Toxicological and Sensitization Studies of Novel Vascular Prostheses Made of Bacterial Nanocellulose Modified with Chitosan (MBC) for Application as the Tissue-Engineered Blood Vessels

Tissue-engineered blood vessels (TEBV) represent an attractive approach for overcoming reconstructive problems associated with vascular diseases in humankind by providing small caliber vascular grafts. The study evaluates biocompatibility and bioaffinity of vascular prostheses made from chitosan-modified bacterial cellulose (MBC) as potential scaffolds for TEBV. During the study, acute oral toxicity, up-and-down procedure (UDP), OECD test No. 425 on 10 Imp:WIST rats, intradermal reactivity on three Imp:BN albino rabbits, and sensitization on 15 Imp:DH guinea pigs were performed. The local effects were determined 1 month after intramuscular implantation of prostheses in 30 Imp:WIST rats. Histopathological and pathomorphological studies were conducted following complete removal of implants with peri-implant tissue. There were no signs of toxicity; the median lethal oral dose (LD50) was greater than 2 g/kg body weight for the rats. No allergic reactions were observed in the case of the guinea pig maximization test. Vascular grafts did not induce significant reactive changes in intradermal reactivity test (Main Irritation Index value 0.03) and do not induce inflammatory changes or hyperplasia of the muscle tissue surrounding the implant. Histopathological examination revealed ingrown vascular-connective bands. Tubes made of MBC offer strong potential for use in future TEBV programs for vascular surgery. Currently, the number of autologous grafts for coronary artery disease and for peripheral artery disease is limited. Particularly materials that will have contact with blood must comply with certain requirements such as mechanical strength, biocompatibility, and no potential to evoke adverse reactions. Bacterial nanocellulose modified with chitosan (MBC) due to its mechanical and biological properties is a promising material for replacing small-diameter vessels grafts. Although previous studies have not shown the toxicity of nanocellulose, we want to check whether medical products based on MBC will be safe when testing in vivo. Thirty Imp:WIST rats and 15 Imp:DH guinea pigs were subject of thorough analysis of potential toxicological and sensitization effect that may develop after applying vascular prostheses made from MBC to living organism. The analysis involved also histopathological and pathomorphological studies following complete removal of implants with peri-implant tissue. The results show that MBC prostheses do not cause any allergic, intradermal reactions and finally, do not display acute toxicity towards the organism in which it is implanted. Moreover, they had not induced inflammatory changes or hyperplasia of the muscle tissue surrounding the implantation sites, thus showing good biocompatibility. Obtained results were discussed with other available studies investigating various aspects of bacterial cellulose or modified bacterial cellulose influence on cells and tissues in both in vitro and in vivo studies. This is the first study analyzing the toxicological and sensitization effect which MBC may evoke and confirm the strong potential for use in future TEBV programs for vascular surgery.


