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Mechanically- and electrically-enhanced CNT-collagen hydrogels as potential scaffolds for engineered cardiac constructs Hongsheng Yu, Hui Zhao, Chenyu Huang, and Yanan Du ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00620 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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ACS Biomaterials Science & Engineering

Mechanically- and electrically-enhanced CNTcollagen hydrogels as potential scaffolds for engineered cardiac constructs Hongsheng Yu, 1 Hui Zhao,2 Chenyu Huang,3, † Yanan Du1,2, † 1

Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing

100084, China, 2Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, Tsinghua University, Beijing, 100084, China, 3Department of Dermatology, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing 102218, China. †

e-mail: [email protected] (Yanan Du); [email protected] (Chenyu Huang)

ABSTRACT: With the development of biomimetic scaffolds for engineered cardiac constructs, a considerable number of biomaterials has been evaluated. However, in most previously reported cardiac constructs, the function of the cardiomyocytes (CMs) is restricted due to mismatches in the mechanics, conductivity, and sub-micron structure of the matrix. In this work, type I collagen hydrogels were combined with carbon nanotubes (CNTs) to assess potential improvements in hydrogel strength and conductivity and potential effects on the hydrogel structure. CMs seeded within the CNT-collagen hybrid hydrogels showed improved cardiac cell functions compared to

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those within pure collagen hydrogels, which suggested great promise of CNT-collagen hydrogels as functional scaffold materials for cardiac construct engineering.

KEYWORDS: Cardiac tissue engineering; Cardiomyocytes; Scaffold; Carbon nanotubes; Collagen hydrogel

Introduction Biomaterial plays an important role in the establishment of engineered cardiac constructs. The mechanical property, electrical performance and sub-micron structure of biomaterials all significantly influence the functions of cardiomyocytes (CMs) in an engineered tissue construct.5,6 Recently, amounting attentions have been paid to applying natural or synthetic materials as scaffolds for cardiac tissue engineering. However, most of these materials have incompatible mechanical property with the native myocardium. For example, stiff polymeric materials often suppress the contractility of CMs, while soft hydrogel-based scaffold might not support the mechanical loading during systolic cycles. The stiffness of normal myocardial tissue is as high as 10 - 20 kPa. Matched mechanical property has to be considered in biomaterial design for the scaffolds of engineered cardiac constructs.7,8,13 In addition, myocardium has a conductivity of around 0.1 S/m at the biologically relevant frequencies (DC to a few hertz), which cannot be achieved by majority of commonly-applied biomaterials in tissue engineering.6 Currently, micro/nano materials, such as gold nanoparticles, are used to reduce the mismatch in electrical performance, as well as to enhance the contractility of cardiac cells.9,10 Furthermore, the extracellular matrix (ECM) of ventricular myocardium has complex sub-micron (10 - 100 nm) fibrous structure which is vital for controlling the cell behaviors. The ideal scaffold materials for cardiac tissue engineering should have biomimetic structure as native ECM.


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It is well known in tissue engineering and regenerative medicine that type I collagen is one of the most thoroughly studied natural biomaterials. Additionally, the type I collagen hydrogel, as the major ECM constituent of native cardiac tissue, has long been used as scaffold materials for cardiac tissue constructs because of its good biocompatibility.11,12 However, its poor mechanical supportability and electrical performance limit its further applications.6,13 To improve the mechanical strength and electrical conductivity of collagen hydrogels, varied optimizations in its chemical and physical aspects have been performed, including blending with other materials such as graphene oxide.9,14 But there is still no satisfactory method to simultaneously improve the mechanical and electrical performance of collagen hydrogel while maintaining its native submicron fibrous structure. Carbon nanotubes (CNTs) are well-known for their excellent electrical conductivity, mechanical strength and high aspect ratio. They are widely used in composite materials to improve mechanical and electrical performance.15 Blending with 1-5 wt.% CNTs has been demonstrated to significantly improve the electrical conductivity and mechanical strength in various polymer and hydrogel materials, such as gelatin methacrylate (GelMA).5,16-19 In this work, the mechanical strength and electrical performance of type I collagen hydrogels were improved, while the original sub-micron fibrous structure was maintained by compounding collagen hydrogels with CNTs. It is hypothesized that the CNT-collagen hydrogel could support cardiac cell functions. And the correlation between loading levels of CNTs and physiological characteristics of cultured CMs was further evaluated. The improvements in CMs functions indicated that the CNT-collagen hydrogel be a promising scaffold material for the construction of engineered cardiac tissue. It can be further applied as in vitro cardiac disease model for pathological study and drug screening.1,2


