CN213910722U - Artificial biological biliary tract - Google Patents

Abstract

The utility model provides an artificial biological biliary tract, which has a length of 1-5cm, a thickness of 0.2-1.6mm and a diameter of 0.6-2.6 cm. The artificial biological biliary tract can be used for replacing the damaged biliary tract in the operation so as to solve the repair or reconstruction of the biliary tract and realize the purpose of surgical treatment of biliary tract injury.

Description

Artificial biological biliary tract

Technical Field

The utility model relates to the technical field of medical equipment, concretely relates to artificial biological biliary tract.

Background

With the development of laparoscopic cholecystectomy and living liver transplantation, the incidence of biliary tract injury is as high as 0.3% -0.7%. At present, the main treatments for biliary tract injury are: firstly, the end of the biliary tract is anastomosed; secondly, performing chole-intestinal anastomosis; (iii) hepatectomy; liver transplantation and bile duct substitute. Although the end-to-end biliary anastomosis is most applied, the incidence rate of biliary tract stenosis after the operation is still as high as 20% -50%. The chole-intestinal anastomosis is one of common operation modes for treating biliary tract injury, is simple to operate, cuts off the injured bile duct, and performs choledochodystic jejunum end-side anastomosis. However, the operating mode changes the physiological anatomical pathway, the sphincter of Oddis is in open position, the adverse flow of intestinal contents and the infection of biliary tract are easy to occur, and secondary biliary cirrhosis and portal hypertension finally cause liver failure and endanger life. The operation is complex, the wound is large and the application is less in clinic of liver resection and liver transplantation.

The artificial biliary tract for implantation can replace the damaged biliary tract, and is matched with normal biliary tracts on two sides of the damaged biliary tract at the end, so that the physiological function of the sphincter of Oddis is completely kept, and the recurrent cholangitis caused by intestinal bacteria entering the biliary tracts is prevented. Except for being applied to the biliary tract injury aspect, when the difference between the pipe diameters of a donor bile duct and a recipient bile duct is large in the auxiliary liver transplantation, the forced end-to-end anastomosis of the biliary tract is faced with the complication of anastomotic fistula caused by overlarge anastomotic tension, and the artificial biliary tract with different pipe diameters (the pipe diameters of the upper end and the lower end are not consistent) can effectively solve the problem. The research and development of the artificial biliary tract become the best method for solving the problem of biliary tract injury. Therefore, the development of artificial biliary tracts is still in the way of animal experiments due to limitations in biocompatibility, material compliance and the like.

In 2002, Nstor A.G Yao mez opened up a precedent of artificial biliary tract. The reinforced artificial blood vessel made of tetrafluoroethylene puffed perfluoroethylene propylene copolymer material is used as an artificial biliary tract. The experimental animals after operation have cholema and a plurality of long-term complications. Zhengjiawei et al found that the intimal hyperplasia implant at the anastomotic site was displaced 3 months after implantation using an artificial Gore-Tex blood vessel for reconstruction of the common carotid artery as an artificial biliary tract substitute. In 2007, Susumus in Japan succeeded in animal experiments with a composite material made of collagen sponge and polypropylene. However, the flexibility and compliance of the material are poor, and the polypropylene promotes fibrosis to cause long-term serious complications, so far, the material cannot be applied to clinic. In 2011, Peter Nau and the like select and utilize an absorbable material mainly comprising polyhydroxy acid polymer and trimethylene carbonate to repair common bile duct in the process of exploring a new method for reconstructing extrahepatic biliary tract, and serious complications such as biliary obstruction and the like occur in half of experimental animals. In 2012, copolymers made from polycaprolactone and polylactic acid, such as Mitsuo Miyazawa in Japan, and absorbable pipelines reinforced by polylactic acid fibers, are used as artificial biliary tract substitutes, and the copolymers are primarily successful in animal experiments, and cannot be clinically used due to poor mechanical strength. In 7 months in 2012, a drumbeat hospital affiliated to the medical college of Nanjing university, a patch (a built-in plastic stent support) made of a mixed biomaterial of fibroblast growth factor (bFGF) and collagen membrane extracted from cow skin is used for animal experiments for repairing biliary tract defects (2cm, two-thirds biliary tract wall defects) of the breeding pigs, and the animal experiments are initially successful. However, the preparation method is complicated, the preoperative operation requires the soaking of physiological saline, and the patients in the operation need to prepare the tubular biliary tract by self, and the like, and the preparation method cannot be used clinically. The 5 th month Alonso in 2012 applies three-dimensional collagen to prepare degradable artificial biliary tracts, and the three-dimensional collagen is successful in animal experiments. However, the preservation of the finished product requires a humid incubator which is kept at 37 ℃, and further long-term observation and animal experiments similar to human anatomy are lacked. Some scholars successfully apply the autologous venous blood vessels to manufacture the artificial biliary tract in rat animal experiments, but the artificial biliary tract can not be applied due to the problems of complex manufacture, different sizes of the blood vessels and the like. The scholars also adopt the gastric wall with the vascular pedicle at the greater curvature of the stomach to prepare a tubular structure to replace the biliary tract, but the method is very complicated and has larger side injury.

