Injectable hydrogel-loaded acellular matrix as well as preparation method and application thereof

Abstract

The invention discloses a preparation method and application of an injectable hydrogel-loaded acellular matrix, belongs to the technical field of biological materials and biomedical engineering, and solves the problem that in the prior art, the effect of applying BMSCs to cartilage injury repair is not ideal enough. The method comprises the following steps: under the action of fluid shear force, inducing mesenchymal stem cells to carry out chondrogenic differentiation by using TGF-beta3, and carrying out decellularization treatment on a cell aggregate to obtain a decellularized matrix; the modified hyaluronic acid and chitosan are cross-linked, and HPCC hydrogel is obtained; and mixing the acellular matrix with the HPCC hydrogel to obtain the injectable hydrogel loaded acellular matrix. According to the invention, in-vitro directional induction of cartilage differentiation of BMSCs is carried out through combination of fluid shear force and TGF-beta3, and the curative effect is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials and biomedical engineering, and more specifically to a preparation method and application of an injectable hydrogel-loaded decellularized matrix. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation, and its pathological features are hyperplastic synovitis, articular cartilage destruction and joint osteophyte formation. The current clinical treatment methods for RA include early non-surgical treatment and late surgical treatment. Non-surgical treatments such as physical and drug therapy are mainly aimed at relieving pain and controlling symptoms, but the therapeutic effect is limited and cannot completely prevent joint tissue damage or the progression of osteoarthritis, which will eventually lead to joint pain, dysfunction and even disability, so the final surgical intervention is inevitable. Therefore, new strategies for cartilage repair should be explored to find more ideal seed cells. Stem cell therapy is a research hotspot for cartilage damage repair in the field of tissue engineering, and has broad prospects for clinical application.

[0003] Bone mesenchymal stem cells (BMSCs) have multidirectional differentiation potential. They can not only escape the host's immune clearance in vivo and in vitro, but also regulate most major immune cells to achieve powerful immunosuppression and anti-inflammatory functions. However, BMSCs have three-line differentiation potential, namely differentiation into osteoblasts, adipocytes and chondrocytes. Therefore, how to induce BMSCs to differentiate in the desired direction is a challenge faced by the field of tissue engineering. Methods for inducing directional differentiation of BMSCs include: chemical drug induction, cytokine and growth factor induction, physical methods such as mechanical force (such as fluid shear force, tensile force, compression force, hydrostatic pressure, etc.), electromagnetic field, etc. Among them, transforming growth factor-β (TGF-β) helps BMSCs modify the local environment and activate endogenous precursor cells, promote the synthesis of extracellular matrix, and play an important role in the chondrogenic differentiation of BMSCs.

[0004] Hydrogels are often used as carriers for the delivery of therapeutic cells, drugs and other bioactive molecules in the field of biomedical engineering. Hyaluronic acid is an important component of the extracellular matrix and has good biocompatibility and degradability. Chitosan has cartilage protection function, which can enhance chondrocyte proliferation, increase the expression level of cartilage matrix components, and inhibit cartilage degradation and synovial inflammation. Summary of the invention

[0005] The present invention provides a preparation method and application of an injectable hydrogel-loaded acellular matrix, which is used to solve the problem in the prior art that the effect of BMSCs applied to cartilage damage repair is not ideal.

[0006] In a first aspect, the present invention provides a method for preparing an injectable hydrogel-loaded decellularized matrix, comprising the following steps: under the action of fluid shear force, inducing bone marrow mesenchymal stem cells to differentiate into chondrocytes using transforming growth factor β3 (TGF-β3) to obtain cell aggregates; decellularizing the cell aggregates to obtain a decellularized matrix; cross-linking modified hyaluronic acid and chitosan to obtain an HPCC hydrogel; and mixing the decellularized matrix with the HPCC hydrogel to obtain the injectable hydrogel-loaded decellularized matrix.

