Poli(Bütilenadipat-Ko-Tereftalat) (Pbat)-Bazlı Nanotopografik Fibröz Doku İskelelerinin Üretimi ve Hücresel Davranışların İncelenmesi

Abstract

The aim of the study is to investigate the effects of poly(butylenadipate-co-terephthalate) (PBAT) based fibers and fibrous matrices with nanotopographic surface properties on the mechanotransduction mechanism and cell behaviour of stem cells. Nanotopographic fibers were produced with electrospinning method supported by phase separation mechanisms. Followed by the physical, chemical and morphological structures of the matrices were characterised. The interaction of human umbilical cord mesenchymal stem cells (UC-MSCs) with matrices and single fibers was studied in cell culture. Cell culture studies investigating the mechanotransduction mechanism, ε-polycaprolactone (PCL) nanotopographic single fibers were compared with PBAT single fibers to determine the effect of polymer chemical structure as well as nanotopography. Four different phase separation mechanisms, non-solvent-induced phase separation (NIPS), vapor-induced phase separation (VIPS), breath figure (BF), and thermally induced phase separation (TIPS) mechanisms, were applied to create roughness on the fiber surfaces. Tetrahydrofuran (THF), dichloromethane (DCM), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were selected as good solvents and dimethylsulfoxide (DMSO) was chosen as a non-solvent for PBAT. In the binary composition, THF was paired with DMSO and DCM was paired with HFIP. For the THF/DMSO group, the polymer concentration was 12% (w/v) and the solvent volume ratio was 9/1 (v/v), while for the DCM/HFIP group, the polymer concentration was 10% (w/v) and the solvent volume ratio was 8/2 (v/v). It was determined by scanning electron microscopy (SEM) that the fibers were produced as a result of these compositions had porous, wrinkled and grooved morphology, while the fibers in the groups using HFIP solvent had smooth morphology. The average fiber diameters of these combinations were calculated from the SEM images and ranged from 2141 ± 860 nm and 867 ± 487 nm, respectively for random and aligned formulations. The biological performances of PBAT tissue scaffolds with two different surface nanotopographies (THF/DMSO-crumpled and HFIP-smooth) were investigated for 14 days. Cell culture studies, the morphology, adhesion and proliferation behaviours of UC-MSCs on these scaffolds were investigated by SEM, F-actin/DAPI immunofluorescence staining (ICC), Alizarin Red staining and MTT analysis. The result of this investigation show that the adhesion-spread behaviour of the cells on the wrinkled nanotopography. These scaffolds was more mineralisation. In addition, the results of RT-PCR analysis showed that the relative gene expressions for RUNX2, OPN and OCN, which are markers representing osteogenic differentiation, reached a statistically higher expression level in these scaffolds. Within the scope of mechanotransduction studies, PBAT and PCL single fibers were patterned on their basement membranes by phase separation assisted electrospinning method. The response of UC-MSCs to nanotopographies of polymeric single fibers was revealed by F-actin/DAPI, fibronectin and integrin ICC staining. ICC staining was performed to visualise YAP (Yes-associated protein), which plays an important role in the mechanotransduction mechanism. ImageJ-FIJI software was used to obtain numerical data for nucleus and cytoplasm aspect ratios, YAP signal ratios and integrin-fibronectin areas from the fluorescence images of the samples. The results of the analyses were statistically compared. As a result, the prominence of lamellipodia structures due to strong cell-surface interaction, the increase in the amount of integrin and fibronectin, and especially the transition of YAP from the cytoplasm to the nucleus were clearly observed on PBAT-based wrinkled nanotopographic surfaces. This was supported by actin staining and larger aspect ratio of the nucleus. In the smooth fibers used in the study, the cells showed less elongation and spreading, the cytoskeleton was mostly localised around the nucleus. The results emphasize that, the morphological changes and the synergistic effect of chemical structure induce many cellular functions such as cell adhesion and spreading. The most effective polymeric structure on cellular activities and mechanotransduction was determined to be PBAT, and the best morphology was determined to be wrinkled nanotopography. It is expected that the findings obtained will contribute to the design and production of 3D nanofibrous scaffolds that can be used in clinical studies, thus contributing to the relevant literature and applications.

