Acellular matrix artificial blood vessel or membrane material for realizing inflammation regulation and preparation method of acellular matrix artificial blood vessel or membrane material

Abstract

The invention belongs to the field of biological materials and tissue engineering, and particularly relates to an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation and a preparation method of the acellular matrix artificial blood vessel or membrane material. The preparation method comprises the following steps: 1) modifying alkynyl molecules on the surface of an acellular matrix artificial blood vessel or a membrane material; 2) modifying an azide group on the surface of the stem cell; and 3) carrying out mixed culture on the stem cells of which the surfaces are modified by the azide groups obtained in the step 2) and the acellular matrix artificial blood vessel or membrane material of which the surfaces are modified by the alkynyl molecules obtained in the step 1) to obtain the acellular matrix artificial blood vessel or membrane material for realizing inflammation regulation. According to the technical scheme, the biological orthogonal reaction is used for mediating rapid and stable combination between the surface modified artificial blood vessel or membrane material and the azide group labeled stem cells, so that the acellular matrix artificial blood vessel / membrane material stably loads the stem cells before transplantation; after being implanted into a body, the artificial blood vessel graft becomes an ideal anti-inflammatory and regeneration-promoting artificial blood vessel graft.

Description

Technical Field

[0001] The present invention belongs to the biological field, and in particular relates to an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation and a preparation method thereof. Background Art

[0002] Immune cells are recruited to the biomaterial early after implantation of tissue-engineered blood vessels and establish a microenvironment for subsequent tissue regeneration. Excessive inflammatory responses can lead to thrombosis and stenosis in vascular grafts, hindering rapid remodeling of the scaffold. Therefore, regulating immune cells to establish a pro-regenerative environment is crucial. Mesenchymal stem cells (MSCs) are mesoderm-derived cells that reside in the stroma of solid organs and function as precursors of non-hematopoietic connective tissue. They possess the ability to differentiate into both mesenchymal and non-mesenchymal lineages and have potential clinical applications in repairing damaged tissues. Furthermore, MSCs have been extensively studied for their role in modulating immune responses, treating allograft rejection and acute graft-versus-host disease, and alleviating experimental autoimmune encephalomyelitis, collagen-induced arthritis, and autoimmune myocarditis. Currently, loading acellular matrices with active substances such as growth factor VEGF into artificial blood vessels has been shown to promote vascular graft regeneration by recruiting stem cells in vivo. However, in vivo stem cell recruitment strategies suffer from limited duration and low recruitment efficiency, limiting their translation into clinical practice.

[0003] Bioorthogonal chemistry exhibits excellent biocompatibility, specificity, and rapidity under physiological conditions. It is an advanced method that forms covalent bonds in a short time and can be applied to living cells. Combined with the cell modification methods of metabolic glycoengineering, bioorthogonal azide groups are non-destructively anchored to the cell surface, providing a foundation for bioorthogonal reactions. Therefore, the combination of bioorthogonal chemistry and metabolic glycoengineering of living cells is expected to become a chemical "bridge" for stem cell implantation in artificial blood vessels, enabling stem cell implantation in acellular matrix artificial blood vessels. Improving the immunomodulatory activity of acellular matrix artificial blood vessels is also key to solving the difficulties in the development and translation of acellular matrix artificial blood vessels. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and to provide an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation and a preparation method thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation comprises the following steps:

[0007] 1) Modifying alkyne molecules on the surface of acellular matrix artificial blood vessels or membrane materials;

[0008] 2) modifying the surface of the stem cells with azide groups; steps 1 and 2) are performed in any order;

[0009] 3) Mixing and culturing the stem cells with modified azide groups on the surface obtained in step 2) and the acellular matrix artificial blood vessels or membrane materials with modified alkyne molecules on the surface obtained in step 1) to obtain acellular matrix artificial blood vessels or membrane materials that can achieve inflammation regulation.

