CN113855859A

CN113855859A - Small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization

Info

Publication number: CN113855859A
Application number: CN202110593207.4A
Authority: CN
Inventors: 谷涌泉; 王聪; 蔡志文
Original assignee: Xuanwu Hospital
Current assignee: Xuanwu Hospital
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-12-31

Abstract

The invention discloses a small-caliber tissue engineering blood vessel which is constructed by a acellular vascular matrix and can promote rapid endothelialization, belonging to the technical field of biological materials and tissue engineering. The tissue engineering blood vessel is prepared by the following method: preparing acellular vascular matrix from the carotid artery of the pig by using detergent TritonX-100 in combination with sodium dodecyl sulfate, reacting in EDC/NHS solution containing heparin for 24 hours to perform crosslinking and grafting of heparin, immersing in bovine albumin serum solution containing hepatocyte growth factor, and incubating for 2 hours to obtain the small-caliber in-situ tissue engineering blood vessel. The in-situ tissue engineering blood vessel has mechanical properties similar to those of a natural artery, and effectively avoids the formation of aneurysm; after heparin is combined, early thrombosis can be inhibited; HGF promotes rapid endothelialization and reduces the thickness of neointimal tissue. The tissue engineering blood vessel has good application prospect in the field of medical clinical blood vessel transplantation.

Description

Small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization

Technical Field

The invention relates to a small-caliber tissue engineering blood vessel which is constructed by a decellularized blood vessel matrix and can promote rapid endothelialization, belongs to the technical field of biological materials and tissue engineering, and particularly relates to a small-caliber in-situ tissue engineering blood vessel prepared by combining the decellularized blood vessel matrix with heparin and hepatocyte growth factor and a preparation method thereof.

Background

Cardiovascular disease has become the "first killer" in humans. Coronary artery bypass grafting is one of the most important procedures for reconstructing cardiac blood flow. Although the coronary bypass surgery adopts autologous veins or arteries to replace diseased vessels, has no immunological rejection reaction and high postoperative patency rate, the sources are limited, and about 30 percent of patients do not have autologous vessels for surgery. The large-caliber (>6mm) artificial blood vessel constructed by terylene and poly (expanded tetrafluoroethylene) (e-PTFE) is widely used in the replacement reconstruction of large blood vessels due to the good long-term patency rate and stability of the artificial blood vessel. However, the small-caliber artificial blood vessels made of these materials have 5-year patency rate of only about 30% due to the lack of vascular endothelium, poor vascular compliance and other reasons, and are difficult to meet the requirements of clinical application.

The proposal and practice of the tissue engineering principle provides a new idea for constructing the small-caliber artificial blood vessel with bioactivity. The core of the method is that seed cells cultured in vitro are planted on a prefabricated porous biomaterial scaffold which is good in biocompatibility and degradable to form a cell-biomaterial compound, after the seed cells are dynamically cultured and matured in a bioreactor, the constructed engineered artificial blood vessel is implanted into a body and then is fused with host vascular tissues for growth and tissue remodeling to restore the continuity of the blood vessel and the smoothness of blood flow, and finally the repair of the blood vessel is finished.

