CN111870739A

CN111870739A - Preparation method and application of multifunctional modified chitosan self-healing hydrogel

Info

Publication number: CN111870739A
Application number: CN202010532756.6A
Authority: CN
Inventors: 项琪; 黄亚东; 于凤麟
Original assignee: Taiyuan Guangzhou Biotechnology Co ltd; Guangzhou Jinan University Medical Biotechnology Research And Development Center Co ltd
Current assignee: Taiyuan Guangzhou Biotechnology Co ltd; Guangzhou Jinan University Medical Biotechnology Research And Development Center Co ltd
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2020-11-03

Abstract

The invention discloses a preparation method and application of multifunctional modified chitosan self-healing hydrogel. In order to realize the functions of different growth factors at different time stages, the self-healing hydrogel prepared by the preparation method introduces microspheres as an encapsulating agent of a post-release drug (TGF beta 3) and is isolated from bFGF directly loaded in the hydrogel to form an inner chamber structure, thereby achieving the purpose of sequentially releasing different growth factors. The hydrogel prepared by the invention has good physical and chemical properties and good injectability, can be used for in-situ injection for bone injury repair, repairs damaged bone tissues by sequentially releasing different growth factors, and provides a new solution and a repair means for basic research in the field of bone tissue engineering.

Description

Preparation method and application of multifunctional modified chitosan self-healing hydrogel

Technical Field

The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a preparation method and application of multifunctional modified chitosan self-healing hydrogel, which can be particularly used for repairing and treating bone defects.

Background

Bone defects refer to diseases in which the integrity of the bone structure is destroyed due to trauma, infection, tumor, congenital disease, etc., and are clinically common symptoms. Often, severe bone defects do not heal and repair themselves and must be effectively restored with the aid of bone repair materials. Statistically, over 2200 million patients requiring bone grafting surgery every year worldwide, with surgery costs over $ 25 million; the number of patients needing transplantation operation due to bone defect or fracture can reach more than 300 ten thousand each year in China.

Stem cells are seeds for the treatment and repair of bone defects and play a role in the production of bone. However, transplanted stem cells are generally difficult to directionally induce differentiation in the microenvironment of the injury site for active repair. Therefore, in combination with exogenous growth factors as a guide for stem cell differentiation, it is particularly important to promote the repair of bone injury by seed stem cells.

At present, the more studied biological scaffold materials comprise chitosan, collagen, silk fibroin and the like. Among them, chitosan is a positively charged natural basic polysaccharide, and is widely used in tissue engineering due to its excellent biocompatibility, biodegradability, non-immunogenicity, and antibacterial and antifungal properties. The natural biological derivative material can be prepared into various forms and applied to bone defect repair. Such as gels, microspheres, sponges, films, and electrospinning, among others. Among them, hydrogels have the most similar structure to native ECM. The injectable hydrogel can be directly injected to an affected part without an operation process, so that the operation wound is reduced to the maximum extent, and the comfort level of a patient is improved; secondly, the hydrogel can adapt to various injury environments and changes along with the change of the shape and the size of a wound, so that the body injury caused by irregular shapes is avoided; moreover, the drug-loaded hydrogel can continuously and slowly release the drug, so that the administration frequency and the side effect of the drug are reduced, and the treatment targeting property is improved. However, conventional hydrogels have encountered application challenges such as the change in mechanical force after injection can cause the stent to deform and even break, imparting the hydrogel with the ability to self-heal while maintaining its structural and functional integrity.

Disclosure of Invention

Aiming at the defects in the prior art, the invention mainly aims to provide the multifunctional modified chitosan self-healing hydrogel. The second purpose of the invention is to provide a preparation method of the multifunctional modified chitosan self-healing hydrogel. The third purpose of the invention is to provide the application of the multifunctional modified chitosan self-healing hydrogel.

Accordingly, the present invention provides the following:

1. a preparation method of multifunctional modified chitosan self-healing hydrogel comprises the following steps:

1) preparing chitosan microspheres by using chitosan as a raw material and adopting an emulsion crosslinking method;

2) preparing a chitosan microsphere solution, and adding TGF beta 3 into the chitosan microsphere solution to form chitosan microspheres containing TGF beta 3;

3) chitosan glutamate (CSG) and four-arm polyethylene glycol benzaldehyde (PEG-BA) are used as raw materials, Schiff alkali reaction is adopted, and self-healing hydrogel is prepared; and

4) mixing the self-healing hydrogel prepared in the step 3) with bFGF, and then mixing the self-healing hydrogel with the chitosan microspheres containing TGF beta 3 prepared in the step 2) to obtain the multifunctional modified chitosan self-healing hydrogel.

2. The method according to the above 1, wherein in the step 1), the chitosan microspheres are obtained by using an aqueous solution of chitosan as an aqueous phase, using petroleum ether, liquid paraffin, Span80, or a mixture thereof as an oil phase, adding the aqueous phase to the oil phase under stirring, and adding glutaraldehyde for crosslinking.

3. The method according to the above 1, wherein in the step 2), the chitosan microspheres contain a combination comprising one or more of TGF β 3 or other active factors.

