CN111588913A

CN111588913A - Self-crosslinking hyaluronic acid and hydrogel injection of composite collagen thereof and application of hydrogel injection

Info

Publication number: CN111588913A
Application number: CN202010412099.1A
Authority: CN
Inventors: 孙勇; 樊渝江; 姚涯; 蒋青
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2020-08-28

Abstract

The invention discloses a self-crosslinking hyaluronic acid and hydrogel injection of composite collagen thereof and application thereof, the structure of the injection is shown as formula I,

the injectable double-crosslinking composite hydrogel injection loaded with bioactive substances is prepared by mixing a natural material or a synthetic polymer modified by sulfydryl and a compound containing double bonds with a natural material or a synthetic polymer modified by the compound without other crosslinking agents, and loading bioactive substances such as cells, medicines, protein active factors and the like into the natural high molecular material or the derivative of the natural high molecular material, and under the action of Michael addition reaction. The modification method is simple and easy to implement, and the obtained modified substance has good biocompatibility. The injection type double-crosslinking composite hydrogel has good reaction rate controllability, mechanical property and biocompatibility, lays a good foundation for the application of the injection type hydrogel, and simultaneously embodies the application prospect in the aspect of bone/cartilage defect filling.

Description

Self-crosslinking hyaluronic acid and hydrogel injection of composite collagen thereof and application of hydrogel injection

Technical Field

The invention relates to the technical field of medical biomaterials, in particular to a self-crosslinking hyaluronic acid and a hydrogel injection of composite collagen thereof and application thereof.

Background

Tissue engineering has been developed rapidly from the present, not only various tissue engineered tissues are constructed in animal bodies, but also the tissue engineering can be widely applied clinically, and a lasting curative effect is obtained. The tissue engineering technology can avoid the situations of immunological rejection reaction and insufficient donor source in the traditional autologous or allogeneic tissue and organ transplantation treatment, and hopefully realize the reconstruction of tissues and organs so as to solve the clinical difficulty. Research in tissue engineering includes seed cells, scaffold materials, and methods and techniques for constructing tissues and organs. The scaffold material plays an important role in the regeneration process as one of important factors of tissue engineering. However, synthetic polymers are mostly used as early scaffold materials, and although the scaffold materials are biocompatible, the materials have no benefit for cells and do not have cell adhesion sites, so that the cells cannot grow normally.

The hyaluronic acid used as an extracellular matrix has good biocompatibility and high water content, but has simple structure, single function, high degradation speed and poor mechanical property, and cannot meet the application requirements. The hydrogel is a polymer network formed by physical or chemical crosslinking of single polymer chains, can absorb a large amount of water but is not dissolved in the water, is used as a tissue engineering scaffold, has certain mechanical support, can provide a growing environment for cells by a three-dimensional network structure, and is favorable for transmission of nutrients and oxygen and discharge of metabolites by a certain pore structure. Meanwhile, the intercellular matrix has a biological induction effect and can directionally and induce the specific repair of tissues. For example, sodium hyaluronate has biological functions of managing cell adhesion and migration, regulating cell division and differentiation, and the like, so that the preparation of hydrogel achieves more and more attention in the field of biomedicine. In recent years, due to the environmental friendliness, bioinert and controllable reaction speed of the Michael addition reaction, the Michael addition reaction is widely used in-situ crosslinking hydrogel applications in organisms.

Disclosure of Invention

The invention aims to provide a self-crosslinking hyaluronic acid and a hydrogel injection of composite collagen thereof and application thereof, and the injection has the following basic chemical principles: under certain conditions, the rapid chemical crosslinking reaction between the sulfydryl and the maleimide group is carried out, and the protein natural materials and the bioactive substances with different proportions are mixed. The injection of the invention has the advantages of good biocompatibility, no byproduct, good stability, convenient use, low cost and the like.

