Poli (2-Hidroksietil-Metakrilat) Temelli Jelatin ve Amino Asit İçeren Süpermakrogözenekli Kriyojel Doku İskelelerinin Geliştirilmesi

Abstract

Cryogels offer the advantage of being used as tissue scaffold in tissue engineering studies due to their large interconnected pores, hydrophilic nature, they do not require toxic substances as solvents and pores forming agents when they synthesis, they are flexible and mechanically durable.

In this study, gelatin containing cryogels were prepared using 2-hydroxyethylmethacrylate (HEMA) as a basic monomer, N-methacryloyl- (L) -histidine methyl ester (MAH) as a comonomer and poly (ethylene glycol) diacrylate (PEGDA) as the crosslinker. The aim of the study is to combine supermacroporous PHEMA cryogels with a natural material such as gelatin and histidine amino acid derived comonomer MAH, to make a tissue scaffold for cells can attach, survive and proliferate. In order to better understand the effects of MAH comonomer and gelatin together, cryogels were prepared to contain different amounts of gelatin and MAH. Accordingly, four different features; poly (2-hydroxyethyl methacrylate) [PHEMA], poly (2-hydroxyethyl methacrylate)-gelatin [PHEMA/GEL], poly (2-hydroxyethyl methacrylate N-methacryloyl- (L) -histidine methyl ester) [PHEMAH] and poly ( hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester)-gelatin [PHEMAH/GEL] and a total of eight cryogel groups were formed depending on the varying gelatin ratio. The usability of prepared cryogels prepared as a scaffold was evaluated. The effects of RGD (Arginine-Glycine-Aspartic acid) sequence in the gelatin structure and effect of histidine amino acid derived MAH comonomer (which is used for the first time in tissue scaffold fabrication studies) to cell proliferation, cell adhesion and cell viability were investigated by in vitro cell culture experiments.

Synthesis of cryogels was carried out by free radical polymerization at -18 ° C. Gelatin and MAH were added to the structure during polymerization to give PHEMA cryogel function. Within the scope of characterization studies of prepared cryogels; swelling properties were evaluated by calculating the degree of macroporosity, gel fraction yield, swelling degree and swelling ratio. In PHEMA-based cryogels, the degree of macroporosity, swelling degree and swelling ratio were decreased due to the gelatin ratio. Whether gelatin and MAH were incorporated into the structure of the cryogels was examined by FTIR (Fourier transform infrared spectroscopy) and as a result, they were successfully incorporated into the structure of cryogels. Three-dimensional characterization analyzes of MAH and gelatin containin cryogels performed by microcomputed tomography (micro-ct) and the porosity was found 80,13% for PHEMAH/GEL. Cell viability and adherence behavior of the cells for different cryogels were examined by scanning electron microscopy (SEM), the results showed that PHEMAH/GEL cryogels showed better cell attachment and cell distribution. Cell viability and cell biocompatibility experiments were performed by using 3- [4,5-dimethylthiazol-2-yl] -diphenyltetrazolium bromide (MTT) assay using L929 mouse fibroblast cells at 24 and 48 hours for all cryogels prepared. It was found that cell viability was higher for PHEMA/GEL ve PHEMAH/GEL cryogels functionalised with gelatin and MAH comonomer than PHEMA cryogels. Among the cryogels prepared, it was concluded that the histidine amino acid derived MAH had an enhancing effect on the cell viability as PHEMAH / GEL cryogels gave higher cell viability. As a result, it was concluded that PHEMAH / GEL cryogels prepared with MAH and gelatin can be used as tissue scaffold.