3D Carbon Nanotube-Based Composites for Cardiac Tissue

Subscriber access provided by Kaohsiung Medical University

Article

3D Carbon Nanotube-Based Composites for Cardiac Tissue Engineering Valentina Martinelli, Susanna Bosi, Brisa Peña, Gabriele Baj, Carlin S Long, Orfeo Sbaizero, Mauro Giacca, Maurizio Prato, and Luisa Mestroni ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00440 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

3D Carbon Nanotube-Based Composites for Cardiac Tissue Engineering Valentina Martinelli, Susanna Bosi, Brisa Peña, Gabriele Baj, Carlin S. Long, Orfeo Sbaizero, ¶†

¶‡

¶§

‡

#§

£§

Mauro Giacca, Maurizio Prato,* Luisa Mestroni* †

‡,&,^

§

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; Department of ‡

†

Chemical and Pharmaceutical Sciences, Life Sciences and Engineering, University of Trieste, Italy; £

Department of Engineering and Architecture, University of Trieste, Italy; University of Colorado §

Cardiovascular Institute, University of Colorado AMC, Aurora, Colorado, USA; University of #

California San Francisco, San Francisco, USA; Carbon Nanobiotechnology Laboratory, CIC &

biomaGUNE, San Sebastián and Basque Foundation for Science, Bilbao, Spain ^

KEYWORDS: Carbon nanotubes, nanoscaffolds, PDMS, tissue engineering, cardiomyocytes, calcium signaling, cardiac repair.

ACS Paragon Plus Environment

1

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 24

ABSTRACT. Heart failure is a disease of epidemic proportion, and is a leading cause of mortality in the world. Since cardiac myocytes are terminally differentiated cells with minimal intrinsic ability to selfregenerate, cardiac tissue engineering has emerged as one of the most realistic therapeutic strategies for cardiac repair. We have previously proven the ability of carbon nanotube scaffolds to promote cardiomyocytes proliferation, maturation and long-term survival. Here, we tested if 3-dimensional scaffolds of carbon nanotube-based composites can also promote cardiomyocyte growth, electrophysiological maturation, and formation of functional syncytia. To this purpose, we developed an elastomeric scaffold which consists of a microporous and self–standing material made of polydimethylsiloxane (PDMS) containing micrometric cavities, and integrated multi-wall carbon nanotubes (MWCNTs) into the scaffold. We combined microscopy, cell biology and calcium imaging, to investigate whether neonatal rat ventricular myocytes (NRVMs) cultured on the 3DPDMS+MWCNT acquire a more viable and mature phenotype compared to control. We found that, when cultured in the 3D-PDMS+MWCNTs, NRVMs showed improved viability (p < 0.005 at day3) and more defined and mature sarcomeric phenotype compared to 3D PDMS control. These modifications were associated with an increase of connexin-43 gene expression, gap junction areas (p < 0.005 at day 3), and a more mature electrophysiological phenotype of syncytia and calcium transients. Finally, 3D-PDMS+MWCNT boosted NRVMs proliferation (p < 0.005 at day 3) while hindering cardiac fibroblasts proliferation compared to control PDMS. Thus, 3D-PDMS+MWCNT has the ability to promote viability, proliferation and functional maturation of cardiac myocytes. These properties are essential in cardiac tissue engineering and offer novel perspectives in the development of innovative therapies for cardiac repair.


2

Page 3 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Cardiovascular disease is the leading cause of death in the European Union (EU). Indeed, in 2014 there were 1.83 million deaths resulting from diseases of the circulatory system in the 28 EU Member States, representing 37.1 % of all deaths — considerably higher than the second most prevalent cause of death, cancer. Likewise, heart disease is the leading cause of death for people of most racial/ethnic groups in 1

the United States, including African Americans, Hispanics, and whites, while for Asian Americans or Pacific Islanders and American Indians or Alaska Natives, heart disease is the second cause after cancer. Heart disease costs the United States about $200 billion each year, which includes the cost of 2

