Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant

Accepted Manuscript Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant

Magdalena Kołaczkowska, Piotr Siondalski, Maciej M. (Michał) Kowalik, Rafał Pęksa, Aldona Długa, Wacław Zając, Paulina Dederko, Ilona Kołodziejska, Edyta Malinowska-Pańczyk, Izabela Sinkiewicz, Hanna Staroszczyk, Agata Śliwińska, Alicja Stanisławska, Marek Szkodo, Paulina Pałczyńska, Grzegorz Jabłoński, Andrzej Borman, Piotr Wilczek PII: DOI: Reference:

S0928-4931(18)31182-2 https://doi.org/10.1016/j.msec.2018.12.016 MSC 9126

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

29 April 2018 19 October 2018 6 December 2018

Please cite this article as: Magdalena Kołaczkowska, Piotr Siondalski, Maciej M. (Michał) Kowalik, Rafał Pęksa, Aldona Długa, Wacław Zając, Paulina Dederko, Ilona Kołodziejska, Edyta Malinowska-Pańczyk, Izabela Sinkiewicz, Hanna Staroszczyk, Agata Śliwińska, Alicja Stanisławska, Marek Szkodo, Paulina Pałczyńska, Grzegorz Jabłoński, Andrzej Borman, Piotr Wilczek , Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant. Msc (2018), https://doi.org/10.1016/j.msec.2018.12.016

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ACCEPTED MANUSCRIPT Title Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant.

authors 1. Magdalena Kołaczkowska, MD, PhD

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Department of Cardiac and Vascular Surgery, Medical University of Gdańsk Ul. Dębinki 7 80-211 Gdańsk

[email protected]

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2. Piotr Siondalski, MD, PhD, D.Sc.

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Department of Cardiac and Vascular Surgery, Medical University of Gdańsk

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Ul. Dębinki 7 80-211 Gdańsk

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Maciej M. (Michał) Kowalik, M.D., Ph.D. Department of Cardiac Anesthesiology Medical University of Gdańsk, Ul. Dębinki 7 80-211 Gdańsk [email protected]

Rafał Pęksa, MD, PhD Department of Patomorphology Medical University of Gdańsk, Ul. Dębinki 7, 80-211 Gdańsk

[email protected]

ACCEPTED MANUSCRIPT 5. Aldona Długa PhD student / MSc Bowil Biotech Sp. z.o.o. Ul. Skandynawska 7, 84-120 Władysławowo

[email protected]

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6. Wacław Zając

Wetprom, Veterinary Clinic, Zwycięstwa 333, 75-001 Koszalin, Poland [email protected] 7. Paulina Dederko, M.Sc., [email protected]

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8. Ilona Kołodziejska, Prof.,

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Edyta Malinowska-Pańczyk, Ph.D., D.Sc., [email protected] 10.

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Izabela Sinkiewicz , Ph.D., [email protected] 11.

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Hanna Staroszczyk, Ph.D., D.Sc., [email protected] 12

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Agata Śliwińska, M.Sc. [email protected]

Gdańsk University of Technology Chemical Faculty,

G. Narutowicza Street 11/12 80-233 Gdańsk, Poland

13. M.Sc.Eng. Alicja Stanisławska, Gdansk University of Technology, Department of Materials Science and Welding Engineering,

ACCEPTED MANUSCRIPT Narutowicza 11/12, 80-233 Gdańsk, Poland [email protected] 14 Marek Szkodo, prof. Gdansk University of Technology, Department of Materials Science and Welding Engineering, Narutowicza 11/12, 80-233 Gdańsk, Poland, [email protected]

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15 Paulina Pałczyńska, MSc , Department of Animal and Human Physiology, University of Gdansk, ul. Wita Stwosza 59 80-308 Gdańsk, Poland,

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Grzegorz Jabłoński, MSc, Department of Animal and Human Physiology, University of Gdansk

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Andrzej Borman prof

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ul. Wita Stwosza 59 80-308 Gdańsk [email protected]

Department of Animal and Human Physiology, University of Gdansk

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ul. Wita Stwosza 59 80-308 Gdańsk [email protected]

18 Piotr Wilczek prof

Professor Zbigniew Religa Foundation of Cardiac Surgery Development ul. Wolności 345a 41-800 Zabrze [email protected]

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Title Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant. Abstract

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Bionanocellulose (BNC) is a clear polymer produced by the bacterium Gluconacetobacter xylinus. In our current study, ’“Research on the use of bacterial nanocelluloze (BNC) in regenerative medicine as a function of the biological implants in cardiac and vascular surgery", we carried out material analysis, biochemical analysis, in vitro tests and in vivo animal model testing. In stage 1 of the project, we carried out physical and biological tests of BNC. This allowed us to modify subsequent samples of bacterial bionanocellulose. Finally, we obtained a sample that was accepted for testing on an animal model. That sample we define BNC1. Patches of BNC1 were then implanted into pigs' vessel walls. During the surgical procedures, we evaluated the technical aspects of sewing in the bioimplant, paying special attention to bleeding control and tightness of the suture line and the BNC1 bioimplant itself. We carried out studies evaluating the reaction of an animal body to an implantation of BNC1 into the circulatory system, including the general and local inflammatory reaction to the bioimplant.

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These studies allowed us to document the potential usefulness of BNC as a biological implant of the circulatory system and allowed for additional modifications of the BNC to improve the properties of this new implantable biological material.

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1. Introduction

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Gluconacetobacter xylinus E25 bacteria cells that are growing and proliferating in a medium produce cellulose nanofibres that are approx. 1.5 nm thick and approx. 9 nm long. The nanofibres intertwine with each other, and after several days of culture, they create a tight, strongly hydrated bionanocellulose mat (membrane). The Bionanocellulose BNC structure is based on a 20-100 nm mesh of microfibrils. Depending on the applied culture method, type of medium and other factors affecting BNC, we can obtain bionanocellulose of various endurance properties and shapes. BNC can also be used to create a flat structure, similar to a piece of a silk-like material. Theoretically, BNC is impermeable to even the smallest cellular elements of blood (thrombocyte diameters are in the range of 1000–2000 nm). The surface of biocellulose is completely smooth for blood, which decreases the probability of thrombus formation. This property should also make it impossible for BNC to merge into the cells of the host's body. The usefulness of BNC in healing various types of wounds was confirmed as early as the 1990s. Compared to dressings and other superficial solutions, BNC is a non-toxic, nonirritant, hypo-allergic, non-pyrogenic and biocompatible material. Furthermore, mammals do not have enzymes that break down BNC. Therefore, after implantation, BNC should not be subject to biodegradability. The production of bionanocellulose is simple and relatively cheap, which can make it competitive in terms of the currently available bioprostheses. All these theoretical considerations allow us to presuppose that BNC can safely be used in the circulatory system as a new bioimplant, and the properties of BNC seem to indicate that it may be preferable to the currently available prostheses.

