CN115487358A

CN115487358A - Gel composite scaffold for cartilage tissue repair and preparation method thereof

Info

Publication number: CN115487358A
Application number: CN202210940356.8A
Authority: CN
Inventors: 许泽川; 韦佼君; 黄宏杰; 陈铭
Original assignee: Nuclear Industry 416 Hospital
Current assignee: Nuclear Industry 416 Hospital
Priority date: 2022-08-05
Filing date: 2022-08-05
Publication date: 2022-12-20
Anticipated expiration: 2042-08-05
Also published as: CN115487358B

Abstract

The invention provides a preparation method of a gel composite scaffold for cartilage tissue repair, which comprises the following steps: (1) Loading KGN into PELA electrospun fiber by using an electrostatic spinning method to obtain KGN-PELA fiber; loading GDF-5 into PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the lengths of the two electrospun fibers to be not higher than 100 mu m; (2) Adding aldehyde hyaluronic acid and succinylated chitosan into PBS solution, adding KGN-PELA fiber and GDF-5-PCL fiber, and obtaining the gel composite scaffold after reaction. The gel composite scaffold has good mechanical property and can be injected; meanwhile, KGN and GDF-5 in the invention have synergistic effect in inducing cartilage differentiation of bone marrow mesenchymal stem cells; the invention has good application prospect in the aspect of treating cartilage defect repair.

Description

Gel composite scaffold for cartilage tissue repair and preparation method thereof

Technical Field

The invention belongs to the technical field of biological materials, relates to the field of cartilage tissue repair biological materials, and particularly relates to a gel composite scaffold for cartilage tissue repair and a preparation method thereof.

Background

Articular cartilage tissue has no blood vessels, nerves and lymph tissues, has low cell content, and the nutrition supply is from the moistening of peripheral joint synovial fluid, so that the joint cartilage tissue cannot or is difficult to repair by self after being damaged. At present, the main methods for clinically treating articular cartilage damage have many risks and defects, such as the problems of difficult integration of fibrotic cartilage repair, transplanted cartilage and autologous cartilage matrix of a receptor, immune rejection, non-arrangement of related cells according to tissue structures, wound bacterial infection and the like, and finally the failure of cartilage repair and even pathological changes are caused.

At present, the tissue engineering technology is considered to be one of ideal methods for repairing cartilage defects, and the tissue engineering scaffold not only can simulate the extracellular matrix of chondrocytes to promote the growth and differentiation of the chondrocytes, but also can provide physical signals for the chondrocytes and enhance the adhesion and migration capacity of the chondrocytes. In practical application, the injectable scaffold has advantages over a surgical implantable scaffold, and the injectable hydrogel scaffold can be used for filling irregular-shaped defects based on in-situ polymerization, so that the complexity of a surgical operation is reduced. Wang (Wang) ^[1] And the like prepares the four-arm star-shaped polyethylene glycol hydrogel modified by vinyl sulfone, and realizes the injection filling treatment of cartilage defects. The gel is in a porous structure after cross-linking, which is beneficial to exchange gas and substances to promote cell migration and adhesion and promote the tight combination of the new cartilage and surrounding cartilage tissues. Therefore, the injection type hydrogel provides a new treatment idea for cartilage tissue repair, can be used for in-situ filling treatment, and reduces the operation risk and secondary injury.

