RU2711545C1 - Method for producing a bioresorbed tube based on methacrylized gelatine and methacrylized fibroin and a method for increasing the consistency of intestinal anastomosis using such a tube - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology and medicine. In more detail, the invention refers to surgery, namely to methods for reducing the incidence of intestinal anastomosis. Invention relates to a method of producing a bioresorbable tube based on methacrylized gelatine and methacrylized fibroin, involving the following steps: a) dissolving in hexafluoroisopropanol of macromonomers: methacrylized gelatine (GMA) at rate of 10 wt% and methacrylized fibroin (FMA) in amount of 4 wt% at 55±5 °C for 18–36 hours; b) obtaining a mixture which includes equal parts of solutions of GMA and FMA and a photoinitiator of diphenyl(2,4,6-trimethylbenzoyl)phosphine in amount of 5 wt% by weight of macromonomers; c) preparing a guide for forming a tube by degreasing, followed by drying a surface of a glass rod with a circular cross-section of 2–15 cm in diameter; d) preparing the reinforcing thread by holding untwisted 8/0-4/0 silk thread in the mixture obtained in step b); e) forming the first layers of the bioresorbable tube on the guide by immersing it in the mixture obtained at step b) for 5–10 seconds with subsequent rotation and exposure of the obtained workpiece in the ultraviolet lamp light for at least 2 minutes; f) reinforcing the end sections of the tube by laying the thread in turns at distance of 2–5 mm from the ends of the formed tube to the workpiece obtained at step e); g) forming the outer layers of the bioresorbable tube by immersing the workpiece obtained at step f) into the mixture obtained at step b) for 5–10 seconds, followed by rotating and exposing the obtained tube in the ultraviolet lamp light for at least 2 minutes; h) processing the formed tube by holding in distilled water for 1–1.5 hours, followed by holding in 96 % ethanol for 12–18 hours and repeated holding in distilled water for 1–1.5 hours, treating internal surface of the formed tube with chloroform.EFFECT: obtaining articles having a programmable rate of degradation, low immunogenicity, ease of modification; developing a new approach to solving the problem of anastomosis inconsistency, where, in addition to mechanical protection of the anastomosis area, the implant is actively built into the regeneration process.11 cl, 3 dwg, 2 ex

Description

Technical field

The invention relates to biotechnology and medicine. In more detail, the invention relates to the field of creation and use in surgery of bioresorbable, biocompatible biopolymer structures in the form of tubes obtained by photocuring a mixture of a methacrylated gelatin derivative and a methacrylated Bombyx mori silk fibroin derivative and intended to close the anastomosis in order to increase solvency.

State of the art

Thanks to the achievement of modern anesthesiology and intensive care, the number of reconstructive operations on the abdominal organs is growing every day. But, despite this, the percentage of complications, such as the failure of anastomoses reaches 20%, and mortality with this complication can reach 80%. Currently, to solve this problem, a large number of preventive measures have been proposed, such as preventive stomas, protection of the anastomosis inside and out, intubation of the anastomosis zone. The essence of all these methods is to increase the strength, tightness of the anastomosis and reduce the load on the formed anastomosis.

Given the wide range of preventive measures for the failure of anastomoses of the gastrointestinal tract, the use of regenerative medicine approaches to solve this problem is promising.

The first mention of suturing an intestinal wound appeared about 2000 BC. The first operations in Europe, with intestinal damage, were associated with the use of glove technique. The essence of this method consisted in suturing the intestinal walls with a continuous twisting seam. At the same time, the ends of the threads were not cut off, but were removed through the wound of the abdominal wall, and the stitched fragment was fixed to the parietal peritoneum. The thread was removed when the barber considered it safe [Gordinsky MB By the centenary of the Lemberg intestinal suture. New Surgical Archive, 1926, 11, 3, 270-273]. In the XVII century, to restore the passage through the intestines, in addition to stitching the edges of the intestinal tube, they tried to improve the treatment results by introducing intraluminal stents (Lanfrank). Intestinal anastomosis at that time was a very rare and dangerous operation. The main treatment at that time was the formation of an intestinal fistula with its subsequent closure (Paracelsus, La Peyronie, B. Bell). This approach remained until the middle of the XIX century [Single-row continuous suture of anastomoses in abdominal surgery. Edited by Egiev V.N. - M .: Media practice-M, 2002, p. 6-12].

