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UDC 616-089.819.84-092.4:615.468.6
ENHANCEMENT OF SMALL INTESTINAL SUTURE BY MEANS OF BACTERIAL NANOCELLULOSE: EXPERIMENTAL-MORPHOLOGICAL STUDY
1 Altai State Medical University, Barnaul
2 IPCET SB RAS, Biysk
3 Regional Clinical Hospital, Barnaul
A.N. Zharikov1, V.G. Lubyansky1, E.K. Gladysheva2, E.A. Skiba2, V.V. Budayeva2, E.N. Semenova3
In experimental studies on 5 dogs, there was studied the possibility of sealing the small-intestine suture using new materials. Plates of wet (99%) bacterial nanocellulose were used, obtained by cultivating the inoculum in a synthetic nutrient medium of an enzymatic hydrolyzate of miscanthus (laboratory of byconversion IPСET SB RAS, Biysk). The producer was a culture of Medusomyces gisevii, known as "Kombucha", which is a symbiotic culture consisting of different types of acetic acid bacteria and yeast. During laparotomy, a plate of bacterial cellulose was fixed to the suture of the wound of small intestine, and then after 14 and 45 days, relaparotomy was performed, during which the fixed material and its interaction with small intestine were visually evaluated. Later resection of the intestine for morphological studies was performed. There was determined a good fixation of the material on small intestine with the formation around the capsule, the absence of purulent complications, the presence of active reparative processes in the region of the intestinal suture, increasing to the 45th day of the postoperative period. Key words: intestinal suture, bacterial cellulose.
In abdominal surgery, situations arise when intestinal sutures have to be placed with a high risk of insolvency: inter-intestinal anastomoses by peritonitis, intestinal obstruction and pancreatic anastomoses in patients with pancreatic cancer or chronic pancreatitis [1]. In this regard, various methods of strengthening the intestinal suture are used in surgery: fibrin-collagen plates "Tachocomb", glue "Sulfacrylate", glue "Tissucol Kit", adhesives on cryoprecipitate basis, etc. Their use is limited due to some negative properties or high cost. Search and use of new nanomaterials can help create additional sealing of dysfunctional intestinal sutures. Bacterial nanocellulose (BNC) is an organic material synthesized extracellularly by microorganisms. The molecular formula and nanostructure of BNC correspond to cellulose isolated from vegetable raw materials, but at the same time, due to the three-dimensional structure formed by fibers, BNC has a higher crystallinity, modulus of tensile strength, water-absorbing ability, plasticity [2]. It is widely used in the textile, paper and food industries [3, 4, 5]. Some properties of bacterial cellulose, such as porosity, moisture, non-toxicity and biocompatibility, high strength make these materials suitable for biomedical applications, including bone tissue engineering [6], blood vessel formation in microsurgery [7, 8, 9] and vascular stents [10 ], for the treatment of burns [11] and skin restoration [12], for replacing ear cartilage tissue [13], nerve regeneration [14], treating trophic ulcers [15, 16], replacing the dura mater [17], treating periodontal diseases [18].
Objective: to study the possibility of using bacterial cellulose to seal the inter-intestinal sutures at different periods of the postoperative period. 44
Materials and methods
Plates of wet (99%) bacterial nanocellulose (BNC) were used as material for experimental studies, which was obtained by cultivating an enzymatic miscanthus hydrolyzate in a synthetic nutrient medium (laboratory of bioconversion IPCET SB RAS, Biysk). The nanostructure of the BC is presented in Figure 1.
The producer was a culture of Medusomyces gisevii, known as "Kombucha", which is a symbiotic culture consisting of different types of acetic acid bacteria and yeast. Under general anesthesia in compliance with international standards of humane treatment of animals (European Convention "On the Protection of Vertebrate Animals Used for Experiments or for Other Scientific Purposes", Strasbourg, 1986) under sterile conditions of the operating unit of the Department of General Surgery, Operative Surgery and Topographic Anatomy of Altai State Medical University, 5 dogs underwent a laparotomy, a loop of small intestine was placed in the median wound, on which a wound was applied in the transverse direction up to 1 cm in length with futher suturing its two interrupted sutures (Vicril 4,0). For additional closure of intestinal sutures, BNC section with a size of 1.5x2.5 cm was used. Since BNC is a wet gel film (dry polymer/water ratio is about 1/100), self-attachment to the intestine was difficult due to its low adhesion. In order to ensure fixation, the material was fixed to the serous membrane of small intestine by imposing 6 serous-muscular sutures (Figures 2a, b).
