CN105688284B

CN105688284B - In-situ hydrogel capable of imitating extracellular matrix injection and preparation method and application thereof

Info

Publication number: CN105688284B
Application number: CN201610049493.7A
Authority: CN
Inventors: 田猛; 游潮
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2014-09-29
Filing date: 2014-10-23
Publication date: 2019-12-06
Anticipated expiration: 2034-10-23
Also published as: CN104307049A; CN105688284A; CN104307049B

Abstract

The invention discloses a raw material box for preparing hydrogel, which comprises the following raw materials: sulfhydrylated gelatin, sulfhydrylated polysaccharide, polyethylene glycol diacrylate and solvent. By using the raw material box, the proportion of different thiolated gelatins and thiolated polysaccharides can be selected according to requirements, multiple hydrogels with different degradation properties and/or release characteristics can be prepared, the hydrogel can be suitable for repairing different tissues with different purposes, the trouble of re-screening raw materials is avoided, the hydrogel is simple and easy to implement, the time and the labor are saved, the cost is reduced, and the hydrogel has good economic value and is very suitable for industrial application.

Description

In-situ hydrogel capable of imitating extracellular matrix injection and preparation method and application thereof

Technical Field

The invention relates to an extracellular matrix-imitated injectable in-situ hydrogel and a preparation method and application thereof.

Background

In the past, human beings have explored the repair and regeneration of body tissues. In recent years, with the development of biomaterials, tissue engineering, and stem cell technologies, research in the field of tissue repair and regeneration has been breaking through. The current research is mainly focused on two aspects: one is the administration of a drug treatment such as growth factors to induce tissue remodeling of endogenous cells at the affected site; another is to implant cells to exert a therapeutic effect on the affected area. Of these two strategies, the design and development of carrier materials undoubtedly play an increasingly important role.

First, the degradation of the carrier material should be matched to the tissue growth. Secondly, carriers for drug delivery must also have the property of controlled release of the drug (V elevator e.santo, Manuela e.gomes, f.mano et al.tissue Engineering Part B2013, 19, 308-326); the carrier for cell delivery should have the function of controlling cell fate (MP Lutolf, PM Gilbert, HM Blau. Nature 2009,462, 433-441; J Thiele, Y Ma, S Bruekers et al. adv. Mater 2014,26, 125-148).

The in situ hydrogel used as a drug or cell transfer carrier has obvious advantages, and can be directly injected into an implantation site, or cells can be implanted into the body after being cultured in vitro for a period of time. Therefore, there is an increasing interest in developing such in situ hydrogel carriers. Various in situ hydrogel gelling mechanisms have been reported including: ultraviolet light initiated free radical polymerization; michael addition reaction; enzymatic crosslinking, and thermally-induced physical crosslinking, among others. Ultraviolet light-initiated free radical polymerization is the most reported one, and after double bonds are introduced on a molecular chain, an initiator is subjected to crosslinking under ultraviolet light irradiation. However, the use of uv light has been limited due to its limited transparency. The Michael addition reaction can be carried out under physiological conditions, an initiator and other initiation conditions are not needed, and the reaction is crosslinked through sulfydryl and double bonds.

Chinese patent CN101864178A discloses an injectable protein/polypeptide in-situ hydrogel for drug sustained release and cell culture, but the hydrogel is composed of natural or synthetic protein/polypeptide with crosslinkable phenolic hydroxyl groups, horseradish peroxidase and hydrogen peroxide, and a chemical crosslinking hydrogel is rapidly formed under physiological conditions.

Chinese patent CN102718991A discloses a hydrogel formed by Michael addition reaction between double bond of polyethylene glycol diacrylate (PEGDA) and sulfydryl on sulfhydrylation natural polymer, and nanoparticles of triblock copolymer of polyethylene glycol and polycaprolactone (PEG-PCL-PEG) are used as reinforcing agent, Chinese patent CN103665397A discloses a hydrogel and a preparation method thereof, wherein polyethylene glycol and hyaluronic acid with high molecular weight are used as raw materials, so that the hydrogel has strong flexibility and certain mechanical strength, the Michael addition reaction is adopted for preparing in-situ hydrogel, but the prepared hydrogel has no functions of controllable degradation, controllable release and/or cell behavior regulation. Chinese patent CN103467755A discloses a drug sustained-release hydrogel and a preparation method and application thereof, wherein a high-voltage electrostatic liquid drop method is adopted to prepare monodisperse drug-containing calcium alginate microspheres, and the drug-loaded calcium alginate microspheres are placed in a hydrogel network structure.

Different release rates of different active ingredients, different symptoms or sites, are often required in order to enhance the therapeutic effect. For example, for osteogenic growth factor bone morphogenetic proteins, the effect of bone regeneration is required to be achieved by hydrogel to gradually and slowly release the bone morphogenetic proteins so as to prevent the occurrence of burst release; for vascular endothelial growth factor, however, the requirement for angiogenesis promotion requires that the hydrogel rapidly release the growth factor at the initial stage.