In 1824, Jobert A. proposed a single-row nodular, through invaginating intestinal suture. Later Lambert A., in 1826 - a single-row nodal, inverting suture with knots on serosa.

One of the first to upgrade this seam was Pirogov N.I. and described the technique for precision serous-muscular-submucous extramucous intestinal suture [Burch, J.M. Single-layer continuous versus two-layer interrupted intestinal anastomosis: a prospective randomized trial / J.M. Burch, R.J. Franciose, E.E. Moore // Ann. Surg. - 2000. -Vol. 231, N 6. - P. 832-837].

A single-row intestinal suture went through many modifications, but the poor quality of the suture material of that time led to frequent complications.

In parallel with the methodology for improving a single-row suture, a technique for mechanically linking organs developed. In the 18th century, Du Verger used goose trachea for stenting the anastomosis, in the 19th century cardboard cylinders moistened with oil or essence (Sabatier) were used, Watson proposed a rubber stent.

Later, compression mechanisms were developed and applied in the clinic, these devices gave good results when used on the small intestine. When using this technique on the colon, mortality reached 50%. In 1908, V. Fisher developed the first stapler. N. Hult in 1908 reported the use of this device for crossing the stomach with the formation of a linear brace suture [Gordinsky MB By the centenary of the Lemberg intestinal suture. New Surgical Archive, 1926, 11, 3, 270-273]. In 1951, the Scientific Research Institute of Surgical Apparatus and Instruments was opened in the USSR, where AKA compression devices were created [Kanshin N.N., Gorsky V.A., Kuprikov S.V. and others // Modern achievements and new technologies in surgery: Sat. scientific tr - M., 2005. - S. 67-72]. Currently, staplers have occupied a certain niche in modern surgery, but at present they cannot completely cover the needs of abdominal surgery.

In connection with this factor, at the beginning of the 20th century, a two-row intestinal suture technique appeared, which became the main method of joining hollow organs. Further studies of two-row interintestinal anastomoses showed that their healing occurs by secondary intention [Semenov G.M. Surgical suture / G.M. Semenov, V.L. Petrishin, M.V. Kovshova. - 2nd ed. - St. Petersburg: Peter, 2006. - 256 p.]. Disputes about the size of the intestinal suture remain relevant to the present, since none of the proposed methods gives a 100% absence of complications.

"Intestinal suture" is a term that combines all types of sutures used to restore the hollow organs of the abdominal cavity.

The reasons for the development of intestinal suture failure are diverse, the leading ones are: structural features of the wall of anastomosed organs, impaired suturing technique, imperfection of suture material, exposure to pathogenic microflora, patient age, impaired homeostasis, and the presence of concomitant pathology.

Given the high relevance of the problem of anastomotic insolvency and the high mortality in the development of this complication, various methods for the prevention of anastomotic insolvency are being actively developed and put into practice. All preventive measures are aimed at increasing the strength, tightness of the formed anastomoses and reducing the load on the formed anastomosis.

To achieve these goals, the following measures are currently used: preventive stomas, products aimed at reducing intraluminal pressure in the anastomosis, protecting the latter from the inside, strengthening the intestinal suture from the outside, using synthetic suture material that enhances the regeneration of stitched tissues [Method for the surgical prevention of intestinal failure seam / A.G. Semenov, V.L. Martynov, M.G. Ryabkov [et al.] // Honey. almanac. - 2013. - No. 5 (28). - S. 138-144].

The formation of a classic preventive stoma is fraught with a number of problems. Such as: difficult care in the postoperative period, partial social isolation of the patient, the occurrence of parastomic complications, the need for re-hospitalization and surgery. Summarizing the above, it can be said that in 19% of cases the temporary stoma becomes permanent [Chow A., Tilney H.S., Paraskeva P. et al. The morbidity surrounding reversal of defunctioning ileostomies: a systematic review of 48 studies including 6,107 cases. Int. J. Colorectal Dis. - 2009. - No. 24 (6). - p. 711-23].