Figure 1a - photo of mesh nanostructure of BNC (zoom x 20 000), b - distribution of the values of microfibrille diameter
of BNC sample obtained on a synthetic nutrient medium
Figure 2 - Experimental study: a - location of BNC on the small intestinal suture, b - fixation of BNC to small intestine
with interrupted sutures
In some cases, BNC plate fixation on intestinal arrow) and a solution comprising 150 units NI /ml
sutures was achieved without suturing with thrombin and 10 ml of aminocaproic acid (Figures
material reinforcement using a fibrin clot formed 3a, b). when mixing dry cryoprecipitate (indicated by
Figure 3 - Experimental study: a - location of BNC on small intestine using dry cryoprecipitate (shown by arrow) and thrombin solution, b - final appearance of the formed socket in the area of the small intestinal suture.
It is known that cryoprecipitate contains high-adhesive plasma proteins (fibronectin, fibrinogen, blood coagulation factors), and in dry lyophilized form, the activity of these substances is greater than in liquid. When these two components were mixed, a stable fibrin clot was formed, which, taking into account further polymerization, fixed BNC plate on the serous membrane of small intestine. After that, the loop of small intestine was immersed in the abdominal cavity, and layer-by-layer suturing of the postoperative wound was performed.
Evaluation of the results of fixing BNC to small intestine was carried out in 14 and 45 days after surgery. After the relaparotomy and the entrance into the abdominal cavity, a loop of small intestine was lead into the median wound. It turned out that 14 days after the first experiment, part of the omentum was fixed to BNC located on the gut. It kind of wrapped it, "plunged into the capsule." Separation of the omentum was possible in a loose way. After separation of the omentum from the intestine and dissection of the tissue, the walls of BNC were well differentiated (Figure 3b).
Figure 3 - Experimental study. 14th day after the operation: a - capsule formation (shown by dotted line) in the area of BNC fixed on small intestine, b - the front wall of the capsule above BNC is formed by an omentum (trapped by forceps), the bottom of BNC is loosely fixed to the serous surface of small intestine (indicated by the arrow).
At the same time, BNC was very tightly fixed at the top, where the omentum was attached, and more freely separated from below from the serous membrane of small intestine. At this point, its fusion with the gut was small. However, the previously imposed intestinal suture was closed with granulation tissue and was not visible. By the conclusion of the experiment, resection of small intestine was performed. The continuity of the gastrointestinal tract was restored by the imposition of an enteric-enteroanastomosis "side-to-side". Small intestine with BNC sample was sent for histological examination. Microscopic examination revealed that on the 14th day of the postoperative period, at the border of BNC and the serous membrane of small intestine there was moderate leukocyte infiltration due to the migration of cellular elements represented by neutrophils and lymphocytes to this area (Figures 4a, b).
45 days after the implantation of BNC by visual assessment of the state of small intestine, we also did not notice signs of infection or the formation of fluid accumulations in this area. The material was well covered and tightly fixed on the serous
membrane of small intestine (Figure 5a). When dissecting the upper section where BNC was located, there were structures in the thickness of the formed tissues resembling loose collagen fibers (Figure 5b). There were no signs of insolvency of the intestinal suture during the test with water load.
Histological examination of the material after 45 days using fibrin (Picro-Mallory) staining at the border of BNC and the small intestinal serous membrane along with a decrease in leukocyte infiltration, two waves ("young" and "mature") fibrin were observed (Figure 6), which was tightly fixed to the serous membrane of small intestine, which indicated reparative processes occurring in this area.
