CN114796620B

CN114796620B - Interpenetrating network hydrogel used as medical implant material and preparation method and application thereof

Info

Publication number: CN114796620B
Application number: CN202210434186.6A
Authority: CN
Inventors: 黄灿锋; 姜涛; 许杰盛
Original assignee: Guangdong Shunde Industrial Design Institute
Current assignee: Guangdong Shunde Industrial Design Institute
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2023-09-29
Anticipated expiration: 2042-04-24
Also published as: CN114796620A

Abstract

The application discloses interpenetrating network hydrogel used as a medical implant material, a preparation method and application thereof, and relates to the field of hydrogel materials. The interpenetrating network hydrogel is formed by alternately inserting a main network and a secondary network in an aqueous medium, wherein the main network is formed by repeatedly freezing and thawing and crosslinking polyvinyl alcohol, and the secondary network is formed by crosslinking an amino-containing natural high-molecular polymer and aldehyde polysaccharide. The interpenetrating network gel prepared by the application has more stable crosslinking effect and no toxicity, the two gel networks are formed by physical crosslinking such as electrostatic effect, hydrogen bonding effect and the like, the mechanical strength is high, the porous structure of the hydrogel also provides conditions for cell adhesion, has good biocompatibility, low swelling and anticoagulation function, can be used as an effective biological material for injury repair of human tissues/organs, and is used in the field of implant materials such as bone repair, cartilage repair, vascular repair promotion, artificial blood vessels and the like.

Description

Interpenetrating network hydrogel used as medical implant material and preparation method and application thereof

Technical Field

The application relates to the field of hydrogel materials, in particular to interpenetrating network hydrogel used as a medical implant material, and a preparation method and application thereof.

Background

Realizing the repair of injury to human tissues/organs is a key problem in clinical medicine. At present, the repair means for clinically treating human tissues/organs still mainly comprise autologous tissues, allogenic tissues or xenogenic tissues. Taking vascular access repair as an example, common treatment modes for severe patients mainly include: (1) The antithrombotic drug is taken, but only has the effect of relieving; (2) constructing a vascular stent, but the reocclusion probability is high; (3) vascular grafting. Autologous vascular transplantation is a golden treatment means for vascular repair, but autologous vascular acquisition is difficult, allogeneic vascular transplantation and xenogeneic vascular transplantation are easy to generate immunogenicity, and the immune system of a patient needs to be subjected to drug suppression after transplantation, so that complications, other organ injuries and the like are frequently generated.

In recent years, tissue engineering, which is a technology of combining artificial biomaterials, bioactive factors (growth factors, cytokines, etc.), and stem cells having a multi-differentiation function or functional cells differentiated from specific tissues, is rapidly developed as a branch of regenerative medicine, and an advanced technology of crossing subjects having functional tissues/organs is combined and constructed by engineering methods. The construction of artificial tissue/organ repair by using tissue engineering technology is possible, and the clinical application effect is highly expected, however, as an emerging technology only developed for more than thirty years, many problems still exist in clinical application. The key technical difficulties include: (1) Lack of artificial biological material systems for comprehensively replacing extracellular matrixes of human tissues and bionic extracellular microenvironments; (2) The living active factors are difficult to produce in large quantities and lack of carrier materials capable of being controlled slowly; (3) Limited cell sources, great difficulty in controlling the in vivo differentiation of stem cells, and difficult establishment of a cell extraction and expansion to transplantation treatment period and a clinical quality control system.

It is apparent that biological materials are the basis for tissue engineering, which is satisfactory both as a surrogate extracellular matrix property and as a carrier for biological signaling molecules. The hydrogel is used as a 'soft substance' biological material with rich three-dimensional network structure, can keep a certain shape in water without being dissolved by the water, and meanwhile, the porous structure can also allow biological macromolecules to spread through the network. Typical hydrogels can form stable macrostructures and strengths through intermolecular bonding, forming a micro/nano porous network in which cells are encapsulated, and providing a platform for cells to adhere, spread, migrate and proliferate. For the chemically crosslinked hydrogel, the cells can be blended with the hydrogel prepolymer solution, and then chemical crosslinking/polymerization is initiated to realize three-dimensional immobilization of the cells.

However, the problem still exists: (1) The physical crosslinking hydrogel is based on weak hydrogen bond formation, and has poor mechanical properties; (2) The chemical crosslinking hydrogel needs to be gelled through chemical reaction, and is usually cytotoxic, so that the survival rate of cells after embedding is affected; (3) Hydrogels often incorporate cross-linking agents, but most cross-linking agents are small molecules and are similarly more cytotoxic; (4) The gel network of a single component is difficult to control the crosslinking degree, and the porosity and the mechanical property often cannot meet the requirements of medical implant materials. Therefore, the development of a novel hydrogel system with good biocompatibility and high mechanical strength has important significance for the repair of the damage of clinical human tissues/organs.

Disclosure of Invention

The application provides interpenetrating network hydrogel used as a medical implant material, and a preparation method and application thereof, so as to solve the technical problems of poor biocompatibility and poor mechanical property of the hydrogel.

In order to solve the technical problems, one of the purposes of the application provides an interpenetrating network hydrogel used as a medical implant material, which is formed by alternately crossing a main network and a secondary network in an aqueous medium, wherein the main network is formed by repeatedly freezing and thawing and crosslinking polyvinyl alcohol, the secondary network is formed by crosslinking an amino-containing natural high molecular polymer and aldehyde polysaccharide, the amino-containing natural high molecular polymer is one or more of carboxymethyl chitosan, chitosan oligosaccharide and polylysine, and the aldehyde polysaccharide is one or more of aldehyde dextran, aldehyde pullulan, aldehyde cyclodextrin, aldehyde carboxymethyl cellulose, aldehyde hydroxyethyl starch, aldehyde hydroxyethyl cellulose, aldehyde sodium alginate and aldehyde chondroitin sulfate.