3

health care services, medications, and lost productivity. Cardiovascular diseases are a heterogeneous group of disorders that affect the heart muscle and the circulatory system, but the most common and clinically important are ischemic heart disease and cardiomyopathies, conditions that may progress towards heart failure, with heart enlargement, decreased pump function, sudden cardiac death and need for heart transplantation. In ischemic heart disease and 4

cardiomyopathies, pathological changes, such as cardiac fibrosis, apoptosis and cardiomyocyte death are always irreversible. Structural and functional changes in the heart lead to the formation of stiff ventricular walls, conduction disorders, reduced oxygen diffusion and eventually heart failure. To date, 5

even the effect of coronary revascularization, widespread used for the treatment of myocardial ischemia, is still unsatisfying.

6

Since adult cardiomyocytes are terminally differentiated cells and have minimal intrinsic ability to self-regenerate, cardiac tissue engineering is an emerging approach in cardiac regeneration and repair research. The aim of cardiac tissue engineering is to develop materials to support or replace the damage cardiac tissue in such a way that the cardiac tissue can maintain contractile force. This can be achieved by integrating CMs or stem cells to compensate for the cellular loss during an injury. The heart also 7,8

functions as an electrical syncytium with a complex electrical network, which is responsible for stimulating the coordinated contraction of the heart. Dysfunction of this electrical network, e.g. due to ACS Paragon Plus Environment

3


Page 4 of 24

loss of cardiomyocytes and replacement fibrosis (scar), interrupts the organized passage of electrical signals across the chambers, promoting arrhythmias which compromise heart function and can cause sudden cardiac death. Therefore, biomaterials for cardiac repair need to be designed to fully integrate 9

within the host myocardium to support normal heart electrical function. Engineered cardiac tissues, developed with electrically conductive nanomaterials, featuring biomimetic heart electrical cues, have recently received attention, and different conductive materials have been tested to be incorporated into cardiac scaffolds to restore the disorder electrophysiological function of the damaged heart.

10-14

An ideal

conductive scaffold should be able to promote the biological function of seeded cardiomyocytes (such as synchronous contraction, differentiation and maturation). Since their discovery in 1991, carbon nanotubes (CNTs) have become the leading edge of nanotechnology due to their unique electrical, mechanical and thermal properties. Their high 15

mechanical strength and low weight combined with their electron conductivity and stability made them useful materials for biomedical applications; more specifically, CNTs are one of the most promising materials to interface with electrically active tissues such as neuronal and cardiac tissues.

10,11,13,16

CNTs can

be functionalized by binding other bioactive molecules, and since the initial stages of investigations, it became clear that this feature was of particular importance for biological applications. In particular functionalized CNTs can modulate neuronal behavior and, multiple investigations have demonstrated that when cultured on functionalized CNTs (f-CNTs), neurons increase the number and lengths of their neurites. In that respect, our previous research has demonstrated the application of CNT for neuronal 17

tissue engineering and succeeded in the construction of 2D and 3D scaffolds.

18-20

In particular, we have

recently developed a novel elastomeric scaffold able to instruct 3D growth of living primary neurons

18

and we reported the ability of 3D geometry to improve functional organization and synchronization in small neuronal assemblies. Entrapping carbon nanotubes in the scaffolds remarkably boosted synaptic 21

activity, thus allowing for the first time to exploit nanomaterial/cell interfacing in 3D growth support.


4

Page 5 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We hypothesized that the behavior observed in neurons could be reproduced in cardiac myocytes, which share several electrical properties and ion channels with neurons.

11,13

Here, we report the unique effect of our 3D elastomeric-CNT scaffold on cardiomyocyte survival, structural and electrical maturation, and spontaneous calcium signaling, which is superior to the effect of 2D CNTs scaffolds,

11,13

as previously seen in neural networks. We suggest that, the unique 18

effects of CNTs on cardiomyocytes are further boosted by the three-dimensional environment and have potential clinical impact in the field of cardiac regeneration and repair.