4. The method according to the above 1, wherein in the step 3), the self-healing hydrogel is prepared as follows: weighing chitosan glutamate (CSG) powder, stirring in a PBS (phosphate buffer solution) with the pH of 7.4 to completely dissolve the chitosan glutamate (CSG), transferring the chitosan glutamate (CSG) powder into a dialysis bag containing the PBS with the pH of 7.4, dialyzing until the pH of the obtained chitosan glutamate solution is 6.8, and storing at room temperature for later use; meanwhile, weighing four-arm polyethylene glycol benzaldehyde (PEG-BA) powder, and completely dissolving in ultrapure water, and storing at-20 ℃ for later use; and finally, mixing the prepared chitosan glutamate (CSG) solution with a four-arm polyethylene glycol benzaldehyde (PEG-BA) solution to obtain the self-healing hydrogel.

5. The method according to the above 4, wherein the volume ratio of the chitosan glutamate (CSG) solution to the four-arm polyethylene glycol benzaldehyde (PEG-BA) solution is 1: 0.01-1: 1 (e.g., 1:0.025, 1:0.05 and 1:0.1), the chitosan glutamate solution has a mass volume concentration of 5-50%, and the PEG-BA solution has a mass volume concentration of 1-30%.

6. The method according to 1 above, wherein in step 4) the self-healing hydrogel comprises bFGF or a combination of one or more of the other active factors, such as Platelet Derived Growth Factor (PDGF).

7. The method according to 1 above, wherein the concentration of the chitosan microspheres in the hydrogel is 0.1-10mg/mL (such as 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mg/mL).

8. A multifunctional modified chitosan self-healing hydrogel, comprising: 1) self-healing hydrogel which contains one or a combination of more of bFGF or other active factors and is prepared by taking chitosan glutamate (CSG) and four-arm polyethylene glycol benzaldehyde (PEG-BA) as raw materials and adopting Schiff base reaction; and 2) chitosan microspheres dispersed in the self-healing hydrogel described in 1), wherein the chitosan microspheres contain one or a combination of more of TGF beta 3 or other active factors.

9. The multifunctional modified chitosan self-healing hydrogel according to the above 8, which can be prepared by the method according to any one of the above 1-7.

10. Use of the multifunctional modified chitosan self-healing hydrogel according to 8 or 9 above for the preparation of a medicament for the repair of bone injuries (e.g. tooth injuries, alveolar bone injuries), optionally/preferably in combination with periodontal ligament stem cells (hPDLSCs) for the repair of alveolar bone injuries.

Detailed Description

The inventor finds that different growth factors have space-time specificity in the process of osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), namely bFGF promotes proliferation and dryness maintenance, TGF beta 3 promotes osteogenic differentiation, so that a self-healing hydrogel which releases bFGF and TGF beta 3 in sequence is designed, injected to the defect part of alveolar bone in situ, and used for repairing damaged alveolar bone tissue by releasing different growth factors in sequence, and a new solution and a new repairing means are provided for basic research in the field of periodontal tissue engineering.

The specific technical scheme is as follows:

a preparation method of multifunctional modified chitosan self-healing hydrogel is characterized by comprising the following steps: the method comprises the following steps:

2) adding TGF beta 3 into the chitosan microsphere solution to form chitosan microspheres containing TGF beta 3;

3) the self-healing hydrogel is prepared by taking chitosan glutamate and four-arm polyethylene glycol benzaldehyde as raw materials and adopting Schiff base reaction;

4) mixing the self-healing hydrogel prepared in the step 3) with a certain amount of bFGF, and then mixing the self-healing hydrogel with the chitosan microspheres containing TGF beta 3 in the step 2) to obtain the multifunctional modified chitosan self-healing hydrogel.

Chitosan is a positively charged natural basic polysaccharide, and is widely used in tissue engineering due to its excellent biocompatibility, biodegradability, non-immunogenicity, and antibacterial and antifungal properties. bFGF is released, cell proliferation can be promoted, growth and development of blood vessels are promoted, sufficient nutrients are provided for the subsequent repair process, TGF beta 3 can induce migration of original mesenchymal stem cells, and osteogenic differentiation of the mesenchymal stem cells can be promoted under the auxiliary action of the bFGF.

Unlike conventional hydrogels, self-healing hydrogels can maximize the ability to avoid leakage of embedded drug molecules by virtue of self-healing properties. In addition, the microsphere carrier has stable property and good biocompatibility, can load various medicaments (especially polypeptides and protein medicaments with easy loss of biological activity), and prolongs the action time of the medicaments by slow degradation in vivo. In the invention, chitosan is used as a raw material, and a gel system capable of sequentially releasing growth factors is designed by combining the advantages of two drug carriers, namely gel and microspheres. TGF beta 3 is loaded in the microspheres, the TGF beta 3-loaded chitosan microspheres are suspended in a self-healing hydrogel system containing bFGF, the bFGF is quickly released in the early stage to promote cell proliferation and the generation of micro-blood vessels, the TGF beta 3 is persistently released by utilizing the slow degradation characteristic of the chitosan microspheres, mesenchymal stem cells are recruited in low concentration in the early stage, and osteogenic differentiation of the stem cells is stimulated in high concentration in the later stage.

Preferably, the preparation process of the chitosan microspheres by the emulsion method in the step 1) is as follows:

taking 1-10mL of 1-5% chitosan solution as a water phase by using a 5mL syringe; mixing petroleum ether (0-30mL), liquid paraffin (0-30mL), and span80(0-3mL) to obtain oil phase. The aqueous phase was added dropwise to the oil phase using a 5ml syringe with stirring. After 0-10h, adding 5-50% glutaraldehyde into the solution by using a 1mL syringe for three times, wherein 0-5mL of glutaraldehyde is added each time, crosslinking the solution for 1-10 h, standing the solution overnight, and then removing the supernatant. And finally, washing with petroleum ether, methanol, ethanol and ultrapure water, washing each solution for at least 2 times, and freeze-drying the washed precipitate to obtain the chitosan microspheres.

