Collagen is a widely used biomaterial in cardiac tissue engineering studies. However, as a natural material, it suffers from variability between batches that can complicate the standardization of culture conditions. In contrast, synthetic materials are modifiable, have well-defined structures and more homogeneous batches can be produced. In this study, several collagen-like synthetic self-assembling nanofiber hydrogels were examined for their suitability for cardiomyocyte culture in 2D and 3D. Six different nanofiber coatings were used in the 2D format with neonatal rat cardiomyocytes (NRCs) and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). The viability, growth, and functionality of the 2D-cultured cardiomyocytes were evaluated. The best-performing nanofiber coatings were selected for 3D experiments. Hydrophilic pH-sensitive nanofiber hydrogel coassembled with hyaluronic acid performed best with both NRCs and hESC-CMs. Hydrophilic non-pH-sensitive nanofiber hydrogels supported the growth of NRCs; however, their ability to promote attachment and growth of hESC-CMs was limited. NRCs also grew on hydrophobic nanofiber hydrogels; however, the cell-supporting capacity of these hydrogels was inferior to that of the hydrophilic hydrogel materials. This is the first study demonstrating that hydrophilic self-assembling nanofiber hydrogels support the culture of both NRCs and hESC-CMs, which suggests that these biomaterials hold promise for cardiac tissue engineering.
Heart failure arising from myocardial loss is a leading cause of morbidity and mortality worldwide [
An ideal biomaterial scaffold for cardiac repair would allow cardiomyocytes to be grown
Beating cardiac constructs have been obtained using collagen patches [
Previously, we tested the commercially available amphiphilic self-assembling peptide PuraMatrix (BD Biosciences). Although PuraMatrix was shown to be less effective than collagen at supporting the growth and survival of cardiomyocytes, it performed well enough to attract our interest in this class of materials [
The aim of this study was to screen different self-assembling nanofiber hydrogels as 2D nanofiber coatings for cardiomyocyte attachment and growth and to then further evaluate the best candidates for use as 3D nanofiber hydrogels. To assess the performance of our nanofiber materials we used NRCs and hESC-CMs, and we considered that an optimal biomaterial should support routine cardiomyocyte attachment, growth, and function. NRCs were used because they can be obtained fairly easily in large numbers, which enabled us to perform large-scale comparison experiments [
Three different types of self-assembling nanofiber hydrogels were examined: hydrophilic pH-sensitive, hydrophilic non-pH-sensitive, and very hydrophobic non-pH-sensitive, hydrogels. We also tested the effect of fiber thickness on cell adhesion for the hydrophilic nanofiber hydrogels. In addition, we tested the effects of pH-sensitive nanofiber hydrogels coassembled with hyaluronic acid (HyA) [
NRCs were isolated as described previously [
We used the H7 human embryonic stem cell line (WiCell, USA). H7 cells were cultured on top of mouse embryonic fibroblasts (MEFs, Millipore, France/USA) in KSR medium (Knockout DMEM (Invitrogen), 20% serum replacement (SR, Invitrogen), 2 mM GlutaMax (Invitrogen), 1% nonessential amino acids (NEAA, Lonza), 50 U/mL P/S, 0.1 mM 2-mercaptoethanol (Invitrogen), and 8 ng/mL basic fibroblast growth factor (bFGF, R&D Systems, USA)) to maintain their pluripotency [
Differentiation was induced by coculturing the stem cells with END-2 cells [
Cells were plated at equal numbers onto nanofiber hydrogels and control wells. The cells were observed daily using a phase-contrast microscope (Nikon Eclipse TS100, Nikon, Japan) and several qualitative parameters were scored to determine the suitability of nanofiber hydrogels for supporting cardiomyocyte culture. These parameters included cell attachment, spreading, morphology, viability, detachment, and beating. Staining for cardiomyocyte markers was used to evaluate the alignment and spreading of cardiomyocytes. Cell attachment was also evaluated quantitatively by counting troponin T positive cells. With NRCs, cell attachment was evaluated by measuring the confluency of troponin-positive cells/well, whereas with hESC-CMs all troponin-positive cells from every replicate were calculated.
The LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes, Inc., Invitrogen), which contains calcein AM to stain live cells green and ethidium homodimer-1 to stain dead cells red, was used to assess viability. The stained cells were observed using phase contrast and fluorescence microscopy (Olympus IX51, Olympus, Japan) and photographed using an Olympus DP30BW camera (Olympus, Japan).