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Experimental Methods Preparation of CNT-collagen I hydrogel. Acid-solubilized rat tail type I collagen (10 mg/ml) was diluted to 4 mg/ml with acetic acid solution (0.02 N). Carboxyl-functionalized multiwalled carbon nanotubes (30 ± 15 nm in diameter and 5-20 µm in length; 95% purity) were purchased from NanoLab Inc. 2 mg CNTs was then added into 1 ml diluted collagen solution (4 mg/ml) and sonicated (SCIENTZ-II D, China, 8%, 2s on and 1s off) for 1 h in ice-bath to gain a black opaque CNT-collagen solution. The CNT-collagen solution was used within 1 month to avoid the coagulation of CNTs. CNTs concentration in collagen hydrogel was expressed in weight percentage, i.e. the weight of CNTs per weight of dry collagen. To prepare CNT-collagen I hydrogel, type I collagen was diluted with Dulbecco minimum essential medium (DMEM) to achieve the final concentration of 2 mg/ml (or 4 mg/ml in dense collagen group) and then neutralized with 1 M sodium hydroxide. After that, the CNT-collagen solution was added into the mixture to harvest suitable loading level of CNTs (up to 10 wt. %). Then, this mixture was poured into the mould or culture plate and incubated in 37 °C for 15 min for gelation. Transmission Electron Microscopy (TEM). CNT-collagen solution and CNTs without collagen coating were diluted respectively with acetic acid solution (0.02 N) to get collagencoated CNTs TEM sample and bare CNTs TEM sample with a CNTs concentration of 0.2 mg/ml. TEM sample solutions were loaded onto holey carbon film-supported grids with uranyl acetate staining. TEM images were acquired by H7650 microscope (Hitachi, Japan) with an acceleration voltage of 80 kV. Fiber structure characterization of CNT-collagen I hydrogel. The fiber structure of collagen I hydrogel with/without CNTs was examined using scanning electron microscope


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(SEM). The SEM samples were prepared by freezing in liquid nitrogen and lyophilization. After that, the samples were cut to expose the cross sections and then coated with Au. SEM images were acquired with a scanning electron microscope (SEM, FEI Quanta 200, Netherlands) at 20,000 magnifications. The pore size and fiber thickness of hydrogel samples were measured by ImageJ software from 3 randomly selected areas of each group. In the statistics of fiber thickness, >200 collagen fibers were randomly selected in each area. Atomic force microscope (AFM). For mechanical property measurements of the collagen I hydrogel with/without CNTs, CNT-collagen hydrogel samples (type I collagen concentration of 2 mg/ml) with CNTs loading levels of 0 wt.% (pure collagen group), 0.2 wt.%, 2 wt.% and dense collagen hydrogel sample (type I collagen concentration of 4 mg/ml) were prepared. Hydrogel samples (1 mm in thickness and 8 mm in diameter) were tested on AFM module CellHesion 200 (JPK Instruments, Germany) which was mounted on an inverted optical microscope (Zeiss Observer A1 stand). Silicon tipless cantilever (ARROW-TL1-50, NANOWORLD) with a nominal spring constant of 0.03 N/m was attached with a plain microsphere (6µm in diameter) to indent the hydrogels. At least 10 points were measured for each sample and the force versus indentation curves were analyzed using Hertz model by JPKSPM Data Processing software (JPK Instruments, Germany) to calculate the Young’s modulus. The indentation was restricted under 1µm to ensure the validity of Hertz model. Electrical testing. To measure the impedance of collagen I hydrogel with/without CNTs, CNTcollagen hydrogel samples (type I collagen concentration of 2 mg/ml) with CNTs loading levels of 0 wt.% (pure collagen group), 1 wt.%, 2 wt.%, 5 wt.%, 10 wt.% and dense collagen hydrogel sample (type I collagen concentration of 4 mg/ml) were prepared. Hydrogel samples were placed between tailor-made electrodes (see Supporting Information, Figure S2). An AC bias sweeping


