阅读说明：本技术 一种功能化双网络水凝胶及其制备方法和应用 (Functionalized double-network hydrogel and preparation method and application thereof ) 是由 余婷婷 王星 韩冰 柳大为 张云帆 张凌云 于 2020-11-27 设计创作，主要内容包括：本发明涉及一种功能化双网络水凝胶及其制备方法和应用。该功能化双网络水凝胶由壳聚糖交联网络和四臂聚乙二醇交联网络相互穿插形成；所述壳聚糖交联网络由偶联有KGN的壳聚糖离子交联而成；所述四臂聚乙二醇交联网络由第一前体和第二前体经化学反应而成,所述第一前体为Tetra-PEG-NH-2,所述第二前体为Tetra-PEG-NHS。本发明提供的功能化的DN水凝胶不仅具有良好的力学性能和细胞亲和性,为骨再生提供力学支撑；还能缓慢释放KGN,持续为种子细胞成骨分化提供必要的生物活性因子。本发明制备过程简单,价廉易得,可以通过比较简单的步骤制得双网络水凝胶,并且无需使用有毒的引发剂。(The invention relates to a functionalized double-network hydrogel and a preparation method and application thereof. The functional double-network hydrogel is prepared by cross-linking chitosanThe connecting network and the four-arm polyethylene glycol cross-linked network are formed by mutual interpenetration; the chitosan crosslinking network is formed by crosslinking chitosan ions coupled with KGN; the four-arm polyethylene glycol cross-linked network is formed by chemical reaction of a first precursor and a second precursor, wherein the first precursor is Tetra-PEG-NH 2 And the second precursor is Tetra-PEG-NHS. The functionalized DN hydrogel provided by the invention has good mechanical property and cell affinity, and provides mechanical support for bone regeneration; can also slowly release KGN, and continuously provides necessary bioactive factors for osteoblast differentiation of seed cells. The preparation method has the advantages of simple preparation process, low price and easy obtainment, can prepare the double-network hydrogel through simpler steps, and does not need to use toxic initiators.)

1. A functionalized double-network hydrogel is characterized in that the double-network hydrogel is formed by mutually interpenetrating a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network;

the chitosan crosslinking network is formed by crosslinking chitosan ions coupled with KGN;

the four-arm polyethylene glycol cross-linked network is formed by chemical reaction of a first precursor and a second precursor, wherein the first precursor is Tetra-PEG-NH₂The second precursor isTetra-PEG-NHS。

2. The functionalized double-network hydrogel according to claim 1,

the mass ratio of the first precursor to the second precursor is (0.5-2): (0.5-2), more preferably 1: 1; and/or

The mass ratio of the chitosan coupled with KGN to the first precursor is 1 (1-5), and more preferably 1 (2-3).

3. The functionalized double-network hydrogel according to claim 1 or 2,

the ionic crosslinking is achieved by a polyvalent anion; optionally, the polyvalent anion is any one or more of citrate, phosphate, phosphite, sulfate, sulfite, persulfate, borate; and/or

The chitosan coupled with KGN is obtained by reacting chitosan with KGN activated by EDC/NHS; preferably, the mass ratio of the chitosan to KGN before activation is (30-40): 1.

4. A method for preparing the functionalized double-network hydrogel according to any one of claims 1 to 3, wherein the method comprises the following steps:

(1) mixing chitosan and KGN activated by EDC/NHS and reacting to obtain a chitosan-KGN compound;

(2) mixing the chitosan-KGN compound obtained in the step (1) and Tetra-PEG-NH₂Preparing a first precursor solution;

(3) preparing Tetra-PEG-NHS into a second precursor solution;

(4) and mixing the first precursor solution and the second precursor solution, standing to form gel to obtain the composite hydrogel, and activating the composite hydrogel to obtain the functionalized double-network hydrogel.

5. The production method according to claim 4,

in the step (2), chitosan-KGN compound with Tetra-PEG-NH₂According to 1 (1-5), more preferably according to 1: (2-3) mixing in a mass ratio; and/or

Tetra-PEG-NH₂The mass ratio of the Tetra-PEG-NHS to the Tetra-PEG-NHS is (0.5-2): (0.5-2), more preferably 1: 1.

6. The production method according to claim 5,

the concentration of the first precursor solution is 60-100mg/mL, more preferably 70-80mg/mL, and most preferably 75 mg/mL; and/or

The concentration of the second precursor solution is 150-250mg/mL, more preferably 180-220mg/mL, and most preferably 200 mg/mL.