[0010] The specific steps of step 1) are: modifying the surface of the decellularized matrix artificial blood vessel or membrane material with alkynyl molecules through EDC / NHS cross-linking reaction; the grafting density of the alkynyl molecules on the decellularized matrix artificial blood vessel or membrane material is 0.075 nmol / mg to 0.30 nmol / mg, preferably 0.15 nmol / mg.

[0011] The alkynyl molecule includes at least one of dibenzocyclooctene DBCO, bicyclononyne DIBAC, 4-dibenzocyclooctanol DIBO, or 2-cyclooctyne-acetic acid, preferably dibenzocyclooctene DBCO.

[0012] The acellular matrix artificial blood vessels or membrane materials are obtained by decellularizing natural blood vessels, or by decellularizing biological conduits constructed by in vivo engineering technology, or by decellularizing tissue-engineered blood vessels obtained by in vitro cell culture.

[0013] Step 2) is to modify the cell surface of stem cells with azide groups based on sugar metabolism technology; preferably, the azide group is tetraacetyl-N-azidoacetylmannosamine, Ac4ManNAz; preferably, it specifically includes the following steps: continuously culturing the stem cells in a stem cell culture medium supplemented with azide groups, and performing sugar metabolism modification to obtain stem cells with surface modified azide groups.

[0014] The stem cells include at least one of mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or human umbilical cord mesenchymal stem cells; preferably, the stem cells are mesenchymal stem cells.

[0015] The specific steps of step 3) are: injecting the stem cell suspension with surface modified azide groups into the surface of the polymer artificial blood vessel / membrane material with surface modified alkyne groups, and incubating in a cell culture incubator at 37°C for 1-2.0 hours to achieve efficient and stable stem cell coverage to obtain acellular matrix artificial blood vessels or membrane materials that can achieve inflammation regulation.

[0016] The present invention also includes an acellular matrix artificial blood vessel or membrane material obtained by the preparation method and capable of achieving inflammation regulation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Traditional decellularized artificial blood vessels have limited inflammatory regulation functions. The technical solution of this application utilizes bioorthogonal reactions to mediate rapid and stable binding between surface-modified artificial blood vessels or membrane materials and azide-labeled stem cells, so that the decellularized matrix artificial blood vessels / membrane materials can be stably loaded with stem cells before transplantation. After implantation into the body, they become ideal artificial vascular grafts with anti-inflammatory and pro-regeneration properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation process of acellular matrix artificial blood vessels or membrane materials for achieving inflammation regulation;

[0020] Figure 2 The figure and scanning electron microscope image of the PCL fiber skeleton reinforced acellular matrix artificial blood vessel constructed in Example 3.

[0021] Figure 3 The anti-scouring performance of the azide-modified MSCs cells planted in Comparative Example 2 and Example 3.

[0022] Figure 4 This is a cross-sectional view of a patent blood vessel 2 weeks after implantation into the mouse carotid artery in Example 8. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0024] Example: Method for preparing acellular matrix artificial blood vessels or membrane materials for achieving inflammation regulation ( Figure 1 ), comprising the following steps:

[0025] 1) Modifying alkyne molecules on the surface of acellular matrix artificial blood vessels or membrane materials;

[0026] S1) A polycaprolactone (PCL) fiber skeleton is prepared on a silicone tube / membrane using melt spinning technology. The silicone tube or membrane together with the PCL fiber skeleton is implanted subcutaneously in an animal as a template for in vivo engineering culture.

[0027] S2) After the cells and extracellular matrix have completely filled the pores of the fibrous skeleton, they are removed and excess tissue is trimmed.

[0028] S3) removing the silicone tube / membrane and performing a decellularization process to obtain an artificial blood vessel or membrane material with an effective integration of the PCL fiber skeleton and the decellularized matrix, which is called a PCL fiber skeleton reinforced decellularized matrix artificial blood vessel / membrane material.