SUMMERY OF THE UTILITY MODEL

In view of this, the present invention provides an artificial biological biliary tract, which can replace the damaged biliary tract by an operation, and repair or reconstruct the biliary tract to solve the problems of biliary tract injury, etc.

In order to realize the aim, the utility model provides an artificial biological biliary tract, which has the length of 1-5cm, the thickness of 0.2-1.6mm and the diameter of 0.6-2.6 cm.

Preferably, the artificial biological biliary tract is an animal-derived tube.

Preferably, the animal-derived tube is a ureter of a ruminant.

Preferably, the animal-derived tube is a bovine ureter, a equine ureter, a donkey ureter or a sheep ureter.

Compared with the prior art, the utility model discloses following beneficial effect has:

1. the utility model discloses broken through the bottleneck of artifical biological biliary tract material selection and immunogenicity, developed immune compatibility good, in the bile environment stable for a long time and be convenient for the anastomotic artificial biological biliary tract of operation for solve biliary tract repair or biliary tract reconstruction through the impaired biliary tract of operation replacement and become the new direction of research of surgery treatment biliary tract damage.

2. The utility model discloses adopt the ox ureter as raw and other materials for the first time, experiment trial in advance is succeeded through preliminary dog animal model, and the pathology proves that artificial biological biliary tract has flesh layer tissue regeneration and endothelial cell to spread into, does not see rejection. The application of the choleintestinal anastomosis is greatly reduced by replacing the damaged biliary tract, the treatment principle of biliary tract injury is overturned, the conventional treatment guidelines are even changed, the application prospect is very wide, and the good news can be brought to thousands of patients with biliary tract injury.

3. The utility model discloses an animal experiment confirms that artifical biological biliary tract immunity compatibility is good, and when human self biliary tract is impaired, utilize this biological biliary tract to replace impaired biliary tract with the anastomotic mode of end, can remain oddis sphincter, prevent palirrhea cholangitis, the material compliance is good, is close human self tissue, is favorable to the art to coincide to the emergence of postoperative complications such as prevention cholangitis and can effectively treat biliary tract complications such as biliary tract injury.

Drawings

FIG. 1 is a schematic structural view of an artificial biological biliary tract provided by the present invention;

FIG. 2 is a graph of artificial biological biliary HE staining X200 after being implanted in an animal for 1 year according to example 2 of the present invention;

FIG. 3 shows the staining of artificial biological biliary CK-19 1 year after the implant of example 2 of the present invention into animals.

Fig. 4 shows Masson staining of an artificial creature 1 year after implantation in an animal, according to example 2 of the present invention.

Fig. 5 is a biliary tract angiography picture 1 year after the implantation of the embodiment 2 of the present invention into an animal.

Detailed Description

The present invention provides an artificial biological biliary tract, and the following embodiments are provided to explain the present invention, it should be understood that the embodiments described herein are only for explaining the present invention, and are not intended to limit the present invention.

As shown in fig. 1, an artificial biological biliary tract 10, which is a bovine ureter, can also be derived from other ruminant animals such as horse, donkey or sheep ureters, but is preferably a bovine ureter in specific implementations; removing tissue protein, nucleic acid and lipid molecules by decellularization, tissue crosslinking and anti-calcification treatment, further removing immunogenicity of tissue, making into tissue with length of 1-5cm, thickness of 0.2-1.6mm and diameter of 0.6-2.6cm, and performing xenotransplantation. For example, in practice, considering the size of the biliary tract of a patient, an artificial biological biliary tract having a length of 2cm, a thickness of 0.2mm, and a diameter of 0.6cm can be prepared.