7. The production method according to claim 4,

in step (4), the activation process is performed as follows:

soaking the composite hydrogel in a solution containing polyvalent anions; optionally, the multivalent anion is any one or more of citrate, phosphate, phosphite, sulfate, sulfite, persulfate, borate.

8. The production method according to any one of claims 4 to 6,

the step (1) is carried out according to the following method:

(11) mixing chitosan, activated KGN and a solvent, and then stirring for reaction at 30-40 ℃; alternatively, KGN is activated as follows: mixing KGN, EDC, NHS and a solvent, adjusting the pH of the mixed solution to 5-6, and then stirring for reaction;

(12) dialyzing the reactant obtained in step (11), preferably by using a dialysis bag with molecular weight cut-off of 20 kD;

(13) and (4) freeze-drying the reactant dialyzed in the step (12) to obtain the chitosan-KGN compound.

9. A functionalized double-network hydrogel prepared by the preparation method of any one of claims 4 to 8.

10. Use of the functionalized double-network hydrogel of any one of claims 1 to 3 and/or the functionalized double-network hydrogel of claim 9 in a bone regeneration scaffold material.

Technical Field

The invention relates to the technical field of new medical materials, in particular to a functionalized double-network hydrogel and a preparation method and application thereof.

Background

Bone defects caused by inflammation, tumor, trauma and the like are common diseases and frequently encountered diseases of human beings, and the physiological function, physical and mental health and life quality of patients are seriously affected. At present, the common methods for clinically repairing bone defects comprise allogeneic bone, autologous bone transplantation, bone lengthening, and the like, but the common methods have poor effects due to the problems of immunological rejection, infectious diseases, limited sources, and the like. The bone tissue engineering material constructed by the seed cells and the biological scaffold is used for repairing bone defects by utilizing a tissue engineering technology, and is an effective treatment means.

In tissue engineering, a good scaffold material is not only used as a cell carrier, but also is a place for promoting cell biological activity and directional differentiation to specific tissues. Growth factors, as an important component of tissue engineering microenvironments, play a key role in promoting the directed differentiation of Mesenchymal Stem Cells (MSCs). Research shows that growth factors such as Bone Morphogenetic Proteins (BMPs), transforming growth factors (TGF-beta), Insulin-like growth factors (IGFs) and the like all have the effect of inducing osteogenic differentiation on MSCs. Although the growth factor has good induction effect on cells, the problems of short biological half-life period, lack of long-acting effect, poor stability in vivo and poor tissue selection specificity and the like of the growth factor cannot be effectively solved, and the defects limit the further application of the growth factor in clinic. In 2012, Johnson et al screened a novel small molecule compound kartogenin (kgn) from 22000 various heterocyclic drug-like molecules with different structures, and found that the compound has the effect of promoting cartilage differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs). Recently, research shows that KGN can not only participate in the regeneration of cartilage tissues mediated by BMMSCs, but also has the function of inducing the osteogenic differentiation of the BMMSCs through in vitro experiments, which shows that KGN has great potential in the field of bone tissue engineering.

Hydrogel is a typical biological scaffold material, and is widely applied to the field of tissue engineering due to a highly hydrated three-dimensional cross-linked structure and good biocompatibility. When the hydrogel is used as a scaffold material for bone regeneration, the mechanical property of the hydrogel is important. Since the hydrogel not only provides the microenvironment for seed cell growth, proliferation and differentiation, but also provides the three-dimensional structure necessary for mechanical support and maintenance of the chondrocyte phenotype. However, most of the hydrogels prepared by conventional methods for bone repair are soft or brittle and do not sufficiently cope with complex mechanical environments, thereby preventing their application as load-bearing scaffolds in bone tissue engineering. The Double-Network (DN) hydrogel takes a hard and brittle heterogeneous polyelectrolyte as a first Network and a soft and tough neutral polymer as a second Network, and the interpenetrating networks with larger difference of physical properties can realize the balance of mechanical properties between rigidity and toughness, thereby endowing the DN hydrogel with excellent mechanical properties. As a novel material with high water content, high mechanical strength and high toughness, the mechanical property of the material is obviously superior to that of the traditional hydrogel, the material can bear a continuous and high-strength loading-unloading process, and has the capability of self-healing after damage, so the material is increasingly used as a substitute material or a biological material for repairing bone damage.

The construction of traditional DN gels typically involves complex preparation steps, toxic initiators and acrylamide starting materials. At the same time, most DN hydrogels are not degradable, which may limit cellular infiltration and matrix deposition and distribution, thus hindering their application in cartilage tissue engineering.

