Preparation method and application of gel bio-ink for 3D printing of small-diameter artificial blood vessel

Abstract

The invention discloses a preparation method and application of gel bio-ink for 3D printing of a small-diameter artificial blood vessel. The preparation method comprises the following steps: providing methylacryloylated recombinant collagen; providing methacrylated hyaluronic acid; the gel bio-ink for preparing the 3D printing small-diameter artificial blood vessel is obtained by taking methylacryloylated recombinant collagen and methylacryloylated hyaluronic acid as raw materials. The gel bio-ink has a strong photocuring characteristic and has a shear thinning characteristic required by bio-printing, an artificial blood vessel stent with the diameter of 6 mm and the height of 1 cm can be successfully prepared through a photocuring 3D printing technology, and the artificial blood vessel stent meets the size requirement of small-diameter blood vessels. An intravascular stent printed by using the gel bio-ink has the advantages of good blood compatibility, effective promotion of rapid endothelialization of the stent, prevention of vascular restenosis and the like, is a potential candidate of a small-caliber blood vessel substitute in the future, and has a wide application prospect in the artificial blood vessel market.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a preparation method and application of gel bio-ink used for 3D printing of small-diameter artificial blood vessels. Background Art

[0002] Blood vessels are mainly composed of collagen fibers, elastic fibers, glycosaminoglycans and other macromolecules. As the main channel for blood circulation, they undertake important functions such as providing oxygen and nutrients to various tissues and cells of the body, regulating body temperature and participating in immune response. In the process of treating cardiovascular diseases, due to the limitations of autologous blood vessels, artificial blood vessels are considered to be an important substitute for autologous grafts. Large-diameter vascular grafts (D>6mm) have been used clinically, however, there are great limitations in the application of small-diameter vascular grafts (D<6mm).

[0003] In the field of small-diameter vascular stents, the problem of restenosis has always been a key difficulty in the research process of small-caliber artificial blood vessels. Due to the narrow lumen of small-diameter vascular stents, the flow of blood in them is easily hindered, thereby increasing the risk of thrombosis and vascular occlusion. The key to solving this problem is to achieve rapid endothelialization. Endothelial cells are an important component of the inner wall of blood vessels. They can secrete a variety of bioactive substances to maintain the normal function of blood vessels. By promoting the rapid growth and coverage of endothelial cells on the surface of small-diameter vascular stents, the occurrence of thrombosis and restenosis can be effectively reduced. How to achieve rapid endothelialization is the key to solving this problem.

[0004] 3D printing has been widely used in regenerative medicine and has promoted significant innovations. In addition, in clinical treatment, the success rate of surgery and the patient's recovery effect are often limited to a certain extent due to the lack of ideal biomimetic natural blood vessel components. Hydrogel materials with excellent biocompatibility have become a hot topic in this research field because their physical and chemical properties are very similar to those of the extracellular matrix. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels in view of the deficiencies of the above-mentioned prior art. The gel bio-ink prepared by the present invention has strong photocuring properties and possesses the shear thinning properties required for bioprinting. Through photocuring 3D printing technology, an artificial blood vessel stent with a diameter of 6 mm and a height of 1 cm can be successfully prepared, which meets the size requirements of small-diameter blood vessels (D<6 mm). The blood vessel stent has good blood compatibility, can effectively promote the proliferation and migration of endothelial cells, and promote the rapid endothelialization of the stent, thereby preventing restenosis of the blood vessel.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels, characterized in that it comprises the following steps:

[0007] Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain a liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain a liquid B;

[0008] Step 2: Mix the liquid A and liquid B in step 1 to obtain a gel solution;

[0009] Step 3: Place the gel solution in step 2 in a 4°C refrigerator for 8 to 36 hours to obtain the gel bio-ink used for 3D printing of small-diameter artificial blood vessels.

[0010] The above-mentioned method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the preparation method of methacryloyl recombinant collagen described in step one specifically includes: adding glycidyl methacrylate to the recombinant collagen solution, stirring and reacting in a 37°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain methacryloyl recombinant collagen.

[0011] The above-mentioned method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the amount of glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen.

[0012] The above-mentioned method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the preparation method of methacryloyl hyaluronic acid described in step one specifically includes: adding glycidyl methacrylate to the hyaluronic acid solution, stirring and reacting in a 60°C water bath for 6h to 8h, dialyzing, and freeze-drying to obtain methacryloyl hyaluronic acid.

