CN119680005A

CN119680005A - Decellularized tissue engineered ureter and preparation method thereof

Info

Publication number: CN119680005A
Application number: CN202411948252.7A
Authority: CN
Inventors: 邱雪峰; 郭莹; 王博
Original assignee: Haimai Medical Technology Suzhou Co ltd
Current assignee: Haimai Medical Technology Suzhou Co ltd
Priority date: 2024-12-27
Filing date: 2024-12-27
Publication date: 2025-03-25

Abstract

The invention discloses a decellularized tissue engineering ureter and a preparation method thereof, wherein the preparation method of the decellularized tissue engineering ureter comprises the steps of taking a nickel-titanium alloy bracket as a supporting layer, fixing a polymer tubular bracket on the supporting layer, wherein the density of the polymer tubular bracket is 100-1000mg/cm ³, the thickness of the polymer tubular bracket is 50-5000 mu m, planting seed cells on the supporting layer and the polymer tubular bracket, culturing in a bioreactor to obtain a tissue engineering ureter, the wall thickness of the tissue engineering ureter reaches 50 mu m-2mm, removing cells from the obtained tissue engineering ureter, enabling DNA to be smaller than 100ng/mg dry weight, enabling alpha-Gal clearance to be more than 90%, performing crosslinking treatment through a crosslinking agent, and sterilizing to obtain the decellularized tissue engineering ureter, wherein the crosslinking agent is glutaraldehyde and/or epoxide 1, 4-butanediol diglycidyl ether.

Description

Acellular tissue engineering ureter and preparation method thereof

Technical Field

The invention relates to the technical field of medical instruments, in particular to a cell-free tissue engineering ureter and a preparation method thereof.

Background

Ureteral stenosis is a common disorder of urinary surgery, and is mainly divided into primary and secondary, with secondary ureteral stenosis being most common with iatrogenic injuries, mainly by surgical treatment. Surgical treatment of ureteral stenosis is challenging because the proximal ureter is fed by branches inside the renal artery, the medial ureter is fed by branches posterior to the common iliac artery, the distal ureter is fed by branches outside the superior bladder artery, and the three vessels are vulnerable to blood supply, which is prone to interruption and thus to ureteral ischemic necrosis. The repair of ureteral stenosis is therefore a challenge for urologists.

In recent years, ureteral stenosis surgical modes are endless, so that the choices of urologists are more diversified. Ureteral stenosis is mainly a lumen stenosis, so that relieving obstruction and unobstructed ureter is beneficial to relieving patient symptoms and improving the life quality of a patient. Among them, there are many options for ureteral stenosis repair, such as endoluminal surgery, ureteroplasty of the bladder wall muscle flap, and autologous patch treatment, however, the above treatment methods have drawbacks and cannot completely solve the patient's needs, so there is no optimal method for treating ureteral stenosis or injury at present.

Taking endoluminal surgical treatment as an example, endoluminal surgical treatment includes balloon dilation, and ureteral stents. Wherein, the balloon dilation treatment of ureteral stenosis is to use a balloon which is evenly dilated to open a section of ureter of the stenosis so as to achieve the purpose of treatment. For ureteral stenosis less than 2cm, the endo-balloon dilation under the ureteroscope is feasible, and a plurality of clinical research results show that the balloon dilation under the ureteroscope direct vision can effectively promote the treatment effect of secondary ureteral stenosis, can not obviously influence postoperative rehabilitation efficiency, postoperative complication are few. However, the method has better effect of treating the short-segment ureteral stenosis, and has higher incidence of operation failure for the long-segment ureteral stenosis. Ureteral balloon dilation is prone to mechanical forces or ischemic injury, which can lead to further ureteral injury, leading to stenosis.

