Journal Pre-proof

Multichannel hydrogel based on a chitosan–poly(vinyl alcohol)

composition for directed growth of animal cells

Anastasia V. Sochilina, Nikita Y. Budylin, Alina M. Gamisonia,

Anatoly E. Chalykh, Vitaly P. Zubov, Alexander A. Vikhrov

PII: S0927-7765(19)30639-3
DOI: https://doi.org/10.1016/j.colsurfb.2019.110495
Reference: COLSUB 110495

To appear in: Colloids and Surfaces B: Biointerfaces

Received Date: 27 May 2019

Revised Date: 9 August 2019
Accepted Date: 5 September 2019

Please cite this article as: Sochilina AV, Budylin NY, Gamisonia AM, Chalykh AE, Zubov VP,
Vikhrov AA, Multichannel hydrogel based on a chitosan–poly(vinyl alcohol) composition for
directed growth of animal cells, Colloids and Surfaces B: Biointerfaces (2019),
doi: https://doi.org/10.1016/j.colsurfb.2019.110495

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© 2019 Published by Elsevier.

Multichannel hydrogel based on a chitosan–poly(vinyl alcohol) composition for directed
growth of animal cells

Anastasia V. Sochilina1,2*, Nikita Y. Budylin3, Alina M. Gamisonia1,4, Anatoly E.

Chalykh3, Vitaly P. Zubov1, Alexander A. Vikhrov1

1
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of
Sciences, Miklukho-Maklaya str., 16/10, Moscow 117997, Russia
2
Federal Scientific Research Centre “Crystallography and Photonics” RAS, Leninsky prospect,
59, Moscow, 119333, Russia
3
Frumkin Institute of Physical chemistry and Electrochemistry of Russian Academy of
Sciences, Leninsky prospect, 31, bld.4, Moscow, 119071, Russia
4
National Medical Research Center for Obstetrics, Gynecology and Perinatology named after
Academician V.I. Kulakov of the Ministry of Healthcare of Russian Federation, Akademika Oparina
str., 4, Moscow 117997, Russia

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*Corresponding author: ddraig@yandex.ru

Graphical abstract

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Highlights:
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 New multichannel chitosan-poly(vinyl alcohol)-based hydrogels have been

developed.
 Poly(vinyl alcohol) promotes formation of hydrogels with an oriented channel
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system.
 The developed hydrogels can provide directed growth of animal cells.
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Abstract
In this study, a new method for production of hydrogels with oriented multichannel
structure based on chitosan–poly(vinyl alcohol) compositions was developed. Microscopic
and biological studies of the obtained hydrogels were conducted to determine the optimal
composition, which would ensure that structure of the material mimics that of the epineurium
and perineurium in a nerve. Structure of the hydrogels was adjusted by variation of the initial
concentration of the precipitant, poly(vinyl alcohol), and acid in the chitosan compositions. A
single cycle of freezing and thawing of the produced hydrogels resulted in lower structural
heterogeneity, which is promising for the production of a scaffold that simulates the structure
of the native peripheral nerve. In vitro cytotoxic assays showed biocompatibility of the
manufactured hydrogels.

Keywords: multichannel hydrogel, oriented channel system, chitosan, poly(vinyl alcohol),

directed growth of animal cells

1. Introduction

Development of new materials for biological application continues to be one of the

urgent tasks of modern polymer chemistry, which allows expanding the possibilities of
medicine in treatment of various complex pathologies. One of these areas is the
regeneration of peripheral nerves, damage to which is observed, for example, in more than
500,000 people in the United States of America every year, and it is a common clinical
problem throughout the world [[1],[2]]. End-to-end neurorrhaphy is the most popular

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method of treatment when a nerve defect is less than 5 mm [3]. Transplantation of
autologous nerve grafts is considered to be the current surgical therapeutic approach of the
first choice, or the "gold standard", to repair longer nerve defects [[4],[5]]. However, nerve