ACCEPTED MANUSCRIPT 1.1. Aim of the study The aim of the study was to prepare a sample of BNC with physical and biological properties that would allow it to qualify for studies as a potential circulatory system implant. 2. Material and methods

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2.1. Method of BNC production The technological process of BNC production is two-stage and involves the preincubation of biomass in the submerged culture and the proper superficial culture of a specially selected G. xylinus E25 strain on a chemically well-composed and optimised medium. We attempted to obtain stable composites of BNC with PVA (vinyl alcohol) and HA (hyaluronic acid) with the following methods:  in situ – addition of PVA/HA at a concentration of 1-4% to the culture medium (all tested culture mediums are described below) (Table 1).  ex situ – impregnation of the cellulose material obtained in a standard SH medium with a water solution of PVA or HA, a. with impregnation (immersion of BNC in a given solution for 2 hours at a temp. of 80°C), b. with sterilisation (placing BNC in a solution of appropriate polymers and autoclaving for 20 minutes at a temp. of 121°C).

Schramm Hestrin (SH) medium containing 2% glucose and 1% aminobak

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Schramm Hestrin (SH) medium containing 2% glucose with a 2, 4 or 8% solution of polyvinyl alcohol

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To obtain an optimal cellulose material for use in cardiac surgery, we cultured bionanocellulose membranes with use of the G.xylinus E25 bacterial strain on the following culture mediums [1]: a. Standard Schramm Hestrin (SH) medium containing Glucose 2% +/- 0,5%, yeast extract 0,5% +/- 0,1%, aminobak 0,5%+/- 0,1%, citric acid 0,1% +/- 0,05%, magnesium sulfate 0,05% +/- 0,025% and disodium phosphate 0,30% +/- 0,01%. The material was used as a starting sample, representing a BNC membrane not subject to any modifications, and as a material for modifications ex situ, i.e., after the production process. b. Modified mediums (Table 1)

Schramm Hestrin (SH) medium containing 2% glucose with 0.5% aminobak Schramm Hestrin (SH) medium containing 2% glucose with 1% sterile hyaluronic acid added at the stage of pouring out onto the tray

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Schramm Hestrin (SH) medium containing 2% glucose with 1% sterile polyvinyl alcohol added at the stage of pouring out onto the tray

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Schramm Hestrin (SH) medium containing 2% glucose with 0.5% aminobak; after being cleaned, bionanocellulose was dried and soaked in demineralised water for 2 hours

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modifications of ready bionanocellulose material produced with the use of Schramm Hestrin (SH) medium, with an addition of 2,4 and 8% polyvinyl alcohol and sterilisation

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modifications of ready bionanocellulose material produced with the use of Schramm Hestrin (SH) medium, with an addition of 2,4 and 8% polyvinyl alcohol and impregnation

Table 1. Characteristics of modified mediums used for the culture of BNC with use of the Gluconacetobacter xylinus E25 strain 2.2. Method of BNC testing: In vitro tests

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2.2.1. Evaluation of the mechanical properties of BNC 2.2.1.1. BNC stretching test

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We evaluated a non-modified BNC (referred to as native BNC), chemically modified BNC and physically modified BNC (BNC 1). We do not provide the methods of physical modification in this paper, due to an ongoing patent application of the obtained BNC. We compared the biomechanical properties of bionanocellulose with the following natural pig tissues: aortic wall, aortic valve cusps and fragments of the pericardial sac. Animal tissues were stored in 0.9% NaCl from their collection until the performance of the stretch tests. Additionally, we analysed tissues that were washed for 10 minutes with a 0.5% solution of glutaraldehyde before the stretch test. The stretch tests were performed on a fatigue test machine model 1112 by INSTRON. The uniaxial stretching velocity was 5 mm/s. The distance between the jaws for the stretched samples was 50 mm. We analysed 15-20 10x1.5-cm patches of each type of bionanocellulose and three other animal tissue fragments (aortic valve cusps, pericardium, and aortic wall).

2.2.1.2. BNC tear tests and fatigue tests.

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It was essential for the test to select bionanocellulose with a resistance to stretching that would be equal to or higher than that of the pig's natural circulatory system tissues. The tear tests were performed for the material of the highest resistance to tear, that is, BNC1. Natural tissues including the aorta, aortic valve and fragments of the pericardial sac constituted a reference material. The tear test was carried out in accordance with the standard PN-EN ISO 9073-4:2002, Textiles. Methods of fibre testing. Section 4: Determination of tear resistance. BNC1 patches 10x1.5 cm in size were subject to fatigue testing. At further stages of the experiment, the physically modified BNC (BNC1) were used. 2.2.2. Evaluation of the biological properties of BNC1 2.2.2.1. Evaluation of BNC1 biodegradability in the presence of S. aureus C. albicans and A. fumigatus Tests aimed at evaluating the susceptibility of bacterial cellulose to in vitro degradation were carried out in a simulated body fluid at a temperature of 37°C. The degradability changes were monitored by analysing changes in the dry and wet mass, measuring the content of biomaterial hydrolysis products with thin-layer chromatography (TLC) and identifying the thermal stability of BNC1. We evaluated the surface of the biomaterial with a scanning electron microscope (SEM) and monitored the growth of microorganisms intentionally introduced to the environment in which BNC1 was being incubated. The microorganisms included S. aureus, C. albicans and A. funigatus. The observation lasted for up to 6 months. [2]

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2.2.2.2. Evaluation of the interaction between the host's cells and BNC1 The cellular material to be grown on fragments of BNC1 included: a) L929 fibroblastic cell line (ATCC® CCL-1™), b) human umbilical vein endothelial cells (HUVEC), and c) mesenchymal stromal cells isolated from cardiac muscle.