Although the research of in vitro tissue engineering artificial blood vessels has made great progress, many technical bottlenecks still remain difficult to overcome so far, such as seed cell source, cell differentiation and amplification culture time and scale, product storage, transportation, use and price, and the like, and the problems can not meet the practical clinical requirements. Therefore, research on in-situ renewable small-caliber artificial blood vessels based on the regenerative medicine principle is carried out. Compared with the traditional tissue engineering blood vessel construction, the method has the greatest characteristic that the degradable absorption stent which is loaded with various cell growth factors, adhesion polypeptides and anticoagulant drugs but not planted with seed cells is directly used for replacing the diseased blood vessel. Then the human body is used as a seed cell source and a bioreactor, and autologous new blood vessels with physiological activity and function are formed in situ by the recruitment, homing, differentiation and proliferation of various stem cells, cell progenitor cells and the like in blood and surrounding tissues and depending on the regeneration capacity of the human body. The method can eliminate the risk of in vitro cell and tissue culture, reduce treatment cost, and the stent product is convenient for storage, transportation and use, and is a practical artificial blood vessel manufacturing method.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: overcomes the defects of the existing small-caliber blood vessel, and provides a small-caliber in-situ tissue engineering blood vessel which can inhibit thrombosis and promote rapid endothelialization and a preparation method thereof. The small-caliber tissue engineering blood vessel is prepared by combining the decellularized blood vessel with heparin and hepatocyte growth factor after cross-linking.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, a small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization is provided, which is prepared by the following method: preparing acellular vascular matrix from the carotid artery of the pig by using detergent TritonX-100 in combination with sodium dodecyl sulfate, reacting in EDC/NHS solution containing heparin for 24 hours to perform crosslinking and grafting of heparin, immersing in bovine albumin serum solution containing hepatocyte growth factor, and incubating for 2 hours to obtain the small-caliber in-situ tissue engineering blood vessel.

The in-situ tissue engineering blood vessel has mechanical properties similar to those of a natural artery, and effectively avoids the formation of aneurysm; after heparin is combined, early thrombosis can be inhibited; HGF promotes rapid endothelialization and reduces the thickness of neointimal tissue. The tissue engineering blood vessel has good application prospect in the field of medical clinical blood vessel transplantation.

A method for preparing a small-caliber tissue engineering blood vessel which is constructed by acellular vascular matrixes and can promote rapid endothelialization comprises the steps of preparing the acellular vascular matrixes by using a pig carotid artery through a detergent TritonX-100 and combining with sodium dodecyl sulfate, reacting for 24 hours in EDC/NHS solution containing heparin to carry out crosslinking and grafting on the heparin, and immersing the blood vessel in bovine albumin serum solution containing hepatocyte growth factors to incubate for 2 hours to obtain the small-caliber in-situ tissue engineering blood vessel.

After the acellular blood vessel is crosslinked, the small-caliber in-situ tissue engineering blood vessel is prepared by combining heparin and hepatocyte growth factor.

The decellularized blood vessel is selected from the group consisting of an animal aorta, carotid artery, internal mammary artery, radial artery, and a gastrointestinal artery.

The cell removal treatment is carried out by adopting TritonX-100 and SDS.

Crosslinking is carried out with a solution of EDC/NHS containing heparin and at the same time grafting of the heparin.

The obtained heparinized decellularized blood vessels were sterilized using gamma rays.

HGF is bound by incubating heparinized decellularized blood vessels by immersion in HGF solution.

In a second aspect of the present invention, there is provided a method for preparing a small-caliber in situ tissue-engineered blood vessel capable of inhibiting thrombosis and promoting rapid endothelialization of the blood vessel, the method comprising the steps of:

(1) preparing a vascular substrate: carefully dissecting the neck tissue of the pig under the aseptic condition, taking out the carotid artery, immediately placing the carotid artery into a PBS ice box at 4 ℃, wherein the hot ischemia time is not more than 1 hour, repeatedly washing the carotid artery of the pig by using the PBS to remove blood clots, and stripping the adventitia and connective tissue of the blood vessel by using a surgical instrument to avoid damaging the blood vessel; the porcine carotid artery was immersed in a PBS solution containing 100U/ml penicillin and 100g/L streptomycin and stored at-20 ℃ until use.

(2) Preparing acellular vascular matrix;

(3) preparing a heparinized acellular vascular matrix;

(4) preparing the small-caliber in-situ tissue engineering blood vessel which can inhibit thrombosis and promote rapid endothelialization.

