A degradable bone filling material and its preparation method and application

Abstract

The present invention discloses a degradable bone filling material, a preparation method and an application thereof, and belongs to the field of biomedical technology. The preparation method comprises: mixing a silk fibroin solution, a chitosan solution and a base gel, adding biphasic calcium phosphate, freezing and freeze-drying after mixing under heating conditions, taking out and soaking in water and placing it in a mold for freeze-drying to obtain a core skeleton structure; taking another silk fibroin solution, a chitosan solution containing methacryloylated RGD peptide and a base gel, mixing them, adding biphasic calcium phosphate, freezing and freeze-drying after mixing under heating conditions, covering the surface of the core skeleton structure, freezing and freeze-drying, taking out and soaking in water and freezing and freeze-drying again to obtain a porous skeleton structure; covering the surface of the porous skeleton structure with hydrogel, and obtaining. The present invention also discloses a degradable bone filling material and an application thereof. The degradable bone filling material prepared by the present invention has high mechanical strength, no biological toxicity and has the effect of promoting osteogenesis, and can be used to prepare bone tissue organoids and drugs or preparations for treating bone defects and skeletal system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a degradable bone filling material and a preparation method and application thereof. Background Art

[0002] Bone defects refer to lesions in which the integrity of bone structure is destroyed due to congenital or acquired factors. Trauma, severe infection, malignant tumors, surgical debridement of osteomyelitis, and various congenital diseases are the main causes of bone defects. Clinically, the most common site of bone defects is the tibial shaft, which presents as pain and limited movement at the site of the bone defect. When combined with infection, symptoms such as fever, redness, swelling, and even pus discharge may occur in the limbs, eventually leading to limb shortening, amputation, and disability. Relevant surveys and studies have shown that patients with bone defects have a long recovery period, a high disability rate, and require high postoperative treatment costs, which seriously affects the quality of life of patients with bone defects. Finding an effective means of treating bone defects has always been an important problem plaguing orthopedic clinics.

[0003] Bone filling materials have become an indispensable means of treating bone defects. They can fill bone defects, provide mechanical support, and promote bone formation. Bone filling materials generally require good biocompatibility, appropriate biodegradability, and a certain degree of biomechanical strength and plasticity. They also need to have a certain ability to induce bone or guide tissue regeneration. However, existing bone filling materials have disadvantages such as poor mechanical properties and the inability of support and porous structure to coexist. To improve their mechanical strength and support performance, the existing technology often achieves this by adding crosslinking agents. Traditional chemical crosslinkers themselves may be biotoxic and may be released after the material is implanted in the body, causing adverse effects on surrounding tissues, such as inducing inflammatory reactions and cytotoxicity. At the same time, the use of crosslinkers will reduce the biosafety and degradability of the overall material. For bone filling materials that need to be gradually absorbed and replaced by the human body, too low biodegradability may cause the material to remain in the body for a long time, which is not conducive to the regeneration and repair of bone tissue.

[0004] Therefore, developing a bone filling material with high biosafety, rapid molding, good biodegradability and excellent mechanical strength is of great significance for the treatment of bone defects. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a degradable bone filling material and a preparation method and application thereof, so as to solve the problems of poor mechanical properties, safety and biodegradability of bone filling materials.

[0006] The technical solution adopted to solve the technical problem is to provide a method for preparing a degradable bone filling material, comprising the following steps:

[0007] (1) mixing a silk fibroin solution, a chitosan solution, and a base gel and adding biphasic calcium phosphate, mixing under heating conditions, freezing, and freeze-drying, taking out, soaking in water, and placing the solution in a mold and freeze-drying again to obtain a core skeleton structure;

[0008] (2) Separately, a silk fibroin solution, a chitosan solution containing methacryloyl-RGD peptide, and a base gel are mixed and biphasic calcium phosphate is added. After mixing under heating conditions, the mixture is covered on the surface of the core skeleton structure and then frozen and freeze-dried in sequence. After being taken out and soaked in water, the mixture is frozen and freeze-dried again to obtain a porous skeleton structure.

[0009] (3) The surface of the porous skeleton structure is covered with hydrogel to obtain a degradable bone filling material.

