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Production of bacterial cellulose tubes for biomedical

applications: Analysis of the effect of fermentation time on
selected properties

D.R. Corzo Salinas, A. Sordelli, L.A. Martinez, G. Villoldo, C.

Bernal, M.S. Perez, P. Cerrutti, M.L. Foresti

PII: S0141-8130(21)01670-6
DOI: https://doi.org/10.1016/j.ijbiomac.2021.08.011
Reference: BIOMAC 19073

To appear in: International Journal of Biological Macromolecules

Received date: 8 April 2021

Revised date: 1 August 2021
Accepted date: 2 August 2021

Please cite this article as: D.R.C. Salinas, A. Sordelli, L.A. Martinez, et al., Production of
bacterial cellulose tubes for biomedical applications: Analysis of the effect of fermentation
time on selected properties, International Journal of Biological Macromolecules (2018),
https://doi.org/10.1016/j.ijbiomac.2021.08.011

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© 2018 © 2021 Published by Elsevier B.V.

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Production of bacterial cellulose tubes for biomedical applications: analysis of the effect of

fermentation time on selected properties

D.R. Corzo Salinasa,b, A. Sordellic, L.A. Martinezd, G. Villoldoc, C. Bernale,f, M.S. Perezf,g,h*, P.

Cerruttia,b, M.L. Forestia,f*

a. Grupo de Biotecnología y Materiales Biobasados, Instituto de Tecnología en Polímeros y

Nanotecnología (ITPN-UBA-CONICET), Facultad de Ingeniería, Universidad de Buenos

Aires, Las Heras 2214 (CP 1127AAR), Buenos Aires, Argentina. Tel/fax: 0054 11

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52850285. *mforesti@fi.uba.ar

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b. Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos

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Aires, Argentina (UBA), Av. Intendente Güiraldes 2620 (CP 1428BGA) – Pabellón de
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Industrias, Ciudad Universitaria, Buenos Aires, Argentina.

c. Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB), Hospital Italiano de

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Buenos Aires (HIBA), CONICET, Instituto Universitario HIBA, Potosí 4240 (CP 1199),
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Buenos Aires, Argentina.

d. Centro IREN, Universidad Tecnológica Nacional, Buenos Aires, Argentina.

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e. Grupo de Ingeniería en Polímeros y Materiales Compuestos, Instituto de Tecnología en

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Polímeros y Nanotecnología (ITPN-UBA-CONICET), Facultad de Ingeniería,

Universidad de Buenos Aires, Las Heras 2214 (CP 1127AAR), Buenos Aires, Argentina.

f. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

g. Instituto de Ingeniería Biomédica, Universidad de Buenos Aires, Buenos Aires,

Argentina.

h. Department of Electrical and Computer Engineering, Florida International University,

Miami, Florida 33174, US. *maxperez@fiu.edu

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Corresponding authors: mforesti@fi.uba.ar, maxperez@fiu.edu

Abstract

Biosynthesis of bacterial cellulose (BC) in cylindrical oxygen permeable molds allows

the production of hollow tubular structures of increasing interest for biomedical applications

(artificial blood vessels, ureters, urethra, trachea, esophagus, etc.). In the current contribution a

simple set-up is used to obtain BC tubes of predefined dimensions; and the effects of

fermentation time on the water holding capacity, nanofibrils network architecture, specific

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surface area, chemical purity, thermal stability, mechanical properties, and cell adhesion,

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proliferation and migration of BC tubes are systematically analysed for the first time. The results

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reported highlight the role of culture time on key properties of the BC tubes produced, with
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significant differences arising from the denser and more compact fibril arrangements generated

at longer fermentation intervals.

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Keywords: bacterial cellulose; hollow tubes; biomedical applications; fermentation time;

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nanofibrils arrangement.

1. Introduction
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Cellulosic materials with any external dimension in the 1-100 nm interval are known as
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nanocelluloses [1]. Besides typical isolation from plant sources, nanocellulose may also be

produced by microbial routes. Microbial cellulose nanofibrils (generally called “bacterial

nanocellulose”, or more commonly “bacterial cellulose”, BC) are characterized by nanometric

rectangular cross-sections and micrometric lengths, for which they are frequently called cellulose

nanoribbons. BC production has been mainly studied in Gluconacetobacter xylinus (formerly

Acetobacter xylinum, and more recently Komagataeibacter xylinus), although some other

microorganisms such as species of Agrobacterium tumefaciens, Rhizobium spp., and gram-

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positive Sarcina ventriculli have also showed the ability to secrete this biopolymer as an

extracellular primary metabolite [2]. In static culture, the interwoven twisting cellulose

nanoribbons produced by these aerobic bacteria form highly hydrated pellicles that grow at the

air-liquid interface. BC production is known to mainly depend on the bacterial strain used,

culture composition, reactor design, temperature, oxygen availability, pH, additives presence,

and fermentation time [3].