[0013] The above-mentioned method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate.

[0014] The above-mentioned method for preparing gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the photoinitiator in step one is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

[0015] The above-mentioned method for preparing the gel bio-ink used for 3D printing of small-diameter artificial blood vessels is characterized in that the mass-volume concentration of methacryloyl recombinant collagen in the gel solution in step 2 is 5% to 10%, the mass-volume concentration of methacryloyl hyaluronic acid is 1.5% to 2%, and the mass-volume concentration of the photoinitiator is 0.1% to 0.25%.

[0016] Furthermore, the present invention also provides an application of the gel bio-ink prepared by the above method in preparing a 3D printed stent for preparing an artificial blood vessel.

[0017] The above-mentioned application is characterized in that the preparation method of the 3D printed stent for preparing an artificial blood vessel comprises: loading the gel bio-ink into the barrel of a 3D printer for 3D printing, while printing, the gel bio-ink is extruded and exposed to ultraviolet light and irradiated for 3min to 5min for photo-crosslinking and curing, and sterilizing to obtain a 3D printed stent for an artificial blood vessel.

[0018] The above application is characterized in that the nozzle specification of the 3D printer is 23G-26G, the printing speed is 10mm / s-30mm / s, the printing temperature is 25°C, and the extrusion amount is 4%-8%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The gel bio-ink of the present invention has strong photocuring properties and possesses the shear-thinning properties required for bioprinting. Through photocuring 3D printing technology, an artificial vascular stent with a diameter of 6 mm and a height of 1 cm can be successfully prepared, which meets the size requirements of small-diameter blood vessels (D<6 mm). The vascular stent has good blood compatibility, can effectively promote the proliferation and migration of endothelial cells, and promote the rapid endothelialization of the stent, thereby preventing vascular restenosis.

[0021] 2. The present invention uses recombinant collagen modified with glycidyl methacrylate, and its photosensitivity can effectively promote the ultraviolet curing process of biological ink to obtain the target 3D printed hydrogel scaffold; the hyaluronic acid modified with glycidyl methacrylate can give full play to its excellent rheological properties, so that the biological ink has fluidity and mechanical stability, which is convenient for the extrusion of the biological ink and the molding of the extrudate.

[0022] 3. The present invention utilizes the unique properties and functions of recombinant collagen and hyaluronic acid to effectively achieve rapid endothelialization. Recombinant collagen can provide a good scaffold structure for the growth of endothelial cells. Its unique three-dimensional reticular structure can simulate the extracellular matrix environment of natural blood vessels and create suitable conditions for the adhesion, migration and proliferation of endothelial cells. Hyaluronic acid participates in the signal transduction process of cells in vascular tissue, regulates the growth, migration and differentiation of endothelial cells, and can promote the endothelialization of artificial blood vessels. Hyaluronic acid and recombinant collagen cooperate with each other to provide strong support for the growth of endothelial cells and vascular endothelialization.

[0023] 4. The recombinant collagen and hyaluronic acid in the present invention can mimic the collagen fibers and glycosaminoglycans in blood vessels, serving as a scaffold to simulate the natural extracellular matrix (ECM), support cell growth and guide tissue regeneration.

[0024] 5. The preparation method of the present invention has a reliable principle and is conducive to popularization and application. The prepared gel bio-ink has great potential and broad application prospects in the artificial blood vessel market.

[0025] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the process for preparing methacrylylated recombinant collagen according to Example 1 of the present invention.

[0027] Figure 2 This is a schematic diagram of the gelation of the methacrylylated recombinant collagen prepared in Example 1 of the present invention after being irradiated with ultraviolet light under the action of a photoinitiator.

[0028] Figure 3 This is a Fourier transform infrared absorption spectrum analysis diagram of the methacrylylated recombinant collagen prepared in Example 1 of the present invention.

[0029] Figure 4 The methacrylylated recombinant collagen prepared in Example 1 of the present invention 1 H-NMR analysis results.

[0030] Figure 5 This is a schematic diagram of the process for preparing methacrylylated hyaluronic acid according to Example 1 of the present invention.

[0031] Figure 6 This is a schematic diagram of the gelation of the methacryloyl hyaluronic acid prepared in Example 1 of the present invention after being irradiated with ultraviolet light under the action of a photoinitiator.

