Aylin ÜRKMEZ ŞENDEMİR, Umut Doğu SEÇKİN, Cansu GÖRGÜN, YİĞİT UYANIKGİL

Deri Doku Mühendisliği lı Üç Boyutlu Biyobaskı ve Keratinosit Kültürü

Amaç: Bu çalışmada üç boyutlu biyoyazıcı için uygun bir biyomürekkep (hidrojel - hücre karışımı) üretilerek keratinosit hücrelerinin biyobaskısı hedeflendi.Elde edilen hidrojel yapılı epidermis doku kültüründe hücre proliferasyonu, canlılık analizi, hidrojel içinde hücre dağılımı ve morfolojisi belirlendi. Yöntemler: Hücre kültürlerinde derinin üst tabakası olan epidermiste bulunan keratinosit hücreleri kullanılmıştır. Karakterizasyonu daha önce tamamlanmış olan HS2 insan keratinosit hücre hattı, farklı biyopolimerhidrojeller (jelatin, aljinat, kitosan) ve karışımları içinde süspanse edilip biyobaskı için en uygun hidrojel bulundu. 3B keratinosit kültürleri deney başlatılmasından 1, 4 ve 7 gün sonra canlılık analizi için MTT testine ve hücre dağılımı ve morfolojisinin görüntülenmesi için Hematoksilen- Eozin boyamasına tabi tutuldu. Bulgular: Elde edilen MTT sonuçlarına göre hücre canlılıkları iki boyutlu (2B) kültürde elde edilen keratinosit canlılıklarının %50'sinden yüksek çıkmıştır. MTT sonuçları keratinositlerin üretilen hidrojel yapısı içerisinde tutunarak canlılıklarını sürdürebildiklerini göstermektedir. Elde edilen hücre içerikli polimerik hidrojelin histoloji için kesit alımına uygun olduğu ve alınan kesitlere uygulanan hematoksilen/eosin boyaması sonucunda da hücrelerin hidrojel içinde homojen olarak dağıldıkları ve canlılıklarını korudukları belirlenmiştir. Sonuç: Bu çalışmada oluşturulan epidermis benzeri doku kesitleri üç boyutlu biyoyazıcı ile üretilmiş ve keratin ositlerin hidrojeller içinde canlılıklarını sürdürüp doku iskelelerine tutundukları belirlenmiştir. Üretilen hidrojel biyomürekkeplerin deri doku mühendisliğinde ve özellikle yanıklarda epidermis tabakasının onarımında hızlı ve kişiye özel tedavi seçenekleri sunma potansiyeli vardır.

3D Bioprinting and Cultivation of Keratinocytesfor Skin TissueEngineering

Objective: Inthisstudy, production of a printablebio-ink (cell-hydrogelmixture), and bioprinting of keratinocytes were aimed. Cell proliferation, viability, distribution and morphology were evaluated within the epidermis-likehydrogels. Methods: Keratinocytes from epidermis layer,that is the upperlayer of skin tissue, wereused. Human keratinocyte cellline (HS2) that had been characterized before we resuspen ded with indifferent hydrogels (gelatin, alginate, chitosan) and the irmixtures to obtain the most suitable hydrogel for bioprinting. Bioprinted skin tissues were tested by MTT assay for viability analysis; and hematoxylin- eosin staining was performed for determination of cell distribution and morphology 1, 4 and 7 days after the beginning of the culture. Results: Accordingtothe MTT results, cellviabilitywasabove 50% of that of control 2-dimensional cultures. MTT results areindic ative of cell attachment and viability within the hydrogel structure. The resulting cell-laden polymeric hydrogel constructs were determined to be suitable for histological cross-sectioning, and hematoxylin/eosin histological staining indicated that cells were distributed homogenously within the hydrogels; and they retained their viability. Conclusion: It was shown that epidermis-like tissues were successfully produced using 3D bioprinting, and keratinocytes were able to attach the hydrogels and retain their viability. The se hydrogel bioinks have the potential for skin tissue engineering applications, and particularly in fast and personalized treatment of burns in dermislayer.

