A hydrogel scaffold material for venous valves and its preparation method and application

Abstract

The application discloses a kind of venous valve hydrogel scaffold material and its preparation method and application.This method uses polyethylene oxide and other viscoelastic non-Newtonian fluid capable of experiencing weissenberg effect as liquid soft template, when polyethylene oxide solution experiences weissenberg effect similar to valve structure, a layer of fast-crosslinking hydrogel material precursor is evenly covered on the liquid surface by spraying, so that the hydrogel is quickly crosslinked, and a venous valve hydrogel scaffold material is obtained.The application has the advantages of simple device, fast and convenient manufacturing method, adjustable venous valve scaffold material structure, etc., and can realize continuous manufacturing of venous valve hydrogel scaffold material with low cost.The application can promote the further development of valve-related physiology and pathology research.The prepared venous valve hydrogel scaffold material can realize the one-way flow function of venous valve, and can be used to construct an in-vitro venous valve model, so it has good medical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional materials, and particularly relates to a method for manufacturing a hydrogel stent material for a venous valve and application thereof. BACKGROUND

[0002] The venous valve model is an important model for studying blood vessel-related diseases, and can be used to study the pathogenesis of venous-related diseases. The parallel flow chamber constructed by using a microfluidic chip is a common in-vitro venous valve model. Because it is manufactured by using a photolithography technology, the structure for simulating the valve in the model is composed of two separate sheets, and the fluid flows through a rectangular space. The model can be used to study the influence of fluid shear stress on endothelial cells and thrombosis, and some research results have been obtained. However, the venous valve model fails to well replicate the physiological structure of the venous valve. On the one hand, the rectangular fluid channel is different from the circular channel of the blood vessel, and on the other hand, the shape of the valve is different from the actual one, resulting in that the fluid shear force on the endothelial cells in the model is different from that in the body. Related studies have shown that the fluid shear force generated by the blood flow plays an important role in the pathogenesis of vascular diseases. A more effective in-vitro venous valve model should be able to simulate the physiological morphology and function of the natural valve. The 3D bioprinting technology has a significant advantage in manufacturing complex three-dimensional structures, and can also be used to prepare the valve structure. However, relatively complex equipment and technical support are required, and the valve structure can only be printed point by point or surface by surface, which is relatively time-consuming. Therefore, the present application hopes to develop a new method for quickly and efficiently manufacturing a low-cost hydrogel stent material for a venous valve. SUMMARY

[0003] The present application proposes to use the Weissenberg effect (the phenomenon that a non-Newtonian fluid climbs along a rotating rod) for the preparation of a hydrogel stent material for a venous valve. The inventors have found in experiments that the liquid surface formed when a viscoelastic non-Newtonian fluid climbs upward along a rotating rod has a shape similar to that of a valve. Therefore, we propose to use the polyethylene oxide solution in which the Weissenberg effect occurs as a dynamically controllable liquid template, and to perform blue light crosslinking of the methacrylated gelatin having excellent biocompatibility above the liquid surface. After removing the lower layer of the non-polymerizable polyethylene oxide solution, a crosslinked structure-controllable hydrogel stent material for a venous valve is obtained.

[0004] The preparation method of the hydrogel stent material for a venous valve provided by the present application comprises the following steps:

[0005] 1) A viscoelastic non-Newtonian fluid capable of undergoing the Weissenberg effect is loaded into an open container, and bubbles in the viscoelastic non-Newtonian fluid solution in the container are removed to make the liquid surface flat; an oil-repellent and water-repellent agent is applied to the rotating shaft of a stirring device, and the rotating shaft is inserted below the liquid surface of the viscoelastic non-Newtonian fluid;

[0006] Or, on the rotating shaft of the stirring device, apply the oil and water repellent agent, insert the rotating shaft into the open container, pour the viscoelastic non-Newtonian fluid capable of generating the Weissenberg effect into the open container, insert the rotating shaft below the liquid level of the viscoelastic non-Newtonian fluid, and remove the bubbles in the viscoelastic non-Newtonian fluid solution in the container;

[0007] 2) After the liquid level is flat, start the rotating shaft to make the solution generate stable Weissenberg effect until the climbing rod shape is similar to the valve structure and a stable liquid level is formed, and then stop the rotating shaft;

[0008] 3) Spray a solution of a material capable of rapid photo-crosslinking above the liquid level, so that the solution of the material capable of rapid photo-crosslinking forms a uniform liquid film above the liquid level;

[0009] 4) Irradiate with a light source capable of rapidly crosslinking the hydrogel material, so that the upper liquid film is rapidly crosslinked to form a venous valve hydrogel scaffold material.

[0010] Further, the method further comprises the following steps: pouring out the lower layer of non-Newtonian fluid without photo-crosslinking properties, and washing the obtained venous valve hydrogel scaffold material with PBS.

