Crosslinked high polymer material, artificial periosteum and bone regeneration material as well as preparation method and application of crosslinked high polymer material and artificial periosteum and bone regeneration material

Abstract

The invention provides a cross-linked high polymer material, which is prepared by carrying out a cross-linking reaction on (A1) a conductive polymer A1 and (A2) a biocompatible polymer A2 containing at least one cross-linkable functional group, and the cross-linked high polymer material at least comprises two layers of structures: (L1) an oriented layer l1 and (L2) a porous layer l2. The cross-linked polymer material has good electrical properties and biocompatibility, and is suitable for being prepared into artificial periosteum and bone regeneration materials for treating bone defects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and relates to a cross-linked polymer material, artificial periosteum and bone regeneration material, and a preparation method and application thereof. Background Art

[0002] A bone defect is a condition where bone tissue is absent in an area of the body where bone should normally be. This can occur due to various bone diseases, such as developmental abnormalities, bone tumors, osteomyelitis, trauma, chronic joint disease, high-energy injuries, delayed healing after fractures, loosening of artificial joints, or surgical removal of the affected area during treatment. Common bone defects include tibial defects, femoral defects, and skull defects.

[0003] The existing means of treating bone defects mainly include: (1) autologous bone transplantation, which is widely used in clinical practice and has good bone conduction and bone induction effects, but the autologous source is limited and may cause damage to the source site; (2) allogeneic / xenogeneic bone transplantation, which has sufficient donor sources and good bone conductivity, but there are risks of disease transmission and immune rejection; (3) bone tissue engineering scaffolds, the scaffold materials have good biocompatibility, degradability and certain mechanical strength, but currently have problems such as poor vascular anastomosis and low osteogenesis efficiency; (4) artificial periosteum, the existing artificial periosteum lacks neural induction function, or has problems such as poor bone conduction and osteoinduction properties, poor anti-soft tissue invasion ability, and biomimetic properties. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a cross-linked polymer material, artificial periosteum and bone regeneration material, as well as preparation methods and applications thereof, which have good effects in repairing bone defects.

[0005] In a first aspect, the present invention provides a cross-linked polymer material, which is a cross-linked product of a mixture comprising the following components:

[0006] (A1) conductive polymer A1 0.1-5 wt%;

[0007] (A2) 0.5-30 wt% of a biocompatible polymer A2 containing at least one cross-linkable functional group;

[0008] (A3) solvent A3: 50-99.4 wt%;

[0009] The cross-linked polymer material comprises at least two layers:

[0010] (L1) layer l1 having orientation;

[0011] (L2) A porous layer l2.

[0012] As a preferred solution, in the cross-linked polymer material of the present invention, the density ρ1 of the oriented layer l1 is greater than the density ρ2 of the porous layer l2.

[0013] As a preferred embodiment, the preparation method of the cross-linked polymer material of the present invention comprises:

[0014] (S1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group, and then performing ice crystal recrystallization to obtain an ice crystal polymer material A3;

[0015] (S2) cross-linking the ice crystal polymer material A3 to obtain the cross-linked polymer material.

[0016] As a preferred solution, the step (S1) includes:

[0017] (s1-1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group and freezing the mixture to form an oriented layer l1;

[0018] (s1-2) applying a mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group on the surface of the oriented layer L1 to form a porous layer L2 to obtain a double-layer polymer material;

[0019] (s1-3) The double-layer polymer material is subjected to ice crystal recrystallization to obtain ice crystal polymer material A3.

[0020] As a preferred embodiment, the freezing temperature is -30°C to -200°C.

[0021] As a preferred solution, the ice crystal recrystallization is as follows: heating the frozen double-layer polymer material to a temperature for ice crystal recrystallization and performing recrystallization at this temperature, wherein the temperature for ice crystal recrystallization is -30°C to 0.5°C.

[0022] As a preferred embodiment, in the cross-linked polymer material of the present invention, the conductive polymer A1 is PEDOT:PSS.

[0023] As a preferred embodiment, the biocompatible polymer is a biometabolizable and / or degradable polymer, more preferably at least one selected from gelatin, silk fibroin, and hyaluronic acid.

[0024] As a preferred embodiment, the crosslinkable functional group can be crosslinked by light; more preferably, the light crosslinking is ultraviolet light crosslinking; most preferably, the crosslinkable functional group comprises an ethylenically unsaturated functional group.

[0025] In a second aspect, the present invention provides a method for preparing the cross-linked polymer material, comprising:

[0026] (S1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group, and then performing ice crystal recrystallization to obtain an ice crystal polymer material A3;

[0027] (S2) cross-linking the ice crystal polymer material A3 to obtain the cross-linked polymer material.