MATERIALS & METHODS Synthesis of Multiwall Carbon Nanotubes and fabrication of PDMS scaffolds. The scaffolds were prepared as previously described with minor modifications. Briefly, pristine multi-wall CNTs 18

(MWCNTs) (Nanoamor Inc., Stock# 1237YJS, 100 mg) are suspended in 100 mL of DMF and sonicated in a water bath for 20 min. Sarcosine (600mg, 6.73 mmol) and heptanal (588 mg, 5.14 mmol) are added portion-wise over 5 hours (1 addition per hour) upon heating at 140°C and the reaction mixture is then stirred at 140 °C for 16 hours. MWCNTs are then washed several times by filtration (pore size = 0.45 μm) with DMF, MeOH and Et2O and dried under high vacuum. The amount of organic functionalization, determined by thermogravimetric analysis, is 13 umol/mg. Functionalized MWCNTs (15 mg) are mixed with sugar (500 mg, granulometry 225-250 micron) in dry conditions and are shaken overnight. Then 20 μL of water are added and mixed, obtaining a homogeneously wet mixture. The mixture should have the appearance of a moist paste but must not be too wet because sugar granules have to sinter without being dissolved (altering their dimensions). The granule sintering allows the formation of connected pores in final spongy structure. The mixture is molded into the desired shape (a parallelepiped with dimensions 20x5x5 mm), gently pressed and dried at 60°C. SYLGARD® 184 Silicone Elastomer from Dow Corning (PDMS) is prepared and layered with a thickness of 5 mm in a glass dish. The sugar/MWCNT mixture is removed ACS Paragon Plus Environment

5


Page 6 of 24

from the mold and placed onto the dish with the layered PDMS, then it is saturated with PDMS under vacuum (thanks to the vacuum, the PDMS impregnate the sugar/MWCNT cube). The cubes are then cured in an oven at 85°C for 1 hour and then cooled at r.t. Excess PDMS is trimmed away, and the cubes are dipped in distilled water to dissolve the sugar. The optimal ratio between sugar and MWCNTs was established by the structural and chemical requirements of the composite material. At lower concentrations, MWCNTs were insufficient to cover the inner walls of the pores to interact with cardiomyocytes. Conversely, at higher concentrations, the polymerization of the PDMS was prevented and the final compactness compromised. Isolation and culture of ventricular cardiomyocytes from neonatal rats. Neonatal rat ventricular cardiomyocytes (NRVM) were isolated from p0-p1-day-old Wistar rats as previously described. In 1,2,3

detail, ventricles from neonatal rats were minced and collected in calcium and bicarbonate-free Hanks with HEPES (CBFHH) buffer containing 1.75 mg/ml trypsin 1:250 (BD Difco), 10 mg/ml DNase II (Sigma) and 5ug/mL Gentamycin (SIGMA). Tryptic digestions were performed at 37°C under gentle agitation for a maximum of 3 hours. Collected cardiomyocytes were immediately used after isolation and seeded onto either 3D-PDMS or 3D- PDMS+ MWCNT scaffolds at the appropriate density of 5x10

5

cells/ml and cultured in complete Dulbecco’s modified Eagle medium 4.5 g/l glucose (DMEM, Life Technologies) supplemented with 5% fetal bovine serum (FBS, Gibco), 20 mg/ml vitamin B12 (Sigma), 5ug/ml of Gentamycin (Sigma). Cells were finally cultured for up to 72 hours in vitro. Cardiomyocytes retention. Cells retention was evaluated by counting the number of alpha-actinin positive cardiomyocytes in adhesion on 3D scaffolds three days after seeding. Nuclei were stained with Hoechst 33342 (Invitrogen, 1:2000 dilution) in PBS for 30 min at room temperature. Fluorescent images were taken from at least three independent experiment and then analyzed by using Image J software. Cell proliferation and viability assay. AlamarBlue® assay (ThermoFisher Scientific) was used according to the manufacturer’s instruction. To determine cell proliferation and cytotoxicity, cells were