[0032] Figure 7This is a Fourier transform infrared absorption spectrum analysis diagram of the methacryloyl hyaluronic acid prepared in Example 1 of the present invention.

[0033] Figure 8 The methacryloyl hyaluronic acid prepared in Example 1 of the present invention 1 H-NMR analysis results.

[0034] Fig. 9 Schematic diagram of the extrudability test results of the stents prepared in Examples 1 and 2 of the present invention.

[0035] Fig.10 Schematic diagram of the stability test results of the stents prepared in Examples 1 and 2 of the present invention.

[0036] Fig.11 This is a cross-sectional scanning electron micrograph of the stent A prepared in Example 1 with a scale bar of 100 μm.

[0037] Fig.12 This is a surface scanning electron micrograph of the bracket A prepared in Example 1 with a scale bar of 100 μm.

[0038] Fig.13 This is a morphology diagram of the bracket A prepared in Example 1 of the present invention.

[0039] Fig.14 The results show the effect of the gel bio-ink prepared in Example 1 of the present invention on the cell viability of human umbilical vein endothelial cells.

[0040] Fig.15 The scratch area in the test of the cell migration promoting ability of the gel bio-ink prepared in Example 1 of the present invention.

[0041] Fig.16 The photographic results of the test of the cell migration promoting ability of the gel bio-ink prepared in Example 1 of the present invention.

[0042] Fig.17 The photographic results of the blood compatibility test of the gel bio-ink prepared in Example 1 of the present invention are shown.

[0043] Fig.18 This is the quantitative result of the blood compatibility test of the gel bio-ink prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0045] The present invention provides a method for preparing a gel bio-ink used for 3D printing of small-diameter artificial blood vessels, comprising the following steps:

[0046] Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain a liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain a liquid B;

[0047] The preparation method of the methacryloyl recombinant collagen specifically comprises: adding glycidyl methacrylate to a recombinant collagen solution, stirring in a water bath at 37°C for 6h to 8h, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen; the recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the amount of glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen;

[0048] The preparation method of the methacryloyl hyaluronic acid specifically comprises: adding glycidyl methacrylate to a hyaluronic acid solution, stirring in a water bath at 60° C. for a reaction for 6 h to 8 h, dialyzing, and freeze-drying to obtain the methacryloyl hyaluronic acid; the hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate;

[0049] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate;

[0050] Step 2, mixing the liquid A and the liquid B in step 1 to obtain a gel solution; the mass-volume concentration of the methacryloyl recombinant collagen in the gel solution is 5% to 10%, the mass-volume concentration of the methacryloyl hyaluronic acid is 1.5% to 2%, and the mass-volume concentration of the photoinitiator is 0.1% to 0.25%;

[0051] Step 3: Place the gel solution in step 2 in a 4°C refrigerator for 8 to 36 hours to obtain the gel bio-ink used for 3D printing of small-diameter artificial blood vessels.

[0052] The method for preparing the methacryloyl recombinant collagen described in step 1 specifically comprises: adding glycidyl methacrylate to the recombinant collagen solution, reacting in a 37° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.

[0053] The gel bio-ink prepared by the present invention can be used to prepare a 3D printed stent for preparing an artificial blood vessel. The specific method includes: loading the gel bio-ink into a barrel of a 3D printer for 3D printing, while printing, extruding the gel bio-ink and contacting it with ultraviolet light and irradiating it for 3min to 5min for photo-crosslinking and curing, sterilizing it, and obtaining a 3D printed stent for the artificial blood vessel; the nozzle specification of the 3D printer is 23G to 26G, the printing speed is 10mm / s to 30mm / s, the printing temperature is 25°C, and the extrusion amount is 4% to 8%.

[0054] The present invention is specifically described below in conjunction with embodiments, but the following description is not intended to limit the present invention.

