Multilayered Hollow Tubes as Blood Vessel ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Multilayered Hollow Tubes as blood vessel substitutes Joana M. Marques Silva, Catarina A. Custódio, Rui L. Reis, and João F. Mano ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00499 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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 free 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 accessible to all readers and 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.

ACS Biomaterials Science & Engineering 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 39

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 Biomaterials Science & Engineering

Multilayered Hollow Tubes as blood vessel substitutes Joana M.Silva,1,2 Catarina A. Custódio, 1,2, Rui L. Reis, 1,2, João F. Mano1,2*#

AFFILIATIONS 1

3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of

Minho, Headquarters of the European Institute of Excellence of Tissue Engineering and Regenerative Medicine, Avepark – Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR, Portugal. 2

ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

* Corresponding author # Current address: Department of Chemistry, CICECO - Aveiro Institute of Materials University of Aveiro 3810-193 Aveiro, Portugal. E-mail: [email protected]

KEYWORDS: layer-by-layer, nanobiomaterials, endothelial cells, Smooth muscle cells, tissue engineering, blood vessel substitutes

1 ACS Paragon Plus Environment


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

ABSTRACT The available therapies for cardiovascular pathologies often require the replacement of diseased vascular grafts. However, the current blood vessel substitutes are unsuitable for small-diameter blood vessel replacements. Herein, we propose the creation of multilayered hollow tubes as blood vessel substitutes. Hollow tubes were obtained by building-up multilayers of marine-derived polysaccharides (i.e., chitosan and alginate) on sacrificial tubular templates using layer-by-layer technology and template leaching. A cross-linking degree of ≈ 59 % was achieved using genipin, which is reflected in an increase of the mechanical properties and a decrease on the water uptake. To further improve the cell adhesive properties of the multilayers, fibronectin (FN) was immobilized on the surface of the hollow tubes. In vitro biological performance of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were assessed. In addition, to perform the culture of HUVECs on the inner side and the HASMCs on the outer side of the tubes, an in-house developed apparatus was created that allowed us to feed cells with their respective culture medium. The developed hollow tubes showed to be a suitable structure to promote cell adhesion, spreading, and proliferation. It is our belief, that the creation of these functional structures will open a new research field in order to develop innovative multilayered tubular structures for cardiovascular TE applications.

ACS Paragon Plus Environment

Page 2 of 39

Page 3 of 39

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 Blood vessels systems are elements of cardiovascular systems that play an important role during the tissue’s exchange of nutrients, oxygen and metabolites with other tissues, maintaining the homeostasis.1-3 A blood vessel is generally composed of three distinct layers: a monolayer of endothelial cells (ECs) as the inner surface (the intima), smooth muscle cell (SMCs) layers (the media), and a fibroblast cell outer-layer (the adventitia).2, 4-6 The number of layers of SMCs varies depending on the size of the vessel and its anatomical location.5 Therefore, the development of blood vessel substitutes should mimic their native structure, present a selective permeable barrier, regulate blood pressure and angiogenesis, and achieve integration with the native vasculature.7 Therapies for cardiovascular diseases often require the replacement of damaged vessels by a vascular graft. The best replacement strategy would be the use of autologous grafts, which is not possible in some patients.8-10 Synthetic vascular grafts have been successfully used in the treatment of large arteries (diameter > 6 mm). Clinically, two of the most successful synthetic vascular graft substitutes are expanded polytetrafluoroethylene and polyethylene terephthalate.9,

11-12

However, their high

thrombogenic and poor mechanical properties limit their use in microvascular grafts.1315

In routine clinical practice, no microvascular grafts (diameter ≈1-2 mm) for vessels

have been fully accepted.11, 13 Current strategies that attempt to generate in vitro tissue engineering (TE) living blood vessels possess substantial limitations such as blood coagulation and a high diameter.16 Thus, the replacement of smaller sized blood vessel substitutes is still a 3



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 4 of 39

challenge. Bearing in mind such limitations, the main goal of this work was the use of layer-by-layer methodology (LbL) and template leaching to create tubular structures that will mimic the in vivo microenvironment of blood vessels. LbL is a powerful and elegant bottom-up approach started by Decher and coworkers to produce multilayered films based on multimode of intermolecular interactions.17-18 Basically, this assembly technique is based on the sequential adsorption of complementary multivalent molecules via electrostatic and non-electrostatic interactions or a combination of both.19-21 By far the majority of the works reported are mainly focused on LbL via electrostatic interactions, often referred as electrostatic selfassembly (ESA).19,

22

For ESA, polyelectrolyte multilayers (PEMs) can be obtained

through the alternate deposition of oppositely charged polyelectrolytes on the top of a surface, which is a consequence of substrate charge overcompensation during the previous polyelectrolyte nanosized layer.19, 21 This technique has witness a tremendous popularity due to the ability to form highly tuned structures with micro and nanometer control over their properties by simple adjustment of processing parameters, such as concentration of biopolymers, pH, ionic strength, nature of biopolymers among others.21-27 The generated multilayered structure present diverse properties when compared to the polyelectrolyte solution or to the bulk crystalline phase of the individual component.28 This template assisted assembly has an invaluable potential of merge the functionalities of the multicomponents presented in the film.29 So far, LbL technique appears as a promising candidate to produce nanostructured films, both planar and convoluted three-dimensional (3D) features, driven by the spontaneous sequential adsorption of multilayer thin films into a myriad 4


Page 5 of 39

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


of substrates.19, 21, 24 The multifunctionality of LbL systems can be further improved through the inclusion of different materials during the multilayers buildup or postassembly.19,

21, 30

Among several strategies, the incorporation of extracellular matrix

(ECM) proteins has been addressed, since they are capable of instructing specific cellular responses.31-32 Among the ECM proteins, fibronectin (FN) is often used to increase cell adhesion. FN is a high molecular protein (∼ 450 kDa) whose subunits contain three types of repeating units (FN I, II, and III) that mediate the interactions with other ECM proteins, cell surface receptors and regulate FN specific interactions.3336

Concerning post-assembly treatments, chemical cross-linking is a common alternative

to improve the stability and the mechanical properties of PEMs.37-38 Between, the most explored polyelectrolyte combinations, LbL assemblies with nature-inspired materials stand out, namely, chitosan (CHI) and alginate (ALG).39-40 To further improve the properties and the stability of these natural origin PEMs genipin has been used as crosslinker.38, 41 Genipin is a naturally derived chemical from gardenia fruit with ability to bridge the amine groups, presenting low cytotoxicity and anti-inflammatory effects.42 Although, LbL has been most commonly associated with two-dimensional (2D) surfaces, in the last years it has been reported their use in the fabrication of CHI microspheres

43

, in freeform assembly of leachable spherical templates

44-45

, to

agglomerate beads 46 and to produce liquefied capsules 47. Recently, it was reported the successful production of multilayered hollow tubular structures.48 The hollow tubes showed to be a suitable structure to promote cell adhesion and spreading, opening a new research field in the development of innovative tubular structures. 48

5



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 6 of 39

Despite the constant advances in the field, multilayered tubular structures have not been explored for cardiovascular purposes. Herein, our goal is to evaluate the potential of these multilayered hollow tubes obtained by LbL and template leaching as suitable blood vessel hybrid substitutes. A schematic representation is given in Scheme 1. A series of physicochemical and biological tests were performed to validate our hypothesis.