In order to achieve the above purpose, the specific technical scheme of the invention is as follows:

a self-crosslinking hyaluronic acid and its hydrogel injection of compound collagen protein has a structure shown in formula I,

wherein

Is a natural material or a synthetic polymer modified by sulfydryl,

is a natural material or a synthetic polymer modified by a compound containing double bonds,

is a protein natural material or a derivative thereof,

is a bioactive substance.

Preferably, the natural material or the synthetic polymer has a carboxyl functional group, including but not limited to any one or more of hyaluronic acid, carboxymethyl chitosan, sodium alginate, chondroitin sulfate, polycarbonate and poly-cystine; the protein natural material or the derivative thereof comprises any one or more of collagen, gelatin and silk fibroin; the bioactive substances include, but are not limited to, one or more of cells, drugs and protein active factors.

The preparation method of the injection comprises the following steps:

mixing natural materials or synthetic polymers modified by sulfydryl and compounds containing double bonds with protein high molecular materials or derivatives thereof in different proportions, loading bioactive substances such as cells, medicines, protein active factors and the like, and carrying out chemical crosslinking under the action of Michael addition reaction to prepare the injectable double-crosslinked composite hydrogel injection loaded with the bioactive substances, wherein the reaction formula is as follows:

wherein, the grafting ratio of double bond groups in the natural material or the synthetic polymer modified by the compound containing double bonds is 1 to 99 percent; the mercapto grafting rate in the mercapto modified natural material or synthetic polymer is 1-99%.

Preferably, the natural material or the synthetic polymer to be modified is hyaluronic acid, the protein polymer material or the derivative thereof added thereto is collagen, the loaded cell is chondrocyte, and the specific steps are as follows:

step A, liquid preparation: respectively preparing a maleimide hyaluronic acid solution and a thiolated hyaluronic acid solution;

step B, Michael addition reaction: and (2) mixing the maleimide hyaluronic acid solution and the thiolated hyaluronic acid solution which are respectively prepared in the step (1), adding a collagen solution and a chondrocyte suspension, uniformly mixing, and standing to enable the maleimide hyaluronic acid solution and the thiolated hyaluronic acid solution containing collagen and chondrocytes to form the hydrogel injection under the action of Michael addition reaction.

Preferably, the thiol-modified hyaluronic acid solution, the maleimide hyaluronic acid solution and the collagen solution are disposed at an equal volume ratio.

Preferably, the thiol-modified hyaluronic acid and the maleimide-modified hyaluronic acid are used in a molar ratio of: 1-50: 1-50.

The structural formula of the maleimide hyaluronic acid is shown as a formula II, and the structural formula of the thiolated hyaluronic acid is shown as a formula III.

Preferably, the thiolated hyaluronic acid solution has a pH of 7.0 to 8.0 and a concentration of 0.1 wt% to 10 wt%, the maleimidoylated hyaluronic acid solution has a pH of 7.0 to 8.0 and a concentration of 0.1 wt% to 10 wt%, the collagen solution to be added has a pH of 7.0 to 8.0 and a concentration of 0.1 wt% to 10 wt%, and the chondrocyte suspension has a concentration of 5 × 10 to 10 wt%²～5×10¹⁰cells/mL。

Preferably, the volume ratio of the hyaluronic acid derivative solution to the collagen is controlled to be 1: 0.1-0.1: 1.

Preferably, the molecular weight of the hyaluronic acid is 1-6000KDa, and the collagen is type I or type II collagen.

Preferably, the Michael addition reaction is carried out at room temperature.

Preferably, the preparation method of the thiolated hyaluronic acid comprises the following steps: dissolving hyaluronic acid in a Mes buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate for 1-2 h, then adding cysteamine hydrochloride to react for 10-15 h at room temperature, and dialyzing to obtain thiolated hyaluronic acid; the molar ratio of the sodium hyaluronate, the N-hydroxysuccinimide, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the cysteamine hydrochloride is regulated and controlled according to requirements.