In addition, for the current research, no research and products exist for modifying KGN on DN hydrogel scaffold materials by a simple and efficient chemical synthesis method so as to promote the repair of large-area bone defects.

Disclosure of Invention

The first purpose of the invention is to provide a functionalized double-network hydrogel grafted with KGN;

the second purpose of the invention is to provide a preparation method for preparing the functionalized double-network hydrogel, which is simple and does not need to use a toxic initiator.

The third purpose of the invention is to provide the application of the functionalized double-network hydrogel.

In order to solve the technical problems, the invention provides the following technical scheme:

a functionalized double-network hydrogel is formed by mutually interpenetrating a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network;

the chitosan crosslinking network is formed by crosslinking chitosan ions coupled with KGN;

the four-arm polyethylene glycol cross-linked network is formed by chemical reaction of a first precursor and a second precursor, wherein the first precursor is Tetra-PEG-NH₂And the second precursor is Tetra-PEG-NHS.

Preferably, the mass ratio of the first precursor to the second precursor is (0.5-2): (0.5-2), more preferably 1: 1; and/or

The mass ratio of the chitosan coupled with KGN to the first precursor is 1 (1-5), and more preferably 1 (2-3).

Preferably, the ionic crosslinking is achieved by polyvalent anions; optionally, the polyvalent anion is any one or more of citrate, phosphate, phosphite, sulfate, sulfite, persulfate, borate; and/or

The chitosan coupled with KGN is obtained by reacting chitosan with KGN activated by EDC/NHS; preferably, the mass ratio of the chitosan to KGN before activation is (30-40): 1.

A preparation method of the functionalized double-network hydrogel comprises the following steps:

(1) mixing chitosan and KGN activated by EDC/NHS and reacting to obtain a chitosan-KGN compound;

(2) mixing the chitosan-KGN compound obtained in the step (1) and Tetra-PEG-NH₂Preparing a first precursor solution;

(3) preparing Tetra-PEG-NHS into a second precursor solution;

(4) and mixing the first precursor solution and the second precursor solution, standing to form gel to obtain the composite hydrogel, and activating the composite hydrogel to obtain the functionalized double-network hydrogel.

Preferably, in step (2), the chitosan-KGN compound is reacted with Tetra-PEG-NH₂According to 1 (1-5), more preferably according to 1: (2-3) mixing in a mass ratio; and/or

Tetra-PEG-NH₂The mass ratio of the Tetra-PEG-NHS to the Tetra-PEG-NHS is (0.5-2): (0.5-2), more preferably 1: 1.

Preferably, the concentration of the first precursor solution is 60-100mg/mL, more preferably 70-80mg/mL, most preferably 75 mg/mL; and/or

The concentration of the second precursor solution is 150-250mg/mL, more preferably 180-220mg/mL, and most preferably 200 mg/mL.

Preferably, in step (4), the activation process is performed as follows:

soaking the composite hydrogel in a solution containing polyvalent anions; optionally, the multivalent anion is any one or more of citrate, phosphate, phosphite, sulfate, sulfite, persulfate, borate.

Preferably, the step (1) is performed as follows:

(11) mixing chitosan, activated KGN and a solvent, and then stirring for reaction at 30-40 ℃; alternatively, KGN is activated as follows: mixing KGN, EDC, NHS and a solvent, adjusting the pH of the mixed solution to 5-6, and then stirring for reaction;

(12) dialyzing the reactant obtained in step (11), preferably by using a dialysis bag with molecular weight cut-off of 20 kD;

(13) and (4) freeze-drying the reactant dialyzed in the step (12) to obtain the chitosan-KGN compound.

The invention provides a functional double-network hydrogel which is prepared by adopting the preparation method provided by the invention.

The invention provides application of the functionalized double-network hydrogel and/or the functionalized double-network hydrogel prepared by the preparation method provided by the invention in a bone regeneration scaffold material.

Advantageous effects

The technical scheme of the invention has the following advantages:

the invention selects Chitosan (CHI) and polyethylene glycol (PEG) which are approved by the Food and Drug Administration (FDA), have no toxicity to human bodies, good biocompatibility and are biodegradable as raw materials. CHI is used as the only basic polysaccharide, has good biocompatibility and biodegradability, and can form a CHI ion cross-linked network through coordination and secondary action in the presence of polyvalent anions. The PEG has the advantages of no immunogenicity, no toxicity, biocompatibility and the like, the four-arm PEG hydrogel can be gelled in situ only by mixing two precursor solutions, and the preparation process is simple and convenient.