[0029] S4) Modifying dibenzocyclooctene groups (DBCO for short) on the PCL fiber skeleton reinforced acellular matrix artificial blood vessel / membrane material by an EDC / NHS cross-linking reaction, specifically: fully activating the carboxyl groups on the inner cavity surface of the PCL fiber skeleton into NHS groups in a 0.1 M EDC / NHS reaction solution prepared in MES buffer, and then cross-linking and modifying them with dibenzocyclooctene groups (DBCO for short) with amino terminals to ensure that the final DBCO density in the artificial blood vessel / membrane material is 0.038±0.005nmol / mg (Example 1), 0.075±0.009nmol / mg (Example 2), 0.15±0.021nmol / mg (Example 3), 0.30±0.032nmol / mg (Example 4), 0.60±0.054nmol / mg (Example 5), and 0nmol / mg (Comparative Example 1, i.e., unmodified DBCO groups).

[0030] The 1.0mm diameter PCL fiber skeleton reinforced acellular matrix artificial blood vessels were used for mouse carotid artery transplantation; the 2.0mm diameter PCL fiber skeleton reinforced acellular matrix artificial blood vessels were used in in vitro anti-scour experiments; the PCL fiber skeleton reinforced acellular matrix membrane material prepared by combining silicone sheet with melt-spun PCL fiber skeleton was used in membrane-related experiments. Figure 2 As shown, in the 1.0 mm and 2.0 mm caliber PCL fiber skeleton reinforced acellular matrix artificial blood vessels prepared in Example 3, the acellular matrix completely filled the pores of the PCL fiber skeleton.

[0031] 2) Modifying the surface of stem cells with azide groups: After MSCs were treated with 20 μM Ac4ManNAz for 48 hours, their surfaces were modified with azide groups. The cells were then collected to obtain stem cells with azide groups on their surfaces.

[0032] 3) Injecting the stem cell suspension modified with azide groups onto the surface of the polymer artificial blood vessel / membrane material modified with alkyne groups, and incubating in a cell culture incubator at 37°C for 1-2.0 hours can achieve efficient and stable stem cell coverage to obtain acellular matrix artificial blood vessels or membrane materials that can achieve inflammation regulation.

[0033] 3.1, Modification of membrane materials

[0034] The membrane materials obtained in Examples 1-5 and Comparative Example 1 in step 1) were punched and placed in a 48-well plate in DMEM / F12 complete medium. The stem cells with surface modified azide groups obtained in step 2) were plated at 3×10 5 pieces / cm 2The cells were inoculated at a density of 1.5 on the membrane materials of Examples 1-5 and Comparative Example 1 to obtain acellular matrix artificial blood vessels or membrane materials that achieve inflammation regulation. After the cells were incubated for 2.0 hours, the unbound cells were washed away. At the same time, Comparative Example 2 was set up, and the seeded cells used were MSCs that had not been treated with sugar metabolism. The other conditions were the same as Comparative Example 1.

[0035] 1. Cell Adhesion Test: DAPI staining was used to visualize adhesion and statistical analysis showed that the cell adhesion to the membranes of Example 1, Comparative Example 1, and Comparative Example 2 was extremely low, with no significant difference between the three. The number of cells adhering to the membranes of Examples 2 and 3 increased with increasing surface azide group modification concentrations, with both groups significantly exceeding the number of cells adhering to the membranes of Examples 1, Comparative Example 1, and Comparative Example 2. The number of cells adhering to the membranes of Examples 4 and 5 was comparable to that of Example 3, with no further improvement. These results demonstrate that azide-modified MSCs can rapidly undergo bioorthogonal reactions with the DBCO on the membranes, enabling rapid and stable cell seeding. Furthermore, the DBCO modification density on the surface of the material of Example 3 enables saturated cell adhesion.

[0036] 2. Cell proliferation test: CCK8 assay was performed after 1, 3, and 5 days of culture. The results showed that the cells on the membrane materials of Examples 1-3, Comparative Example 1, and Comparative Example 2 continued to proliferate. Among them, the proliferation of Example 1, Comparative Example 1, and Comparative Example 2 was comparable, the proliferation of Examples 2-4 was significantly better than that of Example 1, and the optimal cell proliferation occurred in Example 3. The cells on the membrane material of Example 5 basically did not proliferate, and the proliferation rate on the 5th day was lower than that of Comparative Example 1, indicating that the excessively high DBCO modification density caused cytotoxicity.