Introduction
Bacterial cellulose (BC) represents an interesting emerging biocompatible nanomaterial which can be used in a wide range of biomedical applications, such as production of artificial blood vessels or microvessels and artificial skin [1]. Microbial cellulose is biosynthesized by the Gramnegative bacterium named Gluconacetobacter xylinus, formerly Acetobacter xylinum [2]. Bacterial cellulose is susceptible to modifications that significantly improve its properties and/or functionality. As a result, new BCbased nanocomposites are obtained, for example, BC/chitosan, BC/collagen, BC/gelatin, or BC/fibroin [1]. These nanocomposites are a combination of the good properties of BC with specific features of other materials for specific applications [3]. Chitosan modification is an example of improving the properties of bacterial cellulose. Chitosanbased biomaterials are considered antimicrobial, functional, renewable, nontoxic, biocompatible, bioabsorbable, and biodegradable biopolymer agents [4]. Many factors and qualities of chitosan derivatives make the BC/chitosan nanoparticles adequate materials for implantation. Chitosan is also characterized by good mechanical properties in wet state, high ability to maintain moisture, high bioaffinity, bacteriostatic, and bactericidal activity against bacteria Escherichia coli G(−) or Staphylococcus aureus G(+). Moreover, BC/chitosan molecules show susceptibility to specific hydrolytic enzymes, for example, lysozyme. During the enzymatic degradation of chitosan, bioactive mono-and oligosaccharides are being released, which stimulate angiogenesis and tissue regeneration [5]. These types of nanoparticles are promising to advance strategies to treat vascular disease. It has been proven that MBC provides a good scaffold for the host cells and promotes the regeneration of tissues by the body. It has been found that due to its mechanical and biological properties, bacterial cellulose modified with chitosan is a promising material for replacing small-diameter vessels grafts. In recent years, bioengineering of artificial blood vessels is focused mainly on discovery and development of new artificial vascular substitutes, alternative materials that integrate with the patient's native tissue to restore physiological function [6]. The main challenge for tissue engineering is to develop new biologically active small-diameter grafts. Currently expanded-polytetrafluoroethylene (ePTFE; synthetic fluorocarbon polymer), polyethylene terephthalate (Dacron; polymer containing ester functional group), and polyurethanes (formed through a reaction between isocyanates and polyol) are the most commonly used synthetic graft materials for artificial blood vessels [6]. However, these commercial prosthesis-materials are unsuitable for microsurgical requirements, because they have larger diameter than 3 mm. Therefore, they cannot be used in microvessels surgeries. Tissue-engineered blood vessels (TEBVs) represent an innovative approach for overcoming reconstructive problems associated with vascular diseases by providing small-caliber vascular grafts. Moreover they confirm that BC has high mechanical strength, promotes in situ vascular tissue regeneration, can be shaped into three-dimensional structures, and does not require pre-treatment in the form of cell seeding. The ideal vascular prosthesis should be resistant to thrombosis, inflammation, and excessive thickening of the intima. Numerous publications suggest that BC seems to be the best candidate among those possible to construct novel vascular prosthesis and substitutes of small blood vessels [7,8].
In this study, a novel vascular prostheses made of bacterial nanocellulose modified with chitosan (MBC) will be tested for their potential toxicity and adverse sensitization effects. The toxicology study of nanocellulose and nanocellulosebased biocomposites is still restricted at a very preliminary stage (mainly on the level of cytotoxicity) [9]. Although previous studies have not shown the toxicity of nanocellulose, we want to check in vivo whether our product will be safe for patients in the future. Long-term stability and suitability to replace small-caliber blood vessels without significant thrombogenicity must be investigated. Study of compliance between the graft and the surrounding native vessels and tissues, together with the postoperative complications, is now warranted and is the subject of current study.

In Vitro Testing
The assessment of cytotoxic activity in vitro was conducted in the Cell Culture Laboratory, Department of Histology and Embryology of the Wrocław Medical University. The study was conducted on a reference cell line, mouse fibroblasts 3T3/ Balb obtained from the Tissue Bank, Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław. The cells were stored in liquid nitrogen, with the addition of about 10% DMSO. After thawing, the cells were passed through twice with a solution of 0.05% trypsin + 0.02% EDTA in PBS, pH 7.2 (provided by SIGMA, USA).
The cells were cultured under the conditions recommended for tissue culture in an atmosphere of 5% CO2, at 37°C, with constant chamber moisturization.

In vivo Testing
The present study was conducted in accordance with the prior consent of the No. 9

Tested Materials
The research material was modified bacterial cellulose in tubular form (BCTube). Samples for the study were provided by the Institute of Biopolymers and Chemical Fibres, Lodz, Poland. The BC was synthesized using an acetic strain of bacteria, Acetobacter sp. (CCM 2360), from the Czech Collection of Microorganisms, Masaryk University, Brno, Czech Republic. Bacterial cellulose was modified with chitosan at the stage of biosynthesis-by adding chitosan oligomers to the culture medium. As presented previously [5], chitosan chains are physically incorporated into the cellulose network. Modified Hestrin-Schramm medium was used, composed of the following (on 1000 cm3): glucose 20.0 g, yeast extract 5.0 g, soy peptone 5.0 g. disodium phosphate 2.7g, acetic acid 1.2 g, ethanol 20.0 cm3, and chitosan oligomers (ChitoOligo-100, Aminogen, Korea) 2.0 g. Modificator-ChitoOligo-100 is a water-soluble oligomer of β-1,4-linked D-glucosamine, produced from crab chitosan by an enzymatic process using a chitosanase, developed by the Aminogen company (Korean Patent No. 0227040). Product characteristics are as follows: molecular weight >5kDa, degree of acetylation 92%, molecular weight distribution of 1 to 1.5, an intrinsic viscosity of 0.020 to 0.250 g/D-sec, a moisture content >1%, and an inorganic content of 0 to 1%. Samples of the BC tubes were analyzed by Fourier transform infrared spectroscopy (FTIR). BC tubes were obtained by biosynthesis for 7 days at 30°C in an incubator ( Fig. 1.1). Bacterial cellulose in tubular form, BC Tube ( Fig. 1.2), was obtained by biosynthesis in single silicone tubes. The silicone tube (internal diameter 10 mm, wall thickness 1.4 mm) was filled with HS culture medium modified with chitosan oligomers; the ends of the tube were tightly closed with plastic plugs. The external diameter of the BC tube depended on the internal diameter of the silicone tube. For testing biological properties, BC tubes with an external diameter of 10 mm and wall thickness of 1.7-1.8 mm were used (  The obtained BC tubes were washed with distilled water until the complete removal of culture medium components. Next, the tubes were soaked in 1% NaOH, autoclaved at 121°C for 15 min, and washed with distilled water until reaching neutral pH and conductivity <20 μS.