[0010] The beneficial effects of the above technical solution adopted in the present invention are as follows: in the present invention, the raw materials are first mixed and then heated to mix, and then frozen for the first freeze-drying. After being taken out and soaked in water, they are immediately freeze-dried for the second time in a limited space (mold) without freezing. Under this condition, water will quickly form large ice crystals. Due to the limited freeze-drying space, the material can form a core skeleton structure with a diameter of 0.7 to 3 mm under the squeezing of ice crystals. On this basis, there is no need to add a cross-linking agent. According to the characteristics of the material's own components, a core skeleton structure with sufficient mechanical support properties can be formed by relying on physical cross-linking and protein covalent binding, thereby avoiding the risk of the use of cross-linking agents affecting the biocompatibility and safety of the material. Another raw material is then heated and mixed, and then covered on the surface of the core skeleton structure for freezing and freeze-drying. Among them, the methacryloylated RGD peptide is added to the chitosan to prepare a porous skeleton structure, which helps bone formation-related cells adhere to the porous structure of the material. This is conducive to the gradual fusion of the material with the in situ bone tissue of the bone defect during the growth, differentiation and metabolism of the bone formation-related cells after implantation, ultimately completing the effect of bone defect repair. At the same time, the methacryloylated RGD peptide can play a cross-linking role between the material skeleton / porous structure and the hydrogel material, making the material stronger. After being taken out and fully infiltrated, it is frozen and freeze-dried again. This step can form a randomly distributed porous structure with a pore size of 50 to 250 μm on the surface of the core skeleton structure, which is similar to trabecular bone. The surface of the porous skeleton structure is then covered with hydrogel, which makes the degradable bone filling material of the present invention have modular loading capacity, which can realize modular drug delivery or adapt to different application requirements.

[0011] Preferably, the mass concentration of the silk fibroin solution in step (1) and step (2) is 6-8%; the mass concentration of the chitosan solution in step (1) is 1-3%; and the mass concentration of the chitosan solution containing methacryloyl-RGD peptide in step (2) is 1-3%.

[0012] More preferably, the mass concentration of the silk fibroin solution in step (1) and step (2) is 7%; the mass concentration of the chitosan solution in step (1) is 2%; and the mass concentration of the chitosan solution containing methacryloyl RGD peptide in step (2) is 2%.

[0013] More preferably, a silk fibroin solution with a mass concentration of 7% is prepared by the following steps: 3 g of silk and 4.6 g of anhydrous sodium carbonate powder are boiled in 6 L of deionized water for 1 hour, washed with running water overnight, and dried; the silk is then dissolved in alcoholic calcium chloride, and polyethylene glycol is added to precipitate it. The obtained product is freeze-dried and dissolved in water to obtain a silk fibroin solution with a mass concentration of 7%.

[0014] More preferably, a chitosan solution with a mass concentration of 2% is prepared by the following steps: adding 0.8 g of chitosan to 40 mL of deionized water, then adding 160 μL of glacial acetic acid and stirring magnetically, and stirring evenly to obtain a chitosan solution with a mass concentration of 2%.

[0015] More preferably, the chitosan solution containing the methacryloyl RGD peptide is prepared by the following steps: weighing the methacryloyl RGD peptide in proportion and dissolving it in the chitosan solution.

[0016] More preferably, the biphasic calcium phosphate is prepared by ultrasonically mixing hydroxyapatite powder and tricalcium phosphate powder in a mass ratio of 55:45.

[0017] More preferably, in step (1), the material-liquid ratio of the silk fibroin solution, chitosan solution, base glue and biphasic calcium phosphate is 0.7-0.8 mL: 1 mL: 1 mL: 2-4 g; in step (2), the material-liquid ratio of the silk fibroin solution, chitosan solution containing methacryloylated RGD peptide, base glue and biphasic calcium phosphate is 0.7-0.8 mL: 1 mL: 1 mL: 2-4 g; the mass concentration of methacryloylated RGD peptide in the chitosan solution containing methacryloylated RGD peptide is 0.01-1%.

[0018] More preferably, in step (1), the material-liquid ratio of the silk fibroin solution, chitosan solution, base glue and biphasic calcium phosphate is 0.75 mL: 1 mL: 1 mL: 3 g; in step (2), the material-liquid ratio of the silk fibroin solution, chitosan solution containing methacryloylated RGD peptide, base glue and biphasic calcium phosphate is 0.75 mL: 1 mL: 1 mL: 3 g; the mass concentration of methacryloylated RGD peptide in the chitosan solution containing methacryloylated RGD peptide is 1%.

[0019] Preferably, step (1) comprises the following steps: mixing the silk fibroin solution, chitosan solution and base glue and adding biphasic calcium phosphate, mixing under heating conditions in a water bath at 55 to 75° C., freezing at -55 to -200° C. for 20 to 60 minutes and then freeze-drying, taking out and soaking in water for 1.5 to 2 minutes, then placing it in a mold and freeze-drying it again to obtain a core skeleton structure.

[0020] More preferably, step (1) includes the following steps: mixing the silk fibroin solution, chitosan solution and base glue and adding biphasic calcium phosphate, mixing under heating conditions of 65°C water bath, freezing at -80°C for 30 minutes and then freeze-drying, taking out and soaking it in water for 2 minutes, then placing it in a mold and freeze-drying it again to obtain the core skeleton structure.