Scheme 1. Production steps of hollow tubes: (i) Construction of PEMs on paraffin-coated tubes using ALG and CHI as polyelectrolytes (LbL); (ii) Leach out the entire template using an organic solvent, such as DCM; (iii) Cross-link the tubular structure with genipin; (iv) Immobilization of FN using EDC/NHs chemistry; (v) Cellular tests with HUVECs and HASMCs to mimic the hierarchical structure of blood vessels.

MATERIALS AND METHODS Production of tube-like elements. The templates were produced as previously reported

48

. Briefly, the templates were fabricated by dip coating glass tubes

6


Page 7 of 39

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


(Ø 0.5 mm) in molten paraffin, which upon solidification produced a homogeneous coating of paraffin (≈ 800 µm). The templates were modified with polyethylenimine (PEI) in sodium acetate buffer (0.1 M with additional 0.15 M NaCl, pH 5.5) at 0.5 mg mL-1. Upon 1 hour (h) of immersion the tubular structures were extensively washed in sodium acetate buffer. Afterward, the tubular templates were immersed in polyelectrolytes solutions prepared in a sodium acetate buffer at 2 mg mL-1 concentration, with intermediate washing steps (sodium acetate buffer). The two polyelectrolytes used to process the multilayers were CHI medium molecular weight (Mw = 190.000-310.000 Da, Degree of Deacetylation (DD) = 82.6 %, ref. 448877, Sigma Aldrich, USA) and low viscosity ALG (538 kDa ≈ 250 cP, ref. 71238, Sigma Aldrich, USA). CHI was purified by a series of filtration and precipitation in water and ethanol steps. The CHI solution were deposited in the templates during 8 minutes (min), then rinsed for 4 min, before depositing ALG for 8 min, also followed by rinsing steps. The steps were repeated until the desired number of layers (100 bilayers) is reached, using a home-made dipping robot. The coated structures were placed in dichloromethane (DCM) to leach out the paraffin layer present between the glass substrate and the PEMs. After leaching, the structures were dried using critical point dryer (CPD). Afterward, hollow tubes were cross-linked overnight (12 hours) at 37 °C with genipin 3.5 mg mL-1, using a protocol already reported.48-49 Prior to cell culture, FN was immobilized on the surface of hollow tubes,

using

a

protocol

previous

reported.50

Briefly,

1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to pre-activate the proteins by reaction with their carboxyl groups (intermediate 7



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 8 of 39

reaction). This step was performed during 20 min at room temperature (RT) with shaking. The protein solution (20 µg mL-1) was prepared in a phosphate-buffered solution (PBS) with EDC and NHS at concentrations of 4 mM and 10 mM, respectively. After 12 h of incubation the hollow tubes were washed in PBS.

Morphology. The morphology of the hollow tubular structures was observed by Scanning electron microscopy (SEM), using a Jeol JSM-6010LV microscope operated at 15 kV accelerating voltage. All the samples were sputtered with a conductive gold layer, using a sputter coater 108A (Cressington, UK). For the crosssections, the tubes were opened up and the resulting freestanding membranes were immersed in liquid nitrogen until free fracture.

Atomic force microscopy imaging. The inner and outer sides of hollow tubes were imaged in a physiologic-like mode (37 °C, PBS), using a dimension Icon microscope controlled by the NanoScope 9.1 (Bruker, France). A ScanAsyst-Air cantilever (Bruker, France) with a resonance frequency of 70 kHz and a spring constant of 0.4 N m -1 was used. Substrate topographies were imaged with 512 × 512 pixels2 at line rates of 1 Hz. For surface roughness analysis, 5 × 5 µm2 AFM images were obtained and the root mean squared roughness (RRMS) and average height value (Hav) were calculated. The analysis of the images was performed using Gwyddion. At least three measurements were performed in different specimens.

8


Page 9 of 39

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


Quartz crystal microbalance with dissipation monitoring. The buildup of PEMs was followed in situ by quartz crystal microbalance (QCM-Dissipation, QSense, Sweden), using gold coated sensor excited at seventh overtone (n= 7, 35 MHz). The crystals were cleaned in an ultrasound bath at 30 °C using successively acetone, ethanol and isopropanol. Adsorption of the different solutions took place with a constant flow rate of 100 µL min-1. The polyelectrolyte solutions were freshly prepared at a concentration of 2 mg mL-1. For the adjustment of pH, a sodium acetate buffer (0.1 M) was prepared at pH 5.5 in the presence of additional salt (0.15 M NaCl, pH 5.5). Prior to the buildup of CHI/ALG multilayers an initial layer of PEI (0.5 mg mL-1, sodium acetate buffer 0.15 M, pH 5.5) was adsorbed during 10 min, followed by a washing step of 10 min. Afterward, an ALG solution was flushed standing for 8 min to allow the adsorption equilibrium at the crystal surface. After rinsing with sodium acetate buffer/0.15 M NaCl (4 min), the same procedure was followed for CHI deposition. These steps were repeated to five bilayers. The conditions used for the assembly were optimized in previous works.48, 51

After the buildup, the multilayers were flushed during 1 h with a FN solution of

20 µg mL-1 prepared in PBS/EDC/NHS, as aforementioned. After the immobilization of FN, PBS was used as a washing solution and flushed during 1 h. The thickness of viscoelastic films from QCM-D data was estimated using the Voigt-based model. Thus, the obtained frequency and dissipation values were converted to their estimated thickness using this model, which is integrated into the QTools software from Q-Sense. Iterations of the model were performed using at least three overtones and different sensors and experiments. The model requires 9



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 10 of 39

three parameters to be fixed: solvent density, solvent viscosity and film density. The solvent viscosity was fixed at 0.001 Pa s (same as water) and the film density at 1200 kg m-3 (often assumed to return the lowest calculation error).52-54 The solvent density was varied by trial and error until the total error, χ2, was minimized. The solvent density values that allowed obtaining the minimum error was determined to be within 1200 kg m-3. Additionally, for the area density, a rough approximation of the mass adsorbed on top of a gold-coated quartz sensor was calculated by multiplying the thickness by the films density (1200 kg m-3).

Determination of cross-linking degree. The cross-linking of hollow tubular structures was evaluated using the trypan blue method which has affinity to free amines.55 The test was performed immersing the non-cross-linked and the crosslinked hollow tubes in trypan blue 0.4 % (Invitrogen, USA) diluted 50-fold in sodium acetate buffer (0.15 M NaCl, pH 5.5) overnight at 37 °C. The supernatant absorbance was measured at 580 nm in a microplate reader (Sinergy HT, Bio-Tek, USA). A standard curve was prepared by measuring the absorbance for a series of trypan blue solutions at different concentrations. The cross-linking degree was calculated as followed: CL ( % ) =

( NH 3+ non - Xlinked solution) - ( NH 3+ Xlinked solution) ( NH 3+ non - Xlinked solution)

Where NH3+ non-Xlinked and NH3+ Xlinked solution are the free charge amines in non-cross—linked and cross-linked tubes, respectively. 10


Page 11 of 39

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


Water uptake. The water uptake ability was measured by soaking hollow tubes with known weight in PBS (Sigma, USA) at pH = 7.4. Briefly, the containers with the hollow tubes immersed in PBS buffer were placed in a water bath at 37 °C with agitation (60 rpm). At predetermined time points, the excess of solution was removed from the samples using filter papers (Filter Lab), and the hollow tubes were weighed with an analytical balance (Denver Instrument). The water uptake was calculated as followed:

Water uptake ( % ) =

Ww - Wd × 100 Wd

where Ww and Wd are the weights of swollen and dried hollow tubes, respectively.