[0055] Example 1

[0056] This embodiment provides a method for preparing a gel bio-ink used for 3D printing of a small-diameter artificial blood vessel and a method for preparing a 3D printed stent of an artificial blood vessel using the gel bio-ink, specifically comprising:

[0057] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0058] Step 101, dissolving 1g of recombinant collagen in 100mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97KDa;

[0059] Step 102, add 30 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 6 h, and collect the product;

[0060] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0061] Step 2, providing methacrylylated hyaluronic acid, specifically comprising:

[0062] Step 201, dissolving 1 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 10 KDa;

[0063] Step 202, adding 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 6 h, and collecting the product;

[0064] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0065] Step 3: Provide gel bio-ink for 3D printing of small-diameter artificial blood vessels, including:

[0066] Step 301, placing 500 mg of methacrylated recombinant collagen and 12.5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 mL of deionized water, sealing with tin foil to avoid light, and stirring at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0067] Step 302, placing 100 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 2 h;

[0068] Step 303, mixing the solution A in step 301 and the solution B in step 302, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0069] Step 304, placing the gel solution in a refrigerator at 4°C for about 12 hours to obtain gel bio-ink; placing the gel solution at 4°C for about 12 hours to allow the gel solution to be fully cross-linked and defoamed;

[0070] Step 4: Using the gel bio-ink as a raw material, preparing a 3D printed stent for preparing an artificial blood vessel, the specific method comprising: loading the gel bio-ink into a barrel of a 3D printer, performing 3D printing, and obtaining a 3D printed stent; the 3D printer model is FOODBOT-S2; while printing, the bio-ink is extruded and exposed to ultraviolet light (365nm, 10mW / cm 2 ) and then irradiated for 3 minutes for photo-crosslinking and curing, and sterilized by irradiation with Co60 to obtain a 3D printed stent for preparing an artificial blood vessel, which is denoted as stent A; the stent A has a diameter of 6 mm and a height of 1 cm;

[0071] The nozzle specification of the 3D printer is 25G;

[0072] Printing speed is 10mm / s;

[0073] Printing temperature is 25℃;

[0074] The extrusion amount is 5%.

[0075] Comparative Example 1

[0076] This comparative example investigates the factors affecting the bio-inking of the gel solution, and the preparation method includes:

[0077] Step 1: Place 500 mg of the methacrylylated recombinant collagen described in Example 1 and 12.5 mg of the photoinitiator LAP in 2.5 mL of deionized water, cover with tin foil to avoid light, and stir at room temperature until completely dissolved to obtain solution A;

[0078] Step 2: Place 25 mg of the methacryloyl hyaluronic acid described in Example 1 in 2.5 mL of deionized water and stir in a 37° C. water bath until completely dissolved to obtain Solution B;

[0079] Step 3, mixing liquid A and liquid B evenly to obtain a mixed system C;

[0080] Step 4: Place the mixed system C in a UV curing box and irradiate it at 365 nm for 0.5 min, then sterilize it by Co60 irradiation to obtain a hydrogel.

[0081] The hydrogel in this comparative example is not printable, which may be due to the excessive fluidity of the mixed system C and the lack of shear thinning properties.

[0082] Example 2

[0083] This embodiment provides a method for preparing a gel bio-ink used for 3D printing of a small-diameter artificial blood vessel and a method for preparing a 3D printed stent of an artificial blood vessel using the gel bio-ink, specifically comprising:

[0084] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0085] Step 101, dissolving 1.5 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97 KDa;

[0086] Step 102, add 15 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 7 h, and collect the product;

[0087] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0088] Step 2, providing methacrylylated hyaluronic acid, specifically comprising:

[0089] Step 201, dissolving 2 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 200 KDa;

[0090] Step 202, adding 20 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 7 h, and collecting the product;

[0091] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0092] Step 3: Providing 3D printing bio-ink for preparing artificial blood vessels, specifically including:

[0093] Step 301, placing 400 mg of methacrylated recombinant collagen and 10 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 mL of deionized water, sealing with tin foil to avoid light, and stirring at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0094] Step 302, placing 80 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 2 h;

[0095] Step 303, mixing the solution A in step 301 and the solution B in step 302, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0096] Step 304, placing the gel solution in a refrigerator at 4°C for about 12 hours to obtain gel bio-ink; placing the gel solution at 4°C for about 12 hours to allow the gel solution to be fully cross-linked and defoamed;

[0097] Step 4: Using the gel bio-ink as a raw material, a 3D printed stent for preparing an artificial blood vessel is prepared. The specific method includes: loading the gel bio-ink into the barrel of a 3D printer, performing 3D printing, and obtaining a 3D printed stent; the model of the 3D printer is FOODBOT-S2; while printing, the bio-ink is extruded and exposed to ultraviolet light (365nm, 10mW / cm2), and then irradiated for 4min for photo-crosslinking and curing, and sterilized by irradiation with Co60 to obtain a 3D printed hydrogel stent for preparing an artificial blood vessel, which is recorded as stent B; the stent B has a diameter of 6mm and a height of 1cm;

[0098] The nozzle specification of the 3D printer is 26G;

[0099] Printing speed is 20mm / s;

[0100] The printing temperature is 25℃;

[0101] The extrusion amount was 4%.