As well as plant-derived nanocelluloses, BC is recognized for a number of attractive

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properties such as biobased origin, high strength and stiffness, low density, high surface area and

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low thermal expansion [4]. In the last two decades, the mentioned properties of nanocelluloses

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have triggered the development of a number of high-tech applications of interest in the fields of
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packaging, adhesives, special papers, biomedicine, adsorbent products, flexible electronics,

rheology modifiers, drug delivery vehicles, speaker and battery membranes, filtration
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membranes, cosmetic products, etc. [5-6]. Among them, BC is particularly well-suited for
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biomedical applications like those involving wound dressings, skin substitutes, blood vessels

replacement, dental tissue regeneration, urethral implants, artificial cornea and retina, neural
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implants, tympanic membranes, and cartilage and bone regeneration [2, 7-8]. Biomedical uses of
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BC are triggered by a number of outstanding and particular properties of this nanocellulose,

including a unique fibrillar 3D nanostructure that resembles that of normal extracellular matrix,

an entangled nanofibrous network which provides the BC with extraordinary mechanical

properties, high chemical purity (BC is obtained free of lignin and hemicelluloses),

biocompatibility, the possibility of producing it in various shapes and sizes and to control its

porosity during the biosynthesis, and its high water holding capacity derived from its high

surface area and hydrophilic nature [2, 7-9]. On the other hand, in the absence of cellulolytic
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enzymes, the relatively high thermal, mechanical, and chemical stability of native BC render it

resistant to biodegradation in the human body and ensures prolonged structural integrity upon

implantation [9-10].

In this context, in the last years BC has proved useful for the production of non-

resorbable hollow three-dimensional tubes to be used in the human body as artificial blood

vessels and other tubular structures like urethra, trachea, and esophagus [8]. Even if most studies

devoted to the development of BC tubes have been focused on their use as artificial blood vessels

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of small diameter (diameters < 5-6 mm), research on tubular microbial cellulose scaffolds of

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higher diameters to be used in urethral reconstruction (diameters ≈ 9-12 mm [11]) is also of high

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importance in the context of the still unresolved search for an ideal biomaterial for such
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application [12-13]. Irrespectively of their intended biomedical use, (and although rolling of flat

bidimensional BC pellicles followed by lyophilization and rehydration [14], as well as

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perforation of rectangular cross-section BC blocks with a sharp needle [15] have been seldom
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proposed), based on the strictly aerobic nature of producing strains, BC tubes have generally

been obtained by means of tube-shaped oxygen-permeable fermenter designs. In the last years,
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fermenters made of either one or two concentric tubes of oxygen-permeable materials such as
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polyvinyl chloride, silicone, cellophane and polydimethylsiloxane (PDMS), have all proved

useful to create the air/culture medium interface required by the bacterial strains for the

production of BC tubular structures [16-20]. Bioreactor set-ups have generally included an

oxygen flow inlet, and supports were often necessary to keep the fermenters in vertical form.

In the current manuscript BC tubes of different fermentation time with diameters typical

of average human urethra were obtained by use of oxygen-permeable self-standing cylinder-

shaped PDMS bioreactors, which were kept in closed sterile glass beakers with no air/oxygen
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flow inlet requirements. Given their recognized importance in biomedical applications, the effect

of fermentation time on the BC tubes´ water holding capacity, nanoribbons arrangement,

resulting specific surface area, chemical purity, thermal stability, maximum possible load and

stress in both lengthwise and breadthwise directions, and cell adhesion, proliferation and

migration, was systematically assayed. At the authors´ best knowledge this is the first

contribution that makes such analysis devoted to infer the impact of BC tubes age on chosen

properties relevant for the intended biomedical uses.

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2. Materials and Methods

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2.1. Materials

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Materials for the production of fermenters: PDMS precursor and crosslinking agent
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(Sylgard 184 Silicone Elastomer Kit bought from Dow Corning GmbH, Wiesbaden, Germany).

Strain for BC biosynthesis: Gluconacetobacter xylinus (syn Komagataeibacter xylinus) NRRL

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B-42 gently provided by Dr. Luis Lelpi (Fundación Instituto Leloir, Buenos Aires, Argentina).
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Medium formulation for the production of BC tubes: anhydrous dextrose (Biopack), meat

peptone (Laboratorios Britania S.A.), yeast extract (Laboratorios Britania S.A.), anhydrous
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disodium phosphate (Anedra), citric acid (Merck), glycerol (Stanton). Materials used in cells
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isolation, seeding and histological analysis: Dulbecco's Modified Eagle Medium (DMEM,

Dulbecco), collagenase type IV enzyme (Gibco), fetal bovine serum (FBS, Gibco), antibiotic

solution (penicillin and streptomycin, Gibco). All other reagents used were of analytical grade

and they were used as received.

2.2. PDMS fermenters production

The molds used to produce the oxygen permeable bioreactors involved two concentric

tubes (an inner crystal PVC hose (length = 130 mm, external diameter = 11 mm) stiffened by use
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of an aluminium tube placed inside, and an outer polypropylene tube (length = 120 mm, internal

diameter = 13 mm)), whose bottom was sealed with a rubber stopper (Figure 1A). In order to

favour subsequent fermenter demolding, the mold was silanized with trichlorosilane in vacuum

for one hour.