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The cells were cultured in culture containers with a growth surface of 25 cm2 in a medium appropriate for a given type of cell at 37°C and in a 5% CO2 incubator. The medium was changed every 2 days. When a confluent monolayer was obtained, the cells were trypsinised and suspended in a full culture medium. The cells were then left to grow on BNC1. Beforehand, fragments of BNC1 had been washed in PBS (0.9% NaCl) and preincubated in a medium. Then, the investigated material was transferred to multi-well plates, and the previously prepared cellular suspension was transferred onto the BNC1 by sedimentation. The cells were cultured for 2 weeks. The culture medium was changed every 2 days, to avoid the interference of surplus cells on the growth ability and this way on the actual results. Evaluation of the cellular growth, adhesion and vitality was carried out after a) 3 days, b) 1 week, and c) 2 weeks. We used fluorescence microscopy and the following dyes: FDA – fluorescein diacetate (FDA), with green emission, to stain vital cells and PI – propidumiodide, with red emission, to stain dead cells.

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2.2.2.3. Evaluation of the interaction between fresh blood and BNC1 – modified Schima's test

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The test was conducted based on the descriptions by Schima et al. [3], with the aim of identifying the haemolytic and thrombogenic potential of various types of blood pumps. To identify the haemolytic and thrombogenic potential of BNC1, we slightly modified Schima's test. The first 6 experiments were conducted with the use of an axial pump without BNC1. Then, 6 consecutive experiments were carried out in identical conditions but with fragments of BNC1 (10x2 cm) freely moving in the bloodstream inside the system. The test was performed with use of fresh heparinised pig's blood obtained during standard animal slaughter in a slaughterhouse. There were 3.3 units of heparin added per mL of blood (3.3 IU/mL). Blood was transported in containers with a regulated temperature. The testing system was filled with it immediately after delivery to the laboratory. We used the same volume of blood (1 litre) every time. The test was initiated when the ACT (activated clotting time) was at least 1.5 times higher than normal. Blood was pumped at a constant velocity of 5 L/min at a constant temperature of 37°C and pressure of 100 mmHg. The test continued until the ACT was within the norm (200-420 minutes). We carried out a total of 12 experimental sessions: 6 control sessions and 6 with BNC1, inside the system with blood (experimental). The experiment was carried out based on the ASTM F1841 standard[4]. Blood samples for analysis were collected every 30 minutes and were tested for the following:  activated clotting time (ACT)  Hb – total haemoglobin concentration in blood  freeHb – free haemoglobin in blood ΔfreeHb – shows the difference in free haemoglobin concentration (Haemoglobin in plasma) between fresh blood and blood after the pumping time (T [min]) HTC – haematocrit expressed in [%]

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Based on these parameters, we calculated the modified index of haemolysis (taking into account not only haematocrit but also haemoglobin) [5]. Differences in the MIH between the control and experimental (with addition of BNC1) groups were compared statistically using Student's t-test. 2.2.2.4. Pyrogenicity testing of BNC

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The tests were conducted in the Institute of Occupational Medicine of Prof. Nofer in Łódź, based on the approval issued by the local Ethics Committee no. 9 for Experiments on Animals in Łódź (resolution no. 35/ŁB 667/2013 dated 13 May 2013). To test the pyrogenicity of the samples, we used live laboratory rabbits. We prepared samples of normal saline with immersed fragments of BNC1. Then, a solution of 0.5-10 mL/kg of body mass was injected during 4 minutes into the marginal vein of the ear of three selected rabbits. We then measured the animals' body temperature and the temperature and humidity of the room. The first temperature measurement was made 90 minutes before the administration of the investigated solution (to identify the baseline temperature); we then measured it every 30 minutes for a minimum of 180 minutes after solution administration (to identify the maximum temperature). The difference between the baseline and maximum temperature marked the rabbit's pyrogenic reaction. It was presupposed that the investigated solution may be considered free of pyrogens if the sum of the maximum temperature increases does not exceed 1.15°C and that presence of pyrogens can be confirmed if it exceeds 2.65°C. The test was carried out in the Laboratory for the Testing of Medicinal and Veterinary Products in a GMP Quality System, in accordance with the requirements of Polish Pharmacopoeia and European Pharmacopoeia [6,7].

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2.3. Evaluation of the effect of BNC1 with hyaluronic acid on an animal organism - in vivo testing

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2.3.1. Surgical procedures on pigs and the evaluation of a potential inflammatory reaction

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We performed surgical procedures on 4 pigs weighing 70-80 kg. Each pig had a unique number ascribed that was placed on an earring in the animal's ear. For the study we used BNC1 with the addition of hyaluronic acid (BNC1-HA), which has a healing-promoting effect [8,9,10] The surgeries were conducted in operating rooms, in keeping with the principles of aseptics and antisepsis. All animals had the skin in the operated area shaved and washed three times with alcohol and chlorhexidine mixed at a proportion recommended for skin disinfection before surgeries on humans. The operative field was covered with sterile drapes and selfadhesive surgical foil. The surgeries were conducted with animals under intravenous general anaesthesia. At stage one, we intramuscularly administered 500 mg of ketamine and azaperone at a dose calculated according to each animal's weight. Once the surgical access to the posterior jugular vein at the right side was obtained, we started administering the anaesthetics. Anaesthesia was continued by the administration of ketamine at a dose of 5–10 mg per kg of body mass and xylazine at a dose of 2 mg/kg of body mass, approximately every 30 minutes. The surgeries started with an administration of 5,000 heparin units. We dissected the jugular vein and jugular artery with a longitudinal incision made at the left side. The diameter of the vein was approx. 1 cm, slightly larger than the diameter of the artery (approx. 5 mm). Once each of the blood vessels had been clamped with haemostatic forceps, a longitudinal incision of the vascular wall was performed, long enough to fit the BNC1-HA patch. Oval BNC1-HA patches of 2x0.5 cm were sutured into the holes of both vessels. Prolen 6-0 sutures were

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used for a running suture in accordance with the standards of the vascular surgery. Once the suturing was finished, we released the haemostatic forceps and allowed blood to fill the lumen of the vessel with the BNC1-HA patch. The vessel was deaerated by puncture. In all cases, the normal patency of the vessels and tightness of the anastomosis was confirmed. The BNC1-HA patch always remained fully tight. (Pic. 1).