Preferably, the aqueous medium is deionized water, physiological saline, buffer solution or low-concentration acetic acid solution.

Preferably, the aldehyde group substitution degree of the aldehyde group polysaccharide is more than 0.8 mol/mol.

As a preferable scheme, the mass concentration of the polyvinyl alcohol is 5-12%, the mass concentration of the natural high molecular polymer is 2-6%, and the volume ratio of the polyvinyl alcohol to the natural high molecular polymer is (6-9): (1-4).

As a preferred scheme, the mass ratio of the aldehyde polysaccharide to the natural high molecular polymer containing amino is 1: (2-4).

Preferably, the preparation method of the aldehyde group polysaccharide comprises the following steps: dissolving polysaccharide in deionized water, wherein the polysaccharide is one or more of dextran, pullulan, cyclodextrin, sodium carboxymethyl cellulose, hydroxyethyl starch, hydroxyethyl cellulose, sodium alginate and chondroitin sulfate, the mass concentration is 3% -5%, adding sodium periodate, stirring for reaction in a dark place, adding ethylene glycol, continuing stirring in a dark place to terminate the reaction, dialyzing, and freeze-drying to obtain aldehyde polysaccharide.

As a preferable scheme, sodium periodate is added and stirred for reaction time of 6-10h in dark.

As a preferred scheme, the molar ratio of periodate of sodium permanganate to sugar ring groups containing adjacent hydroxyl groups of polysaccharide is 1:1.

preferably, the mass concentration of the polysaccharide dissolved in deionized water is 3% -5%.

Preferably, the aldehyde polysaccharide is one of aldehyde cyclodextrin, aldehyde sodium alginate and aldehyde hydroxyethyl starch.

In order to solve the above technical problems, a second object of the present application is to provide a method for preparing an interpenetrating network hydrogel used as a medical implant material, comprising the following steps:

(1) Dissolving PVA in an aqueous medium to obtain a PVA solution, dissolving an amino-containing natural high-molecular polymer in the aqueous medium to obtain an amino-containing natural high-molecular polymer solution, and mixing the PVA solution and the amino-containing natural high-molecular polymer solution to obtain pre-gel;

(2) Adding aldehyde polysaccharide into the pregel, and uniformly mixing to obtain a prepolymer;

(3) Injecting the prepolymer into a mould, rapidly stirring and exhausting, reacting for 2-4 hours at 35-50 ℃, and repeatedly freezing and thawing to obtain the interpenetrating network hydrogel.

Preferably, in the step (1), the temperature of the PVA when dissolved in the aqueous medium is 90 to 100℃and the temperature of the amino group-containing natural polymer when dissolved in the aqueous medium is 45 to 60 ℃.

In the step (3), the freezing temperature is-20 ℃, the freezing time is 8-12h, and the thawing time is 1-3h in the repeated freezing and thawing process.

Preferably, the number of repeated freeze thawing is 3-8.

In order to solve the above technical problems, the third object of the present application is to provide an application of interpenetrating network hydrogel used as a medical implant material in preparing an implant material for human tissues or organs, such as an implant material for bone repair, vascular repair promotion, artificial blood vessel, etc.

Compared with the prior art, the embodiment of the application has the following beneficial effects:

1. the interpenetrating network gel prepared by the application is formed by alternately inserting a main network and a secondary network in an aqueous medium, the main network is formed by repeatedly freezing and thawing polyvinyl alcohol to form intermolecular hydrogen bonds and crystallization crosslinking, the interpenetrating network belongs to physical crosslinking, the crosslinking effect is more stable, the interpenetrating network is nontoxic, the secondary network is formed by C=N imine bonds by amino groups and aldehyde-based polysaccharide through aldehyde groups and amino groups, the interpenetrating network gel belongs to dynamic covalent crosslinking, the phenomenon that other crosslinking agents are introduced to generate larger cytotoxicity is avoided, the interpenetrating gel network has the effects of electrostatic effect, hydrogen bond effect, molecular chain winding and the like, the mechanical strength is high, and the porous structure of the hydrogel also provides conditions for cell adhesion.

2. The interpenetrating network hydrogel has good biocompatibility, can induce cell adhesion, adopts low-toxicity PVA to improve mechanical strength, has the cell compatibility and blood compatibility meeting application requirements, and simultaneously further improves bioactivity through natural high molecular polymers, avoids introducing expensive bioactive components, and endows the hydrogel material with good biocompatibility from raw materials.

3. The interpenetrating network hydrogel prepared by the application has the advantages of high mechanical strength, low swelling rate, good biocompatibility, excellent anticoagulation performance and the like, the preparation method is simple, the clinical popularization is facilitated, the raw materials are low, the interpenetrating network hydrogel is suitable for mass production, can be used as an effective biological material for repairing the injury of human tissues/organs, and is used in the field of implant materials such as bone repair, cartilage repair, vascular promotion repair, artificial blood vessels and the like.

Drawings

Fig. 1: a reaction schematic diagram of an interpenetrating network hydrogel used as a medical implant material;

fig. 2: an infrared spectrum result of aldehyde carboxymethyl cellulose in the preparation example 1 of the application;

fig. 3: an infrared spectrum result of an aldehyde dextran in the preparation example 2 of the application;

fig. 4: an infrared spectrum result of the aldehyde hydroxyethyl starch in the preparation example 3 of the present application;

fig. 5: an infrared spectrum result of the aldehyde hydroxyethylcellulose in preparation example 4 of the present application;

fig. 6: an infrared spectrum result of aldehyde sodium alginate in preparation example 5 of the application;

fig. 7: an infrared spectrum result of aldehyde chondroitin sulfate in preparation example 6 of the application;

fig. 8: an infrared spectrum result of an aldehyde group pullulan in preparation example 7 of the present application;

fig. 9: an infrared spectrum result of the aldehyde cyclodextrin in the preparation example 8;