[0007] Fluid shear force is a biomechanical form that is ubiquitous in organisms and an important component of the cell microenvironment. It plays an important regulatory role in various biological behaviors of cells, including maintaining morphology, proliferation, apoptosis, secretion, etc. In in vitro research models, the characteristic phenotype of chondrocytes can be restored to a certain extent under the condition of the presence of fluid shear force, which plays an important role in maintaining the phenotype of chondrocytes. However, in the prior art, the mechanism of the effect of fluid shear force on the cell microenvironment is not fully understood. The present invention provides the application of fluid shear force and provides a theoretical basis for studying the effect of biomechanical factors on the directional differentiation of bone marrow mesenchymal stem cells, which is of great significance for the prevention and treatment of diseases such as rheumatoid arthritis.

[0008] As a possible implementation method, the chondrogenic differentiation process includes the following steps: amplifying bone marrow mesenchymal stem cells to the 2nd to 4th generation and then inoculating them into the 3D microporous system; culturing them in DMEM medium containing 8 to 12 ng / mL TGF-β3 for 80 to 90 hours, and loading 0.5 dyn / cm 2 The fluid shear force was applied for 60 minutes to induce the cell aggregates.

[0009] The present invention uses 3D suspended culture of BMSCs to simulate the in vivo physiological environment and prepares them into acellular aggregates, which can effectively preserve bioactive proteins and regulatory growth factors, while removing cellular components (DNA, lipids, etc.) that may cause immunogenicity in the body, and effectively promote cartilage damage repair.

[0010] As a possible implementation method, the decellularization process includes the following steps: freezing treatment: freezing the induced cell aggregates in liquid nitrogen for 2 to 3 minutes; thawing treatment: thawing treatment in PBS at 35 to 40°C for 4 to 6 minutes; preparing inactivated cells: repeating the freezing treatment and the thawing treatment for 2 to 4 cycles to obtain inactivated cells; incubation and enzyme treatment: incubating the inactivated cells in an incubation solution, then treating them with a mixed enzyme for 50 to 70 minutes, and washing to obtain the decellularized matrix; wherein the incubation solution contains 0.25% Triton X-100 and 10mM NH4 OH solution, wherein the mixed enzyme is a mixture of 5 mg / mL deoxyribonuclease I and 10 mg / mL ribonuclease A.

[0011] As a possible implementation method, the preparation process of the HPCC hydrogel includes the following steps: adding sodium periodate solution dropwise to a hyaluronic acid solution, dialyzing the reaction solution after sufficient reaction to obtain oxidized hyaluronic acid; coupling the oxidized hyaluronic acid and 3-aminophenylboronic acid at room temperature, pH 6 to 7, and under the action of a coupling agent, dialyzing to obtain an oxidized hyaluronic acid-phenylboronic acid polymer conjugate; coupling dihydrocaffeic acid with the oxidized hyaluronic acid-phenylboronic acid polymer conjugate in PBS at a pH of 7 to 8, dialyzing to obtain modified hyaluronic acid; and mixing and cross-linking the modified hyaluronic acid and chitosan at a mass ratio of 1:3 to 5 to obtain the HPCC hydrogel.

[0012] As a possible implementation manner, the mass concentration of the modified hyaluronic acid is 6%; and / or the mass concentration of the chitosan is 5%.

[0013] As a possible implementation manner, the amplification culture medium is a DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin.

[0014] In a second aspect, the present invention provides an injectable hydrogel-loaded decellularized matrix prepared by the preparation method described in any possible implementation of the first aspect.

[0015] In a third aspect, the present invention provides a hydrogel, which is the injectable hydrogel described in any possible implementation of the first aspect.

[0016] The present invention develops a chitosan hyaluronic acid-based multifunctional hydrogel (HPCC) dynamically cross-linked by Schiff base bonds to eliminate ROS at the injury site and regulate the inflammatory microenvironment, thereby promoting the therapeutic effect of rheumatoid arthritis.