The prior hydrogel carrier materials, which usually have only a single release rate, are often limited in practical applications, and when the required release rate is changed, the type and amount of the carrier material need to be selected again, and the type and amount of the carrier material may be changed, so that people need to spend time, effort and cost on the carrier material when facing active ingredients with different release rate requirements.

Therefore, if a hydrogel carrier material with controllable release rate and a definite rate regulation mechanism can be provided, the requirements of different active ingredients in practical application can be conveniently met.

disclosure of Invention

The invention aims to provide an extracellular matrix-imitated injectable in-situ hydrogel which has controllable degradation in performance, has a clear degradation rate regulation mechanism, and can conveniently meet the requirements on different release rates of growth factors or medicaments; meanwhile, the method also meets the operation requirements of clinical injection and in-situ molding, and has good application prospect in tissue repair and regeneration.

Specifically, the in-situ hydrogel imitating the extracellular matrix injectable mainly comprises the following components in percentage by weight:

Further selecting that the hydrogel mainly comprises the following components in percentage by weight:

Still further, the hydrogel mainly comprises the following components in percentage by weight:

The polysaccharide is formed by condensing and dehydrating a plurality of monosaccharide molecules, and is a carbohydrate substance with a complex and huge molecular structure. Carbohydrates and their derivatives, which meet the concept of high molecular weight compounds, are called polysaccharides. The thiolated polysaccharide refers to a polysaccharide in which a thiol group is introduced into a polysaccharide molecule by a thiolation method.

the mercapto content of the thiolated gelatin is 0.4-0.6 mmol/g; the mercapto content of the thiolated polysaccharide is 0.4-0.6 mmol/g; the molecular weight of the polyethylene glycol diacrylate is 2-10 KDa, and the molecular weight of the polyethylene glycol diacrylate is further selected to be 3-8 KDa.

The in-situ hydrogel has the molar content of the polyethylene glycol diacrylate being 1/4 of the total molar content of sulfhydrylated gelatin and sulfhydrylated polysaccharide.

In the in-situ hydrogel, the thiolated polysaccharide is any one or more of thiolated sodium alginate, thiolated hyaluronic acid, thiolated heparin and thiolated chondroitin sulfate;

the solvent is one or more of normal saline, phosphate buffer and cell culture medium solution.

The thiolated gelatin or thiolated polysaccharide of the present invention may be prepared by a conventional thiolation reaction, and, in one embodiment of the present invention, the thiolation reaction is performed by:

Dissolving gelatin or polysaccharide, adding cystamine dihydrochloride, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH to 4-5, reacting completely at room temperature, dialyzing, adding dithiothreitol, adjusting the pH to 8.5, reacting completely, adjusting the pH to 4.0, dialyzing under the protection of nitrogen, filtering for sterilization, and freeze-drying to obtain the compound.

The in-situ hydrogel regulates and controls the degradation rate of the hydrogel by regulating the proportion of the thiolated gelatin and the thiolated polysaccharide, wherein the specific gravity of the thiolated gelatin is increased, the degradation rate is increased, the specific gravity of the thiolated gelatin is reduced, and the degradation rate is reduced.

in the pancreatin degradation liquid with the concentration of 0.01mg/ml in vitro, the degradation rate of the hydrogel prepared from the thiolated gelatin and the thiolated polysaccharide in different proportions is 0-80% when the hydrogel is degraded for 60 minutes.

Another object of the present invention is to provide an in situ hydrogel comprising growth factors, wherein the composition of the said extracellular matrix-simulated injectable in situ hydrogel further comprises growth factors.

The in-situ hydrogel containing the growth factors has the content of 0.005-0.02 mu g/mu l in the hydrogel; further optionally, the content of the growth factor in the hydrogel is 0.01 μ g/μ l.

The in-situ hydrogel comprises growth factors, wherein the growth factors are any one or more of bone morphogenetic protein, basic fibroblast growth factors and vascular endothelial growth factors.

in a specific embodiment of the present invention, when the growth factor is bone morphogenetic protein-2 (BMP-2) and/or basic fibroblast growth factor (bFGF), the mass ratio of the thiolated gelatin to the thiolated polysaccharide is 1: 1.

according to the invention, the proportion of the thiolated gelatin and the thiolated polysaccharide is regulated, so that the prepared in-situ hydrogel has controllable growth factor and/or drug release performance, and can meet various requirements on different release rates in actual application.

The in-situ hydrogel disclosed by the invention has the characteristics that in 32 days of in-vitro release, the initial release at 1 day is less than 15%, and the later release shows linear release, and the release percentage at 32 days is 68-76%. The BMP-2-loaded hydrogel is used for bone regeneration experiments, and the content of new bones at 4 weeks is about 90%.

It is still another object of the present invention to provide an in situ hydrogel comprising RGD-containing cell adhesion peptide, wherein the composition of the above-mentioned extracellular matrix-mimicked injectable in situ hydrogel further comprises RGD-containing cell adhesion peptide.

The in-situ hydrogel containing RGD-containing cell adhesion peptide has the molar content of the polyethylene glycol diacrylate which is the sum of 1/4 of the molar content of total sulfydryl in the sulfhydrylated gelatin and the sulfhydrylated polysaccharide and the molar content of the RGD-containing cell adhesion peptide.