Preferably, the preparation method of the maleimide hyaluronic acid comprises the following steps: dissolving hyaluronic acid in a Mes buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for activation for 1-2 h, then adding N- (2-aminoethyl) maleimide hydrochloride, reacting for 10-15 h at room temperature, and dialyzing to obtain maleimido hyaluronic acid; the molar ratio of the sodium hyaluronate, the N-hydroxysuccinimide, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N- (2-aminoethyl) maleimide hydrochloride is regulated and controlled according to requirements.

Preferably, the hydrogel injection can be used as an injection for promoting cartilage/bone regeneration and repair, and can also be used as a carrier of drugs/proteins/genes or/and a biological scaffold material.

Still more preferably, the biomaterial is a tissue engineering three-dimensional cell scaffold.

Preferably, the preparation method of the tissue engineering three-dimensional cell scaffold comprises the following steps:

step A, dissolving maleimide hyaluronic acid with a structural formula shown as a formula II and a maleimide group grafting rate of 8-50% by using a culture medium to form a maleimide hyaluronic acid solution with a concentration of 1-5 wt%;

dissolving thiolated hyaluronic acid with a structural formula shown as a formula III and a sulfhydryl content of 10-70% by using a culture medium to form a thiolated hyaluronic acid solution with a concentration of 1-5 wt%;

dissolving type I collagen with diluted acetic acid after the biological filter head is finished to form type I collagen solution with the concentration of 1 wt% -5 wt%.

And B, respectively adding an equal volume of type I collagen solution into the prepared thiolated hyaluronic acid solution and the maleimide hyaluronic acid solution, uniformly mixing, adjusting the pH value to 7.0-8.0, adding the cell suspension, and uniformly mixing.

Further, immediately injecting the mixed solution after the pH value is adjusted to a part to be repaired in a living body to form hydrogel, and obtaining the tissue engineering three-dimensional scaffold; or injecting into a mold, standing to form gel, taking out the hydrogel from the mold, immersing in culture medium, and placing in incubator at 37 deg.C and 5% CO₂Culturing for at least 1 day to obtain the tissue engineering three-dimensional cell scaffold, and periodically replacing the culture medium during the culture period.

The culture medium is obtained by adding a mixed solution of penicillin and streptomycin, ascorbic acid and fetal calf serum on the basis of an alpha-MEM basic culture medium, wherein the concentration of the mixed solution of penicillin and streptomycin in the alpha-MEM basic culture medium is 0.8-1.2%, the concentration of the ascorbic acid is 0.15-0.25%, and the concentration of the fetal calf serum is 8-12%.

Wherein, preferably, the cell suspension is added in an amount of 5 × 10⁵～5×10⁶cells/mL ratio to the injectable double cross-linked composite hydrogel mixture based on the natural material hyaluronic acid.

Compared with the prior art, the positive effects of the invention are as follows:

the injection prepared by the invention has scientific design, simple and convenient operation, good injectability, biocompatibility, degradability, no byproduct generation, good stability, low cost and the like, and is an ideal tissue engineering biological filling and repairing material.

The injectable double-crosslinking composite hydrogel takes hyaluronic acid as a raw material, is subjected to functional modification, and is formed by mixing thiolated hyaluronic acid and maleimide hyaluronic acid with collagen under the action of Michael addition reaction. Both hyaluronic acid and collagen are the major components of the extracellular matrix, and the material has good biocompatibility and is degradable. The cells are wrapped in the hydrogel precursor solution, the injectable property of the hydrogel precursor solution can fill tissue defect parts with any shapes, and meanwhile, the rapid gelling can prevent the cells from losing, so that the operation is simpler and more convenient, the controllability is strong, and the hydrogel precursor solution has important application value in the field of tissue defect repair and filling.