Hemocompatibility of BC Tubes
The hemocompatibility tests of a modified bacterial cellulose blood vessel prosthesis were prepared on human blood O Rh+ taken into CPD preservative (sodium citrate, citric acid, glucose dihydrogen phosphate). The scope of work included the following: 1. Hemolytic activity study: (a) After temporary contact with blood (plasma Hb determination) (b) After temporary contact of the extract with the red blood cell suspension (determination of the hemolysis rate) 2. Tests of the plasma clotting system after temporary contact of full blood with the prosthesis:

In vitro Testing
Mouse fibroblast cells were cultured on NUNC 12-well plates in density of 0.5·10 6 . After 24 h, the cells adhered to the plate's surface and divided, covering about 50% of the plate surface. After this time, the culture medium was removed and a new medium was added to each well. Samples of 5×5 mm in size were applied to the cell cultures (the samples covered about 10% of the culture surface) and plates were incubated at 37°C, in 5% CO2 atmosphere. Quantitative and morphological changes, after contact with the examined materials, were evaluated after 24, 48, and 72 h under a reversed contrast-phase microscope. Trypan blue staining was used to determine the number of dead cells. The degree of toxicity of the materials was assessed on the basis of changes in cell morphology, their survival, and ability to proliferate according to the criteria given in Table 1.  2) to individual animals at 24-h intervals. If the first animal survives, the next one is given a higher dose. If after the next dose the animal dies or is in agonal condition, a lower dose is administered, between the lethal and non-lethal dose. Statistical evaluation of the test results is performed using the "Statistical Programme-AOT 425 StatPgm, Version: 1." The study used a suspension of fragmented lyophilizate of MBC. Biological-modified bacterial cellulose vascular prostheses were homogenized in distilled water, freeze-dried to remove water, ground, and then dried for 20 h at 105°C. The material for testing was placed in polypropylene packages and secured against re-absorption of moisture by the material with parafilm. Thus prepared prostheses for testing were suspended in water for injections, thus forming a dense, gelatinous suspension. The suspension intended for administration with a stomach probe was obtained after adding up to 1000 mg of cellulose 12.5 ml of water. Injection water "aqua pro injection" was used for the doses. The study was conducted on ten white Imp:WIST rats (five males and five females). Prior to the application of doses, the body mass of the animals was accurately determined.
Bacterial cellulose suspension at a dose of 2 g/kg b.w was administered through a probe into the stomach. Due to the fact that the quantity of the administered suspension exceeded 2 ml/100 g b.w. (OECD guidelines), the substance was administered in two equally divided portions at an interval of four hours (0900 and 1300). The total amount of administered cellulose dispersion was 2.5 ml/100 g b.w. = 0.2 g/100 g m.c, which corresponds to a dose of 2 g/kg b.w.
Consecutive observations were performed twice a day. The body weight of rats was determined 3, 7, and 14 days after treatment, as stated in OECD guidelines No. 425. The scope of animal observations was as follows: autonomic and central nervous system, respiratory system, and circulatory system, as well as the skin, fur, eyes, and mucous membranes. Attention was drawn to the occurrence of tremors, convulsions, salivation, diarrhea, lethargy, and coma. Somatomotor activity and behavior were also evaluated.