6

Page 7 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incubated with alamarBlue reagent for 4h at 37C. The released absorbance was measured with a UV-Vis spectrophotometer at 570nm EdU pulse labelling and detection of DNA synthesis. To assess cardiomyocytes proliferation, after 2 to 3 days in culture, cardiac myocytes were treated with 10mM of ethynyl-29-deoxyuridine (Click-iT® EdU, Life Technologies Invitrogen cat # C10339, Molecular probes) for a period of 20 hours. At 48 and 72 hours after EdU pulse, cells were fixed. At least three independent experiments were performed to measure cell proliferation. Immunofluorescence (IF). Cultured cells were fixed in 4% paraformaldehyde (PFA) for 15min at room temperature followed by 0,1M Glycine quenching. After fixation, each cell sample were washed 3 times in PBS and treated with 1% Triton X-100 for 35 min and blocked in 10% goat serum for 2 hours at room temperature. The samples were incubated with three kinds of primary antibodies: anti-mouse monoclonal Sarcomeric Alpha-Actinin (EA-53 AbCam, ab 1:400 dilution), anti-rabbit monoclonal cardiac Troponin I (AbCam, ab47003, 1:100 dilution) and monoclonal anti-rabbit Connexin-43, (GJA1 AbCam, 1:2000 dilution) all in 10% goat serum, overnight at 4°C. After 3 washes in 0,2% Tween 20X, fluorescent-labeled secondary antibodies, anti-mouse IgG Alexa Fluor 488 (Life Technology, cat#21206) and/or anti-rabbit IgG Alexa Fluor 594 were added to the samples for 1 hour at room temperature (1:500 dilution). After 3 more washes in PBS, the samples with EdU pulse were stained with Click-iT EdU Alexa Fluor® 594 Imaging Kit (Invitrogen cat # C10339, Molecular probes) for 30 min at room temperature. After 3 round washes, the nuclear staining was performed by using Hoechst 33342, (Invitrogen, 1:2000 dilution) in PBS for 30 min at room temperature protected from light. After twice washes in PBS, all the samples were mounted in MOWIOL 4-88 mounting medium (SIGMA), and prepared for confocal microscopy. Confocal microscopy. Upon immunofluorescence staining, cardiomyocyte cultures were imaged using a confocal microscope (Nikon C2). Detailed 3D fixed samples were investigated at lower magnification (20X) and serial confocal planes (z-stacks) were acquired every 150nm across the entire 3D (n=35÷38 ACS Paragon Plus Environment

7


Page 8 of 24

z-stacks maximum) sections. Reconstructions of the images were performed offline using the imageprocessing package Fijj. MWCNTs were visualized by using the reflection mode property during the 22

confocal acquisition. Calcium imaging and data analysis. Intracellular calcium signaling of CMs growing in the 3DPDMS+MWCNT scaffolds and the 3D-PDMS scaffold was evaluated using Cell-permeant fluo 4, AM according to manufacture instructions. Briefly, each sample was loaded with 1 mM fluo4 AM solution and incubated for 30 min. Samples were washed three times with warm media before imaging. Calcium transients during spontaneous beating of NRVMs was recorded using a Zeiss LSM780 confocal and was measured for 20 s (150 images). Experiments were performed in triplicate from three independent experiments and averaged. Data were corrected for background epifluorescence. Statistical Analysis. Statistical analysis was measured by one-way ANOVA followed by KruskallWallis test using GraphPad Prism Software (V.5). All the data are presented as mean ± standard deviation (SD). The significant p-value was fixed to p < 0.05.

RESULTS AND DISCUSSION Fabrication of a solid 3D elastomeric-MWCNT scaffold for cardiac tissue engineering. Our preliminary proof-of-concept investigations in cardiac cells grown on 2D MWCNT substrates, have shown that NRVMs cultured on carbon nanotubes exhibit improved viability, proliferation, maturation, and electrical properties compared with control substrates (2D gelatin coated tissue culture dishes).