fig. 10: protein adhesion results for interpenetrating network hydrogels used as medical implant materials in one of examples 1-3 and comparative example 2 of the present application;

fig. 11: platelet adsorption test results for interpenetrating network hydrogels used as medical implant materials in examples 1-3 and comparative example 2 of the present application;

fig. 12: the results of the cyclic tensile test of the interpenetrating network hydrogel used as the medical implant material in the embodiment 1, 10-12;

fig. 13: blood compatibility test results for interpenetrating network hydrogels used as medical implant materials in examples 1-3 and comparative example 2 of the present application;

fig. 14: blood compatibility test results for interpenetrating network hydrogels used as medical implant materials in one of examples 1, 13 to 15 and comparative example 1 of the present application (note: comparative example 1 is 0% group in the drawing);

fig. 15: the results of the cell compatibility test for an interpenetrating network hydrogel used as a medical implant material in examples 1 to 3 and comparative example 2 of the present application;

fig. 16: the results of the cell compatibility test for interpenetrating network hydrogels used as medical implant materials in examples 1, 13-15 of the present application.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Common high strength hydrogels mainly include: sliding hydrogel, DN interpenetrating hydrogel and composite hydrogel. The sliding hydrogel is a polyrotaxane hydrogel synthesized by utilizing a supermolecular chemical technology, has good expansion performance, but has high swelling rate, is not suitable for medical implant materials, and has complex synthesis route and preparation process. The composite hydrogel is hydrogel formed by polymerizing and crosslinking multipolymer, and metal nano particles and nano fibers are generally introduced to reduce the swelling rate and improve the mechanical strength of the hydrogel, but most of the crosslinking of a polymerized network and a nano structure substance depends on physical crosslinking, namely hydrogen bonding crosslinking, and the metal nano particles and nano fibers are easy to fall off to generate cytotoxicity in the application process. DN interpenetrating hydrogel is an ideal choice, and realizes high-strength mechanical property only through two interpenetrating polymer networks.

However, in order to realize the application in medical implant materials, the hydrogel needs to be endowed with good biocompatibility. Ultraviolet crosslinking photoinitiation crosslinking is mostly adopted in the conventional DN double-penetrating hydrogel flexible polymer network crosslinking process, so that cytotoxicity is easy to cause, and embedding cells is difficult to realize in the preparation process. In addition, to improve the biocompatibility of DN interpenetrating hydrogels, most studies have employed the introduction of bioactive components, which are expensive and unstable in performance; there are also studies to improve biocompatibility by coating a coating on the surface of gel, but the process route is complicated and the coating is easily peeled off.

The application provides an interpenetrating network hydrogel used as a medical implant material, which is prepared by preparing porous high-strength low-swelling hydrogel with good biocompatibility based on a physical-chemical double-crosslinking mode, wherein the interpenetrating network hydrogel is formed by alternately inserting a main network and a secondary network in an aqueous medium, the main network is made of synthetic polymer materials, in particular, polyvinyl alcohol is repeatedly frozen and thawed and crosslinked to form the interpenetrating network hydrogel, the secondary network is formed by crosslinking an amino-containing natural polymer or derivative and aldehyde polysaccharide, and the amino-containing natural polymer or derivative can be carboxymethyl chitosan (CMCS), chitosan (CS), chitosan Oligosaccharide (COS) and Polylysine (PL), and is preferably carboxymethyl chitosan (CMCS); the biological crosslinking agent is selected from high molecular weight aldehyde polysaccharide crosslinking agent, which can be aldehyde dextran (ODex), aldehyde pullulan (OPul), aldehyde cyclodextrin (OCD), aldehyde carboxymethyl cellulose (OCMC), aldehyde hydroxyethyl starch (OHES), aldehyde hydroxyethyl cellulose (OHEC), aldehyde sodium alginate (OSA), aldehyde chondroitin sulfate (OCS) and the like, preferably aldehyde cyclodextrin, wherein the substitution degree of aldehyde groups in the aldehyde polysaccharide is more than 0.8 mol/mol. Can be used as an effective biological material for repairing human tissue/organ injury, such as bone repair, vascular repair promotion, artificial blood vessel and other implantation material fields.

The application specifically selects soft chain polyvinyl alcohol (Professional Video Assistant, PVA) and rigid chain carboxymethyl chitosan (Carboxymethyl chitosan, CMCS) as main materials of interpenetrating networks. PVA forms a stable crystallization area under the physical crosslinking action through freeze thawing circulation, the crosslinking effect is more stable, the PVA is nontoxic, and meanwhile, the PVA hydrogel has good elasticity and compression performance, so that the PVA hydrogel is an ideal biological material. CMCS is taken as a carboxymethylation derivative of chitosan, has good biocompatibility, contains abundant hydroxyl, carboxyl and amino, can be crosslinked with a biological crosslinking agent to form a porous structure network, is crosslinked with aldehyde polysaccharide through amino aldehyde Schiff base, is formed by physical crosslinking such as electrostatic interaction, hydrogen bonding interaction and the like between interpenetrating gel networks, and the porous structure of the hydrogel also provides conditions for cell adhesion. The hydrogel specifically comprises the following preparation steps:

(1) Preparing a mixed solution: dissolving PVA and CMCS in water to prepare mixed solution, dissolving PVA in deionized water at 90-100 deg.C, preferably 5-12%; dissolving CMCS in deionized water at 45-60deg.C, preferably at a concentration range of 2% -6%; mixing PVA and CMCS solution for 2-4 hr to obtain pre-gel in the preferable ratio of 9 to 1-6 to 4;

(2) Preparation of a biological crosslinker: different polysaccharides are selected for oxidation to obtain aldehyde polysaccharides, and the selected polysaccharides comprise: CMC (hydroxymethyl cellulose), dex (dextran), HES (hydroxyethyl starch), HEC (hydroxyethyl cellulose), SA (sodium alginate), CS (chondroitin sulfate), pul (pullulan), CD (cyclodextrin), etc.; dissolving polysaccharide in deionized water, adding sodium periodate according to a group molar ratio of 1:1, stirring in a dark place for reaction for 6-10h, adding ethylene glycol, stirring in a dark place for 2h to terminate the reaction, placing in a dialysis bag for dialysis for 3 days, and finally freeze-drying to obtain white solid product aldehyde polysaccharide;

(3) Adding aldehyde polysaccharide into the pregel, and uniformly mixing to obtain a prepolymer;

(4) Injecting the prepolymerization solution into a mold, rapidly stirring and exhausting, reacting for 2-4h at 35-50 ℃, freezing for 8-12h at-20 ℃, thawing for 1-3h at room temperature, and repeatedly freezing and thawing for 3-8 times to obtain PVA/CMCS hydrogel.