Improving the mechanical strength of the hydrogel to obtain the same viscoelasticity and mechanical magnitude as those of normal cartilage is a key problem for repairing cartilage defects. The common gel crosslinking methods at present are response type crosslinking and chemical crosslinking. The response type crosslinking is related to factors such as temperature, pH, light intensity, enzyme substrate and the like, the mechanical property of the response type crosslinking is easily influenced by energy and the substrate, so that the response is poor, and the problems of toxic and side effects of an initiator, uncontrollable energy of an external source and the like are caused. Chemical crosslinking is mainly reversed by collisions of chemical groupsIt should be used. The mechanical strength of the gel is related to the density of chemical groups and the concentration of the gel, and the gel has good controllability. Tan is a derivative of Tan ^[2] And crosslinking the prepared aldehyde Hyaluronic Acid (HA) and chitosan by using Schiff base reaction to form gel. The gel has good viscoelasticity, is beneficial to cell adhesion, and can shorten the hydrogel crosslinking time by adjusting the reaction ratio of aldehyde groups and amino groups. However, the gel has a narrow mechanical property regulation range and few optimized conditions, and cannot realize accurate regulation of ideal stent parameters. In order to solve the problems, the regulation range of the mechanical property of the gel is expanded by adopting methods such as gel double crosslinking, crosslinking of the gel and rigid nano particles, forming of a composite scaffold by the gel and a solid porous scaffold and the like. Supanasa ^[3] The fibroin fiber and the fibroin hydrogel are used as raw materials to design a fibroin-hydrogel composite material similar to reinforced concrete, the fibroin fiber penetrates through the internal structure of the gel, the mechanical property of the fibroin-hydrogel composite material is obviously improved, but the block-shaped composite material can only be implanted into a defect part through an operation, and the secondary damage of an organism is easily caused.

To solve the problems of stent implantation, there have been designs to dope injectable materials with gel composite, hong ^[4] And the like, the collagen-coated polylactic acid microcarrier and the chitosan are adopted to prepare the injectable hydrogel in a crosslinking manner, so that the mechanical property of a composite system is obviously improved, and the physiological function of chondrocytes is regulated and controlled. However, nanofibers of the same size have a larger specific surface than microspheres; from the aspect of appearance, the contact area between the microspheres is small; the steric hindrance of the microspheres is large and the voids formed after aggregation are small, which may reduce the porosity of the gel. Wei ^[5] And the mechanical strength of the gel is improved by doping short fibers, and KGN is loaded to induce MSC (mesenchymal stem cells) to differentiate into chondrocytes, so that the repair effect of cartilage defects is obtained.

However, loading an inducer into electrospun fibers has a problem that the drug loading is small and it is difficult to achieve good induction of chondrocyte differentiation. That is, although short fibers have a distinct advantage in improving gel strength, the effect of inducing MSC differentiation into chondrocytes is limited by the problem of drug loading. In order to solve this problem, a drug having a higher inducibility or a drug which can exert a synergistic effect with a conventional drug is sought and used together.

KGN (Kartogenin) is found for the first time in 2012 as a small molecule cartilage differentiation inducer, and KGN is dose-dependent and has an ideal inducing effect on the differentiation of MSC into chondrocytes. Currently, the research on agents that can produce a synergistic effect with KGN in inducing differentiation of the relevant cells into chondrocytes is very limited, and only a few sporadic reports have been made. Such as Yanhong ZHao ^[6] The inventors find that KGN and TGF-beta 3 have synergistic effect in inducing the differentiation of human umbilical cord mesenchymal stem cells into chondrocytes; likewise, wenshuai Fan ^[7] The inventors also found that KGN and TGF-beta 3 also have a synergistic effect in inducing differentiation of endogenous mesenchymal stem cells; in addition, zhaofeng Jia ^[8] It has also been found that KGN and TGF-beta 3 have the same synergistic effect in promoting the differentiation of SF-MSCs chondrocyte.

To the best of the inventors' knowledge, the active substances reported to have a synergistic effect with KGN in promoting chondrocyte differentiation have mainly been focused on TGF- β 3. Therefore, this poses a great limitation to the loading of KGN into short fibers and the preparation of corresponding injectable gels to achieve the repair of cartilage defects.

In summary, in the preparation of injectable gels with better mechanical properties for cartilage defect repair, there is still a great need in the prior art to find substances as active ingredients that can have a synergistic effect with KGN in inducing MSC differentiation into chondrocytes.

Reference:

[1]J Wang,F Zhang,W P Tsang et al.Fabrication of injectable high strength hydrogel based on 4-arm star PEG for cartilage tissue engineering[J].Biomaterials,2017,120:11-21.

[2]H Tan,C R Chu,K A Payne et al.Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering[J].Biomaterials,2009,30:2499-2506.

[3]S Yodmuang,S L McNamara,A B Nover et al.Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair[J].Acta Biomaterialia,2016,11:27-36.

[4]Y Hong,Y Gong,C Gao et al.Collagen-coated polylactide microcarriers/chitosan hydrogel composite:Injectable scaffold for cartilage regeneration[J].Part A:Journal of Biomedical Materials Research,2008,85A:628-637.