10,11,13

In

contrast , cardiac fibroblasts showed a decrease of their proliferative capacity on these substrates which allowed for a favorable and stable cardiomyocyte/cardiac fibroblast ratio over time.

11,13

Based on these preliminary studies, we have investigated novel, clinically applicable CNTengineered scaffolds. Biomaterials for cardiac tissue engineering can be synthetic, natural, or a hybrid, and can be developed as injectable polymers, patches, and cell sheets, all with different clinical applications. For heart failure applications, where a diffuse heart muscle damage is present and 8


8

Page 9 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


minimally invasive approach are ideal, we generated a novel injectable reverse thermal gel (RTG) functionalized with MWCNTs (RTG-CNT) that transitioned from a solution at room temperature to a 3D gel-based scaffold after reaching body temperature.

10,23

Our RTG-CNT injectable scaffold, also due to

the more physiological 3D structure, further supported long-term cardiomyocyte survival, promoted cell alignment and proliferation, and improved cardiomyocyte function when compared with 2D gelatin controls and 3D RTG system without CNTs. However, under different circumstances, such as congenital heart diseases or large fibrotic areas, a solid scaffold may be more appropriate. To address this need, we developed an elastomeric solid scaffold for cardiac tissue engineering, which was based on our previous investigation of biocompatibility with neurons, and the biological similarity of neurons and cardiomyocytes we 18

previously exploited. This elastomeric scaffold consists of a microporous and self–standing material 11

made of polydimethylsiloxane (PDMS) with micrometric cavities generated by dissolving a sugar template embedded with PDMS.

18,24

This scaffold has also the capability to integrate CNTs into the

polymeric matrix to further tailor its morphological (e.g. porosity), mechanical (e.g. stiffness), physical (e.g. conductivity) and chemical (e.g. surface modification) properties. PDMS was chosen for its established biocompatibility, extensive clinical applications,

25,26

tissue engineering applications and its 27

stability and chemical structure, which allows integration of nanoparticles such as CNTs. The 18

hydrophobicity of the pristine PDMS due to the presence of methyl-chemical groups has been shown to be efficiently transformed into hydrophilic by oxygen plasma treatment, which introduces hydroxyl groups and improves cell attachment.

18,28

Our PDMS scaffolds are generated by dissolving sugar crystals:

this allows to manufacture 3D structures with a spongy appearance characterized by pores of irregular shape and dimension, interconnected by variable paths of connectivity (Figure 1). The maximal diameter of the pores in the scaffold network is in the range of 20 to 150 µm leading to 40% final porosity and a corresponding bulk density of 0.58 g/cm3, as determined by gravimetric measurements. SEM indicates that the incorporation of sugar crystals generates interconnected pores which allow ACS Paragon Plus Environment

9


Page 10 of 24

networks of channels within the PDMS matrix. The material stiffness can be tailored by controlling the material porosity: the wider the pores the lower the material stiffness. Scaffolds for cardiac tissue engineering require porosity in the range of 50 to 100 um that allow cardiomyocytes seeding integration, permeation and migration and have stiffness in the range of 6 to 18 kPa which are the stiffness values for normal and infarcted myocardium, respectively. As mentioned, 29

the porosity of the scaffold has to be large enough to allow permeation and migration of cells, but not be too large to phase out scaffold mechanical features. We have previously reported that our 3D PDMS with MWCNTs presents stiffness values of 2.35 MPa for the bulk material, and of 45 kPa for the porous scaffold, making this scaffold suitable for cardiac tissue engineering.

18

Figure 1. SEM images of the 3D-PDMS+ MWCNT scaffold. It is possible to observe a quite homogeneous porosity and the roughness of the pore walls given by the presence of the nanotubes that seem to hang out towards the inner cavity.