In order to make the technical content of the present application more clearly understood, the following describes the actual effect of the present application scheme in combination with specific examples. In the present application, the components or materials involved are all conventional commercial products or can be obtained by conventional technical means in the art.

Preparation examples 1 to 8

An aldehyde polysaccharide comprising the following preparation steps: 2.1389g NaIO was taken ₄ Dissolving in 30mL of pure water, stirring and dissolving, wherein the molar ratio of periodate of sodium permanganate to sugar ring group containing adjacent hydroxyl of polysaccharide is 1:1 ratio to NaIO ₄ Adding polysaccharide, which is dextran, pullulan, cyclodextrin, carboxymethyl cellulose, hydroxyethyl starch, hydroxyethyl cellulose, sodium alginate or chondroitin sulfate, stirring in a dark place for reaction for 8 hours, adding 200 mu L of ethylene glycol, continuously stirring in a dark place for 2 hours to terminate the reaction, placing in a dialysis bag of 500Da for dialysis for 3 days, removing a small amount of sodium periodate, ethylene glycol, sodium iodate byproducts, formaldehyde and the like which are remained in the reaction, and finally freeze-drying to obtain white solid product aldehyde polysaccharide.

In order to meet the daily expansion, contraction and stretching requirements of the artificial blood vessel prepared by the hydrogel, 8 kinds of aldehyde polysaccharides are screened for mechanical property, swelling property and structural stability:

1. the degree of substitution of the aldehyde polysaccharide obtained by titration detection of hydroxylamine hydrochloride-sodium hydroxide is shown in table 1, and the infrared spectrum characterization result of the aldehyde polysaccharide is shown in fig. 2-9.

TABLE 1 polysaccharide and degree of substitution of aldehyde groups selected for the aldehyde group polysaccharide in preparation examples 1 to 8

2. CMCS/aldehyde-based hydrogel network stability data (rheology): taking 4mL CMCS (6%) solution in a beaker, adding 700 mu L of the preparation 1-8 aldehyde-based polysaccharide (5%) solution, rapidly stirring uniformly, and placing in a 40 ℃ oven for crosslinking for 4 hours to obtain CMCS/aldehyde-based polysaccharide hydrogel. The storage modulus and loss modulus of the various aldehyde-based polysaccharide crosslinked CMCS hydrogels were determined using the frequency sweep mode of a rotational rheometer, with a strain of 1% and a frequency range of 1-100rad/s, and the test results are shown in Table 2.

TABLE 2 modulus of different aldehyde-based polysaccharide/CMCS hydrogels

Detecting items	Storage modulus/KPa	Loss modulus/Pa
			CMCS/OCMC	0.58	8.4
CMCS/ODex	5.49	19.8
			CMCS/OHES	6.85	22.1
CMCS/OHEC	1.16	6
			CMCS/OSA	7.10	21.9
CMCS/OCS	3.75	13.5
			CMCS/OPul	6.53	18.9
CMCS/OCD	7.49	27

3. Swelling ratio of CMCS/aldehyde-based polysaccharide hydrogel:

taking 4mL CMCS (6%) solution in a beaker, adding 700 mu L of the preparation 1-8 aldehyde-based polysaccharide (5%) solution, rapidly stirring uniformly, and placing in a 40 ℃ oven for crosslinking for 4 hours to obtain CMCS/aldehyde-based polysaccharide hydrogel. Analyzing the dynamic swelling behavior of the CMCS/aldehyde-based polysaccharide hydrogel by adopting a weighing method; cutting hydrogel into wafers with the diameter of 8mm, weighing after freeze-drying, soaking the dry gel in PBS buffer solution, taking out at intervals, and sucking the surface moisture by filter paper, and weighing the mass of the hydrogel until the weight of the hydrogel is constant; record mass m of dry gel ₁ Mass m at constant weight swelling ₂ The equilibrium swelling ratio SR of the hydrogel was calculated by the formula (1.1), with the smaller SR, the massThe smaller the amount and volume changes, the more satisfactory the application requirements of the medical implant material, and the detection results are shown in table 3.

TABLE 3 swelling ratio SR of different aldehyde-based polysaccharide/CMCS hydrogels

Detecting items	SR
		CMCS/OCMC	170.57
CMCS/ODex	127.26
		CMCS/OHES	116.69
CMCS/OHEC	232.89
		CMCS/OSA	88.25
CMCS/OCS	821.40
		CMCS/OPul	132.15
CMCS/OCD	64.04

As can be seen from the results of tables 1 to 3, the aldehyde group substitution degree of the aldehyde group carboxymethyl cellulose and the aldehyde group hydroxyethyl cellulose is lower than 0.8mol/mol, the substitution degree is too low, the density of cross-linking between the aldehyde group polysaccharide and the CMCS is reduced, the hydrogel structure is unstable, and the strength is insufficient; the modulus of aldehyde carboxymethyl cellulose, aldehyde hydroxyethyl cellulose and aldehyde chondroitin sulfate is low, so that the stability of the hydrogel crosslinked network is reduced, and the requirement of structural stability cannot be met; the swelling ratio of the aldehyde dextran, the aldehyde pullulan, the aldehyde sodium carboxymethyl cellulose, the aldehyde hydroxyethyl cellulose and the aldehyde chondroitin sulfate is overlarge, the mass and volume changes in the swelling process of the hydrogel are larger, the larger swelling ratio can greatly influence the regulation and control of the size, the strength can be influenced to a certain extent, and the performance requirements of the medical implant material are difficult to meet. Therefore, aldehyde cyclodextrin, aldehyde sodium alginate and aldehyde hydroxyethyl starch are selected as biological cross-linking agents, and the prepared hydrogel can simultaneously meet the requirements of high modulus, low swelling ratio and high structural stability.

