Ruptured vessels and burnt skin, these are all painful injuries that cause damages and degenerations of soft tissues. The most common treatment is transplantations from donors to recipients.
But it is riddled with problems; most prominently severe inflammatory responses and transplant rejections, which can be fatal. Transplantations from patients’ other body parts are also not always possible.
Tissue engineering allows the regeneration of damaged soft tissues. Cells from patients are seeded onto a biocompatible material (dubbed scaffold) which act as the physical template for the needed tissue. After transplantation into the patient’s body, the scaffold degrades gradually.
Synthetic polymers [i.e. polycaprolactone, poly(l-lactic acid)] are one of the most used materials for soft tissue scaffolds. They can be easily moulded to resemble the extracellular matrix (ECM), i.e. collagen fibres and glycoproteins, which is the non-cellular component that structurally and biochemically supports the surrounding cells. The problem is, most synthetic polymers are hydrophobic, meaning they hate water.
Since 60% of our weight is water, and cells make up our bodies, meaning it is not easy for cells to attach to their scaffolds. Therefore, scientists at CEITEC try their hands on the possible solution that is amine plasma polymer (PPs).
“In our previous study (1), we coated the surfaces of materials with amine PPs thin films, which are hydrophilic.” Lucie Blahová, a researcher from the Plasma Technologies research group at CEITEC MU, explains.
To do so, gas with amine groups is fed into a reactor to be fragmented by plasma into many different molecular species and integrated on the scaffold surfaces.
“Using the C2C12 mouse myoblasts cells (a type of cells that form muscle cells in mice), we discovered that the cells adhere so strongly to the surfaces that trypsinisation (the process of using enzymes to separate the cells and scaffold) is rendered useless.” Therefore, a collaboration between the Plasma Technologies research group, the CEITEC MU Nanotechnologies Core Facility, CEITEC BUT, along with researchers from Masaryk University, ICG SB RAS and Institute of Physiology of the Czech Academy of Sciences ensued.
A huge project with many researchers involved!
The aim is to find out if there is any difference in adhesion and proliferation between several types of cells on different compositions of amine PPs.
7 types of cells - endothelial cells which line blood vessels and lymphatic vessels (HUVEC, HSVEC, CPAE), as well as myoblasts C2C12, outer layer skin cells HaCaT, rat vascular smooth muscle cells VSMC, and connective tissue cells LF fibroblasts, are all subjected to trypsinisation after successful cultivation.
A control group of surfaces is used, that is standard polystyrene (PS) culture dishes. On the other hand, three groups of PS dishes are coated with amine PPs. They are prepared by using relatively low (< 30 W), medium (< 100 W), and high (< 150 W) average power of the electrical discharge in the plasma polymerisation process.
The higher the average power, the more insoluble the film becomes in water, leading to slight swelling when immersed in water. Higher average power also means a lower amount of amine groups located on the surface.
All of the non-endothelial cells studied behave similarly: even after an hour of trypsin treatment, cells hold firmly onto the amine PPs. The cells are also very alive and kicking. This observation is consistent across all amine PPs films. However, the endothelial cells fall off the amine PPs after a much shorter time of trypsinisation, though with some discrepancy: some types of endothelial cells resist trypsin a bit longer on amine PPs prepared by low average power. At the same time, another one prefers PPs made by higher average power.
Sadly, an increase in cell adhesion dampers cell proliferation. The numbers of all cells replicated on all types of amine PPs after days are less than those of cells on the uncoated PS surfaces. However, that doesn’t mean amine PPs are bad for cell proliferation, as PS culture dish is the finest surface for cells to replicate. Nonetheless, out of all, the films prepared by higher average power have shown to be the most suitable for cell replication.
F-actin (red fluorescence) is a type of threadlike protein forming actin cytoskeleton. Actin cytoskeleton alters the shape and motility of the cell for its structural growth. After 2 hours, LF fibroblasts placed on a surface without amine PPs (control group) are round and occupying a small area. The actin cytoskeletons are thick and condensed. In contrast, cells on amine PP coated surface (100W) are spread out, covering a large area of the surface, with a stretch-out actin cytoskeleton. After 2 days, actin cytoskeletons are evenly well-development on both surfaces (1)
As the amine PPs reduces the hydrophobicity of materials surfaces, it is understandable that non-endothelial cell adhesion increases.
Trypsin resistance could be due to the glycocalyx (glycoproteins and glycolipids surrounding the cell membrane). The glycocalyx is negatively charged, creating electrostatic interaction (resulting in attraction or repulsion between objects with electric charges) between non-endothelial cells and the positively charged amine groups.
As for the endothelial cells, researchers theorise that inconsistencies in glycocalyx and ECM (i.e. thickness and biochemical compositions) across types of endothelial cells result in discrepancies in trypsin resistance of cell types on different amine PPs groups. Though, the overall adhesion is much weaker than that of non-endothelial cells.
As for the weakened cell proliferation, the researchers propose the possible cause of strengthened adhesion: adhesion deprivation leads to cell deaths as they detach from ECM. In contrast, overly strong adhesion pushes the cells to skip proliferation and jump into differentiation. That is why medium adhesion strength is most suitable for cell proliferation.
“Of course, these are just hypotheses. We now have to test them.”
“Our findings uncover the existence of different degrees of cell attachment to amine PPs. The next step is to understand the molecular mechanism. It will help to design various tissue replacements, by increasing biocompatibility and bioactivity of the materials.”
Imagine having tissues tailor-made to avoid all the negative impacts of traditional transplantations.
Knowing if the cells will hold tight onto the shape of the scaffolds, without impeding the cell proliferation efficiency, provides tremendous help for advancing soft tissue regeneration. Many patients who might not be lucky enough to find donors can be benefited - ultimately, this would lead to an improvement in public healthcare.
“Besides tissue regeneration, we think amine PPs coatings can be used for many purposes, such as immobilising protein for microarrays, as well as glueing cells together (all organs are composed of specific cells lining up to one another). They can also be incorporated in molecular biosensing. Going beyond biological applications, amine PPs coatings can also increase hydrophilicity and wettability of textile surfaces for painting and dyeing - the possibilities are endless!”
Written by Sophia Man
Publication date: 21.08.2020