Discussion
Thus, in the experimental study, an approach was made to the possible use of new materials for sealing the sutures of small intestine. It was shown that after applying BNC plates on the suture of small intestine on the 14th and 45th days of the postoperative period, infection of the material does not occur. On the 14th day, immature connective
Figure 4 - Morphology of BNC plate and small intestine (SI) 14 days after implantation. Coloring by hematoxylin -Zoom x10 (a), x20 (b). Leukocyte infiltration at the border between BNC and SI (indicated by the arrow).
Figure 5 - Experimental study. 45 days after surgery: a - fixing BNC in small intestine, b - by dissection of BNC, tissue resembling collagen is seen integrated into the serous membrane of the intestine
Figure 6 - Morphology of BNC and small intestine (SI) films after 45 days. Fibrin staining (indicated by arrow) by Picro-Mallory, zoom x10. There is a wave of formation of the "young" fibrin (arrow) on the border of the serous membrane
of small intestine and BNC.
tissue is formed around BNC and small intestine in the form of light fibrinous adhesions on the border with the material and elements of borderline aseptic inflammation, which is determined by histological examination. When analyzing the fixation of the material on the 45th day, active signs of the formation of more dense fibrous structures in small intestine with BNC, the appearance of a young "fibrin" with a simultaneous decrease in borderline leukocyte infiltration are noted.
Conclusions:
1. In experimental studies after the implantation of BNC plates on the seam of small intestine on the 14th and 45th days of the postoperative period, there are no signs of infection and rejection of the material, the failure of the seam of small intestine.
2. The process of biointegration of BNC by the 45th day of the postoperative period is accompanied by tight fixation of the material to the serous membrane of small intestine, a decrease in leukocyte infiltration at the border of their contact and the appearance of fibrinous structures recorded during morphological examination.
References
1. Gorsky V.A., Shurkalin B.K., Faller A.P., Leonenko I.V., Medvedev S.S., Andreev S.S. The problem of the reliability of intestinal suture with peritonitis and intestinal obstruction. Difficult Patient. 2005; 4:12-15.
2. Dahman Y. Nanostructured Biomaterials and Biocomposites from Bacterial Cellulose Nano-fibers. Journal of Nanoscience and Nanotechnology. 2009; 9(9): 5105-5122.
3. Gromovykh T.I., Fan Mi Han, Biryukov E.G., Danilchuk T.N., Abdrashitova G.G. Prospects for the use of bacterial cellulose in meat products. Meat Industry. 2013; 4: 32-35.
4. Hioki N, Hori Y, Watanabe K, Morinaga Y, Yoshinaga F, Hibino Y, Ogura T. Bacterial cellulose as a new material for papermaking. Jpn TAPPI J. 1995; 49: 718-723.
5. Iguchi M, Yamanaka S, Budhiono A. Bacterial cellulose— a masterpiece of nature's arts. J Mater Sci. 2000; 35: 261-270.
6. Saska S, Barud HS, Gaspar AMM, Marchetto R, Ribeiro SJL, Messaddeq Y. Bacterial cellulose-hydroxyapatite nanocomposites for bone regeneration. International Journal of Biomaterials. 2011; 175362: 1-8.
7. Klemm D, Schumann D, Udhard U, Marsch S. Bacterial synthesized cellulose: Artificial blood vessels for microsurgery. Prog. Polym. Sci. 2001; 26: 1561-1603.
8. Zhang J. Glutaraldehyde treatment of bacterial cellulose/fibrin composites: Impact on morphology, tensile and viscoelastic properties. Cellulose. 2012; 19: 127-137.
9. Wippermann J, Schumann D, Klemm D, Kosmehl H, Satehi-Gelani S, Wahlers T. Preliminary results of small arterial substitute performed with a new cylindrical biomaterial composed of bacterial cellulose. Eur J Vasc Endovasc. 2009; 37(5): 592-596.
10. Fink H, Faxalv L, Molnar GF, Drotz K, Risberg B, Lindahl TL, Sellborn A. Real-time measurements of coagulation on bacterial cellulose and conventional vascular graft materials. Acta Biomater. 2010; 6: 1125-1130.