Fermenters were made of polydimethylsiloxane (PDMS), which is an inert highly

transparent and oxygen permeable material. PDMS has been recently proposed for the

manufacture of tubular BC bioreactors whose extremes were closed with stoppers [19]. The

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PDMS precursor and the crosslinking agent were mixed for a minute in a 10:1 mass ratio, poured

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into the interspace between the two concentric tubes of the mold, left in vacuum for 0.5 h to help

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eliminate entrapped air bubbles, and finally put in the oven at 60 °C for 48 h for curing. After
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this period, PDMS tubes (length x internal diameter x wall thickness: 110 mm x 11 mm x 1 mm)

were easily separated from the mold. To avoid the use of stoppers and aiming to develop a more
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practical design, a base of the same material was added to obtain self-standing bioreactors. With
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this purpose, the bottom of the PDMS tubes produced were placed in the centre of plastic caps

with a diameter of 30 mm, which were filled with liquid PDMS to form a 2 mm-high base. The
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complete system was then oven-cured at 60 °C for 24h, and finally demolded. In this way,
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several PDMS bioreactors as the one shown in Figure 1B were prepared. The bioreactors were

left in 2 % w/v NaOH aqueous solution at room temperature for 30 min and then washed with

sterile distilled water till neutrality.

2.3. Production of BC tubes

Inocula were statically incubated at 28 °C for 72 h in 250 mL in Erlenmeyers flasks

containing 50 mL of Hestrin and Schramm (HS) medium (% w/v): anhydrous dextrose 2.0; meat

peptone 0.5; yeast extract 0.5; anhydrous disodium phosphate 0.27; citric acid 0.15; pH 6.0 [21].
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After incubation, the flasks were vortexed for 1 minute and the supernatants were used as

inoculum (10% v/v). The fermentations were carried out in the PDMS bioreactors filled with HS

media using glycerol as C source instead of glucose. Once inoculated, the fermenters were

placed into closed 1.5 L beakers and statically incubated at 28 °C under sterile air atmosphere

during 3, 6, 12 or 18 days (Figure 1C). After incubation the closed BC cylinders obtained were

removed and their ends were cut off in order to drain the fermentation medium retained inside.

The BC tubes formed were extensively rinsed with distilled water to remove the medium trapped

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into the BC structure, boiled in a water bath at 100°C in 2% w/v NaOH solution for 30 minutes

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to eliminate bacterial cells, rinsed with distilled water till neutralization, and autoclaved for 15

minutes at 121 °C.

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2.4. Characterization of BC tubes

Water holding capacity (WHC): The wet weight of cut-opened purified never-dried BC tubes
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was determined after vertically draining during 1 min followed by shaking once. Tubes were
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then dried at 110°C till constant weight. Water holding capacity (WHC) was determined as the

weight of the water removed during drying divided by the dry weight of the tubes (g water/g dry
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cellulose) [22].
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Field emission scanning electron microscopy (FESEM): The morphology and microstructure of

the inner and outer surfaces of the BC tubes was observed by use of a scanning electron

microscope Zeiss Supra 40 (Dresden, Germany) with a field emission gun operated at 3 kV.

Aiming to promote better conservation of their original structure rectangular pieces of never-

dried purified BC tubes of different fermentation times were solvent exchanged from water into

aqueous ethanol solutions of increasing alcohol concentration (i.e. 25, 50, 75%, 100% v/v) prior

to freeze-drying. In each step, BC pieces were soaked for 1 h with shaking. Solvent-exchanged
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samples were then sprayed with liquid nitrogen, lyophilized for 6 h at -60 °C and 0.003 bar

(Labconco FreeZone lyophilizer), and sputtered coated with a thin layer of gold before

observation.

Specific surface area measurement by Congo red adsorption method (SSA): The specific surface

area of the never-dried BC tubes corresponding to different fermentation times was determined

using the Congo red adsorption method [23-25]. To this end, never-dried samples of the BC

tubes were placed in 100 mL of 0.1 M pH 6 phosphate buffer (dry BC content: 0.033% w/v) and

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contacted with various amounts of Congo red that guaranteed dye concentrations of 5, 10, 15 and

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20% w/w with respect to dry BC. Systems were incubated in a shaken water bath at 60ºC under

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120 rpm for 4 days, time at which adsorption equilibrium had been achieved. BC pieces were
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then removed, samples were centrifuged at 12000 rpm for 10 min and the Congo red

concentration in the remaining supernatants was evaluated from the absorbance measured at 𝜆 =
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500 nm on a Spectrum SP 1105 Spectrophotometer. Based on Langmuir isotherms, the

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maximum concentration of the dye adsorbed was calculated by use of Equation (1):

[E]/[A] = 1/(Kad Amax) + [E]/Amax Equation (1)

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where [E] is the concentration of Congo red at adsorption equilibrium in mg/mL, [A] is the
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amount of Congo red adsorbed on the BC surface in mg/g, Amax is the maximum concentration

of dye adsorbed in mg/g, and Kad is the adsorption equilibrium constant. The specific surface

area (SSA) of the BC tubes was then determined by use of Equation 2:

SSA = Amax N SA / (MW 1021) Equation (2)

where N is Avogadro’s constant, SA is the area occupied by a single dye molecule (1.73 nm 2),

and MW is the molecular weight of Congo red (696 g/mol).

Fourier transform infrared spectroscopy (FTIR): Fourier transform infrared spectra of dried
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powdered samples of BC tubes were collected with an IR Affinity-1 Shimadzu Fourier

Transform Infrared Spectrophotometer in absorbance mode. Carefully dried (110 ºC, overnight)

thoroughly milled samples were mixed with KBr at 1:100 ratio and pressed into discs at 7 kg/cm2

which were further dried during 2 h at 110 °C. The IR spectra were recorded between 4000 and

650 cm−1 with 40 scans and at a resolution of 4 cm−1. Spectra were baseline corrected and

normalized against the intensity of the absorption at 1160 cm−1 corresponding to the (C–O–C)

link of cellulose [26].