The method for preparing the acellular vascular matrix in the step (2) comprises the following steps: placing the obtained carotid artery of the pig in distilled water, placing the carotid artery of the pig on a decoloration shaking table (100r/min) for treatment for 24 hours to crack blood cells in a lumen; then put into a 1% (m/v) TritonX-100 solution and treated for 24h (100r/min) by shaking. After being washed for 2h by PBS, the blood vessel is put into 0.25-1.0% (m/v) SDS solution, and is continuously placed on a decoloring shaker (100r/min) for processing for 24-72h, and the SDS solution is replaced every 24 h; finally washing with PBS for 72h (100r/min) with shaking to remove the detergent residue; the cell removing process is carried out in a sterile environment at room temperature; the obtained acellular vascular matrix is placed in PBS for preservation at the temperature of minus 20 ℃;

the method for preparing the heparinized acellular vascular matrix comprises the following steps: preparing 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, sequentially adding heparin sodium, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (1g of acellular vascular matrix (dry weight) needs 200mg of heparin, 108mg of EDC, 67mg of NHS and 40ml of 30mmol/LMES buffer solution), and preparing heparin activation crosslinking solution after ice bath for 2 hours; placing the acellular vascular matrix into a heparin activated cross-linking solution, incubating for 24 hours at 37 ℃, and then washing for 5 times and 5 min/time by using a PBS solution; sealing and packaging the obtained heparinized acellular vascular matrix, and performing gamma ray irradiation sterilization.

The method for preparing the small-caliber in-situ tissue engineering blood vessel capable of inhibiting thrombosis and promoting rapid endothelialization comprises the following steps: preparing a Hepatocyte Growth Factor (HGF) solution (200 ng/ml) containing 1mg/ml bovine serum albumin, sterilizing by a filtration membrane, placing the heparinized acellular vascular matrix in the HGF solution, and incubating for 2 hours at 37 ℃ under oscillation (100 r/min); wash 5 times with PBS 5 min/time. The small-caliber in-situ tissue engineering blood vessel which can inhibit thrombosis and promote rapid endothelialization is obtained.

Compared with the small-caliber tissue engineering blood vessel in the prior art, the invention simplifies the cell removing step, can completely remove the cell components only by using a chemical method, has small influence on extracellular matrix, has simple cell removing step, and is beneficial to control and industrialization; the invention cross-links the decellularized blood vessel to enhance the mechanical property, combines the heparin to inhibit the thrombosis, combines the hepatocyte growth factor to promote the endothelialization, and improves the long-term patency rate.

Endothelial cells are a key factor for maintaining the long-term patency of the transplanted blood vessel, and once the lumen surface of the transplanted blood vessel is covered by the endothelial cells, the endothelial cells have the function of inhibiting the formation of thrombus. Endothelial cells inhibit thrombosis by isolating endothelial collagen from blood and secreting anti-coagulant substances such as thrombomodulin, protein S and prostacyclin. Endothelial cells release anti-proliferative factors such as nitric oxide, prostaglandins, and C-type natriuretic peptide to inhibit proliferation of smooth muscle cells, thereby preventing intimal hyperplasia in the chronic phase. The endothelialization of blood vessels needs a certain time, the combined heparin can realize early anticoagulation, the functional endothelial layer maintains long-term anticoagulation, and theoretically perfect combination. Previous studies have demonstrated that vascular endothelial growth factor, in combination with growth factors such as fibroblast growth factor, can promote endothelialization of transplanted vessels. However, these growth factors simultaneously promote smooth muscle cell proliferation, resulting in intimal hyperplasia and a decrease in long-term patency.

The invention has the most innovative invention point that the acellular vascular matrix is combined with the hepatocyte growth factor to construct the small-caliber tissue engineering blood vessel, the HGF is an endothelial specific cell growth factor secreted by mesenchymal cells, acts on vascular endothelial cells, activates nitric oxide synthase, promotes the migration, proliferation and differentiation of the endothelial cells, and has no induced proliferation effect on smooth muscle cells. HGF acts on endothelial cells 1.48 times as much as bFGF and 1.25 times as much as VEGF.

HGF has a strong affinity for heparin, which may play a role in the storage of growth factors in the extracellular matrix, enabling its sustained release. The physical adsorption of heparin prevents the early degradation of growth factors, thereby maintaining the biological activity of the growth factors. In addition, heparin-binding growth factors enhance the recognition of cellular receptors and enhance the mitogenic activity of endothelial cells. Heparin coatings serve two purposes in grafting blood vessels: one is inhibition of thrombosis; the second is to provide a vehicle for heparin binding growth factors.