[0028] As a preferred solution, the step (S1) includes:

[0029] (s1-1) mixing the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group and freezing the mixture to form an oriented layer l1;

[0030] (s1-2) applying a mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group on the surface of the oriented layer L1 to form a porous layer L2 to obtain a double-layer polymer material;

[0031] (s1-3) The double-layer polymer material is subjected to ice crystal recrystallization to obtain ice crystal polymer material A3.

[0032] As a preferred embodiment, the freezing temperature is -30°C to -200°C.

[0033] As a preferred solution, the ice crystal recrystallization is as follows: heating the frozen double-layer polymer material to a temperature for ice crystal recrystallization and performing recrystallization at this temperature, wherein the temperature for ice crystal recrystallization is -30°C to 0.5°C.

[0034] In a third aspect, the present invention provides an artificial periosteum comprising any one of the cross-linked polymer materials of the present invention.

[0035] As a preferred embodiment, the artificial periosteum of the present invention has:

[0036] The length is 0.005-50 cm; and / or the width is 0.002-50 cm; and / or the thickness is 0.001-50 mm.

[0037] In a fourth aspect, the present invention provides a bone regeneration material comprising:

[0038] (A) any one of the artificial periosteum of the present invention; and

[0039] (B) Bioactive cells B.

[0040] As a preferred embodiment, the bioactive cells B are selected from at least one of epithelial cells, nerve cells, astrocytes, podocytes, pancreatic islet cells, endothelial cells, mesenchymal cells, stem cells, osteoblasts, myocytes, fibroblasts, hepatocytes, and chondrocytes.

[0041] As a preferred embodiment, the bioactive cells B are selected from at least one of epithelial cells, neural cells, astrocytes, endothelial cells, mesenchymal cells, stem cells, osteoblasts, fibroblasts, and chondrocytes.

[0042] In a fifth aspect, the present invention provides use of any of the above-mentioned artificial periosteum or bone regeneration materials of the present invention in the preparation of a medicine or product for treating bone defects.

[0043] Preferably, the bone defect is at least one of a tibial defect, a femoral defect, or a skull defect.

[0044] The present invention achieves the following beneficial effects: BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Infrared spectrum test chart of samples A-1 and A-3 of the present invention.

[0046] Figure 2 : Scanning electron microscope (SEM) images of samples A-1(a), A-2(b) and A-3(c) of the present invention.

[0047] Figure 3 : Schematic diagram of the in vitro swelling rate test results of samples A-1 and A-3 of the present invention.

[0048] Figure 4 : Schematic diagram of water contact angle test results of samples A-1 and A-3 of the present invention.

[0049] Figure 5 : Schematic diagram of the in vitro degradation test results of samples A-1, A-2 and A-3 of the present invention.

[0050] Figure 6 : Schematic diagram of the conductivity test results of samples A-1, A-4, A-5, A-6, and A-7 of the present invention.

[0051] Figure 7 : Conductive performance test of sample A-1 of the present invention: (a) empty sample; (b) dry sample; (c) wet sample.

[0052] Figure 8 : Schematic diagram of the absorbance results of CCK-8 test of samples A-1 and A-3 of the present invention and the blank group.

[0053] Figure 9: Schematic diagram of the cell viability results of CCK-8 test of samples A-1 and A-3 of the present invention and the blank group.

[0054] Figure 10 : Schematic diagram of the Live / Dead test results of samples A-1 and A-3 of the present invention and the blank group. Where 1, 3, and 5 represent the number of days of culture, a is the blank group, b is the extract A-3 group, and c is the extract A-1 group.

[0055] Figure 11 : Schematic diagram of the cell adhesion SEM morphology of samples A-1(b) and A-3(a) of the present invention.

[0056] Figure 12 : Schematic diagram of the results of neurotrophic factor gene expression detection after co-culture of samples A-1, A-4, A-6 of the present invention and the blank group with SCs cells.

[0057] Figure 13 : Schematic diagram of the immunofluorescence staining test results of promoting neurogenesis after co-culture of samples A-1 and A-3 blank groups and SCs cells of the present invention.

[0058] Figure 14 : Schematic diagram of the immunofluorescence detection results of the repair phenotype expression of samples A-1 and A-3 blank groups after co-culture with SCs.

[0059] Figure 15 : Schematic diagram of the expression levels of cell repair phenotype genes after co-culture of samples A-1 and A-3 blank groups with SCs cells.

[0060] Figure 16 : Schematic diagram of the immunofluorescence detection results of promoting osteogenesis after co-culture of samples A-1 and A-3 blank groups with BMSCs.

[0061] Figure 17 : Schematic diagram of the rat skull defect model.

[0062] Figure 18 : Schematic diagram of Micro-CT results of samples A-1, A-2, A-3 of the present invention and the blank group 4 weeks after the rat experiment.