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Pic. 1. Implantation of a BNC1-HA patch into cervical vessels and a defect of a muscular fascia.

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From the muscular fascia we cut out a 3x3-cm square fragment and replaced it with a BNC1HA patch of appropriate size with an interrupted suture (Prolene 5.0). After haemostasis, a loose BNC1-HA fragment (1x3 cm) was left within the fatty tissue under the skin. The integuments were closed in layers. The wound was marked with a tattoo – 4 dots marked a rectangle with its midline along the surgical incision. The control pig was subject to identical anaesthesiological and surgical procedures: incision and suturing of the cervical vessels, muscular fascia and fatty tissue. The only difference was that it did not have the BNC1-HA sample implanted. This was considered a control animal. All surgeries were non-problematic. After the surgery, all the pigs were transported (still asleep) to the stalls and laid on their side. After approximately 1-3 hours, all the pigs were fully awake, presenting motor activity and no pain reactions. The pigs were taken to a pig farm after approximately 1 hour upon non-problematic recovery from anaesthesia. The surgeries were carried out in accordance with the binding legislation (Act of 15 January 2015 on the protection of animals used for scientific and educational purposes, along with secondary legislation), based on the approval of the local Ethics Commission. The animals were subject to post-operative observation, with special attention paid to the course of wound healing, general signs of infection or disturbances in the general condition. Before the surgery (day 0) and in the post-operative course (post-op. day 1, 2, 7, 21, 35, 49, 63, 77, 91), we collected blood samples from the right jugular vein in order to carry out laboratory tests: CBC with differential (white blood cells), haematocrit, fibrinogen, CRP and some pro- and anti- inflammatory cytokines (TNF-α, IL-6 and IL-10 ).

ACCEPTED MANUSCRIPT Three months after the surgery, the animals were subject to euthanasia in accordance with the protocol approved by the local Ethics Committee. An autopsy was performed for macroand microscopic evaluation of the reaction of an animal organism to the presence of BNC1HA. The tissue samples that were collected during the autopsies were prepared according to standard procedure, in 10% formalin for approximately 48 hours, then dehydrated by appropriate alcohol and xylene concentrations, and finally immersed in soft wax. The specimens were cut to 5-µm thick pieces that were dyed with haematoxylin and eosin (H&E).

3. Results and discussion 3.1. Cell-culture-based production of BNC

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Approval for the conduct of the experiment was issued by the local Ethics Committee for Experiments on Animals in Gdansk (resolution no. 40/2013).

As a result of the culture from the above described mediums (Table 1), we obtained composite materials consisting of BNC and PVA (vinyl alcohol), BNC and aminobak, and BNC and HA (hyaluronic acid). IR spectroscopy recorded bands on the spectra characteristic for all appropriate structures (BNC, PVA and HA).

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Modifications of the composition of the medium were limited to the use of only the polymers that are highly biocompatibile with the human body, improve the mechanical properties, bind permanently with cellulose fibre structure and are not of animal origin. The above-listed conditions eliminated chitosan, gelatine, collagen (animal origin), glycerine, agar and alginians (no permanent binding with BNC fibres) from potential modifiers of the biocellulose membrane. The use of NaOH solution, PMMA or PLA after the production process did not produce the desired effect because modifications with the solution of NaOH significantly increased the stiffness of the cellulose material, while PMMA and PLA are water-insoluble and the addition of an organic solvent to the SH medium suppressed the production of the cellulose membrane.

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3.2. In vitro BNC testing. 3.2.1. Evaluation of the mechanical properties of BNC 3.2.1.1. Results of the stretch test

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Fig. 1 Resistance to stretching of natural pig tissues – left coronary cusp, right coronary cusp, non-coronary cusp, pericardium, and aortic wall. [11]

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Fig. 2 Resistance to the stretching of native and modified BNC. Composites: BNCpolyvinyl alcohol (PVA), BNC- hyaluronic acid, BNC with addition of ammonia, polyvinyl alcohol, aminobak (8 days and 9 days), and BNC1 (physically modified).

Studies show that the BNC-PVA composite is characterised by lower resistance to stretching than the native BNC, while the BNC-HA composite is no different from the native BNC in terms of resistance to stretching. The analysed property was improved after the applied modification. Modification of BNC with aminobak (8- and 9-day culture) improved the resistance of the sample to stretching, compared to the native BNC. However, the best results were observed in the case of sample BNC1. BNC1 was subject to modifications that made it almost six times more resistant to stretching compared to the native BNC. Samples of modified BNC1 also manifested better resistance to stretching than the natural tissues collected from pigs' circulatory systems. Resistance to stretching of the native BNC and composite materials is approx. 5 Mpa. Resistance to stretching of the modified BNC1 is approx. 22 Mpa.

ACCEPTED MANUSCRIPT 3.2.1.2. Results of BNC terat tests and fatigue tests Based on the results of the tests, the structural resistance of BNC1 was calculated as 85.8 ± 18.45 J/cm2. The obtained value was compared to the structural resistance of a pig's aorta: 16 ± 0.9 J/cm2. The fatigue test was repeated for the BNC1 sample at the following parameters of the test: force F=45N, vibration amplitude A=15N, vibration frequency f=3 Hz (equivalent of 180 heart beats per minute). The sample resisted this load for approximately 24 hours (260 000 cycles). Physically modified BNC1 has higher structural resistance than the pig's aorta and can resist a load of 45 ± 15 N with a frequency of 3 Hz for approximately 24 hours.