[0021] Preferably, step (2) includes the following steps: separately taking a silk fibroin solution, a chitosan solution containing methacryloylated RGD peptide and a base glue, mixing them and adding biphasic calcium phosphate, heating them in a water bath at 55 to 75° C. and covering the mixture on the surface of the core skeleton structure, freezing it at -55 to -200° C. for 20 to 60 minutes and then freeze-drying it, taking it out and soaking it in water for 2 to 15 minutes, then freezing it at -55 to -200° C. for 20 to 60 minutes and then freeze-drying it to obtain a porous skeleton structure.

[0022] More preferably, step (2) includes the following steps: separately taking a silk fibroin solution, a chitosan solution containing methacryloyl RGD peptide and a base glue, mixing them and adding biphasic calcium phosphate, heating them in a 65°C water bath and covering the core skeleton structure surface, freezing them at -80°C for 30 minutes and then freeze-drying them, taking them out and soaking them in water for 3 minutes, freezing them at -80°C for 30 minutes and then freeze-drying them, to obtain a porous skeleton structure.

[0023] Preferably, the hydrogel in step (3) is a hydrogel that can carry and / or slowly release nucleic acid drugs, small molecule drugs, or can allow cells to grow, crawl, adhere, and migrate in a directional manner.

[0024] The beneficial effects of the above-mentioned technical solution adopted in the present invention are: by carrying and releasing nucleic acid drugs (including related carriers) or small molecule drugs through hydrogels, the degradable bone filling material has modular loading capabilities; by covering the hydrogels that allow cells to grow, crawl, adhere and migrate in a directional manner, the surface or interior of the degradable bone filling material can be loaded with cells, giving it the potential to serve as bone tissue organoids.

[0025] More preferably, the hydrogel in step (3) comprises methacrylated hyaluronic acid, methacrylated gelatin or thermosensitive hydrogel.

[0026] The present invention also provides a degradable bone filling material prepared by the above preparation method.

[0027] The beneficial effects of the above technical solution of the present invention are as follows: the degradable bone filling material of the present invention includes a core skeleton structure, a porous skeleton structure based on the core skeleton structure, and a hydrogel covering its surface; the skeleton structure is prepared by mixing a silk fibroin solution, a chitosan solution, a base glue, and a biphasic calcium phosphate, and undergoing freeze-drying, crystallization, and extrusion. First, a core skeleton structure with a supporting function and strong mechanical properties is generated in a limited space, and then the above mixture is coated on the surface of the core skeleton structure, that is, a randomly distributed porous structure similar to trabeculae is generated based on the core skeleton structure; finally, the surface is covered with a hydrogel for loading drugs or carrying cells, and can be used for the preparation of drugs for skeletal system diseases and bone defects; the degradable bone filling material can be quickly degraded and absorbed by bone tissue after the action, and has good biodegradability; the bone filling material of the present invention has good compressive resistance, safety, and biodegradability.

[0028] The present invention also provides the use of the above-mentioned degradable bone filling material in preparing bone tissue organoid materials.

[0029] Preferably, the use of the degradable bone filling material in preparing bone tissue organoid material comprises the following steps:

[0030] Bone tissue organoids are obtained by loading bone-forming cell lines or primary bone-forming related cells into the hydrogel of degradable bone filling material and culturing them through a microfluidic system.

[0031] More preferably, loading bone-forming cell lines or primary bone-forming-related cells into the hydrogel of the degradable bone filling material comprises the following steps: digesting the cultured bone-forming cell lines or primary bone-forming-related cells with trypsin and then resuspending them in culture medium to obtain a cell suspension; then dissolving the hydrogel monomer in the cell suspension and covering it on the porous skeleton structure of the degradable bone filling material for photocrosslinking.

[0032] More preferably, the bone-forming cell lines or primary bone-forming related cells include the pre-osteoblast cell line MC3T3-E1, the mesenchymal stem cell line C3H10T1 / 2, primary adipose-derived mesenchymal stem cells ADSC, and primary bone marrow-derived mesenchymal stem cells BMSC.

[0033] The present invention also provides the use of the above-mentioned degradable bone filling material in the preparation of medicines or preparations for treating skeletal system diseases.

[0034] The present invention also provides the use of the above-mentioned degradable bone filling material in the preparation of medicines or preparations for treating bone defects.

[0035] The present invention has the following beneficial effects:

[0036] (1) The present invention provides a degradable bone filling material designed based on natural bone tissue. Its chemical composition and morphological structure are highly similar to the trabecular structure of the human body, and it has the advantages of high mechanical strength, no biotoxicity and good biodegradability. In addition, it has the effect of promoting osteogenesis and can be completely absorbed and replaced by the patient's own bones in a short period of time. It is suitable for the preparation of drugs or preparations for treating bone defects and skeletal system diseases.