[0011] Pouring out the lower layer of non-Newtonian fluid, the test tube bottom can be directly cut with a glass tube cutter to facilitate the outflow of the solution. The non-Newtonian fluid remaining on the valve is washed with a PBS solution.

[0012] In the above method step 1), the viscoelastic non-Newtonian fluid capable of generating the Weissenberg effect can be selected from at least one of the following: a polyethylene oxide aqueous solution, a polyisobutylene polybutene solution, and a polyacrylamide aqueous solution. Among them, the polyethylene oxide aqueous solution can be specifically a polyethylene oxide aqueous solution with a concentration of 7-20% (wt / vol), more specifically a polyethylene oxide aqueous solution with a concentration of 10-15% (wt / vol).

[0013] Polyethylene oxide is a polymer and is difficult to dissolve. Continuous stirring, heating, and vacuum extraction are used to assist in dissolution, but attention should be paid to prevent concentration errors caused by solvent water evaporation during the dissolution process. After the solute is completely dissolved, the obtained polyethylene oxide non-Newtonian fluid is poured into a test tube with an inner diameter of 3-16 mm. Because the non-Newtonian fluid has high viscoelasticity and poor fluidity, it is difficult to pour into the test tube, and it can be added in small amounts and multiple times. A large amount of bubbles can be easily retained in the non-Newtonian fluid solution after being poured into the test tube. The bubbles in the low-concentration solution are removed by standing, and the bubbles in the high-concentration solution are removed by centrifugation. For the polyethylene oxide solution, the polyethylene oxide solution can be left to stand for half an hour until the bubbles disappear. The bubbles that do not disappear are removed by centrifugation at a speed of 1000-1500 rpm for 3-20 min.

[0014] In the step 1) of the method, the open container can be a test tube of various materials, such as a glass tube; the diameter of the glass tube is preferably 3-16 mm in inner diameter.

[0015] Specifically, a polyethylene oxide solution with a concentration of 7-20% (wt / vol) or other viscoelastic fluid capable of experiencing the Weissenberg effect can be filled into a glass tube with an inner diameter of 3-16 mm. The gas bubbles in the non-Newtonian fluid solution in the glass tube are removed by standing and centrifugation to make the liquid surface flat.

[0016] In the step 2) of the method, the diameter of the rotating shaft can be 0.5-3 mm, and the material thereof can be a plastic rod, an aluminum rod, a stainless steel rod, an alloy rod, or other materials that are not easily deformed.

[0017] The aluminum rod, stainless steel rod, plastic rod, etc. can be turned into a cylinder with a diameter of 0.5-3 mm by using a lathe. According to the schematic diagram, it is assembled into a rotating shaft. The motor is fixed on the lifting clamp, and the lifting clamp can adjust the height of the rotating shaft. The viewing direction of the stereoscopic mirror is adjusted to be horizontal. The phenomenon of Weissenberg effect is observed. The height of the stereoscopic mirror is adjusted by the knob for adjusting the focal length. Figure 1

[0018] In the step 2) of the method, the rotating speed of the rotating shaft can be 180-1800 r / min, and the specific rotating speed can be 500 r / min, 800 r / min, etc.

[0019] In the step 2) of the method, the stable liquid surface refers to the upper liquid surface of the non-Newtonian fluid when it experiences the Weissenberg effect.

[0020] In the step 3) of the method, the material capable of rapid photo-crosslinking is methacrylated gelatin or polyethylene glycol bisacrylate.

[0021] Further, the solution of the material capable of rapid photo-crosslinking is a methacrylated gelatin solution with a concentration of 7%-20% (wt / vol).

[0022] The spraying rate is about 5 ul / s, the spraying time is 4-20 s, and the thickness of the formed venous valve hydrogel scaffold material (i.e. the thickness of the liquid film) is about 0.4-2 mm. The preparation method of the methacrylated gelatin solution is as follows:

[0023] (1) Take 0.05 g of a photo initiator LAP, add 20 mL of PBS, and place it in a 45°C water bath for 15 min. Shake several times during the process. After complete dissolution, a 0.25% (wt / vol) initiator standard solution is obtained;

[0024] ​(2) Take an appropriate amount (e.g., 1g) of methacrylic anhydride-modified gelatin and place it in a light-protected centrifuge tube. Add the initiator standard solution (2.333-19mL) and place the tube in a 65℃ water bath for 30 minutes, shaking several times during the process to ensure the methacrylic anhydride-modified gelatin is fully wetted and completely dissolved, obtaining a methacrylic anhydride-modified gelatin solution with a concentration range of 5-30% (wt / vol). While still hot, filter the dissolved methacrylic anhydride-modified gelatin solution through a 0.22μm sterile needle filter for sterilization. Place the obtained methacrylic anhydride-modified gelatin solution in a 37℃ water bath for later use to prevent low-temperature gelation.

[0025] In step 4) of the above method, the light source can be blue light or ultraviolet light.