[5]Wei J,Ran P,Li Q,et al.Hierarchically structured injectable hydrogels with loaded cell spheroids for cartilage repairing and osteoarthritis treatment[J].Chemical Engineering Journal,2022,430:132211-.

[6]Synergistic Effects of Kartogenin and Transforming Growth Factor-β3on Chondrogenesis of Human Umbilical Cord Mesenchymal Stem Cells InVitro[J].Orthopaedic Surgery,2020,12(3).

[7]Fan W,Yuan L,Li J,et al.Injectable double-crosslinked hydrogels with kartogenin-conjugated polyurethane nano-particles and transforming growth factorβ3for in-situ cartilage regeneration[J].Materials Science and Engineering:C,2020,110:110705-.

[8]Jia Z,Wang S,Liang Y,et al.Combination of kartogenin and transforming growth factor-β3supports synovial fluid-derived mesenchymal stem cell-based cartilage regeneration[J].American Journal ofTranslational Research,2019,11(4).

disclosure of Invention

Aiming at the defects of the prior art, the core purpose of the invention is to search other substances which have synergistic effect with KGN in inducing MSC to differentiate into chondrocytes except TGF-beta 3 so as to overcome the defect that KGN has insufficient effect on repairing cartilage defects in an injectable hydrogel preparation; on the basis, the invention also aims to provide a composite gel scaffold similar to reinforced concrete, improve the mechanical property of the scaffold and further improve the application value in the aspect of cartilage defect repair.

Aiming at the above purpose, the invention provides the following technical scheme:

a preparation method of a gel composite scaffold for cartilage tissue repair comprises the following steps:

(1) Loading KGN into PELA electrospun fiber by using an electrostatic spinning method to obtain KGN-PELA fiber; loading GDF-5 into PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the lengths of the two types of electrospun fibers to be not more than 100 mu m;

(2) Adding aldehyde hyaluronic acid and succinylated chitosan into PBS solution, adding KGN-PELA fiber and GDF-5-PCL fiber, and obtaining the gel composite scaffold after reaction;

wherein, the aldehyde hyaluronic acid is obtained by reacting hyaluronic acid with sodium periodate; the succinylated chitosan is obtained by reacting chitosan with succinic anhydride;

in the step (2), the weight ratio of KGN to GDF-5 in the KGN-PELA fibers and the GDF-5-PCL fibers is 1.

GAG and Col II are characteristic secretions of chondrocytes, and can be used as a proof of the effect of cartilage differentiation. As shown in the related examples and experimental examples of the present invention, the gel composite scaffold of the present invention can significantly increase the secretion amounts of GAG and Col II, while the secretion amounts of GAG and Col II in the comparative examples are less. This shows that KGN and GDF-5 have a synergistic effect in promoting cartilage differentiation in the gel composite scaffold of the present invention.

The inventors have appreciated that although GDF-5 has also been used to induce cartilage differentiation, it is also rarely used in conjunction with its ability to exert a synergistic effect on chondrogenesis. For example b.appel ^[9] It has been found that GDF-5 and insulin have a synergistic effect in cartilage formation; s oa font tellado ^[10] It has been found that TGF-b2 and GDF5 increase the expression of cartilage markers and type II collagen; wangJiang's discovery of GDF-5 ^[11] The combination of icaritin can induce rat BMSCs to chondrogenic differentiation, wherein the icaritin plays a promoting role. Similarly, GDF-5 has limited use in cartilage defect repair due to its lack of substances that act synergistically in inducing cartilage differentiation.

It will be appreciated that the present invention has found that, in addition to TGF-. Beta.3, another substance which has a synergistic effect with KGN in promoting cartilage differentiation. Meanwhile, the gel composite scaffold is injectable gel, so that the gel composite scaffold can be better used for repairing cartilage defects.