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autografts suffer from many shortcomings, including limited availability of donor tissue,
damage to the functional nerve, a fragment of which is used as an autograft or allograft, the
possibility of neuroma formation, and dimension mismatch of damaged and donor nerves
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[5]. Therefore, the development of artificial nerve grafts (or nerve guidance conduits,
NGCs) is a promising alternative to autologous nerve grafts.
Many works are devoted to the development of methods for producing various types of
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NGCs. Their properties and requirements imposed thereon are summarized in a number of
reviews [[6],[7]]. Briefly, NGCs must protect the regenerating nerve from the formation of
connective tissue barriers, while ensuring the adsorption and proliferation of Schwann cells
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and formation of Büngner bands that promote axon growth through diffusion of growth
factors and nutrient exchange. Herewith, NGCs should be biocompatible and preferably
biodegradable.
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Currently, there are several types of NGCs for the regeneration of nervous tissue that
have undergone clinical trials and are available for medical use, e.g. NeuraGen, Neurotube,
Neurolac, SaluTunnelTM, and Reaxon. They are based on type I collagen, polyglycolic
acid, poly-D,L-lactide-co--carprolactone, poly(vinyl alcohol), or chitosan [[7],[8]]. These
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NGCs are mainly recommended for the regeneration of gaps and defects less than 3 cm
[[7],[9]], since they are single-lumen hollow tubes that do not have a microstructure typical of
nervous tissue. For a more adequate simulation of the microstructure of the nervous tissue,
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many options for modification of these tubular NGCs have been proposed, e.g., micro-
grooved luminal design, surface functionalization, intraluminal guidance, and combination of
the approaches [[10],[11]]. A number of articles showed that longitudinally oriented
microstructures, mimicking the orientation of the perineurium/epineurium of native
peripheral nerve tissue, promote formation of Büngner bands, Schwann cells migration, and
axon elongation [[12],[13]]. Another type of the more advanced NGCs are biomimetic
hydrogels with an oriented channel system (HOCS), structurally similar to the
perineurium/epineurium (layers around the nerve and between fascicles, Fig. S1) of the native
peripheral nervous tissue [[14],[15]].
 Chitosan (CH) demonstrates favorable biological properties, including biodegradation
and significant adhesion to living tissues. It has excellent affinity for neuroglial cells and is
almost non-toxic with respect to Schwann cell growth [16]. Moreover, CH has been shown to
stimulate the production of extracellular matrix proteins (collagen IV, fibronectin and
laminin), which promotes adhesion, migration, and differentiation of nerve cells [17]. At the
same time CH interferes with the adhesion and proliferation of fibroblasts [16], which can
prevent the formation of fibrous tissue. In addition, CH degradation products stimulate axonal
growth [18].
Several methods to produce HOCS’s based on CH have been reported thus far. For
instance, Liangliang et al. [19] proposed frontal bottom-up freezing of the CH solution for
this purpose; however, this method requires complex homemade cryo-equipment to be used
to produce HOCS’s, which limits its widespread application.
A number of works [[20],[21],[22],[23]] describe alternative ways to form a system of
oriented channels. For example, diffusion of a precipitant, layered over a chitosan solution of

of
certain viscosity, into the CH solution results in frontal precipitation of CH (a process termed
frontal gelation) accompanied by formation of elongated channels perpendicular to the
gelation front.

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Rivas-Araiza et al. [20] describe HOCS’s produced from 0.01 to 1% (w/w) CH (Mw
from 200  20 to 540  54 kg/mol) solutions. However, the channel diameter is below 5 µm,
therefore the HOCS cannot be used for directed growth of animal cells (dcell  10 µm).
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Sereni et al. [23] report HOCS’s produced from CH solutions (Mw 170 and 570 kg/mol,
dispersity 1.7 ± 0.2 and 1.5  0.3, respectively) with a concentration range from 0.75 to
4.00% (w/w). These are characterized by high structural heterogeneity across the depth of the
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gels. Only a thin layer (~3–4 mm) of the gel, located under the upper dense layer, contains
channels of desired diameter (dchannel from 10 to 35 μm), which excludes their application for
reparation of long nerve defects (l  5 mm).
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Although providing for the dimensions necessary for reparation of long defects (l > 5
mm, dchannel 150–500 µm), HOCS’s described by Ran et al.[21], similarly to the system
described in an earlier paper [22], are formed exclusively in the presence of toxic cross-
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linking agents, which may negatively influence the growth and proliferation of animal cells.
Thus, the search for effective methods to produce HOCS’s based on chitosan fit for
recovery of nervous tissue defects with l  5 mm without the use of potentially dangerous
cross-linking agents remains an urgent task.
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Under certain conditions, intermacromolecular non-covalent interactions between