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Thermogravimetric analysis (TGA): Thermogravimetric analysis of dried powdered samples (2

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mg) of BC tubes was performed in a TGA-50 Shimadzu instrument after preconditioning (110

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°C, 2 h). Samples were heated from 25 °C to 600 °C at a constant heating rate of 10 °C/min

under an inert nitrogen atmosphere (30 mL/min, 2 kg/cm2). Tonset values were calculated as the
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temperature of 5% weight loss (after moisture removal), whereas Tmax accounts for the
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temperature of greatest rate of change on the weight loss curve. DTG data was normalized with
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respect to the initial mass of the sample.

Mechanical characterization in tensile mode: Mechanical characterization was carried out in an

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Instron dynamometer 5982 on samples directly cut from the never-dried BC tubes of varying
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fermentation time. Dumbbell samples (type IV ASTM D 638) cut from the tubes in the axial

direction were stretched lengthwise (Figure 2A), while rings of 10 mm in width were cut and

stretched breadthwise (radial direction) by use of two U-shaped screws which were inserted

through the BC rings (Figure 2B). In order to simulate the hydration conditions to which the BC

tubes would be exposed in the human body, and also to prevent significant change in wall size

due to water loss during stretching, the ISO 7198: 2016 standard for vascular prostheses and

tubular grafts was adapted by using specially designed devices that allowed immersing and
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keeping the samples in distilled water all throughout the tests (Figure 2). Measurements were

performed in triplicate at room temperature (no significant changes between results at RT≈ 20°C

and at human body temperature ≈37°C were expected) using a crosshead speed of 5 mm/min.

Load-displacement curves were obtained and average values of compliance (displacement/load

ratio for the radial direction), maximum load and maximum stress along with their deviations

were calculated.

Adipose tissue-derived stem cells (ADSCs) isolation, seeding and histological analysis: Tests

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were carried out following standardized protocols approved by the Internal Committee for

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Laboratory Animal Care and Use (CICUAL, Comité Institucional para el Cuidado y Uso de

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Animales de Laboratorio) of Hospital Italiano de Buenos Aires. The adipose tissue extracted
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from the abdomen of anesthetized male Wistar rats was deposited in Dulbecco's Modified Eagle

Medium (DMEM), washed with physiological solution, and residual blood vessels and
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connective tissues were removed and discarded under sterile conditions. The adipose tissue was
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then minced with Mayo and Metzenbaun scissors to facilitate its subsequent contact with

collagenase type IV enzyme (0.1%). A volume of 2 cm3 of the minced adipose tissue was
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digested with 2 cm3 of enzyme solution at 37°C with shaking (2000 rpm) during 45 min, after
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which the action of the enzyme was stopped by dilution with 2 cm3 of DMEM. The suspension

was then centrifuged at 1200 rpm during 10 min. The supernatant was removed and the cell

pellet was resuspended in DMEM enriched with fetal bovine serum (FBS) and 1% antibiotic

solution (penicillin and streptomycin).

Before cell seeding, never-dried sterilized BC tubes were immersed in physiological

solution during 2 h. After this time, 5 knots were made with nonabsorbable suture polypropylene

4.0 thread at one end of each tube, 1.5 mL of the ADSCs suspension were poured inside, and the
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other end of each tube was closed with another 5 knots. The excess ends of the tubes were

trimmed and held in DMEM for later use as histological blanks. The seeded tubes were left to

stand on a Petri dish and incubated in a humidified atmosphere of 95% air/5% CO2 at 37 °C for 2

h. Afterwards, the seeded BC tubes were rotated and returned to the incubator until the next day,

favouring the seeding of the cells on their entire inner surface. To allow daily culture medium

refreshing tubes sutures were trimmed, and the cell seeded scaffolds were cultured for 4 and 7

days. After this period, 0.5 cm-in-width-rings of each sample were cut, fixed with neutral

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buffered formalin (12 h), coated with paraffin, sliced, deparaffinized and finally stained with

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hematoxylin and eosin (H&E) for examination using a light microscope (Nikon Eclipse E400).

3. Results and discussion

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The PDMS fermenters shown in Figure 1B were used in the production of BC tubes

during different fermentation times, i.e. 3, 6, 12 and 18 days. Figure 3 shows the BC tubes
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obtained using the simple set-up shown in Figure 1C and schematized in Figure 3A. The
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oxygen permeable material used in the fermenters production allowed BC formation at the

culture medium/PDMS interface (i.e., inner walls and base of the bioreactor) and at the upper
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culture medium/air interface (Figure 3B). Closed BC hollow tubes with the culture medium
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inside were recovered from the fermenters at the predefined fermentation times (Figure 3C), and

medium removal was allowed by cutting off their ends (Figure 3D). BC tubes of 90 mm-in-

length and with an external diameter of 11 mm were thus obtained. The atmospheric oxygen

available in the sterilized beakers containing the fermenters proved enough for BC tubes

formation without any additional gas flow inlet requirement. Besides, the self-standing PDMS

cylindrical fermenters produced showed to be practical for obtaining a simple and stable set-up

whose transparency allowed visual inspection of the evolution of cellulose production.