[0063] Figure 19 : Schematic diagram of Micro-CT results of samples A-1, A-2, A-3 of the present invention and the blank group 8 weeks after the rat experiment.

[0064] Figure 20 : Schematic diagram of the test results of bone density, trabecular number and trabecular thickness of samples A-1, A-2, A-3 of the present invention and the blank group 4 weeks after the rat experiment.

[0065] Figure 21: Schematic diagram of the test results of bone density, trabecular number and trabecular thickness of samples A-1, A-2, A-3 of the present invention and the blank group 8 weeks after the rat experiment.

[0066] Figure 22 : Schematic diagram of HE staining results of bone tissue of samples A-1, A-2, A-3 of the present invention and the blank group 4 weeks after the rat experiment.

[0067] Figure 23 : Schematic diagram of Masson staining results of bone tissue of samples A-1, A-2, A-3 of the present invention and the blank group 4 weeks after the rat experiment.

[0068] Figure 24 : Schematic diagram of the results of immunofluorescence detection of bone tissue of samples A-1, A-2, A-3 of the present invention and the blank group 4 weeks after the rat experiment. DETAILED DESCRIPTION

[0069] In the following description, numerous specific details are provided to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these details. In other instances, certain technical features well known in the art are not described to avoid confusion with the present invention.

[0070] The following describes the implementation of the present invention in detail with reference to the definitions of terms:

[0071] Unless otherwise specified, "%" in the present invention refers to mass percentage.

[0072] I Cross-linked polymer materials

[0073] The cross-linked polymer material of the present invention is a cross-linked product of a mixture comprising the following components:

[0074] (A1) conductive polymer A1 0.1-5 wt%;

[0075] (A2) 0.5-30 wt% of a biocompatible polymer A2 containing at least one cross-linkable functional group;

[0076] (A3) Solvent: 50-99.4 wt%.

[0077] (i) Conductive polymer A1

[0078] The conductive polymer A1 of the present invention refers to any organic polymer, organic copolymer, or semiconductor polymer capable of conducting electricity, and having a certain electrical conductivity. In one embodiment, the conductive polymer A1 of the present invention is soluble in one or more solvents. In one embodiment, the conductive polymer A1 of the present invention has no / or low biotoxicity and good biocompatibility, and can be, for example, selected from at least one of polyaniline, polypyrrole, polyacetylene, polyphenylene, polyphenylene vinylene, polythiophene, and polyethylenedioxythiophene / polystyrene sulfonic acid (PEDOT:PSS).

[0079] In one embodiment, in order to obtain a cross-linked polymer material with good electrical conductivity and biocompatibility, the mass percentage of the conductive polymer A1 in the cross-linked polymer material can be 0.2-3%, 0.5-2% or 0.9-1.5%.

[0080] In one embodiment, the viscosity of the conductive polymer A1 at 20° C. may be 5-200 cp, 10-150 cp, or 30-100 cp.

[0081] (ii) a biocompatible polymer A2 containing at least one cross-linkable functional group

[0082] The "biocompatible polymers" of the present invention are polymers that are non-toxic, chemically inert, and substantially non-immunogenic in the amounts employed when used internally in a mammalian body (e.g., a human patient). In one embodiment, the "biocompatible polymers" of the present invention can be biometabolizable and / or degradable polymers.

[0083] "Biometabolizable and / or degradable polymer" refers to a polymer that can be metabolized, absorbed, degraded, dissolved or "decomposed" when exposed to body fluids. For example, body fluids can be or include water, blood, plasma, serum, serum-blood fluid and / or purulent exudate, etc. In some embodiments, the biocompatible polymer is metabolized or decomposed by dissolving in an aqueous environment, organelles, enzymatic degradation, chemical processes, etc. In one embodiment, the biocompatible polymer of the present invention can be a synthetic or natural polymer. In one embodiment, a suitable biocompatible polymer can be a synthetic polyester or polyamide or polyamino acid, such as PLA, PLGA, PHA, PBAT, or PCL. In one embodiment, a suitable biocompatible polymer can be a natural polymer, such as a protein (such as gelatin, collagen, fibrin, silk, polypeptide), a polysaccharide (such as hyaluronic acid, starch, cellulose, chitosan, xanthan gum, gellan gum, pectin, alginate, gellan gum, dextran), etc. Preferably, the "biocompatible polymer" of the present invention is selected from at least one of gelatin, silk, and hyaluronic acid.

[0084] In one embodiment, the weight average molecular weight of the "biocompatible polymer" of the present invention may be 10,000 to 50,000,000, 20,000-10,000,000, 30,000-1,000,000, 40,000-500,000, or 50,000-200,000.