functional groups of synthetic polymers can result in network-like supramolecular structures
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[24]. This effect could be used to simulate conditions critical for the formation of HOCS’s
according to Ran et al. [21], i.e. the presence of low-degree covalent cross-linking of CH
macromolecules before gelation. Therefore, we chose a biocompatible synthetic polymer
poly(vinyl alcohol) (PVA), widely used in various medical and biological applications
[[25],[26],[7]], as a CH cross-linking agent. PVA is known to effectively form complexes
with CH, e.g. as in thermosensitive hydrogels [27].
We have not found any reports on the methods to produce HOCS’s from CH–PVA
compositions. In the vast majority of works, materials based on CH–PVA compositions were
produced using the PVA property to form macroporous cryogels during several freeze–thaw
cycles. In such a case, CH is neutralized after the formation of the PVA cryogel structure or
during the last freezing step, which results in a microporous cellular structure, with geometry
mainly determined by the parameters of the PVA cryogel, incompatible with formation of
HOCS’s.
Thus, the purpose of this work was to obtain HOCS’s suitable for directional growth of
animal cells based on CH–PVA compositions without using toxic cross-linking agents.

2. Materials and methods

2.1. Materials

Chitosan (CH, Mw 3 × 105–5 × 105 g/mol, 91% degree of deacetylation, Shijiazhuang

Yishengtang Medical Product Co. Ltd. China), poly(vinyl alcohol) (PVA, Mw 8.9 × 104–9.8 ×
104 g/mol, 99% hydrolyzed), DMEM (Dulbecco’s Modified Eagle Medium), FCS (fetal calf
serum, heat inactivated), P/S/N (Penicillin/Streptomycin/Neomycin sterile solution with 5000

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units penicillin, 5 mg streptomycin and 10 mg neomycin/mL), MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), isopropyl alcohol, hydrochloric acid (HCl),
sodium hydroxide (NaOH) were used. All materials and reagents were purchased from

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Sigma-Aldrich unless otherwise mentioned.

2.2. Methods
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2.2.1. Preparation of CH-based hydrogels with oriented channel system (HOCS’s)
CH was dissolved in HCl solution (1.5 vol.%) at room temperature with vigorous
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agitation for 15 min. Then, PVA solution (10.0%) and Milli-Q water (hereafter H2OmQ) were
added into the CH solution and the mixture was stirred for another 20 min yielding a 2% CH
solution containing 0.36–0.54 vol.% HCl. The CH–PVA solution was cast into 24-well plates
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(SPL Life Sciences) or open hollow cylinders (dinner 12 or 21 mm, h 50 or 60 mm) with a
piston and stored at room temperature until complete removal of air bubbles. Then, 0.5–6.0%
NaOH solution (1:1 v/v with respect to the CH–PVA solution) was cast onto the surface of
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test solutions for pervasion from the hydrogel top to the bottom. Specimens were incubated at
room temperature for 8 h. CH–PVA hydrogels with various concentrations, 0, 0.375, 0.75,
1.5, 3.0, and 6.0%, of poly(vinyl alcohol), hereafter denoted as CH/PVA-0, CH/PVA-0.375,
CH/PVA-0.75, CH/PVA-1.5, CH/PVA-3 and CH/PVA-6, respectively, were prepared. Some
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of the hydrogel samples were then frozen–thawed (–20°C/room temperature) and washed
with H2OmQ until pH 6.5. References to percentages are by weight (% w/w), unless
otherwise mentioned.
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2.2.2. Microstructure observation

All longitudinal or transverse cross-sections of hydrogels and gelation dynamics of
CH/PVA compositions were observed using an inverted microscope (Nikon TE-2000, Nikon,
Japan) equipped with a ToupCam camera connected to a personal computer with the
ToupView software. Gelation dynamics were observed and filmed in 1-mm cuvette;
videodata were used to plot gelation front speed dependence on time. Morphologies of
lyophilized samples were observed by utilizing a JSM-U3 (JEOL, Japan) scanning electron
microscope at an accelerating voltage of 15 kV and electrical current 1 nA. Dry samples were
sputter-coated with carbon and then observed with SEM. Morphometric data, including
quantity and diameters of channels and thickness of surface and channel zones, were
collected using an image manipulation program ImageJ (http://rsb.info.nih.gov/nih-image).
All diagrams were constructed in Excel Microsoft Office software.

