Short communicationHigh resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure
Graphical abstract
Introduction
Polymer nanofibers play a unique role in the development of regenerative medicine [1], [2], [3], [4], [5]. Scaffolds based on such fibers are used successfully for the construction of implants designated for the replacement of soft and elastic tissues [6], [7]. Polymer matrices for tissue culture are often produced using the electrospinning process [8], [9], [10]. They may have various architectures [11] and physical features, such as elasticity [12], [13] and electrical conductivity [14], [15]. In the “smart” scaffolds, polymers also can be loaded with biologically active molecules which are released either spontaneously or in response to external stimuli [16], [17].
The interaction of the cell with the scaffold adds substantially to the degree of freedom of the cell-fiber system, making it even more multidimensional. Consequently, such properties as the mechanical elasticity and robustness of the entire cell-substrate system will depend on the cell-fiber connection as much as on the polymer features. Also, the electrical conductivity of the scaffold would only provide an electrical link between the cells if it is electrically isolated from the cell-surrounding tissue’s ionic solution. The release of the biologically active molecules from the scaffold would only work if their concentration reaches physiologically relevant values which would depend on the tissue architecture. Furthermore, nanoscale features of cell-scaffold interface may significantly influence and regulate cell growth, migration, shape and functionality, and it is especially important if we aim for proper cardiac cell organization in the engineered tissue [18]. Previous studies have suggested that fibroblasts [19] and cardiomyocytes [20] may encompass nanofibers, but no detailed high-resolution microscopy study was performed to reveal nanoscale features of cell-fiber interactions. In order to build a detailed and mechanistic model of the cell-fiber nanointerface, we studied it with the aid of three types of microscopy: confocal laser scanning microscopy (CLSM) [21], scanning probe nanotomography (SPNT) [22] and transmission electron microscopy (TEM) [23].
The necessity of combining these three methods is based on their individual strengths and weaknesses. CLSM gives the most intact image of the living cell, appropriate for three-dimensional (3D) reconstruction, although its resolution is restricted by the light’s wavelength, which is about 500 nm. TEM provides the highest possible resolution, typically resolving objects of a few nanometers size; however, it works best for the samples which are the more distant from the living state, and it is hardly appropriate for the 3D reconstruction. The recently designed SPNT, which combines scanning probe microscopy (SPM) and ultramicrotomy in an integrated “slice-and-view” manner, achieves resolution approaching that of TEM and is specially designed to restore the 3D structure of the biological sample, although also in the far from living state. SPNT is based on the direct measurement of the sample surface with the SPM tip immediately after sample sectioning with the ultramicrotome diamond knife. Successive SPM measurements of the object’s surface after removing a layer of material by ultrathin sectioning enable 3D tomographic reconstruction of the nanoscale sample structure by software integration of the layer-by-layer SPM images obtained [24], [25], [26], [27].
The combination of the above methods allowed us to discover some unusual features of the interface between the polymer fiber and the cell, which were quite different from the well-known focal adhesion of the cell attaching to the plane substrate [28], [29]. It appears that cardiomyocytes tend to create a sheathing shape and envelop the polymer fiber. The fiber is not “swallowed” by the cells, but rather is pushed deeply inside, remaining separated by the cell membrane from the inner cell space, as we will show below.
Section snippets
Electrospinning of suspended PLA/FN nanofibers
Poly(l-lactic acid) (PLA, Mw ∼ 700 000, Polysciences Inc., USA, 21512) was dissolved in the hexafluoroisopropanol (HFP, Sigma-Aldrich Co., USA, 105228) at a concentration of 25 mg/ml. In order to enable confocal fluorescent microscopy studies the PLA solution was supplemented with Rhodamine 6G fluorescent dye at a final concentration of 0.01 µg/ml. Polydimethylsiloxane (PDMS, Dow Chemical, USA) blocks with dimensions of 10 × 20 × 3 mm were used as a substrate for electrospun fibers. Each block
Results
In the present work, we have studied the cell-fiber interface of neonatal rat cardiac cells seeded and cultured on suspended electrospun polylactide/fibronectin (PLA/FN) nanofibers (prepared as shown on the scheme in Fig. 1(a)) with an average diameter of 616 ± 101 nm (according to SEM measurements; see Fig. 1(b)).