[0026] The wavelength of the blue light can be selected as 405nm. The duration of blue light irradiation can be 5-15s (the longer the irradiation time, the higher the valve stiffness).

[0027] To continuously manufacture multiple venous valve hydrogel stent materials, a viscoelastic non-Newtonian fluid (such as PEO solution) can be added to the already cross-linked venous valve hydrogel stent material, including the following steps:

[0028] (1)Use Figure 1 The steps shown complete the preparation of the first venous valve hydrogel stent material;

[0029] (2) Add a viscoelastic non-Newtonian fluid to the prepared venous valve hydrogel stent material and wait for it to form a stable liquid surface;

[0030] (3) Restart the rotating shaft so that the non-Newtonian fluid will undergo the Weissenberg effect again and form a liquid surface similar to the structure of a venous valve.

[0031] (4) Spray a methacrylic anhydride gelatin solution that can be rapidly photocrosslinked;

[0032] (5) By using blue light or ultraviolet light to irradiate, the upper liquid film is rapidly cross-linked to form a venous valve hydrogel scaffold material, while the lower non-Newtonian fluid cannot be cross-linked, thus maintaining the properties of the solution; thereby generating a new venous valve hydrogel scaffold material.

[0033] (6) Similarly, by adding viscoelastic non-Newtonian fluid again and repeating steps (2) to (5), venous valve hydrogel stent material can be continuously generated.

[0034] The venous valve hydrogel stent material prepared by the above method is also within the scope of protection of this invention.

[0035] This invention also provides the application of hydrogel stent materials for venous valves.

[0036] The application of the venous valve hydrogel scaffold material provided by the present application includes at least one of the following aspects:

[0037] 1) application in the preparation of an in vitro venous valve model;

[0038] 1) application in the pathological and pharmacological study of the valve.

[0039] Further, the venous valve hydrogel scaffold material can be used to study the influence of blood flow and flow rate on the physiological structure and function of the valve. Or, to study the one-way flow function of the valve.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] The preparation method of the venous valve hydrogel scaffold material of the present application first proposes a dynamically controllable liquid template method based on the morphological characteristics of the venous valve. Unlike other microfluidic methods for preparing valve structures through photolithography technology, the present application forms a deformable mold through the Weissenberg effect of non-Newtonian fluid, and prepares a venous valve hydrogel scaffold material with controllable structure. The preparation of the venous valve structure hydrogel using the present application has the advantages of low cost, simple equipment and operation, and controllable valve structure. Compared with solid templates, the present application uses liquid as a template and does not rely on complex and expensive molds and molding equipment. The step of removing the template is quite simple. The obtained venous valve hydrogel scaffold material is similar to the in vivo venous valve in geometry and function. This new method and strategy provides a new choice for the preparation of in vitro venous valve structure and has good application prospects in the construction of physiological / pathological models of venous valves. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is the structure of the rotating shaft device built by the method of the present application.

[0043] Figure 2 is a flowchart of the preparation of a single venous valve hydrogel scaffold material by the method of the present application.

[0044] Figure 3 is a flowchart of the continuous preparation of multiple venous valve hydrogel scaffold materials by the method of the present application.

[0045] Figure 4 is a physical diagram of the venous valve hydrogel scaffold material prepared by the method of the present application.

[0046] Figure 5 shows the one-way flow under the condition of a single bionic venous valve and three bionic venous valves arranged side by side.

[0047] Figure 6Cell Tracker Red CMTPX staining results of human umbilical vein endothelial cells in three regions of venous valve under static culture condition and dynamic flow condition. DETAILED DESCRIPTION

[0048] The application will be further described in connection with the following specific examples, but the application is not limited to the following examples. The methods are all conventional methods unless otherwise specified. The raw materials are all commercially available unless otherwise specified.

[0049] The preparation method of the venous valve hydrogel scaffold material provided by the application comprises the following steps:

[0050] (1) A polyethylene oxide solution with a concentration of 7-20% (wt / vol) or other viscoelastic non-Newtonian fluid capable of generating the Weissenberg effect is loaded into a glass tube with an inner diameter of 3-16 mm. Bubbles in the viscoelastic non-Newtonian fluid solution in the glass tube are removed by standing and / or centrifugation to make the liquid surface flat.

[0051] (2) A rotating shaft coated with an oil-repellent and water-repellent agent is inserted below the liquid surface of the non-Newtonian fluid. After the liquid surface is restored to be flat, the rotating shaft is started and its speed is controlled. After the non-Newtonian fluid climbs up due to the Weissenberg effect (the climbing rod shape is similar to the valve structure) and the resultant force on the solution is 0, a stable liquid surface is formed.

[0052] (3) A rapidly photo-crosslinkable methacrylated gelatin solution is sprayed on the liquid surface by using a spray gun to form a uniform methacrylated gelatin liquid film.