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Within the incubation period assayed longer fermentation times resulted in BC tubes of

increasing wall thickness, reaching ≈ 0.4 mm for the oldest tube (the difficulties associated with

the accurate measurement of highly swollen BC samples thicknesses are well-known [18, 20,

27]). Likewise, the dry and wet weight of the tubes produced also increased with fermentation

time. Weight data was used to calculate the water holding capacity (WHC) of the tubes (Figure

4). Even if WHC values are known to depend strongly on the method used to stabilize the wet

BC sample prior to weighing [22], results included in Figure 4 are in the order of those

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previously reported for other BC tubes (i.e. ≈ 300 g/g as inferred from the reported water and BC

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content data; [28]), and also for flat BC samples (e.g. 60-400 g/g; [3, 22, 29-31]). The high

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characteristic WHC values of BC result from the hydrophilic nature of cellulose and from the
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well separated nanofibrils of microbial cellulose which create an expanded surface area and a

highly porous matrix [30, 32]. The 3D network formed by mechanically entangled cellulose
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nanoribbons contributes to the entrapment of water in BC [31]. Water molecules reside inside the
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pores and they also bound to the cellulose fibrils surface through hydrogen bonding [33].

Results summarized in Figure 4 illustrate a progressive reduction in WHC values as

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longer incubation times were used. Previous contributions have shown that the WHC of
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microbial cellulose is not constant, and that it actually depends (positively) on its porosity and

specific surface area [30]. If there are plenty of empty spaces among the BC fibrils, then more

water can penetrate and adsorb onto the material, resulting in higher WHC values [30]. The

porosity and surface area of BC in turn have proved to depend on the arrangement of the fibrils

in the BC network, which has already been shown to vary with the producing strain, the culture

media formulation, the inoculum size and the fermentation time and conditions; as well as with

the BC purification treatment and drying method [30, 34-37]. The reduction of WHC with the
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age of BC tubes shown in Figure 4 suggests that the nanoribbons arrangement must have

significantly varied with incubation time, conditioning the weight of water held per unit weight

of cellulose. Previous investigations on flat BC pellicles have shown that increasing fermentation

times resulted in membranes with a less porous and more compact nanoribbons network

structure [36, 38], which in turn affected the water holding capacity and thermal stability of the

products, as well as their mechanical properties in the dried state [38]. The key effect of culture

time on the porosity and mechanical properties of recycled paper resulting from the in-situ

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production of BC with recycled fibers has also been pointed out [37].

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SEM images of BC tubes revealed an entangled web of twisting cellulose fibrils of

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nanometric sections with no visible orientation (Figure 5). No significant variation in terms of
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fibrils width distribution could be observed among samples of different culture time. Based on at

least 20 measurements per sample the image analysis performed showed that: i) in all cases
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fibrils widths fell within the 12-90 nm interval, ii) more than 70% of the measurements were in
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the 43-56 nm interval, and iii) for all culture times mean width values were in the 43-48 nm

range (standard deviations: 17-21 nm). On the other hand, SEM images confirmed that culture
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time had a significant impact on cellulose nanoribbons networks packaging, as revealed by the
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much looser nanofibrils arrangements observed on both sides of the BC tube of 3 days when

compared with the corresponding sides of the tube of 18 days (Figure 5A and Figure 5B versus

Figure 5C and Figure 5D, respectively).

Micrographs collected in Figure 5 also show that the inner surfaces of the tubes have a

more open nanoribbons network than their outer surfaces, which were the ones in contact with

the oxygen permeable bioreactor surface (Figure 5A and Figure 5C versus Figure 5B and

Figure 5D, respectively). The outer sides of BC tubes show smoother surfaces resulting from
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denser and more compact network structures than the inside of the tubes, the latter accounting for

BC nanoribbons layers which were formed first. Accordingly, Bodin et al. (2007) also observed

that BC tubes had a denser surface in the side in contact with the oxygen permeable material

(i.e., the inner surface of the BC tubes which were in contact with the silicone tubing) and a

porous surface on the glass side, which authors associated with the increase in the production of

cellulose at the silicone/medium interface, as well as with a slight drying and a subsequent

contraction onto the smooth silicone tubing [16]. Denser nanofibrils networks at the tubes

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surfaces in contact with air were also observed by Hong et al. (2015) whose set-up involved two

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concentric oxygen permeable silicone tubes [20]. A denser nanofiber network structure in the BC

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surface in contact with air/air permeable bioreactor material has been previously attributed to a
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differential distribution of oxygen and thus also of active BC-producing bacteria [18, 39].

Observations are also in line with the characteristic morphology of flat BC pellicles whose
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nanofibrils networks are much denser close to the upper culture medium/air transition zone than
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on the opposite side [3, 36, 40].