[0037] (2) The preparation process of the degradable bone filling material of the present invention is simple and low-cost. No cross-linking agent is required during the preparation process. The core skeleton structure with sufficient mechanical support performance is formed only by physical cross-linking and protein covalent binding, avoiding the risks of cross-linking agents on the biocompatibility and safety of the material.

[0038] (3) The degradable bone filling material of the present invention has modular loading capabilities, which can realize modular drug delivery or adapt to different application requirements.

[0039] (4) The bone filling material of the present invention has the ability to carry cells in situ and can be used as a potential bone tissue organoid for different application needs such as bone defect treatment, drug screening and in situ inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figures 1 and 2 show the microstructures of the degradable bone filling material during preparation in Example 1. Figure A shows the microstructure of the organic collagen composed of silk fibroin, chitosan, and base glue. Figure B shows the microstructure of the core skeleton structure. Figure C shows the microstructure of the porous skeleton structure. Figure D shows the microstructure of the degradable bone filling material.

[0041] Figure 2 The following are mechanical characterization diagrams of different bone filling materials;

[0042] Figure 3 Figure 1 is a diagram showing the sustained release effect of degradable bone filling materials on small molecule drugs and nucleic acid drugs; wherein A is a diagram showing the sustained release effect of small molecule drugs; and B is a diagram showing the sustained release effect of nucleic acid drugs.

[0043] Figure 4 The biosafety results of the degradable bone filling material are shown in Figure 1. A is the relative cell activity graph; B is the relative cell cycle graph; C is the hemolysis test result graph; and D is the hemolysis rate graph.

[0044] Figure 5 Figure 1 is a diagram showing the regulatory effect of degradable bone filling materials on osteogenic differentiation; A is the staining result; B is the effect on the expression level of Alp in hMSCs; C is the effect on the expression level of Runx2 in hMSCs;

[0045] Figure 6Figure 3 is a diagram showing the long-term regulatory effect of degradable bone filling materials on osteogenic differentiation through sustained-release drugs; A is the staining result after 2 weeks of drug loading; B is the effect of drug loading on the Alp expression level of hMSC after 2 weeks of drug loading; C is the effect of drug loading on the Runx2 expression level of hMSC after 2 weeks of drug loading; D is the staining result after 4 weeks of drug loading; E is the effect of drug loading on the Alp expression level of hMSC after 4 weeks of drug loading; F is the effect of drug loading on the Runx2 expression level of hMSC after 4 weeks of drug loading;

[0046] Figure 7 Figure 1 is a diagram of the modular modification function of the degradable bone filling material; A is a diagram of the thermosensitive phase transition of the hydrogel surrounding the degradable bone filling material; B is a diagram of the sustained release of nucleic acid drugs in the presence of nucleic acid targeting vectors; C is a diagram of the biosafety results; and D is a diagram of the miR-381-3p expression level.

[0047] Figure 8 Figure 2 shows the activity and osteogenic differentiation of hMSC cells loaded with biodegradable bone filling materials. Figure A shows the biosafety results of hMSC cells during in vitro culture; Figure B shows the biosafety results of hMSC cells during in vivo culture; Figure C shows the effect on the expression level of Alp in hMSCs; and Figure D shows the effect on the expression level of Runx2 in hMSCs.

[0048] Figure 9 The effect of biodegradable bone filling materials loaded with primary ADSCs on promoting osteogenesis is shown in Figures AB and AB. Figures AB and C show the distribution of ADSCs in the material after 72 hours of in vitro culture. Figure C shows the immunofluorescence staining of osteogenic marker genes in cells after 10 days of in vitro culture. Figure D shows the immunofluorescence staining of osteogenic marker genes in cells 10 days after the material was implanted in mice.

[0049] Figure 10 The diagram shows the therapeutic effect of biodegradable bone filling materials on tibial bone defects; A is the micro-CT result of mouse tibia; B is the bone density diagram; C is the relative bone volume diagram;

[0050] Figure 11 Figure 1 shows the results of promoting bone formation in skull bone defects with biodegradable bone filling materials; A shows the results of calcein labeling in the skull bone defect adnexa; and B shows the bone mineralization deposition rate.

[0051] Figure 12 These are diagrams showing the effects of biodegradable bone filling materials on fracture treatment; A is the micro-CT result of mouse tibia; and B is the relative bone volume diagram. DETAILED DESCRIPTION

[0052] The features and properties of the present invention are described in further detail below in conjunction with the examples to facilitate understanding of the present invention by those skilled in the art. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, these modifications are obvious, and all inventions and creations utilizing the present invention are protected. Unless otherwise specified, the reagents, instruments, etc. used in the present invention are all commercially available products.