[0053] (4) The upper methacrylated gelatin solution is rapidly crosslinked by blue light (such as blue light with a wavelength of 405 nm) to form a venous valve hydrogel scaffold material; the lower non-Newtonian fluid does not have the characteristic of photo-crosslinking and remains in a solution state.

[0054] (5) The lower non-Newtonian fluid is poured out, and the glass tube cutter can be directly used to cut the bottom of the test tube to facilitate the outflow of the solution. The viscoelastic non-Newtonian fluid solution remaining on the venous valve hydrogel scaffold material is cleaned in a PBS solution. The experimental flow chart is shown in Figure 2 .

[0055] The preparation method of the venous valve hydrogel scaffold material provided by the application comprises the following steps:

[0056] (1) 0.05 g of a photo initiator LAP is added to 20 mL of PBS, and placed in a 45°C water bath for 15 min. The solution is shaken several times during the dissolution process. After complete dissolution, a 0.25% (wt / vol) initiator standard solution is obtained;

[0057] (2) Take an appropriate amount (e.g., 1g) of methacrylic anhydride-modified gelatin and place it in a light-protected centrifuge tube. Add the initiator standard solution (e.g., 2.333-19mL) and place it in a 65℃ water bath. Dissolve the gelatin in the water for 30 minutes, shaking several times during the process to ensure that the methacrylic anhydride-modified gelatin is fully wetted and completely dissolved. Filter the hot methacrylic anhydride-modified gelatin solution through a 0.22μm sterile needle filter for sterilization. Place the resulting methacrylic anhydride-modified gelatin solution in a 37℃ water bath for later use to prevent low-temperature gelation.

[0058] To continuously manufacture multiple venous valve hydrogel stent materials, PEO solution can be added to the already cross-linked venous valve hydrogel stent material, including the following steps:

[0059] (1)Use Figure 1 The steps shown complete the preparation of the first venous valve hydrogel stent material.

[0060] (2) Add a viscoelastic non-Newtonian fluid to the prepared venous valve hydrogel stent material and wait for it to form a stable liquid surface.

[0061] (3) Restart the rotating shaft to cause the non-Newtonian fluid to undergo the Weissenberg effect again, and form a liquid surface similar to the structure of a venous valve.

[0062] (4) Spray a methacrylic anhydride gelatin solution that can be rapidly photocrosslinked.

[0063] (5) Irradiation with blue light at a wavelength of 405 nm causes the upper layer of methacrylic anhydride-modified gelatin solution to rapidly crosslink, forming a venous valve hydrogel scaffold material. The lower layer of non-Newtonian fluid does not crosslink, maintaining the properties of the solution. This generates a second venous valve hydrogel scaffold material.

[0064] (6) Similarly, by adding a non-Newtonian fluid again and repeating steps (2) to (5), the hydrogel scaffold material for venous valves can be continuously generated. The preparation steps are as follows: Figure 3 As shown.

[0065] In addition to aqueous solutions of polyethylene oxide, the non-Newtonian fluid used in the above preparation method can also be other viscoelastic non-Newtonian fluids, such as polyisobutylene solutions and polyacrylamide aqueous solutions. The methacrylamide gelatin used can be other materials capable of rapid photocrosslinking, such as polyethylene glycol diacrylate.

[0066] The rotating rod in the above preparation method can be a plastic rod, an aluminum rod, a stainless steel rod, an alloy rod, and various materials that are not easy to deform. Since the non-Newtonian fluid is coated with an oil-repellent and water-repellent agent, static climbing does not occur when the non-Newtonian fluid is at rest, and the hydrophilic and hydrophobic properties of the material do not affect the phenomenon of the Weissenberg effect. Due to surface tension, the contact angle between the viscoelastic fluid and the rod is 90° at all rotation speeds. The method of coating the oil-repellent and water-repellent agent eliminates the influence of static climbing while maintaining the dynamic influence of surface tension on the Weissenberg effect.

[0067] The application of the vein valve hydrogel scaffold material prepared by the above method can adhere the vein valve hydrogel scaffold material to a pipe material, such as a silica gel pipe, a glass pipe, a rubber pipe, a polyurethane pipe, etc. Artificial blood with a constant flow rate, a constant flow volume, or pulses is introduced into one side of the pipe material, and the influence of the flow volume and the flow rate of the blood on the physiological structure and function of the valve is studied through the flow volume and the shape of the valve on the other side. By changing the direction of the introduction of the artificial blood, the one-way flow function of the valve can be studied. The flow experiment can be used for pathological and pharmacological studies of the valve.

[0068] The application process includes the following steps:

[0069] (1) The gap between the vein valve and the pipe material (such as a glass pipe) is filled with a methyl methacrylate gelatin solution, and then the methyl methacrylate gelatin solution is irradiated with blue light of a wavelength of 405 nm, so that the vein valve hydrogel scaffold material is connected to the pipe material;

[0070] (2) Artificial blood or cell culture medium is perfused on one side of the pipe material. The flow volume and the inlet pressure of the liquid are controlled, the flow volume and the pressure of the outlet under the corresponding conditions are obtained, and the shape of the valve is observed.