The small-caliber tissue engineering blood vessel prepared by the invention can inhibit early thrombosis, avoid aneurysm, promote endothelialization and inhibit intimal hyperplasia, has high long-term patency rate and has satisfactory effect.

The invention has the following advantages: the invention firstly tries to construct the small-caliber in-situ tissue engineering blood vessel by combining the porcine acellular vascular matrix with heparin and hepatocyte growth factor, and mainly has the following advantages: (1) the detergent is adopted to combine the decellularization, so that the cell components can be completely removed, the damage to extracellular matrix is small, the mechanical property of the decellularized blood vessel matrix is similar to that of a natural blood vessel after the decellularized blood vessel matrix is crosslinked, and the degradation of the decellularized blood vessel matrix is slowed down, so that the formation of aneurysm is avoided; (2) the bound heparin has the function of inhibiting early thrombosis; (3) can stably release hepatocyte growth factor, promote the rapid endothelialization of blood vessels, and endothelial cells can be seen to be paved on the cavity surface after being implanted in vivo for 1 month; (4) the preparation steps are simple and controllable, and the finished product is easy to store and suitable for industrial production.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows histological staining of porcine cervical arteries and decellularized blood vessels. H & E staining of porcine carotid (fig. 1a), DAPI staining (fig. 1c), EVG staining (fig. 1E), Masson staining (fig. 1 g); decellularized blood vessels H & E staining (FIG. 1b), DAPI staining (FIG. 1d), EVG staining (FIG. 1f), Masson staining (FIG. 1H)

FIG. 2 is a scanning electron microscope showing the morphological features of porcine carotid artery, decellularized blood vessels, and heparinized decellularized blood vessels. Porcine carotid lumen (fig. 2a), vessel wall (fig. 2 d); decellularized vascular lumen (fig. 2b), vessel wall (fig. 2 e); heparinized decellularized vascular lumen (fig. 2c), vessel wall (fig. 2 f);

FIG. 3 is a graph showing the release profile of heparinized decellularized vascular in vitro heparin.

Fig. 4 is a graph of HGF release profile in heparinized decellularized blood vessels binding HGF in vitro.

FIG. 5 shows the mechanical properties of porcine carotid artery, decellularized blood vessel and heparinized decellularized blood vessel. FIG. 5 a: a maximum tensile strength; FIG. 5 b: breaking strength; FIG. 5 c: elongation at break; FIG. 5 d: the stitching strength; FIG. 5 e: and (5) blasting pressure.

FIG. 6 is Doppler blood vessel ultrasound images of heparinized decellularized blood vessels bound to HGF and heparinized decellularized blood vessels not bound to HGF at 1,3,6 months after rabbit carotid artery replacement.

FIG. 7 is H & E staining of heparinized decellularized blood vessels bound to HGF and heparinized decellularized blood vessels not bound to HGF at 1,3,6 months after rabbit carotid replacement.

FIG. 8 is an immunohistochemical staining of CD31 at 1,3,6 months after rabbit carotid replacement of heparinized decellularized blood vessels bound to HGF and heparinized decellularized blood vessels not bound to HGF.

Fig. 9 is SEM images of heparinized decellularized blood vessels bound to HGF and heparinized decellularized blood vessels not bound to HGF at 1,3,6 months after rabbit carotid artery replacement.

Detailed Description

The SDS referred to in the invention is an abbreviation for sodium dodecyl sulfate, EDC is an abbreviation for 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, NHS is an abbreviation for N-hydroxysuccinimide, MES is 2- (N-morpholine) ethanesulfonic acid, SEM is an abbreviation for scanning electron microscope.