[0102] The performance of the 3D printed hydrogel scaffold for preparing an artificial blood vessel in this embodiment is substantially consistent with that in Example 1.

[0103] Example 3

[0104] This embodiment provides a method for preparing a gel bio-ink used for 3D printing of a small-diameter artificial blood vessel and a method for preparing a 3D printed stent of an artificial blood vessel using the gel bio-ink, specifically comprising:

[0105] Step 1: providing methacrylylated recombinant collagen, specifically comprising:

[0106] Step 101, dissolving 2 g of recombinant collagen in 100 mL of deionized water to obtain a recombinant collagen solution; the recombinant collagen is obtained by reverse transcription of human collagen mRNA to generate cDNA, and then expressed in Escherichia coli BL21. Its structure and acquisition method can be referred to, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method"; the recombinant collagen has a relative molecular mass of 97 KDa;

[0107] Step 102, add 40 mL of glycidyl methacrylate dropwise to the recombinant collagen solution, stir in a water bath at 37° C. for 8 h, and collect the product;

[0108] Step 103, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated recombinant collagen; the dialysis bag has a molecular weight cutoff of 3500Da;

[0109] Step 2, providing methacrylylated hyaluronic acid, specifically comprising:

[0110] Step 201, dissolving 1.5 g of sodium hyaluronate in 100 mL of deionized water to obtain a hyaluronic acid solution; the relative molecular mass of the sodium hyaluronate is 500 KDa;

[0111] Step 202, adding 30 mL of glycidyl methacrylate dropwise to the hyaluronic acid solution, stirring in a water bath at 60° C. for 8 h, and collecting the product;

[0112] Step 203, dialyzing the product in deionized water for 7 consecutive days, and freeze-drying to obtain methacrylylated hyaluronic acid; the dialysis bag has a molecular weight cutoff of 3500 Da;

[0113] Step 3: Providing 3D printing bio-ink for preparing artificial blood vessels, specifically including:

[0114] Step 301, placing 250 mg of methacrylated recombinant collagen and 5 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 mL of deionized water, sealing with tin foil to avoid light, and stirring at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature may be 12 hours;

[0115] Step 302, placing 75 mg of methacryloyl hyaluronic acid in 2.5 mL of deionized water, stirring in a 37° C. water bath until completely dissolved, to obtain liquid B; the stirring time in the water bath may be 2 h;

[0116] Step 303, mixing the solution A in step 301 and the solution B in step 302, and continuing stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0117] Step 304, placing the gel solution in a refrigerator at 4°C for about 12 hours to obtain gel bio-ink; placing the gel solution at 4°C for about 12 hours to allow the gel solution to be fully cross-linked and defoamed;

[0118] Step 4: Using the gel bio-ink as a raw material, preparing a 3D printed stent for preparing an artificial blood vessel, the specific method comprising: loading the gel bio-ink into a barrel of a 3D printer, performing 3D printing, and obtaining a 3D printed stent; the 3D printer model is FOODBOT-S2; while printing, the bio-ink is extruded and exposed to ultraviolet light (365nm, 10mW / cm 2 ) and then irradiated for 5 minutes for photo-crosslinking and curing, and sterilized by irradiation with Co60 to obtain a 3D printed hydrogel scaffold for preparing an artificial blood vessel, which is denoted as scaffold C; the scaffold C has a diameter of 6 mm and a height of 1 cm;

[0119] The nozzle specification of the 3D printer is 23G;

[0120] Printing speed is 30mm / s;

[0121] Printing temperature is 25℃;

[0122] The extrusion amount is 8%.

[0123] The performance of the 3D printed hydrogel scaffold for preparing an artificial blood vessel in this embodiment is basically the same as that in Example 1.

[0124] Example 4

[0125] This embodiment is the same as Embodiment 1, except that, in step 4, the nozzle specification of the 3D printer is 23G, the printing speed is 20 mm / s, the extrusion volume is 4%, and the cross-linking time is 1 min.

[0126] Example 5

[0127] This embodiment is the same as embodiment 1, except that in step 4, the nozzle specification of the 3D printer is 26G, the printing speed is 20 mm / s, the extrusion volume is 8%, and the cross-linking time is 1 min.