[0037] The adhesion and growth analysis capabilities of MSCs cells with different modification densities of azide groups on PCL fiber skeleton reinforced acellular matrix membrane materials are shown in Table 1.

[0038] Table 1

[0039]

[0040] 3.2 Modification of artificial vascular materials

[0041] MSCs were treated with 20 μM Ac4ManNAz for 48 hours, and then the surface of MSCs was modified with azide. The cells were collected and DiR-labeled, and 3×10 5 pieces / cm 2 The artificial vascular materials prepared in Examples 1-5 and Comparative Example 1 were seeded at a density of 1:1 and incubated for 2.0 hours, rotating 90° every 15 minutes to achieve uniform seeding. Comparative Example 2 was also set up: DiR-labeled MSCs without glucose metabolism treatment were used as the seeding cells, and all other conditions were the same as in Comparative Example 1.

[0042] 1. Blood flow resistance test: The flow rate of the culture medium in the in vitro flow culture device was set to 10 cm / s to simulate the blood flow velocity of the human coronary artery. The artificial blood vessels seeded with MSCs cells were perfused at this flow rate for 12 hours. Finally, the fluorescence intensity at 0 hours and after flushing was observed and quantitatively compared under in vivo fluorescence imaging. Based on this, the ratio of the fluorescence after flushing to that before flushing was calculated as the cell retention rate. Figure 2 As shown in the fluorescence imaging images of the blood vessels before and after flushing in Comparative Example 2 and Example 3, as well as the flux data calculated based on the fluorescence images, it can be seen that the cell retention rate of the artificial blood vessel in Example 3 is significantly higher than that in Comparative Example 2. Further analysis shows that the cell retention rate on the artificial blood vessel material of Example 1 is comparable to that of Comparative Examples 1 and 2; the cell retention rates of Examples 2-5 are all significantly higher than those of Comparative Example 1, Comparative Example 2, and Example 1; among them, the cell retention rate of the artificial blood vessel in Example 3 is the highest.

[0043] The results of the analysis of the anti-blood flow scour ability of MSCs cells seeded on PCL fiber skeleton reinforced acellular matrix artificial blood vessels with different DBCO modification densities are shown in Table 2. Figure 3 The anti-scouring performance of the azide-modified MSCs cells planted in Comparative Example 2 and Example 3.

[0044] Table 2

[0045] Group Cell retention rate% Comparative Example 1 9.32±1.12 Comparative Example 2 8.56±1.45 Example 1 13.12±2.45 Example 2 49.64±5.34 Example 3 73.34±7.63 Example 4 58.11±8.43 Example 5 43.61±7.02

[0046] 2. Vascular transplantation test: After the inoculation, the cells were transplanted into the carotid artery of nude mice. Two weeks after implantation, the patency rates of Comparative Examples 1 and 2 were 0%, the patency rate of Example 1 was 0%, the patency rate of Examples 2 and 4 was 50%, the patency rate of Example 3 was 90%, and the patency rate of Example 5 was 10% (Table 3).

[0047] 3. Inflammation regulation test: In order to detect the inflammatory regulation of transplanted MSCs, we performed co-staining tests of CD68 and iNOS as well as CD68 and CD206. iNOS positivity represents M1 (pro-inflammatory) macrophages in the transplant, CD206 positivity represents M2 (anti-inflammatory) macrophages in the transplant, and CD68 positivity represents all macrophages in the transplant, including M1 and M2 macrophages. The results showed that the ratios of iNOS-positive cells to CD68-positive cells in Comparative Example 1, Example 1 and Example 5 were 78.22±4.13%, 80.43±6.67% and 76.26±3.38%, respectively; the ratios of CD206-positive cells to CD68-positive cells were 18.93±5.16%, 15.74±6.25% and 16.42±6.16%, respectively; the ratios of iNOS-positive cells to CD68-positive cells in Example 2 were 71.33±3.56%, and the ratios of CD2 The ratio of iNOS-positive cells to CD68-positive cells was 28.90±4.36%, the ratio of iNOS-positive cells to CD68-positive cells in Example 3 was 38.10±5.61%, and the ratio of CD206-positive cells to CD68-positive cells was 51.45±6.43%, and the ratio of iNOS-positive cells to CD68-positive cells in Example 4 was 63.14±3.56%, and the ratio of CD206-positive cells to CD68-positive cells was 33.56±5.16% (Table 3). The above co-staining results indicate that the stem cells implanted under the conditions of Example 3 are most effective in reducing graft inflammation and maintaining graft patency.