Preferably, the chitosan microspheres in step 2) contain one or more of TGF β 3 and other active factors.

Preferably, in step 3), the preparation method of the self-healing hydrogel is as follows: weighing a certain amount of chitosan glutamate (CSG) powder, stirring the chitosan glutamate (CSG) powder in a PBS solution with the pH value of 7.4 to completely dissolve the chitosan glutamate (CSG) powder, transferring the chitosan glutamate (CSG) powder into a dialysis bag containing the PBS solution with the pH value of 7.4, dialyzing the chitosan glutamate (CSG) solution until the pH value of the chitosan glutamate (CSG) solution is 6.8, and storing the chitosan glutamate (CSG) solution at room temperature for standby. Meanwhile, a certain amount of four-arm polyethylene glycol benzaldehyde (PEG-BA) powder is weighed and completely dissolved in ultrapure water, and the solution is stored at the temperature of-20 ℃ for standby. And finally, mixing the prepared chitosan glutamate (CSG) solution and the PEG-BA solution according to a certain proportion to obtain the self-healing hydrogel with different proportions.

Preferably, the self-healing hydrogel in step 4) may be mixed with one or more of bFGF or other active factors. The chitosan microspheres loaded with the active factors can separate the loaded active factors from an external self-healing hydrogel system containing active ingredients, so that an inner chamber structure is formed, and the aim of sequentially releasing different growth factors can be fulfilled.

Preferably, the volume ratio of chitosan glutamate (CSG) to PEG-BA is 1: 0.01-1: 1, the mass volume concentration of chitosan glutamate is 5-50%, and the mass volume concentration of PEG-BA is 1-30%. The concentration of the chitosan microspheres is 0.1-10 mg/mL. The optimal volume ratio of chitosan glutamate (CSG), PEG-BA and chitosan microspheres in the self-healing hydrogel is screened by characterization of gelation time, rheological property, SEM, swelling property, in-vitro degradation performance research, mechanical property of gel, self-healing capability of gel and the like.

The multifunctional modified chitosan self-healing hydrogel can be used for in-situ injection for bone injury repair, and different growth factors (bFGF and TGF beta 3) are sequentially released to repair damaged bone tissues.

Compared with the prior art, the invention has the following advantages:

the active factors bFGF and TGF beta 3 involved in the invention have space-time specificity, and the purpose of repairing damaged bone tissues is realized by designing the double-factor-loaded self-healing hydrogel according to the discovery. In the design, bFGF is placed on the outer layer of the hydrogel and is released first; TGF beta 3 is wrapped in microspheres and suspended in hydrogel and released in later period. The hydrogel has the self-healing characteristic and is convenient for injection administration in narrow space of oral cavity.

The self-healing hydrogel prepared by the invention has good biocompatibility and self-healing performance, can realize the sequential release of various growth active factors, and can be applied to the fields of bone tissue engineering and the like.

The raw materials of the invention have wide sources, and can be industrially produced, and the preparation method of the invention is simple and convenient, and is easy to realize the value of popularization.

Drawings

FIG. 1 is a diagram showing the appearance and particle size distribution of Chitosan Microspheres (CM) of the present invention. A is the appearance of CM under a scanning electron microscope. B is the particle size distribution of CM.

Fig. 2 is a study of the drug loading performance of CM. A. Protein adsorption capacity of CM in BSA solutions of different concentrations; B. protein adsorption of CM in 350. mu.g/mL TGF-beta 3 protein solution.

Fig. 3 is a scanning electron micrograph of self-healing hydrogels with different concentrations. A-F are 1% CSG, 1% CSG + CM, 2% CSG, 2% CSG + CM, 3% CSG, 3% CSG + CM in sequence. Wherein, A1-F1 are photographs of the upper surface structure of the hydrogel, A2-F2 are photographs of the cross-section structure, and A3-F3 are photographs of the longitudinal-section structure of the hydrogel. The concentration of microspheres was 5 mg/mL.

Fig. 4 is a test of injectability and self-healing capacity of self-healing gels. And A-B, detecting the injectability performance test of the self-healing hydrogel by using a 1mL syringe. And C, adding dyes with different colors into the self-healing hydrogel, cutting the self-healing hydrogel, and then carrying out combined observation to observe that the self-healing hydrogel can be gelled again, namely the self-healing hydrogel has self-healing capability.

Fig. 5 is an in vitro release profile of a self-healing hydrogel.

FIG. 6 is an experiment of induced osteogenic differentiation of self-healing hydrogel carrying TGF beta 3 microspheres. The self-healing hydrogel loaded with the TGF beta 3 microspheres and the hPDLSCs is cultured in an osteogenesis induction culture medium for 7 days, and alkaline phosphatase staining is carried out for 14 days. The scale in the figure is 200. mu.m.

FIG. 7 shows the growth conditions of hPDLSCs cultured in MTT detection self-healing hydrogel leaching solution for 24h, 48h and 72 h.

