Extracellular Matrix Microfiber Papers for Constructing Multilayered 3D

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Extracellular Matrix Microfiber Papers for Constructing Multi-Layered 3D composite tissues Hirotaka Nakatsuji, and Michiya Matsusaki ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00090 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Biomaterials Science & Engineering

Extracellular Matrix Microfiber Papers for Constructing Multi-Layered 3D composite tissues Hirotaka Nakatsuji1 and Michiya Matsusaki1,2,3* 1Joint

Research Laboratory (TOPPAN) for Advanced Cell Regulatory Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

2Department

of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

3JST-PRESTO,

4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Corresponding Author: Michiya Matsusaki, PhD., Associate Professor Department of Applied Chemistry, Graduate School of Engineering, Osaka University *E-mail: [email protected] Tel: +81-6-6879-7357, Fax: +81-6-6879-7359

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Abstract In vitro construction of highly organized three-dimentional (3D) tissues is still a key challenge for tissue engineering. In this study, we fabricated multi-layered tissues composed of extracellular matrix (ECM) layer and cell layer by stacking cell-seeded ECM papers. Paper like scaffold was prepared by simply casting dispersion of micro-fibered ECM. We showed paper like scaffold was superior material for constructing 3D tissue due to their high permeability and cell migration ability and our method can be control the thickness and component of ECM in multi-layered 3D tissues. It can contribute to construction of normal and disease tissue model.

Keywords: Extracellular matrix, Paper, Fiber, Blood Vessel, Tissue engineering

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In vivo biological tissues have a highly organized structure composed of extracellular matrix (ECM) and multiple cells. Cell-cell and Cell-ECM interactions strongly affect the cellular functions (e.g. proliferation, differentiation and migration) and the arrangement is an important factor for the expression of tissue function.

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multi-layered structure is one of the most common structures in biological tissues. For example, the aorta wall comprises three distinct areas: the intima is composed of a single layer of endothelial cells (EC) and basement membrane; the media have alternately laminated smooth muscle cell (SMC) layers and elastin layers; and the adventitia is a composite of fibroblasts and type I collagen.1 This structure affects the permeability of molecules, vascular constriction and other vascular functions. In addition, lesions from vascular diseases such as arteriosclerosis cause structural changes in the vascular wall. Therefore, in vitro construction of highly organized three-dimensional (3D) tissues for mimicking the structure of biological tissues is still a key challenge in tissue engineering. However, conventional methods could not construct multi-layered structure, which have alternately laminated ECM layer and Cell layer having a thickness of a micrometer order. One approach for constructing such a multi-layered structure is controlling cell accumulation. As an example, coating the cell surface with nanometer-sized ECM films to control cell-cell attachment enabled construction of a multi-layered blood vessel wall model composite of an EC layer and underlying multi-SMC layers.2-4 However, this tissue has a much lower ECM concentration than that in natural aorta (70 wt% in dry weight)5 due to its only nanometer-sized coating. ECM production of accumulated cells can reproduce the ECM environment6 in 3D tissue. However, over 4 month culture is necessary to achieve the same ECM concentration as natural tissues. 3



It is also difficult to control component, quantity and structure of ECM using these methods. Previously, a construction method of multi-layered structure by stacking paper scaffold7-9 was reported. Paper scaffold have high permeability because of the fibrous structure and thin structure enabled to construct precisely controlled multi-layered structure. However, these methods were also unable to mimic in vivo ECM environments, because the paper was made of cellulose fibers or synthetic polymer. In addition, they need precursor hydrogel to attach cells on paper scaffold and the thickness of scaffold layer is higher than ECM layer in aorta media. If papers consisting of natural ECM could be fabricated, it would be valuable for the construction of precisely organized multi-layered 3D tissues. We considered that micro-fibered elastin would enable us to fabricate high density ECM paper without chemical modification. Herein, we report for the first time the preparation of natural elastin and collagenbased ECM papers and construction of multi-layered composite tissues (Figure 1a). In aorta wall, approximately 60 % of ECM is elastin.1,

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However, natural elastin is

difficult to use in many of the fabrication techniques for cell scaffolds due to its insolubility.10-14 Most elastin based biomaterials have therefore been constructed using elastin mimetic polymer, elastin-like polypeptide15, hydrolyzed elastin16 and crosslinked tropoelastin.17 In this study, we discovered a novel way to fabricate insoluble elastin based biomaterials by microfibrillization. We prepared the dispersion of elastin microfiber (EMF) by microfibrillization of natural elastin to make the ECM papers. ECM papers were formed by simple casting of ECM dispersion in a silicone rubber 4


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framework. The obtained papers showed high tensile strength, 1.8-13.3 MPa, depending on the mixing ratio of EMF and type I collagen (Col I), cell adhesion properties, and stacking properties to prepare the multi-layered tissues. The ECM papers reported in this study will be a valuable novel biomaterial for tissue engineering and regenerative medicine. To prepare the EMF, elastin from bovine aorta and neck ligament were fragmented in a homogenizer for 5 min at 1 wt% in deionized water. Phase contrast microscopic images of homogenized elastin (Figure S1) revealed the aggregation of elastin were well defibrated by homogenizer. Elastin derived from the aorta was thinner and shorter than that from neck ligament (aorta: 1.3 ± 0.4 and 9.2 ± 4.8 µm, neck ligament: 10.1 ± 2.7 and 59.9 ± 44 µm). The thickness of each EMF was similar to that of natural elastin in each tissue, indicating that the homogenized elastin kept its natural fiber structure.18 ECM papers were fabricated by an air-dry casting method.13,

