Decellularized tissue engineering ureter and preparation method thereof

Abstract

The invention discloses a decellularized tissue engineering ureter and a preparation method thereof.The preparation method of the decellularized tissue engineering ureter comprises the steps that a nickel-titanium alloy support serves as a supporting layer, a polymer tubular support is fixed to the supporting layer, the density of the polymer tubular support ranges from 100 mg / cm < 3 > to 1000 mg / cm < 3 >, and the thickness of the polymer tubular support ranges from 50 micrometers to 5000 micrometers; after seed cells are planted in the supporting layer and the polymer tubular bracket, culturing in a bioreactor to obtain the tissue engineering ureter, and enabling the thickness of the wall of the tissue engineering ureter to reach 50 mu m-2mm; the obtained tissue engineering ureter is subjected to cell removal treatment, so that the dry weight of DNA is smaller than 100 ng / mg, the alpha-Gal clearance rate is larger than 90%, cross-linking treatment is conducted through a cross-linking agent, sterilization is conducted, the acellular tissue engineering ureter is obtained, and the cross-linking agent is glutaraldehyde and / or epoxide 1, 4-butanediol diglycidyl ether.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a decellularized tissue engineering ureter and a preparation method thereof. Background Art

[0002] Ureteral stenosis is a common disease in urology, which is mainly divided into primary and secondary. Among them, secondary ureteral stenosis is most commonly caused by iatrogenic injury and is mainly treated by surgery. The surgical treatment of ureteral stenosis is challenging because the proximal ureter is supplied by the branches on the inner side of the renal artery, the middle ureter is supplied by the branches on the back of the common iliac artery, and the distal ureter is supplied by the branches on the outer side of the superior vesical artery. The blood supply of these three blood vessels is very fragile and prone to interruption, which can easily cause ureteral ischemia and necrosis. Therefore, the repair of ureteral stenosis is a challenge for urologists.

[0003] In recent years, there have been many new surgical methods for ureteral stenosis, making the choices for urologists more diverse. Ureteral stenosis is mainly caused by lumen stenosis, so relieving obstruction and unblocking the ureter can help relieve patients' symptoms and improve their quality of life. Among them, there are many options for repairing ureteral stenosis, such as intracavitary surgical treatment, bladder wall muscle flap ureteroplasty, and autologous patch treatment. However, the above treatment methods all have disadvantages and cannot completely meet the needs of patients. Therefore, there is currently no best way to treat ureteral stenosis or injury.

[0004] Taking endoluminal surgical treatment as an example, endoluminal surgical treatment includes balloon dilatation and ureteral stents. Among them, balloon dilatation for the treatment of ureteral stenosis is to use a uniformly expanded balloon to open a narrow section of the ureter to achieve the purpose of treatment. For ureteral stenosis less than 2 cm, ureteroscopic endoincision balloon dilatation can be performed. The results of many clinical studies have shown that balloon dilatation under direct vision of the ureteroscope can effectively improve the treatment effect of secondary ureteral stenosis, will not significantly affect the efficiency of postoperative rehabilitation, and has few postoperative complications. However, this method is more effective in treating short-segment ureteral stenosis, while the incidence of surgical failure is higher for long-segment stenosis. Ureteral balloon dilatation can easily cause mechanical force or ischemic damage, which can lead to further damage to the ureter and the formation of stenosis.

[0005] Ureteral stent placement is different from other endoscopic treatments for ureteral stenosis. Ureteral stent implantation can effectively relieve acute urinary tract obstruction, and the extent of stenosis does not affect its use. The Allium stent, which has been on the market, can effectively relieve ureteral stenosis and relieve hydronephrosis. It has mild adverse reactions such as bladder irritation and body pain, less urinary tract infection, low invasiveness, and patients do not need external drainage, which improves the quality of life of patients. However, the Allium stent is currently expensive, has formed stones and infections, which limits its clinical application. One of the main problems with long-term stent placement of ureteral stents is that the probability of bacterial colonization in the stent is high, and it is easy to form a resistant bacterial group. Therefore, ureteral stents cannot be permanently placed. It must be removed or replaced at a specific time. When the ureteral stent is removed, it needs to be done in the hospital, causing physical discomfort to the patient, damaging the urethra, and increasing the economic burden on the patient. Although the indwelling ureteral stent is more convenient, the biggest problem is that the ureteral stenosis is not fundamentally relieved, and most of them are a preventive or palliative treatment.

