Nanotopografik Fiber Matrikslerin Doku İskelesi Olarak Üretimi ve İn-Vitro Etkinliklerinin İncelenmesi

Abstract

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit (BAP) graduate project entitled "Production Of Nanotopographic Fiber Matrices As Tissue Scaffold And Investigation Of Their In-Vitro Effectiveness." (18725).

In this thesis, it was aimed to produce fiber matrices with nanotopographic details and to examine the cellular behavior on them. For this purpose, random and aligned fiber matrices with nanotopographic patterns were produced by electrospinning using a biodegradable polyester polycaprolactone (PCL). Characterization studies were carried out and then, cellular behaviors on the fibers were examined in terms of adhesion, proliferation and morphology, using two different cell lines in the fibroblastic (human dermal fibroblast cell line-BJ) and epithelial (human dermal keratinocyte cell line-HS2) morphology.

PCL solution with 11% (w/v) concentration was prepared by using solvent/non-solvent combinations including 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP), chloroform (CF)/N, N-dimethylformamide (DMF): 9/1, (v/v), CF/dimethyl sulfoxide (DMSO):8/2 (v/v), dichloromethane (DCM)/ DMSO:8/2 (v/v), acetone (ACT)/DMSO:8/2 (v/v) and tetrahydrofuran (THF)/DMSO:9/1 (v/v). Various nanotopographic details on the fiber matrices were obtained via the phase separation mechanisms and observed by scanning electron microscope (SEM). No phase separation was observed in the random and aligned samples with smooth morphology produced using the HFIP solvent. Random fiber matrices with wrinkled morphology were obtained by non-solvent induced phase separation mechanism (NIPS) using CF/DMF and DCM/DMSO solvent systems. On the other hand, CF/DMSO and THF/DMSO solvent systems were used in the production of random fiber matrices with porous morphology. In this system, besides the NIPS mechanism, vapour-induced phase separation (VIPS) was occured as a results of the relative humidity in the closed system. Random fiber matrices with crater-like morphology were obtained via breath figures (BF) mechanism using the ACT/DMSO solvent system. Aligned fibers with groove morphology were obtained by thermally induced phase separation mechanism (TIPS) in fiber matrices by the effect of the rotating drum in all solvent systems.

The chemical structures of the fibers were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and it was revealed that the samples had characteristic peaks of PCL. X-Ray Diffraction (XRD) analysis was performed to confirm the chemical structures of the fiber matrices and determine the phases in the structure. Solvents used in fiber matrix production led to an increase in the crystallinity of the semi-crystalline PCL polymer and two intense peaks were observed at 2θ (°) = 21.4 and 23.7 for each group. The thermal properties of the fibers were determined by Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA). According to the results, it was observed that the characteristic melting peaks of PCL fiber matrices ranged from 56°C to 61°C, and the initial degradation temperatures for each group were around 388°C. By surface roughness analysis, it was determined that the highest roughness was found in fiber matrices with crater-like morphology and produced with ACT/DMSO-R solvent system.

Within the scope of cell culture studies, the cellular behaviors of cell lines with 2 different phenotypes, fibroblastic (BJ) and epithelial (HS2), on the nanofibers were investigated. For this purpose, percentage of cell adhesion was calculated by hemocytometric counting, cell viability was followed by MTT analysis and cell morphology on fibers, cell-cell, cell-material interactions were determined by SEM analysis. Finally, the organization of the cytoskeleton was visualized by F-actin/nucleus dual staining. When the results were evaluated, it was observed that for both cell lines, the most effective cell attachment occurred on the fibers with the wrinkled morphology. Considering the cell proliferation, it was determined that the viability of BJ cells did not change much during the culture period. On the other hand, it was found that the viability of HS2 cells increased with time on all fibers. When the SEM images were examined, it was seen that the cell elongation on the aligned fibers followed the fiber direction, while the cells spread in all directions on the random fibers for both cell types. It was observed that the topographic structures on the fibers increased cell-cell and cell-surface interactions for both cells. This result was supported by fluorescent staining and it was indicated that especially wrinkled and crater-like morphology increased the number of actin filaments. When the two cell groups were compared, it was determined that keratinocyte cells proliferated in groups on the fibers, while dermal fibroblasts spread over larger areas on the surface with a denser skeletal organization.

The results emphasize that, it is necessary and important to evaluate the fiber properties (diameter, surface nanotopography and organization) together with cell morphology in the production of nanfibrous scaffolds that are planned to be used in tissue engineering.