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At further stages of the experiment, the physically modified BNC (BNC1) were used. 3.2.2. Evaluation of the biological properties of BNC1 3.2.2.1. Biodegradability testing

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During the entire 6-month storage period of BNC1 samples in a simulated body fluid (SBF) and phosphate-buffer saline (PBS), we observed no changes in the dry mass. Similarly, the dry mass of cellulose samples was almost unchanged in the presence of S. aureus and C. albicans. A distinct decrease in the dry mass of BNC1 was observed only in the samples incubated for 6 months in the presence of A. fumigatus (decrease by 41%). In the case of the wet mass, we observed a significant increase as early as after the second month of storage, independently of the conditions (PBS or SBF in the presence of microorganisms and without them). [2]

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We also observed that during the storage, the number of all investigated microorganisms grew. After 1 month, the number of S. aureus, C. albicans and A. fumigatus cells increased from approx. 103 to approx. 105 CFU/cm3 and remained unchanged up until month 6, which indicates that they were at a stationary growth phase. An increase both in the number of microorganisms and the BNC1 wet mass indicates that the material is subject to degradation processes, even though the dry mass does not change significantly (except for the samples which contained A. fumigatus). We also observed that when the samples were stored on the surface of BNC1, only A. fumigatus created a biofilm on the polymer's surface. The concentration of saccharides, products of BNC1 hydrolysis, in post-incubation fluids was so low that they could not be detected by thin layer chromatography. After concentrating these fluids 20 times, in tests with and without S. aureus and C. albicans, no products of BNC1 hydrolysis were found. However, they were present (in small quantities) in the concentrated post-incubation fluid after one month of storage of BNC1 with A. fumigatus. Analysis of the results leads to the conclusion that investigating hydrolysis products of cellulose in the post-incubation fluids is not a good method of measuring polymer biodegradation. This is because microorganisms may metabolise the produced monosaccharides, disaccharides and oligosaccharides. Storage of the samples in PBS and sterile SBF for 5 and 6 months decreased the BNC1 decomposition temperature by approx. 10°C (which indicates there were degradation processes taking place) and reduced the quantity of water absorbed on its surface by an average of 50%. Similar changes were observed in the samples stored for the same time along with S. aureus and C. albicans, while samples stored for 6 months with A. fumigatus manifested a decrease in thermal stability by approx. 20°C. Thermography of the samples incubated for 6 months in SBF, with and without microorganisms, revealed that chemically bound water was lost. Microscopic analysis showed that after incubation in simulated body fluids for 1-6 months, the morphology of BNC1 membranes did not change significantly, but it did become more porous.

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Analysis of cellular growth showed that the cells used in the experiment did not have appropriate adhesion to the BNC1 surface. Only a small number of necrotic cells were seen on the investigated surface at particular time points (Fig. 3). The most effective growth was observed for fibroblasts – there were rare clusters of living cells (Fig. 4). However, the cells were poorly adherent to the medium (Fig. 5). After gentle washing in a stream of PBS, the cells were torn away from the medium, and only necrotic cells were left.

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Fig. 3 Estimation of growth and viability of endothelial cells cultured on BNC1 in particular time: A- 3 days of culture, B- 1 week, C- 2 weeks, D- 3 weeks, E- 4 weeks, F – 5 weeks. FDA – green fluorescent; viable cells, PI – red fluorescent; necrotic cells

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Fig. 4 Estimation of growth and viability of fibroblast cells cultured on BNC1 in particular time: A- 3 days of culture, B- 1 week, C- 2 weeks, D- 3 weeks, E- 4 weeks, F – 5 weeks. FDA – green fluorescent; viable cells, PI – red fluorescent; necrotic cells.

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Fig. 5 After 3 days of culture, the cells were gently rinsed in a shaking conditions, to check the adhesion properties. It was observed that the majority of cells showed poor adhesion to the BNC1. The remaining cells observed on BNC1 were classified as necrotic. It seems, however, that necrosis is not the result of BNC1 toxicity but rather the results of limited cell adhesion to the substrate. BNC1 is characterised by low adhesion. Superficial modification of BNC1 with the use of natural proteins of the extracellular matrix could improve the adhesiveness of the cells and their characteristics of growth [12]. Low cellular adherence is a desired property of an intravascular implant. 3.2.2.3. Modified Schima's test [13]

ACCEPTED MANUSCRIPT Concentration of free haemoglobin (fHb) in serum remains in close correlation with haemolysis, taking place in the investigated circulating medium. During the tests (both the control and those with BNC1), the fHb concentration gradually and systematically increased. Comparison of the dynamics of the fHb increase seen during the follow-up and experimental test allowed us to draw some conclusions on the effect of BNC1 on haemolysis. MIH was calculated based on the Hb, fHb and HTC results (Table 2.)

No. of experiments

control

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174

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174.7

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173.5

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175.1

5.

175.5

HTC [%]

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220

4.07

290

43

255

4.32

680

38

407

6.91

110

40

377

1.17

480

39

270

7.23

175

115

45

360

1.17

175

680

41

420

6.37

175.5

310

48

330

3.26

172.4

380

42

345

4.26

10.

171.1

290

38

270

4.44

11.

176.4

300

45

360

3.06

174.7

290

43

255

4.32

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Tab. 2. Results from 6 control and 6 experimental series.

We compared the results of laboratory tests for all 12 sessions. One of the parameters analysed was the flow time in the experiments. Large differences in the flow time would affect the level of red blood cell damage. The flow time of the circulating medium for the control tests was T = 325.57±75.08 min., and for the experimental tests, T = 330.00±60.75 min. – the difference was statistically insignificant. The mean MIH for the control group was 4.145, and for the study group, 4.285. The difference was statistically insignificant.

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Conclusions: Schima's test of BNC1 showed its low haemolysis index and low thrombogenicity.

3.2.2.4. Pyrogenicity testing results

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The differences between the baseline and maximum temperature, identifying the rabbit's pyrogenic reaction were 0.1°C; 0.2°C and 0.05°C, which allowed us to determine a total temperature increase of 0.35°C. It was presupposed that the investigated solution may be considered free of pyrogens since the sum of the maximum temperature increases did not exceed 1.15°C. The test was carried out in the Laboratory for the Testing of Medicinal and Veterinary Products in a GMP Quality System, in accordance with the requirements of the Polish Pharmacopoeia and the European Pharmacopoeia [6,7].

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Conclusions: There were no pyrogenic bodies found in the BNC1 samples.