[0085] The "crosslinkable functional group" of the present invention refers to a group that can form a crosslinked structure through a crosslinking reaction. The crosslinking reaction can be a physical crosslinking, chemical crosslinking, or enzymatic crosslinking reaction between the groups, preferably a chemical crosslinking. In one embodiment, the chemical crosslinking can be heat-induced crosslinking (thermal crosslinking), light-induced crosslinking (photocrosslinking), chemical reaction crosslinking, or crosslinking with a crosslinking agent. Preferably, the chemical crosslinking of the present invention is preferably photocrosslinking.

[0086] In one embodiment, the photocrosslinking of the present invention is UV crosslinking. The UV crosslinking can be carried out in the presence of a photoinitiator. In one embodiment, the UV crosslinking temperature of the present invention is -30°C to 30°C, or -10°C to 10°C, or -5°C to 0°C. In one embodiment, the UV crosslinking time of the present invention is 0.01-60 min, 0.1-30 min, 0.2-10 min, or 0.5-6 min.

[0087] The crosslinkable functional groups of the present invention can be groups inherent to the biocompatible polymer, such as amino, carboxyl, or hydroxyl groups, or can be chemically modified to impart crosslinkable functional groups to the biocompatible polymer, such as alkenyl, alkynyl, or thiol groups. In one embodiment, the "crosslinkable functional groups" of the present invention are preferably ethylenically unsaturated functional groups, such as alkenyl groups. For example, gelatin with alkenyl groups can be prepared through an amidation reaction between methacrylic acid and amino groups on gelatin.

[0088] In one embodiment, in order to obtain a cross-linked polymer material with good conductivity and biocompatibility, the mass percentage of the biocompatible polymer A2 containing at least one cross-linkable functional group in the cross-linked polymer material can be 1-20 wt%, 2-10 wt%, or 3-6 wt%.

[0089] (iii) Solvent A3

[0090] The solvent of the present invention is solely water, or a mixed solvent consisting of water and an organic solvent, wherein the content of the organic solvent in the solvent (water and organic solvent) does not exceed 10 wt%, for example, not more than 8 wt%, 5 wt%, 3 wt%, 2 wt%, or 1 wt%. The organic solvent is preferably a water-soluble organic solvent, and illustratively, the organic solvent includes, but is not limited to, water-miscible solvents such as ethanol, acetone, and dimethyl sulfoxide.

[0091] (iv) Double-layer structure

[0092] In order to better promote bone repair through biomimetic methods, the cross-linked polymer material of the present invention has a multi-layer structure, comprising at least the following two layers:

[0093] (L1) layer l1 having orientation;

[0094] (L2) A porous layer l2.

[0095] In one embodiment, the density ρ1 of the oriented layer l1 is greater than the density ρ2 of the porous layer l2. Thus, a double-layer structure with a dense outer layer and a loose inner layer can be formed.

[0096] In one embodiment, ρ1 can be 0.001-5 g / cm 3 , 0.01-1g / cm 3 , or 0.05-0.1g / cm 3 .

[0097] In one embodiment, ρ1 / ρ2 may be 1.001-10, preferably 1.5, 2, 3, 4, 5, 6, 7, 8 or 9, more preferably 3-5.

[0098] In one embodiment, the thickness of the oriented layer 11 in the cross-linked polymer material of the present invention can be 0.001-50 mm, 0.01-25 mm, 0.1-10 mm, 0.2-5 mm, 0.3-2 mm, or 0.5-1 mm. The orientation in "oriented layer 11" means that the molecules or chain segments in the polymer are fully or partially aligned in a direction parallel to the layer surface.

[0099] In one embodiment, the porous layer 12 of the cross-linked polymer material of the present invention has through pores and a porosity of 50-99.99%, 70-99%, or 90-95%. In one embodiment, the average pore size of the porous layer 12 is 10-400 μm, 50 to 300 μm, and more preferably 70 to 200 μm. In one embodiment, the porous layer 12 of the cross-linked polymer material of the present invention can have a thickness of 0.001-50 mm, 0.01-25 mm, 0.1-10 mm, 0.2-5 mm, 0.3-2 mm, or 0.5-1 mm.

[0100] (v) Preparation method

[0101] The preparation method of the cross-linked polymer material of the present invention comprises:

[0102] (S1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group, and then performing ice crystal recrystallization to obtain an ice crystal polymer material A3;

[0103] (S2) cross-linking the ice crystal polymer material A3 to obtain the cross-linked polymer material.

[0104] Preferably, the step (S1) comprises:

[0105] (s1-1) mixing the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group and freezing the mixture to form an oriented layer l1;

[0106] (s1-2) applying a mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group on the surface of the oriented layer L1 to form a porous layer L2 to obtain a double-layer polymer material;

[0107] (s1-3) The double-layer polymer material is subjected to ice crystal recrystallization to obtain ice crystal polymer material A3.