The in-situ hydrogel comprises RGD-containing cell adhesion peptide, and the concentration of the RGD-containing cell adhesion peptide in the hydrogel is 100-1000 mu mol/L.

The in-situ hydrogel comprises RGD-containing cell adhesion peptide, wherein the mass ratio of the thiolated gelatin to the thiolated polysaccharide is (5/3-3): 1.

In one embodiment of the invention, the mass ratio of thiolated gelatin to thiolated polysaccharide is 3: 1.

The cell adhesion peptide containing RGD refers to a polypeptide of which the molecule contains RGD and one end of the molecule is cysteine.

In one embodiment of the present invention, the RGD-containing cell adhesion peptide is GRGDSPC or cgrgdsp.

The in-situ hydrogel added with the cell adhesion peptide containing RGD also has the performance of regulating and controlling cell behaviors, and the cell behaviors are regulated and controlled through the degradation of the hydrogel and the cell adhesion peptide containing RGD; the thiolated gelatin content and the concentration of the RGD-containing cell adhesion peptide in the hydrogel affect the morphology of the cells, and in addition, the concentration of the RGD-containing cell adhesion peptide significantly affects the migration of the cells.

The invention also provides a preparation method of the in-situ hydrogel.

The method for preparing the extracellular matrix-simulated injectable in-situ hydrogel comprises the following steps:

(1) Respectively dissolving thiolated gelatin, thiolated polysaccharide and polyethylene glycol diacrylate in the solvent to respectively prepare a thiolated gelatin solution, a thiolated polysaccharide solution and a polyethylene glycol diacrylate solution;

(2) Respectively adjusting the pH values of the thiolated gelatin solution and the thiolated polysaccharide solution to 7.0-8.0;

(3) And (3) uniformly mixing the thiolated gelatin solution, the thiolated polysaccharide solution and the polyethylene glycol diacrylate solution to obtain the adhesive.

A method of preparing an in situ hydrogel comprising growth factors, comprising the steps of:

(i) Respectively dissolving thiolated gelatin, thiolated polysaccharide and polyethylene glycol diacrylate in the solvent to respectively prepare a thiolated gelatin solution, a thiolated polysaccharide solution and a polyethylene glycol diacrylate solution;

(ii) Respectively adjusting the pH values of the thiolated gelatin solution and the thiolated polysaccharide solution to 7.0-8.0;

(iii) And (3) uniformly mixing the thiolated gelatin solution, the thiolated polysaccharide solution, the polyethylene glycol diacrylate solution and the growth factor to obtain the growth factor.

a method of preparing an in situ hydrogel comprising RGD-containing cell adhesion peptides, comprising the steps of:

(a) respectively dissolving thiolated gelatin, thiolated polysaccharide, polyethylene glycol diacrylate and RGD-containing cell adhesion peptide into the solvent to respectively prepare a thiolated gelatin solution, a thiolated polysaccharide solution, a polyethylene glycol diacrylate solution and an RGD-containing cell adhesion peptide solution;

(b) respectively adjusting the pH values of the thiolated gelatin solution and the thiolated polysaccharide solution to 7.0-8.0;

(c) Reacting the RGD-containing cell adhesion peptide solution with the polyethylene glycol diacrylate solution for 5-10min, adding the thiolated gelatin solution and the thiolated polysaccharide solution, and uniformly mixing to obtain the product.

In the invention, polyethylene glycol diacrylate, cell adhesion peptide containing RGD and growth factor can be purchased from the market; RGD-containing cell adhesion peptides can also be synthesized using a polypeptide synthesizer.

compared with the prior art, the invention has the following beneficial effects:

(1) The hydrogel has the following functions: controllable degradation and controllable release;

The release performance of the in-situ hydrogel is adjusted by adjusting and controlling the proportion of the thiolated gelatin and the thiolated polysaccharide, when the specific gravity of the thiolated gelatin is increased, the degradation rate is increased, the release rate is also increased, otherwise, the specific gravity of the thiolated gelatin is reduced, the degradation rate is reduced, and the release rate is also reduced;

(2) When the hydrogel is used as a carrier material of the bone morphogenetic protein-2, the hydrogel has the characteristic of slowly releasing growth factors, avoids the phenomenon of explosive release of the bone morphogenetic protein-2, and thus achieves the effect of continuously and efficiently playing a role; the experiment for bone regeneration shows that the bone regeneration agent has strong osteogenesis capacity;

(3) The hydrogel has the function of regulating cell behaviors, and can realize regulation and control of cell morphology, migration and the like by controlling the composition of the hydrogel; not only can be used for in vitro cell culture, but also can meet the requirement of being implanted into the body in cell therapy, and has wide clinical application prospect.