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Fig. 6. Body temperature changes in the investigated rabbits during BNC1 pyrogenicity testing

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3.3. In vivo tests of BNC1 with hyaluronic acid

3.3.1. Analysis of the inflammatory reaction after BNC1-HA implantation

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The animals were observed for 3 months after the implantation of BNC1-HA fragments. They all manifested normal post-operative wound healing. They all had good appetites, with normal behaviour and without pyrexia. They were gaining weight and growing normally. None of the pigs presented an inflammatory reaction. The body temperature was between 37.8 and 38.9°C (norm: 38.0–39.5°C). The laboratory parameters of blood collected from the animals were as follows: In healthy pigs, the total number of leukocytes ranged between 10-22 x 103/μL. In the postoperative course, the animals manifested a two-way fluctuation of WBC in the range of 9.44– 31.7 x 103/μL. Two pigs (1636 and K1639) presented temporary leucocytosis (from 18.2 before the surgery to 26.3 x 103/μL and from 21.0 to 31.7 x 103/μL, respectively). Leucocytosis was an expected effect of surgical intervention. Then, during the next several days, we observed a gradual decrease in WBC correlated with progressive scarring of the post-operative wounds.

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The count and proportion of particular white cell populations was measured for the first 3 weeks post-op. One day after the operation, three experimental pigs presented evident lymphopenia accompanied by neutrophilia. The control pig (no. 1639) did not manifest this effect. One week after the surgery at the latest, the proportion of particular white cell populations returned to normal (Figure 7). The temporary lymphopenia observed in the pigs was consistent with a phenomenon referred to in the literature as stress lymphopenia [14,15].

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WBC [%]

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Figure 7. Changes in the WBC during 30 days post-op.

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The normal range for PLT in pigs falls within 200-800 x 103/μL. Within 3 weeks after the surgery, we observed fluctuations in the platelet count, which exceeded the lower limit (mainly pig 1637) or, periodically, the upper limit (pig K1639). An increase in platelet count to more than 800x103/μL would indicate an inflammatory reaction caused by the presence of BNC1-HA. However, the investigated group presented thrombocytopenia. The reduced platelet count directly after the procedure could have been caused by heparin [16]. It was administered to the animals that had BNC1-HA implanted into the blood vessel wall. Administration of an anticoagulant was necessary due to temporary arterial closure. Thrombocytopenia was not observed in the control pig, in which the platelet count increased to the upper limit of normal. This was in accordance with the theory stating that the activity and number of platelets grow in response to an inflammatory stimulus [17,18]. We also analysed CRP and fibrinogen levels as parameters of an inflammatory reaction. On days 2–5 post-op, all animals presented slight and temporarily elevated CRP and fibrinogen levels in blood. However, this was still within the norm. After 21 days, the CRP level reached its previous value before the surgery. In pigs with implanted BNC1-HA fibrinogen increased temporarily around post-operative day 2-5. On day 21, pig no. 1638 presented a secondary increase of this protein. In the control pig (1639), the fibrinogen was increased initially, in sample 0. All these changes were within the norm for pigs.

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Figure 8. The diagrams illustrate cytokine values (TNF-alpha, IL6, IL10) on consecutive postoperative days, for each of the operated pigs (pigs' numbers are provided at the top of the graphs).

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Cytokines affect all phases of the inflammatory reaction. Therefore, they are its key mediators, both in the pro- and anti-inflammatory phase. For monitoring in the post-operative course, we selected TNF-alpha, IL6 and IL10. TNF-alpha and IL6 are primary mediators in the pro-inflammatory phase, and the TNF-alpha level correlates with mortality in septicaemia [13,19]. To characterise the anti-inflammatory phase, we measured the IL10 level in the observation phase. We can see on the graph for animal K1639 that on post-operative day 1, there was an increase in the pro-inflammatory cytokine level, and then on day 2, the antiinflammatory phase cytokines increased while the others decreased [Fig. 8]. Such a phenomenon reflects an inflammatory reaction to a non-specific stimulus – i.e., a surgical intervention. The animals that had BNC1-HA fragments implanted did not present a post-operative increase in typical inflammatory proteins. Thus, it could be concluded that BNC1-HA suppresses the inflammatory reaction. Even the initially high level of IL-6 in pig no. 1637 eventually decreased.

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Conclusions: 1. Stress lymphopenia is a typical sign of initiation of the hypothalamic–pituitary–adrenal axis. This effect, observed within 24 hours post-op, can be associated with a temporary inflammatory reaction that may have been caused by a surgical intervention. Such a conclusion is confirmed by the neutrophilia and increased CRP and fibrinogen levels in the experimental animals' blood within the first days after the surgery. 2. A more than 2-month observation of the behaviour of all pigs, their body temperature and selected blood parameters explicitly showed no chronic inflammations related to BNC1-HA implantation.

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3.3.2. Results of the surgeries and autopsies - macroscopic evaluation and histopathological examination

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During 3 months of observation, the animals doubled their baseline weight. After euthanasia, performed in accordance with the protocol approved by the Ethics Committee, we excised tissue blocks containing blood vessels, fascia and fatty tissue with the implanted BNC1-HA (from the pig's nape of the neck, at the point marked with a tattoo). There were severe macroscopic difficulties in identifying the areas where BNC1-HA had been implanted. There was no evidence of an inflammatory infiltration. In only one case did we manage to recognise the area where the patch was sutured into the cervical vein wall – the area was slightly bulging compared to the rest of the vessel, with a properly healed running suture. From the inside of the vessel, there was evidence of a macroscopically normal endothelium that did not differ from other fragments of the vessel, as shown in Pic. 2.

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Pic. 2. A tissue fragment with a longitudinally cut venous vessel. On the external surface, there was a healed-in fragment of BNC1-HA covered with a macroscopically normal vascular endothelium.

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On histopathological examination, we observed infiltrations of mononuclear cells around the implanted BNC1-HA, indicating that there was a moderate to severe inflammatory reaction taking place (Pic.3). Similar changes were observed both in animals with implanted bionanocellullose and the control animals (surgery with opening and closure of blood vessels, suturing of the fascia and fatty tissue, but no implantation). Thus, the observed changes were classified as a normal healing process of the post-operative wound and not a pathological effect of the implant.

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Pic. 3. a) On the left: infiltrations of the mononuclear cells. b) On the right: a chronic inflammation with a granulomatous reaction. Homogeneous mass – BNC1-HA.