2.2.3. Sterilization
Samples of CH/PVA hydrogels were boiled 3 times for 30 min in 200 ml of H2OmQ
(hydrogel–H2OmQ, 1:20 v/v) to produce sterile materials.

2.2.4. Gravimetric measurement

Change in the PVA content in the hydrogels was determined by gravimetric method
using cylindrical specimens (h = 20 mm) produced in an open hollow cylinder (dinner 21 mm,
h 60 mm) with a piston to minimize losses during sample extraction. Briefly, after
sterilization samples of the hydrogels were lyophilized at –60°C using the FreeZone 1 Liter

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Benchtop Freeze Dry System (Labconco, USA) until they maintained constant weight. The
residual PVA content in hydrogels (%) was calculated using the following formula:
% PVA = (WCH/PVA − WCH)/WPVA × 100%, (1)

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where WCH/PVA and WCH were weights of CH/PVA hydrogel and CH hydrogel (CH/PVA-0),
respectively. WPVA is the mass of the initial weighed quantity of PVA.

2.2.5. Compression test -p

Compression tests were conducted on an ElectroPuls E-1000 Dynamic Test System
(Instron, USA) at a displacement rate of 0.5 mm/min (room temperature, deformation = 15%)
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in vertical direction and compressive modulus of the scaffolds was calculated (Bluehill 3
TESTING Software). At least 5 samples (h = 10  0.10 mm, d = 16  0.16 mm) were tested
for each fabrication condition.
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2.2.6. Cell culture

C6 glioma cells were cultured in DMEM media supplemented with 10% FCS and 1%
P/S/N (hereafter DMEM culture media) in 5% CO2/air, at 37°C. 1 × 104 cells per well were
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seeded in a 96 well tissue culture plates (Nunc, Thermo Fischer) with 150 μL of the medium.
Before cell cultivation on HOCS the top layer of the gel (h ~ 1 mm) was removed and
these HOCS samples were sterilized and incubated in DMEM culture media (HOCS:DMEM
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= 1:4 v/v) for 24 h. 2 × 104 cells per sample were applied to the surface of the HOCS. HOCSs
with cells were incubated for 1 h (5% CO2/air, at 37°C) in 24-well tissue culture plates
(Nunc, Thermo Fischer) after that DMEM culture media was added in wells until it covered
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the samples completely. The samples with cells were cultured for 7 days. Growth patterns
were visualized using MTT solution (0.5 mg/mL).
Medium was refreshed each 3 days.

2.2.7. Cytotoxicity tests

Cytotoxicity of the hydrogel samples was assessed by test on extracts and test by direct
contact (ISO 10993-5) using the C6 glioma cells as model cells.

2.2.7.1. Extract cytotoxicity

 Samples of sterile hydrogel were incubated in DMEM culture media (10 mg per 1 mL)
at 37°C and supernatants were collected at 24, 48, and 72 h. Cells demonstrating viability of
more than 91% were plated in a 96-well plate (104 cells/well, DMEM culture media) and
transferred to the CO2-incubator (37°C, 5% CO2/air). Then, in 24 h the medium in each well
was replaced with 150 μl of extract. Cell viability was assessed after 24, 48, and 72 h using
the MTT assay.

2.2.7.2 Direct contact cytotoxicity

Cells were seeded into a 24-well plate (5 × 104 cells/well) and incubated for 24 h
(DMEM culture media, 37°C, 5% CO2/air). Then, samples of sterile hydrogel were placed in
the wells, with each fragment occupying approximately 1/10 of the surface of the cell layer.
Before use, the samples were incubated in DMEM culture media for 12 h. Cells were
incubated for 24, 48, and 72 h, then cell viability was assessed using the MTT assay.