As a preferred technical scheme of the invention, when preparing KGN-PELA fiber, KGN is dissolved in acetone, PELA is dissolved in a mixed solvent of acetone and DMF with the volume ratio of 6; when preparing GDF-5-PCL fiber, dissolving GDF-5 in acetone, dissolving PCL in a mixed solvent of chloroform and acetone with the volume ratio of 1;

the spinning conditions for electrostatic spinning of KGN-PELA spinning solution and GDF-5-PCL spinning solution are as follows: the advancing speed was 1.6mL/h, the distance between the injector nozzle and the receiver was 15cm, the voltage was 20kV, the solid drum was used as the receiving device, and the rotational speed was 2500r/min.

As a preferred technical scheme of the invention, in the KGN-PELA fiber, the drug-loading rate of KGN is 0.1wt%; in the GDF-5-PCL fiber, the drug loading of GDF-5 is 0.2wt%.

As a preferred technical scheme of the invention, the preparation method of the aldehyde hyaluronic acid comprises the following steps: adjusting the pH value of deionized water to 6.0 by using dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150ml of deionized water with the pH value of 6.0; stirring for 24 hours by magnetic force to fully dissolve the mixture; then 16.5ml of sodium periodate solution with the concentration of 0.25mol/L is added, magnetic stirring is carried out for 3h at the temperature of 40 ℃, and then 30ml of ethylene glycol is added to stop the reaction; and dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.

As a preferred technical scheme of the invention, the preparation method of the succinylated chitosan comprises the following steps: dissolving 1.0g of chitosan in dilute hydrochloric acid with the volume concentration of 0.37%, and adding glucosamine to ensure that the concentration of the chitosan is 6.20mmol/L to obtain a chitosan solution; 0.63g of succinic anhydride was dissolved in 5ml of pyridine to obtain a succinic anhydride solution. Dropwise adding the succinic anhydride solution into the chitosan solution at room temperature (25-30 ℃) in a fume hood under the stirring condition; adjusting the pH value to 7.0, reacting for 4 hours, and adding 20w/v% NaCl solution to terminate the reaction; adding ethanol, filtering, washing the precipitate with diethyl ether, and drying.

As a preferred technical scheme of the invention, the KGN-PELA fibers and the GDF-5-PCL fibers have the length of 50 mu m.

In the preferred embodiment of the present invention, in the step (2), the reaction is carried out at 37 ℃ for 12 hours.

In the present invention, the gel composite scaffold for cartilage tissue repair is injectable in vivo and reacts under body temperature conditions.

Another object of the present invention is to provide a gel composite scaffold for cartilage tissue repair prepared by the above preparation method.

The invention also aims to provide application of the gel composite scaffold in preparation of a preparation for cartilage tissue repair.

The invention has the beneficial effects that:

the gel composite scaffold for cartilage tissue repair provided by the invention has good mechanical properties and can be injected; meanwhile, KGN and GDF-5 in the invention have synergistic effect in inducing cartilage differentiation of bone marrow mesenchymal stem cells, and the defect of low cartilage differentiation effect in the prior art is overcome; the invention has good application prospect in the aspect of treating cartilage defect repair.

Reference documents:

[9]Appel B,Baumer J,Eyrich D,et al.Synergistic effects of growth and differentiation factor-5(GDF-5)and insulin on expanded chondrocytes in a 3-D environment[J].Osteoarthritis&Cartilage,2009,17(11):1503-1512.

[10]Tellado,Sonia,Font,et al.Heparin functionalization increases retention of TGF-beta 2and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering[J].Acta Biomaterialia,2018.

[11] GDF-5 in combination with icaritin induces differentiation and mechanism of BMSCs into cartilage-like cells.

Drawings

FIG. 1 shows the results of swelling ratio and compression resistance of gel composite scaffolds doped with different concentrations of short fibers (part a in FIG. 1), and the rheology curve (part b in FIG. 1);

FIG. 2 is an SEM photograph of the gel composite scaffold obtained in example 1 of the present invention.

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Unless otherwise specified, abbreviations in the present invention have the following meanings:

KGN: kartogenin,2- ([ 1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid;

PELA: polylactic acid-polyethylene glycol copolymers;

PCL: polycaprolactone;

GDF-5-PCL fiber: PCL fibers loaded with GDF-5;

KGN-PELA fiber: PELA fibers loaded with KGN;

BMSC: bone marrow mesenchymal stem cells; example 1

1. Experimental materials

PELA (Mw =50kda, mw/Mn = 1.23), gifted to the institute of wonderful medicine; PCL (Mw =80 kDa), available from shanghai source leaf technologies ltd; hyaluronic acid (Mw =38 kDa), purchased from santa seifura bio-pharmaceutical company; KGN (Kartogenin) and GDF-5 were purchased from Alantin; chitosan (degree of deacetylation 96.0%), purchased from bio-pharmaceutical company of eastern Xiforda of mountain; other reagents were available commercially from the laboratory.