[0017] In a fourth aspect, the present invention provides an injectable hydrogel-loaded decellularized matrix prepared by the preparation method described in any possible implementation of the first aspect, or the use of the injectable hydrogel-loaded decellularized matrix described in any possible implementation of the second aspect in the preparation of a drug for repairing cartilage damage.

[0018] As a possible implementation manner, the cartilage damage is caused by rheumatoid arthritis.

[0019] In view of the problems that BMSCs have tri-lineage differentiation potential and will differentiate into osteoblasts, chondrogen and adipogenesis respectively under different induction conditions, the present invention adopts fluid shear force and TGF-β3 to synergistically induce BMSCs into chondrogenesis in vitro; in view of the problems of cellular immune rejection, low cell survival rate and infection risk in direct injection of stem cells to treat diseases, the present invention adopts 3D suspended culture of BMSCs to simulate the in vivo physiological environment and prepares them into acellular aggregates, which can effectively preserve bioactive proteins and regulatory growth factors, while removing cellular components (DNA, lipids, etc.) that may cause immunogenicity in the body, and effectively promote cartilage damage repair; in view of the problems that conventional hydrogel materials lack the ability to regulate the inflammatory microenvironment, which limits their application in the treatment of rheumatoid arthritis, a chitosan hyaluronic acid-based multifunctional hydrogel dynamically cross-linked by Schiff base bonds is developed to eliminate ROS at the injury site and regulate the inflammatory microenvironment, thereby promoting the treatment effect of rheumatoid arthritis.

[0020] Decellular products such as decellularized matrix (dECM) and cell secretions are becoming increasingly popular for cartilage regeneration due to their inherent compositional similarity and regulatory ability to support tissue growth and differentiation. These decellularized products can effectively preserve bioactive proteins and regulatory growth factors while removing cellular components (DNA, lipids, etc.) that may cause immunogenicity in vivo. In addition, the degradation products of decellularized matrix can recruit endogenous stem cells and progenitor cells as chemotactic agents to regulate innate immune responses. Compared with tissue-derived decellularized matrix, matrix and secretions derived from stem cells have specific functions and signaling capabilities that are not easily replicated. The rich and complex set of molecules secreted by stem cells retains the capabilities and functions of their original cells and mimics a microenvironment suitable for regeneration through physical interactions and paracrine signals. Therefore, decellularized extracellular matrix derived from stem cells brings new strategies for cartilage tissue engineering repair.

[0021] Hydrogels have important applications in the field of biomedical engineering, especially as carriers for the delivery of therapeutic cells, drugs and other bioactive molecules. Hyaluronic acid (HA) is a linear polysaccharide and an important component of the ECM (extracellular matrix). Its structure and biological properties mediate its role in cell signaling, wound repair, morphogenesis and matrix organization. To date, HA and its derivatives have been widely used in medical products. Chitosan is composed of a copolymer of glucosamine and N-acetylglucosamine units, connected by β-1,4-glycosidic bonds. It has good biocompatibility, progressive degradation, non-toxicity, biological activity, anti-inflammatory effect and antibacterial activity and is considered to be an ideal material for hydrogels. Studies have shown that chitosan has cartilage protection function, can enhance chondrocyte proliferation, increase the expression level of cartilage matrix components, and inhibit cartilage degradation and synovial inflammation. Dynamic hydrogels with appropriate strength, softness and self-healing properties can be tightly wrapped around damaged articular cartilage, creating a moist, elastic and deformation-resistant local environment, while providing appropriate mechanical support and protective barriers for damaged cartilage, thereby reducing adverse immune responses, preventing synovial endothelial cells from infiltrating and growing, and providing a good regeneration environment for the damaged area. Therefore, coupling hyaluronic acid with chitosan for use in hydrogels can have both anti-inflammatory, antioxidant and tissue regeneration effects, and will be more conducive to the repair of rheumatoid arthritis damaged areas.

