How to grow a patch for the heart?
MIPT investigated nanocarc for heart cells
Phys Tech blog on Naked Science website
Biophysicists have studied the structure of a polymer nanofiber substrate and the mechanism of its interaction with rat heart cells. These studies are conducted to create regenerative heart tissue. Scientists have found out that muscle cells – cardiomyocytes – envelop nanofibers during growth, and connective tissue cells – fibroblasts – rely on nanofibers on one side. The article with the results is published in the journal Acta Biomaterialia.
The work was carried out in the Laboratory of Biophysics of Excitable Systems of MIPT in collaboration with colleagues from the V. I. Shumakov National Medical Research Center for Transplantology and Artificial Organs and the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences in Pushchino.
The head of the Laboratory of Biophysics of excitable Systems at MIPT, Professor Konstantin Agladze, says: "Using three independent methods, we have shown that cardiomyocytes, developing on a substrate of nanofibers, cover them from all sides and in most cases take the form of a "case". Fibroblasts, on the other hand, have a more rigid structure and a smaller area of interaction with nanofibers, since they rely on them only on one side."
The task of regenerative medicine is to restore damaged or lost organs of the human body. Tissue engineering is often the only way to restore the functions of such an important organ as the heart and achieve human rehabilitation. Scientists, when creating tissue for "patches" of organs, need to investigate not only the properties of the tissue cells themselves, but also their interaction with the substrate, the surrounding nutrient solution and neighboring cells.
The right support is the key to success
A fundamental role in the growth, development and formation of regenerating tissue is played by the substrate on which the cells are grown. Researchers grow heart tissue cells on a matrix of polymer nanofibers. The latter may have different elasticity, electrical conductivity and additional "smart" functions that allow the release of active substance molecules at a certain point in cell development. Nanofibers are designed to mimic the extracellular matrix – the outer surface of cells that provides structural support. In addition, substances can be injected through them for biochemical effects on surrounding cells. Therefore, in order to correctly select the properties of nanofibers that bring an artificial system closer to structures in vivo (that is, "inside a living organism"), it is necessary to study the mechanism of their interaction at the nanoscale.
What's under the microscope?
To determine the structure and mechanism of interaction between cardiac cells and nanofibers, three stages of research were carried out sequentially.
First, the scientists examined the structure of cardiomyocytes and fibroblasts grown on a nanofiber substrate using confocal laser scanning microscopy. This method is based on spot illumination of the smallest segments of the cell, giving images of micrometer parts, and gradual "scanning" along its entire perimeter. The structures of cardiomyocytes and fibroblasts (nucleus, components of the cytoskeleton of eukaryotic cells) and nanofibers were pre-labeled with fluorescent antibodies. Scientists obtained 3D images of cells and saw that both types of cells are elongated along the nanofibers and have a fusiform shape (Fig. 1). However, the data obtained did not allow us to directly consider the surface of the interaction of nanofibers with cells.
Figure 1. Images obtained using confocal laser scanning microscopy of cardiac cells when examining 1) cardiomyocyte, 2) fibroblast.
Next, the researchers made ultrathin slices perpendicular to the direction of the nanofibers and took "photos" by transmission electron microscopy. During the study, a beam of electrons was passed through the cut samples, and the receiver located behind the object recorded the electrons that reached it. The number of electrons reaching the receiver depends on the properties and thickness of the material. Different cellular structures absorb the passing electron beam differently. Biophysicists have seen that cardiomyocytes cover nanofibers from all sides, leaving them in the middle of the cell. However, the nanofibers are still completely separated from the cellular cytoplasm by a membrane (Fig. 2).
Figure 2. The image of a cardiomyocyte enveloping the nanofiber of the substrate in the section. The image was obtained by transmission electron microscopy. 1 – cardiomyocyte, 2 – nanofiber in the section.
Fibroblasts do not envelop nanofibers, but only rely on them on one side. Also, electron microscopy micrographs show that fibroblast nuclei are less elastic compared to other cellular structures, which reduces the plasticity of cells and the ability to stretch along nanofibers (Fig. 3).
Figure 3. Cross-sectional image of fibroblast obtained by transmission electron microscopy. 1 – fibroblast cell, 2 – nanofiber, er – endoplasmic reticulum, N – nucleus.
Transmission electron microscopy allowed us to see what is happening on the slice. With the help of probe tomography, scientists have created a full-fledged 3D model. The cells grown on a nanofiber substrate were cut into plates 120 nm thick. The structure of their surfaces was studied using a silicon probe, and then virtually recreated (Fig. 4).
Figure 4. 3D model of a cardiomyocyte enveloping nanofibers obtained by probe tomography of cell nanosections.
Increased adhesion of cardiomyocytes
The researchers identified several important aspects of the mechanism of interaction of cells with the substrate.
Firstly, the increased mechanical adhesion – the adhesion of the substrate of nanofibers and cardiomyocytes – contributes to the stability of cells on the substrate. This means that the heart tissue (cardiomyocytes) will hold on to the substrate more firmly during growth. Fibroblast tissue will be less stable on the substrate.
The second thing that follows from the results of the study: the use of additional functions of the substrate, such as the emission of regulatory molecules (proteins that activate the process of cell growth) will also differ in cardiomyocytes and fibroblasts. In the case of cardiomyocytes enveloping nanofibers, the emitted substance will diffuse completely and losslessly through the cell membrane into the cytoplasm. And for fibroblasts, it is necessary to take into account losses due to diffusing into the environment surrounding the cells during growth.
And third: cardiomyocytes completely envelop the nanofibers and isolate them from the fluid in which they develop. Therefore, the complete immersion of nanofibers into the cells of cardiomyocytes responsible for the transmission of electromagnetic waves and, accordingly, for the contractions of the heart, will allow testing the electrical conductivity of cells.
This study and further understanding of the mechanism of interaction of cardiac cells with the substrate will allow us to successfully create nanofibers to form the necessary properties of cells and, accordingly, regenerative (regenerating) tissues.
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