The ureteral stent is placed differently from other methods for treating ureteral stenosis under an endoscope, and the ureteral stent implantation can effectively relieve the acute obstruction of the urinary tract, so that the use of the ureteral stent is not influenced in a narrow range. The Allium stent on the market can effectively relieve ureteral stenosis, relieve adverse reactions such as hydronephrosis, bladder irritation, somatic pain and the like, has little urinary tract infection and low invasiveness, does not need external drainage for patients, and improves the life quality of the patients. However, the current Allium stent has high cost, and stones and infection are formed, so that the clinical application of the Allium stent is limited. One of the main problems of the long-term stent placement of the ureteral stent is that the probability of bacterial colonization in the stent is high, and drug-resistant bacterial groups are easy to form, so that the ureteral stent cannot be permanently placed. It must be removed or replaced at a specific time. When the ureteral stent is taken out, the ureteral stent needs to be carried out in a hospital, so that the patient is uncomfortable to body, the urethra is damaged, and the economic burden of the patient is increased. Although the manner of indwelling ureteral stents is relatively convenient, the biggest problem is that ureteral narrowing is not relieved from the root, and most of them are preventive or palliative treatments.

Taking the example of urinary surgery, the urinary surgery includes ureteral anastomosis and ureteroplasty of the bladder wall muscle flap. Ureteral anastomosis is a relatively simple surgical procedure for repairing ureteral lesions, short stenoses can be repaired by a urinary anastomosis, and longer stenoses may not be possible to achieve tension-free anastomosis. The length and location of the ureteral stenosis determines the feasibility of ureteral anastomosis. There is a certain risk after ureteral end anastomosis, and there may be insufficient blood supply after the operation to cause anastomotic stenosis or anastomotic obstruction, thereby causing hydronephrosis and damage to renal function.

The ureteroplasty of the wall muscle valve of the bladder uses the wall muscle valve of the bladder with the pedicle in the related clinical study, spirally winds the bracket, reserves the upper bladder artery of the branch of the bracket, is anastomosed to the proximal end of the ureter and is used for reconstructing the defect of the full-length ureter, the patient has mild bladder stimulation symptoms and lower urinary tract symptoms, the patient does not have treatment related complications such as urine leakage, renal colic, high fever and the like during the perioperative period, obvious hydronephrosis and anastomotic stenosis are not seen after the operation, and no immunological rejection reaction is caused. However, when the ureter is narrowed beyond 15cm, it is difficult to ensure tension-free repair.

Taking autologous patch treatment as an example, autologous patch treatment includes ileal appendicular ureteroplasty. The ileum and appendiceal ureteroplasty adopts the ileum to replace ureters, has the advantages that long sections of ureters and even full-length ureters can be rebuilt, and clinical research results show that most patients with ureter replacement in the ileum, the colon or the appendiceal sections have good comprehensive conditions after operation, no ureter expansion and effusion are seen, and the ureter can be safely and successfully used for treating the ureter stenosis to cause light and medium renal function loss, and the renal function of most cases is maintained or improved in the long term.

However, due to the particularity of the intestinal canal, the surgical mode is easy to cause electrolyte disturbance, metabolic acidosis, ureteral dilatation and other complications, and also easy to cause anastomotic leakage, thereby causing urine extravasation, and long-time extravasation causes scar fibrosis and hyperplasia around the intestinal wall, and then local stenosis recurrence is caused.

Disclosure of Invention

The invention mainly aims to provide a preparation method of a cell-free tissue engineering ureter and a preparation method thereof, and aims to solve the problems.

In order to achieve the above object, the present invention provides a method for preparing a ureter by decellularized tissue engineering, which comprises:

Taking a nickel-titanium alloy bracket as a supporting layer, and fixing a polymer tubular bracket on the supporting layer, wherein the density of the polymer tubular bracket is 100-1000mg/cm ³, and the thickness is 50-5000 mu m;

planting seed cells on the supporting layer and the polymer tubular bracket, and culturing in a bioreactor to obtain a tissue engineering ureter, wherein the thickness of the wall of the ureter reaches 50 mu m-2mm;

