Injectable hydrogel loaded with acellular matrix and preparation method and application thereof

Abstract

The application discloses a preparation method and application of injectable hydrogel loaded with acellular matrix, and belongs to the technical field of biomaterials and biomedical engineering, and solves the problem that the effect of BMSCs applied to cartilage injury repair is not ideal in the prior art. The method comprises the following steps: under the action of fluid shear force, inducing bone marrow mesenchymal stem cells to differentiate into chondrocytes by using TGF-beta 3, and performing acellular treatment on cell aggregates to obtain an acellular matrix; crosslinking modified hyaluronic acid and chitosan to obtain HPCC hydrogel; and mixing the acellular matrix and the HPCC hydrogel to obtain the injectable hydrogel loaded with the acellular matrix. The application promotes the curative effect by jointly inducing BMSCs to differentiate into chondrocytes in vitro through fluid shear force and TGF-beta 3.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials and biomedical engineering, and more particularly to a preparation method and application of injectable hydrogel loaded with acellular matrix. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation, and its pathological features are hyperplastic synovitis, joint cartilage destruction and joint osteophyte formation. The current clinical treatment methods for RA include early non-surgical treatment and late surgical treatment. Non-surgical treatment such as physical and drug therapy is mainly aimed at relieving pain and controlling symptoms, but the treatment effect is limited and cannot completely prevent joint tissue damage or the progression of osteoarthritis, ultimately leading 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 in the field of tissue engineering for cartilage damage repair, and has a broad clinical application prospect.

[0003] Bone mesenchymal stem cells (BMSCs) have multilineage differentiation potential, and can not only escape from the host's immune clearance in vivo and in vitro, but also regulate most major immune cells to achieve powerful immunosuppression and anti-inflammatory function. However, BMSCs have three-lineage differentiation potential, i.e., differentiation into osteogenic, adipogenic and chondrogenic directions, so how to induce BMSCs to differentiate in the desired direction is a challenge in the field of tissue engineering. The methods for inducing BMSCs to differentiate 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 to modify the local environment and activate endogenous precursor cells, and promotes the synthesis of extracellular matrix, which plays an important role in the chondrogenic differentiation of BMSCs.

[0004] Hydrogel is often used as a carrier 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, can enhance chondrocyte proliferation, increase the expression level of cartilage matrix components, and inhibit cartilage degradation and synovial inflammation. SUMMARY

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

[0006] In a first aspect, the present application provides a preparation method of an injectable hydrogel loaded with an acellular matrix, comprising the following steps: inducing chondrogenic differentiation of bone marrow mesenchymal stem cells under the action of fluid shear force by using transforming growth factor β3 (TGF-β3), and obtaining cell aggregates; performing acellular treatment on the cell aggregates to obtain an acellular matrix; crosslinking modified hyaluronic acid and chitosan to obtain an HPCC hydrogel; and mixing the acellular matrix and the HPCC hydrogel to obtain the injectable hydrogel loaded with the acellular matrix.

[0007] Fluid shear force is a form of biomechanics commonly existing in organisms and is an important component of cell microenvironment, and has important regulatory effects on various biological behaviors of cells, including maintaining morphology, proliferation, apoptosis, secretion, etc. In an in vitro research model, the presence of fluid shear force can restore the characteristic phenotype of chondrocytes to a certain extent, and has 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 completely understood, and the present application provides the application of fluid shear force and provides a theoretical basis for studying the directional differentiation effect of biomechanical factors on bone marrow mesenchymal stem cells, which has important significance for the prevention and treatment of diseases such as rheumatoid arthritis.

[0008] As a possible implementation manner, the chondrogenic differentiation process comprises the following steps: after the bone marrow mesenchymal stem cells are expanded to the 2th-4th generation, the cells are inoculated into a 3D micropore system; the cells are cultured in DMEM medium containing 8-12 ng / mL TGF-β3 for 80-90 h, and 0.5 dyn / cm 2 Fluid shear force is applied for 60 min to obtain the cell aggregates.