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Conclusions:

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1. BNC1-HA implanted into the fatty tissue was subject to partial healing-in and did not cause any systemic or macroscopically confirmed inflammatory reaction. In the vessel in which we managed to recognise the BNC1-HA implant site, it did not cause clotting and it was covered by the endothelium. However, a microscopic examination revealed infiltrations of mononuclear cells, indicating that there was a moderate to severe inflammatory reaction taking place (Pic. 3). 2. The implanted BNC1-HA contained hyaluronic acid, which was to facilitate the healing-in of the bionanocellulose. This factor may have triggered the inflammatory reaction around the bioimplant. It seems that this constitutes a considerable advantage of the material and is an additional factor that can facilitate the process of BNC1-HA healing-in while protecting the implanted BNC1-HA against infection. However, this effect could also have adverse effects (e.g., intravascular clotting) if BNC1-HA was used as a prosthesis in the cardiovascular system. Therefore, in subsequent stages of the study, we will use BNC1 modified with hyaluronic acid and a non-modified one.

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4. DISCUSSION

The described method of production and preparation of the BNC Gluconacetobacter xylinus E25 (BNC GxE25) sample [1] allows a biological material of the desired properties to be obtained. The production system used by the manufacturer (Bowil) allows a chemically clean cellulose polymer to be yielded, devoid of pyrogenic factors such as endotoxins and other chemical molecules such as proteins, carbohydrates or fats. The legitimacy of medium modifications made with PVA (polyvinyl alcohol) [20] and hyaluronic acid [21,22] has been confirmed in the literature reports. These two polymers are appropriate substances to modify the mechanical properties of the cellulose membrane for cardiovascular applications. PVA is water-soluble, biocompatible, non-toxic and has good mechanical properties. The BNC/PVA composites described in scientific papers are dedicated to biomedical applications such as tissue reconstruction and cardiovascular stent implantation. An effective connection of BNC fibres with PVA allows a composite material that is characterised by good resistance

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(properties of bionanocelullose) and appropriate elasticity (properties of PVA) to be obtained. The hyaluronic acid is a biopolymer, which occurs in all living organisms. It is used for the filling of tissue defects and wrinkles and in orthopaedics and dentistry. Use of the BNC-HA composite can potentially improve the biological integration properties and increase the mechanical resistance of the implant. However, it seems that presence of the hyaluronic acid promotes formation of adhesions, which will constitute additional reinforcement and sealing of a vessel. During bacterial culture, we used conditions that allowed for the elimination of any kind of undesired organic or non-organic additions to the biomaterial. Physical modifications, on the other hand, give the final product an appropriate thickness, resistance to stretching and tearing, appropriate non-permeability and smoothness for blood. These properties result from the unique structure of BNC1 GxE25, consisting of 1.5-nm thick and approx. 9-nm long fibres woven together into a 20-100 nm mesh. Such a structure is subject to hydration [23,24]. The applied modifications of this process allowed us to obtain specific physical properties of the material such as resistance to stretching and tearing at a low sample thickness (300–500 micrometres), lack of stretchability and exceptionally high elasticity (susceptibility to change of shape and ability to adapt to a fluid stream). Comparison to natural tissues showed significant similarities of BNC1 to natural tissues in respect of thickness and flexibility, as well as higher resistance of BNC1 GxE25 compared to the natural tissues such as the valve cusps, the pericardial sac or the aortic wall. In the in vitro tests, we also showed that BNC1 is not biodegradable by pathogenic bacteria. Only in the case of moulds did we observe a decreased dry mass of BNC1 and the presence of the products of its hydrolysis. BNC1 does not reveal any haemolytic potential. The discussed properties of BNC1 that make its surface unfavourable for clot formation in the blood flow were confirmed with Schima's test. The in vivo tests showed that BNC1-HA GxE25 is useful in surgery as a material that can be used as patches implanted to arteries and veins. Its surgical usefulness was confirmed based on the ease with which BNC1 can be punctured with surgical needles and the fact that the material can be easily and safely sutured. Moreover, we observed that it is tight – i.e., non-permeable for blood. It was also shown that the bioimplant can heal-in to a host's body with no adverse inflammatory reactions. Reports of the attempts to use bionanocellulose in periodontology were published as early as in the 90s [25]. We showed a lack of pyrogenic activity, which indirectly indicates that the investigated material is very clean, biologically. Attempts to use this material in neurology lead to its application as a patch to cover defects in the pachymeninx. The authors also prove that its presence does not increase the risk of adhesion formation [26]. Research on bionanocellulose indicates that this material can also be shaped in various ways – for example, into the shape of a tube [27,28,29]. The process of the healing-in of BNC1--HA samples in an animal body showed that the host's cells do not penetrate the bioimplant’s structure. On histopathological examination, there were noticeable borders between the implant and the host's cells. Earlier evaluation of the interaction between the host's cells and the BNC1 in in vitro tests (section 3.2.2.2.) showed poor adhesion. It can be concluded that this property will increase the material's resistance to degradation in a living organism by preventing the degeneration caused by the body's own cells or the cells of potential biological pathogens. A smooth surface that results in lack of adhesion of living cells and prevents the morphotic elements of blood to be deposited is an exceptionally desirable property of materials implanted in the cardiovascular system. Evaluation of the possibility of the adhesion of cells to BNC1 and their proliferation showed that only an appropriate modification would lead to the BNC1 surface being covered with new cells, thus creating a new tissue. The study presented in this paper was carried out on bacterial nanocellulose (BNC) produced with use of a patented Gluconacetobacter xylinus E25 strain. So far, nowhere in the world has bacterial nanocellulose been produced on such a large scale. The manufacturing facility meets the Polish and European pharmacological standards, which

ACCEPTED MANUSCRIPT allows us to use BNC1 as a reproducible and uniform material that meets the norms for bioimplants. Appropriate modification allows a material of desirable properties to be obtained. The conducted research resulted in production of new BNC (BNC1) with greater durability than animal tissue, with good biocompatibility, and apyrogenicity. Additional features, as low thrombogenicity, and low haemolysis index justify eventual application as an arterial prosthesis, heart valve prosthesis or a patch in the pericardial sac in human reconstructive surgery. However, this requires further research

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Acknowledgements

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at url of journal website). The authors declare no competing financial interests.