Upon water uptake stabilization, the hollow tubes with and without cross-linking in aqueous environment (PBS) were also evaluated using a stereomicroscope (Zeiss, Germany) with a color camera (Nikon G12).

Mechanical tests. The dynamic mechanical analysis measurements were performed using a TRITEC8000B DMA (Trinton Technology, UK) to evaluate the mechanical/viscoelastic properties of hollow tubes without and with cross-linking. Such assays were performed at physiological-like conditions, i.e., immersed in PBS at 37 °C placed in a Teflon® reservoir. The distance between the clamps was 5 mm. The hollow tubular structures were immersed in PBS until equilibrium was reached (≈ 12 h). After measuring the geometry the samples were clamped in the DMA apparatus and immersed in the PBS bath. The DMA spectra were recorded during a 11



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 12 of 39

frequency scan between 0.1 to 20 Hz with constant strain amplitude of 20 µm. A static pre-load of 1 N was applied to keep the hollow tube tight.

Cell culture. To evaluate the biological performance of cross-linked hollow tubes, cell culture studies were performed with primary cultures, human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs). Both types of cells were selected in order to mimic the structure of blood vessels. HUVECs (Gibco, USA) were cultured in M199 (Sigma, USA) supplemented with sodium bicarbonate, 1% antibiotic/antimycotic (Invitrogen, Scotland), 20% FBS (Invitrogen, USA), 0.34% glutamax (Gibco, USA), 50 µg/mL Endothelial Cell Growth Supplement (ECGS) (BD Biosciences, USA) and 50 µg/mL Heparin (Sigma, USA). HASMCs (Invitrogen, Scotland) were cultured in M231 (Invitrogen, Scotland) supplemented with smooth muscle growth supplement (SMGS) (Invitrogen, Scotland) and 1 % antibiotic-antimicotic. Both cells were used between P2-P6. Prior to cell seeding, the hollow tubes (diameter 0.5 cm and length 1 cm) were sterilized as previously mentioned, and FN was immobilized using a protocol above reported. Individual cellular tests with each cell type were performed on hollow tubes using a home-made Teflon® mold with wells (containing approximately the dimensions of the tubes) – see scheme 2. Cell seeding was performed by adding 35.000 cells per cm2 (HASMCs or HUVECs). The morphology and proliferation of both cell types were evaluated after 1, 3 and 7 days. The main objective of this work is to co-culture HUVECs and HASMCs on hollow tubes. Therefore, HUVECs and HASMCs were co-cultured in the fabricated tubes. Briefly 12


Page 13 of 39

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


HUVECs and HASMCs cells were re-suspended in their respective mediums. Afterward, HUVECs were seeded in the inner side of the hollow tubes with the help of a syringe, and HASMCs were seeded on the outer side of the hollow tube – see scheme 2.

Scheme 2. Schematic representation of the co-culture studies on the hollow tubular structures: (i) HUVECs in their respective medium were injected in the inner side of the tube; (ii) HASMCs suspension was added to the outer side of the hollow tubes. A top view of the hollow tubes in the homemade apparatus was also represented.

Morphological characterization: DAPI-phalloidin and Scanning electron microscopy. After pre-determined time points, cells were fixed with 10% (v/v) formalin, and stained with fluorescein isothiocyanate labelled phalloidin from Amanita phalloides dyes (phalloidin, 10 mg mL-1, Sigma-Aldrich, USA) and 4,6Diaminidino-2-phenylindole-dilactate (DAPI, 20 mg mL-1, Sigma-Aldrich, USA) to visualize F-actin filaments and cell nuclei respectively (n = 2 samples per well, in 13



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 14 of 39

triplicate). Briefly at each time point, culture medium was removed and the samples fixed in 10 % (v/v) formalin for 1 h, and replaced by PBS. Afterward, the cells were permeabilized with 0.2 % Triton X (Sigma, USA) for 5 min. Then, the multilayered tubes were incubated in 30 mg mL-1 of bovine albumin serum (BSA, Sigma, USA) for 30 min to block non-specific binding. Upon PBS washing, 1 mL of PBS containing 5 µL of phalloidin and 1 µL of DAPI was added for 40 min at RT and protected from light. Upon PBS washing the samples were visualized by confocal laser microscopy (Leica TCS SP8). For SEM analysis samples were dehydrated, dried using CPD, sputtered coated with gold, and visualized using a Jeol JSM6010LV microscope.

Cell viability. Cell viability was assessed after 24 hours in culture using the live/dead assay (calcein AM/propidium iodide (PI) staining) (n= 3 samples per well, in triplicate). Briefly, the tubes were incubated for 10 min with 2 µL calcein AM (1 mg mL-1, Molecular Probes, Invitrogen, USA) and 1 µL PI (1 mg mL-1, Molecular Probes, Invitrogen, USA) in 1 mL PBS protected from light. The tubes were washed with PBS to remove residual fluorescent and visualized by confocal laser microscopy.

DNA assay. The double-stranded DNA (dsDNA) content was quantified after each time point, being the cells lysed by osmotic and thermal shock and the supernatant used for dsDNA content analysis. Cell proliferation was evaluated by quantifying the DNA content on HUVECs or HASMCs seeded and cultured separately on 14


Page 15 of 39

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


hollow tubular structures using a fluorimetric dsDNA quantification kit (PicoGreen dsDNA Quantification Kit), according to the manufacteur’s instructions. This assay allows measurement of the fluorescence produced when PicoGreen dye is excited by UV light while bounded to dsDNA. Prior to DNA quantification, samples were thawed and sonicated for 15 min. Samples and controls were vortexed and 28.7 µL of each, plus 71.3 µL of PicoGreen solution and 100 µL of Tris-EDTA buffer was transferred into an opaque 96-well plate. The plate was incubated for 10 min in the dark and the fluorescence was measured, using an excitation of 485 nm (excitation of the dye) and an emission wavelength of 528 nm (when the dye is bounded to the dsDNA). A standard curve was created (λDNA standard ranging between 0 and 2 µg mL-1) and the DNA values of the samples were read off from the standard graph. The DNA amount was calculated from a standard curve.

Histological analysis. The tubes were collected at specified time points and fixed with 10 % (v/v) formalin. The constructs were included in methacrylate and sections of 30 µm of thickness were obtained using a microtome and mounted in a microslide glass. Haematoxylin & Eosin (H&E) staining was performed, following standard histological procedures using the automatic stainer (Micron HMS 740, Thermo Scientific, Germany). The slides were then mounted for observation under a light microscope (Zeiss HAL 100/HBO 100).