The functionalized DN hydrogel provided by the invention has good mechanical property and cell affinity, and provides mechanical support for bone regeneration; can also slowly release KGN, and continuously provides necessary bioactive factors for osteoblast differentiation of seed cells.

Because KGN is a micromolecular compound, adverse factors such as short biological half-life period, instability and the like of the growth factor can be avoided, and the KGN has wide application prospect in the field of bone defect repair.

The DN gel prepared by the invention has a good microstructure.

The preparation steps of the existing double-network hydrogel are relatively complex, but the preparation process of the invention is simple, cheap and easy to obtain, and the double-network hydrogel can be prepared by relatively simple steps.

Drawings

FIG. 1 is a SEM image of PEG-CHI-KGN DN hydrogel;

FIG. 2 is a graph of rheological measurements of oscillation frequency sweep experiments to determine the shear properties of PEG-CHI and PEG-CHI-KGN composite gels and DN gels; g 'is the storage modulus and G' is the dissipation modulus;

FIG. 3 is a LIVE/DEAD staining method for observing the activity of cells in PEG-CHI and PEG-CHI-KGN DN hydrogels, wherein green represents LIVE cells and red represents DEAD cells;

FIG. 4 shows the micro-CT detection of the capacity of PEG-CHI-KGN DN hydrogel to repair skull apical bone defects of mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

First aspect

The invention provides a functionalized double-network hydrogel in a first aspect, wherein the double-network hydrogel is formed by mutually interpenetration of a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network; the chitosan crosslinking network is formed by crosslinking chitosan ions coupled with KGN; the four-arm polyethylene glycol cross-linked network is formed by chemical reaction of a first precursor and a second precursor, wherein the first precursor is Tetra-PEG-NH₂The second precursorIs Tetra-PEG-NHS.

Functionalized double-network hydrogel

KGN (Chinese name is 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid) is introduced into the double-network hydrogel, and the KGN is grafted into the double-network hydrogel by a chemical method, so that the material can slowly release KGN, necessary bioactive factors are continuously provided for osteoblast differentiation of seed cells, and a tissue engineering scaffold material with the capability of directionally inducing osteogenesis is provided.

Chitosan crosslinked networks

The chitosan cross-linked network is formed by cross-linking chitosan ions coupled with KGN.

The above-mentioned ionic crosslinking is preferably effected by means of polyvalent electronegative molecules and/or polyvalent anions. Chitosan has univalent positive charges, and polyvalent anions have polyvalent negative charges, and can form a physical network structure through adsorption (or ionic complexation in some cases) of the positive charges and the negative charges.

Examples of the polyvalent anion include citrate, phosphate, phosphite, sulfate, sulfite, persulfate, and borate, and any one or more of the polyvalent anions can be selected.

The chitosan coupled with KGN can be obtained by reacting chitosan with KGN activated by EDC/NHS. Preferably, the mass ratio of the chitosan to KGN before activation is (30-40: 1, e.g. 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40: 1.

Four-arm polyethylene glycol cross-linked network

The four-arm polyethylene glycol cross-linked network is formed by chemical reaction of a first precursor and a second precursor, wherein the first precursor is Tetra-PEG-NH₂The second precursor was Tetra-PEG-NHS.

The two kinds of four-arm PEG adopted by the invention are hyperbranched polymers, have a large number of terminal functional groups, high branching and three-dimensional spherical structures, and are simple and convenient to synthesize. Compared with common PEG, the four-arm PEG has more terminal functional groups, and provides a large amount of inversions for the modification of polymersThe application site also expands the diversity of the biological application. The present invention adopts-NH₂and-NHS modified two kinds of four-arm PEG to form a four-arm polyethylene glycol cross-linked network, wherein the cross-linked network has the advantages of good toughness, short gelling time, proper degradation speed and excellent biological safety. Note that Tetra-PEG-NH₂And Tetra-PEG-NHS are both prior art materials.

The mass ratio of the first precursor to the second precursor is preferably (0.5-2): (0.5-2), and more preferably 1: 1.

The mass ratio of the chitosan to which KGN is coupled to the first precursor is preferably 1 (1-5), more preferably 1 (2-3).

In summary, the functionalized double-network hydrogel provided by the invention has the following advantages:

the invention selects Chitosan (CHI) and polyethylene glycol (PEG) which are approved by the Food and Drug Administration (FDA), have no toxicity to human bodies, good biocompatibility and are biodegradable as raw materials. CHI is used as the only basic polysaccharide, has good biocompatibility and biodegradability, and can form a CHI ion cross-linked network through coordination and secondary action in the presence of polyvalent anions. The PEG has the advantages of no immunogenicity, no toxicity, biocompatibility and the like, the four-arm PEG hydrogel can be gelled in situ only by mixing two precursor solutions, and the preparation process is simple and convenient.