[0053] Example 1

[0054] A method for preparing a degradable bone filling material comprises the following steps:

[0055] (1) 750 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution, and 1000 μL of Corning 354480 base gel were mixed and 3 g of biphasic calcium phosphate was added. The mixture was heated in a 65°C water bath and mixed. The mixture was frozen at -80°C for 30 min and then freeze-dried at -80°C and a vacuum of 20 Pa. The mixture was taken out and soaked in water for 2 min, then placed in a mold and freeze-dried again at -80°C and a vacuum of 20 Pa to obtain a core skeleton structure.

[0056] (2) Take another 750 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution containing 1% methacryloyl RGD peptide and 1000 μL of Corning 354480 base glue, mix them and add 3 g of biphasic calcium phosphate, mix them in a 65°C water bath and cover the surface of the core skeleton structure, freeze them at -80°C for 30 minutes, and then freeze them at -80°C and 20 Pa vacuum, take them out and soak them in water for 3 minutes, freeze them at -80°C for 30 minutes, and then freeze them at -80°C and 20 Pa vacuum to obtain a porous skeleton structure;

[0057] (3) Covering the surface of the porous skeleton structure with methacryloylated hyaluronic acid hydrogel to obtain a degradable bone filling material.

[0058] A 7% silk fibroin solution was prepared by boiling 3g of silk and 4.6g of anhydrous sodium carbonate powder in 6L of deionized water for 1 hour, washing with running water overnight, and air-drying. The silk was then dissolved in alcoholic calcium chloride and polyethylene glycol was added to precipitate the solution. The resulting product was freeze-dried and dissolved in water to obtain a 7% silk fibroin solution.

[0059] A chitosan solution with a mass concentration of 2% was prepared by the following steps: 0.8 g of chitosan was added to 40 mL of deionized water, and then 160 μL of glacial acetic acid was added and magnetically stirred, and the chitosan solution with a mass concentration of 2% was obtained after stirring evenly;

[0060] Biphasic calcium phosphate was prepared by ultrasonically mixing hydroxyapatite powder and tricalcium phosphate powder in a mass ratio of 55:45.

[0061] This embodiment also provides a degradable bone filling material prepared by the above preparation method. The degradable bone filling material includes a core skeleton structure, a porous skeleton structure based on the core skeleton structure, and a hydrogel covering the surface thereof.

[0062] The microstructure of the biodegradable bone filling material was observed by scanning electron microscopy (SEM). Figure 1 As shown. During the preparation process, silk fibroin, chitosan, and base glue can form a shaped organic collagen ( Figure 1 Figure A, Organic), and after adding biphasic calcium phosphate, it can be extruded and rapidly freeze-dried to form a core skeleton structure ( Figure 1 The core skeleton is covered with the above mixture, frozen after immersion in water, and then freeze-dried to form a randomly distributed porous skeleton structure similar to trabecular bone ( Figure 1 Middle C, Frame), and finally the surface is covered with HAMA hydrogel and encapsulated into a complete degradable bone filling material ( Figure 1 (D in the middle, Artificial bone).

[0063] Example 2

[0064] A method for preparing a degradable bone filling material comprises the following steps:

[0065] (1) 700 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution, and 1000 μL of Corning 354480 base gel were mixed and 2 g of biphasic calcium phosphate was added. The mixture was heated in a 55°C water bath and mixed. The mixture was frozen at -55°C for 60 min and then freeze-dried at -80°C and a vacuum of 20 Pa. The mixture was taken out and soaked in water for 1.5 min, then placed in a mold and freeze-dried again at -80°C and a vacuum of 20 Pa to obtain a core skeleton structure.

[0066] (2) Take another 700 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution containing 0.01% methacryloyl RGD peptide and 1000 μL of Corning 354480 base glue, mix them and add 2 g of biphasic calcium phosphate, mix them in a 55°C water bath and cover the surface of the core skeleton structure, freeze them at -55°C for 60 minutes, and then freeze them at -80°C and a vacuum of 20 Pa to obtain a porous skeleton structure.

[0067] (3) Covering the surface of the porous skeleton structure with methacryloyl gelatin to obtain a degradable bone filling material.

[0068] A 7% silk fibroin solution was prepared by boiling 3g of silk and 4.6g of anhydrous sodium carbonate powder in 6L of deionized water for 1 hour, washing with running water overnight, and air-drying. The silk was then dissolved in alcoholic calcium chloride and polyethylene glycol was added to precipitate the solution. The resulting product was freeze-dried and dissolved in water to obtain a 7% silk fibroin solution.

[0069] A chitosan solution with a mass concentration of 2% was prepared by the following steps: 0.8 g of chitosan was added to 40 mL of deionized water, and then 160 μL of glacial acetic acid was added and magnetically stirred, and the chitosan solution with a mass concentration of 2% was obtained after stirring evenly;

[0070] Biphasic calcium phosphate was prepared by ultrasonically mixing hydroxyapatite powder and tricalcium phosphate powder in a mass ratio of 55:45.