And (3) removing cells from the obtained tissue engineering ureter to enable DNA to be smaller than 100ng/mg dry weight and the alpha-Gal clearance to be more than 90%, and performing crosslinking treatment and sterilization through a crosslinking agent to obtain the cell-removed tissue engineering ureter, wherein the crosslinking agent is glutaraldehyde and/or epoxide 1, 4-butanediol diglycidyl ether.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the step of taking the nickel-titanium alloy stent as a supporting layer and fixing the polymer tubular stent on the supporting layer, wherein the polymer tubular stent covers the surface of the supporting layer and is fixed by a surgical suture, or

The supporting layer is arranged in the middle of the pipe wall of the polymer tubular bracket.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the step of taking the nickel-titanium alloy stent as a supporting layer and fixing the polymer tubular stent on the supporting layer is carried out, wherein the polymer tubular stent is prepared by using one or more of PCL, PGA, PLA, PGA-LA and collagen biodegradable materials.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the step of using the nickel-titanium alloy stent as a supporting layer and fixing the polymer tubular stent on the supporting layer is performed by one or more processes of 3D printing, electrostatic spinning, solution casting, near-field direct writing and braiding.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the step of using a nickel-titanium alloy stent as a supporting layer and fixing a polymer tubular stent on the supporting layer is performed, wherein the radial direction of the polymer tubular stent after the polymer tubular stent is fixed on the supporting layer is circular or elliptical;

When the radial direction of the polymer tubular bracket after being fixed on the supporting layer is circular, the diameter is 0.8-5mm;

When the radial direction of the polymer tubular bracket fixed on the supporting layer is elliptical, the long axis is 0.8-7mm, and the short axis is 0.5-2mm.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the obtained tissue engineering ureter is subjected to cell removal treatment to ensure that the DNA is less than 100ng/mg dry weight and the alpha-Gal clearance rate is more than 90%, and is subjected to crosslinking treatment and sterilization by a crosslinking agent, so that the obtained decellularized tissue engineering ureter has large inner diameters at two ends and small inner diameter in the middle, wherein the inner diameters of the two ends are 1-10mm, and the inner diameter in the middle is 0.5-8mm.

Preferably, in the preparation method of the decellularized tissue engineering ureter, the obtained tissue engineering ureter is subjected to cell removal treatment, so that the DNA is less than 100ng/mg dry weight and the alpha-Gal clearance rate is more than 90%, and the obtained tissue engineering ureter is subjected to crosslinking treatment and sterilization by a crosslinking agent, wherein the inner diameter of the obtained decellularized tissue engineering ureter is the same, and the inner diameter is 1-10mm.

Preferably, in the preparation method of the decellularized tissue engineering ureter, after the supporting layer and the polymer tubular stent are planted with seed cells, the seed cells are cultured in a bioreactor to obtain the tissue engineering ureter, and the wall thickness of the seed cells reaches one or more of humanized vascular smooth muscle cells, ureter smooth muscle cells and fibroblasts.

Preferably, in the preparation method of the decellularized tissue engineering ureter, when the decellularized tissue engineering ureter is used as a ureteral stent to be placed at a ureter stenosis part through a conveyor, the wall thickness of the decellularized tissue engineering ureter is 50-100 μm, or

When the decellularized tissue engineering ureter is used as a partial or full segment replacement of the ureter and is anastomosed with an autologous ureter, the thickness of the wall of the decellularized tissue engineering ureter is 100-200 um.

In order to achieve the above purpose, the invention also provides a decellularized tissue engineering ureter, which is prepared by adopting the preparation method of the decellularized tissue engineering ureter.

Preferably, in the decellularized tissue engineering ureter, after the decellularized tissue engineering ureter is implanted into a body, a lumen of the ureter is endothelialized, a fibroblast is attached to an outer membrane of the ureter, and a wall of the ureter is re-cellularized by smooth muscle cells, so that a nascent ureter with a nickel-titanium alloy stent support is formed.