The prepared functional DN hydrogel has good mechanical property and cell affinity and provides mechanical support for bone regeneration; can also slowly release KGN, and continuously provides necessary bioactive factors for osteoblast differentiation of seed cells.

And the KGN is a micromolecular compound, so that adverse factors such as short biological half-life period, instability and the like of the growth factor can be avoided, and the KGN has wide application prospect in the field of bone defect repair.

The second aspect

The invention provides a method for preparing a functionalized double-network hydrogel in a second aspect, by which the functionalized double-network hydrogel provided in the first aspect of the invention can be prepared, the method comprising the following steps:

(1) mixing chitosan and KGN activated by EDC/NHS and reacting to obtain a chitosan-KGN compound;

(2) mixing the chitosan-KGN compound obtained in the step (1) and Tetra-PEG-NH₂Preparing a first precursor solution;

(3) preparing Tetra-PEG-NHS into a second precursor solution;

(4) and mixing the first precursor solution and the second precursor solution, standing to form gel to obtain the composite hydrogel, and activating the composite hydrogel to obtain the functionalized double-network hydrogel.

Step (1): the step (1) is a step of chemically coupling KGN to Chitosan (CHI) to obtain a CHI-KGN compound (i.e., the above-mentioned chitosan-KGN compound), and specifically, coupling KGN to CHI by coupling KGN to CHI using carbodiimide chemistry to form a chemically linked amide bond between a carboxyl group at the terminus of KGN and a CHI amine group, thereby obtaining a CHI-KGN compound.

In order to secure the coupling effect, step (1) of the present invention may be carried out as follows:

(11) mixing chitosan, activated KGN and a solvent, and then stirring for reaction at 30-40 ℃; alternatively, KGN is activated as follows: mixing KGN, EDC, NHS and a solvent, adjusting the pH of the mixed solution to 5-6, and then stirring for reaction;

(12) dialyzing the reactant obtained in step (11), preferably by using a dialysis bag with molecular weight cut-off of 20 kD;

(13) and (4) freeze-drying the reactant dialyzed in the step (12) to obtain the chitosan-KGN compound.

In the above step, the activated KGN used can be obtained by activating KGN as follows:

mixing KGN, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and a solvent, adjusting the pH of the mixed solution to 5-6, and then stirring for reaction to fully activate the carboxyl of KGN to obtain the activated KGN.

Step (2)): the step (2) is a step of preparing a first precursor solution. The invention combines the CHI-KGN compound prepared in the step (1) with Tetra-PEG-NH₂Mixing, adding solvent (such as PBS buffer solution, phosphate buffer solution) to dissolve thoroughly to obtain first precursor solution.

Chitosan-KGN compound and Tetra-PEG-NH₂Preferably, the components are mixed according to a mass ratio of 1 (1-5), more preferably according to a mass ratio of 1: (2-3) mixing in a mass ratio.

The concentration of the first precursor solution is preferably 60-100mg/mL, more preferably 70-80mg/mL, and most preferably 75 mg/mL. It should be noted that the concentrations used herein refer to the chitosan-KGN compound and Tetra-PEG-NH₂The concentration of both in the solvent.

And (3): the step (3) is a step of preparing a second precursor solution. Tetra-PEG-NHS can be mixed with a solvent (e.g., PBS buffer) to give a second precursor solution.

The concentration of the second precursor solution is preferably 150-250mg/mL, more preferably 180-220mg/mL, and most preferably 200 mg/mL. The concentration herein refers to the concentration of Tetra-PEG-NHS in the solvent.

It should be noted that the order of preparing the first precursor solution and preparing the second precursor solution has no influence on the preparation process. For the sake of convenience and description of the present invention, the preparation of the first precursor solution will be described as step (2), and the preparation of the second precursor solution will be described as step (3). It is understood that the preparation effect of preparing the first precursor solution after preparing the second precursor solution and simultaneously preparing the first precursor solution and the second precursor solution has no influence on the preparation process compared with the preparation effect of preparing the first precursor solution before preparing the second precursor solution.

And (4): the step (4) is a step of obtaining a functionalized hydrogel using the first precursor solution and the second precursor solution. The method comprises the steps of mixing a first precursor solution and a second precursor solution, standing for gelling to obtain a composite hydrogel, and activating the composite hydrogel to obtain the functionalized double-network hydrogel.

The activation process may be performed as follows: soaking the composite hydrogel in a solution containing a valence anion; optionally, the multivalent anion is any one or more of citrate, phosphate, phosphite, sulfate, sulfite, persulfate, borate.