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The EMF

dispersions were cast in a silicone rubber framework at 1, 2 and 3 mg/cm2 and incubated at 30 °C for 12 h in a drying oven. At the same time, we used hydrolyzed elastin solution for comparison. After incubation, EMFs were peeled off the framework as an unclear paper-like scaffold (Figure 1b). This EMF paper was flexible enough to bend and sufficiently processable to punch out the shape without tearing (Figure 1b and S2). In contrast, dried hydrolyzed elastin had a clear film-like appearance and was too fragile to peel from the framework. The microstructures of papers were evaluated by Elastica van gieson stained images (Figure 1c) and scanning electron microscopic images (Figure S3). Elastica van gieson can stain elastin and 5



collagen as purple and red colors, respectively. EMF papers maintained their fiber structure after the paper forming process and had pores of around 10 µm between the EMF. In previous reports12, scaffolds made only of natural elastin particle could not be prepared because they were too fragile. Our data indicate that the microfiber structure enhanced the strength and softness of the elastin scaffold. Col I paper was prepared by using aqueous solution of Col I from pig’s skin. Col I aqueous solution and the mixed solution of Col I and EMF at a weight ratio of 1:1 (Mix) were dried in the same manner as EMF paper. Col I paper had a clear film-like appearance and smooth surface (Figure 1d, e), while, Mix paper had an unclear (Figure S4) film-like appearance (Figure 1f). Elastica van gieson stained images showed that EMF were dispersed uniformly and collagen filled in the gap of EMF (Figure 1g). In addition, compositions of ECM papers were evaluated by Fourier transform infrared (FT-IR) spectroscopy (Figure S5).

EMF paper and Col I paper both showed a characteristic

amide II peak20-21 (elastin: 1,530 cm-1, collagen: 1,548 cm-1), suggesting the existence of EMF and Col I in Mix paper. The spectra of ECM papers showed no peak shift between the paper forming process, suggesting non-denaturation during our procedure. The thickness of the ECM papers was measured by digital micrometer (Figure1h). The thickness of aortic EMF paper and Mix paper ranged from 10 to 30 µm in direct proportion to ECM content. It was suggested that the thickness of papers is controllable by adjusting the concentration of ECM dispersion. When we used an ECM solution of less than 1 mg / cm2, ECM was unable to fabricate uniform paper. On the other hand, Col I paper was thinner than the other two ECM papers due to its close-packed structure. EMF paper derived from bovine neck ligament was thicker than 40 µm (Figure S6) because of its larger fiber shape. In subsequent experiments, 6


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we employed elastin from bovine aorta because the thickness of the ECM layer in the natural aorta wall was similar to EMF paper using elastin from aorta.18, 22 Strength of ECM papers was evaluated by tensile test. The tensile strength of EMF paper was 1.8 MPa which was comparable to human aorta23, however, the paper showed less elasticity, probably due to the non-covalent bonding of EMF (Figure 2a and S7). Tensile strength was increased as the Col I ratio in ECM paper (Col I: 13.3 MPa, Mix: 5.8 MPa), may be due to the densely packed structure of Col I resulted in a higher tensile strength than EMF paper. (Figure S8) To evaluate the stability of ECM papers under physiological conditions, ECM papers were incubated in phosphate buffered saline (PBS) and culture medium at 37°C and time-dependent changes in dry weights were evaluated (Figure 2b and S9). After 1 week, all of the ECM papers retained more than 90 % of their weight. These data suggest that ECM papers were stable under physiological conditions. The surface properties of the ECM papers were measured by static contact angle measurement. All of the ECM papers showed hydrophobic properties (Figure 2c). EMF paper was more hydrophobic than the other ECM papers. The roughness of EMF paper contributed to it having the highest contact angle.24-25 Mix paper showed an intermediate value between Col I and EMF. Interestingly, a droplet on EMF paper immediately passed through even though the paper had the most hydrophobic surface properties. In addition, a redropped droplet showed a lower contact angle (47.4°) than the first drop. These data indicated that the hydrophilic groups of wet EMF papers were exposed in wet conditions, in first drop leading to higher water permeability than other ECM papers. Mix papers in any component ratio have not show high 7