Intradermal Reactivity Test
The study used three rabbits Imp:BN. Two extracts were prepared: nonpolar in cottonseed oil and polar in isotonic sodium chloride solution (which are substances of choice in our lab for this procedure), according to ISO 10993-12:2012. Intracutaneous injections with 0.2 ml of the extract obtained with polar or non-polar solvent at five sites were performed on the left part of each rabbit. Similarly, the right side was injected with polar or non-polar solvent control as shown in Fig. 4. Observations were performed immediately after injection and at 24, 48, and 72 h, as well as 4 and 5 days after injection.  [12]. On the day before investigation, the fur was removed from dorsal area of the guinea pigs on both sites of the trunk, without injuring the skin. Then, on the first day of the induction period, three pairs of intradermal injections (0.1 ml/site) were performed in the scapular area (Fig. 5). On day 8 of the experiment, 0.5 g of 10% sodium lauryl sulfate in vaseline was applied on the right site of all guinea pigs to evoke local inflammation, and these areas were washed after 24 h. Next, a sterile gauze compress with 0.5-ml analyzed water extract was applied to the exposed skin, stabilized with a bandage and left for 48 h. On day 22 of the study, a sensitization reaction was induced in all guinea pigs.
Checking test for sensitization was performed after 24 h. The effects of sensitization were evaluated according to the Magnusson-Kligman Classification 48, 72, and 96 h following the sensitization reaction.

Implantation
The examination was carried out in accordance with the requirements of ISO 30993:6 2009. Thirty male white rats were used in the vascular prosthesis testing. With the animals under deep anesthesia (80-100mg/kg ketamine hydrochloride i.p.; Sigma Aldrich Sp. z.o.o, Poland), the tested implants were inserted into the pocket of panniculus camosus muscles along the dorsal midline, and kept there for 1 month (vascular prosthesis). Following this time, a block measuring 2 cm × 2 cm from the point of entry of the implant (cervical-interscapular region) was resected. This sample covered the entire thickness of the back muscles.

Histopathological Evaluation After Implantation of Vascular Prosthesis of Chitosan-Modified Bacterial Cellulose
The examination was carried out in accordance with the requirements of ISO 30993:6; 2009. The muscle sections were dehydrated in increasing concentrations of alcohol and imbued with paraffin in an RVG/1 computer tissue processor (Belair Instrument Company). Microscopic samples of 4-6μm thickness were prepared on an MH-325 rotary microtome (Thermo Scientific™) and stained with hematoxylin and eosin in a Varistain Gemini staining machine (Varistain™ Gemini ES Automated Slide Stainer A78000014). In addition, samples were stained in acidic fuchsin in picric acid in order to visualize collagen fibers.
After removal of the implants, two assessments of the rats' entrails were conducted: evaluation of the internal organs and their blood supply and a macroscopic examination of the muscles surrounding the implants and of the stomach, liver, and kidneys.

Statistical Analysis
The statistical analysis of in vitro testing results was performed using the Student's t-test. The correlation coefficient was assumed to be significant at *p <0.05, **p<0.01, ***p<0.001.
In comparing the results obtained by in vivo testing, the chi-square test with Fisher's exact test were used to determine whether the differences in scores between the tested animals or sites and respective controls were significant. The differences in the number of responses of the test site/group versus the control site/group were compared using the Mann-Whitney U test. Differences were considered significant for p<0.05. Statistical analysis of the results was performed with Statistica software for Windows (v.12 StatSoft, Inc., Tulsa, OK, USA). Figure 3 shows the spectra for bacterial cellulose without modification; chitosan oligomers used to modify BC tubes and modified bacterial cellulose-BC tube. A wide band at 3400 cm −1 , attributed to the stretching vibration of the OH (connected by hydrogen bond) and/or NH group, could be observed in the all spectra. A band within the range 2800-2950 cm −1 corresponding to the stretching vibration of the C-H bond-characteristic for both polysaccharides-also could be observed. Because during biosynthesis the nutrition medium was modified by chitosan oligomers, also the bands characteristic for chitosan oligomers corresponding to the stretching vibration of the C=O bond in primary amides detected in 1630-1660 cm −1 , was observed also in BC tubes structure. A band at 1320 cm −1 observed in chitosan oligomers and BC tube corresponds to C-N stretching of amide III. A band at 1550 cm −1 corresponding to N-H bending of amide II was observed in chitosan oligomers, but not in BC tube. Probably it was overlapped by other bands.

Physical-Mechanical Properties of BC Tubes
The BC tubes were found to possess the following physical-mechanical properties: -Length 10-12 cm -Surface density of about 10 g/m 2 (of dry cellulose) -Selected molecular parameters of bacterial cellulose (GPC/SEC analysis according to the Turbak procedure [10,11] Fig. 6).