Statistical analysis. The experiments were carried out in triplicate otherwise specified. The results were presented as means ± standard deviation (SD). Statistical 15



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 16 of 39

analysis was performed by Shapiro Wilk normality test using Graph Pad Prism 5.0 for Windows. After this analysis, non-parametric (Mann Whitney test) or parametric tests (one way ANOVA followed by Bonferroni’s test) were used depending if the samples were from normally distributed populations or not, respectively.

RESULTS AND DISCUSSION Nanostructured hollow tubes: Morphology. The lack of functional blood vessel substitutes to ensure the exchange of nutrients, oxygen, metabolites with other tissues may jeopardize tissue regeneration. Despite the recent advances and developments in engineering vascularization, the successful creation of efficient substitutes of blood vessels is still a challenge.5, 39, 56 Herein, we propose the use of multilayered hollow tubes, co-cultured with HUVECs and HASMCs as a new strategy to engineer blood vessel substitutes. Using LbL assembly combined with a sacrificial template, multilayered hollow tubes can be obtained, as we previously reported 48. Using this strategy, multilayered hollow tubes with potential to be used as blood vessel substitutes can be obtained by monitoring several properties, such as diameter size, length, wall thickness, nature of inner and outer layer, bioactivity, and mechanical properties. In this work, marine-derived polysaccharides were used to produce a homogeneous polymeric coating. To increase the stability and the mechanical strength, the tubes were crosslinked using genipin. The morphology of both tubular structures was evaluated by SEM – see micrographs in Figure 1.

16


Page 17 of 39

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


Figure 1. Morphology of the external and inner side of ALG/CHI (native tubes) and ALG/CHI/G/FN (cross-linked tubes with FN immobilized) tubes. Cross-sections micrographs of both formulations were also performed.

The SEM analysis of dried tubes reveals a noticeable hollow tubular imprint of the wax-coated template used. The morphology of the inner and external side of both tubular formulations is homogeneous, revealing a uniform deposition of multilayers and the absence of defects which also corroborated the efficacy of the leaching process. It should be pointed out that the substrate side (side in contact with the substrate prior to the detachment) is smoother in both formulations, as previously 17



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 18 of 39

reported for freestanding membranes.51 The thickness of the hollow tubular structures was determined using cross-section images. Native tubes present a dry thickness of 35.7 ± 1.7 µm. On the other hand, cross-linked tubes with protein immobilized have a slightly non-significant increase on the thickness up to ca. 36.9 ± 2.9 µm. These results are in accordance with earlier studies, where the cross-linking only limits polyelectrolyte diffusion inside the bulk structure and increase its stiffness, without significant changes of PEMs thickness in the dry state.38,

41

The cross-sections also reveal a homogeneous morphology and some

porosity, which is important for the diffusion across the multilayers. The diffusion properties of these multilayered films were already reported in previous studies, being their permeability the key strength of these multilayered systems.48, 51 To have a better perception of the films morphology and topography at a lower scale we also performed AFM imaging of the surface of the hollow tubes in physiologic-like conditions (37 °C, PBS) – see Figure 2. AFM imaging reveals that the coating was homogenous and all the formulations present a rough surface, represented by submicrometer-sized islets over the surface. However, the inner side of the tube was smoother when compared with external side. The roughness results were concordant with previous studies, since a decrease in roughness occurs with the cross-linking, which turns the films more uniform.48

18


Page 19 of 39

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


Figure 2. AFM analysis of hollow tubes at 37 °C in PBS: (A) AFM images (5 x 5 µm2) of the inner and external side of both hollow tubular formulations in physiological-like conditions. (B) Root mean squared roughness (RRMS) (C) Average height value (Hav). Significant differences were found for (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 (Mean +SD of three independent experiences).

19



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 20 of 39

Buildup mechanism and fibronectin immobilization. The assembly of five bilayers of polysaccharide-based multilayered films and the immobilization of FN was monitored in situ with QCM-D. QCM-D is a simple and an ultrasensitive technique that detects the adsorbed mass of polyelectrolytes onto a gold-coated quartz sensor and the viscoelastic properties of the surface.57 Figure 3A shows the buildup of five bilayers and also their response to the immobilization process of FN, in terms of variations on normalized frequency, ∆f7/7, and dissipation, ∆D7. A layer of PEI was adsorbed on the surface of the gold sensor as paraffin was also modified with it before the buildup of PEMs. In addition, the use of PEI allows a homogenous deposition of the oppositely charged polyelectrolytes. As expected, during the buildup of five bilayers, the normalized frequency decreases with each polyelectrolyte solution injection, reflecting the increase of mass over the gold sensor. On the other hand, ∆D7 increases due to the soft viscoelastic nature of the adsorbed layered structure of the deposited film.39-40 During the washing step, after the injection of each polyelectrolyte, the change of both ∆f7/7 and ∆D7 are relatively small, indicating a strong association of the layers on the surface of the gold sensor. The immobilization of FN on these multilayered films was also studied. After the construction of the five bilayers the film was first rinsed with polyelectrolyte-free solution. The film in this stage is taken as the reference. It can be seen that ∆f7/7 decreases, when the film is flushed with a solution of activated protein (PBS-EDC-NHS, pH 7.4). The behavior of the measured ∆D7 mirrored what is seen with ∆f7/7, i.e., increases and decreases in ∆D7 corresponded to decreases and increases in ∆f7/7. The decrease in ∆D7 revealed that the multilayers 20


Page 21 of 39

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


became more rigid and less hydrated, which is associated with the presence of EDC/NHS used to activate the protein. The thickness of the film was estimated using the Voigt model instead of Sauerbrey model

58

. For viscoelastic materials, the adsorbed mass does not fully

couple to the oscillation of the crystal and dampens the oscillation, being in this case the use of the Voigt model more appropriated.

39, 58

As previously reported the

ALG/CHI films present a linear growth, because the thickness of ALG/CHI multilayers increases as the number of layers increase – see Figure 3B.39-40 After the deposition of five bilayers, the film had a mean thickness of 90.72 ± 9.52 nm, corresponding to an approximate total area density of 10.88 ± 1.92 μg cm-2 (obtained by multiplying the thickness by the layer density, 1200 kg m-3). The results obtained through QCM-D measurements demonstrate the successful immobilization of FN on ALG/CHI films.

Figure 3. Monitoring of the buildup of the polyelectrolyte multilayered PEI (ALG/CHI)5/FN films using QCM-D. (A) Normalized frequency (∆f7/7) and dissipation changes (∆D7) obtain at 35 MHz (seventh overtone). (B) Cumulative thickness evolution of polymeric film as a function of the number of deposited layers. The line represents a linear regression with R2 = 0.9953.

21



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 22 of 39

Physicochemical characterization. The hollow tubes are composed by polyelectrolytes with abundant hydrophilic groups, such as hydroxyl, amine, and carboxyl groups, which can promote water uptake.59 In this work, the swelling ability of hollow tubular structures was evaluated in PBS at 37 °C during 7 days (Figure 4A).The hollow tubes were cross-linked with genipin to improve their stability over the whole pH range, and also to avoid the collapse upon drying. Genipin is a natural derived product from the gardenia fruit which is well known to overcome the toxicity of the commonly used synthetic cross-linkers. 60-61 It has been reported that genipin acts through a ring opening reaction by nucleophilic attack of the CHI amine groups. Briefly the mechanism can be explained as a nucleophilic attack of CHI C-2 to C-3 genipin, resulting in opening of the hydropyran ring and the formation of a heterocyclic amine which produce aromatic intermediates.60, 62-63 Subsequent steps involve radical-induce polymerization that create genipin heterocyclic conjugates. In addition, the other possible mechanism involved the reaction of the ester groups of genipin with amine groups in CHI and secondary amide linkages can be stablished. This cross-linker present high selectively and it only targets amine groups. Thus, the hydroxyl and carboxylic groups did not react and in the end this reaction cross-linked CHI chains and introduces monomers as well as further dimerizations within the film.