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2.2.8. MTT assay
At each test time point sample of the gel and/or supernatant from each culture well was
replaced with an equal volume of MTT solution (0.5 mg/mL) in DMEM without FCS and

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P/S/N. The plates were incubated at 37°C for 3 h in dark . Then, the MTT solution from each
well was replaced with an equal volume of isopropanol to dissolve the insoluble formazan
crystals and the absorbance was measured at 570 and 620 nm using a Multiscan plate reader
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(Flow Laboratories, USA). Relative cell viability was calculated by normalizing absorbance
results for culture cells incubated with experimental samples to the control:
% Cell viability = (Abs570nm − Abs620nm)Experimental/(Abs570nm − Abs620nm)Control × 100, (2)
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Cells cultivated in the medium without the extracts or samples of the gel were used as
control.
The material was considered non-cytotoxic if cell viability was reduced by no more
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than 30% compared with the control (ISO 10993-5).

3. Results and discussion

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To verify the hypothesis that HOCS’s can be formed by frontal gelation of CH–PVA
mixtures upon neutralization with NaOH, we tested the composition containing 2% CH and
1.5% PVA. We used CCH higher than the optimal concentration (1.5%) for HOCS formation
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proposed by Rivas-Araiza et al. [20], which took into account viscosity of the solution and
the concentration of the base (CNaOH = 1.5%). Our purpose was to level out the decrease in
the viscosity of the CH solution upon introduction of PVA [28] and thus avoid the formation
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of a strongly curved interphase between the CH–PVA phase and the precipitant. Otherwise,
gelation front would be curved, complicating the interpretation of the obtained results.
Tang et al. [27] showed that when “homogeneous” gelation of 1:1 CH–PVA
compositions (all components, including the precipitator, are evenly distributed), a transition
of the structure of the forming gel from macroporous to microporous is observed. To avoid
this, we used CH and PVA in a mass ratio of 1:0.75.

3.1 Structure of a CH–PVA hydrogel (proof of concept)

 Frontal gelation of control CH solution (without additives) yielded a gel structure
lacking channels (Fig. 1a). Meanwhile, replacement of a potentially dangerous cross-linking
agent (like glutaraldehyde [22], forming covalent bonds with functional groups of CH) with a
polymer, specifically PVA, which is capable of forming polycomplexes with CH, ensured the
formation of HOCS during frontal gelation of a CH–PVA composition (Fig. 1b).
Structure of the CH–PVA hydrogel is similar to that of the CH gels described by Sereni
et al. or Enache et al. [[23],[29]]. It has at least two structurally distinct zones (Fig. 1b). Close
to the upper boundary of the hydrogel there is a zone containing no oriented elements
corresponding to the region of initial contact with the precipitant solution (surface zone, SZ).
Hereafter, we show that the thickness of the SZ (hSZ) ranges from 30 μm to 1 mm,
depending on the gelation conditions. Below SZ, the region of formation and prolongation of
channels (channel zone, CZ) is located. Diameter of the channels dchannels is over 10 μm. Thus,
we confirmed the validity of the composition and gelation conditions choice from the point of
view of HOCS production for further directional cell growth.

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Fig. 1. Light-field microscopy: a) CH/PVA-0; b) CH/PVA-1.5; scale bar 100 µm. Surface zone
(SZ), dense membrane without channels; channel zone (CZ), area of formation and
prolongation of channels. Black arrows show the direction of the precipitant (NaOH) flow;
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dashed line shows the approximate border between the SZ and CZ.

Next, the effects of the concentration of the components on HOCS formation were
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studied in order to determine the optimal CH–PVA composition and gelation conditions to
produce HOCS with minimal hSZ suitable (dchannels  10 µm) for directional growth of animal
cells (including nerve cells).
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3.1.1 Effect of the base and acid concentration

Microscopic studies showed that the final value of hSZ depends on CNaOH and CHCl. hSZ
increased with the growth of CNaOH (CHCl = const) (Fig. 2 and Fig. S2) and decreased with the
growth of CHCl (CNaOH = const) (Fig. 2). For example, hSZ in CH/PVA-1.5 varied from 40
(CNaOH = 0.5%) to 300 m (CNaOH = 6%).
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Fig. 2. Dependence of the Zcz (SZ depth) in the CH/PVA-1.5 gel vs NaOH concentration at
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different HCl concentrations. Data expressed as mean ± S.D., n = 3.