[0022] The present invention uses fluid shear force and TGF-β3 to induce BMSCs to differentiate into cartilage in vitro, prepares acellular extracellular matrix, and combines hyaluronic acid and chitosan hydrogel to construct a composite hydrogel with antioxidant, anti-inflammatory, injectable and self-healing properties, which is expected to replace traditional drugs or surgical therapies for the treatment of RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Characterization results of the injectable hydrogel provided in the embodiments of the present invention, wherein A is the SEM image of the injectable hydrogel, B is the gelation ability test result, C is the image of the decellularized matrix under an optical microscope, D is the injectability test result of the injectable hydrogel, and E is the self-healing ability test result of the injectable hydrogel.

[0025] Figure 2 The application performance test results provided by the embodiments of the present invention are as follows. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In order to solve the problem that the effect of BMSCs in the prior art on cartilage damage repair is not ideal, the embodiment of the present invention provides a preparation method and application of an injectable hydrogel-loaded acellular matrix.

[0028] The present invention provides a preparation experiment of an injectable hydrogel loaded with acellular matrix. The hyaluronic acid and chitosan selected in the present invention are both bioactive macromolecules with good biocompatibility, which can provide lubrication and protection of cartilage; phenylboric acid and dihydrocaffeic acid have strong antioxidant properties. In terms of preparation process, the present invention utilizes Schiff base reaction to prepare hydrogel, which has the characteristics of fast gelation speed, double network, low swelling and simple preparation process; compared with single network hydrogel, the dynamically cross-linked double network hydrogel exhibits stronger mechanical properties and better stability. Compared with chemical bonds, the cross-linking in the double network is more flexible than that in the single network hydrogel, and can self-repair after mechanical damage.

[0029] Furthermore, the embodiments of the present invention provide a performance characterization experiment and an application effect experiment of the composite hydrogel. Figure 1-2 The results show that the product prepared in the embodiment of the present invention has good biocompatibility, injectability and self-healing properties, can be used to lubricate joints with bone erosion and reduce wear between joints; in addition, the composite hydrogel has antioxidant ability, promotes the improvement of the inflammatory microenvironment, and may achieve a comprehensive and balanced therapeutic effect in the treatment of rheumatoid arthritis, and is expected to replace traditional drugs or surgical therapies for the treatment of RA.

[0030] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0031] Example 1

[0032] This example provides an experiment for preparing an injectable hydrogel loaded with acellular matrix.

[0033] 1) Fluid shear force and TGF-β3 jointly induce chondrogenic differentiation of BMSCs:

[0034] Bone marrow mesenchymal stem cells (BMSCs) were derived from SD rats. BMSCs were expanded to the third generation using DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. After cell expansion, BMSCs were plated at 2×10 3 The density of 3D Nunclon Sphera microwell system (Thermo Scientific) was seeded into 96-well plates. TM ). cECM is a static culture of cell aggregates, and tECM is a cell aggregate cultured in DMEM medium containing 10 ng / mL TGF-β3 and loaded with 0.5 dyn / cm 2 Fluid shear force was applied for 60 min to induce decellularization of cell aggregates after 7 days.

[0035] 2) Decellularization method:

[0036] At the end of the culture period, the cell aggregates were collected and decellularized. The decellularization method required maximal preservation of biological activity. First, a physical method was used: the cell aggregates were frozen in liquid nitrogen for 2.5 minutes and then thawed in 37°C PBS for 5 minutes, and the cells were inactivated for 3 cycles; then a chemical method was used: the cell aggregates were inactivated in a solution containing 0.25% Triton X-100 and 10 mM NH 4 OH solution at 37 ° C, and then treated with 5 mg / mL deoxyribonuclease I (DNase I) and 10 mg / mL ribonuclease A (RNase A) for 1 hour. After washing with PBS three times, the decellularized cdECM (static culture decellularized matrix, control group) and tdECM (induced differentiation decellularized matrix, treatment group) were obtained and collected in double deionized water (ddH 2 O) and freeze-dried and stored at -20°C for subsequent use.

