Menisküs Doku Mühendisliği İçin Hibrit Doku İskelelerin Geliştirilmesi

Abstract

Treatment of meniscal tears that occur as aging, injuries, and various traumas is one of the most performed in orthopedic surgery. The increasing rate of meniscal injuries indicates the urgent need to develop an effective repair strategy. A biomaterial that can mimic the microscopic and macroscopic properties of natural meniscus tissue is still under investigation. Within the scope of the thesis study, acellular hybrid scaffolds consisting of in situ cross-linkable 3D interpenetrating mesh structures were obtained by decellularization of the meniscus tissues, in which the ultrastructure was preserved and following by integrating the gel system. In the first section of studies, the comparison between decellularization methods was made; supercritical carbon dioxidee (scCO2) technology was tried by the original method for meniscus tissue. By combining physical pretreatment, enzymatic agitation with trypsin agent (0,05%), and SDS (0,5 %) chemical agitation was found out an optimum method that ensures decreased 82% genomic DNA. For scCO2 decellularization protocol, physical pretreatment is performed and after that penetration depth is yielded by trypsin agent. And then decellularization group with diminished 76 % genomic DNA is obtained by 37°C temperature and 4500 psi pressure. Decellularized tissue was confirmed by H&E and DAPI staining also. Masson Trichrome and Safranin/FCF staining viewed collagen fibers and proteoglycans structure in tissue. Biochemical studies that synchronized to histologic studies carried out collagen and GAG amount to determine whether decreased or not. While collagen amount is not changed in decellularized groups according to hydroxyproline assay, GAG amount in conventional group and scCO₂ group was decreased by respectively 48% and 58% seeing that DMMB analysis. Both two processes were caused by decreased wet and dry weight of decellularized tissues, but scCO₂ group has been in a higher decline than conventional. The organization of collagen fibrils was scanned by SEM analysis, and it was observed minor loosen around fibers after decellularization. In the native tissue (22.8 ± 0.91 MPa) compressive modulus was diminished as to 15,26 ± 0.28 MPa in conventional and 14.49 ± 0.48 MPa in scCO₂ group based on GAG reduction. Both processes were obtained beyond 75% cell viability in cytotoxicity analysis via MTT extraction method. In the second section of the study, with the intent of synthesizing hybrid constructs, there was working on optimization of polymer blends and photoinitiators choosing. Firstly, the gelatin molecule was functionalized by adding methacrylic anhydride and the degree of functionalization was found to be 75% by ¹HNMR analysis. Owing to overlaying absorbances of LAP photoinitiator and UVA light spectrum was resulted with provided fast in situ polymerization. GelMA and GelMA supported hydrogels were conducted both of I2959 and LAP. Swelling, mechanical and FTIR analysis were got on hydrogels. It has been observed that 2-blend (GelMA/PEGDMA) hydrogels are more stable and durable structures than single-phase ones. With the incorporating HAMA molecules, 3-blends hydrogels were the highest mechanical strength and were more resistant to swell than other blends. G-Hybrid, PG-Hybrid, and PGH-Hybrid constructs that generated final alternative decellularized scaffolds were produced by LAP photoinitiators. Following pre-gels immersed through sonicator within 5 mm dry scaffolds was incubated 37°C for avoiding gelation before crosslinking. Subsequently, acellular scaffolds that trapped hydrogels are polymerized with UVA light by turning both sides. Chemical changes were determined with FTIR and presented characteristic peaks in native, decellularized and hybrid structures. Water uptake capacities in cross-linked hybrid structures were measured in swelling studies; it was found that hybrid scaffolds are similar to native tissues about swelling phenomena. Enhanced mechanical properties were seen at crosslinked scaffolds by compressive analysis and also in the PGH-hybrid group was found the highest crosslink density due to 3 polymer blends. TGA and DSC analysis were used to establish thermal properties in acellular scaffolds. Protein denaturation where H-bond broke down between triple helix structure in collagen fibers and decomposition transition temperatures were improved by adding hydrogels to acellular scaffolds according to these analyses. Hydrogels in scaffolds that are covering collagen fibrils were monitored with SEM. Cross-linked acellular scaffolds have exhibited a behavior close to native tissues with below 25% mass loss in PBS and enzymatic degradation. Cell viability and proliferation were examined through Alamar Blue, on 1st, 4th, 7th days, with L929 cell line in acellular hybrid scaffolds. On the first day, cell viability in hybrid constructs has been above 80% while on the 7th day it is established to further increase and close to the control group. Such rising that seen in the hybrid structure have proved that GelMA and HAMA biopolymers are attractive for cell migration and proliferation. It was concluded that the created biomaterial could be used in meniscus tissue engineering with its tunable physicochemical and mechanical properties.