Transanal decompression of the anastomotic zone using drainage. The essence of this method is to decompress the anastomosis zone using a silicone tube, placed above the anastomosis zone, the latter is fixed to the perianal region with a nodal suture. On the 5-7th day after surgery, silicone drainage is removed. In a retrospective study, some authors do not see a significant contribution of this method to the incidence of anastomotic failure [Kim MK, Won D-Y., Lee JK. et al. Comparative study between transanal tube and loop ileostomy in low anterior resection for mid rectal cancer: a retrospective single center trial. Ann. Surg. Treat. Res. - 2015. - No. 88 (5). - p. 260-8]. While in other studies, the effectiveness of transanal intubation in reducing the incidence of insolvency of the colorectal anastomosis was reliably proven [E. Hidaka, F. Ishida, S. Mukai et al. Efficacy of transanal tube for prevention of anastomotic leakage following laparoscopic low anterior resection for rectal cancers: a retrospective cohort study in a single institution. Surg. Endosc. - 2015. - No. 29. - p. 863-7]. All of these studies compared patient groups with and without transanal decompression.

Another known method of improving the viability of ananstomosis is Valtrac. This method of prevention consists in conducting an anastomosis zone of a special device in the form of a biofragmented ring (Valtrac) with a condom. This device is fixed inside the intestine 5 cm proximal to the anastomosis with absorbable suture material. The intestinal contents pass inside the condom without touching the formed anastomosis. On average, self-relapse of the device was observed 14 days after surgery. In comparative studies conducted by this method, the authors Ye et al. [Ye F., Chen D., Wang D. et al. Use of Valtrac TMS Secured Intracolonic Bypass in Laparoscopic Rectal Cancer Resection. Medicine - 2014. - No. 93 (29). - p. 224], the effectiveness of the Valtrac method was not proved, since the incidence of anastomotic failure did not differ between the groups using Valtrac and the preventive stoma and only with the formation of the preventive stoma (4.5% and 5.1%, P = 1; 8.3% and 0%, P = 0.5).

Also known in the art is the C-seal technique. The essence of the method is to protect the anastomosis using C-seal (biofragmented polyurethane "bag"). C-seal is attached to the head of the stapler and is held in the lumen of the reduced intestine. After the formation of the anastomosis, the C-seal turns out into the lumen of the rectum, while covering the formed anastomosis from feces, thereby reducing the incidence of insolvency. On average, an independent departure of the device is observed on the 11th day after the operation. According to research [Morks A.N., Havenga K., ten Cate Hoedemaker H.O. et al. Thirty-seven patients with C-seal: protection of stapled colorectal anastomoses with a biodegradable sheath. Int. J. Colorectal Dis. - 2013. - No. 28 (10). - p. 1433-8] it is difficult to judge the effectiveness of the technique, since they were not comparative in nature and, as a rule, a preventive stoma was formed for all patients.

Currently, in experimental and clinical studies, reinforcement of the seam line with membranes that have different chemical composition and structure has become widespread. The essence of the method is to strengthen a single-row seam with a polymer film membrane, which leads to an increase in the tightness of the latter on the 1st day, and therefore reduces the risk of insolvency in the early stages of the experiment. The advantage of the method is the prevention of biological insolvency of the development of purulent-necrotic process and, as a consequence, the failure of the anastomosis. The disadvantages of the method are mechanical failure, which equally leads to failure of the joints of the anastomosis. In addition, not all membranes have good adhesive properties and are easy to use [Kannelos I., Blouhos K., Demetriades N., Pramateftakis MG, Mantzoros I, Zacharakis E, Betsis D. The failed intraperitoneal colon anastomosis after colon resection // Tech. Coloproct. 2004. Vol. 8. P. 53-55].