The aim of the study is to investigate the effects of poly(butylenadipate-co-terephthalate) (PBAT) based fibers and fibrous matrices with nanotopographic surface properties on the mechanotransduction mechanism and cell behaviour of stem cells. Nanotopographic fibers were produced with electrospinning method supported by phase separation mechanisms. Followed by the physical, chemical and morphological structures of the matrices were characterised. The interaction of human umbilical cord mesenchymal stem cells (UC-MSCs) with matrices and single fibers was studied in cell culture. Cell culture studies investigating the mechanotransduction mechanism, ε-polycaprolactone (PCL) nanotopographic single fibers were compared with PBAT single fibers to determine the effect of polymer chemical structure as well as nanotopography. Four different phase separation mechanisms, non-solvent-induced phase separation (NIPS), vapor-induced phase separation (VIPS), breath figure (BF), and thermally induced phase separation (TIPS) mechanisms, were applied to create roughness on the fiber surfaces. Tetrahydrofuran (THF), dichloromethane (DCM), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were selected as good solvents and dimethylsulfoxide (DMSO) was chosen as a non-solvent for PBAT. In the binary composition, THF was paired with DMSO and DCM was paired with HFIP. For the THF/DMSO group, the polymer concentration was 12% (w/v) and the solvent volume ratio was 9/1 (v/v), while for the DCM/HFIP group, the polymer concentration was 10% (w/v) and the solvent volume ratio was 8/2 (v/v). It was determined by scanning electron microscopy (SEM) that the fibers were produced as a result of these compositions had porous, wrinkled and grooved morphology, while the fibers in the groups using HFIP solvent had smooth morphology. The average fiber diameters of these combinations were calculated from the SEM images and ranged from 2141 ± 860 nm and 867 ± 487 nm, respectively for random and aligned formulations. The biological performances of PBAT tissue scaffolds with two different surface nanotopographies (THF/DMSO-crumpled and HFIP-smooth) were investigated for 14 days. Cell culture studies, the morphology, adhesion and proliferation behaviours of UC-MSCs on these scaffolds were investigated by SEM, F-actin/DAPI immunofluorescence staining (ICC), Alizarin Red staining and MTT analysis. The result of this investigation show that the adhesion-spread behaviour of the cells on the wrinkled nanotopography. These scaffolds was more mineralisation. In addition, the results of RT-PCR analysis showed that the relative gene expressions for RUNX2, OPN and OCN, which are markers representing osteogenic differentiation, reached a statistically higher expression level in these scaffolds. Within the scope of mechanotransduction studies, PBAT and PCL single fibers were patterned on their basement membranes by phase separation assisted electrospinning method. The response of UC-MSCs to nanotopographies of polymeric single fibers was revealed by F-actin/DAPI, fibronectin and integrin ICC staining. ICC staining was performed to visualise YAP (Yes-associated protein), which plays an important role in the mechanotransduction mechanism. ImageJ-FIJI software was used to obtain numerical data for nucleus and cytoplasm aspect ratios, YAP signal ratios and integrin-fibronectin areas from the fluorescence images of the samples. The results of the analyses were statistically compared. As a result, the prominence of lamellipodia structures due to strong cell-surface interaction, the increase in the amount of integrin and fibronectin, and especially the transition of YAP from the cytoplasm to the nucleus were clearly observed on PBAT-based wrinkled nanotopographic surfaces. This was supported by actin staining and larger aspect ratio of the nucleus. In the smooth fibers used in the study, the cells showed less elongation and spreading, the cytoskeleton was mostly localised around the nucleus. The results emphasize that, the morphological changes and the synergistic effect of chemical structure induce many cellular functions such as cell adhesion and spreading. The most effective polymeric structure on cellular activities and mechanotransduction was determined to be PBAT, and the best morphology was determined to be wrinkled nanotopography. It is expected that the findings obtained will contribute to the design and production of 3D nanofibrous scaffolds that can be used in clinical studies, thus contributing to the relevant literature and applications.