Hemocompatibility
In the study of hemolytic activity using concentrated red blood cells, which come into contact with both the material under evaluation and its extract in physiological saline, the average value of hemolysis percentage was 0.25 ± 0.06%. The obtained value did not exceed the value allowed by the standard, i.e., 3%; in full blood hemolytic activity tests (CPD), having direct contact with the surface of the material, the mean value of hemolysis percentage was 0.14± 0.03%. The obtained value did not exceed the value acceptable by the standard, i.e., 1%; in tests of plasma clotting system, significant (p<0.05) shortening of prothrombin time (PT) with unchanged values of partial thromboplastin time after activation (APTT) was found within 2 h of blood contact with the material. However, after 24 h, APTT and PT were prolonged. The thrombin time (TT) values were prolonged and fibrinogen concentration decreased. It follows that the grafts made of modified bacterial cellulose activates to some extent the blood clotting process. However, the observed changes in the values of the determined clotting system parameters do not exceed the range of reference values. These changes may be due to the effect of material components on the coagulation factors of both the intra-and extra-transmitted system as well as to the conversion of fibrinogen into fibrin. The activation of the clotting system by the material under assessment is evidenced by shortened clotting time of whole blood after calcification.

Toxicity Test
No pathological signs were observed in the fur, orifices, eyes, muscle tension, or behavior of three males and three females immediately after administration of modified BC suspension. After 1 day, female no. 65 was less mobile; there was a clear tilt of the head to the right and increased tension in the neck muscles. After 2 days, the tested female died. Based on clinical observations and the results of the post-mortem examination, it was assumed that the cause of rat's death was a lesion of the esophagus wall during administration of the test substance. Death was then assessed not be related to the toxic effects of tested implants. All of the remaining animals survived the period of 14 days of observation (five males and four females). Constant weight gain was found (Table 3). No signs of pathology were found regarding animal behavior, evaluation of hair in natural orifices of the body, the appearance of the eyes, or muscle tension. The autopsy found the structure and blood supply to the internal organs to be normal. The macroscopic image of the gastric area and small and large bowel was within normal limits. No pathological signs regarding animal behavior, hair, orifices, the eyes, or muscle tension were noted within 14 days. Detailed results of sectional studies was presented in Table 4.

Intradermal Reactivity Test
The results are presented in Table 5. The non-polar and polar extracts of modified bacterial cellulose implants revealed mild erythema in one out of three rabbits after 24 hours of examination time after 48 hours, this erythema disappeared. No significant changes in intradermal reactivity were observed. The Primary Irritation Index was found to be 0.03: The product is not irritating.

Guinea Pig Maximization Test (GPMT)
The detailed results are presented in Table 6. According to