permeability similar to EMF paper (Figure S10). We guess filling gap of EMF by collagen induce lower permeability than EMF paper. Thus, we consider gap of EMF and surface change of EMF contribute to high permeability. The hydrophobicity of ECM papers is in the ideal range for cell attachment25-26 and the high water permeability is appropriate for constructing 3D tissue because permeability of nutrients and oxygen deep into 3D tissue is an important factor to avoid cell necrosis. Normal Human Dermal Fibroblasts (NHDFs) stained with cell tracker red were seeded on ECM papers and they were well attached to the papers (Figure 3a, b). Moreover, we confirmed endothelial cells and SMC also attached well on ECM papers (Figure S11). Viability and proliferation of NHDFs were evaluated by cell counting and the measurement of extracted DNA from cell-seeded ECM papers (Figure 3c and S12). All of the cultured NHDFs on ECM papers showed high cell viability and proliferation. While Mix paper showed highest cell proliferation rate of the three papers, they showed no significant difference. On the other hand, fluorescence images of them labeled with cell tracker red showed different morphology (Figure S13). NHDFs on EMF and mix papers were dispersed between EMF fibers, wheras NHDFs on Col I paper were aggregated. It is considerable that the stiffness and component of the scaffold affects cell proliferation and attachment.27,28 The cause for these difference requires further investigation.

However, these data suggest that the

cellular function of 3D tissue can be controllable by adjusting the ECM components. NHDF seeded on EMF paper were evaluated cell migration by section image and fluorescence image (Figure 3d,e). Hematoxylin and eosin stained section image showed NHDFs were migrated to opposite side of EMF paper. Fluorescence image 8


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from backside of EMF paper showed NHDFs labelled with cell tracker red were attached around EMF pore. These data demonstrate that EMF paper can use for evaluation of cell migration. Finally, we constructed multi-layered composite tissue by layering NHDF-seeded EMF paper (Figure 3f, g) and mix paper (Figure S13). NHDFs were seeded on the papers at 2 x 105 cells/cm2 and incubated at 37°C in a CO2 incubator for 24 h. The NHDF-seeded papers were layered and centrifuged at 1,100 rpm for 5 min to attach them together. After 3 days, constructed 3D tissues were fixed by formaldehyde solution and observed after staining with hematoxylin and eosin or the elastica van gieson staining method. The section images of the multi-layered tissues using EMF and Mix papers revealed that the tissues had alternately laminated papers and NHDF layers and that the thickness of each layer in both cases was approximately 10 and 20 µm, respectively. In addition, multi-layered 3D tissue composite of mix papers showed higher paper-paper attachment (Figure S14). Our methods achieved construction of precisely controlled multi-layered tissue composed of ECM component and cells. The thickness of each layer was much lower than previous methods7-9, which is comparable to EMF layer in aortic media22. In this study, we demonstrated the construction of precisely controlled multilayered composite tissue by layering cell-seeded ECM papers. The ECM paper based on natural insoluble ECM can be prepared by using ECM microfiber without chemical denaturation. The ECM papers were stable under physiological conditions and sufficiently biocompatible to use as a cell scaffold. By using this method, we can precisely control the ECM component and quantity of ECM in multi-layered 9



composite tissue. Our 3D tissue could be useful model for evaluating cell migration, and cell-cell cell-ECM interaction of biological tissue which have multi-layered structure such as blood vessel wall. In addition, precise control of ECMs can equally well contribute to the construction of normal tissue- as well as disease tissue-models.

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Figures

Figure 1. (a) Schematic illustration of the construction of multi-layered composite tissue. (b, d, f) Photographic images of ECM papers composed of elastin microfiber (b), collagen (d), and elastin microfibers mixed collagen in a weight ratio of 1:1 (f). Scale bar corresponds to 5 mm. (c, e, g) elastica van gieson stained images of ECM papers. Red: Collagen Purple: Elastin Scale bar corresponds to 20 µm. (h) Thickness of ECM papers in various concentrations (1, 2 and 3 mg/cm2).

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Figure 2. Characterization of ECM papers (a) Tensile strength and (b) Time-dependent weight change of ECM papers in culture medium (DMEM, 10% FBS) at 37 °C, (n=3). (c) Representative time-dependent change of contact angles on each paper. Second drop was added on EMF paper at 60 sec.

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Figure 3. (a) 2D fluorescence and (b) 3D reconstructed fluorescence images of NHDF (red) seeded on EMF paper (green). Scale bar corresponds to (a) 50 µm and (b) 200 µm. (c) Cell proliferation of NHDFs cultured on ECM papers (n=3). (d) Section image of NHDF seeded on single EMF paper stained with hematoxylin and eosin (H&E). (e) Fluorescence image of back surface of NHDF seeded EMF paper. Red: NHDFs labeled with cell tracker red Green: Self-fluorescence of EMF. Scale bar corresponds to 300 µm. The arrows indicate migrated cells. Section images of multi-layered composite tissues composed of NHDF and EMF

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papers after 3 days culture stained with (f) H&E and (g) Elastica van gieson. Scale bar corresponds to 100 µm.

Author information Corresponding author *E-mail: [email protected]

Notes The authors declare no competing financial interest.

Acknowledgement This research was supported by JST, PRESTO (15655131), a Grant-in-Aid for Scientific Research (B) (17H02099) and Research Activity start-up (17H06823) of JSPS.

Supporting Information Material and methods, phase contrast image, SEM image, photographic image, UV-vis-NIR spectra, FT-IR spectra fluorescence images and section image are displayed on Supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Extracellular Matrix Microfiber Papers for Constructing Multilayered 3D

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