[0009] The present application simulates the in-vivo physiological environment by using 3D suspension culture of BMSCs, and prepares the BMSCs into an acellular aggregate, which can effectively preserve bioactive proteins and regulatory growth factors, remove cell components (DNA, lipids, etc.) that may cause immunogenicity in vivo, and effectively promote the repair of cartilage damage.

[0010] As a possible implementation manner, the process of the decellularization treatment comprises the following steps: freezing treatment: freezing the induced cell aggregates in liquid nitrogen for 2-3 min; thawing treatment: thawing in PBS at 35-40 DEG C for 4-6 min; preparing inactivated cells: repeating the freezing treatment and the thawing treatment for 2-4 cycles to obtain inactivated cells; incubation and enzyme treatment: incubating the inactivated cells in an incubation solution, and then treating with mixed enzymes for 50-70 min, and obtaining the decellularized matrix after washing; wherein the incubation solution is a solution containing 0.25% Triton X-100 and 10 mM NH4OH, and the mixed enzymes are a mixture of 5 mg / mL deoxyribonuclease I and 10 mg / mL ribonuclease A.

[0011] As a possible implementation manner, the process of preparing the HPCC hydrogel comprises the following steps: adding a sodium periodate solution dropwise into a hyaluronic acid solution, and after sufficient reaction, dialyzing the reaction solution to obtain oxidized hyaluronic acid; coupling the oxidized hyaluronic acid and 3-aminobenzene boronic acid at room temperature, under the condition that the pH is 6-7 and a coupling agent acts, 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 with a pH of 7-8, and dialyzing to obtain modified hyaluronic acid; mixing and cross-linking the modified hyaluronic acid and chitosan at a mass ratio of 1:3-5 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 culture medium for amplification is a DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin.

[0014] In the second aspect, the application provides an injectable hydrogel loaded with a decellularized matrix prepared by the preparation method in any possible implementation manner of the first aspect.

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

[0016] The application develops a chitosan-hyaluronic acid-based multifunctional hydrogel (HPCC) cross-linked dynamically by Schiff base bonds, so as to eliminate ROS at the damaged site and regulate the inflammatory microenvironment, and promote the treatment effect of rheumatoid arthritis.

[0017] In a fourth aspect, the application provides an application of the injectable hydrogel loaded with the acellular matrix prepared by the preparation method of any possible implementation manner of the first aspect or the injectable hydrogel loaded with the acellular matrix of any possible implementation manner of the second aspect in the preparation of a medicine for repairing cartilage injury.

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

[0019] The application adopts fluid shear force and TGF-β3 to cooperatively induce BMSCs to differentiate into chondrocytes in vitro in view of the problems that BMSCs have trilinear differentiation potential, and different induction conditions will respectively differentiate into osteogenesis, chondrogenesis, lipogenesis and the like; the application adopts 3D suspension culture of BMSCs to simulate the in-vivo physiological environment, and prepares the BMSCs into an acellular aggregate in view of the problems that direct injection of stem cells to treat diseases exists cell immune rejection, low cell survival rate, infection risk and the like; the acellular aggregate can effectively preserve bioactive proteins and regulatory growth factors, and remove cell components (DNA, lipids and the like) that may cause immunogenicity in the body, and effectively promote cartilage injury repair; the application develops a chitosan-hyaluronic acid-based multifunctional hydrogel dynamically cross-linked by Schiff base in view of the problems that conventional hydrogel materials lack the ability to regulate the inflammatory microenvironment, and limit the application thereof in the treatment of rheumatoid arthritis, so as to eliminate ROS at the injury site and regulate the inflammatory microenvironment, and promote the treatment effect of rheumatoid arthritis.