The decellularization treatment is preferably performed by a chemical decellularization treatment. For example, fresh tubular tissue of animal origin is used as a starting material, and cells or cell fragments in the tissue are removed by a detergent, such as Triton-X-100 or Sodium Dodecyl Sulfate (SDS), preferably Triton-X-100, at an action concentration of 0.1-10.0 wt%, preferably 2-5 wt%, for an action time of 12h-6d, preferably 12h-72 h; the above-mentioned decellularization method is preferably carried out under conditions such as shaking and stirring. In specific implementation, the fresh tubular tissue of animal origin is added with 2-5 wt% of Triton X-100 Tris-HCl buffer solution (pH7.5) at 4 ℃, and the shaking is continued for 24-72 h to remove cells.

The tissue cross-linking and anti-calcification treatment is to perfuse a tissue preservation solution, such as 0.25-0.5 wt% glutaraldehyde solution or isotonic Hank's solution, preferably 0.4 wt% glutaraldehyde solution, into the lumen to better enhance histocompatibility and facilitate the growth of intimal cells. Fastening two ends of the pipeline, and then performing tissue crosslinking and hydroxychrome anti-calcification treatment for 2h-5 days at 10-60 ℃ and pH 2.5-8.5 by using glutaraldehyde solution with the weight percent of 0.20-2.0 of a crosslinking agent so as to ensure that the tissue thermal shrinkage temperature is more than or equal to 82 ℃; in specific implementation, two ends of the pipeline are fastened, and tissue crosslinking and hydroxychromium anti-calcification treatment is carried out for 2 days at 50 ℃ and pH 6 by 0.5 wt% of glutaraldehyde solution of a crosslinking agent, so that the tissue thermal shrinkage temperature reaches 82 ℃.

Wherein, after the step of perfusing the tissue preservation solution into the tube cavity and before the step of fastening the two ends of the pipeline, the method also comprises a shaping step; the shaping step comprises the following steps: adding a tubular shaping support capable of changing the diameter and the wall thickness of the pipeline into the tubular shaping support after the tissue preservation solution is poured into the lumen; the plastic brace is made of glass, plastic or metal which is transparent and does not react with the tissue preservation solution in the lumen, for example, the plastic brace can be a plastic tube with the outer diameter of 0.6-3.0cm and the thickness of 0.1-0.3cm, preferably a plastic tube with the outer diameter of 2cm and the thickness of 0.2cm, and has wide sources and low cost.

The invention is further described below with reference to specific embodiments.

EXAMPLE 1 preparation of Artificial biological biliary tract

Obtaining materials of the artificial biological biliary tract:

selecting a 1-3 year old bovine ureter with a pipe diameter close to that of a human body, taking renal pelvis parts to bladder parts of the bovine ureters at two sides within 30 minutes of separation in a slaughterhouse, and placing the bovine ureters in an isotonic sodium chloride solution for preservation.

The reason why the bovine ureter is adopted as the raw material of the artificial biological biliary tract is that: 1. both have a three-layer structure of mucosa, muscle layer and adventitia. The main difference is the fine bile duct epitheliumThe cells are columnar epithelium, while the ureters are transitional epithelium. 2. Urine and bile have slightly different pH values and components, but both are in a sterile internal environment. 3. Both have the ability to spontaneously peristalsis, and both have an autonomic phenomenon of smooth muscle. 4. Most importantly, the physiological pressure born by the two is similar, the ureteral pressure: 7-13cmH₂O, and common bile duct physiological pressure is 12cmH₂And O. 5. Both are similar in diameter, wall thickness and compliance.