The invention has at least the following beneficial effects:

The preparation method of the decellularized tissue engineering ureter comprises the steps of taking a nickel-titanium alloy bracket as a supporting layer, fixing a polymer tubular bracket on the supporting layer, wherein the density of the polymer tubular bracket is 100-1000mg/cm ³ and the thickness of the polymer tubular bracket is 50-5000 mu m, planting seed cells on the supporting layer and the polymer tubular bracket, culturing in a bioreactor to obtain the tissue engineering ureter, enabling the wall thickness of the tissue engineering ureter to reach 50 mu m-2mm, removing cells on the obtained tissue engineering ureter, enabling DNA to be smaller than 100ng/mg dry weight and enabling the alpha-Gal clearance to be more than 90%, and conducting crosslinking treatment through a crosslinking agent and sterilizing to obtain the decellularized tissue engineering ureter, wherein the crosslinking agent is glutaraldehyde and/or epoxide 1, 4-butanediol diglycidyl ether, and the decellularized tissue engineering ureter prepared in this way has good biocompatibility, infection resistance and no immunogenicity.

Furthermore, the wall structure of the decellularized tissue engineering ureter prepared by the invention is a decellularized cell matrix (including allogenic or xenogenic) with a nickel-titanium alloy support, has good biocompatibility, is anti-infection and non-immunogenic, and can be implanted into a body to pass through an epithelium and smooth muscle cells to reconstruct the tissue engineering ureter in vivo to form a new ureter with a nickel-titanium alloy support, thereby effectively preventing infection and calculus formation.

Furthermore, the tissue engineering ureter prepared by the invention is cultured in an in vitro standardized way, can be prepared into tissue engineering ureters with different lengths and different inner diameters according to clinical requirements, and can also partially or fully replace diseased ureters.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

Fig. 1 is a schematic flow chart of an embodiment of a method for preparing a decellularized tissue engineering ureter according to the present invention;

fig. 2 is a schematic diagram of an embodiment of a ureter with decellularized tissue engineering according to the present invention;

fig. 3 is a cross-sectional view of the decellularized tissue engineering ureter of fig. 2 in a first embodiment;

fig. 4 is a cross-sectional view of the decellularized tissue engineering ureter of fig. 2 in a second embodiment;

fig. 5 is a cross-sectional view of the decellularized tissue engineering ureter of fig. 2 in a third embodiment;

Fig. 6 is a cross-sectional view of the decellularized tissue engineering ureter of fig. 2 in a fourth embodiment;

FIG. 7 is a schematic view of the support layer of FIG. 2;

fig. 8 is a schematic diagram of another embodiment of an decellularized tissue engineering ureter provided by the present invention;

fig. 9 is a schematic view of the support layer of fig. 8.

1-Composition, 11-polymeric tubular stent, 12-support layer.

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Fig. 1 is a schematic flow chart of an embodiment of a method for preparing a decellularized tissue engineering ureter according to the present invention. Referring to fig. 1, at step S100 in fig. 1, a nitinol stent is used as a supporting layer 12, and a polymer tubular stent 11 is fixed on the supporting layer 12, wherein the polymer tubular stent 11 has a density of 100-1000mg/cm ³ and a thickness of 50-5000 μm. Wherein the structure of the support layer 12 is shown in fig. 7 and 9.

For convenience of description, the present invention refers to the combination of the polymer tubular stent 11 fixed on the supporting layer 12 as composition 1.

Specifically, the polymer tubular stent 11 may be fixed on the supporting layer 12 in a manner that the polymer tubular stent 11 covers the surface of the supporting layer 12 (fig. 2, 3 and 5) and is fixed by a surgical suture, or in a manner that the supporting layer 12 is disposed in the middle of the wall of the polymer tubular stent 11 (fig. 4 and 6).

Wherein, the polymer tubular stent 11 is prepared by using one or more of PCL, PGA, PLA, PGA-LA and collagen biodegradable materials.

The polymer tubular stent 11 is prepared by one or more processes of 3D printing, electrostatic spinning, solution casting, near-field direct writing and braiding.