[0128] Both Examples 4 and 5 can obtain 3D printed hydrogel scaffolds. From the perspective of the scaffold structure, the hydrogel scaffold of Example 1 has a more consistent and symmetrical scaffold structure, indicating that the size of the bio-ink extrusion filaments of the present invention, the extrusion amount per unit time and the printing speed will affect the morphology of the 3D printed hydrogel scaffold.

[0129] Performance Evaluation

[0130] Physicochemical properties of each synthetic component

[0131] Figure 1 This is a schematic diagram of the process of preparing methacryloyl recombinant collagen in Example 1 of the present invention. Glycidyl methacrylate is added dropwise to a hyaluronic acid solution, and uniform and transparent methacryloyl hyaluronic acid can be formed under 37° C. water bath conditions. Figure 2 This is a schematic diagram of the formation of gel after ultraviolet light irradiation under the action of a photoinitiator for the methacrylylated recombinant collagen prepared in Example 1 of the present invention. Figure 2 It can be seen that the methacrylylated recombinant collagen of the present invention can form gel under light conditions. Figure 3 This is a Fourier transform infrared absorption spectrum analysis diagram of the methacrylylated recombinant collagen prepared in Example 1 of the present invention. Figure 3 It can be seen that at 1653cm -1 The peak at the center is the stretching vibration peak of the carbon-carbon double bond, indicating that the recombinant collagen was successfully methacrylated. Figure 4 The methacrylylated recombinant collagen prepared in Example 1 of the present invention 1 H-NMR analysis results. Figure 4 It can be seen that there are olefin double peaks at 6.08 ppm and 5.70 ppm, confirming that the recombinant collagen was successfully grafted with double bond groups.

[0132] Figure 5 This is a schematic diagram of the process of preparing methacryloyl hyaluronic acid in Example 1 of the present invention. Glycidyl methacrylate is added dropwise to a hyaluronic acid solution, and uniform and transparent methacryloyl hyaluronic acid can be formed under a 60° C. water bath condition. Figure 6This is a schematic diagram of the formation of gel after ultraviolet light irradiation under the action of a photoinitiator on the methacryloyl hyaluronic acid prepared in Example 1 of the present invention. Figure 6 It can be seen that the methacryloyl hyaluronic acid of the present invention can form a gel under light conditions. Figure 2 The methacryloyl recombinant collagen and methacryloyl hyaluronic acid of the present invention can be used as the skeleton structure of the hydrogel. Figure 7 This is a Fourier transform infrared absorption spectrum analysis diagram of the methacryloyl hyaluronic acid prepared in Example 1 of the present invention. Figure 7 It can be seen that at 1710cm -1 It is the stretching vibration peak of carbon-carbon double bond, indicating that hyaluronic acid is successfully methacrylated. Figure 8 The methacryloyl hyaluronic acid prepared in Example 1 of the present invention 1 H-NMR analysis results. Figure 8 It can be seen that there are olefin double peaks at 6.17 ppm and 5.74 ppm, confirming that hyaluronic acid is successfully grafted with double bond groups.

[0133] Physicochemical properties of gel bio-ink for preparing artificial blood vessels

[0134] The rheological properties of the scaffolds prepared in Example 1 and Example 2 were tested. The testing method includes: placing the gel bio-ink described in step 303 of Example 1 or Example 2 on the round table of a rotational rheometer, and scanning at a steady rate (0.1 to 1000 s -1 ) to test the extrudability of the gel; the scaffold A described in Example 1 and Example 2 was subjected to an oscillation frequency sweep (0.1-100 Hz, strain 1%) to test the stability of the hydrogel. Fig. 9 Schematic diagram of the test results of the extrudability of the gel bio-ink of Example 1 and Example 2, according to Fig. 9 It can be seen that as the shear rate increases, the viscosity of the samples shows a decreasing trend, indicating that the samples have shear thinning properties and are printable.

[0135] Fig.10 Schematic diagram of the stability test results of the stents of Example 1 and Example 2. Fig.10 As shown, the storage modulus (G') and loss modulus (G") of the sample remain basically unchanged with the change of oscillation frequency, indicating that the scaffold has solid-like elasticity, confirming the stability of the hydrogel scaffold of the present invention.