60, 62-65

. Since ALG does not contain primary amines,

genipin will give rise to semi-interpenetrating polymer networks with free ALG chains entrapped inside cross-linked CHI multilayers, as previously reported in other multilayered systems.66-67 The hollow tubes present a cross-linking degree of 59.1 ± 7.6 %, which was determined by trypan blue assay. Trypan blue is a dye with the ability to bind to free amines, leading to changes in the blue color intensity of the 22


Page 23 of 39

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


supernatant solution and also on the samples, as previously reported.55 The cross-linking with genipin led to lower water uptake due to the smaller intermolecular space between the polyelectrolyte chains, which limits their molecular mobility at the nanoscale level.41 The smaller intermolecular space is related with the introduction of cross-linker conjugates within the films upon genipin cross-linking. Such effects are the ones that are classically reported in hydrogel systems62,

68

and multilayered films41,

61, 69

. In

addition the results show that water uptake of both formulations increases mainly during the first 30 min and then tends to remain in equilibrium. Further structural information in physiological-like conditions was obtained by optical microscopy (Figure 4B). Both formulations maintained the tubular shape in wet conditions, besides their swelling ability. However, the results clearly reveal a more stable and handling structure upon cross-linking because the diameter of the tube was maintained.

23



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 24 of 39

Figure 4. Physicochemical characterization of the hollow tubes: (A) Water uptake of ALG/CHI and ALG/CHI/G/FN in PBS at 37 °C during 7 days, as well as during the first hour. (B) Microscopy image of both formulations in dry and wet state. The scale bar is 1 mm.

The results obtained in water uptake were further corroborated with the ones obtained in DMA analysis. DMA assays were performed in a hydrated environment at 37 °C, allowing the assessment of the mechanical properties in more realistic conditions. The storage modulus (E’) and loss factor (tan δ) were recorded as a function of frequency on the developed hollow tubes – see Figure 5A and Figure 5B. The results show a slight increase in E' and tan δ with increasing frequency. Moreover, the E’ of cross-linked hollow tubes are significantly higher than in the native ones (4.5 fold increase), indicating the stiffening effect generated upon covalent cross-linking with genipin. It is also important to point out that in all the formulations, no evident variation 24


Page 25 of 39

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


of E' was seen along the frequency axis. The tan δ is the ratio between the energy lost by viscous mechanisms and the energy stored in the elastic component.70-71 In cross-linked hollow tubes a decrease of tan δ in cross-linked occurred, which indicates that the multilayers acquired more elastic properties, and also the release of water molecules from the multilayers.

Figure 5 Mechanical properties: Frequency dependence of (A) Storage modulus (E′) and (B) loss factor (tan δ) of tubes with or without cross-linking at 37 °C immersed in a PBS solution.

Biological performance. The in vitro cellular assays with the optimized hollow tubular structures were performed using two primary cell types selected based on blood vessels composition: HUVECs and HASMCs. Other cell types could also be envisaged such as fibroblasts and or perycites. Both, HUVECs and HASMCs, are highly relevant as components of blood vessels. Native hollow tubes were not used due to their reduced stiffness and higher water content, which impair cell adhesion. The tunable nature of multilayered hollow tubes is of paramount importance to guide cell behavior, because inappropriate physicochemical cues may lead to loss of cell phenotype and function. Cell adhesion and morphology were studied by DAPI-

25



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 26 of 39

phalloidin assay, and also by SEM observation (Figure 6A and Figure 6B).

Figure 6. Cell morphology of HASMCs and HUVECs seeded on ALG/CHI/G/FN. (A) DAPI-phalloidin fluorescence assay at 1, 3, and 7 days of in vitro individual cell culture. Cells nuclei were stained blue by DAPI and F-actin filaments in green by phalloidin. Scale bar represents 100 µm. (B) SEM images of HASMCs and HUVECs seeded on hollow tubes after the same time points. Scale bar represents 500 µm and 50 µm in lower and higher magnification images, respectively.

26


Page 27 of 39

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


Both cell types adhere on the surface of hollow tubes, presenting a stretched morphology. Live/dead assay for HUVECs and HASMCs was performed after 24 h in culture, to assess the effect of these hollow tubes on cell viability (Figure 7A). These results confirmed the biocompatibility of the multilayered hollow tubes for cell culture. With DNA quantification it is also possible to verify an increase in cell number with increasing culture time (up to 7 days) – see Figure 7B. These results revealed that both cell types remain adhered, stretched, spread out along the surface, proliferating and, thus, viable.

Figure 7. (A) Cell viability at 24 h of culture on cross-linked ALG/CHI tubes seeded with HASMCs and HUVECs. (B) DNA assay on hollow tubes seeded with HASMCs and HUVECs. Statistical analysis was performed, and data was considered statistically different for p < 0.05: (#) denotes

27



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 28 of 39

significant differences when compared to other cell type; (*) denotes significant differences between different time points (Mean +SD of three independent experiences).

Afterward, hollow tubes were co-cultured with HUVECs and HAMSCs to better mimic the cellular composition, function and structure of a native blood vessel.

72

In an

attempt to recreate this complex microenvironment of blood vessels and for a more comprehensive understanding of the conditions experienced by cells in the body, coculture studies were performed in hollow tubes using a home-made Teflon® apparatus. The Teflon® mold was designed to ensure that inner side of the hollow tubes contact with ECs and their respective culture medium, while the external side contact with HASMCs and their respective medium (see scheme 2). After 7 days of HUVECs/HASMCs co-culture, the adhesion, distribution and morphology of both cellular types in the respective inner and outer layer of the tubes, were evaluated by histological and SEM analysis (Figure 8). The results show that both cell types adhered in the corresponding sides, and maintained their morphologyGathering all the results, this work demonstrates the simplicity, versatility, flexibility of these hollow tubes with well compartmentalized cell populations, as well as their promising use in cardiovascular field.

28


Page 29 of 39

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


Figure 8. (A) Histological cross-sections of hollow tubes seeded with HUVECs and HASMCs stained by H&E after 7 days of culture. The inset image is the view of the hollow tube section. Two magnifications are shown. (B) SEM micrographs at two magnifications of the inner and external side of the hollow tubes upon co-culture of HUVECs and HASMCs.