(4) According to the hydrogel, active molecules containing double bonds or sulfydryl, such as RGD or protein of MMP (metal protease) degradation sites, are introduced by a Michael addition reaction, so that a carrier material of a microenvironment meeting the requirements of tissue repair and regeneration can be designed;

The components of the hydrogel are similar to those of extracellular matrix, so that the hydrogel has good biocompatibility, has the functions of controllable degradation, controllable release and/or regulation of cell behaviors in performance, and can conveniently meet the requirements on different release rates of growth factors or drugs; meanwhile, the method also meets the operation requirements of clinical injection and in-situ molding, and has good application prospect in tissue repair and regeneration.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

the present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

drawings

FIG. 1 schematic of examples 1-5 preparation of thiolated gelatin and thiolated polysaccharide.

FIG. 2 examples 1-5 schematic illustrations of the preparation of an injectable in situ hydrogel of a simulated extracellular matrix.

Fig. 3 is an in vitro degradation curve of the extracellular matrix-simulated injectable in-situ hydrogel of thiolated gelatin-thiolated sodium hyaluronate.

FIG. 4 shows the in vitro release curves of BMP-2 and bFGF from an extracellular matrix-containing sulfhydrylated gelatin-sulfhydrylated heparin injectable in situ hydrogel.

FIG. 5 BMP-2 loaded thiolated gelatin-thiolated heparin-mimetic extracellular matrix injectable in situ hydrogel HE stained sections for bone regeneration (A) and alkaline phosphatase activity (B).

FIG. 6 is a confocal picture of the cell morphology after encapsulation of rMSC by thiolated gelatin-thiolated sodium alginate hydrogel (without the addition of RGD-containing cell adhesion peptide) mixed at different ratios.

FIG. 7 is a confocal picture of cell morphology after rMSC is wrapped by sulfhydrylated gelatin-sulfhydrylated sodium alginate hydrogel with different concentrations of RGD.

FIG. 8 is a graph showing the migration distance of rMSC in thiolated gelatin-thiolated sodium alginate hydrogel complexed with RGD of different concentrations as a function of time.

Detailed Description

the raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

EXAMPLE 1 preparation of an extracellular matrix-simulated injectable in situ hydrogel

(1) Preparation of thiolated gelatin

1g of gelatin was added to 100ml of ultrapure water, heated to 40 ℃ and dissolved with stirring, and then cooled to room temperature. Adding cystamine dihydrochloride, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) according to a molar ratio of 1:2:2:2 (the molar ratio of carboxyl groups in gelatin is 1), adjusting the pH of a reaction solution to 4.75, starting dialysis after reacting for 4 hours at room temperature, replacing the dialysate every 24 hours, adding 0.5g of DTT (dithiothreitol) after dialyzing for 3 days, adjusting the pH of the reaction solution to 8.5, adjusting the pH to 4.0 after reacting for 2 hours, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. After dialysis, filtration sterilization and freeze drying are carried out to obtain the thiolated gelatin, and the preparation schematic diagram is shown in figure 1.

The mercapto content was determined to be 0.51mmol/g by the Ellman method (cf. Butterworth, P.H.W.; Baum, H.; Porter, J.W.Arch.biochem.Biophys.1967,118, 716-723.).

(2) Preparation of thiolated sodium alginate

0.5g of sodium alginate was dissolved in 70ml of a 50mM MES (2- (N-morpholine) ethanesulfonic acid monohydrate) solution and cystamine dihydrochloride, EDC and NHS were added in a molar ratio of 1:2:2:2 (molar ratio of carboxyl groups in sodium alginate: 1), respectively. Adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 24h, starting dialysis, replacing the dialysate every 24h, dialyzing for 3 days, adding 0.5g DTT, adjusting the pH value of the reaction solution to 8.5, reacting for 2h, adjusting the pH value to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. After dialysis, filtration sterilization and freeze drying are carried out to obtain the thiolated sodium alginate, and the preparation schematic diagram is shown in figure 1.

The mercapto content was determined by the Ellman method to be 0.48 mmol/g.

(3) Preparation of RGD-containing cell adhesion peptide

Synthesis of RGD-containing cell adhesion peptide 1 using a polypeptide synthesizer: GRGDSPC.

(4) Preparation of extracellular matrix-imitated injectable in-situ hydrogel

Respectively dissolving thiolated gelatin and thiolated sodium alginate in DMEM (cell culture medium) aqueous solution, and adjusting pH to 8.0; GRGDSPC containing RGD polypeptide and polyethylene glycol diacrylate (PEGDA, molecular weight 3400) are respectively dissolved in PBS (phosphate buffer solution) solution to obtain RGD polypeptide containing solution and PEGDA solution.