[0006] Taking urinary system surgery as an example, urinary system surgery includes ureteral anastomosis and bladder wall muscle flap ureteroplasty. Ureteral anastomosis is a relatively simple procedure for repairing ureteral injuries. Short strictures can be repaired with ureteral end-to-end anastomosis, while longer strictures may not be able to achieve tension-free anastomosis. The length and location of ureteral strictures determine the feasibility of ureteral anastomosis. There are certain risks after ureteral end-to-end anastomosis. Insufficient blood supply may occur after surgery, leading to anastomotic stenosis or anastomotic obstruction, which in turn causes hydronephrosis and renal damage.

[0007] In relevant clinical studies, bladder wall muscle flap ureteroplasty uses a pedicled bladder muscle flap, a spirally wrapped stent, retains its branched superior vesical artery, and anastomoses it to the proximal ureter to reconstruct the full-length ureteral defect. Patients experience mild bladder irritation symptoms and lower urinary tract symptoms. No patient has treatment-related complications such as urinary incontinence, renal colic, and high fever during the perioperative period. No obvious hydronephrosis and anastomotic stenosis were observed after surgery, and there was no immune rejection reaction. However, when the ureteral stenosis exceeds 15 cm, it is difficult to ensure tension-free repair.

[0008] Taking autologous patch treatment as an example, autologous patch treatment includes ileal and appendiceal ureteroplasty. Ileal and appendiceal ureteroplasty uses the ileum to replace the ureter, and its advantage is that it can reconstruct a long segment or even the entire length of the ureter. Clinical research results show that the vast majority of patients who undergo ureteral replacement in the ileum, colon or appendix have a good overall condition after surgery, without ureteral expansion and effusion. It can be safely and successfully used for mild to moderate renal function loss caused by ureteral stenosis. In the long run, renal function in most cases has been maintained or improved.

[0009] However, due to the particularity of the intestinal tract, surgical methods are prone to cause complications such as electrolyte imbalance, metabolic acidosis, ureteral dilatation, and are also prone to cause anastomotic leakage, leading to urine extravasation. Long-term extravasation leads to scar fibrosis and hyperplasia around the intestinal wall, followed by recurrence of local stenosis. Summary of the invention

[0010] The main purpose of the present invention is to provide a preparation method of a decellularized tissue engineered ureter and a preparation method thereof, aiming to solve the above-mentioned problems.

[0011] To achieve the above object, the present invention provides a method for preparing a decellularized tissue-engineered ureter, the method comprising:

[0012] A nickel-titanium alloy stent is used as a support layer, and a polymer tubular stent is fixed on the support layer, wherein the density of the polymer tubular stent is 100-1000 mg / cm 3 , thickness is 50-5000μm;

[0013] After seeding the support layer and the polymer tubular scaffold with seed cells, the cells are cultured in a bioreactor to obtain a tissue-engineered ureter, so that the wall thickness of the ureter reaches 50 μm-2 mm;

[0014] The obtained tissue-engineered ureter is decellularized to make the DNA content less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and is cross-linked with a cross-linking agent and sterilized to obtain a decellularized tissue-engineered ureter, wherein the cross-linking agent is glutaraldehyde and / or epoxide 1,4-butanediol diglycidyl ether.

[0015] Preferably, in the method for preparing the decellularized tissue engineered ureter, in the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent covers the surface of the support layer and is fixed with surgical sutures; or,

[0016] The support layer is arranged in the middle of the tube wall of the polymer tubular support.

