The Effect of physical properties of the ELP-collagen based patterned surfaces on cell attachment and deformation

Download
2013
Antmen, Ezgi
Cell and substrate interactions are important in tissue engineering products especially on the behavior of the cells such as adhesion, migration, proliferation, and differentiation. These have been widely studied using substrates with different physical, chemical, and mechanical properties and form. In this study, elastin-like recombinamers (ELRs) were used blended with collagen or only collagen as the surface material. The ELR used in this study has Valine-Proline-Glycine-X-Glycine aminoacid sequences in its primary structure as the repeating sequence. Collagen was used because it is a biodegradable and biocompatible polymer. The films used for this study were designed to have micropillar covered surfaces with the pillar dimensions involving 4 regions covered with 8x8 µm² and 16x16 µm² pillars separated by either 4 or 8 µm gaps with a height of 5 µ. Saos-2 human osteosarcoma cell line was used to study cell behavior (proliferation, adhesion and conformational change) on these films. Films were observed after 1 day and 14 days of culture by using fluorescence microscopy and SEM and ALP activity of the cells on the micropillar covered surfaces were determined by alkaline phosphatase (ALP) assay after 14 days of culture. Deformation extent and fraction of deformed cells were measured. In terms of adhesion, there was no significant difference in between the different surfaces as a result of ELR presence whereas the micropillar dimensions affected the number of cells and cell numbers were the least at area B which has the lowest pillar size with highest gap (8x8 µm², gap 8 µm) where it was higher for unpatterned surfaces on the 1st day of the culture. Also there was no consistent difference between the three types of films considering the contribution of ELR to the stiffness of the surface. For the 14th day of the culture, it was observed that there is almost no cell on the micropillar surfaces but there were a number of cells on the middle of the films which has no micropillar probably due to the deformation in the micropillar geometries of the films stored in PBS. In terms of ALP concentration, results showed that the highest ALP activity on the films with the highest ELR ratio and higher ALP activity with the cells on the B area but the highest activity with cells on TCP. Lastly, in terms of conformational changes of the cells, it was observed that cells and their nuclei are deformed on the micropillar covered surfaces on all types of films. ELR and collagen content of the films did not seem to affect nuclei deformation. However, pillar placement and dimensions seem to be effective on the nuclei shapes that nuclei of the cells fall in the gaps when the gaps are large enough and when the gaps were smaller, nuclei were observed mostly on the pillar surfaces instead of the gaps. Moreover, there was a consistency between the nucleus and cell deformations in terms of shape. Also there was no cell deformation on the smooth, unpatterned surfaces and the reason of the deformations was the pillars on the film surfaces. In deformation quantification analysis, nuclei deformation frequency, nuclei circularity and nuclei perimeter showed no significant difference between the different films with changing ELR contents. However, considering the pillar dimensions, the highest deformation frequency was on the smallest pillar with largest gap, the least circularity meaning the highest deformation can be seen for the design B (8x8 µm², gap 8 µm) and the highest perimeter for the nuclei was for areas having the highest pillar gaps as area B (8x8 µm², gap 8 µm) and D (16x16 µm², gap 8 µm).

Suggestions

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds
Sayin, Esen; Baran, Erkan Turker; Elsheikh, Ahmed; Mudera, Vivek; Cheema, Umber; Hasırcı, Vasıf Nejat (2021-04-01)
The physiological O-2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of ost...
A high throughput approach for analysis of cell nuclear deformability at single cell level
Ermis, Menekse; Akkaynak, Derya; Chen, Pu; Demirci, Utkan; Hasırcı, Vasıf Nejat (2016-11-14)
Various physiological and pathological processes, such as cell differentiation, migration, attachment, and metastasis are highly dependent on nuclear elasticity. Nuclear morphology directly reflects the elasticity of the nucleus. We propose that quantification of changes in nuclear morphology on surfaces with defined topography will enable us to assess nuclear elasticity and deformability. Here, we used soft lithography techniques to produce 3 dimensional (3-D) cell culture substrates decorated with micron ...
The influence of elastin-like recombinant polymer on the self-renewing potential of a 3D tissue equivalent derived from human lamina propria fibroblasts and oral epithelial cells
Kinikoglu, Beste; Carlos Rodriguez-Cabello, Jose; Damour, Odile; Hasırcı, Vasıf Nejat (2011-09-01)
Three-dimensional epithelial tissue equivalents tend to lose their self-renewing potential progressively during culture as their epithelial cells lose their proliferative capacity with time. Even though the tissue engineered construct can mimic the native tissue well, it rapidly degrades after implantation due to the insufficient number of proliferating cells in the equivalent. In the present study we demonstrate for the first time that the use of an elastin-like recombinant polymer (ELR) engineered to cont...
INVESTIGATION OF THE PERMEABILITY OF THE CELL MEMBRANE FOR DIFFERENT CRYOPROTECTANT AGENTS IN A CONTINUOUS THERMO-FLUIDIC MICRO-CHANNEL SYSTEM
Hatiboğlu, Anıl; Külah, Haluk; Önel, Selis; Department of Micro and Nanotechnology (2021-9-13)
Modeling cell membrane permeability in different solutions is a critical requirement in controlling the response of cells during preconcentration processes in biotechnological applications, such as drug delivery, fluorescence imaging, and cryopreservation . Current multi-step methods employed in loading cells with high concentrations of cryoprotectant agents (CPAs) prior to cryopreservation for long term storage affect cell viability as a result of extended exposure times associated with these methods. One ...
The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum
Kocabıyık, Semra; Russel, RJM; Danson, MJ; Hough, DW (Elsevier BV, 1996-07-05)
In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate s...
Citation Formats
E. Antmen, “The Effect of physical properties of the ELP-collagen based patterned surfaces on cell attachment and deformation,” M.S. - Master of Science, Middle East Technical University, 2013.