Performing Michael addition reaction on the RGD-containing polypeptide solution and a PEGDA solution for 5 minutes, then adding thiolated gelatin and thiolated sodium alginate, wherein the weight percentages of the PEGDA, the thiolated gelatin and the thiolated sodium alginate in the obtained mixed solution (with the density of about 1g/ml) are respectively 2.05%, 3% and 1%, and the content of RGD-containing polypeptide is 1000 mu mol/L, so as to obtain the injectable in-situ hydrogel.

example 2: preparation of extracellular matrix-imitated injectable in-situ hydrogel

(1) Preparation of thiolated gelatin

1g of gelatin was added to 100ml of ultrapure water, heated to 40 ℃ and dissolved with stirring, and then cooled to room temperature. Adding cystamine dihydrochloride, EDC and NHS according to a molar ratio of 1:1:2:2 (the molar ratio of carboxyl groups in gelatin is 1), adjusting the pH of the reaction solution to 4.75, reacting at room temperature for 4 hours, starting dialysis, replacing the dialysate every 24 hours, adding 0.5g of DTT after 3 days of dialysis, adjusting the pH of the reaction solution to 8.5, reacting for 2 hours, adjusting the pH to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the thiolated gelatin.

the mercapto content was determined by the Ellman method to be 0.42 mmol/g.

(2) Preparation of thiolated sodium hyaluronate

0.5g of sodium hyaluronate was dissolved in 50ml of ultrapure water, and cystamine dihydrochloride, EDC and NHS were added in a molar ratio of 1:2:2:2 (molar ratio of carboxyl groups in sodium hyaluronate: 1), respectively. Adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 24h, starting dialysis, replacing the dialysate every 24h, dialyzing for 3 days, adding 0.5g DTT, adjusting the pH value of the reaction solution to 8.5, reacting for 2h, adjusting the pH value to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the sodium thiolated hyaluronate.

the mercapto content was determined by the Ellman method to be 0.43 mmol/g.

(3) Preparation of RGD-containing cell adhesion peptide

Synthesis of RGD-containing cell adhesion peptide 2 using a polypeptide synthesizer: cgrgdsp.

(4) Preparation of extracellular matrix-imitated injectable in-situ hydrogel

Respectively dissolving thiolated gelatin and thiolated sodium hyaluronate in a PBS solution, and adjusting the pH of the two solutions to 7.4; respectively dissolving RGD-containing polypeptide CGRGDSPC and polyethylene glycol diacrylate (PEGDA, molecular weight is 3400) in PBS solution to obtain RGD-containing polypeptide solution and PEGDA solution.

Performing Michael addition reaction on the RGD-containing polypeptide solution and the PEGDA solution for 5 minutes, and then adding 2.91%, 5% and 3% by weight of the PEGDA, the thiolated gelatin and the thiolated sodium hyaluronate into the obtained mixed solution (with the density of about 1g/ml), wherein the RGD-containing polypeptide content is 100 mu mol/L, and thus the injectable in-situ hydrogel is obtained.

Example 3: preparation of extracellular matrix-imitated injectable in-situ hydrogel

(1) Preparation of thiolated gelatin

1g of gelatin was added to 100ml of ultrapure water, heated to 40 ℃ and dissolved with stirring, and then cooled to room temperature. Adding cystamine dihydrochloride, EDC and NHS according to a molar ratio of 1:3:3:3 (the molar ratio of carboxyl groups in gelatin is 1), adjusting the pH of the reaction solution to 4.75, reacting at room temperature for 4 hours, starting dialysis, replacing the dialysate every 24 hours, adding 0.5g of DTT after 3 days of dialysis, adjusting the pH of the reaction solution to 8.5, reacting for 2 hours, adjusting the pH to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the thiolated gelatin.

The mercapto content was determined by the Ellman method to be 0.6 mmol/g.

(2) Preparation of thiolated heparin

1g of heparin was dissolved in 100ml of ultrapure water, and cystamine dihydrochloride, EDC and NHS were added in a molar ratio of 1:4:4:4 (molar ratio of carboxyl groups in heparin is 1), respectively. Adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 24h, starting dialysis, replacing the dialysate every 24h, dialyzing for 3 days, adding 0.5g DTT, adjusting the pH value of the reaction solution to 8.5, reacting for 2h, adjusting the pH value to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the thiolated heparin.

the mercapto content was determined by the Ellman method to be 0.58 mmol/g.

(3) The bone morphogenetic protein growth factor BMP-2 is commercially available.

(4) preparation of extracellular matrix-imitated injectable in-situ hydrogel

Respectively dissolving thiolated gelatin and thiolated heparin in DMEM aqueous solution, and adjusting the pH of the two solutions to 8.0; BMP-2 and polyethylene glycol diacrylate (PEGDA, molecular weight 3400) are respectively taken and dissolved in the PBS solution to obtain a BMP-2 solution and a PEGDA solution.

BMP-2, PEGDA, thiolated gelatin and thiolated heparin solution are uniformly mixed, the weight percentages of the PEGDA, the thiolated gelatin and the thiolated heparin in the obtained mixed solution (the density is about 1g/ml) are respectively 3%, 3% and 3%, and the content of the BMP-2 is 0.01 mu g/mu l, so that the injectable in-situ hydrogel is obtained.