Given the wide range of preventive measures for the failure of anastomoses of the gastrointestinal tract and the lack of agreement of researchers in this direction, the use of regenerative medicine approaches to solve this problem is very promising. This approach uses a biocompatible framework, which serves as a substrate for the migration, proliferation and differentiation of cells of the damaged organ [S.A. Markosyan, N.M. Lysyakov, M.Yu. Belyaev. Study of the effect of the greater omentum on the healing of the intestinal anastomosis in conditions of impaired blood supply to the small intestine. Bulletin of experimental and clinical surgery. Volume 11, No 4 (37), p. 288-292, 2017]. Currently, there is a huge amount of bioresorbable material, such as collagen, PLA, chitazan, etc., but each of them has a number of disadvantages, including immunogenicity, allergenicity, insufficient mechanical strength, toxicity of decay products, etc. Silk fibroin products occupy one of the leading places. Scafolds based on silk fibroin have high biocompatibility, programmed degradation rate, and ease of chemical modification [Kapoor S., Kundu S.C. Silk protein-based hydrogels: Promising advanced materials for biomedical applications // Acta Biomater. - 2016. - Vol. 31. - P. 17-32.]. Silk fibroin products have high tensile strength, which is 740 MPa. while collagen has 0.9-7.4 MPa and PLA - 28-50 MPa. In addition, the rate of decomposition of silk fibroin can be adjusted by changing the treatment regimen of fibroin, which affects the degree of β-folding, and the following dependence is observed - the decomposition rate decreases with an increase in the total content of β-layers. The unique properties of silkworm silk, such as accessibility, ease of chemical modification, the ability to biodegrad in a living organism and the ability to obtain materials of various formats from it from aqueous and organic solutions have attracted the attention of researchers. In this regard, over the past few years, active work has been carried out to study the possibilities of using the natural silk fibroin polymer as the basis of scaffolds for regenerative medicine. High biocompatibility, low immunogenicity, and controlled biodegradation of silk fibroin have been shown. As a result of a number of studies, methods have been developed to obtain different types of scaffolds from silk fibroin. Currently, studies are being conducted on the possibility of their use in tissue regeneration.

The closest solution to the claimed method, known from the prior art, is disclosed in patent document US 4719916 A "Intraintestinal bypass tube" An intestinal transplant is disclosed, formed from a thin-walled latex or elastic tube of various widths and lengths. The upper circular end of the tube and the vertical seam are reinforced with a fabric that contains a radiopaque thread. The graft is connected to the submucosal layer of the intestine proximal to the usual anastomosis, which prevents contact of the digestive tract: esophagus, gastrointestinal tract, food and feces with the closure of the anastomosis. Thus, the use of such a graft prevents leakage of the contents of the gastrointestinal tract from any intestinal anastomosis and ensures healing of the anastomosis, while the graft itself is naturally removed through the rectum.

The disadvantages of this invention is the use of synthetic materials, which can lead to additional postoperative complications. In addition, the materials used in the present inventions are not biodegradable and / or bioresorbable, which can also lead to complications, including fibrosis of surrounding tissues and, in the long term, perforation of the intestinal wall.

Disclosure of Invention

The claimed invention proposes the use of a biodegradable implant based on methacrylated fibroin silk to reduce the likelihood of failure of gastrointestinal anastomoses. Products from this material possess such properties as: programmable rate of degradation, low immunogenicity, ease of modification, structural feature allows you to vitalize this implant.

A new approach is proposed to solve the problem of anastomotic insolvency, where in addition to the mechanical protection of the anastomotic zone, the implant is actively integrated into the regeneration process. The implant becomes the basis for the regeneration of the intestinal wall. In experimental studies in rats, complete regeneration of the intestinal wall was shown, while maintaining the architectonics and function of the organ.

The objective of the invention is to develop a method of using implants based on fibroin to reduce the likelihood of insolvency anastomoses.

The technical result achieved by the claimed invention is the ability to correct a circular bowel defect through the use of a tubular biodegradable implant made of methacrylated silk fibroin and gelatin derivatives. An implant (bioresorbable tube based on methacrylated gelatin and methacrylated fibroin) ensures intestinal integrity during the entire healing period of the defect.

The inventive method is characterized by simplicity, low cost, the absence of the need for additional supplies and special skills, which will quickly and widely introduce it into clinical practice.

The problem is solved in that, the method for producing a bioresorbable tube based on methacrylated gelatin and methacrylated fibroin, according to the technical solution, includes the following steps:

a) dissolution of macromonomers in hexafluoroisopropanol: methacrylated gelatin (LMA) at the rate of 10 mass. % and methacrylated fibroin (FMA) at the rate of 4 mass. % at 55 ° C ± 5 for 18-36 hours;

b) obtaining a mixture comprising equal parts of solutions of FMA and FMA, and the photoinitiator diphenyl (2,4,6-trimethylbenzoyl) phosphine at the rate of 5 wt. % by weight of macromonomers;

c) preparation of the guide for forming the tube by degreasing, followed by drying of the surface of the glass rod with a circular cross section with a diameter of 2-15 cm;

d) preparation of the reinforcing yarn by keeping the silk untwisted yarn 8 / 0-4 / 0 in the mixture obtained in step b);