[0108] In one embodiment, the freezing in step (s1-1) causes the liquid to form ice crystals with a certain orientation. For example, the liquid may be exposed to low temperature at one end face of a template of a specific shape, causing ice crystals to extend outward from the low temperature end face. Preferably, the freezing of the present invention is vertical freezing, which means that the bottom of the liquid in a template of a specific shape (such as a vertical column) is exposed to low temperature and ice crystals are first formed near the bottom. The ice crystals extend vertically upward from the bottom until the entire liquid is formed, and the ice crystals have a vertically oriented structure.

[0109] In one embodiment, the freezing temperature in step (s1-1) is -30°C to -200°C. Exemplarily, the freezing temperature is -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -100°C, -110°C, -120°C, -130°C, -140°C, -150°C, -160°C, -170°C, -180°C, -190°C or -196°C.

[0110] In one embodiment, the mixed solution applied in step (s1-2) can be the unfrozen mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group, as described in step (s1). The "application" is preferably performed by hanging drop, where the mixed solution is dripped onto layer L1 from a height of 0.1-2.5 m, 0.5-2 m, or 1-1.6 m above the surface of layer L1.

[0111] In one embodiment, the ice crystal recrystallization in step (s1-3) is as follows: heating the frozen double-layer polymer material to a temperature for ice crystal recrystallization and performing recrystallization at this temperature, wherein the temperature T for ice crystal recrystallization satisfies T冰晶重结晶 Satisfy 0 <T 溶液的熔点 -T 冰晶重结晶 ≤30℃. For example, 0 <T 溶液的熔点 -T 冰晶重结晶 ≤20℃,0 <T 溶液的熔点 -T 冰晶重结晶 ≤15℃,0 <T 溶液的熔点 -T 冰晶重结晶 ≤10℃. 溶液的熔点 -T 冰晶重结晶 For example, 30° C., 28° C., 26° C., 25° C., 24° C., 22° C., 20° C., 18° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4.5° C., 4° C., 3.5° C., 3° C., 2.5° C., 2° C., 1.5° C., 1° C. or 0.5° C. The melting point refers to the melting point of the solution at normal pressure.

[0112] In one embodiment, the temperature T at which the ice crystals recrystallize is 冰晶重结晶 In some embodiments, the temperature of ice crystal recrystallization T 冰晶重结晶 It is -15°C to -0.5°C, -10°C to -0.5°C, for example, -30°C, -28°C, -26°C, -25°C, -24°C, -22°C, -20°C, -18°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4.5°C, -4°C, -3.5°C, -3°C, -2.5°C, -2°C, -1.5°C, -1°C or -0.5°C.

[0113] In one embodiment, the time for ice crystal recrystallization is the time maintained at the temperature of ice crystal recrystallization, such as maintaining for more than 0.5 hours, for example, maintaining for 1 to 480 hours, and for example maintaining for 1 to 240 hours, such as 1 hour, 2 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 120 hours, 180 hours, 240 hours, 360 hours or 480 hours, preferably 60-90 hours, more preferably 70-80 hours.

[0114] In one embodiment, in the process of heating the frozen double-layer polymer material to the temperature at which ice crystals recrystallize, the heating rate is 0.1°C / min to 15.0°C / min, for example, 0.1°C / min, 0.2°C / min, 0.5°C / min, 0.8°C / min, 1°C / min, 1.2°C / min, 1.5°C / min, 1.8°C / min, 2°C / min, 2.5°C / min, 2.8°C / min, 3°C / min, 3.2°C / min, 3.5°C / min, 3.8°C / min, 4°C / min, 4.2°C / min, 4.5°C / min, 4.8°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min, 10°C / min, 11°C / min, 12°C / min, 13°C / min, 14°C / min or 15°C / min.

[0115] II Artificial periosteum

[0116] The cross-linked polymer material of the present invention has excellent electrical properties and biocompatibility and can be made into or used as an artificial periosteum. Depending on the application scenario, the artificial periosteum has a length of 0.005-50 cm, a width of 0.002-50 cm, and / or a thickness of 0.001-50 mm.

[0117] In one embodiment, the length of the artificial periosteum of the present invention is 0.01-5 cm, 0.05-2 cm, 0.1-1 cm, or 0.2-0.8 cm, or 1-50 cm, 2-30 cm, 5-20 cm, or 1-15 cm.

[0118] In one embodiment, the width of the artificial periosteum of the present invention is 0.005-5 cm, 0.05-2 cm, 0.1-1 cm, or 0.2-0.8 cm, or 1-50 cm, 2-30 cm, 5-20 cm, or 1-15 cm.