Example 1

An interpenetrating network hydrogel for use as a medical implant material comprising the following preparation steps:

(1) PVA is dissolved in deionized water at the temperature of 90-100 ℃ with the mass concentration of 10%; dissolving CMCS in deionized water at 45-60deg.C with mass concentration of 6%;

(2) Mixing PVA solution (10%) and CMCS solution (6%) according to the volume ratio of 9:1 to form pre-gel, wherein the mixing time is 2h;

(3) To 10mL of the pregel was added 0.025g of the aldehyde cyclodextrin (OCD) obtained in preparation 8, at which the mass ratio of aldehyde cyclodextrin to CMCS was 1:2.4, uniformly mixing to obtain a prepolymerization solution;

(4) Injecting the prepolymerization solution into a mold, exhausting, reacting for 4 hours at 40 ℃, freezing for 8 hours at-20 ℃, thawing for 2 hours at room temperature, and repeatedly freezing and thawing for 4 times to obtain the hydrogel.

Example two

An interpenetrating network hydrogel used as a medical implant material, wherein each step and the reagent and the process parameters used in each step are the same as those in the first example, except that aldehyde cyclodextrin (OCD) obtained in preparation example 8 in step (3) is replaced by aldehyde hydroxyethyl starch (OHES) obtained in preparation example 3.

Example III

An interpenetrating network hydrogel used as a medical implant material, wherein each step and the reagent and the process parameters used in each step are the same as those in the first embodiment, except that aldehyde cyclodextrin (OCD) obtained in preparation example 8 in step (3) is replaced by aldehyde sodium alginate (OSA) obtained in preparation example 5.

Example IV

An interpenetrating network hydrogel used as a medical implant material, wherein the steps and the reagents and the process parameters used in the steps are the same as those in the first embodiment, and the difference is that the adding amount of aldehyde cyclodextrin (OCD) obtained in preparation example 8 in the step (3) is 0.015g, and the mass ratio of the aldehyde cyclodextrin to CMCS is 1:4.

example five

The interpenetrating network hydrogel used as the medical implant material has the same steps and the same reagents and process parameters as those used in the first embodiment, except that the added amount of the aldehyde cyclodextrin (OCD) obtained in the preparation example 8 in the step (3) is 0.02g, and the mass ratio of the aldehyde cyclodextrin to the CMCS is 1:3.

example six

The interpenetrating network hydrogel used as the medical implant material has the same steps and the same reagents and process parameters as those used in the first embodiment, except that the added amount of the aldehyde cyclodextrin (OCD) obtained in the preparation example 8 in the step (3) is 0.03g, and the mass ratio of the aldehyde cyclodextrin to the CMCS is 1:2.

example seven

An interpenetrating network hydrogel used as a medical implant material, the reagents and the process parameters used in each step are the same as those in the first embodiment, and the difference is that PVA solution (10%) and CMCS solution (6%) in the step (2) are in a volume ratio of 8:2, and the aldehyde cyclodextrin is added in an amount of 0.05g.

Example eight

An interpenetrating network hydrogel used as a medical implant material, the reagents and the process parameters used in each step are the same as those in the first embodiment, and the difference is that PVA solution (10%) and CMCS solution (6%) in the step (2) are in a volume ratio of 7:3, and the aldehyde cyclodextrin is added in an amount of 0.075g.

Example nine

An interpenetrating network hydrogel used as a medical implant material, the reagents and the process parameters used in each step are the same as those in the first embodiment, and the difference is that PVA solution (10%) and CMCS solution (6%) in the step (2) are in a volume ratio of 6:4, and the aldehyde cyclodextrin is added in an amount of 0.1g.

Examples ten

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform embodiment of the reagents and the process parameters used in each step, except that the steps (4) are repeatedly frozen and thawed for 3 times.

Example eleven

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform embodiment of the reagents and the process parameters used in each step, except that the steps (4) are repeatedly frozen and thawed for 5 times.

Example twelve

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform embodiment of the reagents and the process parameters used in each step, except that the steps (4) are repeatedly frozen and thawed for 6 times.

Example thirteen

An interpenetrating network hydrogel used as a medical implant material, wherein the steps and the reagents and the process parameters used in the steps are the same as those in the first embodiment, except that the concentration of the CMCS solution prepared in the step (1) is 3%, and the adding amount of aldehyde cyclodextrin is 0.0125g.

Examples fourteen

An interpenetrating network hydrogel used as a medical implant material, wherein the steps and the reagents and the process parameters used in the steps are the same as those in the first embodiment, except that the concentration of the CMCS solution prepared in the step (1) is 4%, and the adding amount of aldehyde cyclodextrin is 0.0167g.

Example fifteen

An interpenetrating network hydrogel used as a medical implant material, wherein the steps and the reagents and the process parameters used in the steps are the same as those in the first embodiment, except that the concentration of the CMCS solution prepared in the step (1) is 5%, and the adding amount of aldehyde cyclodextrin is 0.0208g.

Examples sixteen

An interpenetrating network hydrogel used as a medical implant material, the reagents and process parameters used in each step are the same as those in the uniform embodiment, except that the concentration of PVA solution prepared in the step (1) is 8%.

Example seventeen

An interpenetrating network hydrogel used as a medical implant material, the reagents and process parameters used in each step are the same as those in the uniform embodiment, except that the concentration of PVA solution prepared in the step (1) is 9%.