[0048] 4. Test of artificial blood vessel regeneration ability: In order to evaluate the ability of implanted MSCs to promote artificial blood vessel regeneration, we conducted statistical analysis on the regeneration of endothelium (CD31 staining) and smooth muscle (α-SMA staining) of the grafts. The results showed that the endothelial cell coverage of Comparative Example 1, Comparative Example 2 and Example 1 was 0%, and the smooth muscle layer thickness was 0 μm; the endothelial cell coverage of Example 2 was 50.64±11.22%, and the smooth muscle layer thickness was 23±4.2 μm; the endothelial cell coverage of Example 3 was the highest, which was 86.82±10.31%, and the smooth muscle layer thickness was the thickest, which was 41±5.8 μm; the endothelial cell coverage of Example 4 was 56.61±12.38%, and the smooth muscle layer thickness was 25±3.9 μm; the endothelial cell coverage of Example 5 was 0%, and the smooth muscle layer thickness was 0 μm (Table 3).

[0049] Table 3

[0050]

[0051] Based on the above experiments, we screened out the membrane material and tube material of Example 3 as having the optimal DBCO modification concentration. On this basis, we tested the effects of different incubation times on MSCs in vitro. Specific examples and comparative examples are as follows:

[0052] Example 6: Cells were seeded using the DBCO-modified PCL fiber skeleton reinforced acellular matrix artificial blood vessel or tube material prepared in Example 3. After MSCs were treated with 20 μM Ac4ManNAz for 48 hours, the surface of the MSCs was modified with azide. The cells were collected and plated at 3×10 5 pieces / cm 2 For membrane materials, the inoculation density was 3×10 5 / cm 2 The tubes were seeded with azide-modified MSCs at a density of 100 μg / cm2 and incubated for 60 min, rotating the tubes 90° every 15 min to achieve uniform seeding of the tube material.

[0053] Example 7: Same as Example 6, but the cell seeding time is 90 minutes.

[0054] Example 8: Same as Example 6, but the cell seeding time is 120 minutes.

[0055] Example 9: Same as Example 6, but the cell seeding time is 150 minutes.

[0056] Comparative Example 3: Cell seeding was performed using the non-DBCO-modified membrane and tube materials prepared in Comparative Example 1. The azide-modified MSCs were seeded at the same density and method as in Example 6, but the cell seeding time was 150 minutes. Comparative Example 3 relied on natural cell adhesion, which is proportional to the seeding time. For the non-DBCO-modified membrane and tube materials, cell adhesion at 150 minutes was the highest among the 60-150 minute incubation times. Therefore, a single comparative example with a 150-minute incubation time was established to illustrate the advantages of cell seeding based on bioorthogonal reactions.

[0057] Comparative Example 4: Cell seeding was performed using the non-DBCO-modified membrane and tube materials prepared in Comparative Example 1. MSCs were also seeded without azide modification. The seeding density and method were the same as in Example 6, but the cell seeding time was 150 minutes. Comparative Example 4 relies on natural cell adhesion, which is proportional to the seeding time. For the non-DBCO-modified membrane and tube materials, cell adhesion at 150 minutes was the highest within the 60-150 minute incubation period. Therefore, a single comparative example with a 150-minute incubation time is sufficient to illustrate the advantages of cell seeding based on bioorthogonal reactions.