[0119] In one embodiment, the thickness of the artificial periosteum of the present invention is 0.01-20 mm, 0.1-10 mm, 0.2-5 mm, or 0.5-2 mm.

[0120] III Bone regeneration materials

[0121] The bone regeneration material of the present invention comprises:

[0122] (A) artificial periosteum A of the present invention; and

[0123] (B) Bioactive cells B.

[0124] The type and content of the bioactive cells B can be appropriately selected according to the purpose. Specifically, the cell type can be at least one of epithelial cells, neural cells, astrocytes, podocytes, pancreatic islet cells, endothelial cells, mesenchymal cells, stem cells, osteoblasts, myocytes, fibroblasts, hepatocytes, and chondrocytes.

[0125] Preferably, the bioactive cells B are at least one selected from epithelial cells, nerve cells, astrocytes, endothelial cells, mesenchymal cells, stem cells, osteoblasts, fibroblasts, and chondrocytes.

[0126] More preferably, the bioactive cells B of the present invention are at least one selected from the group consisting of neural cells, mesenchymal cells, stem cells and fibroblasts.

[0127] When cells need to be seeded onto the artificial periosteum of the present invention, conventional methods can be used. For example, cells in the form of a suspension are seeded onto the artificial periosteum of the present invention placed in a suitable container. In one example, when the artificial periosteum has a volume of 5 mm × 5 mm × 1 mm, the seeding amount of any one cell type can be 10 3 -10 6 or 10 4 -5×10 5 indivual.

[0128] The bone regeneration material of the present invention can be subjected to 2D or 3D cell culture as required to prepare a material with better biomimetic properties.

[0129] For therapeutic applications, cells derived from the host or allogeneic cells can be used.

[0130] IV Applications

[0131] The artificial periosteum or bone regeneration material of the present invention can be used to prepare drugs or products for treating bone defects. It can also be used as a medicament for bone regeneration therapy in combination with cell transplants or osteoinductive agents. In one embodiment, the bone defects of the present invention include fractures, bone trauma, and bone-related conditions following bone trauma surgery, joint repair surgery, orthopedic surgery, bone chemotherapy / radiotherapy, or dental surgery. Fractures include all types of microscopic and macroscopic fractures. Preferably, the bone defect is at least one of a tibial defect, a femoral defect, or a cranial defect.

[0132] The present invention is described in more detail below with reference to specific embodiments.

[0133] Experimental raw materials

[0134] Porcine skin gelatin A: type A, reagent grade, Sigma-Aldrich, China.

[0135] Methacrylic anhydride: Sigma-Aldrich, USA.

[0136] Phosphate buffered saline (PBS): Hyclone, USA.

[0137] Photoinitiator (2959): 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, 410896; Sigma-Aldrich.

[0138] Conductive polymer (PEDOT:PSS): purchased from Sigma, model: 739332-100G.

[0139] RNA extraction kit: Cell / Tissue Total RNA Isolation Kit V2, RC112; Nanjing Novozymes Co., Ltd.

[0140] Reverse transcription kit: III RT SuperMix for qPCR (+gDNA wiper), R323; Nanjing Novozymes Co., Ltd.

[0141] Taq Pro Universal SYBR qPCR MasterMix: Q712; Nanjing Novozymes Co., Ltd.

[0142] Anti-RUNX2 antibody [2B9] (ab76956); abcam.

[0143] Recombinant Anti-S100 beta antibody [EP1576Y]-Astrocyte Marker (ab52642); abcam.

[0144] Recombinant Anti-Collagen I [EPR24331-53] antibody (ab270993); abcam.

[0145] Monoclonal anti-neurofilament 200 (phosphate and phosphate): N0142; Sigma-Aldrich.

[0146] Test equipment

[0147] Fourier transform infrared spectrometer: VERTEX 70V vacuum microscope Fourier transform infrared spectrometer.

[0148] Scanning electron microscope: ZEISS Sigma 300, Carl Zeiss AG, Jena, Germany.

[0149] Water contact angle tester: OCA50, German dataphysics contact angle meter.

[0150] Conductivity test: Uni-T multimeter UT61E.

[0151] Microplate reader: Microplate spectrophotometer, EPOCH TAKE3.

[0152] Laser confocal microscope: AXR, Nikon Corporation, Japan.

[0153] Micro-CT: AX-2000, Aoying Detection Technology (Ningbo) Co., Ltd.

[0154] Fluorescence microscope: upright fluorescence microscope, Ni-U, Nikon.

[0155] Unless otherwise specified, the statistical analysis of the results in the examples of the present invention was performed using SPSS 22.0 for variance test and t-test. In the figures, ns indicates p>0.05, * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.