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The project is carried out in collaboration of scientific and research institutions including the Medical University of Gdansk, Faculty of Medicine, Bowil Biotech Sp. z o.o. Gdansk University of Technology, Faculty of Chemistry, Mechanical Faculty of Centrum Techniki Okrętowej S.A. The Foundation for Cardiac Surgery of Prof. Zbigniew Religa, University of Gdansk, Faculty of Biology. Name and number of the project: KARDIO BNC PBS2/A7/16/2013 References

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1 Długa, A. & Kaczmarek, H., Characterisation of composites of bacterial cellulose and poly(vinyl alcohol) obtained by different methods. FIBRES & TEXTILES in Eastern Europe 2014; 22, 6(108): 69-74.

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2. Dederko P., et al. In vitro biodegradation of bacterial nanocellulose under conditiona simulating human plasma in the presence of selected pathogenic microorganisms. Polimery 5, 2018, 372-381

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3 Schima, H., et al. In vitro haematological testing of rotary blood pumps: Remarks on standardization and data interpretation. Artif. Organs 1993; 17:103-110. 4 ASTM International, Standard Practice for Assessment of Hemolysis in Continuous Flow Blood Pumps1. Designation: F1841 − 97 (Reapproved 2013) . 5 Kozo Naito, Kazumi Mizuguchi, Yukihiko Nosé, The Need for Standardizing the Index of Hemolysis. Volume 18, Issue 1, January 1994, 7–10. 6 Farmakopea Polska 10th Edition (Warsaw 2016) 7 European Pharmacopoeia 8th Edition; Biological Tests 2.6.8. 8

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ACCEPTED MANUSCRIPT 9 Sabrina Alves de Oliveira, Bruno Campos da Silva, Izabel Cristina Riegel-Vidotti, Alexandre Urbano, et al. Production and characterization of bacterial cellulose membranes with hyaluronic acid from chicken comb. International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.077

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10 Lin-Cui Da, Yi-Zhou Huang, Hui-Qi Xie, Progress in development of bioderived materials for dermal wound healing. Regenerative Biomaterials, 2017, 325–334 doi: 10.1093/rb/rbx025

11. https://www.degruyter.com/view/j/adms.2015.15.issue-3/adms-2015-0017/adms-20150017.xml A. Stanislawska, K. Dawidowska, Influence Of Preservative On The Tensile Strength Of The Tissue Of Porcine Circulatory System, Advances in Materials Science, Volume 15, 2015, Issue 3, Pages 67–75. 12 Grande, C. J., et al., Nanocomposites of bacterial cellulose/hydroxyapatite for biomedical applications. Acta Biomaterialia, vol. 5, 2009, no. 5, pp. 1605–1615.

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13 Schima, H., In-vitro Tests on Hemolysis, Thrombogenicity and Endothelial Cell Cultures, http://www.zmpbmt.meduniwien.ac.at/forschung/cardiovascular-dynamics-artificialorgans/museum/in-vitro-tests-on-hemolysis-and-thrombogenicity/

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14 Hoffman-Goetz, L., Quadrilatero, J., Treadmill exercise in mice increases intestinal lymphocyte loss via apoptosis. ActaPhysiol Scand. 2003 Nov;179(3):289-97

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15 Krüger, K., Mooren, FC., Exercise-induced leukocyte apoptosis. Exerc Immunol Rev. 2014;20:117-34. Review.

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16 Warkentin, TE., Clinical picture of heparin-induced thrombocytopenia. In: Warkentin TE, Greinacher A, eds. Heparin-Induced Thrombocytopenia. 5th ed. Boca Raton, FL: CRC Press 2013; pp. 24–76.

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17 Stokes, K.Y., Granger, D.N. , Platelets: a critical link between inflammation and microvascular dysfunction. J. Physiol. 2012; 590: 1023–1034

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18 Maślanka, K., The role of platelets in inflammatory processes. Journal of Transfusion Medicine 2014, vol. 7, No. 3, 102–109 19 Karpel, E., The place of systemic inflammatory response mediators in intensive care. Anestezjologia Intensywna Terapia 3/2001, s. 181-190 20 Padavan et al., Synthesis, characterization and in vitro cell compatibility study of a poly(amic acid) graft/cross-linked poly(vinyl alcohol) hydrogel. Acta Biomater. 2011;7(1):258-67. 21 Ruiz et al., Differential support of cell adhesion and growth by copolymers of polyurethane with hyaluronic acid. J Biomed Mater Res A. 2013;101(10):2870-82. 22 Ruiz et al., Effect of hyaluronic acid incorporation method on the stability and biological properties of polyurethane-hyaluronic acid biomaterials. J Mater Sci Mater Med. 2014;25(2):487-98.

ACCEPTED MANUSCRIPT 23 Klemm, D., et al., Nanocelluloses: A New Family of Nature-Based Materials. Angew. Chem. Int. Ed. 2011, 50, 5438 – 5466. 24 Zielińska, A., Kołodziejczyk, M., Bielecki, S., Application of bacterial cellulose in implant manufacturing. Military Pharmacy and Medicine 2010, 3, 68-72. 25 Novaes, AB., Novaes, AB., Bone formation over a TiAl6V4 (IMZ) implant placed into an extraction socket in association with membrane therapy (Gengiflex). Clin Oral Implants Res 1993; 4: 106–110.

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26 Mello, L.R., Alcantara, B.B., Bermardes, C.I., Boer, V.H., Late favorable results of duroplasty with biocellulose : clinical retrospective study of 20 cases. Arq Bras Neurocir 31(3):128-34, 2012. 27 Bäckdahl, H., Risberg, B., Gatenholm, P., Observations on bacterial cellulose tube formation for application as vascular graft. Materials Science and Engineering: C Volume 31, Issue 1, 1 January 2011, Pages 14–21. 28 Czaja, W.K., Young, D.J., Kawecki, M., Brown, R.M. Jr., The future prospects of microbial cellulose in biomedical applications.Biomacromolecules. 2007 Jan;8(1):1-12.

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29 Klemm, D., Kramer, F., Ahrem, H., Bacterial nanocellulose for medical applications: potential and examples. Handbook of Green Materials, chapter 15, Materials and Energy vol.5

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Physical modification makes it possible to obtain bionanocellulose with better properties Modified bionanocellulose (BNC1) has higher tensile strength than animal tissues Shima's test of BNC1 showed its low haemolysis index and low thrombogenicity Bionanocellulose as a bioimplant in cardiac surgery and vascular surgery

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