The polymer tubular stent 11 may be, but is not limited to, circular or oval in radial direction after being secured to the support layer 12. As shown in fig. 3 and 4, when the polymer tubular stent 11 is circular in the radial direction after being fixed to the supporting layer 12, the diameter is 0.8-5mm. Preferably, the diameter may also be 1mm, 2mm, or 3mm.

As shown in fig. 5 and 6, when the polymer tubular stent 11 is oval in the radial direction after being fixed to the supporting layer 12, the major axis is 0.8-7mm and the minor axis is 0.5-2mm. Preferably, the major axis may also be 1mm, 3mm, or 5mm, and the minor axis may also be 1mm or 1.5mm.

Preferably, the polymeric tubular stent 11 may also have a density of 200mg/cm ³、500mg/cm³、700mg/cm³, or 900mg/cm ³. The thickness of the polymeric tubular stent 11 may also be 500 μm, 1000 μm, or 2000 μm.

At step S200, after the seed cells are planted in the supporting layer 12 and the polymer tubular stent 11, the seed cells are cultured in a bioreactor to obtain the tissue engineering ureter, so that the thickness of the wall of the tissue engineering ureter reaches 50 μm-2mm. Wherein the incubation time may be 6-12 weeks (e.g., 7 weeks, 8 weeks, or 10 weeks).

Preferably, the wall thickness of the tube may also be 1mm, or 1.5mm.

Wherein the seed cells comprise one or more of humanized vascular smooth muscle cells, ureteric smooth muscle cells, and fibroblasts.

And at the step S300, the obtained tissue engineering ureter is subjected to cell removal treatment, so that the DNA is less than 100ng/mg dry weight and the alpha-Gal clearance rate is more than 90%, and is subjected to crosslinking treatment and sterilization through a crosslinking agent, so that the cell removal tissue engineering ureter is obtained, wherein the crosslinking agent is glutaraldehyde and/or epoxide 1, 4-butanediol diglycidyl ether.

Preferably, the DNA content may also be 90ng/mg (dry weight), or 97ng/mg (dry weight). The alpha-Gal clearance can also be 95%, 97%, or 99%.

The ureter of the decellularized tissue engineering can be similar to the ureter of a human body, and the inner diameters of the ureter of the decellularized tissue engineering can be different at different positions or the same at different positions, and can be determined according to specific situations.

For example, as shown in fig. 8 and 9, the obtained decellularized tissue engineering ureter may have a large inner diameter at both ends and a small inner diameter in the middle, wherein the inner diameter at both ends is 1-10mm and the inner diameter in the middle is 0.5-8mm, or as shown in fig. 3 to 7, the obtained decellularized tissue engineering ureter may have the same inner diameter and an inner diameter of 1-10mm.

Wherein when the ureter in the decellularized tissue engineering is large in inner diameter at two ends and small in inner diameter in the middle, the inner diameters of the two ends can be 3mm, 5mm, 7mm or 8mm, and the inner diameter in the middle can be 1mm, 3mm, 4mm, 5mm or 7mm.

The wall thickness of the decellularized tissue engineering ureter also needs to be determined according to the installation position. For example, when the decellularized tissue engineering ureter is placed as a ureteral stent at a ureteral stenosis via a delivery device, the wall thickness of the decellularized tissue engineering ureter is 50-100 μm (preferably, the wall thickness may also be 60 μm, 70 μm, or 90 μm). When the decellularized tissue engineering ureter is used as a partial or full segment replacement of a ureter and is anastomosed with an autologous ureter, the wall thickness of the decellularized tissue engineering ureter is 100-200 um (preferably, the wall thickness can also be 120um, 160um or 180 um).

More specifically, the obtained decellularized tissue engineering ureter comprises collagen or collagen and elastin, and also comprises a small amount of fibronectin, fibrin, laminin, extracellular matrix related growth protein and glycosaminoglycan.

The invention also provides a cell-free tissue engineering ureter, which is prepared by adopting the preparation method of the cell-free tissue engineering ureter. The embodiment of the acellular tissue engineering ureter comprises the embodiment of the preparation method of the acellular tissue engineering ureter, and the beneficial effects of the preparation method of the acellular tissue engineering ureter can be applied to the acellular tissue engineering ureter.