[0058] 1. The adhesion evaluation of MSCs cells on the PCL fiber skeleton reinforced acellular matrix membrane material with the optimal DBCO modification density at different implantation times is shown in Table 4.

[0059] Cell adhesion in Comparative Examples 3 and 4 relied solely on natural cell adhesion, without the bioorthogonal click reaction to accelerate cell adhesion. The number of cells adhering to Examples 6-8 gradually increased, significantly exceeding that of Comparative Examples 3 and 4. The number of cells adhering to Example 9 was comparable to that of Example 8, with no further significant increase. These results demonstrate that bioorthogonality can promote cell adhesion, reaching saturated adhesion density after 120 minutes of incubation (Table 4).

[0060] Table 4

[0061]

[0062]

[0063] 2. Evaluation of the anti-blood flow scour ability of MSCs cells on PCL fiber skeleton reinforced acellular matrix artificial blood vessels with optimal polypeptide modification density at different implantation times: In order to achieve fluorescent quantitative analysis of the retention ratio, the cells in Examples 6-9 and Comparative Examples 3-4 were labeled with near-infrared fluorescent probe DIR. The inoculated tube material was connected to the in vitro flow culture system, and the flow rate of the culture medium in the in vitro flow culture device was set to 10 cm / s, simulating the human coronary blood flow velocity. The artificial blood vessels inoculated with MSCs cells were perfused at this flow rate for 12 hours, and then the retention efficiency of the cells was monitored at each time point using the IVIS imaging system. The results showed that the cell retention rates of Comparative Examples 3 and 4 were lower than those of the examples. The cell retention rates of Examples 6-8 increased with the increase of adhesion time; the cell retention rate of Example 9 was equivalent to that of Example 8, and there was no further significant increase. The above results show that bio-orthogonal cell adhesion can improve the anti-scour ability of cells, and reach saturated anti-scour ability at incubation for 120 minutes (Table 5).

[0064] Table 5

[0065]

[0066] 3. Vascular transplantation test

[0067] We also performed carotid artery transplantation of nude mice using MSCs cells implanted on PCL fiber skeleton reinforced acellular matrix artificial blood vessels with optimal DBCO modification density after different implantation times. Two weeks after implantation, the patency rates of Comparative Examples 3 and 4 were 20%; the patency rates of Example 6 were both 60%; the patency rates of Example 7 were both 70%; and the patency rates of Examples 8-9 were all 90% (Table 6). Figure 4This is a cross-sectional view of a patent blood vessel 2 weeks after implantation into the mouse carotid artery in Example 8.

[0068] In order to detect the inflammatory regulation of transplanted MSCs, we performed co-staining experiments of CD68 and iNOS as well as CD68 and CD206. iNOS positivity represents M1 (pro-inflammatory) macrophages in the transplant, CD206 positivity represents M2 (anti-inflammatory) macrophages in the transplant, and CD68 positivity represents all macrophages in the transplant, including M1 and M2 macrophages. The results showed that the ratios of iNOS-positive cells to CD68-positive cells in Comparative Examples 3 and 4 were 87.23±6.25% and 86.46±7.51%, respectively, and the ratios of CD206-positive cells to CD68-positive cells were 13.78±2.35% and 16.64±5.21%, respectively. The ratios of iNOS-positive cells to CD68-positive cells in Examples 6, 7, 8, and 9 were 72.13±5.38%, 50.44±9.32%, 38.10±5.61%, and 39.61±7.38%, respectively, and the ratios of CD206-positive cells to CD68-positive cells were 28.82±8.28%, 48.91±4.45%, 51.45±6.43%, and 49.76±5.23%, respectively (Table 6). The above results show that stem cell lines based on bioorthogonal reactions can regulate inflammatory responses, effectively reduce the ratio of pro-inflammatory polarized macrophages, and increase the ratio of anti-inflammatory polarized macrophages, and achieve saturated immune regulation effects after incubation for 120 minutes. Figure 4 As shown, the lumen of the patent vascular graft of Example 8 is smooth and free of thrombosis.