[0071] This embodiment also provides a degradable bone filling material prepared by the above preparation method. The degradable bone filling material includes a core skeleton structure, a porous skeleton structure based on the core skeleton structure, and a hydrogel covering the surface thereof.

[0072] Example 3

[0073] A method for preparing a degradable bone filling material comprises the following steps:

[0074] (1) 800 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution, and 1000 μL of Corning 354480 base gel were mixed and 4 g of biphasic calcium phosphate was added. The mixture was heated in a 75°C water bath and mixed. The mixture was frozen at -200°C for 20 min and then freeze-dried at -80°C and a vacuum of 20 Pa. The mixture was taken out and soaked in water for 2 min, then placed in a mold and freeze-dried again at -80°C and a vacuum of 20 Pa to obtain a core skeleton structure.

[0075] (2) Take 800 μL of 7% silk fibroin solution, 1000 μL of 2% chitosan solution containing 1% methacryloyl RGD peptide and 1000 μL of Corning 354480 base glue, mix them and add 4 g of biphasic calcium phosphate, mix them in a 75°C water bath and cover the surface of the core skeleton structure, freeze them at -200°C for 20 minutes, and then freeze them at -80°C and 20 Pa vacuum, take them out and soak them in water for 15 minutes, freeze them at -200°C for 20 minutes, and then freeze them at -80°C and 20 Pa vacuum to obtain a porous skeleton structure;

[0076] (3) The surface of the porous skeleton structure is covered with a thermosensitive hydrogel to obtain a degradable bone filling material.

[0077] A 7% silk fibroin solution was prepared by boiling 3g of silk and 4.6g of anhydrous sodium carbonate powder in 6L of deionized water for 1 hour, washing with running water overnight, and air-drying. The silk was then dissolved in alcoholic calcium chloride and polyethylene glycol was added to precipitate the solution. The resulting product was freeze-dried and dissolved in water to obtain a 7% silk fibroin solution.

[0078] A chitosan solution with a mass concentration of 2% was prepared by the following steps: 0.8 g of chitosan was added to 40 mL of deionized water, and then 160 μL of glacial acetic acid was added and magnetically stirred, and the chitosan solution with a mass concentration of 2% was obtained after stirring evenly;

[0079] Biphasic calcium phosphate was prepared by ultrasonically mixing hydroxyapatite powder and tricalcium phosphate powder in a mass ratio of 55:45.

[0080] This embodiment also provides a degradable bone filling material prepared by the above preparation method. The degradable bone filling material includes a core skeleton structure, a porous skeleton structure based on the core skeleton structure, and a hydrogel covering the surface thereof.

[0081] Example 4 Mechanical Characterization of Degradable Bone Filling Materials

[0082] Bone filling materials need to have certain supporting and compressive properties. In this example, the elastic moduli of four groups of materials were measured: blank methacryloyl hyaluronic acid (HAMA) hydrogel, HAMA (core) wrapped only with the core skeleton structure, HAMA (peripheral) wrapped only with the porous skeleton structure, and the degradable bone filling material (artificial bone) prepared by the present invention. The results are as follows: Figure 2 As shown. Figure 2It can be seen that the addition of the core skeleton structure significantly improves the compressive resistance of HAMA, and the degradable bone filling material has better elasticity and material strength than the other groups, which can meet the mechanical load-bearing requirements of bone tissue and is expected to be a potential bone filling material.

[0083] Example 5 Sustained release effect of degradable bone filling materials on small molecule drugs and nucleic acid drugs

[0084] A HAMA hydrogel (rhodamine B: 5 μg / mL, FAM-NC: 100 μM, SSAD / HA: 100 μL) was prepared using a solution containing a fluorescent marker and prepared as a degradable bone filling material according to the method of Example 1. The prepared degradable bone filling material was soaked in deionized water for 30 minutes, then immersed in deionized water and placed at 4°C for sustained release. The HAMA hydrogel containing only rhodamine B was placed at room temperature for sustained release. After collecting the sustained-release solution at different time points, the degradable bone filling material was immersed in deionized water again to continue the sustained release. The fluorescence intensity was measured using a multifunctional microplate reader to evaluate the sustained release effect of the degradable bone filling material on small molecule drugs and nucleic acid drugs.

[0085] The fluorescent dye Rhodamine B was used to detect the sustained release characteristics of small molecule drugs. Figure 3 As shown. Figure 3 As can be seen from Figure A, the degradable bone filling material has a certain ability to carry and release small molecule drugs; Figure 3 As shown in Figure B, the biodegradable bone filling material can sustainably release FAM-modified siRNA-NC with the addition of the nucleic acid delivery vehicle PVAm, demonstrating its enhanced ability to carry and sustain nucleic acid release over a long period of time. This capability remains high even on day 14. This result suggests that the biodegradable bone filling material has the potential to sustainably release small molecule drugs and nucleic acid drugs, making it a promising bone filling material for promoting osteogenesis and treating bone defects.