10

Page 11 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To further implement the 3D-PDMS scaffold, and based on our previous investigations showing the effect of CNTs on cardiomyocytes growth and maturations,

11,13

we incorporated MWCNTs

(approximately 20-30 nm diameter, 0.5-2 um length) into the PDMS matrix. As shown in Figure 1, this allows the formation of scaffolds with the pores layered by an irregular carbon nanotube carpet (around 100 nm thick) stably entrapped within the PDMS matrix. The porous environment is similar to the previously described PDMS scaffolds except that MWCNTs are exposed to the inner cavities of the surfaces. We used the pristine 3D-PDMS porous scaffold as control: the two scaffolds (with and without MWCNTs) do not display differences in compressibility.

18

Carbon nanotubes are conductive materials, and conductivity is expected to improve the performance of excitable cells such as neurons and cardiomyocytes. However, in our previous investigations, we found that the 3D-PDMS+MWCNT had a lower conductivity (1025 S/cm), compared to the pristine MWCNTs high conductivity (104 S/cm) and the heart tissue (0.0016 S/cm to 5x10-5 S/cm).

18,30,31

We further quantified the electrical resistance of the 3D-PDMS+MWCNT scaffold, and found

that resistance is indeed high, in the range of 1 - 4 MΩ, significantly higher than 2D MWCNT scaffold (24.3±0.64 KΩ). We believe that the large contact resistances between contiguous carbon nanotubes, the porosity of the material and the low volumetric fraction of MWCNTs may prevent carbon nanotubes from reaching their electrical percolation limit. We also found that resistance measured at constant load 18

exhibited a time dependent behavior with a fast time decay constant (τf 0.68 s) and a slow time decay constant (τs 15 s), which we ascribed to the porous-elastic properties of our scaffolds.

18

Effect of the 3D-PDMS+MWCNT microenvironment on cardiomyocyte viability and organization. In-vitro biocompatibility of 3D-PDMS and 3D-PDMS+MWCNT was demonstrated by evaluating its effect on NRVMs. To assess the suitability of both substrates, cardiac primary cells were seeded to evaluate cell adhesion and retention, cardiomyocytes viability, proliferation and phenotypic organization for a period of 24h, 48h and 72h of culture. As previously demonstrated on 2D scaffolds, MWCNT enriched substrates are responsible for an improvement of cardiomyocytes viability and ACS Paragon Plus Environment

11


Page 12 of 24

retention and is associated with a significantly higher proliferative capacity when compared to those on control (gelatin coated dishes). Here, based on a metabolic activity assay, we found that 3D11

PDMS+MWCNT scaffolds exhibit again a significant improvement in NRVMs cell retention and viability as indicated by the relative absorbance, compared to 3D-PDMS control (Figure 2, Figure S-1). Moreover, the biological effect of 3D-PDMS+MWCNT scaffolds were compared to the pristine 3DPDMS, and the 2D MWCNT scaffolds, which were previously found to boost NRVM growth and maturation. Finally, they were also compared with 2D gelatin which is the established 2D control condition for NRVMs (Figure 2).

11,13

Figure 2. Improved viability of neonatal rat cardiomyocytes cultured on 3D carbon nanotube scaffolds. Neonatal cardiomyocytes cell health and metabolism as determined by AlamarBlue Viability Assay. Cardiomyocyte retention is increased in 3D PDMS substrates enriched by MWCNTs compared to pristine 3DPDMS (**p value< 0.005, n=5; one-way ANOVA, Kruskall-Wallis test).

The phenotype of in-vitro culture on the 3D scaffolds was evaluated by immunostaining for sarcomeric alpha-actinin, troponin I and connexin 43 (Cx-43), three well known cardiac-specific protein