Example 1

A preparation method of a small-caliber in-situ tissue engineering blood vessel capable of inhibiting thrombosis and promoting rapid endothelialization comprises the following steps:

(1) at a nearby slaughterhouse, the porcine neck tissue was carefully dissected using sterile surgical instruments and the carotid artery was removed, with an internal diameter of 2.5mm and a length of approximately 5 cm. Immediately after being taken out, the sample was placed in an ice box of PBS at 4 ℃, the warm ischemia time was not more than 1 hour, and the sample was rapidly returned to the laboratory. The porcine carotid artery is repeatedly washed by PBS to remove blood clots, and the adventitia and connective tissue of the blood vessel are stripped by using surgical instruments to avoid damaging the blood vessel. The porcine carotid artery was immersed in a PBS solution containing 100U/ml penicillin and 100g/L streptomycin and stored at-20 ℃ until use.

(2) The obtained carotid artery of pig was placed in distilled water and treated on a decolorizing shaker (100r/min) for 24h to lyse the blood cells in the lumen. Then put into a 1 percent TritonX-100 solution and treated for 24 hours (100r/min) by shaking. After washing with PBS for 2h, the vessels were placed in 0.3% (m/v) SDS solution and further treated on a destaining shaker (100r/min) for 72h, with the SDS solution being changed every 24 h. Finally, washing was performed with PBS for 72h (100r/min) with shaking to remove the detergent residues. The cell removing process is carried out in a sterile environment at room temperature. The obtained acellular vascular matrix was stored in PBS at-20 ℃.

(3) Preparation of heparin-activated cross-linking solution: 1g of acellular vascular matrix (dry weight) requires 200mg of heparin, 108mg of EDC, 67mg of NHS, 40ml of 30mmol/LMES buffer. Weighing the acellular vascular matrix, and weighing heparin, EDC, NHS and MES according to the proportion. Preparing MES buffer solution, adding heparin, EDC and NHS in sequence, and carrying out ice bath for 2 hours. The acellular vascular matrix is put into a heparin activated cross-linking solution to be incubated for 24 hours at 37 ℃, and then is washed 5 times and 5 min/time by using a PBS solution. Sealing and packaging the obtained heparinized acellular vascular matrix, and performing gamma ray irradiation sterilization.

(4) A solution containing 400ng/ml HGF and 1mg/ml bovine serum albumin was prepared, and after sterilization by filtration membrane, heparinized acellular vascular matrix was placed in a liver growth factor solution and incubated at 37 ℃ for 2 hours with shaking (100 r/min). Wash 5 times with PBS 5 min/time. The small-caliber in-situ tissue engineering blood vessel which can inhibit thrombosis and promote blood vessel reconstruction is obtained.

Example 2

Each of the blood vessels and the relevant materials in example 1 was sampled and tested for performance by the following method

(1) Histological observation

The porcine carotid artery obtained in example 1 and the prepared acellular vascular matrix were fixed with 4% paraformaldehyde, and sections were subjected to HE staining, DAPI staining, EVG staining, and Masson staining after paraffin embedding, and the acellular effect was observed under a microscope.

Histological staining of porcine carotid artery and decellularized blood vessels is shown in fig. 1, and HE staining and DAPI staining results show that no cells and cell nucleus components remain in the blood vessel wall after decellularization treatment. EVG staining and Masson staining showed that the elastic and collagen fibers in the decellularized vessels were well preserved.

(2) Microstructure observation

The porcine carotid artery, decellularized blood vessel and heparinized decellularized blood vessel obtained in example 1 were fixed in 2.5% glutaraldehyde for 2h, washed 3 times with sterile PBS, then sequentially dehydrated and dried with gradient ethanol (50%, 70%, 80%, 95%, 100%), gold sprayed with a plating machine, and finally morphologically observed with a scanning electron microscope.

Scanning electron microscope results (fig. 2) show that the porcine carotid artery lumen is covered with a layer of intact endothelial cells, endothelial cells are not seen on the decellularized vascular luminal surface, and the vascular wall becomes loose and porous. The heparinized decellularized blood vessel has compact lumen surface and vessel wall, and is similar to the carotid artery of a pig.