Example eighteen

An interpenetrating network hydrogel used as a medical implant material, the reagents and process parameters used in each step are the same as those in the uniform embodiment, except that the concentration of PVA solution prepared in the step (1) is 11%.

Examples nineteenth

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform embodiment of the reagents and process parameters used in each step, and is different in that CMCS adopts CS instead, and PVA solution and CS solution in the step (2) are mixed according to the volume ratio of 8:2 to form a pre-gel.

Example twenty

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform embodiment of the reagents and process parameters used in each step, and is different in that CMCS adopts CS instead, and PVA solution and CS solution in the step (2) are mixed according to the volume ratio of 6:4 to form a pre-gel.

Example twenty-one

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform reagent and technological parameters used in the steps, and is characterized in that CMCS is replaced by COS, and the volume ratio of PVA solution to COS solution in the step (2) is 8:2 to form a pre-gel.

Examples twenty two

The interpenetrating network hydrogel used as the medical implant material has the same steps and the uniform reagent and technological parameters used in the steps, and is characterized in that CMCS is replaced by COS, and the volume ratio of PVA solution to COS solution in the step (2) is 6:4 to form a pre-gel.

Examples twenty-three

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform reagent and technological parameters used in the steps, and is characterized in that CMCS adopts PL instead, and PVA solution and PL solution in the step (2) are mixed according to the volume ratio of 8:2 to form a pre-gel.

Examples twenty-four

The interpenetrating network hydrogel used as medical implant material has the same steps and the uniform reagent and technological parameters used in the steps, and is characterized in that CMCS adopts PL instead, and PVA solution and PL solution in the step (2) are mixed according to the volume ratio of 6:4 to form a pre-gel.

Comparative example one

An interpenetrating network hydrogel used as a medical implant material, wherein the steps and the reagent and the process parameters used in the steps are the same as those in the uniform embodiment, and the difference is that the adding amount of CMCS solution in the step (2) is 0.

Comparative example two

(1) PVA is dissolved in deionized water at the temperature of 90-100 ℃ and the mass concentration is 10%, so that a prepolymer liquid is prepared;

(2) And injecting the prepolymerization solution into a mold, then exhausting, reacting for 4 hours at 40 ℃, freezing for 8 hours at-20 ℃, thawing for 2 hours at room temperature, and repeatedly freezing and thawing for 4 times to obtain the hydrogel.

Performance test

1. The hydrogels obtained in examples 1 to 3 and comparative example 2 above were subjected to protein adhesion test using BCA kit, and the test results are shown in fig. 10, comprising the steps of: freeze-drying the hydrogel, sterilizing, placing in PBS solution for swelling for 30min, transferring to a 48-well plate, and adding 500 mu LBSA solution into each well; after incubation for 2h at 37 ℃, the hydrogel was taken out and washed three times with sterile PBS solution; then adding 500 mu L of 2% SDS solution, and incubating for 1h at 37 ℃ in a shaking table to enable proteins adsorbed on the hydrogel to fall into the solution; protein standard curves were drawn by BCA protein assay kit and absorbance was measured at 560nm wavelength by calculating the amount of protein adhered to the hydrogel sheet.

As shown in the results of fig. 10, the adhesion conditions of different aldehyde-based polysaccharide crosslinked PVA/CMCS hydrogels to proteins are different, wherein the hydrogel crosslinked by using aldehyde-based cyclodextrin in example 1 has a certain protein adhesion resistance, and studies show that the lower protein adhesion amount is beneficial to platelet adhesion, so that the occurrence of conditions such as intimal hyperplasia and occlusion is avoided.

2. The hydrogels obtained in examples 1 to 3 and comparative example 2 above were subjected to a platelet adsorption test using a lactate dehydrogenase cytotoxicity detection kit (LDH kit) to determine the relative amounts of platelets adsorbed to the hydrogels, the detection results being shown in fig. 11, comprising the steps of: the hydrogels were lyophilized, sterilized, placed in PBS solution for 30min swelling, transferred to 48 well plates, and 200 μl LPRP was added, incubated at 37 ℃ for 1h, the hydrogel sheets were removed and placed in clean well plates, 200 μl2% Ttiton-X100 was added, incubated at 37 ℃ for 30min, and absorbance was measured at 440nm wavelength as indicated by LDH kit.

As shown in FIG. 11, the adsorption amount of the PVA/CMCS hydrogel crosslinked by different aldehyde groups was significantly lower than that of the PVA crosslinked hydrogel, wherein the PVA/CMCS hydrogel crosslinked by aldehyde group cyclodextrin had the lowest adhesion amount to platelets, which indicates that the hydrogel in example 1 had the best anticoagulation effect when used for preparing artificial blood vessels.

3. The re-calcification times of the hydrogels of examples 1-3, 7-9, 19-24 and comparative examples 1-2 were tested with reference to GB/T14233.2-2005, ISO 10993-4:2017, and ASTM F756-17, and the test results are shown in Table 4 and include the following steps: freeze drying the hydrogel, sterilizing, standing in PBS solution for 30min, transferring to 48-well plate, adding 200 μLPPP, incubating at 37deg.C for 1 hr, collecting 100 μL of the hydrogel in 96-well plate, adding 100 μL of CaCl preheated at 37deg.C ₂ After shaking for 30s (25 mM), absorbance was measured with an enzyme-labeled instrument at 405nm, and observed for 30min for a half-peak corresponding time of the curve as the recalcification time.

TABLE 4 recalcification time of hydrogels of examples 1-3, 7-9, 19-24 and comparative examples 1-2

As shown by the results of examples 1-3 and comparative example 2 in Table 4, the recalcification time of different aldehyde-based polysaccharide crosslinked PVA/CMCS hydrogels was above 3.5min, wherein the recalcification time of PVA/CMCS hydrogels crosslinked by using aldehyde-based cyclodextrin was the longest, and the PVA/CMCS hydrogels crosslinked by using aldehyde-based cyclodextrin were low in both platelet and protein, which proves that the artificial vascular hydrogels prepared in the examples of the present application have excellent anticoagulation effect, wherein the anticoagulation effect of PVA/CMCS hydrogels crosslinked by aldehyde-based cyclodextrin is the best.