Histopathology
Histopathological studies showed that the implanted vascular implants induced an initial inflammatory response which developed into a chronic inflammatory immune response known as host foreign body reaction (FBR). The classic host response is generally divided into several stages, from the formation of the wound during implant placement to the wound healing period. The final host response reaches a relative impasse with most implants, unresolved in chronic inflammation as long as the implant remains. This final condition is defined by the long-term presence of both activated macrophages and the associated giant foreign body cells (FBGC) at the tissue/ implant interface and the formation of a dense fibrous collagen capsule around the implant. These two characteristics-the presence of FBGC and the thickness of the fibrous    Sex: male. Body weight 380 g Clinical data: no signs of pathology were found in animal behavior, hair, natural body orifices, eyes and muscle tension during 14 days of observation. Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucosa is grayish-white in color; the glandular part is grayish-white, light red with a correct picture The small and large intestine contains semi-liquid, brown food, grey-pink mucous membrane Rat Code: 43 Sex: male. Body weight 350 g Clinical data: during 14 days of observation, no signs of pathology were found in animal behavior, assessment of coat, natural body orifices, eyes, and muscle tension Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucosa is grayish-white in color; the glandular part is grayish-white, light red in color with correct picture. The small and large intestine contains semi-liquid, brown food, gray-pink mucous membrane Rat Code: 62 Sex: male. Body weight 395 g Clinical data: no pathological signs were found in animal behavior, hair, natural body orifices, eyes, and muscle tension during 14 days of observation Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting the pre-stomach mucous membrane is grayish-white in color; the glandular part is grayish-white, light red with correct drawing The small and large intestine contains semi-liquid brown food, gray-pink mucous membrane Rat Code:  Sex: male. Body weight 350 g Clinical data: no signs of pathology were found in animal behavior, hair, natural body orifices, eyes, and muscle tension during 14 days of observation Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen of dark red color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucosa is grayish-white in color; the glandular part is grayish-white, light red in color with correct picture. The small and large intestine contains semi-liquid, brown food, gray-pink mucous membrane Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucosa is grayish-white in color; the glandular part is grayish-white, light red in color with correct picture. The small and large intestine contains semi-liquid brown food, gray-pink mucous membrane Rat Code: Thoracic and abdominal organ system normal. Lungs sunken, not very airy, greyish-red surface with clearly marked widening of blood vessels (congestion). The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium sized spleen, dark red in color. The esophagus is empty, after cutting with red, congested mucous membrane. The stomach is filled with a small amount of greenish, nutritious content. After the intersection, the pre-stomach mucosa is grayish-white in color; the glandular part is grayish-white, light red with a correct figure. The small and large intestine is bloated, contains liquid brown food; the mucous membrane is greyish-red, bloodied Rat Code:58 Sex: female. Body weight 250 g Clinical data: no signs of pathology were found in animal behavior, coat evaluation, natural body orifices, eyes, and muscle tension during 14 days of observation Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucosa is grayish-white in color; the glandular part is greyish-white, light red in color with correct picture. The small and large intestine contains semi-liquid brown food, gray-pink mucous membrane Rat Code:60 Sex: female. Body weight 250 g Clinical data: during 14 days of observation, no signs of pathology were found in animal behavior, assessment of hair, natural body orifices, eyes, and muscle tension Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface. The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucous membrane is grayish-white in color; the glandular part is grayish-white, light red with correct drawing. The small and large intestine contains semi-liquid brown food, grey-pink mucous membrane Rat Code:32 Sex: female. Body weight 240 g Clinical data: no signs of pathology were found in animal behavior, hair, natural body orifices, eyes and muscle tension during 14 days of observation. Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organs system is normal. Air lungs, chest filling, gray-pink surface Liver is dark red in color, red on the cross sections with correct lobular pattern. Medium sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting the pre-stomach mucous membrane is grayish-white in color; the glandular part is greyish-white, light red with correct drawing. The small and large intestine contains semi-liquid brown food, gray-pink mucous membrane Rat Code:36 Sex: female. Body weight 245 g Clinical data: no signs of pathology were found in animal behavior, hair, natural body orifices, eyes, and muscle tension during 14 days of observation. Method of euthanasia: ether anesthetic, bleeding through femoral vessels Thoracic and abdominal organ system is normal. Air lungs, chest filling, gray-pink surface. The liver is dark red in color, red on the cross sections with correct lobular pattern. Medium-sized spleen, dark red in color. Stomach filled with greenish, dense food content. After cutting, the pre-stomach mucous membrane is grayish-white in color; the glandular part is grayish-white, light red with correct drawing. The small and large intestine contains semi-liquid brown food, gray-pink mucous membrane introduction. The study was conducted on outbred Stock Han Wistar Imp:WIST rats due to the possibility of extrapolating the results to human populations. In microscopic view, implants of modified bacterial cellulose were visible as an amorphous, fibrillar structure. The implants were placed between the muscles of the back, the epimysium, and intramuscularly ( Fig. 7.1, 7.2, and 7.3). In all animals, the implants were surrounded by connective capsule (Fig. 7.4, 7.5, and 7.6 and Fig. 8.1 and 8.2) formed by histiocytes and fibroblasts (Fig. 7.4 and 7.5) and collagen fibers (Fig. 8.1 and 7.8). The implants placed under the epimysium produced a significant thickening of the connective capsule over the implant (Fig. 7.1). In all cases, vascular-connective tissue strands grew from the inside of the capsule into the implants (Fig. 7.1, 7.2, 7.3, and 7.6). These bands were made of collagen fibers (Fig. 8.2), blood vessels, histiocytes and fibroblasts (Fig. 7.6 and Fig. 7.7). In the The sum, sum of points the severity of redness (erythema) and swelling of the skin (30 readings). Numerous fibroblasts surrounded the vascular-connective bands growing between the capsule and the implant (Fig. 7.4  and 7.6). The implants introduced into the intramuscular area were surrounded with a connective capsule, well-separated from the structure of skeletal muscle. No visible change in the form of reactive inflammatory cells was observed in the muscle area (Fig. 7.4, 7.5, and 7.6). There were no apparent signs of muscle interstitial fibrosis (Fig. 8.1 and 8.2). The macroscopic examination of the muscles surrounding the implants and of the stomach, liver, and kidneys confirmed the correct structure of internal organs and the degree of their blood supply. Macroscopically evaluated appearance of gastric and small and large intestine mucosa was within normal limits.