(3) Detection of heparin binding content and heparin release of heparinized decellularized blood vessels

Preparing heparin standard solution, incubating 2ml toluidine blue solution and 2ml standard solution at 37 deg.C for 20min, adding n-hexane 3ml, incubating for 15min, measuring absorbance of each concentration standard solution at 631nm wavelength with spectrophotometer, and drawing standard curve. 2ml toluidine blue solution, 2ml PBS buffer solution and 5 x 5mm of the heparinized vascular matrix in example 1 were incubated at room temperature for 20min, the light absorbance was measured with the same procedure, and the bound heparin content was obtained from the light absorbance versus a standard curve.

The heparinized vascular matrix of example 1 was cut to 5X 5mm and placed in 10ml centrifuge tubes, 2 specimens per tube were added with 10ml PBS buffer and left at room temperature. And (3) pouring the PBS buffer solution in the corresponding centrifuge tube on

days

1,3,6,10,15,21 and 28 after heparinization, washing the centrifuge tube for 5 times and 5 min/time by using 10ml of PBS, measuring the residual heparin content of the sample, and subtracting the residual amount from the total heparin binding amount of the sample to obtain the release amount. The release curve is obtained by plotting the release amount at the corresponding time points.

The content of heparinized acellular vascular heparin obtained in example 1 is 94.43 +/-10.69 mu g/cm². As shown in fig. 3, the 28 days cumulative heparin release reached 67.6%. The release rate was very fast during the first 3 days and gradually stabilized after 15 days. Suggesting that the heparinized decellularized blood vessel has an important function of inhibiting thrombosis.

(4) Coagulation test

Activated Partial Thromboplastin Time (APTT) assay: the carotid artery, decellularized blood vessel and heparinized decellularized blood vessel of the pig in example 1 were cut into 3X 5mm, placed in a four-well cuvette, 50ul of standard plasma added with citric acid and 50ul of kaolin were added to the cuvette, preheated for 3 minutes, added with 50ul of calcium chloride solution, and the coagulation time was measured using a full-automatic coagulation analyzer.

The APTT (140.5 +/-22.0 s) of the heparinized cross-linked vascular stent is detected to be obviously prolonged compared with the natural artery (37.3 +/-3.1 s, p is 0.014) and the decellularized artery (35.7 +/-2.5 s, p is 0.014).

(5) Detection of HGF binding content and HGF release

Prepare 10ml of 1mg/ml bovine serum albumin solution, add 4ug HGF, take 1ml of solution (sample 1) to preserve at-20 deg.C, put 5X 5mm of heparinized vascular matrix of example 1 in growth factor solution to incubate at 37 deg.C for 2 hours with shaking (100r/min), take out blood vessel, take 1ml of solution (sample 2) to preserve at-20 deg.C. HGF concentrations in HGF samples 1 and 2 were determined according to the Human HGF Pre-Coated Elisa Kit (Peprotech) Kit procedure, with the amount of vascular stroma-bound HGF ═ 9ml (sample 1HGF concentration-sample 2HGF concentration).

The heparinized acellular vascular matrix bound to HGF was cut into 5X 5mm, placed in a 1.5ml EP tube, and 1ml of a 1mg/ml bovine serum albumin solution was added, and the tube was placed at room temperature. The blood vessel is placed in another EP tube containing 1ml of bovine serum albumin solution respectively at

days

1,2,3,5,8,12,17,23 and 30, the HGF concentration in the original EP tube is detected, namely the release amount of HGF at the corresponding time, and the HGF release curve is plotted by taking the time (days) as the abscissa and the corresponding HGF release percentage as the ordinate.

The content of HGF in the heparinized decellularized blood vessel combined with HGF is 51.23 +/-1.83 ng/cm². As shown in fig. 4, HGF was released stably from the blood vessels, with the fastest release on day one, reaching 12.32%, gradually plateauing after 20 days, and the cumulative release amount of 93.59% after 30 days.

(6) Mechanical property detection

Detection of tensile strength and elongation at break: the porcine carotid artery, decellularized blood vessel and heparinized decellularized blood vessel in example 1 were cut into 4mm × 20mm using a mold, the thickness of the sample was measured with a micrometer screw, and the breaking strength and breaking elongation were measured by stretching at a speed of 20mm/min using an electronic tensile tester.