CONCLUSION In this work multilayered hollow tubes were obtained combining LbL and template leaching. Using this technique, it was possible to obtain tubular structures with tuned properties such as diameter, length, layer number, wall thickness, elasticity, and to control the nature of the formed layers. PEMs are naturally flexible and the adjustment of assembly or post-assembly parameters allowed for the control over film 29



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 30 of 39

growth and internal structure, which was translated in nanostructured multilayers with tailored physicochemical and biological properties. The multilayered tubes were produced in order to mimic the structure of blood vessels and, consequently, to support the adhesion of ECs and SMCs on both sides of the tubular structure. Herein, the potential of this technique towards the development of multifunctional tubular hybrid structures was demonstrated and robust, compliant, and flexible hollow tubes were developed. The results showed that the use of chemical cross-linking together with immobilized FN promotes cell adhesion and proliferation of ECs and SMCs. Coculturing ECs and SMCs on hollow tubes indicates the potential of these structures in the cardiovascular field. With the knowledge generated in this research, we believe that new perspectives can be opened namely the use of these multilayered films as reservoirs of bioactive molecules in order to improve the cellular performance, blood compatibility and avoid vessel occlusion, as well as the long-term function of these tubes in physiological like conditions or after implantation. To further control the degradation rate of these stable tubes, enzymes may be further incorporated in the multilayers. The behavior of these tubes upon blood contact would be an important step to assess their long-term anti-coagulation ability. Few studies decouple the effect of endothelialization and hemocompatibility. It has been reported that implants covered by endothelial cells will present improved properties and will not cause thrombotic effects, being the adhesion impaired in the presence of anti-coagulant molecules. 73-75 To conclude, the progress made in this field is impressive but the tubes herein developed may lead to novel blood vessel substitutes with the ability to obtain highly tuned structures with micro and nanometer control over their properties. 30


Page 31 of 39

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


Acknowledgments The authors acknowledge the financial support by the Portuguese Foundation for Science and Technology (FCT) through the Doctoral and Post-doctoral grants with the reference numbers SFRH/BD/81372/2011 (JMS), SFRH/BPD/100594/2014 (CAC), respectively, co-financed by the Operational Human Potential Program (POPH) developed under the scope of the National Strategic Reference Framework (QREN) from the European Social Fund (FSE). The authors would also like to acknowledge PTDC/CTMBIO/4706/2014 financed by FCT.

REFERENCES 1. Rouwkema, J.; Rivron, N. C.; van Blitterswijk, C. A., Vascularization in tissue engineering. Trends in Biotechnology 2008, 26 (8), 434-441. 2. Kim, H. N.; Jiao, A.; Hwang, N. S.; Kim, M. S.; Kang, D. H.; Kim, D.-H.; Suh, K.-Y., Nanotopography-guided tissue engineering and regenerative medicine. Advanced Drug Delivery Reviews 2012, (0). 3. Chen, L.; Han, D.; Jiang, L., On improving blood compatibility: From bioinspired to synthetic design and fabrication of biointerfacial topography at micro/nano scales. Colloids and Surfaces B: Biointerfaces 2011, 85 (1), 2-7. 4. Alberts, B.; Johnson, A.; Lewis, J., Molecular Biology of the cell. 4 ed.; New York, 2002. 5. Yoshida, H.; Matsusaki, M.; Akashi, M., Multilayered Blood Capillary Analogs in Biodegradable Hydrogels for In Vitro Drug Permeability Assays. Advanced Functional Materials 2013, 23 (14), 1736-1742. 6. Aird, W., Phenotypic Heterogeneity of the Endothelium: I. Structure, Function, and Mechanisms. Circulation Research 2007, 100 (2), 158-173. 7. Novosel, E. C.; Kleinhans, C.; Kluger, P. J., Vascularization is the key challenge in tissue engineering. Advanced Drug Delivery Reviews 2011, 63 (4–5), 300-311.

31



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 32 of 39

8. Rayatpisheh, S.; Heath, D. E.; Shakouri, A.; Rujitanaroj, P.-O.; Chew, S. Y.; Chan-Park, M. B., Combining cell sheet technology and electrospun scaffolding for engineered tubular, aligned, and contractile blood vessels. Biomaterials 2014, 35 (9), 2713-2719. 9. Kerdjoudj, H.; Boura, C.; Moby, V.; Montagne, K.; Schaaf, P.; Voegel, J. C.; Stoltz, J. F.; Menu, P., Re‐endothelialization of Human Umbilical Arteries Treated with Polyelectrolyte Multilayers: A Tool for Damaged Vessel Replacement. Advanced Functional Materials 2007, 17 (15), 2667-2673. 10. Wang, Z.; He, Y.; Yu, X.; Fu, W.; Wang, W.; Huang, H., Rapid vascularization of tissue-engineered vascular grafts in vivo by endothelial cells in co-culture with smooth muscle cells. Journal of Materials Science: Materials in Medicine 2012, 23 (4), 1109-1117. 11. Lovett, M.; Cannizzaro, C.; Daheron, L.; Messmer, B.; Vunjak-Novakovic, G.; Kaplan, D. L., Silk fibroin microtubes for blood vessel engineering. Biomaterials 2007, 28 (35), 5271-5279. 12. Briggs, D.; Rance, D.; Kendall, C.; Blythe, A., Surface modification of poly (ethylene terephthalate) by electrical discharge treatment. Polymer 1980, 21 (8), 895900. 13. Kerdjoudj, H.; Berthelemy, N.; Rinckenbach, S.; Kearney-Schwartz, A.; Montagne, K.; Schaaf, P.; Lacolley, P.; Stoltz, J.-F.; Voegel, J.-C.; Menu, P., Small vessel replacement by human umbilical arteries with polyelectrolyte film-treated arteries: in vivo behavior. Journal of the American College of Cardiology 2008, 52 (19), 1589-1597. 14. Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J. C.; Muller, S.; Stoltz, J. F., Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification. Biomaterials 2003, 24 (20), 3521-3530. 15. Hung, H.-S.; Chen, H.-C.; Tsai, C.-H.; Lin, S.-Z., Novel Approach by Nanobiomaterials in Vascular Tissue Engineering. Cell Transplantation 2011, 20 (1), 63-70. 16. Kelm, J. M.; Lorber, V.; Snedeker, J. G.; Schmidt, D.; Broggini-Tenzer, A.; Weisstanner, M.; Odermatt, B.; Mol, A.; Zünd, G.; Hoerstrup, S. P., A novel concept for scaffold-free vessel tissue engineering: Self-assembly of microtissue building blocks. Journal of Biotechnology 2010, 148 (1), 46-55. 17. Tang, Z.; Wang, Y.; Podsiadlo, P.; Kotov, N. A., Biomedical Applications of Layer-by-Layer Assembly: From Biomimetics to Tissue Engineering. Advanced Materials 2006, 18 (24), 3203-3224.