d) the formation of the first layers of the bioresorbable tube on the guide by immersing it in the mixture obtained in step b) for 5-10 seconds, followed by rotation and exposure of the resulting workpiece in the light of an ultraviolet lamp for at least 2 minutes;

f) reinforcing the end sections of the tube with thread by laying the thread in turns at a distance of 2-5 mm from the ends of the formed tube onto the workpiece obtained in step e);

g) the formation of the outer layers of the bioresorbable tube by immersing the workpiece obtained in step e) in the mixture obtained in step b) for 5-10 seconds, followed by rotation and exposure of the obtained tube in the light of an ultraviolet lamp for at least 2 minutes;

h) treatment of the formed tube by aging in distilled water for 1-1.5 hours, followed by aging in 96% ethanol for 12-18 hours and repeated aging in distilled water for 1-1.5 hours, treating the inner surface of the formed tube with chloroform .

The dissolution of PMA and PMA is carried out for 24 hours at 50 ° C. The guide mainly has a diameter of 4 mm, while in step d) it is kept in the mixture for 7 seconds, exposed in the light of a 36 W UV lamp for 2 minutes, rotating the guide at a speed of 15 rpm, and step e) is repeated 4-6 times depending on the formed number of layers. In step e), 10 turns of thread are evenly laid on the end sections of the tube 2.5 mm long, departing from one edge of 2 mm and 7.5 mm, while the density of the reinforcement turns is 10-15 turns per 3 mm of the tube. In step h), the formed tube is kept in distilled water for 1 hour, then 12 hours are kept in 96% ethanol and another 1 hour in distilled water. The formed tube has the following parameters: tube wall thickness: from 150 microns to 700 microns, length of reinforced sections 2-15 mm, length of unreinforced sections: 1-6 mm, number of reinforced sections: 2, location of reinforced sections: at the edges of the tube at a distance of 1 -6 mm from the edge, inner diameter: from 2 mm to 15 cm. The guide is degreased with 70% ethanol. Step g) is repeated 4-20 times depending on the number of layers being formed.

The problem is also solved by the fact that the method of increasing the viability of the intestinal anastomosis, according to the technical solution, includes the following steps:

a) the preparation of a bioresorbable tube obtained in accordance with paragraph 1 to close the anastomosis by incubating it in sterile saline for 15-30 minutes with multiple replacement of saline;

b) preoperative preparation of experimental animals by feeding 10% glucose solution without restriction for 2 days.

c) closing the anastomosis with a tube, for which the animals are fixed in the supine position, ceftriaxone is administered intramuscularly at the rate of 100 mg / kg body weight, an surgical wound is formed, into which the small intestine is removed (7 cm distal to the stomach), and then from the mesenteric edge of the intestine an incision is made into half the diameter of the intestinal tube, a bioresorbable tube is inserted into the intestinal lumen and fixed with a surgical thread, after which a 1-3 mm length of the intestinal tube is removed at the implantation site and the layers are sutured and the placement of the abdominal cavity of the intestine;

g) postoperative care through daily injections of ceftriaxone at the rate of 50 mg / kg body weight for 4 days. The bioresorbable tube used can have the following parameters: inner diameter - 4 mm, length of the unreinforced section - 3 mm, length of the reinforced sections - 2.5 mm, wall thickness - 300 microns. At the implantation site, a 1 mm section of the intestinal tube is removed. Wistar rats were used as experimental animals, and 15 minutes before the start of surgery, the rats were subjected to general anesthesia with sedation of atropine sulfate at a dose of 0.04 mg / kg, subcutaneously.

In the claimed invention can be used fibroin silk frame wire spiders, silk fibroin silkworm and other types of silkworms, fibroin recombinant silk, as well as artificial silk analogues.

Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (97%) was selected as a photoinitiator, which is a highly reactive, water-insoluble compound used for fast photo-curing, for example, for laser stereo lithography. A bioresorbable tube obtained in accordance with the claimed invention can be used to increase the viability of anastomoses of hollow organs.

Brief Description of the Drawings

The invention is illustrated by drawings.

In FIG. 1 presents a bioresorbable tube obtained by the claimed method.

In FIG. 2 shows an anastomosis additionally closed by a bioresorbable tube with a diameter of 4 mm.

In FIG. 3 shows the state of the intestine 2 weeks after surgery.

