 3b rabbits were euthanized 2 months after transplantation, and subgroup 3b, group 1, group 2 corneal blocks were evaluated histopathologically and immunohistochemically. The results were evaluated statistically. Conjunctivalization in group 3 was less severe than in groups 1 and 2. MSCs facilitated and accelerated the regeneration process in group 3. DAPI-positive MSCs were observed in the corneal endothelium, as well as in the inner nuclear layer of the retina in subgroup 3a. Cellular and acellular AM used for transplantation acted as barrier that prevented corneal perforation. MSCs had a beneficial effect on the regeneration process, and DAPI-marked MSCs were observed both in retina and cornea

Keywords

• Amniotic membrane
• cornea
• mesenchymal stem cell
• rabbit

Tavşanlarda korneal alkali yanık ile oluşturulan kök hücre yetmezliğinde amniyotik membran (AM)ile mezenkimal kök hücre (MKH) transplantasyonunun karşılaştırılması

Abstract

Bu çalışmanın amacı tavşana özgü kemik iliği (KI) kaynaklı MKH’lerin kornea epiteline dönüşümü hücresiz AM ile MKH transplantasyonunun karşılaştırılmasıdır. Yirmi dört adet dişi tavşanın gözünde alkali yanık ile 3. derece limbal yetersizlik oluşturuldu (kornea ve limbusu da içeren). 12 mm çapında halka kullanılarak 1N NaOH her bir göze 60 sn boyunca uygulandı ve izotonik NaCl ile yıkandı. 24 adet tavşanın gözleri 3 gruba ayrıldı. Grup 1’e (n=8) plasenta kaynaklı hücresiz AM uygulaması, grup 2’de (n=8) ek bir tedavi uygulaması yapılmadı, grup 3’te ise (n=8) kornea da MKH (AM’a yerleştirilen) transplantasyonu gerçekleştirildi. Grup 3, 2 alt gruba ayrıldı (altgrup 3a’da bulunan MKH’ler DAPI ile işaretlendi (4,6,diamidino-2-phenylindole)). Altgrup 3a’da bulunan tavşanlara transplantasyondan 12 gün sonra ötenazi uygulandı ve bu tavşanlara ait korneal bloklar immunfloresan mikroskopta incelendi. Altgrup 3b’de bulunan tavşanlara ise transplantasyondan 2 ay sonra ötenazi uygulandı ve altgrup 3b ve 2. grubun korneal blokları 2 ay sonra histopatolojik ve immunhistokimyasal muayene ile değerlendirildi. İstatistiksel değerlendirmede tüm grupların erken ve geç dönem oftalmaskopik muayene bulgularında, ikili orantı (two proportional) testi, histopatolojik bulgularında Kruskal Vallis testi, immunhistokimyasal bulgularında ise varyans analizi kullanıldı. Grup 3’te bulunan konjunktivalizasyon grup 1 ve 2 dekine göre daha az şiddetli olarak izlendi. MKH lerin grup 3 te iyileşmeye olumlu etki ettiği ve süreci hızlandırdığı izlendi. DAPI-pozitif hücrelerin kornea endotelinde ve retinada gözlendiği izlendi. Transplantasyonda kullanılan hücreli ve hücresiz AM‘ların bariyer görevi gördüğü ve korneal perforasyonu engellediği ve MKH’lerin rejenerasyona olumlu etki ettiği izlendi. DAPI ile işaretlenen MKH’ ler retina ve kornea epitelinde gözlendi

Keywords

• Amniyotik membran
• kornea
• mezenkimal kök hücre
• tavşan

References

1. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. (1994): Differentiated cells and the maintenance of tissues 966-982. In: B Alberts (Ed), Molecular Biology of the Cell. Garland Publishing, New York.
2. Ballen PH (1963): Mucous membrane grafts in chemical (lye) burns. Am J Ophthalmol, 55, 302-312.
3. Chee KY, Kicic A, Wiffen SJ (2006): Limbal stem cells: The search for a marker. Clin Experiment Ophthalmol, 34, 64-73.
4. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM (1989): Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells, Cell, 57, 201-209.
5. Darnell JE, Lodish HF, Baltimore D (1989): Development of cell specificity 1026-1028. In: H Lodish (Ed), Molecular Cell Biology. W.H. Freeman and Company, New York.
6. Daya SM, Ilari L (2001): Living related conjunctival limbal allograft for the treatment of stem cell deficiency, Ophthalmology, 108, 126-134.
7. Deda H (2008): Stem Cell Therapies for Neurological Diseases. J Nervous Sys Surgery, 1: 142-152.
8. Dong Y, Roos M, Gruijters T, Donaldson P, Bullivant S, Beyer E, Kistler J (1994): Differential expression of two gap junction proteins in corneal epithelium, Eur J Cell Biol, 64, 95-100.

