3 Boyutlu Yazıcı ile Metakrilatlanmış Jelatin/PRP Hidrojel ve Mezenkimal Kök Hücre İçeren Osteokondral Gradyan Sistemlerin Hazırlanması ve in-Vitro Etkinliklerinin Belirlenmesi
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The aim of the presented doctoral thesis is to develop a gradient hydrogel system which loaded adipos tissue derived stem cells (AdMSCs) by using photoactivated platelet-rich plasma (PRP) gradient and 3D bioprinting method for using in the treatment of osteochondral damages. At the first stage of the doctoral thesis, bone tissue engineering studies were carried out. Primarily, differently from the traditional approach, synthesis of highly methacrylated (≥90%) gelatin (Gel-MA) was accomplished with the microwave energy. The bio-ink formed by the mixing of preosteoblastic MC3T3-E1 cells to the Gel-MA was crosslinked with UV (300-500 nm) in the presence of a photoinitiator (Irgacure) to form a stable hydrogel structure after 3D bioprinting. Thus, hydrogels with high mechanical strength, elastic structure, low biodegradation ratio (27%±1.9 at 35 days), and that support in-vitro bone formation were obtained. In the second stage of the thesis, cartilage tissue engineering studies were carried out. In this section, PRP which has growth factors was used as a guiding agent for chondrogenic differentiation. Primarily, PRP was photoactivated by a polychromatic light source (PAC, 600-1200 nm) differently from the traditional activation methods. Thus, controlled and long-term growth factor release was achieved from periodically photoactivated PRP. The PRP bio-ink formed by the addition of Gel-MA and ATDC5 chondrogenic cells to the PRP was photoactivated by PAC after the printing process. Then, the hydrogels were crosslinked with UV in the presence of Irgacure to form a stable hydrogel structure. As a result, hydrogels with high mechanical strength, which release growth factors for long term and constant rate and support in-vitro hyaline cartilage formation were acquired with the periodically PAC application. In the final step of the thesis study, adipose derived mesenchymal stem cells (AdMSCs) were separately mixed to Gel-MA, Gel-MA/PRP1:0.5 and Gel-MA/PRP1:1 solution, and then bio-inks gradually printed and photo crosslinked to form a gradient system. Gradient composite structure formed by the gradual mixing of PRP with Gel-MA has a synergistic effect on the differentiation of AdMSCs into cartilage, interface and bone phases. Mw-Gel-MA with osteoinductive and osteoconductive properties and PRP with activation-dependent release of a wide variety of bioactive agents in its structure have provided a favorable environment for differentiation of AdMSCs into osteochondral tissue. According to the results of cell viability, histology, immunocytochemistry, gene expression and biochemical analysis hydrogel systems which were periodically PAC-treated during cell culture supported the formation of in-vitro osteochondral tissue with progressively hyaline cartilage, hypertrophic cartilage, subchondral bone, and calcified bone. When the related literature is examined, there is no gradient system formed by using 3D bioprinter and AdMSC loaded photoactivated-Gel-MA/PRP composite bioinks and there is no in-vitro osteochondral gradient formation. This study is unique in this respect. Improvement of mechanical strength of Gel-MA with the microwave assisted methacrylation process and photoactivation of PRP/Gel-MA bioink the other unique aspects of the study.