Keratin temelli iskele üretimi ve doku mühendisliği uygulamaları için etkinliğinin değerlendirilmesi
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Tarih
2017
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Çanakkale Onsekiz Mart Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, keratin insan saçından ekstrakte edilmiş (Tescilli patent: TPE 2014 02104) kollajen ise Rhizostoma pulmo türü denizanasından izole edilmiştir. Elde edilen protein ekstraktlar, Lowry protein, hidroksiprolin ve serbest sülfidril grup tayini ve SDS-PAGE ile karakterize edilmiştir. Denizanası kollajeni ve insan saçı keratin ekstraktları % 3 (w/v) konsantrasyonda ultra saf suda çözülmüş ve dondurma-kurutma tekniği kullanılarak kollajen ve kollajen-keratin süngerimsi iskeleler üretilmiştir. Elde edilen süngerimsi üç-boyutlu doku mühendisliği FTIR, TGA ve SEM ile karakterize edilmiştir. Biyoiskelelerin gözenek çapları kollajen ve kollajen-keratin için yaklaşık olarak 68,65 ± 17 µm ve 64,08 ± 17,93 µm'dir. Ayrıca sıvı yerdeğiştirme yöntemi ile yapılan analiz sonucuna göre kollajen iskelelerin gözenekliliği % 89,93 ± 0,83 (n=3), kollajen-keratin iskelerin gözenekliliği ise % 95,14 ± 0,30 (n=3) olarak hesaplanmıştır. İnsan adipoz doku kaynaklı mezenkimal kök hücreler (iAMKH) kültüre edilmiş ve in vitro çoklu soy farklılaşma kapasitelerini belirlemek amacıyla, adipojenik, osteojenik ve kondrojenik indüksiyonlar gerçekleştirilmiştir. Kök hücreler daha sonra süngerimsi üç-boyutlu DMİ'ler üzerine ekilmiştir ve iskeleler üzerinde hücre canlılığı MTT temelli mitokondriyal dehidrogenaz aktivite testi ile değerlendirilmiştir ve sitotoksik etki gözlenmemiştir. Ayrıca iskeleler üzerindeki hücre yoğunluğunun günden güne arttığı mikroskobik gözlemlerle kanıtlanmıştır. Sonuç olarak, kollajen ve kollajen-keratin iskelelerin doku mühendisliği uygulamalarında kullanılabilecek potansiyel adaylar olabilecekleri düşünülmektedir.
In this study, keratin was extracted from human hair (National registrated patent : TPE 2014 02104) and collagen was also isolated from Rhizostoma pulmo species jellyfishes. Obtained protein extracts were characterized by Lowry protein, hydroxyprolin and free sulfhydryl assay and SDS-PAGE. And then jellyfish collagen and human hair keratin extracts were dissolved in distilled water at a concentration of 3 % (w/v). Collagen and collagen-keratin spongy scaffolds were fabricated via freeze-drying technique. The resultant spongy 3D-TES were characterized by FTIR, TGA and SEM. Pore diametres of collagen and collagen-keratin bioscaffolds were approximately 68,65 ± 17 µm and 64,08 ± 17,93 µm, respectively. The porosity of collagen was determined to be 89,93 ± 0,83 % porous (n=3) and the porosity of collagen-keratin scaffolds was also determined to be 95,14 ± 0,30 % porous (n=3) by liquid displacement analysis. Human adipose-derived mesenchymal stem cells (hAMSCs) were expanded and induced into adipogenic, osteogenic and chondrogenic lineages to determine their in vitro multi-linage differentiation capacity. Stem cells were seeded onto these spongy 3D TES and cell viability was assessed by MTT based mitochondrial dehydrogenase activity test and there was no cytotoxic evidence for these scaffolds. Microscopic observations also showed that cell density increased day by day. In conclusion, it is believed that collagen and collagen-keratin scaffolds have the potential candidates to use in tissue engineering applications.
In this study, keratin was extracted from human hair (National registrated patent : TPE 2014 02104) and collagen was also isolated from Rhizostoma pulmo species jellyfishes. Obtained protein extracts were characterized by Lowry protein, hydroxyprolin and free sulfhydryl assay and SDS-PAGE. And then jellyfish collagen and human hair keratin extracts were dissolved in distilled water at a concentration of 3 % (w/v). Collagen and collagen-keratin spongy scaffolds were fabricated via freeze-drying technique. The resultant spongy 3D-TES were characterized by FTIR, TGA and SEM. Pore diametres of collagen and collagen-keratin bioscaffolds were approximately 68,65 ± 17 µm and 64,08 ± 17,93 µm, respectively. The porosity of collagen was determined to be 89,93 ± 0,83 % porous (n=3) and the porosity of collagen-keratin scaffolds was also determined to be 95,14 ± 0,30 % porous (n=3) by liquid displacement analysis. Human adipose-derived mesenchymal stem cells (hAMSCs) were expanded and induced into adipogenic, osteogenic and chondrogenic lineages to determine their in vitro multi-linage differentiation capacity. Stem cells were seeded onto these spongy 3D TES and cell viability was assessed by MTT based mitochondrial dehydrogenase activity test and there was no cytotoxic evidence for these scaffolds. Microscopic observations also showed that cell density increased day by day. In conclusion, it is believed that collagen and collagen-keratin scaffolds have the potential candidates to use in tissue engineering applications.
Açıklama
Fen Bilimleri Enstitüsü, Biyomühendislik ve Malzeme Mühendisliği Ana Bilim Dalı
Anahtar Kelimeler
Biyomühendislik, Bioengineering