Fibrils network architecture is expected to have an outstanding role on the mechanical

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properties of the BC scaffolds produced, as well as on cell adhesion, proliferation and migration.
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The particular importance of adequate nanofibrils compaction for promoting cell attachment,

growth, infiltration, and vascularization; as well as for facilitating nutrients supply, high oxygen

diffusion and waste transport in BC scaffolds for tissue regeneration or grafts, has been

previously recognized [40-41]. Besides, the importance of a proper porous structure of BC tubes

to be used in urethral tissue regeneration has been particularly highlighted [13]. In this context,

the BC fibrils network architecture in the tubes of increasing fermentation times was further

evaluated by determination of their specific surface area (SSA). The Congo red dye adsorption
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method is generally considered an accurate method for the measurement of the SSA of cellulosic

materials in the never-dried state [25]. SSA determination by this method has previously proved

useful for inferring the relative aggregation of dehydrated and later redispersed cellulose

nanofibrils of plant origin [42], as well as for assaying the effect of processing methods on the

SSA of microfibrillated cellulose isolated from hardwood pulp samples [25]. Congo red has a

high degree of specificity for the (1->4)-beta-D-glucopyranosic units of cellulose, being its

adsorption strongly dependent on the pH of the medium [43]. Since measurement of SSA by the

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adsorption of Congo red is performed in aqueous suspension, the original BC tubes nanoribbons

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network structure is expected to be kept as intact as possible. On the other hand, SSA

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determination methods relying on nitrogen adsorption (e.g. BET area determination) require the
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previous drying of the samples, which may lead to cellulose hornification (i.e. the irreversible

formation of hydrogen bonds between fibrils during suspension dehydration [44-45]). In this
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process large agglomerations are produced and significant reduction of the surface area of the
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fibers exposed may take place [42, 44]. Figure 6 summarizes the SSA values determined by the

dye adsorption method for the never-dried BC tubes produced at different fermentation times. In
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accordance with WHC and FESEM results, the specific surface area of BC tubes effectively
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exposed to the dye showed to significantly decrease with fermentation time, a behavior

compatible with BC networks formed by more closely packed cellulose nanoribbons in the tubes

obtained at longer periods of time.

Figure 7A collects the Fourier transform infrared (FTIR) spectra of BC tubes of

increasing fermentation times. Spectra show absorbances characteristic of native cellulose, e.g.

vibrations of hydroxyl groups at 3000-3500 cm-1; C-H stretching (2850-2920 cm−1); absorbed

water molecules (1640 cm−1); CH2 symmetrical bending (1428 cm−1); cellulose C-O-C bridges
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(1160 cm−1); C-O stretching (1108 cm−1); C-O-C (1057 cm−1); and the band at 893 cm−1 typical

of b-linked glucose polymers [46-51]. Moreover, the spectra of BC tubes of 6, 12 and 18 days

shows an additional absorbance centered at 1545 cm-1 which is attributed to the bending

vibration of amide II N-H bonds [51]. The existence of nitrogenated compounds in BC samples

is associated with trapped nutrient medium residues (proteins and amino acids) remaining in the

samples even after extensive washing [38, 51]. As previously reported for BC flat pellicles of

different culture time [38], the mentioned absorbance has been found to increase with the age of

of
the BC tubes, suggesting a higher difficulty for removing nitrogenated compounds from the BC

ro
nanofibrillar structure when thicker pellicles and/or more closely packed intertwined

nanoribbons networks are obtained.

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Figure 7B collects the TG and DTG curves of dried pieces of BC tubes of increasing

fermentation time, illustrating their thermal decomposition patterns. The TG data of all samples
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showed two weight loss events associated with BC dehydration (RT-120 °C, ca. 3 wt.% due to
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moisture remaining after samples preconditioning) and cellulose decomposition (≈ 200-400 °C),

respectively. As expected, data illustrate that thermal decomposition of BC tubes will not take
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place while being used in a physiological environment or during sterilization procedures.

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However, the Tonset of flat BC pellicles has previously been shown to increase with nanofibrils

networks compactness [38], so Tonset and Tmax values were calculated and summarized in Figure

7B. Results show that both, Tonset and Tmax increased with culture time, with 32-40 °C differences

between the samples of 3 and 18 days. Increments in the Tonset and Tmax of BC tubes with

fermentation time are in line with a more densely coagulated nanofibril network of the older

pellicles, where a higher probability of interfibrillar interactions through hydrogen bonding is

expected.
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Samples of never-dried BC tubes of increasing fermentation time were then assayed for

their mechanical properties in tensile mode in both axial and radial configurations. Figures 8A

and 8B collect the load-displacement curves and the compliance values as a function of

fermentation time for the radial configuration (this is the direction that best represents the actual

loading condition that the tubes will have to withstand in the envisaged application). Several

peaks can be seen in all load-displacement curves, indicating that the material successively

“tears” as it breaks (Figure 8A). This behaviour has been sometimes associated with a layered

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structure where the layers building up the tube wall are not fully connected [16]. In accordance

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with the variation of the slope of load-displacement curves, Figure 8B shows that compliance

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values decrease with increasing fermentation time. Lower compliance values indicate lower
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material deformability, which can be explained in terms of the denser and more compact fibril

arrangements generated at longer fermentation intervals.