And (3) testing the stitching strength: the porcine carotid artery, the decellularized blood vessel and the heparinized decellularized blood vessel in example 1 were cut into 4mm × 10mm using a mold, one end was fixed to the lower end of a testing machine, the other end was fixed to a cloth piece by 2 stitches with 7-0 stitches, the stitch length was 2mm, the cloth piece was fixed to the upper end of the testing machine, stretching was performed at a speed of 5mm/min, and the tensile force when the specimen was torn by the stitches was the suture strength.

And (3) testing the blasting pressure: the porcine carotid artery, decellularized blood vessel and heparinized decellularized blood vessel in example 1 were cut to 4cm long, one end was inserted into a balloon and connected to a small air compressor, the other end was ligated and sealed, and gradually pressurized until the specimen was ruptured, and the pressure in the lumen at rupture was recorded as the burst strength of the specimen.

In contrast to the porcine carotid artery, as shown in fig. 5, the maximum strength, rupture strength and suture strength of the decellularized blood vessels were significantly reduced, while the cross-linked heparinized decellularized was similar to the porcine carotid artery. The breaking elongation of the decellularized blood vessel and the heparinized decellularized blood vessel are both obviously reduced, and the explosion pressure of the three blood vessels is not obviously different. The results show that the mechanical properties of the cross-linked heparinized acellular blood vessels are enhanced and are similar to the properties of the carotid arteries of pigs.

(7) Transplantation of rabbit carotid artery

Male Japanese big ear white rabbits, 3-3.5kg body weight, fixed after isoflurane mask anesthesia, were sequentially subjected to paratracheal incision of skin, subcutaneous tissue, muscle layer, exposure and free carotid artery, and occlusion of arterial clamp, then autologous artery was cut to cause 2cm defect, the heparinized decellularized blood vessel bound with HGF in example 1 was subjected to carotid artery replacement with heparinized decellularized blood vessel not bound with HGF (inner diameter 2.5mm, length 2cm, 12 per group), end-to-end anastomosis was intermittently sutured using 7-0Prolene suture, 100U/kg heparin was administered during the procedure, and aspirin was administered at 50mg/d after the operation. The ultrasonic examination is carried out every 2 weeks after the operation to check the patency of the transplanted blood vessel. After the white rabbits are euthanized at 1,3 and 6 months after the operation, the transplanted blood vessels are taken out for observation by an optical microscope and an electronic scanning microscope.

As shown in fig. 6, in the blood vessel ultrasonic examination, both the HGF-bound heparinized decellularized blood vessel and the unbound group in example 1 showed no aneurysm, and the patency rate of the HGF-bound group was 91.67% for 6 months, which was higher than 83.33% of that of the heparin-only group. HE staining (fig. 7) showed that a layer of neointimal tissue was visible on the luminal surface, there was no significant difference in intimal thickness between the two groups at 1 month post-operation, and the intimal thickness in the HGF-bound group was significantly less than that in the heparinized only group at 3 months and 6 months post-operation. At 1 month after surgery, immunohistochemical staining with CD31 (fig. 8) and SEM (fig. 9) showed only a small number of endothelial cells on the luminal surface of the blood vessels in the heparinized group, and an intact layer of endothelial cells was seen in the HGF group. After 3 months of operation, endothelial cells of two groups of transplanted blood vessels are seen to completely cover the cavity surface of the tube, and the endothelial cells combined with HGF group of the cavity surface of the tube are connected more tightly. After 6 months of operation, the endothelial cells of the two groups of transplanted vascular cavity surfaces are completely and tightly covered.

The invention has industrial applicability: the decellularized blood vessel of the present invention which binds HGF and heparin can be used, for example, for the manufacture of materials for medical applications.

The above listing of a series of detailed descriptions is merely a detailed description of possible embodiments of the present invention and is not intended to limit the scope of the invention, and one skilled in the art may devise many other modifications and embodiments that will fall within the spirit and scope of the principles disclosed herein. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure, the drawings and the appended claims. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.