Example 4: preparation of extracellular matrix-imitated injectable in-situ hydrogel

(1) Preparation of thiolated gelatin

1g of gelatin was added to 100ml of ultrapure water, heated to 40 ℃ and dissolved with stirring, and then cooled to room temperature. Adding cystamine dihydrochloride, EDC and NHS according to the molar ratio of 1:2:4:4 (the molar ratio of carboxyl groups in gelatin is 1), adjusting the pH of the reaction solution to 4.75, reacting at room temperature for 4 hours, starting dialysis, replacing the dialysate every 24 hours, adding 0.5g of DTT after 3 days of dialysis, adjusting the pH of the reaction solution to 8.5, reacting for 2 hours, adjusting the pH to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the thiolated gelatin.

The mercapto content was determined by the Ellman method to be 0.48 mmol/g.

(2) Preparation of thiolated chondroitin sulfate

1g of chondroitin sulfate was dissolved in 100ml of ultrapure water, and cystamine dihydrochloride, EDC and NHS were added thereto in a molar ratio of 1:4:4:4 (molar ratio of carboxyl groups in chondroitin sulfate: 1), respectively. Adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 24h, starting dialysis, replacing the dialysate every 24h, dialyzing for 3 days, adding 0.5g DTT, adjusting the pH value of the reaction solution to 8.5, reacting for 2h, adjusting the pH value to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. And after dialysis, filtering, sterilizing and freeze-drying to obtain the sulfhydryl chondroitin sulfate.

The mercapto content was determined by the Ellman method to be 0.56 mmol/g.

(3) Preparation of extracellular matrix-imitated injectable in-situ hydrogel

Respectively dissolving thiolated gelatin and thiolated chondroitin sulfate in a PBS solution, and adjusting the pH of the two solutions to 7.6; dissolving polyethylene glycol diacrylate (PEGDA, molecular weight 8000) in PBS solution to obtain PEGDA solution.

Uniformly mixing PEGDA, thiolated gelatin and thiolated chondroitin sulfate solution, wherein the weight percentages of the PEGDA, the thiolated gelatin and the thiolated chondroitin sulfate in the obtained mixed solution (the density is about 1g/ml) are respectively 4%, 3% and 1%, and thus the injectable in-situ hydrogel is obtained.

EXAMPLE 5 preparation of an extracellular matrix-simulated injectable in situ hydrogel

(1) Preparation of thiolated gelatin

1g of gelatin was added to 100ml of ultrapure water, heated to 40 ℃ and dissolved with stirring, and then cooled to room temperature. Adding cystamine dihydrochloride, EDC and NHS according to the molar ratio of 1:2:2:2 (the molar ratio of carboxyl groups in gelatin is 1), adjusting the pH of the reaction solution to 4.75, reacting at room temperature for 4 hours, starting dialysis, replacing the dialysate every 24 hours, adding 0.5g of DTT after 3 days of dialysis, adjusting the pH of the reaction solution to 8.5, reacting for 2 hours, adjusting the pH to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. Filtering, sterilizing and freeze-drying after dialysis to obtain the thiolated gelatin.

The mercapto content was determined by the Ellman method to be 0.51 mmol/g.

(2) preparation of thiolated sodium alginate

0.5g of sodium alginate was dissolved in 70ml of 50mM MES solution, and cystamine dihydrochloride, EDC and NHS were added in a molar ratio of 1:2:2:2 (molar ratio of carboxyl groups in sodium alginate: 1), respectively. Adjusting the pH value of the reaction solution to 4.75, reacting at room temperature for 24h, starting dialysis, replacing the dialysate every 24h, dialyzing for 3 days, adding 0.5g DTT, adjusting the pH value of the reaction solution to 8.5, reacting for 2h, adjusting the pH value to 4.0, dialyzing for 3 days under the protection of nitrogen, and replacing the dialysate every day. After dialysis, filtration sterilization and freeze drying are carried out to obtain the thiolated sodium alginate, and the preparation schematic diagram is shown in figure 1.

the mercapto content was determined by the Ellman method to be 0.48 mmol/g.

(3) Preparation of extracellular matrix-imitated injectable in-situ hydrogel

Respectively dissolving thiolated gelatin and thiolated sodium alginate in DMEM aqueous solution, and adjusting the pH of the two solutions to 8.0; dissolving polyethylene glycol diacrylate (PEGDA, molecular weight 10000) in PBS solution to obtain PEGDA solution.

And (3) uniformly mixing the PEGDA, the thiolated gelatin and the thiolated sodium alginate solution, wherein the weight percentages of the PEGDA, the thiolated gelatin and the thiolated sodium alginate in the obtained mixed solution (the density is about 1g/ml) are respectively 4.88%, 1% and 3%, so that the injectable in-situ hydrogel disclosed by the invention is obtained.

To illustrate the advantageous effects of the present invention, the present invention provides the following test examples:

Test example 1: controllable degradability of extracellular matrix-simulated injectable in-situ hydrogel

The thiolated gelatin-sodium thiolated hyaluronate was prepared as in example 2, and the extracellular matrix-simulated injectable in-situ hydrogel was prepared by changing the mixing ratio of the thiolated gelatin and the sodium thiolated hyaluronate without adding RGD-containing cell adhesion peptide to obtain 5 kinds of hydrogels with the ratio of 100/0, 70/30, 50/50, 30/70 and 0/100, respectively.