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Given the common difficulties associated with an accurate measurement of highly

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hydrated samples thicknesses, in the literature the strength of BC tubes has been frequently

reported in terms of maximum load (in N) instead of maximum stress (in MPa) [18, 20, 27]. Both
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maximum load and maximum stress values in the axial and radial directions are herein presented
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as a function of fermentation time. Results shown in Figures 8C and 8D highlight the significant

effect of incubation time on the maximum load and/or stress of the BC tubes produced. As it can

be observed in Figure 8C, maximum tensile load values increased with incubation time for both

stretching directions, being the effect more pronounced in the axial direction. The observed trend

can be attributed to more fibrils as well as to the more closely packed structures in the older

tubes. Results are in agreement with those reported by Tang et al. (2017) who reported that the

maximum load of BC tubes increased with prolonged cultivation time during the period of 4–10
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days, a phenomenon attributed to more BC fibers being formed at longer culture times [18].

Maximum load values shown in Figure 8C fall within the range of those reported in the

literature for similar BC tubes [18, 20, 27]; and in particular maximum load in the radial

direction values are comparable with those reported for native tissue blood vessels (0.36–2.2 N)

[27].

On the other hand, -and despite the expectable differences associated with a biological

material produced under varying conditions (culture medium, strain, bioreactor design,

of
fermentation conditions), as well as the difficulties related to tubes thickness measurement-,

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maximum stress values for both axial and radial directions herein obtained (Figure 8D) are in

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the same order of those reported by others [16-17, 40]. Moreover, maximum stress values in
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lengthwise stretching direction are similar to those reported for rabbit urethra [12]. Entangled

nanofibers networks provide the BC scaffolds with strong mechanical properties [20, 52]. In
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reference to the effect of fermentation time on maximum stress values, different trends were
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observed depending on the stretching direction. While maximum stress values in the radial

direction showed to be roughly independent of incubation time, a significant increase in the

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maximum stress in the axial direction was observed with the age of the BC tubes. The latter trend
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can be attributed to a denser and more compact fibril network in the BC tubes obtained at higher

fermentation times which results in stronger structures, at least in the axial direction. Reduction

in the maximum axial stress values of BC tubes of increased porous structure have been

previously reported [13, 52]. Table 1 summarizes maximum load and maximum stress values

determined in the current work, as well as corresponding data reported in the literature for other

BC tubes, rabbit urethra and human and porcine blood vessels.

Finally, adhesion, proliferation and migration of adipose tissue-derived stem cells

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(ADSCs) on never-dried BC tubes were evaluated. ADSCs were the cells chosen to seed the BC

scaffolds based on their recognized ability to differentiate into mature mesenchymal tissues in

vitro, their relatively easy obtention in large numbers through minimally invasive procedures,

and the possibility of being effectively transplanted into the body [53]. The histological images

obtained for the tubes of 6 and 18 days of fermentation are shown in Figure 9, confirming cell

adhesion onto BC. On the other hand, results indicate that the number of cells shown in the

samples was significantly higher for the 6 days-tube. Differently from the cell behaviour

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observed in the BC tubes of 18 days, in the 6 days-sample ADSCs effectively proliferated and

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also migrated from the tubes surface into the nanoribbons network, which may be attributed to its

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more open fibrils structure. Accordingly, Zahedmanesh et al. (2011) studied the infiltration of
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mesenchymal stem cells in BC tubes during 28 days, concluding that more porous surfaces

promoted higher infiltration thicknesses, whereas in highly packed fibrous structures cells
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adhered but no significant infiltration into BC took place [54]. Similarly, Bäckdahl et al. (2006)
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also reported that the nanofibrils arrangement influenced how far human smooth muscle cells

could migrate into the BC. Authors found that in compact BC sides cells were present only on
ur

the material surface, whereas in porous sides cells migrated into the BC which would be
Jo

beneficial for promoting the scaffold integration with the host tissue [40]. Later contributions of

these authors were devoted to improve cells migration into the BC scaffolds by incorporation of

selected porogen materials in the fermentation medium [52]. As previously reported, although

more open nanofibrils networks are desirable for providing better cell infiltration and improved

diffusion of nutrients and waste, reasonable mechanical strength values should be guaranteed,

especially for scaffolds intended to be used for urethral reconstruction which must bear voiding

urine pressures [13, 55-56].

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4. Conclusions

In the current contribution, air permeable self-standing tubular PDMS fermenters were

prepared and used in the biosynthesis of BC tubes of biomedical interest. PDMS proved to be a

versatile material for easily obtaining the required bioreactor shape and size (length, diameter,

wall thickness) at a relative low cost; whereas its transparency allowed continuous monitoring of

the evolution of BC production. Besides, the atmospheric oxygen available in the sterilized

beakers containing the fermenters proved enough for BC tubes formation and no additional

of
air/enriched air flow inlet was required.

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The described set-up was used to systematically assay the effect of fermentation time on

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selected properties of permanent non-resorbable BC tubes. The results obtained illustrated the
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key role that the nanofibrils network structures developed at varying fermentation times had on

the properties of the tubes produced. As higher culture times were set, BC tubes with more
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closely packed nanofibrils networks and smaller specific surface areas were obtained. The
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previous had a marked effect on the mechanical properties of the tubes in the wet state, with

improved values as longer culture times were used. On the other hand, adipose tissue-derived
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stem cells proliferation and migration into BC was promoted by the more open fibrils networks
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of younger tubes.