[0069] In order to evaluate the ability of MSCs to promote artificial blood vessel regeneration, we conducted a statistical analysis of the regeneration of the endothelium (CD31 staining) and smooth muscle (α-SMA staining) of the grafts. The results showed that the endothelial cell coverage of Examples 3 and 4 was 0%, and the smooth muscle layer thickness was 0 μm; the endothelial cell coverage of Examples 6, 7, 8, and 9 was 30.12±7.31%, 50.2±6.87%, 86.82±8.31%, and 87.23±9.12%, respectively. The smooth muscle layer thickness was 16±3.1μm, 20±4.7μm, 41±5.8μm, and 42±5.2μm, respectively. The above results show that endothelial cell planting based on bioorthogonal reaction can increase the rate of artificial blood endothelialization and achieve saturated angiogenesis-promoting effect at 120 minutes of incubation (Table 6).

[0070] Table 6

[0071]

[0072]

[0073] The results show that the inflammatory regulation function of traditional decellularized artificial blood vessels is limited. The technical solution of this application uses bioorthogonal reactions to mediate rapid and stable binding between surface-modified artificial blood vessels or membrane materials and azide-labeled stem cells, so that the decellularized matrix artificial blood vessels / membrane materials can be stably loaded with stem cells before transplantation, and after implantation into the body, they become ideal artificial vascular grafts with anti-inflammatory and pro-regeneration properties.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation, characterized in that: The steps include: 1) Modifying alkyne molecules on the surface of acellular matrix artificial blood vessels or membrane materials; 2) modifying the surface of the stem cells with azide groups; steps 1 and 2) are performed in any order; 3) Mixing and culturing the stem cells with modified azide groups on the surface obtained in step 2) and the acellular matrix artificial blood vessels or membrane materials with modified alkyne molecules on the surface obtained in step 1) to obtain acellular matrix artificial blood vessels or membrane materials that can achieve inflammation regulation.

2. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 1, characterized in that: The specific steps of step 1) are: modifying the surface of the decellularized matrix artificial blood vessel or membrane material with alkynyl molecules through EDC / NHS cross-linking reaction; the grafting density of the alkynyl molecules on the decellularized matrix artificial blood vessel or membrane material is 0.075 nmol / mg to 0.15 nmol / mg.

3. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 2, characterized in that: The alkynyl molecule includes at least one of dibenzocyclooctene DBCO, bicyclononyne DIBAC, 4-dibenzocyclooctanol DIBO, or 2-cyclooctyne-acetic acid, preferably dibenzocyclooctene DBCO.

4. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 1, characterized in that: The acellular matrix artificial blood vessels or membrane materials are obtained by decellularizing natural blood vessels, or by decellularizing biological conduits constructed by in vivo engineering technology, or by decellularizing tissue-engineered blood vessels obtained by in vitro cell culture.

5. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 1, characterized in that: Step 2) is to modify the cell surface of stem cells with azide groups based on sugar metabolism technology; preferably, the azide group is tetraacetyl-N-azidoacetylmannosamine, Ac4ManNAz; preferably, it specifically includes the following steps: continuously culturing the stem cells in a stem cell culture medium supplemented with azide groups, and performing sugar metabolism modification to obtain stem cells with surface modified azide groups.

6. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 1, characterized in that: The stem cells include at least one of mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or human umbilical cord mesenchymal stem cells; preferably, the stem cells are mesenchymal stem cells.

7. The method for preparing an acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation according to claim 1, characterized in that: The specific steps of step 3) are: injecting the stem cell suspension with surface modified azide groups into the surface of the polymer artificial blood vessel / membrane material with surface modified alkyne groups, and incubating in a cell culture incubator at 37°C for 1-2.0 hours to achieve efficient and stable stem cell coverage to obtain acellular matrix artificial blood vessels or membrane materials that can achieve inflammation regulation.

8. An acellular matrix artificial blood vessel or membrane material for achieving inflammation regulation obtained by the preparation method according to any one of claims 1 to 7.

Images