12

Page 13 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


markers. We found that the NRVMs at 48h and 72h days of culture in 3D-PDMS+MWCNT were phenotypically different from the 3D-PDMS control (Figure 3 and 4). In particular, three days after seeding (72h), NRVMs grown on 3D PDMS-MWCNTs scaffolds showed well-defined sarcomeric structures, with an elongated and interconnected sarcomere organization, and better uniaxial alignment. (Figure 3, Figure S-2). Although similar in pattern, cells grown on 3D-PDMS scaffolds displayed a different alpha-actinin staining. In the confocal images in Figure 3, d, some alpha actinin protein-body aggregates were found, suggesting cell aging or a possible cytotoxic effect. We then further investigated Troponin I, known to be involved in contraction and muscle calcium binding and a highly specific marker of cardiomyocytes functional maturity (Figure 4). When cultured in the 3D PDMS-MWCNTs, NRVMs showed a more defined and mature sarcomeric phenotype when compared to 3D PDMS control. 3D-PDMS

3D-PDMS+MWCNTs

3D-MWCNT scaffold 3D-PDMS 3D-PDMS+MWCNTs

Figure 3. Phenotype of cardiomyocytes cells seeded on control 3D-PDMS and 3D-PDMS+MWCNTS scaffolds. A. Immunostaining of sarcomeric alpha-actinin (green) and TOTO-3 (red nuclei) revealed that cardiomyocytes at day 3 of culture on 3D-PDMS control (a, d) and 3D-PDMS+MWCNT (b, e) were phenotypically different. In particular, analyzed under high magnification (bottom, 60X), compared to 3DPDMS control scaffold (d), 3D-PDMS+MWCNTs were shown to develop more elongated and defined sarcomeric structures, together with a partial improvement on uniaxial alignment (e). Moreover, in the 3D PDMS control, some dots-like aggregates of sarcomeric alpha-actinin were present suggesting cell aging process or cytotoxic effects. Representative engineered 3D-PDMS+MWCNTs scaffold are shown (c, f) by reflection mode. Bars: 50 μm. B. Cardiomyocytes (CMs) retention is significantly increased while cardiac fibroblasts retention is decreased in 3D-PDMS+MWCNTs compared to control (*p value< 0.05, n=5; **p value< 0.005, n=5; one-way ANOVA, Kruskall-Wallis test).


13


Page 14 of 24

Effect of the 3D elastomer-MWCNT environment on electrophysiological maturation. Another important aspect of cardiomyocyte culture on 3D scaffolds was their electrophysiological maturation. We previously found that NRVMs grown on carbon nanotube scaffolds, either 2D MWCNT substrates

13

or 3D reverse-thermal polymers with integrated MWCNTs, had a more developed and mature 10

electrophysiological phenotypes as shown by higher gap junction connexin 43 (Cx-43) protein expression content, better organized in structural syncytia. Similarly, the study of intracellular communication and electrical coupling of NRVMS by immunofluorescence revealed an increase in Cx43 expression on 3D-PDMS+MWCNTs compared to 3D-PDMS control with a more homogeneous distribution and a significant higher number of cell-junctions (Figure 4).

3D-PDMS

3D-PDMS+MWCNTs 3D-MWCNT scaffold 3D-PDMS 3D-PDMS+MWCNTs

Figure 4. Phenotype of cardiac cells seeded on 3D-PDMS (control), and 3D-PDMS+MWCNT scaffolds. A. Upper panel: immunostaining of Troponin I (green) and TOTO-3 (nuclei, red) at 3-day culture, showed a significant higher and similar amount of protein expression on both culture condition, with more a defined phenotype on 3D-PDMS+MWCNTs (b) when compared to 3D-PDMS control (a). Representative engineered 3D-PDMS+MWCNTs scaffold is shown in white (c) Lower panel: fluorescence images of intracellular communication of NRVMS growing on both scaffolds. Connexin-43 (red dots), sarcomeric-alpha-actinin (green), and TOTO-3 (nuclei, blue) staining, revealed an increase in Cx-43 distribution on 3DPDMS+MWCNTs, much more homogeneously distributed with a significative high number of cell-junctions (e) compared to 3D-PDMS control (d). Representative engineered 3D-PDMS+MWCNTs scaffold is shown in white (f). Bars: 50 μm. B. Troponin I area (measured according to fluorescence arbitrary units: AU) and connexin 43 (Cnx43) gap junction area (measured as Cnx43 fluorescence-positive area: µm2) are both significantly increased in the 3D-PDMS+MWCNTs compared to control (*p value< 0.05, n=5; **p value< 0.005, n=5; one-way ANOVA, Kruskall-Wallis test).