The implementation of the invention

A portion of methacrylated gelatin (LMA) is dissolved in hexafluoroisopropanol in a water bath at 55 ° C ± 5 for 18-36 hours at the rate of 10 mass. % A portion of methacrylated fibroin (FMA) is dissolved in hexafluoroisopropanol in a water bath at 55 ° C ± 5 for 18-36 hours at the rate of 4 mass. % The resulting solutions of FMA and FMA are mixed in equal volume ratios and the photoinitiator diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO) is added in an amount of 5% by weight of macromonomers: FMA and FMA. The formation of the tube with a diameter of 2-15 mm and a length of 4-36 mm is performed on a pre-processed glass rod with a circular cross section of the corresponding length and diameter. Processing a glass rod involves degreasing with 70% ethanol, drying and treating with a vacuum grease dissolved in chloroform (5 mg / ml). To do this, the stick is immersed in a solution of vacuum grease in chloroform for 10-30 seconds and then dried in air at room temperature for 30-60 minutes. Next, the first 4-6 layers are formed on the guide - treated with the appropriate glass shelf. To do this, immerse it in a ZhMA / FMA / TPO mixture for 5-10 seconds, extract and photopolymerize the layer of ZhMA / FMA / TPO mixture deposited on the guide by means of exposure for at least 2 minutes in the light of a UV lamp. The guide is rotated at a speed of 10-20 rpm. min Depending on the required number of layers, the procedure is repeated 4-6 times. Then, the end sections of the formed tube are reinforced by applying turns of untwisted silk thread, pretreated in a ZhMA / FMA / TPO mixture for at least 2 minutes, at the rate of 10-15 turns per 3 mm at a distance of 2-5 mm from the edge. The number of turns and the distance between them is determined by the required parameters of the resulting tube: the length of the reinforced section and the length of the unreinforced section. Next, the outer layers of the tube are formed. The number of outer layers is determined based on the required wall thickness of the tube, assuming that each layer is 30 μm. The workpiece is placed in a mixture of LMA / FMA / SRW for 5-10 seconds, removed and rotated at a speed of 10-20 rpm. expose in the light of a UV lamp (36 W power) for at least 2 minutes. The formed tube on the mandrel is transferred to distilled water for 1-1.5 hours, then 12-18 hours are kept in 96% ethanol and another 1-1.5 hours in distilled water. The resulting tube is removed from the guide, the inner surface is treated with chloroform and transferred to 96% ethanol for storage.

The obtained bioresorbable tube is used to close the anastomosis. For Wistar rats weighing 190-230 g, a tube with the following parameters is used: diameter - 4 mm, length of the unreinforced section - 3-5 mm, length of the reinforced sections - 2.5 mm, wall thickness - 280-320 μm. Before surgery, the tube is washed from ethanol. To do this, transfer the tube from 96% ethanol to a sterile saline solution, incubate at 37 ° C for 15-30 minutes, replace the saline solution with a fresh one and hold the tube in it for another 30 minutes. The procedure is repeated 3 times. Animals begin to prepare for surgery 2 days before surgery. 2 days before the operation, animals are deprived of solid food and given them a 10% glucose solution without restriction (per os). The operation is performed under general anesthesia (Zoletil + Xylazine), premedication - atropine sulfate at a dose of 0.04 mg / kg, subcutaneously 15 minutes before the start of surgery. The animals are fixed in the supine position. At the beginning of the operation, ceftriaxone is administered intramuscularly at the rate of 100 mg / kg body weight. The surgical field is limited by sterile gauze napkins, a laparotomy is performed. A section of the small intestine (7 cm distal to the stomach) is removed into the surgical wound, after which an incision is made from the mesenteric edge of the intestine to half the diameter of the intestinal tube, a sterile tube is inserted into the intestinal lumen and the implant is hermetically fixed with an implant using a surgical thread (polyester, 4/0 ) Then, at the site of implantation, a section of the intestinal tube 1-3 mm long is removed. The intestine is placed in the abdominal cavity and layered cavity suturing is performed. Within 4 days after the operation, daily injections of ceftriaxone are performed at a rate of 50 mg / kg body weight. Within 2 days, animals receive a 10% glucose solution, do not receive solid food, on the third day they are transferred to a standard diet.