[0020] Acellular matrix (dECM) and cell secretions and other acellular products are increasingly popular for cartilage regeneration due to their inherent ingredient similarity and regulatory ability to support tissue growth and differentiation. These acellular products can effectively preserve bioactive proteins and regulatory growth factors, while removing cell components (DNA, lipids, etc.) that may cause immunogenicity in the body. In addition, the degradation products of the acellular matrix can recruit endogenous stem cells and progenitor cells as chemotactic agents to regulate innate immune responses. Compared with tissue-derived acellular matrix, stem cell-derived matrix and secretions have specific functions and signaling capabilities that are difficult to replicate. The rich and complex molecular set secreted by stem cells retains the ability and function of the original cells, and mimics a suitable microenvironment for regeneration through physical interactions and paracrine signals. Therefore, stem cell-derived acellular extracellular matrix brings a new strategy for cartilage tissue engineering repair.

[0021] Hydrogels have important applications in biomedical engineering, especially as carriers for the delivery of therapeutic cells, drugs and other bioactive molecules. Hyaluronic acid (HA) is a linear polysaccharide, an important component of ECM (extracellular matrix), and its structure and biological properties mediate the role in cell signaling, wound repair, morphogenesis and matrix organization. So far, HA and its derivatives have been widely used in medical products. Chitosan, composed of copolymers of glucosamine and N-acetylglucosamine units, connected by beta-1, 4-glycosidic bonds, is considered to be an ideal material for hydrogels due to its good biocompatibility, gradual degradation, non-toxicity, biological activity, anti-inflammatory effect and antibacterial activity. 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 can tightly wrap around the damaged articular cartilage site, create a local environment that is moist, elastic and resistant to deformation, while providing appropriate mechanical support and protective barriers for damaged cartilage, thereby reducing adverse immune responses, preventing synovial endothelial cells from infiltrating and growing inward, and providing a good regeneration environment for the damaged site. Therefore, coupling hyaluronic acid with chitosan for hydrogels can have anti-inflammatory, antioxidant and tissue regeneration promoting effects, and will be more beneficial to the repair of rheumatoid arthritis damage sites.

[0022] The application prepares an acellular extracellular matrix after the BMSCs are induced to differentiate into chondrocytes in vitro by fluid shear force and TGF-β3, and constructs a composite hydrogel with antioxidant, anti-inflammatory, injectable and self-healing properties by combining with hyaluronic acid and chitosan hydrogel, which is expected to replace traditional drugs or surgical therapy for RA. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 The characterization results of the injectable hydrogel provided by the embodiments of the present application are shown in the table, wherein A is the SEM image of the injectable hydrogel, B is the gel forming capacity experimental result, C is the image of the acellular matrix under the optical microscope, D is the injectable capacity experimental result of the injectable hydrogel, and E is the self-healing capacity experimental result of the injectable hydrogel.

[0025] Figure 2 The application performance detection experimental results provided by the embodiments of the present application are shown in the table. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] To address the issue that the effects of BMSCs on cartilage injury repair in existing technologies are not ideal, this invention provides a method for preparing and applying an injectable hydrogel-loaded decellularized matrix.

[0028] This invention provides an experimental method for preparing an injectable hydrogel loaded with a decellularized matrix. Hyaluronic acid and chitosan, selected in this invention, are both biocompatible bioactive macromolecules that can provide lubrication and protection for cartilage; phenylboronic acid and dihydrocaffeic acid possess strong antioxidant properties. In terms of preparation technology, this invention utilizes the Schiff base reaction to prepare the hydrogel, which features rapid gelation, a dual-network structure, low swelling, and a simple preparation process. Compared to single-network hydrogels, the dynamically cross-linked dual-network hydrogel exhibits stronger mechanical properties and better stability. Compared to chemical bonds, cross-linking in the dual network is more flexible than in single-network hydrogels and can self-repair after mechanical damage.

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

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

[0031] Example 1

[0032] This embodiment provides an experimental preparation of an injectable hydrogel-loaded decellularized matrix.