Drawings

Figure 1 is an injection-extrusion diagram for the determination of six groups of injectable double-crosslinked hyaluronic acid-based hydrogels;

figure 2 is a graph of rheological tests for determining six groups of injectable double-crosslinked hyaluronic acid-based hydrogels;

FIG. 3 is a swelling performance test chart of six groups of injectable dual-crosslinked hyaluronic acid-based hydrogels;

fig. 4 is a test chart of the degradation performance of six groups of injectable double-crosslinked hyaluronic acid-based hydrogels;

FIG. 5 is a mechanical property test chart of six groups of injectable double-crosslinked hyaluronic acid-based hydrogels;

figure 6 is a graph of cell proliferation within six groups of injectable double-crosslinked hyaluronic acid-based hydrogels;

FIG. 7 is a graph showing the changes in the apparent morphology of three hydrogel/cell complexes;

FIG. 8 is a graph of FDA/PI and backbone staining of cells in three sets of hydrogel/cell complexes;

FIG. 9 is a schematic representation of the detection of CCK-8 in cells from three sets of hydrogel/cell complexes;

Detailed Description

In order to facilitate an understanding of the present disclosure, the process described herein will be further described with reference to the accompanying drawings and detailed description. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

wherein

Is a natural material or a synthetic polymer modified by sulfydryl, in particular to sulfydryl modified hyaluronic acid, carboxymethyl chitosan, sodium alginate, chondroitin sulfate, polycarbonate or poly-cystine.

such as hyaluronic acid modified by compound containing double bonds, carboxymethyl chitosan, sodium alginate, chondroitin sulfate, polycarbonate or poly-cystine.

Is collagen, gelatin or silk fibroin.

Is cartilage cell, medicine or protein active factor.

Example 1:

0.4g of sodium hyaluronate (molecular weight is 100000Da) dry powder is weighed and dissolved in 40mL of PBS solution, 0.56g of cysteamine hydrochloride is added into the dissolved solution, the pH value of the solution is adjusted to 5.0, 0.9g of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added into the solution, the pH value is kept stable and is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then freeze-dried to obtain a sample A1 for standby.

0.4g of sodium hyaluronate (molecular weight of 100000Da) is weighed and dissolved in 40mL of PBS solution, 0.6g of N- (2-aminoethyl) maleimide hydrochloride is added to the solution, the pH value is adjusted to 5.0, 0.6g of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added to the solution, the pH value is kept stable and the reaction solution is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then freeze-dried to obtain a sample B1 for later use.

A sample of 0.04g of A1 and 0.04g of B1 was taken; 0.02g of A1 and 0.02g of B1 were separately dissolved in 2mL of PBS solution, and the solutions A1 and B1 were rapidly mixed, and an equal volume of collagen solution was added to the mixture to obtain mixed solutions I and II, i.e., hydrogels I and II, having final concentrations of 20mg/mL and 10mg/mL at room temperature.

Example 2:

0.4g of sodium hyaluronate (molecular weight is 340000Da) dry powder is weighed and dissolved in 40mL of PBS solution, 0.56g of cysteamine hydrochloride and cysteamine hydrochloride are added into the dissolved solution, the pH value of the solution is adjusted to about 5.0 by dripping NaOH and HCl, 0.9g of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added into the solution, the pH value is kept stable and unchanged, the reaction solution is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then freeze-dried to obtain a sample A2 for later use.

0.4g of sodium hyaluronate (molecular weight of 340000Da) is weighed and dissolved in 40mL of PBS solution, 0.6g of N- (2-aminoethyl) maleimide hydrochloride is added to the solution, the pH value is adjusted to 5.0, 0.6g of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added to the solution, the pH value is kept stable, the reaction solution is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then freeze-dried to obtain a sample B2 for later use.

A sample of 0.04g of A2 and 0.04g of B2 was taken; and respectively dissolving 0.02g of A2 and 0.02g of B2 in 2mL of PBS solution, quickly mixing the A2 and B2 solutions, adding an equal volume of collagen solution into the mixed solution, and obtaining mixed solutions III and IV with final concentrations of 20mg/mL and 10mg/mL at room temperature, namely hydrogels III and IV.

Example 3:

0.4g of sodium hyaluronate (molecular weight is 1000000Da) dry powder is weighed and dissolved in 40mL of PBS solution, 0.56g of cysteamine hydrochloride is added into the dissolved solution, the pH value of the solution is adjusted to about 5.0 by adding NaOH and HCl dropwise, 0.9g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added into the solution, the pH value is kept stable and is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then freeze-dried to obtain a sample A3 for later use.