Fig. 8 shows the in vivo compatibility study and in vivo degradation of self-healing hydrogels. A. The self-healing hydrogel is implanted in SD rats subcutaneously for 7 days, 14 days, 28 days and 42 days later, and then the materials are taken and photographed; B. self-healing of the degradation profile of the hydrogel in vivo. The scale in A is 1 cm.

FIG. 9 is a plot of Micro-CT at 1 week and 6 weeks after local injection of each group of hydrogels into the alveolar bone defect site in the periodontal ligament. Normal group, no treatment; a model group which is not subjected to any treatment after molding; blank gel group, after molding, self-healing gel without carrying TGF beta 3 is given; in the low-dose group, after molding, self-healing gel loaded with 1 microgram/mL TGF beta 3 microspheres is given; in the high-dose group, after molding, self-healing gel loaded with 4 mug/mL of TGF beta 3 microspheres is given. Micro-CT graphs of alveolar bones of rats 1 week after administration of each group a-e; f-j Micro-CT image of alveolar bone of rat 6 weeks after administration to each group.

Fig. 10 is a schematic diagram of preparation and drug release of the multifunctional modified chitosan self-healing hydrogel.

Detailed description of the preferred embodiments

The invention provides a double-factor-loaded self-healing hydrogel for realizing the purpose of repairing damaged bone tissues, wherein bFGF is placed on the outer layer of the hydrogel and is released in advance, so that cell proliferation and generation of micro-vessels can be promoted; TGF beta 3 is wrapped in microsphere resuspension hydrogel and released at later stage, and can stimulate stem cells to differentiate into osteogenesis. The preparation method is simple and convenient, and has wide application prospect in the fields of bone tissue engineering and regenerative medicine.

Transforming growth factor-beta is a polypeptide biological factor super family, and plays an important role in embryonic development, bone reconstruction, cell proliferation and differentiation and the like. The research shows that BMP-2 and TGF-beta 3 can remarkably up-regulate the expression level of bone specific markers RUNX-2 and OSX, and TGF-beta 3 can enhance the osteogenesis function of BMP-2, and the TGF-beta 3 is considered to be a possible starting factor for promoting bone formation. Meanwhile, TGF-beta 3 is a homologous double-chain polypeptide protein, is rich in bone tissues, can promote mitosis and protein synthesis of stem cells, and can regulate the expression of osteogenic phenotypes such as ALP activity, OC synthesis, mineralized nodule formation and the like.

In a preferred embodiment, the invention provides a preparation method of a multifunctional modified chitosan self-healing hydrogel, which comprises the following specific steps:

preparing chitosan microspheres:

the preparation process of the chitosan microsphere by the emulsion method comprises the following steps: taking 1-10mL of 1-5% chitosan solution as a water phase by using a 5mL syringe; mixing petroleum ether (0-30mL), liquid paraffin (0-30mL), and Span80(0-3mL) to obtain oil phase. The aqueous phase was added dropwise to the oil phase using a 5ml syringe with stirring. After 0-10h, adding 5-50% glutaraldehyde into the solution by using a 1mL syringe for three times, wherein 0-5mL of glutaraldehyde is added each time, crosslinking the solution for 1-10 h, standing the solution overnight, and then removing the supernatant. Finally washing with petroleum ether, methanol, ethanol and ultrapure water, washing each solution for at least 2 times, and freeze-drying the washed precipitate to obtain the chitosan microspheres

Preparation of loaded (containing) TGF beta 3 chitosan microspheres:

adding the prepared chitosan microsphere particles into a PBS solution, vortexing to fully dissolve the chitosan microsphere particles, centrifuging to remove supernatant, adding a certain amount of TGF beta 3 solution, and carrying out 37 ℃. And oscillating and adsorbing at constant temperature to obtain the TGF beta 3 loaded chitosan microspheres.

Preparation of self-healing hydrogel:

weighing a certain amount of chitosan glutamate powder, stirring in a PBS solution with pH of 7.4 to completely dissolve the chitosan glutamate powder, transferring the chitosan glutamate powder into a dialysis bag containing the PBS solution with pH of 7.4, dialyzing until the pH of the chitosan glutamate (CSG) solution is 6.8, and storing at room temperature for later use. Meanwhile, a certain amount of four-arm polyethylene glycol benzaldehyde (PEG-BA) powder is weighed and completely dissolved in ultrapure water, and the solution is stored at the temperature of-20 ℃ for standby. And finally, mixing the prepared chitosan glutamate (CSG) solution and the PEG-BA solution according to a certain proportion to obtain the self-healing hydrogel with different proportions.

Preparing the bFGF-loaded self-healing hydrogel:

before the self-healing hydrogel prepared in different proportions is gelatinized, a certain amount of bFGF is added into the self-healing hydrogel, and the mixture is swirled to be uniformly mixed. Thus obtaining the self-healing hydrogel loaded with the bFGF.

The invention also aims to provide application of the multifunctional modified chitosan self-healing hydrogel.

The invention provides application of self-healing hydrogel for repairing damaged bone tissues by sequentially releasing different growth factors, which comprises the following specific steps:

(1) preparing multifunctional modified chitosan self-healing hydrogel: the preparation method of the multifunctional modified chitosan self-healing hydrogel is carried out according to the preparation method of the multifunctional modified chitosan self-healing hydrogel provided by the invention;

(2) preparation of bone injury site: the left gum of the maxillary incisor is cut by a sharp-end scalpel under the aseptic condition, the gum flap is slightly turned, after the alveolar bone surface is exposed, a dental drill is used for drilling, the dental drill is intermittently ground off, and water is sprayed at a low speed to prevent osteonecrosis, so that the spherical defects with the diameter of 2mm and the depth of 1mm are unified when the spherical defects reach the operation standard.