[0136] Fig.11 The cross-sectional scanning electron microscope image of stent A at a scale of 100 μm in Example 1 is shown. The testing method includes: freeze-drying the hydrogel used to prepare an artificial blood vessel, cutting it into a sample with a thickness of about 1 mm, adhering it to a sample stage with conductive glue, spraying gold, and observing its microscopic morphology with a scanning electron microscope (SEM). Fig.11 It can be seen that the bracket is printed layer by layer by the 3D printer.

[0137] Fig.12 The surface scanning electron microscope image of the stent A in Example 1 with a scale of 100 μm is shown. The testing method includes: freeze-drying the hydrogel used to prepare the artificial blood vessel, cutting it into samples with a thickness of about 1 mm, adhering it to the sample stage with conductive glue, spraying gold, and observing its microscopic morphology through a scanning electron microscope (SEM). Fig.12 It can be seen that scaffold A has a three-dimensional pore structure, which can provide a microenvironment for endothelial cell growth;

[0138] Fig.13 The topography of the stent A in Example 1 is shown in FIG. 1 . The testing method includes: loading the bio-ink into the barrel of a 3D printer, performing 3D printing, and obtaining a 3D printed stent. Fig.13 It can be seen that the diameter of stent A is 6 mm and the height is 5 mm;

[0139] Cytocompatibility

[0140] Fig.14 The figure is a schematic diagram of the cytotoxicity test results of the scaffold A of Example 1, and the test method thereof includes:

[0141] The DMEMF12 complete culture medium is prepared, specifically comprising: adding fetal bovine serum and double antibodies to the DMEMF12 basal culture medium to obtain the DMEMF12 complete culture medium; in the DMEMF12 complete culture medium, the mass percentage of the fetal bovine serum is 10%, the mass percentage of the double antibodies is 1%, and the double antibodies are streptomycin and penicillin;

[0142] The scaffold extract is prepared, specifically comprising: placing the scaffold A of Example 1 in a 50 mL sterile centrifuge tube, adding 1 mL of DMEMF12 complete culture medium per 0.1 g of scaffold A, and placing the centrifuge tube in an incubator at 37° C. and 5% CO2 for 72 hours to obtain a scaffold extract;

[0143] HUVECs were cultured at 1×10 4 The cells / well were inoculated in a 96-well plate at a density of 1.34 μg / well and cultured in an incubator at 37°C and 5% CO2 for 24 h. The old culture medium was removed and the scaffold extract was added to continue the culture. DMEM complete culture medium was used as the control group. After culturing for 24 h and 48 h, CCK-8 solution was added to the 96-well plate at 10 μL / well. After continuing to culture for 2 to 4 h, the supernatant was removed and the OD value of each well was measured at 450 nm using an enzyme marker. The cell survival rate was calculated based on the OD value. The cell viability was calculated as follows:

[0144]

[0145] The cell viability calculation results are as follows Fig.14 As shown, it can be seen that the 3D scaffold material prepared by the present invention has no obvious cytotoxicity and can be used in organisms.

[0146] Cell migration promoting properties

[0147] Fig.15 and 16 The figure is a schematic diagram of the test results of the cell migration promoting ability of the hydrogel used for preparing artificial blood vessels in Example 1. The test method includes placing the hydrogel and low-serum DMEMF12 culture medium in a 50 mL sterile centrifuge tube to obtain a sample, wherein the hydrogel concentration in the sample is 0.1 g / mL, and the low-serum DMEMF12 culture medium contains DMEMF12 basal culture medium and 2% fetal bovine serum, placing the centrifuge tube containing the sample in an incubator at 37°C and 5% CO2 for 72 hours to obtain a hydrogel extract, and incubating HUVECs at 1.2×10 6 The cells / well were inoculated in a 6-well plate at a density of 1.54 μg / well. After the cells completely covered the bottom of the 6-well plate, a 10 μL pipette tip was used to vertically draw down, the old culture medium was sucked out and washed twice with PBS. After that, the hydrogel extract and low-serum DMEMF12 culture medium were added respectively to obtain the experimental group and the control group. After culturing for 0 h, 12 h and 24 h, three areas were randomly selected using an inverted microscope for photography and quantification using Image J software. The cell migration rate was calculated as follows:

[0148]

[0149] The scratch area is Fig.15 As shown, the photo results are as follows Fig.16 As shown, according to Fig.15 and 16 It can be seen that the migration rate of the hydrogel group of the present invention is 64.21±2 (%), which has a significantly improved ability to promote the migration of HUVECs.