[0156] Synthesis Example 1 Gelatin GelMA containing alkenyl groups and synthesis

[0157] 10 g of pigskin gelatin (Type A, Bloom, weight-average molecular weight Mw = 90 kDa) was added to 100 mL of phosphate buffered saline (DPBS) and dissolved by stirring at 50°C. 5 mL of methacrylic anhydride (MA) was added dropwise to the gelatin solution using a syringe pump to obtain a concentration of approximately 5% (v / v). The mixture was placed on a magnetic hot plate and heated and stirred at 50°C for 2 h to obtain GelMA. The product was diluted 5 times with DPBS to terminate the reaction and dialyzed in a 50°C oven for 7 days, with the water changed every 12 h during the dialysis process. The product was then freeze-dried and stored in a refrigerator.

[0158] Synthesis Example 2 Synthesis of GelMA+PEDOT:PSS Double-layer Cross-linked Polymer Artificial Periosteum A-1

[0159] GelMA freeze-dried powder (the amount of GelMA added is 40 mg / mL) and photoinitiator 2959 (mass ratio of photoinitiator to mass ratio of GelMA = 1:20) were added to 100 mL of a 1.1 wt% PEDOT:PSS aqueous dispersion and stirred to dissolve to obtain a mixed solution. A portion of the mixed solution was injected into a sheet glass mold using a syringe and vertically frozen to form a layer l1 with an oriented structure. The layer with the oriented structure was placed on a low-temperature cold stage in a liquid nitrogen environment. The remaining mixed solution was dripped from a height of about 1.5 m above the cold stage to form a porous layer l2 on the surface of the layer with the oriented structure to obtain a double-layer polymer material. The double-layer polymer material was placed in a low-temperature alcohol pump and heated to -4°C for ice crystal recrystallization for 72 hours to obtain an ice crystal polymer material.

[0160] The ice crystal polymer material was placed under -4 °C and exposed to ultraviolet light (wavelength 365 nm, light intensity 1.8-2 W / cm 2 ) were irradiated and cross-linked for 5 minutes to obtain artificial periosteum A-1, which was about 1 mm thick, with the thickness of layer 11:layer 12 being approximately 3:7.

[0161] Synthesis Example 3 Synthesis of GelMA+PEDOT:PSS Monolayer Cross-linked Polymer Artificial Periosteum A-2

[0162] Different from Synthesis Example 2, there is no step in Synthesis Example 6 of forming a porous layer l2 on the surface of the layer l1 with an oriented structure by using a hanging drop mixture. The layer l1 with an oriented structure is directly recrystallized by ice crystals. In addition, referring to the method of Synthesis Example 2, a GelMA+PEDOT:PSS single-layer cross-linked polymer artificial periosteum A-2 with a thickness of about 1 mm is prepared.

[0163] Synthesis Example 4 Synthesis of GelMA Monolayer Cross-linked Polymer Artificial Periosteum A-3

[0164] Unlike Synthesis Example 3, PEDOT:PSS was not added. 100 mL of an aqueous solution of GelMA freeze-dried powder (concentration 40 mg / mL) and photoinitiator 2959 (mass amount of photoinitiator:mass amount of GelMA = 1:20) were stirred and dissolved to obtain a mixed solution. In addition, a GelMA single-layer cross-linked polymer artificial periosteum A-3 with a thickness of approximately 1 mm was prepared by referring to the method of Synthesis Example 3.

[0165] Synthesis Example 5-8 Synthesis of GelMA+PEDOT:PSS Double-layer Cross-linked Polymer Artificial Periosteum A-4, A-5, A-6, and A-7

[0166] Different from Synthesis Example 2, the ice crystal recrystallization time of Synthesis Examples 5-8 was changed from 72h to 0h, 12h, 24h and 48h. In addition, GelMA+PEDOT:PSS double-layer cross-linked polymer artificial periosteum A-4, A-5, A-6 and A-7 were prepared with reference to the method of Synthesis Example 2.

[0167] Example 1 Characterization of artificial periosteum

[0168] 1. Infrared spectroscopy

[0169] A-1 and A-3 were subjected to FITR test, and the results were as follows Figure 1 As shown in the figure, it can be seen that the double bonds on the gelatin underwent a cross-linking reaction, and the cross-linked polymer material was successfully synthesized.

[0170] 2. SEM morphology observation

[0171] The morphologies of A-1, A-2 and A-3 were observed under SEM along the direction perpendicular to the orientation of the l1 layer. Figure 2 As shown in Figure 2, A-1 forms a structure with dense orientation in the outer layer and loose and porous inner layer.

[0172] 3. Swelling rate

[0173] A-1 and A-3 were made into 5mm×5mm×1mm samples, immersed in phosphate buffered saline (PBS), and then placed in a 37℃ shaker with a speed of 40rpm. The volume changes of the samples were measured at different times and the swelling ratio was calculated. The results are shown in Figure 2. Figure 3 The equilibrium swelling ratio of A-1 is about 200%, which shows good stability.