In order to further verify the beneficial effects of the decellularized tissue engineering ureter provided by the present invention, the following description is made by way of examples and comparative examples.

Example 1

(1) Taking a nickel-titanium alloy bracket as a supporting layer 12, adopting 6% PGA solution for electrostatic spinning to prepare a polymer tubular material with the thickness of 300 mu m and the density of 550mg/cm ³, covering the surface of the nickel-titanium alloy bracket by the polymer tubular material, and fixing by using a surgical suture;

(2) The nickel-titanium alloy-PGA is planted into ureter smooth muscle cells, the ureter smooth muscle cells are cultured in vitro for 12 weeks at 37 ℃, the thickness of the tube wall is 700um, and a tissue engineering ureter is obtained, wherein the tissue engineering ureter is circular, and the inner diameters of two ends of the ureter are consistent with the inner diameter of the middle of the ureter and are 2.5mm;

(3) After 500mmol/L of water, 800mmol/L of sodium cholate and 800mmol/L of sulfamyl betaine are treated for 36 hours, the liquid is replaced every 6 hours, the DNA content after decellularization is 56.3ng/mg, the a-Gal clearance rate is 94.3%, after glutaraldehyde crosslinking for 12 hours, the ureter of the decellularization tissue engineering is 200um, and the ureter of the decellularization tissue engineering does not leak under 100 mmHg.

(4) The acellular tissue engineering urinary catheter is used for replacing a narrow urinary catheter after irradiation sterilization;

Example 2

(1) Taking a nickel-titanium alloy bracket as a supporting layer 12, 3D printing the PCL bracket at 60 ℃, wherein the nickel-titanium alloy bracket is positioned in the pipe wall of the PCL bracket, and the thickness of the degradable bracket is 100 mu m, and the density is 500mg/cm ³;

(2) Planting vascular smooth muscle cells on the surface of the degradable stent, culturing for 6 weeks at 37 ℃, wherein the wall thickness of the vessel is 500um, and obtaining a tissue engineering ureter, wherein the tissue engineering ureter is elliptical, the diameter of the major axis side of the ellipse is 2.2cm, and the diameter of the minor axis side of the ellipse is 1.1mm;

(3) After 300mmol/L Triton and 750mmol/L sodium dodecyl sulfate for 24 hours, the cell is removed until the DNA content is 38.9ng/mg and the a-Gal clearance is 94.3 percent, and 1, 4-butanediol diglycidyl ether is treated for 6 hours, the thickness of the obtained cell-free tissue engineering ureter is 100um, and the cell-free tissue engineering ureter does not leak under 100 mmHg;

(4) The acellular tissue engineering urinary catheter is used as a ureteral stent to dilate a narrow part after irradiation sterilization.

Comparative example 1

(1) The degradable stent is woven by using PLA-GA with the thickness of 50um, the density of the degradable polymer stent is 500mg/cm ³, the thickness is 200 mu m, and the PLA-GA and the nickel-titanium alloy stent are fixed by adopting a suture;

(2) Ureter smooth muscle cells are planted on the surface of the degradable stent, the degradable stent is cultured for 4 weeks at 37 ℃, the wall thickness of the ureter is 600um, the inner diameter of the tissue engineering ureter is circular, and the diameter is 2.3mm;

(3) Treated with 400mmol/L hexadecyl trimethyl ammonium bromide, 650mmol/L N-decanoyl-N-methyl glucosamine for 24h until the DNA content is 102.9ng/mg, the clearance of a-Gal is 78.1%, glutaraldehyde is 0.65% crosslinked for 12h, the thickness of the ureter of the decellularized tissue engineering is 100um, and leakage is 3.5ml/min under 100mmHg of the ureter of the decellularized tissue engineering.