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between 10 Hz and 100 kHz was made by SR780 Dynamic Signal Analyzer (Stanford Research Systems, USA) and the impedance at each frequency was recorded automatically. Cell isolation and culture. Neonatal rat CMs were isolated from one-day-old Sprague-Dawley rats. All procedures followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University. Firstly, heart tissue was surgically acquired and washed in HBSS with 200 unit/ml penicillin-streptomycin. After digestion in 0.25% trypsin overnight at 4 °C, supernatant was removed. Then, 100 U/ml collagenase type II was added for further digestion at 37 °C in a water bath for 5 min. Such collagenase digestion was repeated for four times. Next, CMs were centrifuged to remove residual enzyme and re-suspended in DMEM. CMs were used just after the isolation. CMs (5×106 cells/ml) or LX-2 cells (1×106 cells/ml) were mixed into the CNT-collagen I hydrogel before gelation; and 50 µl of the mixture was poured into each well of a standard 96-well. After incubation in 37 °C for 15 min to gelation, culture medium of DMEM with 10% fetal bovine serum and 100 unit/ml penicillin-streptomycin was added 150 µl/well and the cell-contained hydrogels were cultured in a humidified 5% CO2 incubator (Thermo, USA) at 37 °C. During the culturing, culture medium was changed every other day. Cell characterization. A Calcein-AM/PI Live/Dead assay (WAKO, Japan) was used according to the manufacturer's instructions. The fluorescence image was measured using an inverted fluorescence microscope (Nikon, Eclipse Ti, Japan). Statistical Analysis. Student's t-test was used to analyze statistical significance. A value of P < 0.05 was considered to be statistically significant. Quantitative data were presented as mean ± standard deviation (SD).


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Results and Discussion The aim of this study was to develop stable CNT-collagen hydrogels with suitable mechanical, electrical performance, and native sub-micron fibrous structure. To avoid precipitation of CNTs in solution, CNTs was coated with collagen fiber through ultrasonic treatment (Figure 1A).3,19,20 Optical images indicated that collagen-coated CNTs were uniformly dispersed in CNT-collagen hydrogels with a loading level of CNTs up to 10 wt.% (Figure 1B). The TEM data showed an increase in the diameter of collagen-coated CNTs fibers compared to bare CNTs, indicating that collagen fibers were successfully coated on the surface of CNTs (Figure 1C).

Figure 1. (A) Preparation of CNT-collagen hydrogels. (B) Optical image of hydrogels with different loading levels of CNTs. Pure collagen group and CNT-collagen groups with collagen concentration of 2 mg/ml, dense collagen group with collagen concentration of 4 mg/ml and no CNTs. (C) TEM images of pure CNTs (upper) and CNTs coated with collagen (lower). As shown in the statistical results of SEM, no significant change was observed in pore size (Figure 2A, B, S1) and fiber thickness (Figure 2A, C) of CNT-collagen hydrogels, compared to


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the pure collagen hydrogels. These unchanged pore size and fiber thickness indicated that compounding collagen with CNTs did not alter the native sub-micron fibrous structure of collagen hydrogels, which is proven to be vital in controlling cell behaviors. In contrast, the widely-used methods to increase collagen hydrogel stiffness, such as increasing the gel concentration, resulted in significant changes in both pore size and fiber thickness (Figure 2B, C). The Young’s modulus of collagen hydrogels with/without CNTs were evaluated by AFM to estimate the influence of CNTs on mechanical performance of collagen scaffolds. Through incorporation with CNTs, the CNT-collagen hydrogel with a CNTs loading level of 2 wt.% exhibited a significant improvement in Young’s modulus (1.85±0.44 kPa), which matches the mechanical property of native heart tissue from neonatal rat (4.0-11.4 kPa) better than the pure collagen hydrogels (80±35 Pa) (Figure 2D).8 Since the Young’s modulus of hydrogels plays an important role in regulation of cardiac beating, such a superiority of mechanical performance could benefit its applications in engineering cardiac tissue constructs.6 The electrical testing indicated that all groups had low impedance at high frequencies (above 100 Hz). However, at lower and more biologically relevant frequencies (below 100 Hz), the CNT-collagen hydrogels showed lower impedance compared to collagen hydrogels without CNTs. And the decrease was enlarged as CNTs loading level increased (Figure 2E). Since recent researches have proved that the electrical conduction among cardiac cells could be enhanced by conductive scaffold,6,21,22 improved electrical conductivity among CMs was expected in our CNT-collagen hydrogels. Therefore, we improved the CNT-collagen hydrogels in their mechanical strength, electrical conductivity and sub-micron fibrous structure. In contrast, the traditional modification, such as increased gel concentration, cannot meet such requirements (Figure 2D, E).