[0017] Preferably, in the method for preparing the decellularized tissue engineered ureter, in the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent is prepared using one or more of PCL, PGA, PLA, PGA-LA, and collagen biodegradable materials.

[0018] Preferably, in the method for preparing the decellularized tissue engineered ureter, in the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent is prepared by one or more processes including 3D printing, electrospinning, solution casting, near-field direct writing, and weaving.

[0019] Preferably, in the method for preparing the decellularized tissue engineered ureter, in the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the radial direction of the polymer tubular stent after being fixed on the support layer is circular or elliptical;

[0020] When the polymer tubular stent is circular in radial direction after being fixed to the support layer, the diameter is 0.8-5 mm;

[0021] When the polymer tubular stent is elliptical in radial direction after being fixed to the support layer, the major axis is 0.8-7 mm and the minor axis is 0.5-2 mm.

[0022] Preferably, in the method for preparing the decellularized tissue-engineered ureter, the obtained tissue-engineered ureter is decellularized to make the DNA less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and is cross-linked with a cross-linking agent and sterilized to obtain the decellularized tissue-engineered ureter. The decellularized tissue-engineered ureter has a large inner diameter at both ends and a small inner diameter in the middle, wherein the inner diameter at both ends is 1-10 mm, and the inner diameter in the middle is 0.5-8 mm.

[0023] Preferably, in the method for preparing the decellularized tissue-engineered ureter, the obtained tissue-engineered ureter is decellularized to make the DNA less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and cross-linked with a cross-linking agent and sterilized to obtain the decellularized tissue-engineered ureter. The decellularized tissue-engineered ureter has the same inner diameter of 1-10 mm.

[0024] Preferably, in the method for preparing the decellularized tissue-engineered ureter, in the step of planting seed cells in the support layer and the polymer tubular scaffold and then culturing them in a bioreactor to obtain a tissue-engineered ureter with a wall thickness of 50 μm-2 mm, the seed cells include one or more of human vascular smooth muscle cells, ureteral smooth muscle cells, and fibroblasts.

[0025] Preferably, in the method for preparing the decellularized tissue engineered ureter, when the decellularized tissue engineered ureter is placed in the ureteral stenosis via a conveyor as a ureteral stent, the wall thickness of the decellularized tissue engineered ureter is 50-100 μm; or,

[0026] When the decellularized tissue engineered ureter is used as a partial or full replacement of the ureter and anastomosed with the autologous ureter, the wall thickness of the decellularized tissue engineered ureter is 100um-200um.

[0027] In order to achieve the above object, the present invention further provides a decellularized tissue engineered ureter, which is prepared by the above-mentioned preparation method of the decellularized tissue engineered ureter.

[0028] Preferably, in the decellularized tissue engineered ureter, after being implanted in vivo, the ureteral lumen is endothelialized, fibroblasts are attached to the ureteral adventitia, and the ureteral wall is recellularized by smooth muscle cells to form a new ureter supported by a nickel-titanium alloy stent.

[0029] The present invention has at least the following beneficial effects:

[0030] The method for preparing the decellularized tissue-engineered ureter provided by the present invention comprises using a nickel-titanium alloy stent as a support layer, and fixing a polymer tubular stent on the support layer, wherein the density of the polymer tubular stent is 100-1000 mg / cm 3 , thickness of 50-5000 μm; after seeding the support layer and the polymer tubular scaffold with seed cells, they are cultured in a bioreactor to obtain a tissue-engineered ureter with a wall thickness of 50 μm-2 mm; the obtained tissue-engineered ureter is decellularized to make the DNA less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and cross-linked with a cross-linking agent, sterilized to obtain a decellularized tissue-engineered ureter, wherein the cross-linking agent is glutaraldehyde and / or epoxide 1,4-butanediol diglycidyl ether. The decellularized tissue-engineered ureter prepared in this way has good biocompatibility, is anti-infective and non-immunogenic.