The cells were first fixed with 4% paraformaldehyde (Fluka, Italy) at room temperature for 20 minutes and then blocked at room temperature for 45 minutes prior to labeling with primary antibodies (mouse monoclonal to myosin ventricular heavy chain alpha/beta (MHC) (1 : 100) (Chemicon Temecula, USA), mouse monoclonal to myosin-specific ventricle of the mammalian heart (MLC2v) (1 : 100) (Synaptic Systems, Germany), and goat polyclonal to cardiac troponin T (1 : 2000) (Abcam, UK)) at 4°C overnight. Cells were then labeled with secondary antibodies (Alexa 488 anti-mouse donkey (1 : 400, 1 : 800) (Invitrogen) or Alexa 568 anti-goat donkey (1 : 400, 1 : 800) (Invitrogen)) at room temperature for 2 hours. DAPI (4,6′
Self-assembling nanofiber hydrogels (Figure
Properties of the nanofiber hydrogels and the growth of cardiomyocytes on them. Cell attachment, spreading, morphology, viability, detachment, and beating were evaluated and the comparison was always done compared to control (NRCs: uncoated well plate, hESC-CMs: 0.1% gelatin-coated well plate).
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Nanofiber hydrogel | R1 | R2 | R3 | Fiber surface | Fiber dimensions | 2D NRCs | 2D hESC-CMs | 3D NRCs | 3D hESC-CM |
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Hydrophilic, protic | 4.2 nm [ |
+++ | − | na | + |
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+++ | +++ | ++ | ++ |
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Nanofiber hydrogel | R1 | R2 | Fiber surface | Fiber dimensions | 2D NRCs | 2D hESC-CMs | 3D NRCs | 3D hESC-CM |
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Hydrophilic, cationic | nd | na | − | na | na |
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+ | + | na | na | ||||
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Hydrophilic, protic | 13 nm | +++ | − | na | na |
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Hydrophilic, protic | 13 nm × 50–200 nm | na | +++ | +* | +* |
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Hydrophilic, protic | 13 nm × 100 nm–3 |
+++ | + | na | na |
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H | Very hydrophobic | nd | + | − | na | na |
nd: not defined.
na: not analyzed.
+++: preferable material with as good performance as the control.
++: suitable material with lower performance than the control.
+: sufficient material with inferior performance than the control.
−: unsuitable material because cells did not attach.
*The nanofiber hydrogel degraded too fast.
A schematic representation of gelation through self-assembly.
Several microliters of the nanofiber suspensions were deposited on bare 700 lines/inch mesh copper grids. After excess liquid was blotted away, the grids were plunged quickly into liquid ethane. Frozen-hydrated specimens were mounted on a cryo-holder (Gatan, model 626) and observed using a Philips CM 120 electron microscope operated at 120 kV. Micrographs were recorded under low-dose conditions using a slow-scan CCD camera (Gatan, model 794).
A solution of 10 mg of the HCl salt (Boom B.V., The Netherlands) of the gelator in 3 mL of mQ water was prepared by gentle heating. The solution was neutralized by addition of 1 mL of 100 mM HEPES, pH 8 (Sigma-Aldrich). Aliquots of the 200
A solution of 10 mg of the HCl salt of the gelator in 2.6 mL mQ water was prepared by gentle heating. To this solution we added 0.4 mL of a 0.5% (w/v) solution of hyaluronic acid (Sigma-Aldrich) in mQ water. The resulting solution was neutralized by the addition of 1 mL of 100 mM HEPES, pH 8. Aliquots of the 200
A solution of 10 mg of the gelator in 16 mL of a 95 : 5 ethanol/water mixture was prepared by gentle heating. Aliquots of 750
Based on the results from the 2D experiments, the nanofiber coatings
The gelators were dissolved in DMSO (Sigma-Aldrich) (
Statistical significance for cell attachment was analyzed using The Kruskal-Wallis and Mann-Whitney tests.
The properties of the nanofiber hydrogels have been previously described in detail [
Nanofiber hydrogels have fibers thicknesses that range from nanometers to micrometers (Table
Whereas gelators
Cryo-TEM images of nanofiber hydrogels
The cells were cultured on the nanofiber coatings for seven days, after which they were stained with the LIVE/DEAD kit or fixed and stained with cardiac-specific antibodies (troponin T, MHC or MLC2v). The suitability of nanofiber coatings for cardiomyocyte culture was evaluated by observing cell attachment, spreading, morphology, viability, detachment, and beating in comparison to cells cultured on control surfaces (NRCs on untreated commercial well plates and hESC-CMs on 0.1% gelatin-coated commercial well plates). The results for the evaluation criteria for each material are summarized in Table
Cell attachment of NRCs was evaluated from troponin-T-positive cells.