[0037] 3) Preparation of modified hyaluronic acid:

[0038] 5 mL of 0.108 g / mL NaIO 4 The sodium periodate solution was added dropwise to 5 mL of 1% (w / v) hyaluronic acid solution, stirred and reacted for 4 h at room temperature in the dark, and then 1 mL of ethylene glycol was added dropwise to the reaction system to neutralize the excess NaIO 4 , continue the reaction for 1h, and terminate the reaction. Add 0.25mmol 3-aminophenylboronic acid, carry out cross-linking reaction under the conditions of pH 6-7 and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methyl-chloramine salt as a cross-linking agent, dialyze to obtain oxidized hyaluronic acid phenylboronic acid polymer. Couple dihydrocaffeic acid with oxidized hyaluronic acid phenylboronic acid polymer in PBS at pH 7-8, dialyze to obtain modified hyaluronic acid.

[0039] The dialysis process is as follows: the reaction solution is placed in a dialysis bag with a molecular cutoff of 8 kD and dialyzed for 3 days, with the water being changed 3 to 4 times a day.

[0040] 4) Preparation of decellularized matrix and composite hydrogel:

[0041] 6% (w / v) modified hyaluronic acid and 5% (w / v) chitosan (CS) were mixed and cross-linked at a ratio of 1:4 to obtain injectable (HPCC) hydrogel. Subsequently, 100 μg of cdECM and tdECM were mixed with 1 mL of injectable hydrogel to obtain injectable HPCC@cdECM composite hydrogel and HPCC@tdECM composite hydrogel, respectively.

[0042] Example 2

[0043] This example provides a performance characterization experiment of a decellularized matrix and a composite hydrogel.

[0044] The HPCC@tdECM composite hydrogel prepared in Example 1 was observed for its microscopic morphology and network structure using an Inspect-F field emission scanning electron microscope. Figure 1 The result is shown in A. Figure 1 As shown in A, the HPCC@tdECM composite hydrogel has an interconnected three-dimensional porous network structure.

[0045] The modified hyaluronic acid and chitosan in Example 1 were mixed evenly and then the gel formation was confirmed by the vial inversion method to obtain the following Figure 1 The result shown in B is obtained by Figure 1 From B in the figure, we can see that the two successfully form gel after mixing.

[0046] The HPCC@tdECM composite hydrogel prepared in Example 1 was observed under an optical microscope. Figure 1 The result shown in C in FIG. 1 shows that the morphology is uniform.

[0047] The HPCC@tdECM composite hydrogel prepared in Example 1 was loaded into a syringe to test whether it could be squeezed through a pillow (29G) and complete the writing of a specific pattern after pushing the syringe. Figure 1 The result is shown in D. Figure 1 As shown in D, the writing of English letters is completed, and the surface HPCC@tdECM composite hydrogel has good injectability.

[0048] The self-healing properties of the hydrogel were observed macroscopically. The HPCC@tdECM composite hydrogel prepared in Example 1 was mixed with methyl orange (0.1wt%) and methylene blue (0.1wt%) to prepare hydrogels of different colors. After gelation, the two colored hydrogels were placed in a culture dish and placed at room temperature for 30 minutes. The self-healing ability of the two hydrogels was evaluated by the contact surface state. Figure 1 The result is shown in E. Figure 1 As shown in Figure E, two hydrogels were dyed with dye and placed together, and they healed each other at room temperature, indicating that the hydrogel has good self-healing ability.

[0049] Example 3

[0050] This embodiment provides an application performance testing experiment of an injectable hydrogel loaded with acellular matrix.

[0051] Six-week-old male Wistar rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All animal experiments were performed with the approval of the Animal Ethics Committee.