2. Preparation of drug-loaded short fiber

(1) Dissolving KGN with different concentrations (concentration range of 0.1mg-1mg, gradient of 0.05 mg) in 2mL of acetone, dissolving 500mg of PELA in 3.0mL of a mixed solvent of acetone and DMF with a volume ratio of 6, and then uniformly mixing the KGN solution and the PELA polymer solution to obtain a KGN-PELA spinning solution; carrying out electrostatic spinning on the KGN-PELA spinning solution, wherein the spinning conditions of the electrostatic spinning are as follows: the advancing speed was 1.6mL/h, the distance between the injector nozzle and the receiver was 15cm, the voltage was 20kV, the solid drum was used as the receiving device, and the rotational speed was 2500r/min. Finally, KGN-PELA fiber with the drug loading of 0.1wt% is selected.

The drug-loaded oriented fiber membrane was soaked with distilled water and folded at 1cm intervals in a direction perpendicular to the orientation direction of the fibers, and frozen at-70 ℃ for 5min. The cryomicrotome was set to a cut thickness of 50 μm. And finally, cleaning, centrifugally collecting short fibers, freeze-drying and storing in a dry and cool place for later use.

(2) Dissolving GDF-5 with different concentrations (concentration range is 0.1mg-1mg, gradient is 0.1 mg) in 2mL of acetone, dissolving 450mg of PCL in a chloroform and acetone mixed solvent with the volume ratio of 1; carrying out electrostatic spinning on the GDF-5-PCL spinning solution, wherein the spinning conditions of the electrostatic spinning are as follows: the advancing speed was 1.6mL/h, the distance between the injector nozzle and the receiver was 15cm, the voltage was 20kV, the solid drum was used as the receiving device, and the rotational speed was 2500r/min. Finally selecting GDF-5-PCL fiber with drug loading of 0.2wt%.

3. Preparation of aldehyde hyaluronic acid

Adjusting the pH value of deionized water to 6.0 by using dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150ml of deionized water with the pH value of 6.0; stirring for 24 hours by magnetic force to fully dissolve the mixture; then 16.5ml of sodium periodate solution with the concentration of 0.25mol/L is added, magnetic stirring is carried out for 3 hours at the temperature of 40 ℃, and then 30ml of ethylene glycol is added to stop the reaction; and dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.

4. Preparation of succinylated chitosan

Dissolving chitosan in 1v/v% acetic acid, slowly dripping the chitosan into 20ml of acetone solution dissolved with succinic anhydride, and stirring the mixture in a water bath for reaction for 4 hours; adding excessive acetone for precipitation, performing suction filtration to obtain a solid phase, washing with acetone, and drying for 4h to obtain solid particles, namely succinylated chitosan. .

5. Preparation of composite gel scaffold

Adding the aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the equal amount of 0.2 g/ml; adding KGN-PELA fiber and GDF-5-PCL fiber in equal weight; reacting at 37 ℃ for 12h to obtain hydrogel; regulating and controlling the total weight of KGN-PELA fibers and GDF-5-PCL fibers in the obtained gel scaffold to account for 1.0 percent of the total dry weight of the gel scaffold; and controlling the gel concentration to be 3% which is the total dry weight (w/v) of the aldehyde-modified hyaluronic acid and succinylated chitosan contained in each unit volume of the solution.

Comparative example 1

The procedure of example 1 was followed, except that KGN-PELA fibers were added only when preparing the composite scaffold. Namely, the gel scaffold method of this comparative example was:

adding the aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the equal amount of 0.2 g/ml; adding KGN-PELA fiber, and reacting at 37 ℃ for 12h to obtain hydrogel; in the obtained gel scaffold, the total weight of KGN-PELA fibers accounts for 1.0% of the weight of the gel scaffold.