As shown by the results of examples 7-9, 19-24 and comparative example 1 in Table 4, the mass ratio of PVA solution to CMCS/CS/COS/PL solution was different, and the re-calcification time of the prepared hydrogel was also changed when the mass ratio of PVA solution to CMCS/CS/COS/PL solution was 6:4, the recalcification time is the longest, and the anticoagulation effect is the best.

4. The storage modulus and loss modulus of hydrogels at different OCD contents in examples 1-6, 16-18 and comparative example 2 were measured using a frequency sweep mode of a rotational rheometer, wherein strain was 1%, frequency ranges were 1-100rad/s, and the higher the storage modulus, the better the network stability and rheological mechanical strength were shown in Table 5; in addition, the greater the difference between storage modulus and loss modulus, the better the network stability of the material is demonstrated.

Table 5-modulus results of hydrogels in examples 1-6, 16-18 and comparative example 2

Detecting items	Storage modulus/kPa	Loss modulus/kPa
			Example 1	24.7	7.1
Example 2	23.4	6.8
			Example 3	21.6	6.2
Example 4	13.8	0.3
			Example 5	14.6	1.0
Example 6	15.3	3.6
			Example 16	11.6	2.7
Example 17	13.5	2.9
			Example 18	23.1	6.9
Comparative example 2	10.5	1.9

As shown by the results of examples 1 and 4-6 in Table 5, the mechanical properties of PVA/CMCS/aldehyde-based polysaccharide hydrogels are critically dependent on the crosslinking ability of aldehyde-based polysaccharide and CMCS, and the crosslinking density of aldehyde-based cyclodextrin and CMCS is increased and the modulus is also increased when the addition amount of aldehyde-based cyclodextrin is increased within a certain interval. As the content of aldehyde polysaccharides continues to increase, the storage modulus tends to decrease, and the analysis may be that the aldehyde group utilization is reduced due to the increase of the content of aldehyde polysaccharides, and ideally, the aldehyde groups of the OCD on one molecular chain are all crosslinked with the amino groups of CMCS.

As shown by the results of examples 1, 16-18 in Table 5, the mechanical properties of PVA/CMCS/aldehyde-based hydrogels are critically dependent on the crystalline cross-links formed by PVA during the freeze-thaw cycle, so the higher the PVA solubility in a certain interval, the greater the theoretical cross-link density, the greater the network stability of the hydrogels, and therefore the higher the modulus of the hydrogels.

5. The hydrogels of examples 1-3, 7-9, 19-24 and comparative examples 1-2 were tested for tensile strength, tensile modulus, compressive strength and compressive modulus at different PVA/CMCS ratios using a universal mechanical tester, the test results being shown in Table 6, and comprising the steps of: after the hydrogel is soaked in PBS solution to reach an equilibrium swelling state, the surface moisture is absorbed by filter paper, and a CMT1203 microcomputer is used for controlling an electronic universal tester to test the mechanical property of the hydrogel; test standards reference YY0500-2004/ISO 7198:1998; tensile test: preparing a stretching sample (length 60mm, width 10mm and thickness 2 mm) by using a strip-shaped mould, fixing two ends of a hydrogel spline on clamps of a testing machine, setting an original gauge length between the clamps to be 50mm, setting the stretching rate to be 50mm/min, and stretching the sample on one side until the spline is broken; compression performance test: compressed samples were prepared using a circular mold, the samples being cylindrical (diameter 25mm, height 4 mm); the hydrogel was placed on the jig of the tester and the compression rate was set at 10mm/min. But when the sample was compressed to 80%, its compressive strength was recorded.

TABLE 6 mechanical Property results of hydrogels of different PVA/CMCS ratios in examples 1-3, 7-9, 19-24 and comparative examples 1-2

As shown in the results of examples 1, 7 to 9, 19 to 24 and comparative example 2 in Table 6, the mechanical strength of PVA was improved after being entangled with natural high molecular polymer to form an interpenetrating network, but when the ratio of PVA to natural high molecular polymer was large, the mechanical strength was decreased mainly due to the decrease in the content of PVA, which was reduced by hydrogen bonding and crystallization crosslinking by freeze thawing cycles.

In addition, the enhancement effect of CMCS/CS/COS/PL on PVA interpenetrating is found to be superior, and the CMCS has relatively high hydrophilic functional group, and the carboxyl in the outer molecular chain can strengthen the electrostatic effect between molecules and inside molecules, so as to raise the mechanical performance.

6. The hydrogels of examples 1, 10-12 were subjected to cyclic stretching tests using a universal mechanical tester for different freeze thawing times, wherein the stretching rate was 100mm/min, the original gauge length was 45mm, the tensile strain was 20%, and the number of cyclic stretching times was 100, according to the YY0500-2004/ISO 7198:1998 standard, and the test results are shown in FIG. 12.

As shown in the results of FIG. 12, the more freeze thawing times of the hydrogel, the tensile stress strain curves overlap each other during loading-unloading, showing better fatigue resistance, indicating that the higher the degree of crystallization cross-linking of PVA, the higher the cross-linking density, and the higher the mechanical strength of the final formed hydrogel, and the better the fatigue resistance.

