3B basılı hücre taşıyan gelma hidrojelleri ile kornea stroma mühendisliği.

Tissue engineering is an emerging field which aims to replace missing or damaged tissues and restore their functions. Three dimensional (3D) printing has recently been in the heart of tissue engineering which enables design and production cell loaded or cell carrying scaffolds with shapes, sizes, and porosities specific for the patients. Corneal damages and diseases are the third major cause for blindness after cataract and glaucoma. Transplantation and keratoprostheses are the only acceptable treatments for severe corneal damages despite their limitations. In the current study a 3D bioprinted stromal equivalent was designed to mimic the ultrastructure of the native tissue. The construct was produced by bioprinting a keratocyte loaded GelMA solution, using a model created by Sketchup program resulting in a stable, highly transparent, cell loaded hydrogels to serve as a corneal stroma. In order to carry on physical characterization and study the in vitro and in vivo performance of the constructs “slab” equivalents of the 3D printed constructs were used. GelMA slabs prepared from solutions with different concentrations (5, 8, 10 and 15%, w/v in PBS) showed that water content of the hydrogels decreased with increasing concentration and UV duration. Stability of the hydrogels studied by incubation in PBS and collagenase type II was also enhanced with increased hydrogel concentration. Transparency of the hydrogels was over 90% at 700 nm and comparable with the native cornea. Transparency of the constructs did not change during enzymatic degradation tests. Human keratocytes in the native stroma are elongated and interact with each other. Optimum concentration of the cells in the hydrogels was 1x106 cells/mL enabled interactions between the cells. Live-Dead cell viability assay showed that over 90% of the cells were alive and homogenously distributed in the hydrogels. Alamar Blue cell proliferation assay showed continuous cell proliferation, Draq5 Phalloidin stained cells illustrated network like structures, and immunofluorescence studies showed synthesis of representative collagens (Collagen types I and V) and proteoglycans (decorin and biglycan) of the cells in the hydrogels. HEMA, another hydrogel forming polymer widely used as a biomaterial in contact lenses, was incorporated into the GelMA structure to enhance the mechanical properties of the constructs. Compressive modulus of the constructs significantly increased in the presence of HEMA but number of cells loaded in the hydrogels decreased. Collagen types I and V synthesis by the cells in GelMA-HEMA hydrogels were also lower than in GelMA hydrogels. Pure GelMA hydrogels, therefore, were used in 3D bioprinting and in vivo studies. In order to have pattern reproducibility in 3D printing, the printing conditions were optimized by changing movement speed of the nozzle in x-y direction (Fxy, mm/min) and the spindle speed (R/S, Dots/min). 3D printed hydrogels were very stable in PBS during three weeks of incubation (92% remained). Live-Dead cell viability assay showed 98% cell viability on Day 21 indicating that printing conditions did not harm the cells. Mechanical properties of the cell loaded 3D printed hydrogel increased significantly during three weeks of incubation. Transparency of cell loaded and cell free hydrogels was studied for three weeks and was over 80% (at 700 nm) at all time points which is comparable to that of the native cornea (90% at 700 nm). The in situ and in vitro performances of the three selected 3D printed hydrogels were similar. In vivo performance of the GelMA15-Slab (Cell free) hydrogel was tested on rabbits. It was implanted into a mid-stromal pocket without suture fixation and observed for 8 weeks under slit lamp. No edema, ulcer formation, inflammation or infection was detected in both control (sham) and hydrogel implanted corneas. Slight vascularization on week 3 was treated with one dose of anti-VEGF application. Hematoxylin and Eosin staining showed that the hydrogel was integrated with the host tissue and there was only a minimal foreign body reaction. Moreover, results demonstrated some degradation of the construct in 8 weeks as evidenced by the decrease of its diameter from 4 mm to 2.6 mm. Thus, the 3D printed cell loaded GelMA hydrogels could mimick the native ultrastructure of the corneal stroma with excellent transparency, adequate mechanical strength, high cell viability and proliferation. In vivo studies with cell-free slabs further demonstrated that the hydrogels could be used in corneal tissue engineering applications.Ph.D. - Doctoral Progra

FEN BİLİMLERİ ENSTİTÜSÜ/LİSANSÜSTÜ TEZ PROJESİ

ÇOK KATMANLI KORNEA YAPILARI İÇİN YÖNLENDİRİLMİŞ HÜCRE TABAKALARI VE S-IPN HİDROJELLE

Cell loaded 3D bioprinted GelMA hydrogels for corneal stroma engineering

Tissue engineering aims to replace missing or damaged tissues and restore their functions. Three-dimensional (3D) printing has been gaining more attention because it enables the researchers to design and produce cell loaded constructs with predetermined shapes, sizes, and interior architecture. In the present study, a 3D bioprinted corneal stroma equivalent was designed to substitute for the native tissue. Reproducible outer and inner organization of the stroma was obtained by optimizing printing conditions such as the nozzle speed in the x-y direction and the spindle speed. 3D printed GelMA hydrogels were highly stable in PBS during three weeks of incubation (8% weight loss). Live-Dead cell viability assay showed 98% cell viability on day 21 indicating that printing conditions were suitable for keratocyte printing. Mechanical properties of the cell loaded 3D printed hydrogels increased 2-fold during this incubation period and approached those of the native cornea (ca. 20 kPa vs. 27 kPa, respectively). Expression of collagens types I and V, and proteoglycan (decorin) in keratocytes indicates maintenance of the phenotype in the hydrogels. Transparency of cell-loaded and cell-free hydrogels was over 80% (at 700 nm) during the three week culture period and comparable to that of the native cornea (85%) at the same wavelength. Thus, GelMA hydrogels bioprinted with keratocytes mimic the biological and physical properties of the corneal stroma with their excellent transparency, adequate mechanical strength, and high cell viability

Çok Katmanlı Kornea Yapıları için Yönlendirilmiş Hücre Tabakaları ve S-IPN Hidrojeller

Projenin amacı, kornea doku mühendisliğinde kullanılmak üzere insan keratosit hücrelerinden oluşan yönlendirilmiş hücre katmanları ve metakrile jelatin ile doğal göz yapısına benzer hidrojeller üretmek ve bu yapıları in vitro ve in vivo koşullarda denemektir. Dünya çapında yaklaşık 10 milyon insan kornea hastalıkları ve/veya yaralanmalarından dolayı görme kaybı yaşamaktadır. Geri dönüşsüz görme kaybının günümüzde kabul edilen tek tedavi yöntemi donör doku kullanılarak yapılan doku naklidir. Fakat, iyi kaliteli donör dokusunun sınırlı miktarda oluşu, hastalık bulaşma riskinin bulunması ve dokunun bağışıklık sistemi tarafından reddedilmesi gibi sebeplerden dolayı kornea yerine kullanılabilecek yapay biyomalzemelere acil gereksinim vardır.Bu çalışmada oluşturulacak 3 boyutlu hidrojeller diğer biyomalzemelere oranla önemli avantajlar sunmaktadır. Kollajen korneanın %70’ini oluşturur ve bu kollajen fiberlerinin mikro yapısı korneanın ışık geçirgenliği ve sağlamlığı açısından çok önemlidir. Jelatin kollajenin geri dönüşümsüz olarak hidroliz edilmiş halidir ve bir çok doku mühendisliği çalışmalarında başarıyla uygulanmaktadır. Bu çalışmada metakrile jelatin kullanılması ve aynı zamanda keratositlerin korneanın mikroyapısına uygun şekilde yönlendirilmiş olarak yapının içinde olması projenin avantajlarından bazılarıdır