[0071] The process of preparing the vein valve hydrogel scaffold material by the method of the present application utilizes the Weissenberg effect of the non-Newtonian fluid. The Weissenberg effect has different intensities at different rotation speeds and concentrations, and the climbing height of the Weissenberg effect increases with the increase of the rotation speed and the concentration. The non-Newtonian fluid that exhibits the Weissenberg effect is used as a liquid soft template, and the intensity and shape of the Weissenberg effect are controlled by the concentration of the non-Newtonian fluid and the rotation speed of the rotating shaft. As shown in Figure 2 and Figure 3 The methyl methacrylate gelatin is sprayed above the liquid surface of the non-Newtonian fluid and rapidly cross-linked by blue light to prepare vein valve hydrogel scaffold materials with different opening degrees (as shown in Figure 4 ).

[0072] The device for preparing the vein valve hydrogel scaffold material by the method of the present application is simple, the cost is low, and the manufacturing process is fast. Vein valve structures with different opening degrees can be prepared.

[0073] The following describes an embodiment of the method of the present application:

[0074] The oil and water repellent used in the following examples is Scotchgard.

[0075] The preparation method of the methacrylated gelatin solution used in the following examples is as follows:

[0076] (1) Take 0.05 g of photoinitiator LAP and add it to 20 mL of PBS. Place it in a 45°C water bath and dissolve for 15 min. Shake several times during the process. After complete dissolution, a 0.25% (wt / vol) initiator standard solution is obtained.

[0077] (2) Take an appropriate amount (e.g., 1 g) of methacrylated gelatin and place it in a lightproof centrifuge tube. Add the initiator standard solution (e.g., 2.333-19 mL) and place it in a 65°C water bath for 30 min. Shake several times during the process to ensure that the methacrylated gelatin is fully soaked and completely dissolved. Filter the resulting methacrylated gelatin solution with a 0.22 μm sterile needle filter while it is still hot to remove bacteria. The obtained methacrylated gelatin solution is placed in a 37°C water bath for standby to prevent gelation at low temperature.

[0078] Example 1:

[0079] (1) Use a lathe to turn an aluminum rod into a cylinder with a diameter of 1.5 mm. Assemble it into a rotating shaft according to the schematic diagram. Figure 1

[0080] (2) Fill a glass tube with an inner diameter of 8 mm with a polyethylene oxide solution with a concentration of 15% (wt / vol). Remove the air bubbles in the non-Newtonian fluid solution in the glass tube by standing and centrifugation to make the liquid surface flat.

[0081] (3) Apply an oil and water repellent to the aluminum rotating shaft and insert it into the polyethylene oxide solution below the liquid surface. After the liquid surface is restored, start the rotating shaft and control its speed to 500 r / min. Wait for the non-Newtonian fluid to form a stable liquid surface due to the Weissenberg effect, then turn off the rotating shaft.

[0082] (4) Use a spray gun to spray a methacrylated gelatin solution with a concentration of 10% (wt / vol) onto the surface of the polyethylene oxide solution to form a uniform liquid film of the methacrylated gelatin solution above the polyethylene oxide solution. The spraying rate is about 5 ul / s, and the spraying time is about 10 s. The thickness of the resulting venous valve hydrogel scaffold material is about 1 mm.

[0083] (5) Use blue light with a wavelength of 405 nm for light irradiation (5-15 s) to quickly crosslink the upper layer of methacrylated gelatin solution and form a venous valve hydrogel scaffold material. The lower layer of non-Newtonian fluid does not have the characteristics of photo-crosslinking and remains in a solution state.​

[0084] (6) The lower layer of non-Newtonian fluid is poured out, and the test tube bottom can be directly cut with a glass tube cutter to facilitate the outflow of the solution. The non-Newtonian fluid remaining on the valve is washed with a PBS solution. A clean venous valve hydrogel scaffold material with a diameter of 8 mm is obtained.

[0085] Example 2:

[0086] (1) A stainless steel rod is turned into a cylinder with a diameter of 2 mm using a lathe, and assembled into a rotating shaft according to the schematic diagram of Figure 1 .

[0087] (2) A polyethylene oxide solution with a concentration of 15% (wt / vol) is filled into a glass tube with an inner diameter of 8 mm. Bubbles in the non-Newtonian fluid solution in the glass tube are removed by standing and centrifugation to make the liquid level flat.

[0088] (3) An oil and water repellent is applied to the aluminum rotating shaft, and the rotating shaft is inserted into the polyethylene oxide solution below the liquid level. After the liquid level is restored, the rotating shaft is started and the speed is controlled at 500 r / min. After the non-Newtonian fluid forms a stable liquid level due to the Weissenberg effect, the rotating shaft is turned off.