[0031] Furthermore, the wall structure of the decellularized tissue-engineered ureter prepared by the present invention is a decellularized cell matrix (including allogeneic or xenogeneic) supported by nickel-titanium alloy, which has good biocompatibility, is anti-infective and non-immunogenic. After implantation in the body, the ureteral transitional epithelium and smooth muscle cells reconstruct the tissue-engineered ureter in vivo to form a new ureter supported by a nickel-titanium alloy stent, which can effectively prevent infection and stone formation.

[0032] Furthermore, the decellularized tissue-engineered ureter prepared by the present invention can be cultured in a standardized manner in vitro, and tissue-engineered ureters of different lengths and inner diameters can be prepared according to clinical needs, and diseased ureters can also be partially or fully replaced. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 A schematic diagram of a process of one embodiment of a method for preparing a decellularized tissue engineered ureter provided by the present invention;

[0035] Figure 2 A schematic diagram of an embodiment of the decellularized tissue engineered ureter provided by the present invention;

[0036] Figure 3 for Figure 2 A cross-sectional view of the decellularized tissue engineered ureter in the first embodiment;

[0037] Figure 4 for Figure 2 A cross-sectional view of a decellularized tissue engineered ureter in a second embodiment;

[0038] Figure 5 for Figure 2 A cross-sectional view of the decellularized tissue engineered ureter in the third embodiment;

[0039] Figure 6 for Figure 2 A cross-sectional view of a decellularized tissue engineered ureter in a fourth embodiment;

[0040] Figure 7 for Figure 2 Schematic diagram of the middle support layer;

[0041] Figure 8 A schematic diagram of another embodiment of the decellularized tissue engineered ureter provided by the present invention;

[0042] Fig. 9 for Figure 8 Schematic diagram of the middle support layer.

[0043] 1-composition, 11-polymer tubular scaffold, 12-support layer.

[0044] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0045] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0046] Figure 1 This is a schematic diagram of a process for preparing a decellularized tissue engineered ureter according to an embodiment of the present invention. Figure 1 , Figure 1 In step S100, a nickel-titanium alloy stent is used as a support layer 12, and a polymer tubular stent 11 is fixed on the support layer 12, wherein the density of the polymer tubular stent 11 is 100-1000 mg / cm 3 , with a thickness of 50-5000 μm. The structure of the support layer 12 is as follows Figure 7 and Fig. 9 shown.

[0047] It should be noted that, for the sake of convenience, in the present invention, the polymer tubular stent 11 is fixed to the support layer 12 and then combined together to form a composite material 1 .

[0048] Specifically, the polymer tubular stent 11 is fixed to the support layer 12 in a configuration in which the polymer tubular stent 11 covers the surface of the support layer 12 (eg Figure 2 , Figure 3 , Figure 5 ), and fixed with surgical sutures; the support layer 12 may also be arranged in the middle of the tube wall of the polymer tubular stent 11 (such as Figure 4 and Figure 6 ).

[0049] The polymer tubular stent 11 is prepared using one or more of PCL, PGA, PLA, PGA-LA, and collagen biodegradable materials.

[0050] The polymer tubular stent 11 is prepared by one or more processes including 3D printing, electrospinning, solution casting, near-field direct writing, and weaving.

[0051] The radial shape of the polymer tubular stent 11 after being fixed to the support layer 12 may be, but is not limited to, circular or elliptical. Figure 3 and Figure 4 As shown, when the polymer tubular stent 11 is circular in radial direction after being fixed to the support layer 12, the diameter is 0.8-5 mm. Preferably, the diameter can also be 1 mm, 2 mm, or 3 mm.

[0052] like Figure 5 and Figure 6 As shown, when the polymer tubular stent 11 is elliptical in radial direction after being fixed on the support layer 12, the major axis is 0.8-7 mm and the minor axis is 0.5-2 mm. Preferably, the major axis can also be 1 mm, 3 mm, or 5 mm, and the minor axis can also be 1 mm or 1.5 mm.