The preparation principle of the preparation method is as follows:

the method comprises the steps of firstly coupling KGN on Chitosan (CHI) by a chemical method to obtain a CHI-KGN compound (namely the chitosan-KGN compound mentioned above), and then preparing the functionalized double-network (DN) hydrogel, namely the PEG-CHI-KGN DN hydrogel by using a method combining in-situ gel formation and salt solution soaking ion crosslinking gel formation.

The preparation method provided by the invention has the following advantages:

the PEG-CHI DN gel is prepared by combining in-situ gelling and salt solution soaking ion crosslinking gelling, and the obtained DN gel has good mechanical property and cell affinity and provides mechanical support for bone regeneration; can also slowly release KGN, and continuously provides necessary bioactive factors for osteoblast differentiation of seed cells.

② the DN gel prepared by the invention has good microstructure.

The preparation steps of the existing double-network hydrogel are relatively complex, but the preparation process of the invention is simple, cheap and easy to obtain, and the double-network hydrogel can be prepared by relatively simple steps.

(third aspect)

The functionalized double-network hydrogel provided by the first aspect of the invention or the functionalized double-network hydrogel prepared by the preparation method provided by the second aspect of the invention can be applied to bone regeneration scaffold materials and used for bone defect repair.

The following are examples of the present invention.

Example 1

Preparation of CHI-KGN compounds

And (3) forming an amido bond for chemical connection between the KGN terminal carboxyl and the CHI amido by using a carbodiimide chemical method to prepare the CHI-KGN compound.

The preparation method comprises the following steps:

KGN 120mg, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)726mg, N-hydroxysuccinimide (NHS)434.8mg were weighed and dissolved in 20mL of distilled water, the pH value of the solution was adjusted to 5.7, the solution was stirred at 37 ℃ for 2h, and the carboxyl group of KGN was fully activated to obtain an activated KGN solution.

3600mg of chitosan is weighed and added into the activated KGN solution, distilled water is added to enable the total volume of the solution to reach 150mL, and the reaction is stirred for 24 hours at the temperature of 37 ℃.

And putting the reacted reactant into a dialysis bag with the molecular weight cutoff of 20kD for dialysis, replacing distilled water every 12h, and continuously dialyzing for 72 h.

Freeze-drying the dialyzed reactant to obtain a purified material, namely the CHI-KGN compound, and storing at the temperature of-20 ℃ for later use.

Preparation of PEG-CHI-KGN DN hydrogel

Weighing prepared CHI-KGN compound 200mg, dissolving in 10mL PBS solution, adding 600mg Tetra-PEG-NH₂(purchased from Xylongson, cat. under the trade name 06020700209) was dissolved sufficiently to prepare a precursor solution 1.

Precursor solution 2 was prepared by weighing 300mg of Tetra-PEG-NHS (available from Xiamenocong, cat # 06020702909) in 2mL of PBS solution.

And mixing the precursor solutions 1 and 2, quickly and uniformly mixing by using a circumference shaking instrument, standing to form gel, and obtaining the PEG-CHI-KGN composite hydrogel containing CHI-KGN.

And (3) adding a sodium sulfate solution above the composite hydrogel after the hydrogel is completely gelatinized, and standing overnight to obtain the PEG-CHI-KGN DN hydrogel.

The obtained material is a double-network hydrogel material and is formed by mutually interpenetration of a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network. Compared with the traditional hydrogel, the double-network hydrogel has better mechanical property. The CHI and the two four-arm PEGs are nontoxic to human bodies, good in biocompatibility and biodegradable, and the prepared double-network hydrogel is free of cytotoxicity. KGN is coupled to CHI by a chemical method, so that the double-network hydrogel is functionalized, KGN can be released, and necessary bioactive factors are provided for osteogenic differentiation of seed cells.

Example 2

Preparation of CHI-KGN compounds

And (3) forming an amido bond for chemical connection between the KGN terminal carboxyl and the CHI amido by using a carbodiimide chemical method to prepare the CHI-KGN compound.

The preparation method comprises the following steps:

KGN 120mg, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)726mg, N-hydroxysuccinimide (NHS)434.8mg were weighed and dissolved in 20mL of distilled water, the pH value of the solution was adjusted to 5.7, the solution was stirred at 37 ℃ for 2h, and the carboxyl group of KGN was fully activated to obtain an activated KGN solution.

4800mg of chitosan was weighed and added to the above activated KGN solution, and then distilled water was added to make the total volume of the solution reach 150mL, and the reaction was stirred at 37 ℃ for 24 hours.