[0089] (4) A 10% (wt / vol) methacrylated gelatin solution is sprayed on the surface of the polyethylene oxide solution using a spray gun to form a uniform liquid film of the methacrylated gelatin solution on the polyethylene oxide solution. The spraying rate is about 5 ul / s, and the spraying time is about 10 s. The thickness of the venous valve hydrogel scaffold material formed is about 1 mm.

[0090] (5) Blue light with a wavelength of 405 nm is used for irradiation to rapidly crosslink the upper layer of methacrylated gelatin solution, forming a venous valve hydrogel scaffold material. The lower layer of non-Newtonian fluid does not have the property of photo-crosslinking and remains in a solution state.

[0091] (6) The lower layer of non-Newtonian fluid is poured out, and the test tube bottom can be directly cut with a glass tube cutter to facilitate the outflow of the solution. The non-Newtonian fluid remaining on the valve is washed with a PBS solution. A clean venous valve hydrogel scaffold material with a diameter of 8 mm is obtained.

[0092] Example 3:

[0093] (1) An aluminum rod is turned into a cylinder with a diameter of 1.5 mm using a lathe, and assembled into a rotating shaft according to the schematic diagram of Figure 1 .

[0094] (2) The polyethylene oxide solution with a concentration of 15% (wt / vol) was filled into a glass tube with an inner diameter of 6 mm. The bubbles in the non-Newtonian fluid solution in the glass tube were removed by standing and centrifugation to make the liquid level flat.

[0095] (3) The anti-oil and water-repellent agent was applied on the aluminum rotating shaft, and the rotating shaft was inserted below the liquid level of the polyethylene oxide solution. After the liquid level was restored to be flat, the rotating shaft was started and the rotating speed was controlled at 500 r / min. After the non-Newtonian fluid climbed up to form a stable liquid level due to the Weissenberg effect, the rotating shaft was turned off.

[0096] (4) The methacrylated gelatin solution with a concentration of 10% (wt / vol) was sprayed on the liquid level of the polyethylene oxide solution using a spray gun to form a uniform liquid film of the methacrylated gelatin solution on the polyethylene oxide solution. The spraying rate was about 5 ul / s, the spraying time was about 8 s, and the thickness of the venous valve hydrogel scaffold material formed was about 0.8 mm.

[0097] (5) The upper layer of the methacrylated gelatin solution was quickly crosslinked by irradiation of blue light with a wavelength of 405 nm to form the venous valve hydrogel scaffold material. The lower layer of the non-Newtonian fluid did not have the characteristics of photo-crosslinking and remained in a solution state.

[0098] (6) The lower layer of the non-Newtonian fluid was poured out, and the glass tube cutter was used to cut the bottom of the test tube to facilitate the flow of the solution. The non-Newtonian fluid remaining on the valve was washed with PBS solution. A clean venous valve hydrogel scaffold material with a diameter of 6 mm was obtained.

[0099] Example 4:

[0100] (1) The aluminum rod was turned into a cylinder with a diameter of 1.5 mm using a lathe, and assembled into a rotating shaft according to the schematic diagram of Figure 1 .

[0101] (2) The polyethylene oxide solution with a concentration of 10% (wt / vol) was filled into a glass tube with an inner diameter of 8 mm. The bubbles in the non-Newtonian fluid solution in the glass tube were removed by standing and centrifugation to make the liquid level flat.

[0102] (3) The anti-oil and water-repellent agent was applied on the aluminum rotating shaft, and the rotating shaft was inserted below the liquid level of the polyethylene oxide solution. After the liquid level was restored to be flat, the rotating shaft was started and the rotating speed was controlled at 800 r / min. After the non-Newtonian fluid climbed up to form a stable liquid level due to the Weissenberg effect, the rotating shaft was turned off.

[0103] (4) A 10% (wt / vol) methacrylated gelatin solution was sprayed above the surface of the polyethylene oxide solution using a spray gun to form a uniform liquid film of the methacrylated gelatin solution above the polyethylene oxide solution. The spraying rate was about 5 ul / s and the spraying time was about 15 s. The resulting venous valve hydrogel scaffold material was about 1.5 mm thick.

[0104] (5) The upper layer of methacrylated gelatin solution was rapidly crosslinked using a blue light with a wavelength of 405 nm to form the venous valve hydrogel scaffold material. The lower layer of non-Newtonian fluid did not have the property of photo-crosslinking and remained in a solution state.

[0105] (6) The lower layer of non-Newtonian fluid was poured out, and the test tube bottom was cut directly using a glass tube cutter to facilitate the flow of the solution. The non-Newtonian fluid remaining on the valve was washed with a PBS solution. A clean venous valve hydrogel scaffold material with a diameter of 8 mm was obtained.