[0053] Preferably, the density of the polymer tubular stent 11 can also be 200 mg / cm 3 , 500mg / cm 3 、700mg / cm 3 , or 900mg / cm 3 The thickness of the polymer tubular stent 11 may also be 500 μm, 1000 μm, or 2000 μm.

[0054] In step S200, the support layer 12 and the polymer tubular scaffold 11 are seeded with cells and then cultured in a bioreactor to obtain a tissue-engineered ureter with a wall thickness of 50 μm-2 mm. The culture time may be 6-12 weeks (e.g., 7 weeks, 8 weeks, or 10 weeks).

[0055] Preferably, the tube wall thickness may also be 1 mm or 1.5 mm.

[0056] Wherein, the seed cells include one or more of human-derived vascular smooth muscle cells, ureteral smooth muscle cells, and fibroblasts.

[0057] In step S300, the obtained tissue engineered ureter is decellularized to make the DNA less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and is cross-linked with a cross-linking agent and sterilized to obtain a decellularized tissue engineered ureter, wherein the cross-linking agent is glutaraldehyde and / or epoxide 1,4-butanediol diglycidyl ether.

[0058] Preferably, the DNA content may also be 90 ng / mg (dry weight) or 97 ng / mg (dry weight). The α-Gal clearance rate may also be 95%, 97%, or 99%.

[0059] It should be noted that the decellularized tissue engineered ureter can be similar to the human body's own ureter, and the decellularized tissue engineered ureter can have different inner diameters at different locations, or can have the same inner diameter at different locations, depending on the specific circumstances.

[0060] For example, Figure 8 and Fig. 9 As shown, 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 1-10 mm and the inner diameter in the middle is 0.5-8 mm; Figures 3 to 7 As shown, the decellularized tissue engineered ureters may have the same inner diameter, which is 1-10 mm.

[0061] Wherein the decellularized tissue engineered ureter has a large inner diameter at both ends and a small inner diameter in the middle, the inner diameters at both ends may be 3mm, 5mm, 7mm, or 8mm; the inner diameter in the middle may be 1mm, 3mm, 4mm, 5mm, or 7mm.

[0062] The wall thickness of the decellularized tissue engineered ureter also needs to be determined according to the installation location. For example, when the decellularized tissue engineered ureter is placed in the ureteral stenosis via a conveyor as a ureteral stent, the wall thickness of the decellularized tissue engineered ureter is 50-100 μm (preferably, the wall thickness can also be 60 μm, 70 μm, or 90 μm). When the decellularized tissue engineered ureter is used as a partial or full replacement of the ureter and anastomosed with the autologous ureter, the wall thickness of the decellularized tissue engineered ureter is 100um-200um (preferably, the wall thickness can also be 120um, 160um, or 180um).

[0063] More specifically, the obtained decellularized tissue-engineered ureter includes collagen or collagen and elastin; and also includes a small amount of fibronectin, fibrin, laminin, extracellular matrix-related growth protein, and glycosaminoglycan.

[0064] The present invention also provides a decellularized tissue-engineered ureter, which is prepared by the above-mentioned method for preparing the decellularized tissue-engineered ureter. The embodiment of the decellularized tissue-engineered ureter includes the embodiment of the above-mentioned method for preparing the decellularized tissue-engineered ureter; the beneficial effects of the above-mentioned method for preparing the decellularized tissue-engineered ureter can also be applied to the decellularized tissue-engineered ureter.

[0065] In order to further verify the beneficial effects of the decellularized tissue engineered ureter provided by the present invention, the following is an illustration through examples and comparative examples.

[0066] Example 1

[0067] (1) A nickel-titanium alloy stent was used as the support layer 12, and a 6% PGA solution was used for electrospinning to prepare a 300 μm thick and 550 mg / cm 3 A polymer tubular material, which is covered on the surface of the nickel-titanium alloy stent and fixed with surgical sutures;

[0068] (2) Nitinol-PGA was used to implant ureteral smooth muscle cells and cultured in vitro at 37°C for 12 weeks. The wall thickness of the ureter was 700 μm, and a tissue-engineered ureter was obtained. The tissue-engineered ureter was circular, and the inner diameter of the two ends of the ureter was consistent with that of the middle, which was 2.5 mm.