In vitro degradation was determined by the weight loss of the hydrogel in degradation solution. The degradation liquid is 0.01mg/ml Trypsin/PBS. The sample was taken out at a set time and weighed after sucking off excess degradation liquid on the surface with filter paper. The residual weight ratio is calculated according to the following formula:

The residual weight ratio was Wt/W0X 100%

w0 and Wt represent the initial sample weight and the sample weight measured at the set time, respectively.

The in vitro degradation of the hydrogel is shown in figure 3. In the pancreatic juice enzyme degradation solution, the 100/0 hydrogel showed a fast degradation rate and was completely degraded at 90 min. When thiolated hyaluronic acid is mixed, the degradation rate is significantly slowed. 70/30 the residual weight rate of the hydrogel after 150min degradation in trypsin solution was 54.3. + -. 6.1%. 30/70 did not degrade significantly within 150 min. 50/50 the residual weight ratio of the hydrogel at 150min was 86.3%. The above results show that the degradation rate of the hydrogel can be regulated and controlled by controlling the mixing ratio of the thiolated gelatin and the thiolated sodium hyaluronate.

Test example 2: extracellular matrix-imitated injectable in-situ hydrogel for controlled release of growth factors

Growth factor-loaded simulated extracellular matrix injectable in situ hydrogels were prepared according to example 3. PEGDA, thiolated gelatin, thiolated heparin, and growth factors (bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF), respectively) are mixed to prepare the growth factor-loaded hydrogel. Wherein the mixing ratio of the thiolated gelatin to the thiolated heparin is 1:1, the mixing amount of the growth factors is 2 mu g, and the volume of the prepared growth factor-loaded extracellular matrix-simulated injectable in-situ hydrogel is 200 mu l.

For in vitro growth factor release testing, the prepared growth factor loaded hydrogel was placed in 10ml of PBS buffer containing 1% BSA, 10. mu.g/ml heparin and 1mM EDTA (ethylenediaminetetraacetic acid) and shaken at 37 ℃ and 150 rpm. 50. mu.l of the release solution was aspirated at 1 hour, 6 hours, 12 hours, 1 day, 2 days, 4 days, 8 days, 16 days, and 32 days, respectively, and frozen at-80 ℃ with the same amount of buffer added after each aspiration. The concentration of the growth factor in the absorbed release solution is detected by an Elisa method. The cumulative release rate of growth factor is the percentage of released content divided by the initial amount added, 2 μ g.

The conditions of the two growth factors, BMP-2 and bFGF, released in vitro by the sulfhydrylated gelatin-sulfhydrylated heparin imitating extracellular matrix injectable in-situ hydrogel are shown in figure 4. For both growth factors, the hydrogel exhibited a smaller dose release profile on day 1 of 14.3 (+ -1.5)% and 14.6 (+ -1.5)%, respectively. But exhibited a linear release profile over the late 32 days of release with the percent release at day 32 being 75.7 (+ -6.1)% and 68.3 (+ -3.1)%, respectively.

The above results indicate that the hydrogel has the characteristic of controllable release of growth factors.

test example 3: growth factor-loaded extracellular matrix-simulated injectable in-situ hydrogel for bone tissue regeneration

according to the preparation method of the growth factor-loaded extracellular matrix-simulated injectable in-situ hydrogel in test example 2, the BMP-2-loaded thiolated gelatin-thiolated heparin hydrogel was directly injected into abdominal muscles of rats, and the BMP-2-free hydrogel was used as a control. After 4 weeks the rats were sacrificed and the implant material was removed along with surrounding tissue and evenly divided into 2 parts. After 1 part of the bone is fixed, decalcified, embedded and paraffin-sectioned, the bone formation condition is evaluated by grading the new bone formation index by using HE staining and observing and analyzing photography under a microscope (see figure 5 (A)).

Newborn bone formation index score definition: over 40% new bone is formed and contains bone marrow, beat 5 points; 20% of new bone generation is divided into 4 points; 3 points are given for 10% of new bone generation; only cartilage is scored 2 points; only the fibrous tissue scored 1. The percentage of osteogenesis of the BMP-2 loaded simulated extracellular matrix injectable in situ hydrogel was 89.3%, and no new bone formation occurred in the control group.

After homogenizing another 1 part of the homogenate, the supernatant was collected to examine the alkaline phosphatase (ALP) activity, and the ALP value of the BMP-2-loaded mimic extracellular matrix injectable in situ hydrogel group was 183. + -. 10. mu. mol/mg protein/min, as shown in FIG. 5 (B).

The test result shows that the in-situ hydrogel has strong bone forming capability and high ALP activity.

Test example 4: extracellular matrix-imitated injectable in-situ hydrogel for regulating cell behavior

According to the preparation method of the cell-loaded extracellular matrix-simulated injectable in-situ hydrogel in example 1, rat bone marrow stromal stem cells (rMSCs) are wrapped in the hydrogel, and the behavior of the cells is regulated through the composition of the hydrogel.