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Figure 2. Structural, mechanical and electrical characteristics of CNT-collagen hydrogels (A) SEM images of pure collagen hydrogels and CNT-collagen hydrogels. Pure collagen group and CNT-collagen groups with collagen concentration of 2 mg/ml. Dense collagen group with collagen concentration of 4 mg/ml. (B) Pore size of pure collagen hydrogels and CNT-collagen hydrogels. (C) Fiber thickness of pure collagen hydrogels and CNT-collagen hydrogels. (D) Young’s modulus of pure collagen hydrogels and CNT-collagen hydrogels with different loading levels of CNTs. (E) Impedance of pure collagen hydrogels and CNT-collagen hydrogels. To evaluate the influence of CNTs on cell viability and cell behaviors, LX-2 cells were seeded into collagen hydrogels with/without CNTs. After 3-day’s culture, no obvious cell death or morphologic change could be detected with the incorporation of CNTs (up to 10 wt.%) in the collagen hydrogels (Figure 3A). Statistical results of fibroblast-mediated hydrogel contraction indicated that loading levels of CNTs up to 5 wt.% had no significant impacts on the hydrogel contraction, while traditional methods of enhancing collagen hydrogel stiffness by increasing the gel concentration significantly decreased the contractility of LX-2 (Figure 3B, C).


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Figure 3. Effect of CNTs in fibroblast viability and fibroblast-mediated hydrogel contraction. (A) Live/dead staining of LX-2 cells after culturing for 3 days in pure collagen hydrogels and CNTcollagen hydrogels with different loading levels of CNTs. (B) Optical images of LX-2 mediated hydrogel contraction. (C) Quantification of contracted collagen hydrogel areas. To assess the feasibility of CNT-collagen hydrogels for CMs culture in cardiac tissue constructs, primary neonatal rat CMs were seeded into CNT-collagen hydrogels. After 5-day’s culture, higher proportion of CMs that seeded in CNT-collagen hydrogels exhibited rhythmic contraction ability (Figure 4A, Supplementary video 1). For hybrid hydrogels with CNTs loading level of 2 wt.%, rhythmic contraction of CMs occurred in 36.3 ± 9.4% area of the entire scaffold, which was nearly 3-fold as high as than that in pure collagen group (13.5 ± 4.5%) (Figure 4B). The increase in rhythmic contraction area indicated that CMs are more likely to have better systolic contractility in CNT-collagen hydrogels.


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Figure 4. Rhythmic contraction of CMs within CNT-collagen hydrogels. (A) Rhythmic contraction of CMs after 5-day’s culture in pure collagen hydrogels and CNT-collagen hydrogels with different loading levels of CNTs. Circled areas indicate that rhythmic contraction of CMs could be observed. (B) Quantification of hydrogel areas with rhythmic contraction.

Conclusions In summary, we demonstrated that the incorporation of CNTs with type I collagen hydrogel led to simultaneous improvements in the mechanical strength and electrical performance, meanwhile maintaining the native sub-micron fibrous structure of hydrogels. CMs cultured within the CNT-collagen hybrid hydrogel scaffolds showed increased rhythmic contraction area, suggesting an improvement of cardiac cell function in CNT-collagen hydrogels. We attributed


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this improvement in CMs function to the optimization of mechanical and electrical properties of the CNT-collagen hydrogels, since the vital role of individual mechanical strength or electrical conductivity in cardiac tissue constructs has been proved by previous studies.3,5

-7

However,

further works are still necessary to elucidate the mechanism that mechanical and electrical properties interplayed to impact the cardiac cell behaviors. The improved CMs functions potentiated the CNT-collagen hydrogel as a candidate scaffold material for cardiac tissue engineering. Furthermore, since CMs contractility is widely used as a functional parameter in pathologic study and drug screening for cardiac diseases,1,2,4 the CNT-collagen hydrogel based cardiac constructs with an improved systolic contractility could benefit the drug development and pathologic research in cardiac diseases.

Supporting Information Available. Tailored electrodes, analysis of pore size, and videos of cardiomyocytes contraction. This material is available free of charge via the Internet at http://pubs.acs.org. Acknowledgement. The authors would like to thank Prof. Weiping Gao from Tsinghua University for his assistance in ultrasonic treatment and fruitful discussions. The authors also acknowledge Prof. Jing Liu from Tsinghua University for providing the electrical measuring platform and Prof. Yan Shi from Tsinghua University for providing the AFM measuring platform. Prof. Ali Khademhosseini from Harvard Medical School offered great help in this project especially in CNT preparation. Funding Sources. This work is financially supported by National Natural Science Foundation of China (grant No. 31671036) and Beijing Natural Science Foundation (7162210).


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