[0069] (3) After treatment with 500mmol / L water and 800mmol / L sodium cholate and aminosulfobetaine for 36h, with the liquid replaced every 6h, the DNA content after decellularization was 56.3ng / mg, and the a-Gal clearance rate was 94.3%; after glutaraldehyde cross-linking for 12h, the decellularized tissue-engineered ureter was 200um, and the decellularized tissue-engineered ureter did not leak under 100mmHg.

[0070] (4) Decellularized tissue-engineered urinary catheters are used to replace narrow urinary catheters after irradiation sterilization;

[0071] Example 2

[0072] (1) The nickel-titanium alloy stent is used as the support layer 12, and the PCL stent is 3D printed at 60°C. The nickel-titanium alloy stent is in the PCL stent tube wall. The degradable stent has a thickness of 100 μm and a density of 500 mg / cm 3 ;

[0073] (2) Vascular smooth muscle cells were planted on the surface of the biodegradable stent and cultured at 37°C for 6 weeks. The wall thickness of the ureter was 500 μm, and a tissue-engineered ureter was obtained. The tissue-engineered ureter was elliptical, with a long axis diameter of 2.2 cm and a short axis diameter of 1.1 mm.

[0074] (3) After treatment with 300mmol / L Triton and 750mmol / L sodium dodecyl sulfate for 24h, the cells were decellularized to a DNA content of 38.9ng / mg and an a-Gal clearance rate of 94.3%. After 6h of treatment with 1,4-butanediol diglycidyl ether, the decellularized tissue-engineered ureter was 100um thick and did not leak at 100mmHg.

[0075] (4) After irradiation sterilization, the decellularized tissue-engineered ureteral catheter is used as a ureteral stent to dilate the narrow area.

[0076] Comparative Example 1

[0077] (1) Use 50um PLA-GA to weave a biodegradable stent, and the density of the biodegradable polymer stent is 500mg / cm 3 , with a thickness of 200 μm, the PLA-GA and the nickel-titanium alloy stent were fixed with sutures;

[0078] (2) Ureteral smooth muscle cells were planted on the surface of the biodegradable scaffold and cultured at 37°C for 4 weeks. The wall thickness was 600 μm, and the inner diameter of the tissue-engineered ureter was circular with a diameter of 2.3 mm.

[0079] (3) After treatment with 400mmol / L hexadecyltrimethylammonium bromide and 650mmol / L N-decanoyl-N-methylglucamine for 24h, the DNA content was 102.9ng / mg and the a-Gal clearance rate was 78.1%. After cross-linking with 0.65% glutaraldehyde for 12h, the thickness of the decellularized tissue-engineered ureter was 100um and the leakage of the decellularized tissue-engineered ureter was 3.5ml / min at 100mmHg.

[0080] Comparative Example 2

[0081] (1) PCL was directly written into the biodegradable stent, with a stent density of 500 mg / cm 3 , with a thickness of 500 μm, the PCL and the degradable polymer scaffold were fixed with sutures;

[0082] (2) Ureteral smooth muscle cells were planted on the PCL surface and cultured at 37°C for 4 weeks. The wall thickness was 200 μm and the inner diameter of the tissue-engineered ureter was circular with a diameter of 2.3 mm.

[0083] (3) After treatment with 500mmol / L N-decanoyl-N-methylglucamine, 650mmol / L water and sodium cholate for 24h, the DNA content was 243.9ng / mg and the a-Gal clearance was 81.9%. After cross-linking with 0.65% glutaraldehyde for 12h, the thickness of the decellularized tissue-engineered ureter was 150um and the leakage was 6.3ml / min at 100mmHg.