9. Dua HS (1998): The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol, 82 (12), 1407-1411.
10. Dua HS, Azuara-Blanco A (1999): Allo-limbal transplantation in patients with limbal stem cell deficiency. Br J Ophthalmol, 83, 414-419.
11. Dua HS, Azuara-Blanco A (2000): Limbal stem cells of the corneal epithelium. Surv Ophthalmol, 44, 415–425.
12. Dua HS, Gomes JA , Singh A (1994): Corneal epithelial wound healing, Br J Ophthalmol, 78, 401-408.
13. Espana EM, Grueterich M, Ti SE, Tseng SC (2003): Phenotypic study of a case receiving a keratolimbal allograft and amniotic membrane for total limbal stem cell deficiency. Ophthalmology, 110, 481-486.
14. Guo T, Wang W, Zhong J, Chen X, Li BZ, Li LS (2006): Experimental study on repairing damage of corneal surface by mesenchymal stem cells transplantation. Zhonghua Yan Ke Za Zhi, 42, 246-250.
15. Hosseini H, Nejabat M (2007): A potential therapeutic strategy for inhibition of corneal neovascularization with new anti-VEGF agents. Med Hypotheses, 68, 799-801.
16. Hughes WF (1946): Alkali burns of the eye; clinical and pathologic course. Arch Ophthal, 36, 189-214.
17. Hughes WF (1946): Alkali burns of the eye; review of the literature and summary of present knowledge, Arch Ophthal, 35, 423-449.
18. Kasper M, Moll R, Stosiek P, Karsten U (1988): Patterns of cytokeratin and vimentin expression in the human eye, Histochemistry, 89, 369–377.
19. Kenyon KR, Rapoza PA (1995): Limbal allograft transplantation for ocular surface disorders, Ophthalmology, 102 (suppl), 101-102.
20. Kenyon KR, Tseng SC (1989): Limbal autograft transplantation for ocular surface disorders, Ophthalmology, 96, 709-722.
21. Kim JC, Tseng SC (1995): Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas, Cornea, 14, 473-484.
22. Kirschstein R (2001): Stem Cells: scientific progress and future research directions report. National institutes of health, June ES-2/ES-6.
23. Lauweryns B, Van den Oord J.J, Missotten L (1993): The transitional zone between limbus and peripheral cornea. An immunohistochemical study, Invest Ophthalmol Vis Sci, 34, 1991–1999.
24. Lavker RM, Tseng SC, Sun TT (2004): Corneal epithelial stem cells at limbus: looking at some old problems from a new angle, Exp Eye Res, 78, 433-446.
25. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remsberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringden O (2008): Mesenchymal stem cells for the treatment of steroid-resistant, severe, acute graft-versus- host disease: a phase II study. Lancet, 371, 1579-1586.
26. Li W, Hayashida Y, Chen YT, Tseng SC (2007): Niche regulation of corneal epithelial stem cells at the limbus. Cell Res, 17, 26-36.
27. Lohrum MAE, Vousden KH (2000): Regulation and function of the p53-related proteins: same family, different rules. Trends Cell Biol, 10, 197-202.
28. Ma DHK, Chen JK, Zhang F, Lin KY, Yao JY, Yu JS (2006): Regulation of corneal angiogenesis in limbal stem cell deficiency, Prog Ret Eye Res, 25, 563-590.
29. Matic M, Petrov IN, Chen S, Wang C, Dimitrijevich SD, Wolosin JM (1997): Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity. Differentiation, 61, 251-260.
30. Ocarino NM, Bozzi A, Pereira RD, Breyner NM, Silva VL, Castanheira P, Goes AM, Serakides R (2008): Behavior of mesenchymal stem cells stained with 4', 6- diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures. Biocell, 32, 175- 183.
31. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Bondanza S, Ponzin D, McKeon F, De Luca M (2001): p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A, 98, 3156-3161.
32. Rao SK, Rajagopal R, Sitalakshmi G, Padmanabhan P (1999): Limbal allografting from related live donors for corneal surface reconstruction. Ophthalmology, 106, 822- 828.
33. Ren G, Zhang L, Zhao X, Xu G, Zhang Y, Roberts AI, Zhao RC, Shi Y (2008): Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell, 2, 141-150.
34. Roper-Hall MJ (1965): Thermal and chemical burns. Trans Ophthalmol Soc U K, 85, 631-653.
35. Schmer A, Galvin S, Sun TT (1986): Differentiation- related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol, 103, 49-62.
36. Schlötzer-Schrehardt U, Kruse FE (2005): Identification and characterization of limbal stem cells. Exp Eye Res, 81, 247-264.
37. Shimazaki J, Yang HY, Tsubota K (1997): Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology, 104, 2068–2076.
38. Şaroğlu M (1999): Tavşanlarda deneysel olarak oluşturulan kornea alkali yanıkların sağaltımında bazı antikollajenik ilaçların karşılaştırılması üzerine çalışmalar (Ph.D), İÜ Sağlık Bilimleri Enstitüsü, Cerrahi Anabilim Dalı.
39. Taylor RJ, Wang MX (1998): Rate of re-epithelialisation following amniotic membrane transplantation. Invest Ophth Vis Sci, 39, 1038.
40. Tseng SC, Tsubata K (1997): Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol, 124, 825- 835.
41. Wang DY, Hsueb YJ, Yang VC, Chen JK (2003): Propagation and phenotypic preservation of rabbit limbal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci, 44, 4698-4704.
42. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, Mckeon F (1998): P63 a p53 homolog at 3q27-29, encodes multiple products with transactiving, death-inducing and dominant negative activities. Mol Cell, 2, 305-316.
43. Yang A, Schweitzer R, Sun D, Kagdat M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F (1999): p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature, 398, 714-718.
44. Ye J, Yao K, Kim JC (2006): Mesenchymal stem cell transplantation in a rabbit corneal alkali burn model: engraftment and involvement in wound healing. Eye, 20, 482-490.