0.4g of sodium hyaluronate (molecular weight of 1000000Da) is weighed and dissolved in 40mL of PBS solution, 0.6g of N- (2-aminoethyl) maleimide hydrochloride is added to the solution, the pH value is adjusted to 5.0, 0.6g of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC) and 0.3g of N-hydroxysuccinimide (NHS) are added to the solution, the pH value is kept stable, the reaction solution is reacted for 24 hours at room temperature, and the reaction solution is dialyzed, purified and then lyophilized to obtain a sample B3 for standby.

Weighing 0.04g of A3 and 0.04g of B3; and respectively dissolving 0.02g of A3 and 0.02g of B3 in 2mL of PBS solution, quickly mixing the A3 and B3 solutions, adding an equal volume of collagen solution into the mixed solution, and obtaining mixed solutions V and VI with final concentrations of 20mg/mL and 10mg/mL at room temperature, namely hydrogels V and VI.

Test 1:

mixtures I, II, III, IV, V and VI prepared in examples 1, 2 and 3, respectively, were taken. The injectable extrusion force of each group of samples was measured under a universal material tester, and the specific measurement results are shown in fig. 1. As can be seen from fig. 1, the influence of the concentration of the precursor solution on the injection extrusion force of the hydrogel is found to be much greater than the effect of the molecular weight. The extrusion force of the hydrogel of the 20mg/mL group is distributed in the range of 24-42N, which is much higher than that of the hydrogel of the 10mg/mL group in the range of 17-20N.

And (3) testing 2:

the mixtures I, II, III, IV, V and VI of examples 1, 2 and 3 were taken. The storage modulus G 'was measured under a rotational rheometer, and the loss modulus G' versus time curve is shown in FIG. 2. As can be seen from FIG. 2, the gel time of the 1.0M Da 20mg/mL hydrogel was the shortest, requiring only about 5 seconds, and the longest gel time of the six groups was not longer than two minutes.

And (4) testing:

the mixtures I, II, III, IV, V and VI of examples 1, 2 and 3 were taken. The swelling performance in the PBS solution was measured separately, as shown in FIG. 3. As can be seen from FIG. 3, the maximum swelling ratio of the six groups of hydrogels is 0.1M Da 10mg/mL, the swelling ratio can reach 36%, and the minimum swelling ratio of the six groups of hydrogels is 1.0M Da 20mg/mL, the swelling ratio can reach 23%, which indicates that the hyaluronic acid-based hydrogels have better swelling performance.

And (5) testing:

the mixtures I, II, III, IV, V and VI of examples 1, 2 and 3 were taken. Degradation performance in the DTT solution (a) and the hyaluronidase solution (B) was measured separately, as shown in fig. 4. As can be seen from FIG. 4, under the reductive degradation of 0.1mM DTT, the hydrogel was degraded faster in the first 4 hours, and the quality thereof tended to be stable after 17 hours. The overall degradation trend of the six groups of hydrogels in hyaluronidase is that the degradation rate thereof gradually increases as the molecular weight of hyaluronic acid and the concentration of the precursor solution decrease.

And 6, testing:

the mixtures I, II, III, IV, V and VI of examples 1, 2 and 3 were taken. The storage modulus G 'and the loss modulus G' were measured separately and plotted against frequency as shown in FIG. 5. As can be seen from FIG. 5, the storage modulus (G') of the 1.0M Da 20mg/mL hydrogel can reach 13KPa at a frequency of 10Hz, which is caused by the construction of a skeleton network in the hydrogel by two chemical crosslinking methods, the increase of the molecular weight and concentration of hyaluronic acid, and the superposition of the effects of the increase of intermolecular forces.