Discussion
Constantly rising expectancy of life and progress in the implant technology causes continuous increase in number of implantations in human medicine. Thanks to advance in the field of microscopy, suture materials and microsurgical instruments microsurgical techniques have developed to make surgical interventions at the finest structures of organism possible. In case of vascular prostheses, autogenous vessels are still most widely used due to their biocompatibility; however, this procedure requires additional operation and there are situations in which usage of this kind of prostheses is not possible. Moreover, the number of autologous grafts for coronary artery disease and for peripheral artery disease is limited. Due to this fact, scientists from all around the world started looking for alternatives. The ideal implant material should integrate without complications with tissue of the patient and maintain close connection over the time. Clinical problems such as damage of tissue around the implants, loosening of the implants, pain, and surgical revisions could be avoided. Particularly materials that will have contact with blood must comply with certain requirements such as mechanical strength, biocompatibility, blood compatibility, and sterilizability. Moreover, the inner and outer surface of artificial blood vessel must have different properties: outer should be overgrown with connective tissue whereas inner should be covered with endothelial cells and not stimulate adhesion of blood cells and components [13]. The essential requirement for biomedical materials is biocompatibility, referred as the ability of a foreign material implanted in the body to exist in harmony with tissue without causing deleterious changes. Generally, studies provide various results due to the different methodologies, workflow, and sample preparations [9]. Nowadays the most common clinically used synthetic grafts in peripheral artery disease remain expanded polytetrafluoroethylene, Dacron, and polyurethane [14]. Because of the unsatisfactory patency rates of common clinically used synthetic grafts, risk of stasis and thrombosis and rigidity, the researchers focus mainly on new grafts as small-diameter vessel substitutes [14]. The main challenge for tissue engineering is to develop new biologically active smalldiameter grafts that would not cause serious side effects. Bacterial nanocellulose (BNC) is one of such example which has demonstrated a tempting prospect for applications in substitute of small blood vessels [15]. Bacterial cellulose (BC) synthesized by Gluconacetobacter xylinus has recently been studied as a biocompatible material due to its unique features such as high purity, crystallinity, biodegradability, and tensile strength. Promising mechanical properties and good biocompatibility of BC promote its research and development as substitute/medical biomaterial, such as the replacement of blood vessel-vascular grafts [9]. Currently, BC has very high potential to be used as biomaterial on a larger scale. In our study, new material made from bacterial nanocellulose modified with chitosan used in future as biological vascular prostheses was investigated. In our study, this novel vascular prostheses made of bacterial nanocellulose modified with chitosan (MBC) was tested for their potential toxicity and adverse sensitization effects. The presented results and previously conducted research are consistent that in most of the experimental studies, the lack of toxicity or low toxicity of nanocellulose was observed. However, nanocellulose used as biomedical material in humans should be subject to more specific toxicological control. Chitosan is affirmed as a non-dangerous and biocompatible polymer. It is thought that chitosan-based biomaterials are antimicrobial, functional, renewable, nontoxic, bioabsorbable, and biodegradable biopolymer agents [4]. CS alone is considered to be suitable for oral organization, but its properties may change totally upon modification and combination with nanocellulose [16]. Moreover, it is understood that the pharmacokinetic properties of this kind of nanomaterials can significantly change when incorporated into a nanoparticulate system [16]. It should be emphasized that toxicology studies of nanocellulose and nanocellulose-based biocomposites are still restricted at a very preliminary stage, mainly on the level of cytotoxicity [9]. To confirm the earlier hypothesis about the lack of toxicity of materials based on nanocellulose and chitosan, we performed a number of tests, e.g., Toxicity Test No. 425: acute oral toxicity: up-and-down procedure; intradermal reactivity test; Guinea Pig Maximization Test No. 406: Skin Sensitization of Magnusson and Kligman (GPMT); and histopathological evaluation after implantation of vascular prosthesis of modified bacterial cellulose. Toxicity test showed that modified bacterial cellulose suspension does not exhibit any signs of pathology in animal behavior. Assessment of hair in the body's natural orifices, eye appearance, and muscle tension did not indicate any signs of toxic effects of the test substance. The autopsy confirmed a normal structure and blood supply to internal organs and orifices of the body. In