[0106] Example 5:

[0107] (1) An aluminum rod was turned into a cylinder with a diameter of 1.5 mm using a lathe, and assembled into a rotating shaft according to the schematic diagram of Figure 1 .

[0108] (2) A polyethylene oxide solution with a concentration of 15% (wt / vol) was filled into a glass tube with an inner diameter of 8 mm. Bubbles in the non-Newtonian fluid solution in the glass tube were removed by standing and centrifugation to make the liquid surface flat.

[0109] (3) The rotating shaft was inserted into the polyethylene oxide solution below the liquid surface after applying an oil and water repellent to the aluminum rotating shaft. After the liquid surface was restored to be flat, the rotating shaft was started and its speed was controlled at 500 r / min. After the non-Newtonian fluid formed a stable liquid surface due to the Weissenberg effect, the rotating shaft was turned off.

[0110] (4) A 12% (wt / vol) methacrylated gelatin solution was sprayed above the surface of the polyethylene oxide solution using a spray gun to form a uniform liquid film of the methacrylated gelatin solution above the polyethylene oxide solution. The spraying rate was about 5 ul / s and the spraying time was about 10 s. The resulting venous valve hydrogel scaffold material was about 1 mm thick.

[0111] (5) The upper layer of methacrylated gelatin solution was rapidly crosslinked using a blue light with a wavelength of 405 nm to form the venous valve hydrogel scaffold material. The lower layer of non-Newtonian fluid did not have the property of photo-crosslinking and remained in a solution state.

[0112] (6) The non-Newtonian fluid in the lower layer is poured out, and the bottom of the test tube can be cut directly using a glass tube cutter to facilitate the outflow of the solution. The non-Newtonian fluid remaining on the valve is washed with a PBS solution. A clean venous valve hydrogel scaffold material with a diameter of 8 mm is obtained.

[0113] The polyethylene oxide solution in the above examples is replaced by other other viscoelastic non-Newtonian fluids capable of generating the Weissenberg effect, such as a polybutene solution of polyisobutylene and an aqueous polyacrylamide solution, and similarly, a venous valve hydrogel scaffold material with similar performance can be prepared.

[0114] Application Example 1:

[0115] The application of the venous valve hydrogel scaffold material prepared by the above preparation method can adhere the venous valve hydrogel scaffold material to a pipe material, such as a silicone tube, a glass tube, a rubber tube, a polyurethane tube, etc. A constant flow rate, constant flow or pulse of artificial blood is introduced into one side of the pipe material, and the flow rate and shape of the valve are studied through the other side of the blood flow to study the influence of the blood flow rate and flow rate on the physiological structure and function of the valve. By changing the direction of the artificial blood, the one-way flow function of the valve can be studied. The flow experiment can be used for pathological and pharmacological studies of the valve.

[0116] The application process includes the following steps:

[0117] (1) The gap between the venous valve and the glass tube is filled with a methyl methacrylate gelatin solution, and then the methyl methacrylate gelatin solution is irradiated with blue light of 405 nm wavelength to make the venous valve hydrogel scaffold material adhere to the pipe.

[0118] (2) Artificial blood is perfused on one side of the pipe. The liquid flow rate is controlled to be 5 x 10 -6 m 3 / s, 1 x 10 -5 m 3 / s, 2 x 10 -5 m 3 / s, 3 x 10 -5 m 3 / s, 4 x 10 -5 m 3 / s, 5 x 10 -5 m 3 / s and 6 x 10 -5 m 3 / s. The flow rate at the outlet under the corresponding conditions is obtained, and the shape of the valve is observed. Figure 5 Figure a shows the one-way flow of the biomimetic venous valve (the red solution is a cell culture medium with phenol red). The scale is 2 mm; Figure 5 Figure b shows the one-way flow of three biomimetic venous valves arranged side by side. The scale is 2 mm.

[0119] Application Example 2:

[0120] (1) Fill the gap between the venous valve hydrogel scaffold material and the silicone tube with a methylacrylate gelatin solution, then irradiate the methylacrylate gelatin solution with blue light of 405 nm wavelength to make the venous valve hydrogel scaffold material adhere to the tube.

[0121] (2) Seed endothelial cells on the venous valve hydrogel scaffold material, and after the cell density reaches 80%, perfuse cell culture medium on one side of the tube. Control the inlet pressure to be 25 Pa, 50 Pa, 75 Pa, 100 Pa, 150 Pa, 200 Pa, 250 Pa, 500 Pa, 750 Pa and 1000 Pa, respectively. Obtain the flow rate at the outlet under the corresponding conditions, and observe the shape of the valve and the growth state of the endothelial cells. Figure 6 Fig. 1 is a schematic diagram of the selected area; Figure 6 Fig. 1b is the Cell Tracker Red CMTPX staining results of human umbilical vein endothelial cells in three areas on the venous valve under static culture conditions and dynamic flow conditions. The scale is 100 μm.