A similar trend of hSZ growing with the increase of CCH has been described by Sereni et
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al. [23]. We assume that in either experimental setting (here or in [23]) the increase in the hSZ
is mainly associated not with a change in the viscosity of the solution, but with an increase of
the acid amount needed to protonate more NH2-groups with increasing CCH. In terms of
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producing NGCs, in the first approximation the samples with a minimum hSZ value are
preferable, because surface zone must be removed to ensure cell penetration into the
channels.
Analysis of the dynamics of gelation front speed (Vf) together with the video of the
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frontal gelation process in thin cuvettes at different ratios of CNaOH/CHCl for the first time ever
allowed us to demonstrate that mass initiation of channels (i.e. transition from SZ to CZ)
begins when Vf decreases to a “critical value” (Vf.crit). Interestingly, Vf.crit does not depend on
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the initial CNaOH/CHCl ratio (CPVA = const), but is determined solely by CPVA (Fig. 3).
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Fig. 3. Dynamics of Vf in samples with different CPVA and CNaOH. Black stars show
Vf.crit according to video data.
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Vf is defined by NaOH diffusion speed and its depletion in the process of

neutralization. CNaOH increase results in rise of initial Vf. In turn, the higher the initial Vf the
more time it takes before Vf decreases to Vf.crit, which defines hSZ.

3.1.2 Effect of PVA concentration

The effect of PVA is complex. Channel zone was not observed at CPVA  0.375%,
which was probably due to the low degree of CH complexation that did not support formation
of oriented channels [21]. At CPVA  0.375%, two-layered hydrogels with SZ and CZ were
formed. CZ was structurally not homogeneous: the number and diameter of oriented channels
gradually changed as the distance from surface (Z) increased (Fig. 4a). A similar
phenomenon has been described by Ran et al. [21] for cross-linked CH gels produced using
glutaraldehyde.
Increase of CPVA from 0.375 to 1.5% was accompanied by an increase in the specific
number of channels (per unit area) (Fig. 4b). Further increase in CPVA to 6% resulted in a
decrease in the specific number of channels (Fig. 4b) and an increase in the average diameter
of the channels (Fig. 5). For example, CH/PVA-1.5 at Z = 1 mm dchannel = 18.9 ± 9.3 µm, at Z
= 3 mm dchannel = 39.7 ± 23.5 µm, at Z = 5 mm dchannel = 34.0 ± 20.2 µm, for CH/PVA-6 at Z
= 1 mm dchannel = 58.0 ± 35.3 µm, at Z = 3 mm dchannel = 70.9 ± 39.0 µm, at Z = 5 mm dchannel
= 64.4 ± 39.0 µm. We speculate that PVA forms aggregates (supramolecular complexes) with
CH and the resulting local mechanical stresses lead to nucleation of channels. PVA
concentration determines the size and quantity of aggregates, which consequently affects the
initial diameter and number of channels.

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Fig. 4. a) Optical microscopy of transversal cross-sections of CH/PVA-3.0 (CHCl 0.36

vol.%, CNaOH 3.0%) at varying depths of samples (Z). Zmax, cross-section containing
maximum number of channels. Scale bar 50 µm. b) Diagram of channel number per 100 µm2
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vs depth of cross-section (Z) of HOCS depending on PVA concentration. Data expressed as

mean ± S.D., n = 4. Microscopic observation of deeper cross-sections was prevented by
fragility of deeper gel layers.
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Fig. 5. Distribution of channel diameters in the CH/PVA-1.5 and CH/PVA-6.0 (CHCl 0.36
vol.%, CNaOH 3.0%) HOCS’s at varying depths (Z) of the samples.
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Notably, the dependence of the total number of channels on Z passes through a

maximum (Zmax) (Fig. 4). As shown in Fig. 4b, when CPVA increases, Zmax shifts to deeper
layers of the hydrogel (Fig. 4b), which is partly due to an increase in hSZ (CHCl = const, CNaOH
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= const). For example, hSZ of the CH/PVA-6.0 (CNaOH = 3.0%, CHCl = 0.45 vol.%) gel
exceeded that of the CH/PVA-1.5 and CH/PVA-3.0 gels 3 and 2.3 times, respectively. In the
range of CNaOH from 0.5 to 6%, hSZ of CH/PVA-1.5 changed from 30 to 500 m and that
of CH/PVA-6.0, from 350 m to 1 mm (CHCl = 0.45 vol%). Thus, variation of the gel
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preparation conditions allows us to control hSZ and channel density.