And putting the reacted reactant into a dialysis bag with the molecular weight cutoff of 20kD for dialysis, replacing distilled water every 12h, and continuously dialyzing for 72 h.

Freeze-drying the dialyzed reactant to obtain a purified material, namely the CHI-KGN compound, and storing at the temperature of-20 ℃ for later use.

Preparation of PEG-CHI-KGN DN hydrogel

Weighing 200mg of the prepared CHI-KGN compound, dissolving in 8mL of PBS solution, and adding 400mg of Tetra-PEG-NH₂(purchased from Xylongson, cat. under the trade name 06020700209) was dissolved sufficiently to prepare a precursor solution 1.

Precursor solution 2 was prepared by weighing 400mg of Tetra-PEG-NHS (available from Xiamenocong, cat # 06020702909) in 2mL of PBS solution.

And (3) mixing the precursor solutions 1 and 2, quickly and uniformly mixing by using a circumference shaking instrument, standing to form gel, and obtaining the PEG-CHI-KGN composite hydrogel with the content of CHI-KGN of 2%.

And (3) adding a saturated sodium citrate solution above the composite hydrogel after the hydrogel is completely gelatinized, and standing overnight to obtain the PEG-CHI-KGN DN hydrogel.

The obtained material is a double-network hydrogel material and is formed by mutually interpenetration of a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network. Compared with the traditional hydrogel, the double-network hydrogel has better mechanical property. The CHI and the two four-arm PEGs are nontoxic to human bodies, good in biocompatibility and biodegradable, and the prepared double-network hydrogel is free of cytotoxicity. KGN is coupled to CHI by a chemical method, so that the double-network hydrogel is functionalized, KGN can be released, and necessary bioactive factors are provided for osteogenic differentiation of seed cells.

Example 3

Preparation of CHI-KGN compounds

And (3) forming an amido bond for chemical connection between the KGN terminal carboxyl and the CHI amido by using a carbodiimide chemical method to prepare the CHI-KGN compound.

The preparation method comprises the following steps:

KGN 120mg, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)726mg, N-hydroxysuccinimide (NHS)434.8mg were weighed and dissolved in 20mL of distilled water, the pH value of the solution was adjusted to 5.7, the solution was stirred at 37 ℃ for 2h, and the carboxyl group of KGN was fully activated to obtain an activated KGN solution.

Weighing and adding 4000mg of chitosan into the activated KGN solution, adding distilled water to enable the total volume of the solution to reach 150mL, and stirring and reacting for 24 hours at 37 ℃.

And putting the reacted reactant into a dialysis bag with the molecular weight cutoff of 20kD for dialysis, replacing distilled water every 12h, and continuously dialyzing for 72 h.

Freeze-drying the dialyzed reactant to obtain a purified material, namely the CHI-KGN compound, and storing at the temperature of-20 ℃ for later use.

Preparation of PEG-CHI-KGN DN hydrogel

Weighing 200mg of the prepared CHI-KGN compound, dissolving in 8mL of PBS solution, and adding 400mg of Tetra-PEG-NH₂(purchased from Xylongson, cat. under the trade name 06020700209) was dissolved sufficiently to prepare a precursor solution 1.

Precursor solution 2 was prepared by weighing 400mg of Tetra-PEG-NHS (available from Xiamenocong, cat # 06020702909) in 2mL of PBS solution.

And (3) mixing the precursor solutions 1 and 2, quickly and uniformly mixing by using a circumference shaking instrument, standing to form gel, and obtaining the PEG-CHI-KGN composite hydrogel with the content of CHI-KGN (the content is the ratio of the mass of the CHI-KGN to the volume of the added liquid) of 2%.

And (3) adding a saturated sodium citrate solution above the composite hydrogel after the hydrogel is completely gelatinized, and standing overnight to obtain the PEG-CHI-KGN DN hydrogel.

The obtained material is a double-network hydrogel material and is formed by mutually interpenetration of a chitosan cross-linked network and a four-arm polyethylene glycol cross-linked network. Compared with the traditional hydrogel, the double-network hydrogel has better mechanical property. The CHI and the two four-arm PEGs are nontoxic to human bodies, good in biocompatibility and biodegradable, and the prepared double-network hydrogel is free of cytotoxicity. KGN is coupled to CHI by a chemical method, so that the double-network hydrogel is functionalized, KGN can be released, and necessary bioactive factors are provided for osteogenic differentiation of seed cells.