And 7, testing:

the mixed chondrocytes were cultured for 7 days in the mixed solutions I, II, III, IV, V and VI of examples 1, 2 and 3, and their cell growth was examined, as shown in FIG. 6. As can be seen from FIG. 6, for the 0.1M Da, 10mg/mL hydrogel, the cells were uniformly distributed in the hydrogel, and at 7 days, the cells apparently appeared in an aggregate growth state and existed in the gel interior in the form of cluster growth; for the hydrogel of 1.0M Da and 20mg/mL, the phenomenon of cell aggregation and growth can be observed at 7 days, but the conglomeration density is not as strong as that of the hydrogel of 0.1M Da and 10mg/mL, and the reason for the phenomenon is probably that the inner pore size of the low-molecular gel is large, which is favorable for the proliferation and migration of cells.

The comprehensive test result shows that the hydrogel solution II with the molecular weight of 100000Da and the concentration of 10mg/mL wraps the chondrocytes for co-culture, and the application of the hydrogel solution II in cartilage tissue engineering is better.

Example 4:

the embodiment discloses a preparation method of a tissue engineering three-dimensional cell scaffold, which comprises the following specific steps:

step a. the maleimidoylated hyaluronic acid of example 1 (i.e. sample a1) was dissolved in a culture medium to form a maleimidoylated hyaluronic acid solution with a concentration of 10 wt%;

the thiolated hyaluronic acid of example 1 (i.e., sample B1) was dissolved in a culture medium to form a thiolated hyaluronic acid solution having a concentration of 10 wt%;

dissolving type I collagen with the concentration of 10 wt% by using dilute acetic acid after the type I collagen passes through the biological filter.

And step B, quickly mixing the prepared thiolated hyaluronic acid solution and the maleimide hyaluronic acid solution, adding the type I collagen solution, uniformly mixing (namely the volume ratio of the maleimide hyaluronic acid solution to the thiolated hyaluronic acid solution to the type I collagen solution is 1:1:1), adjusting the pH value to 7.4, and finally adding the cell suspension and uniformly mixing.

Wherein the amount of the cell suspension added is 5 × 10⁵～5×10⁶cell/mL ratio to injectable double-crosslinked composite based on natural material hyaluronic acidAdding cell suspension into the hydrogel mixed solution.

Respectively putting the mixed solution with the adjusted pH value into a mold, standing to form gel, taking out the obtained hydrogel from the mold, immersing the hydrogel in a culture medium, and placing the hydrogel in an incubator at 37 ℃ and 5% CO₂At least 1 day, and periodically replacing the medium every three days during the culturing period. The result shows that the tissue engineering three-dimensional cell scaffold is obtained.

The culture medium is obtained by adding a mixed solution of penicillin and streptomycin, ascorbic acid and fetal calf serum on the basis of an alpha-MEM basic culture medium, wherein the mass concentration of the mixed solution of penicillin and streptomycin in the alpha-MEM basic culture medium is 1%, the mass concentration of the ascorbic acid is 0.2%, and the mass concentration of the fetal calf serum is 10%. The penicillin and streptomycin mixture in this example was supplied by Hyclone.

After 3 days, 7 days, 14 days and 21 days of culture, the three-dimensional cell scaffolds were taken out, and firstly photographed by a digital camera, and the degradation of the gel blocks after different times of culture was observed, as shown in fig. 7. The three-dimensional cell scaffolds were then washed 3 times with PBS buffer, and the washed three-dimensional cell scaffolds were stained for 1min by immersion in a staining solution containing 5. mu.g/mL FDA and 1. mu.g/mL PI. Meanwhile, the washed three-dimensional cell scaffold was immersed in a staining solution containing phalloidin for 3 hours, then washed 3 times with PBS buffer, and the growth state and cytoskeleton of the cells in the three-dimensional scaffold were observed by a Confocal Laser Scanning Microscope (CLSM), as shown in fig. 8. Then, the proliferation of cells in the gel mass was quantitatively determined by using CCK-8 reagent, and the results are shown in FIG. 9. Wherein, the control group is pure hyaluronic acid hydrogel and pure collagen hydrogel.