[0086] Example 6 Biosafety of Degradable Bone Filling Materials

[0087] The degradable bone filling material was placed in human mesenchymal stem cell (hMSC) culture medium and co-cultured with cells to detect the cell proliferation rate. The results were as follows: Figure 4 As shown, Blank represents untreated cells, Artificial bone represents cells treated with the degradable bone filling material prepared in Example 1 of the present invention, HAMA represents cells treated with methacryloyl hyaluronic acid, and Triton represents a positive control (Triton X-100). Figure 4 It can be seen that the degradable bone filling material has no effect on the cell proliferation rate (such as Figure 4Cell cycle analysis showed that the cell cycle ratio of cells treated with the biodegradable bone filler material did not change compared with untreated cells and HAMA-treated cells (e.g. Figure 4 Hemolysis tests also showed that degradable bone filling materials do not cause hemolysis (e.g. Figure 4 (Figure CD). This demonstrates that the material has good in vitro biosafety.

[0088] Example 7 Ability of Degradable Bone Filling Material to Promote Osteogenic Differentiation of Cells after Loading Drugs

[0089] The small molecule drug bergamotrin and the nucleic acid drug recombinant antagomiR-138 (anti138) were introduced into the biodegradable bone filling material, and the material was co-cultured with hMSCs in the logarithmic growth phase. The expression levels of Alp and Runx2 were detected by qPCR, the level of mineralized nodules was detected by staining with alizarin red dye, and the alkaline phosphatase activity of cells was detected by alkaline phosphatase staining. The results are shown in Figure 3. Figure 5 shown. Figure 5 The results showed that after the introduction of the small molecule drug bergamotrin and the nucleic acid drug recombinant antagomiR-138, the biodegradable bone filling material can promote the expression levels of Alp and Runx2 of hMSC, the level of mineralized nodule formation and alkaline phosphatase activity, indicating that the biodegradable bone filling material can promote cell osteogenic differentiation after being loaded with drugs.

[0090] Example 8 Ability of Degradable Bone Filling Material to Sustained-Release Drugs to Promote Cell Osteogenic Differentiation

[0091] The sustained-release liquid of the degradable bone filling material loaded with the small molecule drug bergamotrin and the nucleic acid drug recombinant antagomiR-138 was extracted at different time points and used to transfect hMSCs to detect the ability of the sustained-release liquid to promote cell osteogenic differentiation, thereby detecting the sustained-release ability of the degradable bone filling material. The results are as follows Figure 6 shown. Figure 6 The results showed that after the degradable bone filling material was loaded with drugs for 4 weeks, it was still able to promote the osteogenic differentiation of hMSCs through a sustained release effect, proving that the degradable bone filling material prepared by the present invention has the ability to sustain drug release.

[0092] Example 9 Modular Modification Function of Degradable Bone Filling Material

[0093] By incorporating less than 30% of DNA self-assembly materials into the peripheral HAMA hydrogel of the degradable bone filling material (Li Z, et al, J Am Chem Soc, 2019), the peripheral hydrogel is cross-linked to form a thermosensitive hydrogel material. At 35°C, which is close to the biological body temperature, it is solid, and at 45-55°C, it becomes a viscous liquid due to the denaturation of the DNA material (e.g. Figure 7 The degradable bone filling material can maintain the sustained release of nucleic acid drugs for about 2 weeks in the presence of nucleic acid targeting vectors (e.g. Figure 7 B in the middle), and also has good in vitro and in vivo biosafety (such as Figure 7 Figure C). Degradable bone filling materials modified with thermosensitive hydrogels and nucleic acid targeting vectors were implanted subcutaneously in the skull and in the femoral medullary cavity of mice. The results showed that the degradable bone filling materials can precisely deliver nucleic acid drugs to bone tissue (e.g. Figure 7 (Figure D in the middle).

[0094] Example 10 Ability and biosafety of degradable bone filling materials as bionic organoids

[0095] The biodegradable bone filling material was used to carry hMSC cells, a small molecule drug called bergamotrin that promotes cell osteogenic differentiation, and a nucleic acid drug recombinant antagomiR-138. In vitro osteogenic culture was performed in a culture dish, and the cell activity and cell osteogenic differentiation level of the biodegradable bone filling material were detected. The results are as follows: Figure 8 shown. Figure 8 The results showed that hMSCs can grow normally in degradable bone filling materials regardless of whether the materials are implanted in mice or cultured in vitro, and when the materials are also loaded with nucleic acid drugs or small molecule drugs, it will also affect the cells' osteogenic differentiation ability; this shows that degradable bone filling materials have the potential to serve as bionic bone tissue organoids.