(3) Transplanting the multifunctional modified chitosan self-healing hydrogel: transplanting the self-healing hydrogel with the size being proper to that of the alveolar bone defect part prepared in the step (1) to the alveolar bone defect part in the step (2) in a surgical operation mode, suturing a wound, fixing the self-healing hydrogel to obtain the transplanted part, and sterilizing;

(4) the multifunctional modified chitosan self-healing hydrogel is used for identifying the treatment and repair effects of alveolar bone injury: the alveolar bone injury repair effect is evaluated through Micro-CT and in-vivo compatibility research of the self-healing hydrogel.

Aiming at the problems in the aspects of bone tissue defect and reconstruction, the invention prepares the self-healing hydrogel with multifunctional modified chitosan by adopting the method of mixing the chitosan microspheres loaded with TGF beta 3 and the self-healing hydrogel loaded with bFGF, has good biocompatibility, self-healing property and injectability, and can realize the sequential release of different growth factors for repairing the damaged alveolar bone tissue. The invention belongs to the technical field of bone tissue engineering and regenerative medicine, and has wide application prospect in the field of bone tissue engineering and regenerative medicine.

Examples

The reagents, materials or instruments used in the multifunctional modified chitosan self-healing hydrogel provided by the invention can be purchased from the market, and the percentages are weight percentages unless otherwise indicated or obviously contradicted.

The technical solutions of the present invention are further described below with reference to the accompanying drawings and examples, which are only for better understanding of the present invention and do not limit the scope of the present invention.

Without being bound to a particular theory, the inventors found that bFGF and TGF β 3 have spatio-temporal specificity in the osteogenic differentiation process of hPDLSCs, i.e., bFGF promotes their proliferation and sternness maintenance and TGF β 3 promotes their osteogenic differentiation. In view of the above findings, the inventors designed a gel system capable of sequentially releasing growth factors by using chitosan as a raw material and combining the advantages of two drug carriers, namely gel and microspheres. TGF beta 3 is loaded in microspheres, the TGF beta 3-loaded chitosan microspheres are suspended in a hydrogel system containing bFGF, the rapid release of the bFGF is expected to promote cell proliferation and the generation of micro-blood vessels in the early stage, the slow degradation characteristic of the chitosan microspheres is utilized to release the TGF beta 3 persistently, mesenchymal stem cells are recruited at low concentration in the early stage, and osteogenic differentiation of the stem cells is stimulated at high concentration in the later stage.

Example 1

Preparation of TGF beta 3-loaded chitosan microspheres

The method comprises the following specific steps

Preparing microspheres by an emulsification method, and taking 5mL of 2% chitosan (Mw is 200 ten thousand, Zhengzhou Kerui fine chemical industry) solution as an aqueous phase by using a 5mL injector; petroleum ether (10mL, Mi Europe chemical reagent Co., Ltd., Tianjin Ke), liquid paraffin (14mL, Mi Europe chemical reagent Co., Ltd., Tianjin Ke) and Span80(0.74mL) were mixed to prepare an oil phase. The aqueous phase was added dropwise to the oil phase using a 5ml syringe with stirring.

After 1-2h, adding 25% glutaraldehyde into the solution by using a 1mL syringe for three times, wherein 1.5mL of glutaraldehyde is added each time, crosslinking the solution for 3-4 h, standing the solution overnight, and removing the supernatant.

And finally, washing with petroleum ether, methanol, ethanol and ultrapure water, washing each solution for at least 2 times, freeze-drying the washed precipitate to obtain the chitosan microspheres, and observing with a microscope.

20mg of the prepared chitosan microsphere particles were added to PBS solution, vortexed to dissolve them thoroughly, centrifuged to remove the supernatant, and a certain amount of TGF beta 3 (supplied by medical biotechnology research and development center, Inc., of university of river, Guangzhou) solution was added at 37 ℃. And oscillating and adsorbing at constant temperature to obtain the TGF beta 3 loaded chitosan microspheres.

The results in FIG. 1 show that the Chitosan Microsphere (CM) particles are in the shape of intact spheres, have relatively smooth surfaces and compact structures, but have wrinkles on the surface at some locations and also some microsphere fragments, which may be associated with mechanical agitation and freeze-drying during microsphere preparation. The particle size distribution histogram of CM is shown in FIG. 1B, and the particle size distribution of the prepared microspheres is uniform. The particle size of the microspheres is mainly distributed between 10 and 60 mu m, wherein the number of the microspheres distributed between 20 and 30 mu m is the largest. As shown in fig. 2, a, the amount of adsorption of CM to BSA varied with time. FIG. 2, B shows the adsorption of TGF-beta 3 by CM, and the adsorption amount of TGF-beta 3 by CM is 23.53 + -0.41 mug/mg.

Drug loading experiment of chitosan microspheres

Accurately weighing 20mg of chitosan microsphere powder, adding PBS buffer solution, vortexing to fully swell the chitosan microsphere powder, centrifuging to remove supernatant, adding BSA solution with a certain concentration, carrying out constant-temperature oscillation adsorption at 37 ℃, centrifuging to separate supernatant after preset time (l, 3, 6, 12, 24, 36, 48, 60, 72 and 96 hours) is reached, and measuring the protein content in the BSA solution after adsorption by using a BCA kit (Sigma, USA). And calculating the drug loading rate of the microspheres by taking the BSA stock solution as a control.