Using fresh tubular tissue of animal origin as raw material, removing cell components by continuously shaking with 1 wt% Triton X-100 at 4 deg.C for 48h, centrifuging at 7000rpm, and repeatedly washing with distilled water to remove cells or cell debris in the tissue; then, a tissue preservation solution, namely 0.4 wt% glutaraldehyde solution, is perfused into the lumen to enhance histocompatibility and facilitate the growth of intimal cells. And the two ends of the pipeline are tightened, and then the tissue cross-linking and the hydroxychromium anti-calcification treatment are carried out for 2 days at 50 ℃ and pH 6 by 0.5 wt% of glutaraldehyde solution of a cross-linking agent, so that the tissue thermal shrinkage temperature reaches 82 ℃. Wherein, after the step of perfusing the tissue preservation solution into the tube cavity and before the step of fastening the two ends of the pipeline, the method also comprises a shaping step; the shaping step comprises the following steps: adding a tubular shaping support capable of changing the diameter and the wall thickness of the pipeline into the tubular shaping support after the tissue preservation solution is poured into the lumen; the shaping brace is a plastic tube which is light-permeable and does not react with tissue preservation solution in the tube cavity, the outer diameter is 2cm, and the thickness is 0.2 cm.

Immune compatibility of the artificial biological biliary tract:

the first problem to be solved is the immunity problem of the artificial biological biliary tract developed by using animal-derived tissues. The bovine ureter can be subjected to decellularization, tissue crosslinking and anti-calcification treatment to remove tissue protein, nucleic acid and lipid molecules, thereby achieving the purpose of removing immunogenicity of the tissue. The animal implantation experiment survives for 5 months, and then the autopsy is carried out, the pathological condition shows the regeneration of the muscular layer tissue of the artificial biliary tract, endothelial cells are laid in, a large amount of lymphocytes and inflammatory cells are not infiltrated in the artificial biliary tract, and rejection reaction is not generated.

Example 2 animal model establishment and animal experiments

1. The experimental animals are bred by using 12 Bama miniature pigs with age of 1-2 years, weight of 30-40kg, injured extrahepatic biliary tract replacement end-to-end anastomosis, and miniature pigs fed with feed (special for feed of Australian feed Co., Ltd. of Beijing Ke) for three meals a day.

2. Animal anesthesia: the trachea cannula is full anesthesia. Antibiotic impact: ceftriaxone (Sichuan Colon pharmaceutical), 2g intravenous plus 0.9 wt% sodium chloride 100ml preoperative ballistic.

3. The operation scheme is as follows:

the gallbladder is exposed, and the visceral membrane of the gallbladder attached to the liver is cut off with surgical scissors. Separating the cystic duct and the common bile duct. Ligating cystic duct and gallbladder artery. (if the gallbladder is not cut off, the probability of retrograde cholecystitis after operation is high and the operation formula is inconsistent with the clinic) the position of the common bile duct is confirmed by attention. After circularly cutting off 2cm biliary tract, the artificial biliary tract is treated with absorbable suture of 5, 6-0. The artificial biological biliary tract is anastomosed with the broken ends of the upper and lower common bile ducts by simple intermittent eversion suture.

4. And (3) postoperative management: after operation, ceftriaxone (2g +0.9 wt% sodium chloride 100ml) is stood for 3 days, water is forbidden within 12 hours, and fasting is performed within 24 hours. Biochemical indexes such as liver function, blood amylase and the like of the animals are detected conventionally and analyzed, and obvious abnormality is not found in the conventional blood, liver function and amylase of all the tested animals within 1 week, 1 month and 3 months. The incision was changed 1 time every 3 days. The stitches were removed after 12 days.

After 1 year, images and pathology prove that the test animals have no complications such as biliary calculus, gall sediment deposition, biliary tract infection and the like, the patency is good, no rejection reaction exists, the imaging result (figure 5) indicates that the common bile duct has no stenosis, no calculus is found in the common bile duct, a large amount of lymphocytes and inflammatory cell infiltration are not found in pathology HE staining and CK-19 staining (figures 2 and 3), no rejection reaction is found, the regeneration of CK-19 epithelium is expressed, and excessive proliferation of fibrous tissues is not found in Masson staining (figure 4).

5. Detection of postoperative indexes: 6 animals in the experimental group were dissected respectively 6 months and 12 months after the operation, and no biliary nodules were confirmed by forward-dissecting cholangiography.

6. Post-anatomical histological analysis: 1) the immune histochemistry of the HE stain proves that the near-end and far-end anastomotic stoma of the artificial biliary tract and the biliary tract body have immune rejection and inflammatory reaction, and proves that the artificial biliary tract has no rejection reaction. 2) MASSON trichrome staining analysis was performed on both ends of the anastomotic stoma and the body of the artificial biliary tract, confirming that there was no fibrous tissue hyperproliferation. 3) Detecting the growth and the pavement of the CK-19 expressing epithelial cells in the biliary tract. 4) After being soaked in physiological saline for ten minutes, the mechanical strength thereof was measured by a tensile tester (Tinius, Olsen, Norway) to confirm that the mechanical strength was good.