[0033] 1) Fluid shear stress and TGF-β3 jointly induce BMSCs to differentiate into chondrocytes:

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

[0035] 2) Decellularization method:

[0036] At the end of the culture period, cell aggregates were collected and subjected to decellularization. The decellularization method needs to preserve the bioactivity to the maximum. First, physical method was used: cell aggregates were frozen in liquid nitrogen for 2.5 min and then thawed in PBS at 37 °C for 5 min, repeating 3 cycles to inactivate the cells; then chemical method was used: incubation in a solution containing 0.25% Triton X-100 and 10 mM NH4OH at 37 °C, followed by treatment with 5 mg / mL deoxyribonuclease I (DNase I) and 10 mg / mL ribonuclease A (RNase A) for 1 h. After washing with PBS for 3 times, the decellularized cdECM (statically cultured decellularized matrix, control group) and tdECM (induced differentiated decellularized matrix, treatment group) were collected in double-distilled water (ddH2O) and freeze-dried, stored at -20 °C for subsequent use.

[0037] 3) Preparation of modified hyaluronic acid:

[0038] 5 mL of 0.108 g / mL NaIO4 (sodium periodate) solution was added dropwise to 5 mL of 1% (w / v) hyaluronic acid solution, and after stirring the reaction at room temperature in the dark for 4 h, 1 mL of ethylene glycol was added to the reaction system to neutralize the excess NaIO4, and the reaction was continued for 1 h, and the reaction was terminated. 0.25 mmol of 3-aminobenzene boronic acid was added, and the cross-linking reaction was carried out under the action of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride as a cross-linking agent at pH 6-7, and dialysis was performed to obtain oxidized hyaluronic acid benzene boronic acid polymer. Dihydrocaffeic acid was coupled with oxidized hyaluronic acid benzene boronic acid polymer in PBS at pH 7-8, and dialysis was performed to obtain modified hyaluronic acid.

[0039] The dialysis process is as follows: the reaction solution is loaded into a dialysis bag with a molecular cut-off of 8 kD and dialyzed for 3 days, with water changed 3-4 times per day.

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

[0041] 6% (w / v) of the modified hyaluronic acid and 5% (w / v) of chitosan (CS) are mixed at a ratio of 1:4 and crosslinked to obtain an injectable (HPCC) hydrogel. Subsequently, 100 μg of cdECM and tdECM are mixed with 1 mL of the injectable hydrogel, respectively, to obtain an injectable HPCC@cdECM composite hydrogel and an injectable 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 is observed by Inspect-F field emission scanning electron microscopy for its micro-morphology and network structure, and the results are shown in A of Figure 1 From A of Figure 1 , it can be seen that the HPCC@tdECM composite hydrogel has a three-dimensional porous network structure connected to each other.

[0045] After the modified hyaluronic acid and chitosan in Example 1 are uniformly mixed, the vial inversion method is used to confirm whether they are gelled, and the results are shown in B of Figure 1 From B of Figure 1 , it can be seen that they are successfully gelled after mixing.

[0046] The HPCC@tdECM composite hydrogel prepared in Example 1 is observed under an optical microscope, and the results are shown in C of Figure 1 It can be seen that its morphology is uniform.

[0047] The HPCC@tdECM composite hydrogel prepared in Example 1 is loaded into a syringe, and whether it can be extruded through a pillow (29G) and complete a specific pattern writing after the syringe is pushed is detected, and the results are shown in D of Figure 1 From D of Figure 1 , it can be seen that 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 (0.1 wt%) and methylene blue (0.1 wt%) to prepare hydrogels of different colors, respectively. After gelation, two pieces of dyed hydrogel were pasted together and placed in a culture dish at room temperature for 30 minutes. The self-healing ability was evaluated by the state of the contact surface of the two hydrogels, and the results are shown as E in Figure 1 . As shown in E in Figure 1 , the two pieces of hydrogel were dyed with dyes and placed together, and healed with each other at room temperature, indicating that the hydrogel has good self-healing ability.

[0049] Example 3

[0050] This example provides an application performance detection experiment of an injectable hydrogel loaded with a decellularized matrix.