[0052] 200 mL of an emulsion containing type II collagen and an incomplete adjuvant (volume ratio of 1:1) was subcutaneously injected at the base of the tail, and 100 mL of the same emulsion was injected one week later to enhance the immune effect, so as to establish a rat model of rheumatoid arthritis (RA). After the RA model was successfully established, 20 RA model mice with the same growth conditions were selected and randomly divided into 4 groups (RA group, HPCC group, HPCC@cdECM group and HPCC@tdECM group), and 5 healthy control groups (Health) without model establishment. The corresponding hydrogel samples (HPCC, HPCC@cdECM and HPCC@tdECM) were injected into the joints of each group of mice for treatment. The RA group was not injected, and the mice in the Health group were not treated. At the specified time points (1 and 4 weeks), the joints of the rats were photographed and their swelling volumes were measured, as shown in the following figure. Figure 2 The results show that compared with other groups, the HPCC@tdECM group had the most obvious reduction in swelling, which basically returned to normal levels, while the other groups had varying degrees of swelling. The results show that the HPCC@tdECM composite hydrogel has a significant effect in alleviating arthritis swelling symptoms.

[0053] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0054] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an injectable hydrogel-loaded acellular matrix, characterized in that: The following steps are involved: Under the action of fluid shear force, TGF-β3 was used to induce bone marrow mesenchymal stem cells to differentiate into chondrocytes, and cell aggregates were induced; Decellularizing the cell aggregates to obtain a decellularized matrix; The modified hyaluronic acid was cross-linked with chitosan to obtain an injectable hydrogel; The decellularized matrix is ​​mixed with the injectable hydrogel to obtain the injectable hydrogel-loaded decellularized matrix.

2. The preparation method according to claim 1, characterized in that: The process of chondrogenic differentiation comprises the following steps: After expanding bone marrow mesenchymal stem cells to the 2nd to 4th generation, they were seeded into the 3D microwell system; DMEM medium containing 8-12 ng / mL TGF-β3 and 0.5 dyn / cm 2 The cell aggregates were induced by co-culturing with fluid shear force for 7 days.

3. The preparation method according to claim 1, characterized in that: The decellularization process comprises the following steps: The cell aggregates are incubated in an incubation solution, then treated with a mixed enzyme for 50 to 70 minutes, and washed to obtain the decellularized matrix; wherein the incubation solution is a solution containing 0.25% Triton X-100 and 10mM NH4OH, and the mixed enzyme is a mixture of 5mg / mL deoxyribonuclease I and 10mg / mL ribonuclease A.

4. The preparation method according to claim 1, characterized in that: The preparation process of the injectable hydrogel comprises the following steps: The sodium periodate solution is added dropwise to the hyaluronic acid solution, and after sufficient reaction, 3-aminophenylboronic acid is added to carry out a cross-linking reaction under the condition of pH 6 to 7 and the action of a cross-linking agent, and dialyzed to obtain an oxidized hyaluronic acid phenylboronic acid polymer; Conjugating dihydrocaffeic acid with the oxidized hyaluronic acid-phenylboronic acid polymer conjugate in PBS at a pH of 7 to 8, and dialyzing to obtain modified hyaluronic acid; The modified hyaluronic acid and chitosan are mixed and cross-linked at a mass ratio of 1:3 to 5 to obtain the injectable hydrogel.

5. The preparation method according to claim 1, characterized in that: The mass concentration of the modified hyaluronic acid is 6%; And / or, the mass concentration of the chitosan is 5%.

6. The preparation method according to claim 2, characterized in that: The amplification medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

7. The injectable hydrogel-loaded acellular matrix prepared by the preparation method according to any one of claims 1 to 6.

8. A hydrogel, characterized in that The injectable hydrogel according to any one of claims 1 to 6.

9. Use of the injectable hydrogel-loaded acellular matrix prepared by the preparation method according to any one of claims 1 to 6 or the injectable hydrogel-loaded acellular matrix according to claim 7 in the preparation of drugs for repairing cartilage damage.

10. The use according to claim 9, characterized in that: The cartilage damage is caused by rheumatoid arthritis.

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