As can be seen from the above figures, the cell proliferation is obvious along with the increase of time, and the conglomerate growth appears at the later stage, so that the hydrogel is beneficial to be applied to the three-dimensional cell scaffold for tissue engineering.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A self-crosslinking hyaluronic acid and hydrogel injection of composite collagen thereof is characterized in that the structure of the injection is shown as formula I,

wherein

Is a natural material or a synthetic polymer modified by sulfydryl,

is a protein natural material or a derivative thereof,

is a bioactive substance.

2. The injection of claim 1, wherein: the natural material or the synthetic polymer in the sulfydryl modified natural material or the synthetic polymer and the double bond-containing compound modified natural material or the synthetic polymer respectively have carboxyl functional groups, and include but are not limited to any one or more of hyaluronic acid, carboxymethyl chitosan, sodium alginate, chondroitin sulfate, polycarbonate and poly-cystine; the protein natural material or the derivative thereof comprises any one or more of collagen, gelatin and silk fibroin; the bioactive substances include but are not limited to one or more of cells, drugs and protein active factors.

3. The process for preparing an injection according to claim 1, comprising the steps of:

preparing a sulfydryl modified natural material or a synthetic polymer and a double-bond compound modified natural material or a synthetic polymer into solutions respectively, and then carrying out chemical crosslinking on the solutions and a protein high molecular material or a derivative thereof under the action of Michael addition reaction to load cells, drugs or protein active factors to prepare the injectable double-crosslinked composite hydrogel injection loaded with the bioactive substances; the reaction formula is as follows:

4. A process for the preparation of an injection as claimed in claim 3, characterized by comprising the steps of:

step B, Michael addition reaction: b, mixing the maleimide hyaluronic acid solution and the thiolated hyaluronic acid solution respectively prepared in the step A, then adding a collagen solution and a chondrocyte suspension, uniformly mixing, and standing; forming hydrogel by reacting maleimide hyaluronic acid solution containing collagen and chondrocytes with thiolated hyaluronic acid solution under the action of Michael addition reaction;

the molar ratio of the thiolated hyaluronic acid to the maleimidoylated hyaluronic acid is: 1-50: 1-25.

5. The process for producing an injection according to claim 4,

6. The method for preparing an injection according to claim 4, wherein the thiolated hyaluronic acid solution has a pH of 7.0 to 8.0 and a concentration of 0.1 wt% to 10 wt%;

the pH value of the maleimide hyaluronic acid solution is 7.0-8.0, and the concentration is 0.1-10 wt%;

the pH value of the collagen solution is 7.0-8.0, and the concentration is 0.1-10 wt%;

the concentration of the chondrocyte suspension is 5 × 10²～5×10¹⁰cells/mL。

7. The method for preparing an injection according to claim 4, wherein the molecular weight of each hyaluronic acid is 1 to 6000 kDa; the collagen is type I or type II collagen; the volume ratio of the total hyaluronic acid derivative solution to the total collagen is 1: 0.1-0.1: 1; the temperature of the Michael addition reaction is room temperature.

8. The method of preparing an injection according to claim 4, wherein the thiolated hyaluronic acid is prepared by: dissolving hyaluronic acid in a Mes buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate for 1-2 h, then adding cysteamine hydrochloride to react for 10-15 h at room temperature, and dialyzing to obtain thiolated hyaluronic acid; the molar ratio of the sodium hyaluronate, the N-hydroxysuccinimide, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the cysteamine hydrochloride is regulated and controlled according to requirements.

9. The method of preparing an injection according to claim 4, wherein the maleimidoylated hyaluronic acid is prepared by: dissolving hyaluronic acid in a Mes buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for activation for 1-2 h, then adding N- (2-aminoethyl) maleimide hydrochloride, reacting for 10-15 h at room temperature, and dialyzing to obtain maleimido hyaluronic acid; the molar ratio of the sodium hyaluronate, the N-hydroxysuccinimide, the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N- (2-aminoethyl) maleimide hydrochloride is regulated and controlled according to requirements.

10. The use of the injection according to claim 1 or claim 2 as an injection for promoting cartilage/bone regeneration repair, as a drug/protein/gene carrier, or/and as a biological scaffold material.