Claims

1. A small-caliber tissue engineering blood vessel which is constructed by acellular vascular matrix and can promote rapid endothelialization is characterized by being prepared by the following method: preparing acellular vascular matrix from the carotid artery of the pig by using detergent TritonX-100 in combination with sodium dodecyl sulfate, reacting in EDC/NHS solution containing heparin for 24 hours to perform crosslinking and grafting of heparin, immersing in bovine albumin serum solution containing hepatocyte growth factor, and incubating for 2 hours to obtain the small-caliber in-situ tissue engineering blood vessel.

2. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: after the acellular blood vessel is crosslinked, the small-caliber in-situ tissue engineering blood vessel is prepared by combining heparin and hepatocyte growth factor.

3. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: the decellularized blood vessel is selected from the group consisting of an animal aorta, carotid artery, internal mammary artery, radial artery, and a gastrointestinal artery.

4. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: the cell removal treatment is carried out by adopting TritonX-100 and SDS.

5. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: crosslinking is carried out with a solution of EDC/NHS containing heparin and at the same time grafting of the heparin.

6. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: the obtained heparinized decellularized blood vessels were sterilized using gamma rays.

7. The small-caliber tissue engineering blood vessel constructed by acellular vascular matrix capable of promoting rapid endothelialization according to claim 1, wherein: HGF is bound by incubating heparinized decellularized blood vessels by immersion in HGF solution.

8. The method for preparing a small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization as claimed in claim 1, wherein the method comprises the following steps:

(2) Preparing acellular vascular matrix;

(3) preparing a heparinized acellular vascular matrix;

9. The method for preparing small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization as claimed in claim 8, wherein the small-caliber tissue engineering blood vessel comprises the following steps: the method for preparing the acellular vascular matrix in the step (2) comprises the following steps: placing the obtained carotid artery of the pig in distilled water, placing the carotid artery of the pig on a decoloration shaking table (100r/min) for treatment for 24 hours to crack blood cells in a lumen; then putting the mixture into a 1 percent (m/v) TritonX-100 solution, and carrying out oscillation treatment for 24 hours (100 r/min); after being washed for 2h by PBS, the blood vessel is put into 0.25-1.0% (m/v) SDS solution, and is continuously placed on a decoloring shaker (100r/min) for processing for 24-72h, and the SDS solution is replaced every 24 h; finally washing with PBS for 72h (100r/min) with shaking to remove the detergent residue; the cell removing process is carried out in a sterile environment at room temperature; the obtained acellular vascular matrix was stored in PBS at-20 ℃.

10. The method for preparing small-caliber tissue engineering blood vessel constructed by acellular vascular matrix and capable of promoting rapid endothelialization as claimed in claim 8, wherein the small-caliber tissue engineering blood vessel comprises the following steps: the method for preparing the heparinized acellular vascular matrix comprises the following steps: preparing 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, sequentially adding heparin sodium, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (200 mg of heparin, 108mg of EDC, 67mg of NHS and 40ml of 30mmol/LMES buffer solution are required for 1g of acellular vascular matrix (dry weight)), and preparing heparin activation crosslinking solution after ice bath for 2 hours; placing the acellular vascular matrix into a heparin activated cross-linking solution, incubating for 24 hours at 37 ℃, and then washing for 5 times and 5 min/time by using a PBS solution; hermetically packaging the obtained heparinized acellular vascular matrix, and performing gamma ray irradiation sterilization;

the method for preparing the small-caliber in-situ tissue engineering blood vessel capable of inhibiting thrombosis and promoting rapid endothelialization comprises the following steps: preparing a Hepatocyte Growth Factor (HGF) solution (200 ng/ml) containing 1mg/ml bovine serum albumin, sterilizing by a filtration membrane, placing the heparinized acellular vascular matrix in the HGF solution, and incubating for 2 hours at 37 ℃ under oscillation (100 r/min); washing with PBS for 5 times (5 min/time); the small-caliber in-situ tissue engineering blood vessel which can inhibit thrombosis and promote rapid endothelialization is obtained.