14

Page 15 of 24

Once we established the high expression of Cx-43, we then investigated the functional maturity of the intracellular syncytia by imaging intracellular calcium oscillations. We previously demonstrated that spontaneous calcium transients are found to be in NRVMs by the presence of carbon nanotubes, both in our 2D scaffold and 3D reverse-thermal polymer as reported by Martinelli et al. and Peña et al. 13

10

Indeed, as reported in our previous studies, in the 2D MWCNT substrate, NRVMs showed a more homogenously distributed pattern of oscillations, while in the 3D RTG-MWCNT scaffold, calcium oscillations were more frequent and homogeneous when compared with controls (2D gelatin coated tissue culture dishes and plain 3D RTG). Likewise, here, the MWCNT enriched 3D-PDMS scaffolds exhibited a distinct behavior, with more frequent and homogeneously regular calcium oscillations (Figure 5), indicating electrophysiological maturation toward the natural beating frequency of rat hearts, which is in the range of 300-480 beats/min. These finding further support the enhanced 32

maturation, both structural and electrophysiological, promoted by carbon nanotubes tightly interacting with cardiomyocytes.

10,11,13,33

Studies on neurons

16,18,20

and cardiomyocytes

13,32,33

strongly support the hypothesis

that carbon nanotubes enhances the electrical function of excitable cells.

3D-PDMS+MWCNT

48 h

3D-PDMS

72 h

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 s

Figure 5. Calcium oscillations of NRVMS grown in 3D PDMS with carbon nanotubes and 3D-PDMS controls at different time points. NRVMs cultured in the 3D-PDMS+MWCNT scaffolds present more homogeneous and frequent calcium transients when compared with NRVMs cultured in the 3D-PMDS scaffolds (n=5).


15


Page 16 of 24

Effect of the 3D elastomer-MWCNT environment on cardiomyocyte proliferation. Cardiomyocytes are mainly terminally differentiated cells that do not proliferate sufficiently to recover the loss of cardiomyocytes during an injury. However, it has been shown that in the early stages of neonatal life in vivo and in vitro, they maintain a low level of proliferative capacity. There is evidence from our 34

previous investigations that carbon nanotubes interacting with NRVMs boost their proliferative capacity, as shown by the number of active cell duplications in MWCNT enriched scaffolds both in 2D and 3D environments.

10,11,13

We also observed that carbon nanotubes inhibit cardiac fibroblast

proliferation. To test if the 3D-PDMS+MWCNT scaffold recapitulate our previous findings on different materials, we performed a proliferation assay on NRVMs and cardiac fibroblasts grown on 3DPDMS+MWCNTs and compared them with NRVMs and fibroblasts cultured in native 3D-PDMS. NRVMs were immunostained with 5-ethynyl-2'-deoxyuridine staining (EdU), a nucleotide analogue that 3D-PDMS

3D-PDMS+MWCNTs 3D-PDMS 3D-PDMS+MWCNTs

Figure 6. Proliferation assay of cardiomyocytes cells and cardiac fibroblasts grown on 3D-PDMS control and 3D-PDMS+MWCNTs. A. Immunostaining of sarcomeric alpha-actinin (green), EdU (pink, positive cardiomyocytes nuclei), and TOTO-3 (blue nuclei) at 3-day culture of NRVMs cultured on 3D-PDMS control (a-c) and 3D-PDMS+MWCNTs (b-d). B. Upper panel: Significant differences on % ratio of dividing cardiomyocytes were observed at day 3 on 3D-PDMS+MWCNTs when compared to 3D-PDMS controls (** p value:

3D Carbon Nanotube-Based Composites for Cardiac Tissue

Recommend Documents