32


Page 33 of 39

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


18. Decher, G.; Hong, J. D. In Buildup of ultrathin multilayer films by a self‐assembly process, 1 consecutive adsorption of anionic and cationic bipolar amphiphiles on charged surfaces, Makromolekulare Chemie. Macromolecular Symposia, Wiley Online Library: 1991; pp 321-327. 19. Borges, J.; Mano, J. F., Molecular Interactions Driving the Layer-by-Layer Assembly of Multilayers. Chem. Rev. 2014, 114 (18), 8883-8942. 20. Decher, G.; Hong, J. D.; Schmitt, J., Buildup of ultrathin multilayer films by a self-assembly process: Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces. Thin Solid Films 1992, 210–211, Part 2 (0), 831835. 21. Costa, R. R.; Mano, J. F., Polyelectrolyte multilayered assemblies in biomedical technologies. Chem. Soc. Rev 2014, 43 (10), 3453-3479. 22. Boudou, T.; Crouzier, T.; Ren, K.; Blin, G.; Picart, C., Multiple functionalities of polyelectrolyte multilayer films: new biomedical applications. Advanced Materials 2010, 22 (4), 441. 23. Detzel, C.; Larkin, A.; Rajagopalan, P., Polyelectrolyte Multilayers in Tissue Engineering. Tissue Eng., Part B 2011, 17 (2), 101-113. 24. de Villiers, M. M.; Otto, D. P.; Strydom, S. J.; Lvov, Y. M., Introduction to nanocoatings produced by layer-by-layer (LbL) self-assembly. Adv Drug Delivery Rev 2011, 63 (9), 701-715. 25. Ariga, K.; Hill, J. P.; Ji, Q., Layer-by-layer assembly as a versatile bottom-up nanofabrication technique for exploratory research and realistic application. Physical Chemistry Chemical Physics 2007, 9 (19), 2319-2340. 26. Hammond, P. T., Engineering materials layer-by-layer: Challenges and opportunities in multilayer assembly. AIChE Journal 2011, 57 (11), 2928-2940. 27. Matsusaki, M.; Ajiro, H.; Kida, T.; Serizawa, T.; Akashi, M., Layer-by-Layer Assembly Through Weak Interactions and Their Biomedical Applications. Advanced Materials 2012, 24 (4), 454-474. 28. Kumar, G.; Wang, Y. C.; Co, C.; Ho, C.-C., Spatially controlled cell engineering on biomaterials using polyelectrolytes. Langmuir 2003, 19 (25), 10550-10556. 29. Silva, J. M.; Reis, R. L.; Mano, J. F., Biomimetic Extracellular Environment Based on Natural Origin Polyelectrolyte Multilayers. Small 2016, n/a-n/a. 30. Decher, G., Layer-by-layer assembly (putting molecules to work). Multilayer thin films, 2nd edn. Wiley, Weinheim 2012, 1-21. 33



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 34 of 39

31. Lee, K.; Silva, E. A.; Mooney, D. J., Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. 2011; Vol. 8, p 153-170. DOI: 10.1098/rsif.2010.0223. 32. Zanina, N.; Haddad, S.; Othmane, A.; Jouenne, T.; Vaudry, D.; Souiri, M.; Mora, L., Endothelial cell adhesion on polyelectrolyte multilayer films functionalised with fibronectin and collagen. Chem. Pap. 2012, 66 (5), 532-542. 33. Pytela, R.; Pierschbacher, M. D.; Ruoslahti, E., Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 1985, 40 (1), 191-198. 34. Ruoslahti, E.; Hayman, E. G.; Pierschbacher, M.; Engvall, E., Fibronectin: purification, immunochemical properties, and biological activities. Methods in enzymology 1981, 82, 803-831. 35. Wittmer, C. R.; Phelps, J. A.; Saltzman, W. M.; Van Tassel, P. R., Fibronectin terminated multilayer films: protein adsorption and cell attachment studies. Biomaterials 2007, 28 (5), 851-860. 36. Wierzbicka-Patynowski, I.; Schwarzbauer, J. E., The ins and outs of fibronectin matrix assembly. Journal of Cell Science 2003, 116 (16), 3269-3276. 37. Caridade, S. G.; Monge, C.; Almodóvar, J.; Guillot, R.; Lavaud, J.; Josserand, V.; Coll, J.-L.; Mano, J. F.; Picart, C., Myoconductive and osteoinductive free-standing polysaccharide membranes. Acta Biomaterialia (0). 38. Silva, J. M.; Duarte, A. R. C.; Caridade, S. G.; Picart, C.; Reis, R. L.; Mano, J. F., Tailored Freestanding Multilayered Membranes Based on Chitosan and Alginate. Biomacromolecules 2014, 15 (10), 3817-3826. 39. Alves, N. M.; Picart, C.; Mano, J. F., Self Assembling and Crosslinking of Polyelectrolyte Multilayer Films of Chitosan and Alginate Studied by QCM and IR Spectroscopy. Macromolecular Bioscience 2009, 9 (8), 776-785. 40. Martins, G. V.; Merino, E. G.; Mano, J. F.; Alves, N. M., Crosslink Effect and Albumin Adsorption onto Chitosan/Alginate Multilayered Systems: An in situ QCM-D Study. Macromolecular Bioscience 2010, 10 (12), 1444-1455. 41. Silva, J. M.; Caridade, S. G.; Oliveira, N. M.; Reis, R. L.; Mano, J. F., Chitosanalginate multilayered films with gradients of physicochemical cues. Journal of Materials Chemistry B 2015, 3 (22), 4555-4568. 42. Chaubaroux, C.; Vrana, E.; Debry, C.; Schaaf, P.; Senger, B.; Voegel, J.-C.; Haikel, Y.; Ringwald, C.; Hemmerlé, J.; Lavalle, P.; Boulmedais, F., Collagen-Based

34


Page 35 of 39

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


Fibrillar Multilayer Films Cross-Linked by a Natural Agent. Biomacromolecules 2012, 13 (7), 2128-2135. 43. Grech, J. R.; Mano, J.; Reis, R., Processing and characterization of chitosan microspheres to be used as templates for layer-by-layer assembly. J Mater Sci: Mater Med 2010, 21 (6), 1855-1865. 44. Sher, P.; Custódio, C. A.; Mano, J. F., Layer-By-Layer Technique for Producing Porous Nanostructured 3D Constructs Using Moldable Freeform Assembly of Spherical Templates. Small 2010, 6 (23), 2644-2648. 45. Silva, J. M.; Georgi, N.; Costa, R.; Sher, P.; Reis, R. L.; Van Blitterswijk, C. A.; Karperien, M.; Mano, J. F., Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering. PLoS ONE 2013, 8 (2), e55451. 46. Miranda, E.; Silva, T.; Reis, R.; Mano, J., Nanostructured Natural-Based Polyelectrolyte Multilayers to Agglomerate Chitosan Particles into Scaffolds for Tissue Engineering. Tissue Engineering Part A 2011, 17 (21-22), 2663-2674. 47. Correia, C. R.; Sher, P.; Reis, R. L.; Mano, J. F., Liquified chitosan-alginate multilayer capsules incorporating poly(l-lactic acid) microparticles as cell carriers. Soft Matter 2013, 9 (7), 2125-2130. 48. Silva, J. M.; Duarte, A. R. C.; Custódio, C. A.; Sher, P.; Neto, A. I.; Pinho, A. C. M.; Fonseca, J.; Reis, R. L.; Mano, J. F., Nanostructured Hollow Tubes Based on Chitosan and Alginate Multilayers. Advanced Healthcare Materials 2014, 3 (3), 433440. 49. Borges, J.; Caridade, S. G.; Silva, J. M.; Mano, J. F., Unraveling the Effect of the Hydration Level on the Molecular Mobility of Nanolayered Polymeric Systems. Macromolecular Rapid Communications 2015, n/a-n/a. 50. Custódio, C. A.; Alves, C. M.; Reis, R. L.; Mano, J. F., Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation. Journal of Tissue Engineering and Regenerative Medicine 2010, 4 (4), 316-323. 51. Caridade, S. G.; Monge, C.; Gilde, F.; Boudou, T.; Mano, J. F.; Picart, C., FreeStanding Polyelectrolyte Membranes Made of Chitosan and Alginate. Biomacromolecules 2013, 14 (5), 1653-1660. 52. Costa, R. R.; Custódio, C. A.; Arias, F. J.; Rodríguez‐Cabello, J. C.; Mano, J. F., Layer‐by‐Layer Assembly of Chitosan and Recombinant Biopolymers into Biomimetic Coatings with Multiple Stimuli‐Responsive Properties. Small 2011, 7 (18), 2640-2649.