[0150] Blood compatibility

[0151] Fig.17 and 18The blood compatibility test results of the hydrogel used to prepare artificial blood vessels in Example 1. The test method includes centrifuging the red blood cells (5000rpm, 5min) and washing them three times with normal saline, and then diluting them into a 5% (v / v) solution. Normal saline was added to the sterilized stent sample at a concentration of 0.1g / mL, and the hydrogel extract was obtained by soaking at 37°C for 24h. Subsequently, 800μL of the hydrogel extract and 200μL of the red blood cell suspension were mixed. Deionized water and normal saline were added to the control group, respectively. After incubation at 37°C for 1 hour, centrifugation (2000rpm, 5min) was performed, and the absorbance of the supernatant at 540nm was detected by an enzyme marker. The hemolysis rate is calculated as follows:

[0152]

[0153] Among them, As, An and Ap represent the absorbance values ​​of the hydrogel group, saline (negative group), and deionized water (positive group), respectively.

[0154] The photo results are as follows Fig.17 The quantification results are shown in Fig.18 As shown, according to Fig.17 and 18 It can be seen that the hemolysis rate of the hydrogel group of the present invention is less than 0.2%, which proves the biological safety of the prepared scaffold material.

[0155] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels, characterized in that: The following steps are involved: Step 1: dissolving methacrylylated recombinant collagen and a photoinitiator in deionized water under light-proof conditions to obtain a liquid A; dissolving methacrylylated hyaluronic acid in deionized water to obtain a liquid B; Step 2: Mix the liquid A and liquid B in step 1 to obtain a gel solution; Step 3: Place the gel solution in step 2 in a 4°C refrigerator for 8 to 36 hours to obtain the gel bio-ink used for 3D printing of small-diameter artificial blood vessels.

2. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 1, characterized in that: The method for preparing the methacryloyl recombinant collagen described in step 1 specifically comprises: adding glycidyl methacrylate to the recombinant collagen solution, reacting in a 37° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl recombinant collagen.

3. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 2, characterized in that: The recombinant collagen solution is a recombinant collagen solution obtained by dissolving recombinant collagen in deionized water, and the mass-volume concentration of the recombinant collagen solution is 1% to 2%; the relative molecular mass of the recombinant collagen is 97KDa; the dosage of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of recombinant collagen.

4. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 1, characterized in that: The preparation method of the methacryloyl hyaluronic acid in step 1 specifically comprises: adding glycidyl methacrylate dropwise to the hyaluronic acid solution, reacting in a 60° C. water bath with stirring for 6 to 8 hours, dialyzing, and freeze-drying to obtain the methacryloyl hyaluronic acid.

5. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 4, characterized in that: The hyaluronic acid solution is a hyaluronic acid solution obtained by dissolving sodium hyaluronate in deionized water, and the mass-volume concentration of the hyaluronic acid solution is 1% to 2%; the relative molecular mass of the sodium hyaluronate is 10KDa to 500KDa; and the amount of the glycidyl methacrylate is 10mL to 30mL of glycidyl methacrylate per gram of sodium hyaluronate.

6. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 1, characterized in that: The photoinitiator in step 1 is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

7. The method for preparing a gel bio-ink for 3D printing of small-diameter artificial blood vessels according to claim 1, characterized in that: In the gel solution in step 2, the mass-volume concentration of methacryloyl recombinant collagen is 5% to 10%, the mass-volume concentration of methacryloyl hyaluronic acid is 1.5% to 2%, and the mass-volume concentration of the photoinitiator is 0.1% to 0.25%.

8. Use of the gel bio-ink prepared by the method of claim 1 in preparing a 3D printed stent for preparing an artificial blood vessel.

9. The use according to claim 8, characterized in that: The preparation method of a 3D printed stent for preparing an artificial blood vessel comprises: loading the gel bio-ink into the barrel of a 3D printer for 3D printing, while printing, extruding the gel bio-ink, contacting it with ultraviolet light and irradiating it for 3 minutes to 5 minutes for photo-crosslinking and curing, and sterilizing it to obtain a 3D printed stent for an artificial blood vessel.

10. The use according to claim 9, characterized in that: The nozzle specification of the 3D printer is 23G~26G, the printing speed is 10mm / s~30mm / s, the printing temperature is 25℃, and the extrusion volume is 4%~8%.

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