Preparation of microspheres carrying TGF beta 3: the BSA protein solution was changed to a TGF β 3 protein solution according to the above method.

Example 2

Preparation of TGF beta 3 loaded sericin microspheres

The method comprises the following specific steps

Preparing microspheres by an emulsification method, and taking 5mL of 2% sericin solution as a water phase by using a 5mL syringe; petroleum ether (10mL), liquid paraffin (14mL), and Span80(0.74mL) were mixed to give an oil phase. The aqueous phase was added dropwise to the oil phase using a 5ml syringe with stirring.

And finally, washing with petroleum ether, methanol, ethanol and ultrapure water, washing each solution for at least 2 times, freeze-drying the washed precipitate to obtain sericin microspheres, and observing with a microscope.

Adding the prepared sericin microsphere particles of 20mg into a PBS solution, vortexing to fully dissolve the sericin microsphere particles, centrifuging to remove a supernatant, adding a certain amount of TGF beta 3 solution, and carrying out 37 ℃. Vibrating and adsorbing at constant temperature to obtain the sericin microsphere loaded with TGF beta 3.

Example 3

Preparation of BMP-2-loaded chitosan microspheres

The method comprises the following specific steps

Preparing microspheres by an emulsification method, and taking 5mL of 2% chitosan solution as a water phase by using a 5mL injector; petroleum ether (10mL), liquid paraffin (14mL), and Span80(0.74mL) were mixed to give an oil phase. The aqueous phase was added dropwise to the oil phase using a 5ml syringe with stirring.

Adding the prepared 20mg chitosan microsphere particles into a PBS solution, vortexing to fully dissolve the chitosan microsphere particles, centrifuging to remove supernatant, adding a certain amount of BMP-2 solution, and carrying out 37 ℃. Carrying out constant-temperature oscillation adsorption to obtain the BMP-2 loaded chitosan microspheres.

Example 4

Preparation of multifunctional modified chitosan self-healing hydrogel

The method comprises the following specific steps

1) Weighing 3g chitosan glutamate (CSG) (Technological development, Inc. of far city, Wuhan), adding into 100mL PBS solution with pH of 7.4, and stirring overnight with a stirrer to completely dissolve;

2) the resulting mixture was dialyzed with PBS having a pH of 7.4 and a CSG solution having a pH of about 6.8 in a dialysis bag having a MW of 3000Da, and stored at room temperature under a sealed condition.

3) 200mg of PEG-BA (WUKUHU Peng Biotech Co., Ltd.) powder was precisely weighed, dissolved in 1mL of ultrapure water, vortexed to dissolve it sufficiently, and stored at-20 ℃ for further use.

4) Diluting the prepared CSG with PBS to the concentration of 1%, 2% and 3%, wherein the volume ratio of the CSG to the PEG-BA (20%) solution is 1:0.025, 1:0.05 and 1:0.1 respectively, and mixing to obtain hydrogels with different proportions.

Add 100. mu.g of bFGF (supplied by pharmaceutical Biotechnology research and development center, Inc., of university of river, Guangzhou) or platelet-derived growth factor (100. mu.g of PDGF) (supplied by pharmaceutical Biotechnology research and development center, Inc., of university of river, Guangzhou) to step 4) and vortex to mix well. Thus obtaining the self-healing hydrogel loaded with bFGF or PDGF.

The self-healing hydrogel loaded with bFGF or PDGF is mixed with the chitosan microspheres loaded with TGF beta 3 or BMP-2 in example 1 to obtain the multifunctional modified chitosan self-healing hydrogel.

As shown in fig. 3, the hydrogel prepared under the condition of 3% CSG concentration has relatively fewer ridges on the surface, the ridges on the surface of the hydrogel are increased but are relatively fragile with the decrease of the CSG concentration, the pore size of the surface is relatively larger, the lamellae formed in the hydrogel are thickened with the increase of the CSG concentration, the bonding between the lamellae is increased, the lamellae formed in the hydrogel are thickened with the concentration of 2% CSG concentration, more bonding points exist among the lamellae, the fragments in the hydrogel are reduced, and the distribution of internal meshes is relatively uniform; at a 3.0% CSG concentration, the lamellae formed in the hydrogel further thickened, the bonding between lamellae was tighter, but the internal cell size was not uniform. In addition, it can be seen from the group of microspheres added that 1% CSG, the microspheres are less uniformly distributed, while 2% and 3% CSG, the microspheres are more uniformly distributed, which is advantageous for drug release in microspheres. FIG. 4 is an evaluation of the self-healing ability of the hydrogel, which was picked up with forceps 1h after immersion in a small amount of PBS solution, and it was found that the spliced hydrogel did not come off and separate. The self-healing may be caused by Schiff base reactions in the hydrogel that cross-link together. Therefore, it can be concluded that after the hydrogel is implanted into the alveolar bone defect for further use, if the material is broken and fractured due to external force, the material can be self-healed subsequently, and the treatment and repair tasks are continuously undertaken. In fig. 5, bFGF and TGF β 3 were measured for in vitro release using an ELISA kit (enzyme immunoassay kit, jiangsu enzyme immunoassay limited) (TGF β 3 concentration 4 μ g/ml, bFGF concentration 100ng/ml, CSG concentration 2% optimum, CSG: PEG-BA 1:0.05), and bFGF and TGF β 3 released amounts of 87.3% ± 2.7% and 40.0% ± 4.8% of the total load, respectively, in 7 days of the release test. The bFGF encapsulated in the gel is detected to release in 12h, the release amount accounts for 4.7% + -1.2% of the total amount, and the slow sustained release in the following 6 days is about 82.6%; TGF-beta 3 was encapsulated layer by layer and its release was only detected at 36h, with a release of 4.3% + -2.3% of the total amount and at 7 days a release of 40.0% + -4.8% was achieved, meaning that a significant amount of TGF-beta 3 remained in the hydrogel. Generally speaking, bFGF encapsulated in gel is released rapidly, and the release rate of TGF beta 3 encapsulated in the microsphere and gel double layers is far lower than that of bFGF, so that the aim of sequential release is achieved.