The performance of the functionalized double-network hydrogel provided by the invention is described in detail by taking the material prepared in example 3 as an example. SEM Observation of hydrogel

The hydrogel was freeze-dried at-80 ℃ for 48h, the dried sample was carefully stuck on a conductive resin, and a thin layer of gold was sprayed on the surface thereof, and a field emission SEM image was obtained using JSM-7900FSEM at an accelerating voltage of 3 kV. FIG. 1 shows the morphology of PEG-CHI-KGN DN hydrogel, and it can be seen from FIG. 1 that the material has a good microstructure.

Rheology test

Gel formation times and shear moduli of the 4 hydrogels were measured using a Thermo-Haake rheometer, a conical parallel plate apparatus with a diameter of 35 mm.

The time sweep experiment was performed at a constant strain of 0.05% and an oscillation frequency of 1rad/s, with the sample in solution (0.5mL) placed between two plates, with a gap set at 0.5mm and a gel formation time at the intersection of G 'and G'.

The frequency sweep experiment was performed at a frequency range of 0.1-10rad/s with a constant strain of 0.05% and the shear modulus of the hydrogel was measured by placing the hydrogel (d 35mm, h 3.5mm) between two parallel plates with the gap set at 3 mm.

Figure 2 shows the shear performance of hydrogels of different structures.

It should be noted that:

the preparation idea of the PEG-CHI DN hydrogel is different from that of the embodiment 3 in that KGN is not added, and the specific preparation steps comprise:

weighing CHI 200mg, dissolving in 8mL PBS solution, adding 400mg Tetra-PEG-NH₂(purchased from Xylongson, cat. under the trade name 06020700209) was dissolved sufficiently to prepare a precursor solution 1.

Precursor solution 2 was prepared by weighing 400mg of Tetra-PEG-NHS (available from Xiamenocong, cat # 06020702909) in 2mL of PBS solution.

And (3) mixing the precursor solutions 1 and 2, quickly mixing the mixture by using a peripheral oscillator, and standing the mixture to form Gel to obtain the PEG-CHI composite hydrogel (namely the PEG-CHI composite Gel).

And (3) adding a saturated sodium citrate solution above the PEG-CHI composite hydrogel after the PEG-CHI composite hydrogel is completely gelatinized, and standing overnight to obtain the PEG-CHI DN hydrogel.

LIVE/DEAD staining for observing cell state on hydrogel

After the cell-scaffold complex was cultured for 3d, the excess medium on the scaffold was removed by washing 2 times with PBS solution.

The samples were completely immersed in LIVE/DEAD staining working solution (calcein AM 2 mM; buprenorphine bromide dimer-14 mM) and incubated at 37 ℃ for 1 h.

Fluorescence of calcein AM (live cells green) or buprenorphine bromide dimer-1 (dead cells red) was detected using a confocal laser microscope at excitation wavelengths of 488nm or 568 nm.

The number and cell status of BMMSCs on the hydrogel were observed.

FIG. 3 shows the activity of cells in different hydrogel materials, green for live cells and red for dead cells. As can be seen in fig. 3, the functionalized double-network hydrogel was not cytotoxic.

Mouse skull bone defect repairing animal experiment

(1) Cell inoculation and composite culture

The third generation 1 x 10⁷Dripping the/ml BMMSCs suspension on the surface of the bracket, putting the bracket into a cell culture box, incubating for 2h, and preparing to implant into the experimental animal body.

(2) Constructing a mouse craniocerebral head critical bone defect model and evaluating the bone tissue regeneration curative effect

Selecting 8-week-old C57BL/6 mice, after general anesthesia, preparing skin, sterilizing, longitudinally cutting skin about 1cm along the positive midline of skull, exposing skull parietal bone, removing skull periosteum by using a cotton swab, drilling holes on the left side and the right side of cranial sutures by using a puncher with the diameter of 3mm, timely cooling and removing residual bone powder in the drilling process, and pulling out the drill to stop drilling after the drill penetrates through the whole layer of skull and has a falling feeling so as to prevent the dura mater under the skull from being damaged.

Grouping experiments: a sham operation group, B bone defect blank control group and C bone defect repairing group, and the patients are killed 12 weeks after the operation and are subjected to imaging detection

The tissue was placed in fixative and fully fixed and the cranial parietal bone samples were scanned using Scano μ CT. The pixel size of Scano mu CT is set to be 20 mu m, the kilovolt peak value is set to be 30kVp, the current is set to be 200 mu A, the scanned data are reconstructed by using Scano mu CT software, and the data are poured into Amira software for image acquisition and new bone area analysis. The results are shown in FIG. 4.

What should be noted later is: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.