11. Czaja W, Krystynowicz A, Kawecki M, Wysota K, Sakiel S, Wroblewski P, Glik J, Nowak M, Bielecki S. Biomedical Applications of Microbial Cellulose in Burn Wound Recovery. Cellulose: Molecular and Structural Biology. Selected Articles on the Synthesis, Structure, and Applications of Cellulose. Springer: Dordrecht, 2007: 307-321. https://doi. org/10.1007/978-1-4020-5380-1
12. Fu L, Zhang J, Yang G. Present status and applications of bacterial cellulose-based materials for skin tissue repair. Carbohydr. Polym. 2013; 92: 1432-1442. doi: 10.1016/j.carbpol.2012.10.071
13. Nimeskern L, Avila HM, Sundberg J, Gatenholm P, Mueller R, Stok KS. Mechanical evaluation of bacterial nanocellulose as an implant material for ear cartilage replacement. J Mech Behav Biomed Mater. 2013; 22: 12-21. doi: 10.1016/j.jmbbm. 2013.03.005
14. Kowalska-Ludwicka K, Cala J, Grobelski B, Sygut D, Jesionek-Kupnicka D, Kolodziejczyk M, Bielecki S, Pasieka Z. Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves. Arch Med Sci. 2013; 9: 527-534. doi: 10.5114/ aoms. 2013.33433
15. Portal O, Clark WA, Levinson DJ. Microbial Cellulose Wound Dressing in the Treatment of Nonhealing Lower Extremity Ulcers. Wounds-A Compendium of Clinical Research and Practice. 2009; 21(1): 1-3.
16. Solway DR, Clark WA, Levinson DJ. A parallel open-label trial to evaluate microbial cellulose wound dressing in the treatment of diabetic foot ulcers. International Wound Journal. 2011; 8(1): 69-73.
17. Rosen CL, Steinberg GK, De Monte F, Delashaw JBJr, Lewis SB, Shaffrey ME, Aziz K, Hantel J, Marciano FF. Results of the prospective, randomized, multicenter clinical trial evaluating a biosynthesized cellulose graft for repair of dural defects. Neurosurgery. 2011; 69(5): 1093-1103.
18. Novaes ABJr, Novaes AB. Soft tissue management for primary closure in guided bone regeneration: Surgical technique and case report. Int J Oral Maxillofac Implants. 1997; 12: 84-87.
Contacts
Corresponding author: Zharikov Andrey Nikolayevich, Doctor of Medical Sciences, Professor of the Department of Faculty Surgery named after Professor I.I. Neimark, hospital surgery with the
course of FVE, Altai State Medical University, Barnaul.
656056, Barnaul, ul. Lyapidevskogo, 1. Tel.: (3852) 689574. Email: zhar67@mail.ru
Author information
Lubyansky Vladimir Grigorievich, Doctor of Medical Sciences, Professor, Professor of the Department of Faculty Surgery named after Professor I.I. Neimark, hospital surgery with course of FVE, Altai State Medical University, Barnaul. 656056, Barnaul, ul. Lyapidevskogo, 1. Tel.: (3852) 689574. Email: lvg51@mail.ru
Gladysheva Evgenia Konstantinovna, junior research assistant of the laboratory of bioconversion IPCET SB RAS, Biysk. 659322, Biyskm ul. Sotsialisticheskaya, 1. Tel.: (3854) 301415. E-mail: evg-gladysheva@yandex.ru
Skiba Ekaterina Anatolyevna, Candidate of Technical Sciences, senior research assistant of the laboratory of bioconversion IPCET SB RAS, Biysk. 659322, Biyskm ul. Sotsialisticheskaya, 1. Tel.: (3854) 305955. Email: science@agmu.ru
Budaeva Vera Vladimirovna, Candidate of Chemical Sciences, Associate Professor, Head of the laboratory of bioconversion IPCET SB RAS, Biysk. 659322, Biyskm ul. Sotsialisticheskaya, 1. Tel.: (3854) 305985. Email: science@agmu.ru
Semenova Elena Nikolayevna, pathologist of the Regional Clinical Hospital, Barnaul. 656038, Barnaul, Komsomolsky Prospekt, 73. Tel.: (3852) 245830. Email: science@agmu.ru