Example 5

Research on multi-functional modified chitosan self-healing hydrogel inducing hPDSCs to differentiate into osteogenesis

1) Human periodontal ligament stem cells (hPDLSCs) were taken at passage 5 (see methods for cell extraction: [1]Effect of collagen scaffold on proliferation and osteogenic differentiation of human periodontal ligament stem cells treated with folium Eucommiae extract [ J]Tissue engineering of China 2020,24(16):2537-]Zhangjie, role of Hedgehog signaling pathway in human periodontal ligament stem cell stress osteogenesis and transduction mechanism [ J]Zhejiang Utility medicine 2018,23 (5): 313-316), the trypsin digestion is made to 6X 10⁶Cell suspension/mL.

2) Under the aseptic condition, preparing chitosan microspheres loaded with TGF beta 3 with different concentrations according to the method in the example 1;

3) CSG with the concentration of 2% is prepared according to the method in example 4, and the volume ratio of the CSG to the PEG-BA (20%) solution is 1:0.05 respectively;

4) adding 50 mu L of cell suspension into a multifunctional modified chitosan self-healing hydrogel system (containing 5mg of loaded TGF beta 3 solutions with different concentrations, 1mL of sterile 2% CSG, 50 mu L of 20% PEG-BA and 100ng/mL of bFGF solution), incubating at 37 ℃ for 30min, and adding an induction medium for culturing after the cell suspension is formed.

5) Grouping experiments: blank gel group, 0.25 mu g/mL TGF beta 3 self-healing hydrogel group, 0.50 mu g/mL TGF beta 3 self-healing hydrogel group and 1.00 mu g/mL TGF beta 3 self-healing hydrogel group. After culturing for 7 days and 14 days, staining was performed according to the procedure of ALP staining kit (Shanghai Biyuntian Biotech Co., Ltd., China) and photographs were taken. As shown in FIG. 6, at day 7, the group carrying TGF-beta 3 microspheres was darker than the control group, and the effect was better at a TGF-beta 3 concentration of 1. mu.g/mL than the other groups. The same trend was observed for the 14-day staining results.

In addition, cell viability of hPDLSCs was analyzed in hydrogel extracts (24h, 48h, 72h) using the MTT assay, and low cytotoxicity favors the growth and differentiation of periodontal cells on the hydrogel. As shown in fig. 7, when the control group is used for 1 hour and 24 hours, the proliferation rates of the hydrogel 100% leaching solutions of all groups are greater than 1, but there is no significant difference compared with the control group; at 48h, the proliferation rate of 1% CSG is the highest, and the proliferation rate of the microsphere-loaded group is lower than that of the microsphere-unloaded group in other groups, the proliferation rate of each group is more than 85%, and the proliferation rate of each group is not significantly different from that of a control group (control); the result for 72h was the same as 48 h. Overall, there were no significant differences between the groups compared to the control group, indicating that our hydrogels were not significantly cytotoxic.

Example 6

In vivo compatibility research of multifunctional modified chitosan self-healing hydrogel

The method comprises the following specific steps:

72 SD rats weighing 220 +/-20 g are anesthetized by 3% sodium pentobarbital, shaved at the back part close to the spine, and then 100 mu L of hydrogel material is injected subcutaneously at the left side and the right side respectively according to groups. Grouping experiments: 1% CSG group, 2% CSG group, 3% CSG group, 1% CSG + CM group, 2% CSG + CM group, 3% CSG + CM group (n ═ 12). At

weeks

1, 2, 4, and 6, tissues were collected, photographed, weighed, recorded, and plotted to examine hydrogel degradation in vivo.

From fig. 8, a, it can be seen that the bulge-like skin mound does not disappear rapidly, indicating that the hydrogel is not absorbed rapidly, but is degraded slowly, so that the drug in the hydrogel can be released in situ. In addition, the skin dome part has normal color and does not have the phenomena of red swelling and ulceration, which indicates that no obvious inflammatory reaction occurs at the injection part. It can be seen from the figure that the hydrogel gradually shrinks with time, indicating that most of the hydrogel can be completely degraded in vivo. The gel was taken out of the body and weighed, and the graph shown in fig. 8, B was obtained after counting. As can be seen from the graph, the degradation was fastest for the 1% CSG concentration group and was substantially complete at day 42; the degradation of the 3% CSG concentration group is slow, and about 30% of CSG concentration group is not completely degraded in 42 days; the degradation rate of the 2% CSG concentration group is between the two groups, and the degradation rate is about 80% in 42 days. According to different bone injury parts and different bone repair time, different self-healing hydrogel concentrations are selected, the degradation rate is high, such as 1% or 2% of CSG can be used for repairing alveolar bone injury, and the degradation rate is low, such as 3% of CSG can be used for repairing skull injury.