Three-day old cultures were observed with the aid of phase contrast optical microscopy and optical mapping of the excitation wave and were confirmed to exhibit a usual spontaneous
Discussion
The above data creates a novel prospective view on how scaffold fibers may interact with growing cells. It is well known that developing cells are greatly influenced by their environment: matrix anisotropy impacts cell phenotype and matrix stiffness affects cell migration, proliferation and differentiation [37]. The distribution of focal adhesion sites defines the actin network structure and thus impacts cell morphology. Generally, when cells are interacting with a scaffold matrix (either for
Conclusion
The high resolution study of the filamentous structures formed by cardiac cells interacting with polymer nanofibers revealed that cardiomyocytes and fibroblasts have different behavior on such type of scaffolds. The study was performed using three independent methods: confocal laser scanning microscopy (CLSM), scanning probe nanotomography (SPNT), and transmission electron microscopy (TEM). It was demonstrated that in the sparse nanofiber meshes, having a cell-fiber filamentous structure,
Acknowledgements
We thank Dr. N. Agladze and V. Tsvelaya for the help with tissue culture. We thank G. Semenova, V. Peshenko and S. Khutsyan for the help with transmission electron microscopy. We also like to show our gratitude to T. Starodubtseva for text correction. The work was supported by the Russian Ministry of Education and Science of the Russian Federation grant (state task) 6.9906.2017/BCh.
References (42)
- et al.
Substrates for cardiovascular tissue engineering
Adv. Drug Delivery Rev.
(2011) - et al.
Functional materials by electrospinning of polymers
Prog. Polym. Sci.
(2013) - et al.
Electrospun fine-textured scaffolds for heart tissue constructs
Biomaterials
(2005) - et al.
The significance of electrospinning as a method to create fibrous scaffolds for biomedical engineering and drug delivery applications
J. Drug Delivery Sci. Technol.
(2016) - et al.
Electro spinning: Applications in drug delivery and tissue engineering
Biomaterials
(2008) - et al.
Electrospun nanofibers as a tool for architecture control in engineered cardiac tissue
Biomaterials
(2011) - et al.
Engineered tropoelastin and elastin-based biomaterials
Adv. Protein Chem. Struct. Biol.
(2009) - et al.
The mechanical stress-strain properties of single electrospun collagen type I nanofibers
Acta Biomater.
(2010) - et al.
Three-dimensional imaging of polymer materials by Scanning Probe Tomography
Eur. Polym. J.
(2014) - et al.
Engineering microscale topographies to control the cell-substrate interface
Biomaterials
(2012)
Nuclear envelope breakdown requires overcoming the mechanical integrity of the nuclear lamina
J. Biol. Chem.
Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds
Acta Biomater.
A potential role for integrin signaling in mechanoelectrical feedback
Prog. Biophys. Mol. Biol.
Myocardial tissue engineering using electrospun nanofiber composites
BMB Rep.
Electrospun nanofiber scaffolds: engineering soft tissues
Biomed. Mater.
Polymer-based platforms by electric field-assisted techniques for tissue engineering and cancer therapy
Expert Rev. Med. Devices
Electrospinning: A fascinating method for the preparation of ultrathin fibres
Angew. Chem. Int. Ed.
Analysis of biological compatibility of polylactide nanofibrous matrix vitalized with cardiac fibroblasts in a porcine model
Genes Cells
Developments in conducting polymer fibres: from established spinning methods toward advanced applications
RSC Adv.
Fabrication of conductive polymer-based nanofiber scaffolds for tissue engineering applications
J. Nanosci. Nanotechnol.
Coaxial electrospun fibers: applications in drug delivery and tissue engineering
Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol.
Cited by (12)
Current research trends and challenges in tissue engineering for mending broken hearts
2019, Life SciencesCitation Excerpt :To understand the mechanism by which cells interact with nanofibers, Balashov et al., performed a recent study to understand a single cell-single fiber interaction. They observed that cardiomyocyte with its cytoplasm, covers the entire nanofiber or nanofibers bundles in certain cases, forming a sheath-like structure, which enables the cell attachment over the entire fiber [118]. There are various electrospinning techniques to obtain nanofibers with different dimensions, morphologies, and porosity to most closely match natural ECM as shown in Fig. 2.
Biomimetic Cardiac Tissue Models for In Vitro Arrhythmia Studies
2023, BiomimeticsNovel Molecular Vehicle-Based Approach for Cardiac Cell Transplantation Leads to Rapid Electromechanical Graft–Host Coupling
2023, International Journal of Molecular SciencesPolymer Kernels as Compact Carriers for Suspended Cardiomyocytes
2023, MicromachinesModeling the human heart ex vivo—current possibilities and strive for future applications
2022, Journal of Tissue Engineering and Regenerative MedicineDo Human iPSC-Derived Cardiomyocytes Cultured on PLA Scaffolds Induce Expression of CD28/CTLA-4 by T Lymphocytes?
2022, Journal of Functional Biomaterials