Comparative example 2

The procedure of example 1 was repeated, except that GDF-5-PCL fibers were added only when preparing the composite scaffold. Namely, the gel scaffold method of this comparative example was:

adding the aldehyde hyaluronic acid and succinylated chitosan into PBS solution according to the equal amount of 0.2 g/ml; adding GDF-5-PCL fiber; reacting at 37 ℃ for 12h to obtain hydrogel; in the obtained gel scaffold, the total weight of GDF-5-PCL fiber accounts for 1.0% of the weight of the gel scaffold.

Experimental example 1

1. Culture of Stem cell spheroids

Reference is made to the literature (Wang W, li B, li Y, et al.I.)n vivo restoration of full-thickness cartilage defects by poly(lactide-co-glycolide)sponges filled with fibrin gel,bone marrow mesenchymal stem cells and DNA complexes[J]Biomaterials.2010,31 5953-5965) culturing BMSC stem cell spheres, namely: extracting bone marrow from a rabbit body, and separating BMSC from the bone marrow by a density gradient centrifugation method; rabbits (12kg, 1 month old) were anesthetized systemically (50 mg/kg dose) with 3g/L pentobarbital sodium, on a lateral operating table, with iliac depilatory treatment, and iodophor-sterilized, and joints were anesthetized locally with 2g/L lidocaine. 1mL of heparin sodium (3000 kU/L) was drawn from a 10mL syringe, which was then connected to a 16-gauge bone marrow puncture needle, which was then inserted into the bone marrow cavity, and 34mL of bone marrow fluid was drawn. The marrow fluid was then added to serum-free medium containing lymphocyte isolate and centrifuged at 2000rpm for 30min to remove the supernatant containing monocytes. The cells were resuspended by adding 5mL of serum-free medium, centrifuged at 1500rpm for 5min, the supernatant discarded, and the cells washed again. Resuspending the pelleted cells in low glucose DMEM medium containing 10% fetal bovine serum, 100mg/mL penicillin and 100U/mL streptomycin, placed at 37 ℃,5% ² Culturing under the condition of (3), changing the liquid every 2 days, and carrying out passage amplification after the cells are fused to more than 90%.

BMSC differentiation function test

The effect of the injection process of the composite gel scaffold obtained in example 1, comparative example 1 and comparative example 2 on the BMSC cell spheres was examined. Taking example 1 as an example, KGN-PELA fiber and GDF-5-PCL fiber with equal weight were uniformly dispersed in PBS solution containing aldehydic hyaluronic acid, BMSC cell spheres were dispersed in PBS solution containing succinylated chitosan, and the two solutions were rapidly and uniformly mixed (after mixing, it was ensured that aldehydic hyaluronic acid and succinylated chitosan were both 0.2G/ml, and KGN-PELA fiber and GDF-5-PCL fiber occupied 1w/w% of the gel obtained except BMSC cell spheres), injected into a 48-well plate through a 16G needle syringe, and after complete crosslinking of the gel, the medium was added for culturing, and the solution was changed every two days. Comparative examples 1 and 2 were carried out with reference to the above-mentioned method.

Function test of BMSC cell spheres wrapped in composite gel scaffolds: the secretion amount of GAG was measured by the dimethyl methylene blue (DMMB) method; the content of Col II was determined by Col II ELISA kit. The test results are shown in table 1.

TABLE 1 GAG, colII secretion results

Experimental example 2

Determination of aldehyde group content in aldehyde-ylated hyaluronic acid obtained in example 1: 0.2g of aldehyde-modified hyaluronic acid is weighed and placed in a 100mL beaker, 10mL of deionized water is added to completely dissolve the aldehyde-modified hyaluronic acid, the pH is adjusted to 5.0, 8mL of hydroxylamine hydrochloride reagent (0.05 g/mL, pH = 5.0) is accurately added into the solution, the solution is stirred for 4 hours under the condition of water bath at 40 ℃, finally, 0.01mol/L of NaOH standard solution is used for titration until the pH =5.0, and the volume number of consumed NaOH solution is recorded. Weighing hyaluronic acid with the same mass for blank experiment comparison.

