Osteokondral Doku Mühendisliğinde Tabakalı Hibrit Yapıların Geliştirilmesi

Abstract

In this study, ceramic and decellularized bone inserted Poly(glycerol sebacate) (PGS) elastomer-based scaffolds for use in bone tissue engineering and osteochondral tissue engineering applications were designed, produced and characterized, and their efficiency in bone and osteochondral tissue engineering was determined by in-vitro cell culture studies. In the first part of the thesis, the PGS elastomer was functionalized with different amounts of decellularized bone (deK) extracellular matrix (ECM) to be used as a subchondral bone scaffold. In the second part of the thesis, the PGS elastomer was functionalized with different amounts of β-TCP to be used as a subchondral bone tissue scaffold. In addition, the complex interaction between pore size and PGS elastomer composition in the context of osteogenic differentiation was revealed in the first two chapters of the thesis. In the last part of the thesis, biphasic osteochondral scaffolds were produced and characterized by using ceramic and bone-inserted subchondral scaffolds with proven osteogenic activity on mesenchymal stem cells, and their osteochondrogenic activities were determined by in-vitro cell culture studies. The first two parts of the thesis work were carried out at Trinity College Dublin, Trinity Biomedical Sciences Institute, supported by TÜBİTAK, BİDEB, 2214A- international research scholarship program for doctoral students. The third part of the thesis was supported by the Hacettepe University BAP commission with the project number 19746.

In the first part of the thesis, a biocompatible and bioactive scaffold based on PGS elastomer was developed for bone tissue engineering with a suitable microstructure and containing tissue-specific clues for osteogenic lineage commitment of MSCs. After the PGS elastomer was functionalized with 14% and 28% by weight decellularized bone (deK) ECM source to increase its osteoinductive potential. In order to determine the preferred pore size for in-vitro osteogenesis, PGS/deK scaffolds with two different pore sizes, small (100–150 μm) and large (250–355 μm), were produced by salt removal method. Functionalization of PGS/deK subchondral scaffolds with decellularized bone ECM increased the initial cell adhesion efficiency to 100% and also improved osteogenic differentiation. It also increased the mechanical strength of the scaffold up to 165 kPa. Along with the contribution of bone to the PGS structure, it has also been shown to be successfully adapted with an improved degradation rate/pH variation and wettability. The small pore (KG) PGS/deK scaffold with 28% bone doped lost 12% mass after 28 days of incubation and the pH value of the medium was measured as approximately 7.14. In vitro osteogenic differentiation of MSCs in PGS/deK scaffolds suggests a better commitment to osteogenic lineage of scaffolds with small pore size and 28% (w/w) bone doped, as evidenced by calcium quantification, Alkaline phosphatase (ALP) expression, and alizarin rejection. provided has been demonstrated. In this study, the appropriate pore size and the amount of decellularized bone ECM for osteoinduction of MSCs through PGS/deK scaffolds adapted as bone tissue scaffolds have been demonstrated and it has been demonstrated that they can be used successfully in bone tissue engineering applications.

In the second part of the thesis, a biocompatible and bioactive subchondral scaffold based on PGS elastomer with tissue-specific clues and an appropriate microstructure was developed. Accordingly, the PGS elastomer was functionalized with 14% and 28% β-TCP additives by weight in order to increase its osteoinductive potential. Likewise, to determine the preferred pore size for in-vitro osteogenesis, PGS/β-TCP scaffolds with two different pore sizes, small (100–150 μm) and large (250–355 μm), were produced by salt removal method. The elastic compressive modulus was measured as 30.56 kPa with 14% β-TCP contribution to the PGS structure. No further improvement was observed in the elastic compression modulus of the scaffold with increasing amount of β-TCP in the structure. However, the hydrophilicity of the scaffolds increased significantly with the contribution of β-TCP. With the increase in the amount of ceramic added to the PGS structure, the cell seeding efficiency was measured as 100%. Small-pore scaffold without β-TCP inclusion (PGS-0TCP-KG) showed 40.7% cell adhesion, while PGS-5TCP-KG scaffold showed 58.3% and PGS-15TCP-KG scaffolds with 100% yield. Despite the high β-TCP inclusion in the structure, the cell seeding efficiency in PGS-15TCP-BG scaffold decreased to 32.3% due to the increase in the pore size of the scaffold. SEM analysis of porcine BMSCs seeded on PGS/β-TCP bone scaffolds showed that the small-pore scaffold surface exhibited wide spreading area and the correct contractile cell phenotype could be beneficial for osteogenic lineage commitment. After 21 days of culture in in-vitro osteogenic induction medium, the amount of Ca as a late marker of osteogenic differentiation increased in all tissue scaffolds, while the increase in ALP expression was prolonged until the 21st day. Mineralization of scaffolds was determined with Alizarin red as a further marker of osteogenic differentiation of MSCs. Intense positive Alizarin red was observed especially in small-pore PGS/β-TCP scaffolds. In this study, the appropriate pore size and amount of β-TCP for osteoinduction of MSCs were demonstrated through PGS/β-TCP scaffolds adapted as bone tissue scaffolds, and it was demonstrated that they could be used successfully in bone tissue engineering applications.

In the third part of the thesis, biphasic osteochondral scaffolds were produced by using PGS-15deK-KG and PGS-15TCP-KG scaffolds, which showed the best efficiency in the first two sections as subchondral scaffolds. By keeping the pore size in the range of 0-45 μm for the chondral layer, a certain amount of salt was transferred onto the PGS/deK and PGS/β-TCP subchondral scaffold molds, with a chondral layer thickness above 1 mm, to form a mold of biphasic osteochondral scaffolds, and in-situ polymerization of PGS was created in one go. The biphasic osteochondral scaffolds were named OC-PGS-15deK and OC-PGS-15TCP, while the biphasic control scaffold without ceramic or bone insertions was named OC-PGS-Ktrl. It was also determined that the highly hydrophilic biphasic osteochondral tissue scaffolds were also cross-linked over 90%. Biphasic osteochondral scaffolds had a degradation rate of 13% after 28 days of incubation, while interlayer integrity was preserved, and the media pH value was measured as 7.2. Cell seeding into the chondral layer of biphasic osteochondral tissue scaffolds was accomplished by encapsulation of decellularized and neutralized cartilage ECM pre-gel. Biphasic osteochondral tissue scaffolds were cultured in vitro for 28 days in osteochondrogenic differentiation medium containing both osteogenic and chondrogenic differentiation cues, by co-culture of rat BMSC to the chondral layer and MC3T3-E1 preosteoblastic cells to the subchondral layer. After 28 days of culture, the amount of DNA and GAG in the chondral and subchondral bone layers was measured independently for each layer and also cumulatively for entire biphasic scaffold. Histological analyzes revealed clues to osteochondral tissue formation.