The increase in the total number of channels at Z < Zmax occurs mainly due to the
increase in the number of the channels of smaller diameter (Figs. 4b, 5). Such channels may
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either be formed de novo or branch off from the larger channels. Initiation of secondary
channels was directly observed in the course of videoimaging of gelation in thin (1 mm)
optical cells. As evidenced by the video images, the secondary channels branched off from
primary channels of the largest diameter (Fig. 6). Secondary channel formation starts at
deeper layers with the CPVA increase (e.g. for CH/PVA-1.5 Z = 1.5 mm, for CH/PVA-6.0 Z =
3 mm), which made a major contribution to Zmax shift into deeper layers.
Diameters of secondary channels also enlarge with increase of Z, complicating the
discrimination between primary and secondary channels and thus interfering with primary
channel counting. Therefore, primary channels can be correctly counted and measured only
above zone of secondary channel formation.
Fig. 6. Microscopic photographs of secondary channels: a) initiation of formation, Z = 2 mm
(transversal cross-section), CH/PVA-3.0; b) elongation, Z = 4 mm (transversal cross-section),
CH/PVA-3.0; c) loci of secondary channel formation, Z = 2 mm (longitudinal cross-section),
CH/PVA-1.5. Circles indicate secondary channel location; arrows point at discrete secondary
channels; dotted arrows point at primary channel wall. Scale bar is 30 µm.

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For samples CH/PVA-3 and CH/PVA-6, hydrogel multilayering occurred at Z = 10

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mm, after which no growth of the channels was observed. Multilayered hydrogel structures
are described in the literature and are adequately explained by Liesegang rings phenomenon
[[30],[31]]. Although the HOCS in this work also possess multilayers in lower zones the
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whole process of channel formation cannot be explained by this phenomenon. Study of the
effects observed in the course of HOCS formation is a vast unexplored field of knowledge.
HOCS’s containing multilayers appearing at Z < 15 mm do not meet the requirements
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for NGC’s for the regeneration of large nerve tissue breaks and were not assayed in further
tests. As an optimal variant for biological tests, we chose the HOCS’s produced on the basis
of CH/PVA-1.5 (CHCl = 0.36 vol.%, CNaOH = 1.5%), which was characterized by hSZ  100
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m, dchannels  10 m. This composition enabled production of HOCS’s without multilayering
with hCZ up to 15 mm.
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3.1.3. Cryo-processing of the gels

The CH/PVA HOCS’s thus produced contain branching channels, which is beneficial
for the regeneration of soft tissues rich with blood vessels but reduces the attractiveness of
the hydrogel application in the regeneration of peripheral nerves due to the high probability
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of axon branching.
To reduce branching, the method of additional cryo-processing of HOCS was
developed. It turned out that one cycle of freezing–thawing of the produced hydrogels results
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in an increase in the average diameter of the channels, a sharp narrowing of the channel
diameters distribution (Fig. 7) and an increase in the uniformity of channel distribution vs Z
while maintaining the longitudinal orientation of the channels. For cryo-processed CH/PVA-
1.5 at Z = 1 mm dchannel = 30.7 ± 11.1 µm, at Z = 3 mm dchannel = 53.5 ± 11.0 µm, at Z = 5 mm
dchannel = 57.5 ± 13.6 µm, at Z = 10 mm dchannel = 66.5 ± 14.5 µm.
Notably, PVA at a concentrations of more than 0.75 % also acted as cryoprotector,
apparently due to the formation of cryogel [32] that prevents disruption of the oriented HOCS
structure when ice crystals are formed. Samples containing less than 0.75 % of PVA formed a
spongy system of disordered interconnected pores and were not used in biological tests.
Fig. 7. SEM of a,b) longitudinal and c) transversal cross-sections (Z = 5 mm) of CH/PVA-1.5
(CHCl 0.36 vol.%) after 1 cycle of freezing-thawing.

Thus, the structure of the produced material resembles the structure of a peripheral
nerve: outer walls of HOCS resemble epineurium and channels, perineurium (Fig. 7, Fig S1).
Alongside with unification of channel distribution across Z, one cycle of freezing–

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thawing led to an increase in compression modulus reflecting mechanical stiffness of the
material. For CH/PVA-1.5 (CHCl = 0.36 vol.%, CNaOH = 1.5%), compression modulus after

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freezing–thawing was 54.4 ± 7.5 kPa, which is 2.3 times higher than compression modulus of
the original sample (Table S1). Tang-Schomer et al. [33] reported that the compression
moduli of rat and mouse brain tissues were approximately 46.8 and 49.1 kPa, respectively.
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Thus, the CH/PVA-1.5 can be considered as a suitable candidate for nerve tissue engineering.