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1. Lanza R, Langer R, Vacanti JP. Principles of tissue engineering: Academicpress; 2011.
2. O'brien FJ. Biomaterials & scaffolds for tissue engineering. Mater Today. 2011; 14:88-95.
3. Akdoğan E, Omay SB. Organ Mühendisliğinde Kök Hücre Uygulamaları. Turkiye Klinikleri J Surg Med Sci. 2006; 2:63-8.
4. Kim I-Y, Seo S-J, Moon H-S, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv. 2008; 26:1-21.
5. Murphy SV, Atala A. 3D bioprinting of tissues and organs. NatBiotechnol. 2014;32:773-85.
6. Kolesky DB, Truby RL, Gladman A, Busbee TA, Homan KA, Lewis JA. 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater. 2014;26:3124-30.
7. Bell E, Ehrlich HP, Buttle DJ, Nakatsuji T. Livingtissueformed in vitro and accepted as skinequivalent tissue of full thickness. Science. 1981; 211(4486):1052-4.
8. Billiet T, Vandenhaute M, Schelfhout J, Van Vlierberghe S, Dubruel P. A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials. 2012;33:6020-41.
9. Currie LJ, Sharpe JR, Martin R. Theuse of fibrin glue in skin grafts and tissue-engineered skin replacements. Plast Reconstr Surg. 2001;108:1713-26.
10. Auger FA, Berthod F, Moulin V, Pouliot R, Germain L. Tissue-engineered skin substitutes: from in vitro constructs to in vivo applications. Biotechnol Appl Biochem. 2004;39:263-75.
11. Stanton M, Samitier J, Sanchez S. Bioprinting of 3D hydrogels. Lab Chip. 2015;15:3111-5.
12. Mandrycky C, Wang Z, Kim K, Kim D-H. 3D bioprinting for engineering complex tissues. Biotechnol Adv. 2016;34:422-34.
13. Diduch DR, Jordan LC, Mierisch CM, Balian G. Marrow stromal cells embedded in alginate for repair of osteochondrald efects. Arthroscopy: Arthroscopy. 2000;16:571-7.
14. Priya SG, Jungvid H, Kumar A. Skin tissue engineering for tissue repair and regeneration. Tissue Engineering Part B: Reviews. 2008; 14:105-18.
15. Fragonas E, Valente M, Pozzi-Mucelli M, et al. Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate. Biomaterials. 2000;21:795-801.
16. Yücesan E, Başoğlu H, Göncü B, Kandaş NÖ, Ersoy YE, Akbaş F, Ayşan E. Mikroenkapsüle edilen paratiroid hücrelerinin in-vitro optimizasyonu. Dicle Tıp Derg. 2017;44:373-80.
17. Uslu B, Arbak S. Doku Mühendisliğinde Kitozanın Kullanım Alanları. 2010.
18. Suh J-KF, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 2000;21:2589-98.
19. Kutlu B, Aydın T, Seda R, Akman AC, Gümüşderelioglu M, Nohutcu RM. Platelet-richplasma-loaded chitosan scaffolds: Preparation and growth factor release kinetics. Journal of Biomedical Materials Research Part B: J Biomed MaterRes B Appl Biomater. 2013;101:28- 35.
20. Pandey AR, Singh US, Momin M, Bhavsar C. Chitosan: Application in tissue engineering and skin grafting. J Polym Res. 2017;24:125.
21. Liu X, Smith LA, Hu J, Ma PX. Biomimetic nanofibrous gelatin/apatite composite scaffolds for bone tissue engineering. Biomaterials. 2009;30:2252-8.
22. Hori K, Sotozono C, Hamuro J, et al. Controlledrelease of epidermal growth factor from cationized gelatin hydrogel enhances corneal epithelial wound healing. J Control Release. 2007;118:169-76.
23. Lien S-M, Ko L-Y, Huang T-J. Effect of pore size on ECM secretion and cellgrowth in gelatins caffold for articular cartilage tissue engineering. ActaBiomater. 2009; 5:670-9.
24. Kang H-W, Tabata Y, Ikada Y. Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials. 1999;20:1339-44.
25. Skardal A, Zhang J, Prestwich GD. Bioprinting vessellike constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetra crylates. Biomaterials. 2010; 31:6173-81.
26. Highley CB, Rodell CB, Burdick JA. Direct 3dprinting of shear-thinning hydrogels into self-healing hydrogels. Adv. Mater. 2015;27:5075-9.
27. Hinton TJ, Jallerat Q, Palchesko RN, et al. Threedimensional printing of complex biological structures by free form reversible embedding of suspended hydrogels. Sci. Adv. 2015; 1:e1500758.
28. Colosi C,Shin SR, Manoharan V, et al. Microfluidic bioprinting of heterogeneous 3d tissue constructs using low viscosity bioink. Adv. Mater. 2015,28:677-84.
29. Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol. Biosci. 2006;6:623-33.
30. Duan B, Hockaday LA, Kang KH, Butcher JT. 3D bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. J Biomed Mater Res A.2013; 101:1255-64.
31. Koch L, Deiwick A, Schlie S, et al. Skin tissue generation by laser cell printing. Biotechno Bioeng. 2012; 109:1855-63.
32. Miller JS. The billion cell construct: will threedimensional printing get us there? PLoSBiol. 2014; 12:e1001882.
33. Murphy SV,Atala A. 3D bioprinting of tissues and organs. Nat. Biotechnol. 2014;32:773-85.
34. Blaeser A, Campos DFD, Puster U, Richtering W, Stevens MM, Fischer H. Controlling shear stress in 3d bioprinting is a key factor to balance printing resolution and stem cell integrity. Adv. Healthc. Mater. 2016; 5:326-33.
35. Fedorovich NE, Schuurman W, Wijnberg HM et al. Biofabrication of osteochondral tissue equivalents by printing to pologically defined, cell-laden hydrogel scaffolds. Tissue Eng C Methods.2012;18:33-44.