Thus, the use of a tubular biodegradable implant made of methacrylated silk fibroin and gelatin derivatives provides the possibility of correcting a circular bowel defect. In this case, the implant (bioresorbable tube based on methacrylated gelatin and methacrylated fibroin) ensures intestinal integrity during the entire healing period of the defect. The following are examples of specific implementation. One skilled in the art will appreciate that these examples are not limiting of the invention, but merely show the feasibility of its implementation.

Example 1

Receiving tube

A portion of lyophilized methacrylated gelatin (LMA) is placed in 1.5 ml of hexafluoroisopropanol (HFIP) at the rate of 4 wt. % and placed in a sealed container in a water bath. Incubate the mixture of PMA with HFIP until complete dissolution for 24 hours at 50 ° C. A portion of lyophilized methacrylated fibroin (FMA) is placed in 1.5 ml of HFIP at the rate of 10 mass. % and placed in a sealed container in a water bath. Incubate the mixture of PMA with HFIP until complete dissolution for 24 hours at 50 ° C. Solutions of macromonomers are mixed: 1.5 ml of LMA and 1.5 ml of PMA, and diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO) photoinitiator is added to them in an amount of 5% by weight of the macromonomers. The surface of the glass rod with a diameter of 4 mm is degreased with 70% ethyl alcohol. Next, a grease is prepared for the mandrel - a glass rod with a diameter of 4 mm: 5 mg of vacuum grease is added to 1 ml of chloroform and dissolved in a water bath at 50 ° C. A dry glass stick with a diameter of 4 mm, fat-free with alcohol, is immersed in the lubricant for 20 seconds and then dried in air at room temperature for 30 minutes. A greased glass rod is used as a guide to form the tube. The guide is immersed in a ZhMA / FMA / TPO mixture for 7 seconds, removed from the solution and irradiated in the light of a UV lamp (36 W power) for 2 minutes, rotating the guide at a speed of 15 rpm. Repeat the immersion-exposure procedure 4 times. To reinforce the ends of the tube using a surgical untwisted silk thread soaked in a solution of FMA / FMA / TPO. 10 turns of thread are evenly laid on the end sections of the tube 2.5 mm long, departing from one edge of 2 mm and 7.5 mm. Thus, a workpiece is obtained with the following parameters: the length of the reinforced sections is 2.5 mm, the length of the unreinforced section is 3 mm. The workpiece is again immersed in the ZhMA / FMA / TPO solution for 5 seconds, removed and rotated at a speed of 15 rpm, exposed in the light of a UV lamp (36 W power) for 2 minutes. Repeat the procedure 4 more times. The formed tube on the mandrel is transferred to distilled water for 1 hour, then 12 hours are kept in 96% ethanol and another 1 hour in distilled water. The resulting tube is removed from the guide, the inner surface is treated with chloroform to remove grease residues and transferred to 96% ethanol for storage. The tube thus formed is shown in FIG. 1.

Example 2

Correction of the anastomosis with a bioresorbable tube

A bioresorbable tube with the following parameters: diameter - 4 mm, length of the unreinforced section - 3 mm, length of the reinforced sections - 2.5 mm, wall thickness - 300 μm, washed from ethanol: transfer the tube from 96% ethanol to sterile saline solution, incubated at 37 ° C for 20 minutes, replace the saline with fresh and stand the tube in it for another 30 minutes. The procedure is repeated 3 times. Wistar rats weighing 190-230 g begin to prepare for surgery 2 days before surgery. 2 days before the operation, animals are deprived of solid food and given them a 10% glucose solution without restriction (per os). The operation is performed under general anesthesia (Zoletil + Xylazine), sedation atropine sulfate at a dose of 0.04 mg / kg, subcutaneously 15 minutes before the start of surgery. The animals are fixed in the supine position. At the beginning of the operation, ceftriaxone is administered intramuscularly at the rate of 100 mg / kg body weight. The surgical field is limited by sterile gauze napkins, a laparotomy is performed. A section of the small intestine (7 cm distal to the stomach) is removed into the surgical wound, after which an incision is made from the mesenteric edge of the intestine to half the diameter of the intestinal tube, a sterile tube is inserted into the intestinal lumen and the implant is hermetically fixed with an implant using a surgical thread (polyester, 4/0 ) Then, at the implantation site, a 1 mm section of the intestinal tube is removed. The intestine is placed in the abdominal cavity and layered cavity suturing is performed. Within 4 days after the operation, daily injections of ceftriaxone are performed at a rate of 50 mg / kg body weight. Within 2 days, animals receive a 10% glucose solution, do not receive solid food, on the third day they are transferred to a standard diet. As a control, animals are used that are used to suture the anastomosis using a surgical thread (polyester, 4/0). An anastomosis closed by a bioresorbable tube is shown in FIG. 2. On the second day after surgery, 70% of the animals in the control group died, on the third day another 30% died. Thus, the mortality rate in the control group was 100%. At autopsy of the animals of the control group, characteristic signs of peritonitis were observed, the anastomosis is insolvent. The survival rate of animals from the experimental group was 100%. 2 weeks after the correction of the anastomosis with a bioresorbable tube, the animals were removed from the experiment. In animals of the control group, 2 weeks after the operation, a pronounced adhesive process and insolvency of the anastomosis were not observed. The intestinal state in the area of surgery after 2 weeks is shown in FIG. 3. Thus, the use of bioresorbable tubes described in the present invention ensures the viability of the anastomosis.