The animal experiment adopts the end-to-end anastomosis of the upper common bile duct, the artificial biliary tract and the lower common bile duct. In the operation, a 2cm common bile duct is annularly cut, and common bile duct-artificial bile duct end-end anastomosis is performed by using the artificial bile duct. The postoperative experimental dog recovered well. The upper and lower anastomoses in this animal experiment were stained in fig. 2 below. As shown in figure 2, the artificial biological biliary tract HE staining is multiplied by 200, so that the spreading of the endothelial cells of the artificial biliary tract can be obviously seen, a large amount of infiltration of lymphocytes and inflammatory cells is not seen in the artificial biliary tract, and no obvious rejection reaction is generated. FIG. 3 shows that the regeneration of CK-19 endothelial cells is expressed, the regeneration of subcutaneous tissue capillaries is obvious, and the blood supply around the anastomotic stoma is good. FIG. 5 shows that after the implant is implanted into the animal body for 1 year, no stenosis exists in the extrahepatic biliary tract, and no calculus is formed in the extrahepatic biliary tract. FIG. 4 shows that no excessive fibroplasia is observed in the biliary tract of the artificial creature. The pathological section result fully shows that the artificial biological biliary tract can be kept stable in a bile environment for a long time and in an intestinal tract bacteria environment, has no rejection reaction, has the endothelial cells for expressing CK-19 laid in, has no excessive proliferation of fibrous tissues and shows wide application prospect.

The utility model has the advantages of it is following:

1. the utility model breaks through the bottleneck of drawing materials and immunogenicity of the artificial biological biliary tract, and develops the artificial biological biliary tract which has good immune compatibility, is stable in the bile environment for a long time and is convenient for operation anastomosis.

1) The animal-derived materials of the artificial biliary tract are obtained: because the biliary tract has certain similarity with the ureter in the histology and embryology, the three-layer structure is adopted, the diameter and the thickness of the tube wall are similar, and the most important is that the pressure born by the biliary tract and the ureter is similar, so the selection and the preparation of the raw materials of the artificial biliary tract are the main problems to be solved by the utility model.

2) Chemical modification studies to address immune compatibility: the removal of immunogenicity of bovine ureteral tissue by chemical modification is key to achieving its long-term implantable. The utility model discloses a technique such as Triton-X-100 PBS cell-free method and change cross-linking degree has also solved the immune compatibility problem.

2. The utility model discloses an animal experiment confirms that artificial biological biliary tract immunity compatibility is good to confirm that artificial biological biliary tract is convenient for the operation and coincide, can remain advantages such as oddis sphincter, for solving the best scheme of biliary tract damage problem.

1) Animal experiments and postoperative sample analysis prove that the artificial biological biliary tract has good immune compatibility. The perioperative complications, postoperative pathology and immunological indexes prove that no rejection reaction and other complications occur after the implantation of the artificial biological biliary tract, and the postoperative pathology and immunohistochemical experiments prove that no rejection reaction occurs.

2) The animal experiment postoperative complication comparison and specimen analysis prove that the bioartificial biliary tract treatment on the biliary tract injury is superior to the choleintestinal anastomosis. Because the artificial biological biliary tract retains the function of oddis sphincter, the occurrence of complications such as cholangitis, biliary cirrhosis and the like can be effectively reduced. The appearance of the artificial biological biliary tract can avoid adopting a biliary-enteric anastomosis method. The occurrence of the artificial biliary tract can lead a clinician to completely cut off the narrow and broken biliary tract and replace the narrow and broken biliary tract with the artificial biliary tract, thereby thoroughly solving the complications such as biliary fistula and the like finally caused by biliary tract stenosis.

Finally, it should be noted that: the above-mentioned embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention.

Claims (1)

1. An artificial biological biliary tract is characterized in that the length of the artificial biological biliary tract is 1-5cm, the thickness is 0.2-1.6mm, and the diameter is 0.6-2.6 cm; the artificial biological biliary tract is a bovine ureter, a equine ureter, a donkey ureter or a sheep ureter.

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