After culturing 1X 105 rMSC cells in 100. mu.l of each hydrogel for 1 day and 7 days, the cells were stained with live/dead kit from Invitrogen, and then the morphology of the cells was observed by laser confocal microscopy.

cell clusters were prepared by wrapping 2X 106 rMSCs in 15. mu.l of 3% thiolated gelatin hydrogel. Then, the cell clusters were added to a pre-gel solution of each hydrogel, and cultured in DMEM medium containing 10% FBS after gelation. Cell migration was observed using an inverted microscope and photographed.

Confocal pictures of cell morphology after encapsulation of rMSC by thiolated gelatin-thiolated sodium alginate hydrogel (without RGD-containing cell adhesion peptide) mixed at different ratios are shown in FIG. 6. 100/0 the cells in the hydrogel are in a spread form, and as the culture time increases, more and more cells are spread and some cells are connected. When the thiolated sodium alginate was mixed, only a small fraction of the cells appeared in a spread form after 7 days of cell culture in 70/30 hydrogel. While as the amount of thiolated sodium alginate continued to increase, all cells appeared round within 7 days.

Confocal pictures of the morphology of the rMSC cells in thiolated gelatin-thiolated sodium alginate hydrogels containing RGD-containing cell adhesion peptides at different concentrations are shown in fig. 7. With increasing concentration of RGD-containing cell adhesion peptides, more and more cells were observed in a spread morphology. Wherein, when the concentration of the RGD-containing cell adhesion peptide was 100. mu.M, a large number of cells were already in a spread state by day 7, compared to the hydrogel containing no RGD-containing cell adhesion peptide. When the concentration of the RGD-containing cell adhesion peptide was increased, the number of the spread cells was gradually increased.

The results show that the cell morphology can be regulated and controlled by controlling the mixing ratio of the thiolated gelatin and the thiolated sodium alginate in the hydrogel or by compounding the RGD-containing cell adhesion peptides with different concentrations into the thiolated gelatin-thiolated sodium alginate hydrogel.

In the thiolated gelatin-thiolated sodium alginate hydrogel (without the addition of RGD-containing cell adhesion peptide), the rMSC cells migrated only in 100/0 hydrogel, with a migration distance of 240 μm by day 5. In none of the other hydrogels, there was significant cell migration. When RGD-containing cell adhesion peptides were complexed (see fig. 8), the migration distance of cells was gradually increased as the concentration of RGD-containing cell adhesion peptides was increased. By day 5, migration distances of rMSCs in hydrogels with RGD concentrations of 100, 200, 400 and 800M were 295 + -35, 452 + -36, 640 + -45 and 658 + -35 μ M, respectively.

the above results indicate that cell migration can be regulated by controlling various concentrations of RGD-containing cell adhesion peptides in the hydrogel.

In conclusion, the hydrogel of the present invention has the following beneficial effects:

(1) According to the invention, the degradation rate of the hydrogel can be regulated and controlled by controlling the mixing ratio of the thiolated gelatin and the thiolated sodium hyaluronate;

(2) According to the invention, the in-situ hydrogel prepared by regulating the proportion of the thiolated gelatin and the thiolated polysaccharide has controllable release performance;

when the hydrogel is used as a carrier material of the bone morphogenetic protein-2, the hydrogel has the characteristic of slowly releasing growth factors, so that the phenomenon of burst release of the bone morphogenetic protein-2 is avoided;

(3) The hydrogel containing the growth factors has strong bone forming capability and high ALP activity;

(4) The hydrogel can regulate and control the form and/or migration of cells by controlling the mixing ratio of the thiolated gelatin and the thiolated sodium alginate in the hydrogel or by compounding the RGD-containing cell adhesion peptides with different concentrations into the hydrogel.

Claims

1. The application of the in-situ hydrogel capable of being injected by the simulated extracellular matrix in preparing bone tissue regeneration materials; the in-situ hydrogel for imitating the extracellular matrix injectable consists of the following components in percentage by weight:

The molar content of the polyethylene glycol diacrylate is 1/4 of the total molar content of sulfhydrylated gelatin and sulfhydrylated polysaccharide;

The composition of the hydrogel further comprises growth factors; the growth factor is bone morphogenetic protein-2;

the thiolated polysaccharide is any one or more of thiolated sodium alginate, thiolated hyaluronic acid, thiolated heparin and thiolated chondroitin sulfate.

2. Use according to claim 1, characterized in that: the hydrogel mainly comprises the following components in percentage by weight:

3. Use according to claim 1 or 2, characterized in that: the mercapto content of the thiolated gelatin is 0.4-0.6 mmol/g; the mercapto content of the thiolated polysaccharide is 0.4-0.6 mmol/g; the molecular weight of the polyethylene glycol diacrylate is 2-10 KDa.

4. Use according to claim 1 or 2, characterized in that: the solvent is one or more of normal saline, phosphate buffer and cell culture medium solution.

5. Use according to claim 1, characterized in that: the content of the growth factor in the hydrogel is 0.005-0.02 mu g/mu l.

6. Use according to claim 1, characterized in that: the mass ratio of the thiolated gelatin to the thiolated polysaccharide is 1: 1.