3.2. Biological tests

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Before the in vitro tests, the top layer of the gel (surface zone, SZ) was removed and the
HOCS samples were sterilized (boiled 3 times for 30 min each, H2OmQ–HOCS, 20:1 v/v).
After boiling, according to gravimetric analysis the residual amount of PVA was not more
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than 12.1  3.5% of the initially used amount. Thus, PVA promotes production of HOCS
with dchannels  10 μm and stabilizes their structure during freezing–thawing, while the final
gel contains almost no non-biodegradable synthetic polymer.
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3.2.1. Cytotoxicity analysis

Cytotoxicity is one of the most important factors to consider when choosing materials
for the regeneration of soft tissues. HOCS cytotoxicity was characterized by the change in the
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metabolic activity of cells using MTT analysis. This method is based on the ability of animal
cells to reduce tetrazolium salts to intensely colored formazan derivatives using an
intracellular recovery system, mainly located in the mitochondria of living cells.
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Cytotoxicity of the CH/PVA-1.5 HOCS with respect to the C6 glioma cells was
assayed upon direct contact of the cells with the gel and using the extracts method. The
HOCS did not have any significant effect on the metabolic activity of the cells; cell viability
exceeded 80% after 72 h of incubation (Fig. 8a). Thus, the HOCS can be referred to non-
cytotoxic materials (ISO 10993-5). A slight decrease in viability of cells in direct contact
with hydrogel fragments, observed after 24 h (Fig. 8a), is obviously related to the initial
mechanical effect on the cells when the gel was applied, which could lead to the death of a
fraction of the cells.
Fig. 8. a) Cytotoxicity evaluation performed for CH/PVA-1.5 (CHCl 0.36 vol.%) using the
MTT assay. b) Longitudinal cross-section of CH/PVA-1.5 on days 3, 5, and 7 after C6

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glioma cell culture cultivation after application of a tetrazolium salt. Black arrows point at
formazan crystals inside channels, which confirms the presence of active cells. Scale bar 100
µm. Data expressed as mean ± S.D., n = 4.

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The results of the MTT assay evidence that the hydrogel does not secrete cytotoxic
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compounds and can be regarded as promising for further biomedical applications.
The ability of the developed HOCS to provide oriented cell growth was evaluated using
the C6 glioma cells. Cells were applied to the surface of samples with SZ cut off and were
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cultured for 7 days. Growth patterns were visualized using MTT-solution. During the first
three days, cell growth was observed predominantly on the surface of the HOCS, followed by
penetration/invasion of the cells into the channels on days 5–7 of cultivation. The dark-
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colored areas in Fig. 8b correspond to the formed formazan crystals, which indicate high
cellular activity, both on the gel surface and inside the channels.

4. Conclusions
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A new method to produce hydrogels with oriented channels based on chitosan-

poly(vinyl alcohol) compositions, which can be considered as epineurium/perineurium-
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mimicking materials, was developed. Structure of the hydrogels can be adjusted by changing
the initial concentration of the precipitant, PVA, and acid in the CH compositions. The
developed method enables channel diameter control, as well as the control of relative
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thickness of CZ. Taking into account the requirements for NGCs for the regeneration of
peripheral nerves, we chose the optimal composition of the hydrogel and conditions for its
production. The CH/PVA HOCS is not cytotoxic and ensures directional growth of animal
cells with preservation of cellular activity, both on the surface of the gel and inside the
channels. The positive result of in vitro studies allows us to consider further in vivo studies
aimed at their potential use for the regeneration of peripheral nervous tissue.

5. Acknowledgements
We enclose gratitude to PhD Anna Prostyakova for help in assessment of mechanical
properties of HOCS.
 This work was supported by the Ministry of Science and Higher Education within the
State assignment Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS in the part
of «glioma in vitro and mechanical tests», and Russian Science Foundation (Project No. 17-
19-01416) in the part of «optical microscopy».

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