Comparative example 2

(1) Preparing a degradable bracket by direct write of PCL, wherein the density of the bracket is 500mg/cm ³ and the thickness is 500 mu m, and fixing the PCL and the degradable polymer bracket by adopting a suture;

(2) The smooth muscle cells of the ureter are planted on the surface of the PCL, the culture is carried out for 4 weeks at 37 ℃, the thickness of the tube wall is 200um, the inner diameter of the tissue engineering ureter is circular, and the diameter is 2.3mm;

(3) Treated with 500 mmol/LN-decanoyl-N-methylglucamine, 650mmol/L water and sodium cholate for 24h until the DNA content is 243.9ng/mg, the clearance of a-Gal is 81.9%,0.65% glutaraldehyde crosslinks for 12h, the thickness of the decellularized tissue engineering ureter is 150um, and leakage is 6.3ml/min under 100 mmHg.

Table 1 examples and comparative examples effect comparison

As can be seen from Table 1 and the examples and comparative examples, the decellularized tissue engineering ureter provided by the invention has good biocompatibility, anti-infection property and no immunogenicity.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The preparation method of the decellularized tissue engineering ureter is characterized by comprising the following steps of:

2. The method of claim 1, wherein the step of fixing the nickel-titanium alloy stent as a supporting layer and the polymer tubular stent on the supporting layer, wherein the polymer tubular stent covers the surface of the supporting layer and is fixed by a surgical suture, or

3. The method for preparing a decellularized tissue engineering ureter according to claim 1, wherein the step of using a nickel-titanium alloy stent as a supporting layer and fixing a polymer tubular stent on the supporting layer is performed by using one or more of PCL, PGA, PLA, PGA-LA and collagen biodegradable materials.

4. The method for preparing an acellular tissue engineering ureter according to claim 3, wherein the nickel-titanium alloy stent is used as a supporting layer, and the polymer tubular stent is fixed on the supporting layer, and the polymer tubular stent is prepared by one or more of 3D printing, electrostatic spinning, solution casting, near-field direct writing and braiding.

5. The method for preparing an acellular tissue engineering ureter according to claim 1, wherein in the step of fixing a polymer tubular stent on a supporting layer by using a nickel-titanium alloy stent as the supporting layer, the radial direction of the polymer tubular stent after the polymer tubular stent is fixed on the supporting layer is circular or elliptical;

6. The method for preparing a decellularized tissue engineering ureter according to claim 1, wherein the obtained tissue engineering ureter is subjected to cell removal treatment to ensure that the DNA is less than 100ng/mg dry weight and the alpha-Gal clearance rate is more than 90%, and is subjected to crosslinking treatment and sterilization by a crosslinking agent, so that the obtained decellularized tissue engineering ureter has a large inner diameter at two ends and a small inner diameter in the middle, wherein the inner diameter at two ends is 1-10mm, and the inner diameter in the middle is 0.5-8mm;

Or the inner diameter of the obtained decellularized tissue engineering ureter is the same, and the inner diameter is 1-10mm.

7. The method for preparing a decellularized tissue engineering ureter according to claim 1, wherein after the supporting layer and the polymer tubular stent are planted with seed cells, the seed cells are cultured in a bioreactor to obtain the tissue engineering ureter, and the wall thickness of the seed cells reaches one or more of humanized vascular smooth muscle cells, ureter smooth muscle cells and fibroblasts.

8. The method for producing a decellularized tissue engineering ureter according to claim 1, wherein the wall thickness of the decellularized tissue engineering ureter is 50-100 μm when the decellularized tissue engineering ureter is placed at a ureter stenosis as a ureter stent by a conveyor, or

9. A decellularized tissue engineering ureter prepared by the method of preparing a decellularized tissue engineering ureter according to any of claims 1 to 8.

10. The decellularized tissue engineering ureter of claim 9, wherein after the decellularized tissue engineering ureter is implanted in vivo, the lumen of the ureter is endothelialized, the outer membrane of the ureter is attached with fibroblasts, and the wall of the ureter is decellularized by smooth muscle cells to form a nascent ureter with a nickel-titanium alloy stent support.