Overall, results highlighted the importance of culture time for altering the density and

compactness of the nanofibrils networks in the BC tubes produced, as well as for attaining a

proper balance between cell ingrowth and mechanical properties compatible with the envisaged

use. In view of the known importance of nanofibrils network density and tubes wall thickness on

other key properties of BC tubes for biomedical use such as permeability, suture retention

strength (i.e., force required to pull a suture out of the prosthesis) and burst pressure (i.e.,
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maximum internal pressure that the graft resists before failure) [18, 28, 57]; further research on

the effect of culture time on the mentioned properties of the produced BC tubes is required.

Competing interests’ statement

Authors have no competing interests to declare.

Declaration of interest

None.

5. Acknowledgments

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This work was supported by Ministerio de Ciencia, Tecnología e Innovación de

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Argentina [PICT 2016 0843 and PICT 2016 1021].

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Captions to illustrations

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Figure 1. (A) Scheme of the mold used to cast the PDMS tubes: (1) stopper placed at the bottom

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of the concentric tubes; (2) external polypropylene tube; (3) void area within which PDMS is
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casted; (4) crystal PVC hose; (5) aluminium cylinder used to stiffen the hose; (B) Self-standing

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bioreactors.
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Figure 2. Devices used for the mechanical characterization of samples in tensile mode while

kept in distilled water at room temperature. (A) Lengthwise stretching (axial direction); (B)
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Breadthwise stretching (radial direction).

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Figure 3. (A) Scheme of culture system; (B) PDMS fermenters with the BC tubes already

produced inside; (C) Closed BC tubes; (D) BC tubes obtained after cutting off their closed ends.

Figure 4. Effect of fermentation time on the dry weight and water holding capacity of never-

dried BC tubes.

Figure 5. Representative scanning electron microscopy micrographs of freeze-dried BC tubes

walls of 3 and 18 days of fermentation: (A) 3 days, inner side; (B) 3 days, outer side (in contact

with the oxygen entrance); (C) 18 days, inner side; (D) 18 days, outer side (in contact with the
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oxygen entrance).

Figure 6. Effect of fermentation time on the specific surface area of never-dried BC tubes

determined by Congo red adsorption method.

Figure 7. (A) FTIR spectra of BC tubes of increasing fermentation time; (B) TG and DTG

curves of BC tubes of increasing fermentation time.

Figure 8. Effect of fermentation time on (A) load-displacement curves obtained in radial tests;

(B) compliance values for the radial configuration; (C) maximum tensile load values (axial and

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radial directions); (D) maximum tensile stress values (axial and radial directions).

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Figure 9. Histological images of cross sections of BC tubes with ADSCs cultured for 4 and 7

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days stained with H&E. (A) 6 days-BC tube, blank; (B) 6 days-BC tube, ADSCs cultured for 4
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days; (C) 6 days-BC tube, ADSCs cultured for 7 days; (D) 18 days-BC tube, blank; (E) 18 days-

BC tube, ADSCs cultured for 4 days; (F) 18 days-BC tube, ADSCs cultured for 7 days.
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Figure 1
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Figure 2

A B

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Figure 3

A B C
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Figure 4

WHC
Dry Weight
100 0,16

80

Dry weight [mg]

0,12
WHC [g/g]

60
0,08

of
40

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0,04
20

0 -p 0,00
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3 6 12 18
Fermentation time [days]
Figure 5
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Figure 6

350

300

250
SSA [m /g]

200
2

of
150

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100

50

0
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3 6 12 18
Fermentation time [days]
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Figure 7

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Figure 8

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Figure 9

A B C

50 m 200 m 100 m

50 m
D E F

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200 m 100 m

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Table 1. Maximum load and stress values reported for BC tubes, rabbit urethra and human and
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porcine blood vessels.
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Radial direction
Sample Maximum load Maximum stress Ref.
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values (N) values (MPa)

BC tube - 0,28 - 0,4 [16]
BC tube - 0,37 [17]
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BC tube 1,22 - 1,6 - [20]

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BC tube - 0,15 [40]

BC tube 0,8 - [27]
Human hollow vein 0,36 - [27]
Human abdominal aorta 0,95 - [27]
Human jugular vein 1,6 - [27]
Human carotid artery 2,2 - [27]
Porcine carotid artery - 0,95 [40]
BC tube 0,38 - 1,47 0,04 - 0,19 This work
Axial direction
Sample Maximum load Maximum stress Ref.

values (N) values (MPa)

BC tube - 0,59 [17]
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BC tube 1,3 - 2,8 - [20]

BC tube - 60 - 92 [19]
BC tube - 16,6 - 30,5 [13]
BC tube 1,7 – 18,0 - [18]
BC tube 1,64 - [57]
Rabbit urethra - 0,4 [12]
Human abdominal aorta 8,5 - 13,8 - [58]
Human saphenous vein 19,6 - 31,8 - [59]
Human umbilical vein 16,8 - 27,4 - [59]
BC tube 0,63 - 2,09 0,48 - 0,81 This work

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Author statement
D.R. Corzo Salinas: formal analysis, investigation, writing.
A. Sordelli: investigation; methodology.
L.A. Martinez: methodology; writing.
G. Villoldo: methodology; supervision; funding acquisition.
C. Bernal: formal analysis; methodology; investigation; supervision; writing.
M.S. Perez: methodology; supervision; funding acquisition.
P. Cerrutti: formal analysis; investigation; methodology; conceptualization; supervision; writing.
M.L. Foresti: conceptualization; formal analysis; methodology; funding acquisition; supervision;

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writing.

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