[0084] Table 1 Comparison of effects of embodiments and comparative examples

[0085]

[0086]

[0087] It can be seen from Table 1 and the above examples and comparative examples that the acellular tissue engineered ureter provided by the present invention has good biocompatibility, is anti-infective and non-immunogenic.

[0088] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for preparing a decellularized tissue engineered ureter, characterized in that: include: A nickel-titanium alloy stent is used as a support layer, and a polymer tubular stent is fixed on the support layer, wherein the density of the polymer tubular stent is 100-1000 mg / cm 3 , thickness is 50-5000μm; After seeding the support layer and the polymer tubular scaffold with seed cells, the cells are cultured in a bioreactor to obtain a tissue-engineered ureter, so that the wall thickness of the ureter reaches 50 μm-2 mm; The obtained tissue-engineered ureter is decellularized to make the DNA content less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, and is cross-linked with a cross-linking agent and sterilized to obtain a decellularized tissue-engineered ureter, wherein the cross-linking agent is glutaraldehyde and / or epoxide 1,4-butanediol diglycidyl ether.

2. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: In the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent covers the surface of the support layer and is fixed with surgical sutures; or, The support layer is arranged in the middle of the tube wall of the polymer tubular support.

3. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: In the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent is prepared using one or more of PCL, PGA, PLA, PGA-LA, and collagen biodegradable materials.

4. The method for preparing the decellularized tissue engineered ureter according to claim 3, characterized in that: In the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the polymer tubular stent is prepared by one or more processes including 3D printing, electrospinning, solution casting, near-field direct writing, and weaving.

5. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: In the step of using the nickel-titanium alloy stent as a support layer and fixing the polymer tubular stent on the support layer, the radial direction of the polymer tubular stent after being fixed on the support layer is circular or elliptical; When the polymer tubular stent is circular in radial direction after being fixed to the support layer, the diameter is 0.8-5 mm; When the polymer tubular stent is elliptical in radial direction after being fixed to the support layer, the major axis is 0.8-7 mm and the minor axis is 0.5-2 mm.

6. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: In the step of removing cells from the obtained tissue engineering ureter to make the DNA content less than 100 ng / mg dry weight and the α-Gal clearance rate greater than 90%, cross-linking treatment with a cross-linking agent, and sterilization to obtain the decellularized tissue engineering ureter, the obtained decellularized tissue engineering ureter has a large inner diameter at both ends and a small inner diameter in the middle, wherein the inner diameter at both ends is 1-10 mm and the inner diameter in the middle is 0.5-8 mm; Alternatively, the obtained decellularized tissue engineered ureters have the same inner diameter, which is 1-10 mm.

7. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: In the step of planting seed cells on the support layer and the polymer tubular scaffold, culturing them in a bioreactor to obtain a tissue-engineered ureter with a wall thickness of 50 μm-2 mm, the seed cells include one or more of human vascular smooth muscle cells, ureteral smooth muscle cells, and fibroblasts.

8. The method for preparing the decellularized tissue engineered ureter according to claim 1, characterized in that: When the decellularized tissue engineered ureter is used as a ureteral stent and placed in a ureteral stenosis via a conveyor, the wall thickness of the decellularized tissue engineered ureter is 50-100 μm; or, When the decellularized tissue engineered ureter is used as a partial or full replacement of the ureter and anastomosed with the autologous ureter, the wall thickness of the decellularized tissue engineered ureter is 100um-200um.

9. A decellularized tissue engineered ureter, characterized in that: The decellularized tissue engineered ureter is prepared by the preparation method of the decellularized tissue engineered ureter according to any one of claims 1 to 8.

10. The decellularized tissue engineered ureter according to claim 9, characterized in that: After the decellularized tissue engineering ureter is implanted in the body, the ureteral lumen is endothelialized, fibroblasts are attached to the ureteral adventitia, and the ureteral wall is recellularized by smooth muscle cells to form a new ureter supported by a nickel-titanium alloy stent.

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