The aldehyde group content = C (V1-V2)/m/376 x 100%

Wherein, C, naOH standard solution concentration mol/L; v1 is the volume (L) of NaOH consumed by the sample specimen; v2 is the volume number (L) of NaOH consumed by a hyaluronic acid blank experiment; and m is the mass (g) of the sample specimen.

The aldehyde group content of the aldehyde-based hyaluronic acid in example 1 was calculated to be 62%.

Experimental example 3

According to the method of preparation of succinylated chitosan membrane and pH sensitive study thereof ([ 1] plum as, zhangiuzhuang, zhang Qingxue, succinylated chitosan membrane and pH sensitive study thereof [ J ]. Food industry technology, 2009 (1): 3.), the acylation degree of succinylated chitosan in example 1 is investigated, and the acylation degree of succinylated chitosan in example 1 is 12.1%.

Experimental example 4

The gel composite scaffold obtained in example 1 was tested for swelling ratio, compression resistance, storage modulus G' and loss modulus G ″. As shown in FIG. 1, the gel composite scaffold obtained in example 1 has a better swelling ratio and better mechanical properties.

Claims

1. A preparation method of a gel composite scaffold for cartilage tissue repair is characterized by comprising the following steps:

(1) Loading KGN into PELA electrospun fiber by using an electrostatic spinning method to obtain KGN-PELA fiber; loading GDF-5 into PCL electrospun fiber to obtain GDF-5-PCL fiber; controlling the lengths of the two electrospun fibers to be not longer than 100 mu m;

2. The process according to claim 1, wherein, in the preparation of KGN-PELA fiber, KGN is dissolved in acetone, PELA is dissolved in a mixed solvent of acetone and DMF at a volume ratio of 6; when preparing GDF-5-PCL fiber, dissolving GDF-5 in acetone, dissolving PCL in a mixed solvent of chloroform and acetone with the volume ratio of 1;

the spinning conditions for electrostatic spinning of the KGN-PELA spinning solution and the GDF-5-PCL spinning solution are as follows: the advancing speed was 1.6mL/h, the distance between the injector nozzle and the receiver was 15cm, the voltage was 20kV, the solid drum was used as the receiving device, and the rotational speed was 2500r/min.

3. The method according to claim 1, wherein the KGN-PELA fiber has a KGN drug loading of 0.1wt%; in the GDF-5-PCL fiber, the drug loading of the GDF-5 is 0.2wt%; the total weight of the KGN-PELA fibers and the GDF-5-PCL fibers accounts for 1.0 percent of the total dry weight of the gel scaffold.

4. The method according to claim 1, wherein the aldehyde-modified hyaluronic acid is prepared by: adjusting the pH value of deionized water to 6.0 by using dilute hydrochloric acid, and dissolving 1.5g of hyaluronic acid in 150ml of deionized water with the pH value of 6.0; stirring for 24 hours by magnetic force to fully dissolve the mixture; then 16.5ml of sodium periodate solution with the concentration of 0.25mol/L is added, magnetic stirring is carried out for 3h at the temperature of 40 ℃, and then 30ml of ethylene glycol is added to stop the reaction; and dialyzing for 3 days, changing water every day, and freeze-drying to obtain the aldehyde hyaluronic acid.

5. The method according to claim 1, wherein the succinylated chitosan is prepared by: dissolving chitosan in 1v/v% acetic acid, slowly dripping into 20ml acetone solution dissolved with succinic anhydride, and stirring in water bath for reaction for 4h; adding excessive acetone for precipitation, performing suction filtration to obtain a solid phase, washing with acetone, and drying for 4h to obtain solid particles, namely succinylated chitosan.

6. The method for preparing in accordance with claim 1, wherein the KGN-PELA fibers and GDF-5-PCL fibers have a length of 50 μ ι η.

7. The production method according to claim 1, wherein the reaction is carried out at 37 ℃ in the step (2) for 12 hours.

8. The method for preparing the gel composite scaffold for cartilage tissue repair according to claim 1, wherein the gel composite scaffold for cartilage tissue repair is injectable in vivo and reacts under body temperature conditions.

9. A gel composite scaffold for cartilage tissue repair, which is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the gel composite scaffold of claim 9 in the preparation of a formulation for cartilage tissue repair.