[0051] 6-week-old male Wistar rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Animal Ethics Committee.

[0052] 200 mL of emulsion containing type II collagen and incomplete adjuvant (volume ratio 1:1) was injected subcutaneously through the tail root, and 100 mL of the same emulsion was injected one week later to enhance the immune effect, to establish a rheumatoid arthritis (RA) rat model. After the RA model was successfully established, 20 RA model rats 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) were not established. The corresponding hydrogel samples (HPCC, HPCC@cdECM and HPCC@tdECM) were injected into the joint parts of each group of rats for treatment, and the RA group was not injected, and the Health group of rats was not treated. At the specified time points (1, 4 weeks), the joint parts of the rats were photographed and the swelling volume was measured, and the results are shown as Figure 1 Figure 2 . It can be seen that compared with other groups, the swelling of the HPCC@tdECM group is most obvious and basically returns to the normal level, while the other groups have different degrees of swelling. The results show that the HPCC@tdECM composite hydrogel has a significant effect on relieving the swelling symptoms of arthritis.

[0053] Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all changes and modifications falling within the scope of the present application.

[0054] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A method for preparing an injectable hydrogel loaded decellularized matrix, characterized in that, The preparation method comprises the following steps: After the bone marrow mesenchymal stem cells were expanded to the 2nd to 4th generation, they were inoculated into a 3D micropore system, and were induced to form cell aggregates by co-culturing in DMEM medium containing 8 to 12 ng / mL TGF-β3 and loading 0.5 dyn / cm 2 The co-culturing was performed under the condition of fluid shear force for 60 min, and the cell aggregates were induced for 7 d. subjecting the cell aggregate to decellularization treatment to obtain a decellularized matrix; adding a sodium periodate solution into a hyaluronic acid solution, and after sufficient reaction, adding 3-aminobenzene boronic acid under the action of a crosslinking agent at pH 6-7 to perform crosslinking reaction, and then dialyzing to obtain oxidized hyaluronic acid benzene boronic acid polymer; coupling dihydrocaffeic acid with the oxidized hyaluronic acid benzene boronic acid polymer in PBS at pH 7-8, and then dialyzing to obtain modified hyaluronic acid; crosslinking the modified hyaluronic acid with chitosan to obtain an injectable hydrogel; mixing the decellularized matrix with the injectable hydrogel to obtain the decellularized matrix loaded injectable hydrogel.

2. The production method according to claim 1, characterized by, The decellularization treatment process comprises the following steps: freezing the cell aggregate in liquid nitrogen for 2.5 minutes, and then thawing in PBS at 37 ℃ for 5 minutes, repeating 3 cycles to obtain a treated cell aggregate; incubating the treated cell aggregate in an incubation solution, and then treating with mixed enzymes for 50-70 min, and then washing to obtain the decellularized matrix; wherein the incubation solution is a solution containing 0.25% Triton X-100 and 10 mM NH4OH, and the mixed enzymes are a mixed solution of 5 mg / mL deoxyribonuclease I and 10 mg / mL ribonuclease A.

3. The preparation method according to claim 1, characterized in that, The preparation process of the injectable hydrogel comprises the following steps: mixing and crosslinking the modified hyaluronic acid with chitosan at a mass ratio of 1:3-5 to obtain the injectable hydrogel.

4. The method of claim 1, wherein, The mass concentration of the modified hyaluronic acid is 6%. And / or, the mass concentration of the chitosan is 5%.

5. The preparation method according to claim 1, characterized in that, The expansion culture medium is DMEM culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

6. The decellularized matrix loaded injectable hydrogel prepared by the preparation method in any one of claims 1-5.

7. The application of the decellularized matrix loaded injectable hydrogel prepared by the preparation method in any one of claims 1-5 or the decellularized matrix loaded injectable hydrogel in claim 6 in the preparation of a drug for repairing cartilage injury.

8. Use according to claim 7, characterized in that, The cartilage injury is caused by rheumatoid arthritis.

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