[0096] Example 11: Possibility of using degradable bone filling materials to carry cells as organoids

[0097] Primary mouse adipose-derived mesenchymal stem cells (ADSCs) were embedded in degradable bone filling materials and cultured in a flow medium. Figure 9 As shown in the figure, ACTIN represents β-Actin (cytoskeletal protein), ALP represents alkaline phosphatase, and RUNX2 represents Runt-related transcription factor 2. Figure 9 The results showed that the biodegradable bone filler material can support cell growth within its skeletal structure and maintain its migration and differentiation potential. This conclusion was also confirmed by in vivo experiments in mice. This shows that the biodegradable bone filler material can embed cells without affecting the physiological properties of the embedded cells.

[0098] Example 12 Therapeutic Effects of Degradable Bone Filling Materials on Bone Defect Mice

[0099] A mouse model of skull and tibia bone defects was constructed, and the small molecule drug bergamotrin and recombinant antagomiR-138 were locally implanted into the bone defects of the mouse skull and tibia by introducing degradable bone filling materials, respectively, to study their repair effects on mouse bone defects.

[0100] from Figure 10 The results of micro-CT scan of the mouse tibia showed that the biodegradable bone filling material had a significant filling effect on the bone defect site, and after adding bergamotin and recombinant antagomiR-138, it played a significant role in promoting the repair of the bone defect site. Figure 11 The results of calcein labeling in the accessory area of ​​the mid-skull bone defect showed that the bone formation rate in the bergamotrin and recombinant antagomiR-138 groups was significantly increased, proving that the degradable bone filling material can carry and deliver drugs, thereby promoting bone repair.

[0101] Example 13 The therapeutic effect of degradable bone filling materials on fractures

[0102] A mouse model of tibial fracture was established. The small molecule drug bergamotrin and recombinant antagomiR-138 were respectively introduced into the degradable bone filling material and locally implanted into the fracture end of the mouse tibia to study their effects on tibial bone formation in mice. The results are as follows: Figure 12 shown. Figure 12 Micro-CT results showed that both bergamotrin and recombinant antagomiR-138 groups had a significant promoting effect on fracture repair, proving that the degradable bone filling material can carry and deliver drugs, thereby playing a role in treating fractures.

[0103] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of the present invention.

Claims

1. A method for preparing a degradable bone filling material, characterized in that: The following steps are involved: (1) Silk fibroin solution, chitosan solution, and base glue were mixed and biphasic calcium phosphate was added. The mixture was heated in a water bath at 55-75°C and then frozen at -55--200°C for 20-60 min and then freeze-dried. The mixture was taken out and soaked in water for 1.5-2 min, then placed in a mold and freeze-dried again to obtain a core skeleton structure. (2) Separately, silk fibroin solution, chitosan solution containing methacryloyl RGD peptide, and base glue are mixed and biphasic calcium phosphate is added. The mixture is heated in a water bath at 55-75°C and then covered on the surface of the core skeleton structure. The mixture is frozen at -55--200°C for 20-60 min and then freeze-dried. The mixture is taken out and soaked in water for 2-15 min, and then frozen at -55--200°C for 20-60 min and then freeze-dried to obtain a porous skeleton structure. (3) covering the surface of the porous skeleton structure with hydrogel to obtain a degradable bone filling material; The mass concentration of the silk fibroin solution in step (1) and step (2) is 6-8%; the mass concentration of the chitosan solution in step (1) is 1-3%; the mass concentration of the chitosan solution containing methacryloyl RGD peptide in step (2) is 1-3%; In step (1), the material-liquid ratio of the silk fibroin solution, the chitosan solution, the base glue, and the biphasic calcium phosphate is 0.7-0.8 mL: 1 mL: 1 mL: 2-4 g; in step (2), the material-liquid ratio of the silk fibroin solution, the chitosan solution containing the methacryloylated RGD peptide, the base glue, and the biphasic calcium phosphate is 0.7-0.8 mL: 1 mL: 1 mL: 2-4 g; the mass concentration of the methacryloylated RGD peptide in the chitosan solution containing the methacryloylated RGD peptide is 0.01-1%; The hydrogel in step (3) is a hydrogel that can carry and / or slowly release nucleic acid drugs, small molecule drugs, or can be used for cell growth, crawling, adhesion and directional migration.

2. A degradable bone filling material prepared by the preparation method according to claim 1.

3. Use of the degradable bone filling material according to claim 2 in the preparation of bone tissue organoid materials.

4. Use of the degradable bone filling material according to claim 2 in the preparation of a medicine or preparation for treating skeletal system diseases.

5. Use of the degradable bone filling material according to claim 2 in the preparation of a medicine or preparation for treating bone defects.

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