Cell attachment was determined by calculating the amount of troponin-T-positive hESC-CMs on each material. However, cell attachment was not the only criteria for optimal material and thus despite good attachment some nanofiber coatings were not optimal supporters for cardiomyocyte culture (for details, see Section
The ratio between live and dead cells for both cell types was almost the same on every nanofiber coating. Approximately 70% of cells were alive and 30% were dead (Figures
Cardiomyocytes on different nanofiber coatings stained positively using ((a)–(e), (g)–(k)) cardiac antibodies or ((f), (l)) LIVE/DEAD Kit. Red is ((a)–(e), (g)–(k)) troponin T, green is (g) MLC2v or ((h)–(k)) MHC, and blue is DAPI (nuclei). NRCs grew as well on top of nanofiber coatings (b)
There were no major differences in cell growth among the nanofiber coatings when culturing NRCs. Nanofiber coatings
Nanofiber coatings
Hydrophilic nanofiber coatings
Nanofiber coatings
Nanofiber coatings
Nanofiber coatings
Nanofiber coatings
NRCs grew well both on top of and inside the nanofiber hydrogels. Nanofiber hydrogel
The three nanofiber hydrogels also supported the growth of hESC-CMs. All of the tested nanofiber hydrogels allowed the hESC-CMs to grow and beat on top of the gels as well as inside the gels; however, nanofiber hydrogel
In this paper, we evaluated the suitability of six different self-assembling nanofiber hydrogels for attachment and growth of NRCs and hESC-CMs, first as 2D coatings and then as 3D gels. We examined nanofiber hydrogels that were pH sensitive, non-pH sensitive, hydrophilic, and hydrophobic. Hyaluronic acid was coassembled with the pH-sensitive hydrogels. Two hydrophilic synthetic nanofiber hydrogels were found to support human and rat cardiomyocytes in both 2D and 3D culture, thus providing an alternative platform for
Collagen has been extensively studied as a biomaterial for cardiac tissue engineering [
In our previous study [
In our 2D coating experiments, all nanofiber coatings supported the growth and survival of NRCs. However, two of the nanofiber coatings supported only limited attachment and growth of NRCs: one was the hydrophobic non-pH sensitive nanofiber
For hESC-CMs, the differences in cell attachment and survival among 2D nanofiber coatings were more pronounced. The nanofiber
The nanofiber hydrogel
Nanofiber hydrogels
Some differences in the results between NRCs and hESC-CMs were observed, especially in 2D experiments. It has been reviewed in previous experiments that hESC-CMs are more sensitive to surrounding biomaterial than other cell types [
When evaluating nanofiber materials as in the present study, it is important to note that both NRC- and hESC-derived beating areas contain both cardiomyocytes and noncardiomyocytes. Consequently, when the beating areas are dissociated, there is always a mixture of cell types. As a result, it is currently not possible to obtain pure populations of human cardiomyocytes for testing. However, the other cell types existing in the beating areas, such as fibroblasts, have been shown to support cardiomyocyte growth and functionality [
As cells grow in 3D
Not many 3D biomaterial studies have been performed using hESC-CMs. Our study is the first to demonstrate the growth of hESC-CMs in 3D self-assembling nanofiber hydrogels. The first 3D vascularized human cardiac tissues were created by combining hESC-CMs, ECs, and fibroblasts with PLLA(50%)/PLGA(50%) biodegradable scaffolds [
Based on our study, self-assembling nanofiber hydrogels can be modified to obtain beneficial features that support the growth of cardiomyocytes. To further improve the performance of these synthetic cell-supporting structures, the gelation procedure should be optimized to allow more homogeneous formation of the hydrogels. Another possibility that we are now investigating is decoration of the self-assembling nanofiber hydrogels with functional groups (e.g., the RGD peptide sequence) to improve cell adhesion. Finally, because a main application for 3D cardiac tissue is drug discovery and testing, the possibility of measuring signals from hESC-CMs in a 3D hydrogel using a microelectrode array (MEA) is under investigation. The suitability of MEA platforms for measuring drug responses of hESC-CMs has been shown in 2D culture [
In our previous study, we compared different natural and synthetic biomaterials for cardiomyocyte culture. Collagen type I best supported the growth of cardiomyocytes. However, as a natural material, collagen has batch-to-batch variations. We therefore decided to investigate a synthetic material similar to collagen. In this study, neonatal rat cardiomyocytes and human embryonic stem-cell-derived cardiomyocytes were grown on different synthetic self-assembling nanofiber hydrogels. The pH-sensitive nanofiber hydrogel with hydrophilic and protic fiber surfaces and coassembled with hyaluronic acid best supported the growth of rat and human cardiomyocytes. These nanofiber hydrogels are promising materials for the development of future cardiac tissue models.
The author declare no conflicts of interest.
This study was funded by the Finnish Cultural Foundation-Pirkanmaa Regional Fund, Competitive Research Funding of the Pirkanmaa Hospital District, Tekes–the Finnish Funding Agency for Technology and Innovation and Finnish Foundation for Cardiovascular Research. This work is supported by NanoNextNL, a micro- and nanotechnology consortium of the Government of The Netherlands and 130 partners. The authors would like to thank Ms. Henna Venäläinen and Mr. Markus Haponen for technical assistance, and Ms. Annemarie Tuin for fruitful discussions and Ms. Heini Huhtala for statistical analysis. They would also like to acknowledge the animal facilities of the University of Tampere.