Intradermal Reactivity Test, the non-polar and polar extract prepared from biological vascular prostheses of modified bacterial cellulose have been evaluated. None of the above showed an irritating effect. The maximization test (GPMT) No. 406: Skin Sensitization of Magnusson and Kligman confirmed lack of sensitization effect. During implantation process, correct structure of internal organs as well as the level of their blood supply was reported. Macroscopic view of gastric and small and big bowel was within normal limits. Histopathology assessment showed that in all animals, the implants were surrounded by a connective capsule mainly consisting of histiocytes, fibroblasts, and collagen fibers. In the muscle area, the reactive inflammatory cells were not found. There were no apparent signs of muscle interstitial fibrosis. Summarizing all obtained results, we can conclude that the study confirmed that biological vascular prosthesis of modified bacterial cellulose does not induce inflammatory response and hyperplasia of muscle tissue surrounding the implant. Previous studies on bacterial synthesized cellulose (BASYC®) have been a great success in the field of implantation and long-term maintenance of carotid arteries among rats and pigs. Another study conducted on BASYC® tube prosthesis for blood vessels has shown that BC has a potential to be used as a scaffold in tissue from the implant is sharply demarcated by a thick collagen fiber bag. A vascular and connective tissue band containing collagen fibers grows into the implant. No signs of muscle interstitial fibrosis engineering process and in in vivo tissue-engineered blood vessels as part of cardiovascular surgeries. This work confirms that it will be possible to fabricate BC as in vitro scaffold and implant it as a substitute for a small diameter carotid arteries [17]. Wippermann et al. showed that BC exhibits properties that are promising for its further use in tissue-engineered blood vessels programs. Tissue-engineered blood vessels (TEBVs) represent an innovative approach for overcoming reconstructive problems associated with vascular diseases by providing small-caliber vascular grafts. Moreover they confirm that BC has high mechanical strength, promotes in situ vascular tissue regeneration, can be shaped into threedimensional structures, and does not require pre-treatment in the form of cell seeding. The new BC-engineering technique they used resulted in a production of new stable vascular conduits [7]. Scherner et al. investigated the biocompatibility, blood compatibility, and biostability of a novel biomaterial of bacterially synthesized cellulose (BC) as a potential scaffold for small-diameter TEBV. BC tubes were implanted to replace the carotid arteries of 10 sheep for a period of 3 months. Analysis of BC grafts in a sheep model revealed the development of a three-layered structure of these grafts similar to those of native arteries with a single layer of endothelium with basement membrane followed by a concentric layer of smooth muscle cells and an outer layer with adjacent tissue with ingrowing capillaries after 3 months. The study confirmed that BC grafts support the physical load imposed by hemodynamic environment and allow remodeling, have minimal inflammatory potential, and are functionally stable. All patent grafts displayed confluent luminal endothelialization without neointima or thrombus formation. Scherner et al. came to similar conclusions during the research on in vivo application of tissue-engineered blood vessels of bacterial cellulose. Conducted study confirmed that BC can be a potential scaffold for small-diameter TEBVs and confirmed the statement that in future, TEBV programs will be used for cardiovascular surgeries [8]. Leitão et al. demonstrated that grafts made of BC which was histologically evaluated do not show any signs of chronic inflammation, infection, foreign body responses, clot formation, cell in growth, and diameter via electron microscopy [18]. BASYC® a novel biomaterial introduced to the market by Lee et al. actually is successfully used for implantation and long-term maintenance of carotid arteries. Moreover BASYC® prostheses are non-toxic and have non-immunoreactive effect of BC implantation. Azevedo et al. obtained similar results to ours; he proved that cellulose and chitosan (CELL:CHIT) polymers are biocompatible, nontoxic, and very abundant, which make them good candidates for biomedical applications [19]. Kim et al. investigated the immunoreactivity of BC in vitro in human umbilical vein endothelial cells (HUVECs) and in vivo using BALB/c mice. BC does not induce apoptosis and necrosis in HUVECs and does not stimulate immune response in both HUVECs and BALB/c mice models. These results suggest that BC may be widely used as a biocompatible biomaterial for tissue engineering.

Conclusions
Biological vascular prosthesis of modified bacterial cellulose does not induce inflammatory response and hyperplasia of muscle tissue surrounding the implant. The tested substances do not cause any allergic, intradermal reactions and finally, do not display acute toxicity.

Conflict of Interests The authors declare no competing interests.
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