Example 7

Treatment and repair of alveolar bone injury by multifunctional modified chitosan self-healing hydrogel

The research on the restoration of alveolar bone by the multifunctional modified chitosan self-healing hydrogel comprises the following steps:

preparing multifunctional modified chitosan self-healing hydrogel: prepared according to the method provided in example 4.

Constructing an alveolar bone injury model: 3% sodium pentobarbital (Sigma, USA) anesthetic is prepared, after intraperitoneal injection, SPF grade SD rats (male, purchased from Guangdong province laboratory animal center (qualification number: 44007200069979)) are observed (the rats are raised in an environment with the temperature of 25 ℃, the humidity of 55 +/-10%), the rats are subjected to light for 12 hours each day with the change of dark period, drinking water is freely taken, the animals are adaptively fed for 1 week before the experiment, and the operation is started after about 5-10 min. The rat is fixed in the supine position and the mandible is pulled up to expose the upper jaw dentition as much as possible. The left gum of the maxillary incisor is cut by a sharp-end scalpel under the aseptic condition, the gum flap is slightly turned, after the alveolar bone face is exposed, a dental drill with the diameter of 1.5mm is used for drilling, the dental drill is intermittently ground off, water is sprayed at low speed to prevent osteonecrosis, and finally the spherical defects with the diameter of 2mm and the depth of 1mm are unified to reach the operation standard. The wounds were washed with physiological saline without suturing, and the rats were housed in groups. After surgery, soft food was taken for 3 days, and 100. mu.L of antibiotic was injected 2 times per subcutaneous.

Grouping tests: SD rats were randomly divided into 4 groups of 12 rats each and housed in cages. a. Model group (self-healing group, i.e. inflicting only lesions and no drug); b. blank gel group (control, blank hydrogel only); c. a low-dose group (an experimental group, a bFGF loaded by 100ng/mL + TGF beta 3 microsphere hydrogel group by 1 mu g/mL); d. high dose group (experimental group, 100ng/mL bFGF + 4. mu.g/mL TGF beta 3 microsphere hydrogel group).

Before material taking, SD rats are photographed by Micro-CT (Hitachi Micro-CT: LCT200) at time points of 7 days and 42 days respectively, and the restoration condition of alveolar bone is observed.

As shown in FIG. 9, the alveolar bone loss at the alveolar bone defect part of the model group is severe, and no obvious recovery is seen; the blank gel group alveolar bone defect part is not recovered, but the alveolar bone loss condition is weaker than that of the model group; the TGF beta 3 microspheres in the low dose group and the high dose group both had different degrees of alveolar bone regeneration at the defect site, and both had better recovery effect than the high dose group (FIG. 9). After 6 weeks of administration treatment, the red marked damaged parts of the groups carrying the TGF beta 3 microsphere self-healing hydrogel with high dosage are obviously less than those of other groups, and the effect of regenerating and repairing damaged alveolar bones is achieved.

Claims

2. The method according to claim 1, wherein in step 1), an aqueous chitosan solution is used as an aqueous phase, petroleum ether, liquid paraffin, Span80, or a mixture thereof is used as an oil phase, the aqueous phase is added to the oil phase under stirring, and glutaraldehyde is added for crosslinking to obtain chitosan microspheres.

3. The method of claim 1, wherein in step 2), the chitosan microspheres contain a combination comprising one or more of TGF β 3 or other active factors.

4. The method according to claim 1, wherein in step 3), the self-healing hydrogel is prepared as follows: weighing chitosan glutamate (CSG) powder, stirring in a PBS (phosphate buffer solution) with the pH of 7.4 to completely dissolve the chitosan glutamate (CSG), transferring the chitosan glutamate (CSG) powder into a dialysis bag containing the PBS with the pH of 7.4, dialyzing until the pH of the obtained chitosan glutamate solution is 6.8, and storing at room temperature for later use; meanwhile, weighing four-arm polyethylene glycol benzaldehyde (PEG-BA) powder, and completely dissolving in ultrapure water, and storing at-20 ℃ for later use; and finally, mixing the prepared chitosan glutamate (CSG) solution with a four-arm polyethylene glycol benzaldehyde (PEG-BA) solution to obtain the self-healing hydrogel.

5. The method of claim 4, wherein the volume ratio of the chitosan glutamate (CSG) solution to the four-arm polyethylene glycol benzaldehyde (PEG-BA) solution is 1: 0.01-1: 1, the mass volume concentration of the chitosan glutamate solution is 5-50%, and the mass volume concentration of the PEG-BA solution is 1-30%.

6. The method according to claim 1, wherein in step 4) the self-healing hydrogel comprises bFGF or a combination of one or more of other active factors, such as Platelet Derived Growth Factor (PDGF).

7. The method of claim 1, wherein the concentration of chitosan microspheres in the hydrogel is 0.1-10 mg/mL.

9. The multifunctional modified chitosan self-healing hydrogel according to claim 8, which can be prepared by the method according to any one of claims 1 to 7.

10. Use of the multifunctional modified chitosan self-healing hydrogel according to claim 8 or 9 in the preparation of a medicament for bone injury repair.