Claims (19)

1. A method of obtaining a bioresorbable tube based on methacrylated gelatin and methacrylated fibroin, comprising the following steps: a) dissolution of macromonomers in hexafluoroisopropanol: methacrylated gelatin (LMA) at the rate of 10 mass. % and methacrylated fibroin (FMA) at the rate of 4 mass. % at 55 ° C ± 5 for 18-36 hours; b) obtaining a mixture comprising equal parts of solutions of FMA and FMA and the photoinitiator diphenyl (2,4,6-trimethylbenzoyl) phosphine at the rate of 5 wt. % by weight of macromonomers; c) preparation of the guide for forming the tube by degreasing, followed by drying of the surface of the glass rod with a circular cross section with a diameter of 2-15 cm; d) preparation of the reinforcing yarn by keeping the silk untwisted yarn 8 / 0-4 / 0 in the mixture obtained in step b); d) the formation of the first layers of the bioresorbable tube on the guide by immersing it in the mixture obtained in step b) for 5-10 seconds, followed by rotation and exposure of the resulting workpiece in the light of an ultraviolet lamp for at least 2 minutes; f) reinforcing the end sections of the tube with thread by laying the thread in turns at a distance of 2-5 mm from the ends of the formed tube onto the workpiece obtained in step e); g) the formation of the outer layers of the bioresorbable tube by immersing the workpiece obtained in step e) in the mixture obtained in step b) for 5-10 seconds, followed by rotation and exposure of the obtained tube in the light of an ultraviolet lamp for at least 2 minutes; h) treatment of the formed tube by aging in distilled water for 1-1.5 hours, followed by aging in 96% ethanol for 12-18 hours and repeated aging in distilled water for 1-1.5 hours, processing by internal the surface of the formed tube with chloroform. 2. The method according to p. 1, characterized in that the dissolution of the PMA and PMA is carried out for 24 hours at 50 ° C. 3. The method according to p. 1, characterized in that the guide has a diameter of 4 mm 4. The method according to p. 1, characterized in that in step e) the guide is kept in the mixture for 7 seconds, exposed in the light of a 36 W UV lamp for 2 minutes, rotating the guide at a speed of 15 rpm. 5. The method according to claim 1, characterized in that in step e) 10 turns of thread are laid evenly on the end sections of the tube 2.5 mm long, with a deviation of 2 mm and 7.5 mm from the end of the tube. 6. The method according to p. 1, characterized in that in step h) the formed tube is kept in distilled water for 1 hour, then 12 hours are kept in 96% ethanol and another 1 hour in distilled water. 7. The method according to p. 1, characterized in that the formed tube has the following parameters: tube wall thickness: from 150 μm to 700 μm, the length of the reinforced sections 2-15 mm, the length of the unreinforced section: 1-6 mm, the number of reinforced sections: 2, the location of the reinforced section: along the edges of the tube at a distance of 1-6 mm from the edge, inner diameter: from 2 mm to 15 cm. 8. The method according to p. 1, characterized in that step d) is repeated 4-6 times depending on the number of layers being formed. 9. The method according to p. 1, characterized in that the degreasing of the guide lead 70% ethanol. 10. The method according to p. 1, characterized in that the density of the turns of the reinforcement is 10-15 turns per 3 mm tube. 11. The method according to p. 1, characterized in that step g) is repeated 4-20 times depending on the number of layers being formed.

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