35



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 36 of 39

53. Dutta, A. K.; Belfort, G., Adsorbed gels versus brushes: viscoelastic differences. Langmuir 2007, 23 (6), 3088-3094. 54. Weber, N.; Pesnell, A.; Bolikal, D.; Zeltinger, J.; Kohn, J., Viscoelastic properties of fibrinogen adsorbed to the surface of biomaterials used in blood-contacting medical devices. Langmuir 2007, 23 (6), 3298-3304. 55. Shen, W.-C.; Yang, D.; Ryser, H. J. P., Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation. Analytical Biochemistry 1984, 142 (2), 521-524. 56. Forgacs, G.; Marga, F.; Poltera, C.; Norotte, C., Fully Biological Bioprinted Blood Vessel Substitutes. Biophysical Journal 96 (3), 634a. 57. Marx, K. A., Quartz Crystal Microbalance: A Useful Tool for Studying Thin Polymer Films and Complex Biomolecular Systems at the Solution−Surface Interface. Biomacromolecules 2003, 4 (5), 1099-1120. 58. Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B., Viscoelastic Acoustic Response of Layered Polymer Films at Fluid-Solid Interfaces: Continuum Mechanics Approach. Phys. Scr. 1999, 59, 391-396. 59. Almodóvar, J.; Place, L. W.; Gogolski, J.; Erickson, K.; Kipper, M. J., Layer-byLayer Assembly of Polysaccharide-Based Polyelectrolyte Multilayers: A Spectroscopic Study of Hydrophilicity, Composition, and Ion Pairing. Biomacromolecules 2011, 12 (7), 2755-2765. 60. Chen, H.; Ouyang, W.; Lawuyi, B.; Martoni, C.; Prakash, S., Reaction of chitosan with genipin and its fluorogenic attributes for potential microcapsule membrane characterization. Journal of Biomedical Materials Research Part A 2005, 75A (4), 917-927. 61. Hillberg, A. L.; Holmes, C. A.; Tabrizian, M., Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films. Biomaterials 2009, 30 (27), 4463-4470. 62. Gao, L.; Gan, H.; Meng, Z.; Gu, R.; Wu, Z.; Zhang, L.; Zhu, X.; Sun, W.; Li, J.; Zheng, Y.; Dou, G., Effects of genipin cross-linking of chitosan hydrogels on cellular adhesion and viability. Colloids and Surfaces B: Biointerfaces 2014, 117 (0), 398-405. 63. Mi, F.-L.; Sung, H.-W.; Shyu, S.-S., Synthesis and characterization of a novel chitosan-based network prepared using naturally occurring crosslinker. Journal of Polymer Science Part A: Polymer Chemistry 2000, 38 (15), 2804-2814. 64. Mi, F. L.; Shyu, S. S.; Peng, C. K., Characterization of ring‐opening polymerization of genipin and pH‐dependent cross‐linking reactions between chitosan 36


Page 37 of 39

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


and genipin. Journal of Polymer Science Part A: Polymer Chemistry 2005, 43 (10), 1985-2000. 65. Butler, M. F.; Ng, Y. F.; Pudney, P. D., Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science Part A: Polymer Chemistry 2003, 41 (24), 39413953. 66. Gaudière, F.; Morin-Grognet, S.; Bidault, L.; Lembré, P.; Pauthe, E.; Vannier, J.-P.; Atmani, H.; Ladam, G.; Labat, B., Genipin-Cross-Linked Layer-by-Layer Assemblies: Biocompatible Microenvironments To Direct Bone Cell Fate. Biomacromolecules 2014, 15 (5), 1602-1611. 67. Silva, J. M.; Caridade, S. G.; Oliveira, N. M.; Reis, R. L.; Mano, J. F., Chitosan– alginate multilayered films with gradients of physicochemical cues. Journal of Materials Chemistry B 2015, 3 (22), 4555-4568. 68. Cui, L.; Jia, J.; Guo, Y.; Liu, Y.; Zhu, P., Preparation and characterization of IPN hydrogels composed of chitosan and gelatin cross-linked by genipin. Carbohydrate Polymers 2014, 99 (0), 31-38. 69. Gaudière, F.; Morin-Grognet, S.; Bidault, L.; Lembré, P.; Pauthe, E.; Vannier, J.-P.; Atmani, H.; Ladam, G.; Labat, B. a., Genipin-Cross-Linked Layer-by-Layer Assemblies: Biocompatible Microenvironments To Direct Bone Cell Fate. Biomacromolecules 2014, 15 (5), 1602-1611. 70. Caridade, S. G.; Merino, E. G.; Alves, N. M.; Mano, J. F., Bioactivity and Viscoelastic Characterization of Chitosan/Bioglass® Composite Membranes. Macromolecular Bioscience 2012, 12 (8), 1106-1113. 71. Mano, J. F., Viscoelastic properties of bone: Mechanical spectroscopy studies on a chicken model. Materials Science and Engineering: C 2005, 25 (2), 145-152. 72. Liu, Y.; Lu, J.; Li, H.; Wei, J.; Li, X., Engineering blood vessels through micropatterned co-culture of vascular endothelial and smooth muscle cells on bilayered electrospun fibrous mats with pDNA inoculation. Acta Biomaterialia 2015, 11 (0), 114125. 73. Li, G.; Yang, P.; Liao, Y.; Huang, N., Tailoring of the titanium surface by immobilization of heparin/fibronectin complexes for improving blood compatibility and endothelialization: an in vitro study. Biomacromolecules 2011, 12 (4), 1155-1168. 74. Li, G.; Yang, P.; Qin, W.; Maitz, M. F.; Zhou, S.; Huang, N., The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization. Biomaterials 2011, 32 (21), 4691-4703.

37



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 38 of 39

75. Li, G.; Zhang, F.; Liao, Y.; Yang, P.; Huang, N., Coimmobilization of heparin/fibronectin mixture on titanium surfaces and their blood compatibility. Colloids and Surfaces B: Biointerfaces 2010, 81 (1), 255-262.

38


Page 39 of 39

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


TABLE OF CONTENTS Multilayered Hollow Tubes as blood vessel substitutes Joana M.Silva,1,2 Catarina A. Custódio, 1,2 Rui L. Reis, 1,2, João F. Mano1,2I*#

Multilayered hollow tubes based on marine-derived polysaccharides were obtained, combining layer-by-layer (LbL) and template leaching (leaching of paraffin). Biological tests with human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) showed the potential of the hollow tubes to promote cell adhesion, spreading, and proliferation. These hollow tubular structures promise to be versatile blood vessel substitutes.

39


Multilayered Hollow Tubes as Blood Vessel ... - ACS Publications

Recommend Documents