7. The results of the measurements of examples 1 to 3 and comparative example 2 are shown in FIG. 13, and the results of the measurements of examples 1, 13 to 15 and comparative example 1 (corresponding to the 0% group in FIG. 7) are shown in FIG. 14, referring to GB/T16886.1-2011 for the haemocompatibility of hydrogels with the standard haemolysis rate, comprising the steps of: taking 5mL of sterile PBS solution according to the volume ratio V _{Anticoagulated rabbit blood} ：V _PBS =4: 5, adding fresh anticoagulated rabbit blood, preparing diluted anticoagulated rabbit blood, and preserving in a refrigerator at 4 ℃ for later use; freeze-drying the hydrogel, sterilizing, placing in PBS solution for swelling for 30min, transferring to a centrifuge tube, and adding 10mLPBS solution into each tube; directly adding the negative control group into a 10mL LPBS solution to a centrifuge tube, and directly adding 10mL deionized water into the positive control group; placing all the groups of centrifuge tubes in a 37 ℃ water bath for preheating for 30min, adding 200 mu L of diluted anticoagulated rabbit blood into each tube, and preheating in the 37 ℃ water bath for 1h again; centrifuging the supernatant with a centrifuge at 2500rpm for 15min, carefully sucking the supernatant, adding the supernatant into a quartz cuvette, and measuring the absorbance at a wavelength of 540 nm; the hemolysis rate is calculated by a formula.

6. The in vitro cytotoxicity (cyto-compatibility) of the hydrogels of examples 1-3, 13-15 and comparative example 2 was tested by MTT assay with reference to GB/T16886.1-2011 standard, the results of the assays of examples 1-3 and comparative example 2 are shown in FIG. 15, and the results of the assays of examples 1, 13-15 are shown in FIG. 16, comprising the steps of:

(1) Preparing leaching solution: firstly freeze-drying hydrogel, sterilizing for standby, placing the dry gel in MEM culture medium (containing 10% fetal calf serum and 1% penicillin-streptomycin solution) for swelling for 30min, and placing in MEM culture medium (30 mg/mL) for leaching at 37 ℃ for 24h;

(2) Inoculation of cells and detection of toxicity: digestion of logarithmic phase L929 mouse fibroblasts with pancreatin, modulation of cell density to 1X 10 ⁵ Inoculating the cell suspension into 96-well cell culture plates, adding 100 mu L of the cell suspension into each well, culturing for 24 hours in a culture box containing 5% CO2 at 37 ℃, absorbing and discarding the culture solution, adding gel leaching solution into an experimental group, adding normal culture medium into a control group, setting 5 parallel holes in each group, placing the culture plates into a culture box containing 5% CO2 at 37 ℃ for culturing for 24 hours, adding 50 mu LMTT solution (5 g/L) into each hole, absorbing and discarding the culture solution after 4 hours of conventional incubation, adding 100 mu L of isopropanol, oscillating for 10 minutes at a constant temperature of 37 ℃, and measuring absorbance value at a wavelength of A570nm by using an enzyme-labeled instrument;

(3) Processing data: the relative proliferation rate RGR% of the cells was calculated by the formula.

As shown in FIGS. 13-16, the addition of CMCS component can increase the biocompatibility, cell proliferation rate and blood compatibility of PVA hydrogel, especially blood compatibility, due to the large amount of COO contained in CMCS ^- These negatively charged groups can effectively inhibit the adhesion of platelets and proteins (negatively charged proteins associated with the coagulation mechanism), thereby providing an anticoagulant effect, the more CMCS, the better the anticoagulant effect and the better the blood compatibility.

The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present application, and are not to be construed as limiting the scope of the application. It should be noted that any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art without departing from the spirit and principles of the present application are intended to be included in the scope of the present application.

Claims

1. The interpenetrating network hydrogel used as a medical implant material is characterized by being formed by alternately inserting a main network and a secondary network in an aqueous medium, wherein the main network is formed by repeatedly freezing and thawing and crosslinking polyvinyl alcohol, the secondary network is formed by crosslinking an amino-containing natural high polymer and aldehyde polysaccharide, the amino-containing natural high polymer is carboxymethyl chitosan, the aldehyde polysaccharide is one or more of aldehyde hydroxyethyl starch and aldehyde sodium alginate, and the aldehyde substitution degree of the aldehyde polysaccharide is more than 0.8 mol/mol; the mass concentration of the polyvinyl alcohol is 11% -12%, and the volume ratio of the polyvinyl alcohol to the natural high molecular polymer is 6:4, a step of; the mass ratio of the aldehyde polysaccharide to the natural high molecular polymer containing amino is 1: (2-4).

2. An interpenetrating network hydrogel for use as a medical implant material according to claim 1, wherein said aqueous medium is deionized water, physiological saline or a buffer solution.

3. An interpenetrating network hydrogel for use as a medical implant material according to claim 1, wherein said natural high molecular weight polymer has a mass concentration of 2% to 6%.

4. An interpenetrating network hydrogel for use as a medical implant material according to claim 1, wherein said method of preparing said aldehyde-based polysaccharide comprises the steps of: dissolving polysaccharide in deionized water, wherein the polysaccharide is one or more of dextran, pullulan, cyclodextrin, sodium carboxymethyl cellulose, hydroxyethyl starch, hydroxyethyl cellulose, sodium alginate and chondroitin sulfate, the mass concentration is 3% -5%, adding sodium periodate, stirring for reaction in a dark place, adding ethylene glycol, continuing stirring in a dark place to terminate the reaction, dialyzing, and freeze-drying to obtain aldehyde polysaccharide.

5. A method for preparing an interpenetrating network hydrogel for use as a medical implant material, comprising the steps of:

6. The method of preparing an interpenetrating network hydrogel for medical implant material according to claim 5, wherein in the step (1), the temperature of PVA is 90 to 100 ℃ when dissolved in an aqueous medium, and the temperature of natural high molecular polymer containing amino groups is 45 to 60 ℃ when dissolved in an aqueous medium; in the step (3), in the repeated freezing and thawing process, the freezing temperature is-20 ℃, the freezing time is 8-12h, the thawing time is 1-3h, and the repeated freezing and thawing times are 3-8 times.

7. Use of an interpenetrating network hydrogel according to any of claims 1-4 as a medical implant material for the preparation of a human tissue or organ implant material.