[0122] The above only describes the preferred embodiments of the present application. It should be noted that those skilled in the art can make several improvements and supplements without departing from the method of the present application, and these improvements and supplements should also be considered within the protection scope of the present application.

Claims

1. A method for preparing a venous valve hydrogel scaffold material, comprising the following steps: 1) placing a viscoelastic non-Newtonian fluid capable of undergoing Weissenberg effect into an open container, and removing the air bubbles in the viscoelastic non-Newtonian fluid in the container to make the liquid surface flat; coating an anti-oil and water-repellent agent on the rotating shaft of a stirring device, and inserting the rotating shaft into the liquid surface of the viscoelastic non-Newtonian fluid; or, coating an anti-oil and water-repellent agent on the rotating shaft of a stirring device, inserting the rotating shaft into an open container, then pouring a viscoelastic non-Newtonian fluid capable of undergoing Weissenberg effect into the open container, inserting the rotating shaft into the liquid surface of the viscoelastic non-Newtonian fluid, and removing the air bubbles in the viscoelastic non-Newtonian fluid in the container; 2) after the liquid surface is flat, starting the rotating shaft to make the solution undergo stable Weissenberg effect until the climbing rod shape is similar to the valve structure and a stable liquid surface is formed, then turning off the rotating shaft; 3) spraying a solution of a material capable of undergoing rapid photo-crosslinking above the stable liquid surface formed in step 2) so that the solution of the material capable of undergoing rapid photo-crosslinking forms a uniform liquid film above the liquid surface; 4) irradiating the liquid film with a light source to make the upper liquid film undergo rapid crosslinking to form a venous valve hydrogel scaffold material; in the step 1), the viscoelastic non-Newtonian fluid capable of undergoing Weissenberg effect is selected from a polyethylene oxide aqueous solution wherein the polyethylene oxide aqueous solution is specifically a polyethylene oxide aqueous solution with a concentration of 7-20% wt / vol; in the step 3), the material capable of undergoing rapid photo-crosslinking is methacrylated gelatin; in the step 3), the solution of the material capable of undergoing rapid photo-crosslinking is a methacrylated gelatin solution with a concentration of 7%-20% wt / vol; wherein the preparation method of the methacrylated gelatin solution is as follows: (1) taking 0.05 g of a photo initiator LAP, adding 20 mL of PBS, and placing in a 45 ℃ water bath for 15 min of water bath dissolution, with shaking several times during the process, to obtain a 0.25% wt / vol initiator standard solution after complete dissolution; (2) taking an appropriate amount of methacrylated gelatin, placing in a light-proof centrifuge tube, adding the initiator standard solution, and placing in a 65 ℃ water bath for 30 min of water bath dissolution, with shaking several times during the process, to make the methacrylated gelatin be fully infiltrated and completely dissolved.

2. The method of claim 1, wherein: in the step 1), the air bubbles in the non-Newtonian fluid solution in the container are removed by means of standing and / or centrifugation.

3. The production method according to claim 1 or 2, characterized by: in the step 1), the diameter of the rotating shaft is 0.5-3 mm; in the step 2), the rotating speed of the rotating shaft is 180-1800 r / min.

4. The preparation method according to claim 1, characterized in that: in the step 4), the light source is blue light or ultraviolet light; the irradiation time of the blue light is 5-15 s.

5. The production method according to claim 1 or 2, characterized by: the method further comprises the following step: pouring out the lower viscoelastic non-Newtonian fluid, and washing the obtained venous valve hydrogel scaffold material with PBS.

6. A method for continuously manufacturing a plurality of venous valve hydrogel scaffold materials, comprising the following steps: (1) The first vein valve hydrogel scaffold material is prepared according to the method of any one of claims 1-5; (2) A viscoelastic non-Newtonian fluid is added to the prepared vein valve hydrogel scaffold material, and a stable liquid surface is formed; (3) The rotating shaft is started again, so that the non-Newtonian fluid again experiences the Weissenberg effect, and a liquid surface similar to the vein valve structure is formed again; (4) A solution of a material capable of rapid photo-crosslinking is sprayed, so that the solution of the material capable of rapid photo-crosslinking forms a uniform liquid film above the liquid surface; (5) The liquid film is irradiated by a light source, so that the upper liquid film is rapidly crosslinked to form a vein valve hydrogel scaffold material, thereby generating a new vein valve hydrogel scaffold material; (6) Similarly, a viscoelastic non-Newtonian fluid is added again, and the steps of (2) to (5) are repeated, so that vein valve hydrogel scaffold materials can be continuously generated.

7. The vein valve hydrogel scaffold material prepared by the method of any one of claims 1-6.

8. The application of the vein valve hydrogel scaffold material of claim 7, including at least one of the following aspects: 1) Application in the preparation of an in vitro vein valve model; 2) Application in the pathological and pharmacological study of vein valves.

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