[0174] 4. Water contact angle

[0175] The water contact angles of A-1 and A-3 were tested in a water contact angle tester. The results are as follows: Figure 4 As shown, it can be seen that A-1 has good hydrophilicity.

[0176] 5. In vitro degradation rate

[0177] Nine 5mm×5mm×1mm samples of A-1, A-2, and A-3 were prepared and placed in 27 PBS solutions containing 2μg / ml type II collagenase. One aliquot of each of A-1, A-2, and A-3 was removed at 0h, 12h, 24h, 48h, 72h, 5d, 7d, 14d, and 21d, rinsed three times with distilled water, and freeze-dried. The sample weight was measured and the degradation rate was calculated. The results are shown in the figure. Figure 5 As shown, it can be seen that A-1 has good biodegradability.

[0178] 6. Conductivity and electrical conductivity

[0179] A-1, A-4, A-5, A-6, and A-7 were made into 5mm×5mm×1mm samples. The resistance of each sample was tested with a multimeter and the conductivity was calculated. The results are as follows: Figure 6 It can be seen that the conductivity of the sample increased after ice crystal recrystallization, especially after 72h of ice crystal recrystallization, the conductivity of A-1 reached as high as 0.680S / m.

[0180] A-1 was made into two samples of 5mm×5mm×1mm. One of them was untreated and became a wet sample. The other sample was freeze-dried to obtain a dry sample. The conductivity of each sample was tested in a circuit connected to a small light bulb. The results are as follows: Figure 7 As shown in Figure 2, it can be seen that the wet sample has better electrical conductivity than the dry sample.

[0181] Example 2 Biocompatibility of artificial periosteum

[0182] 1. Cell Source

[0183] (1) Schwann cells (SCs)

[0184] After 2-day-old SD rats were killed, their sciatic nerves and brachial plexus nerves were isolated and dissected. After mincing, 1% collagenase and 0.125% trypsin were added and digested with magnetic stirring for 20 minutes. The resulting tissue suspension was added to DMEM / F12 medium (containing 10% fetal bovine serum and 10ng / mL heregulin-β1) and placed in an incubator at 37°C and 5% CO2 for 2 days. The above medium was then poured out and washed with PBS. Neurobasal B27 purified culture medium was then added, and the cultured Schwann cells were cultured every 3 days. Subsequently, DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% double antibody was used for culture using traditional cell culture methods.

[0185] (2) PC12 cells

[0186] Purchased from American Type Culture Collection (Rockville, MD, USA).

[0187] (3) Dorsal root ganglion

[0188] The lower spine of a one-day-old SD rat was taken and split in the middle. Dark circular tissue between the spinous processes could be observed under a stereomicroscope. The sample was carefully taken with microtweezers and temporarily stored in a low-temperature (4°C) culture medium, and then DRG-specific culture medium was added.

[0189] (4) Bone marrow mesenchymal stem cells (BMSCs)

[0190] 2-week-old SD rats were selected and killed by cervical dislocation. They were then immersed in 75% alcohol for 15 minutes for disinfection. The bilateral femurs and tibias were aseptically dissected in a clean bench and placed in pre-cooled sterile PBS. The ends of the femoral shaft were cut off to fully expose the bone marrow cavity. α-MEM culture medium was extracted with a 1 ml syringe to flush the bone marrow cavity. The bone marrow cavity was fully flushed 4-5 times until the medullary cavity turned white. The flushing fluid was collected, pipetted and mixed, and the excess impurities were filtered through a sterile cell sieve. The cells were centrifuged at 1200 rpm for 5 minutes to collect the precipitated cells. The cells were resuspended in α-MEM complete medium and seeded in a 10 cm culture dish. The cells were placed in a cell culture incubator for routine culture overnight, and then the culture medium was replaced to remove cells that were not attached to the surface. The culture medium was continued, and the cell culture medium was replaced every three days. When the cells grew to 90% fusion, the culture medium was discarded, the cells were rinsed with PBS 2-3 times, trypsin was added for digestion, and then the cells were neutralized by adding complete culture medium. The cells were passaged at a ratio of 1:3 and routine culture passage was continued. P3-P5 generations were used for the experiments in the following invention.

[0191] 2. Cytotoxicity

[0192] (1) Preparation of artificial periosteum extract

[0193] 1 g of cobalt-60-sterilized A-1 and A-3 samples were placed in 15 ml centrifuge tubes, and 15 ml of α-MEM complete culture medium was added. After incubation in a 37°C CO2 incubator for 24 hours, the remaining undissolved substances were removed to obtain culture media containing periosteum materials as extracts A-1 and A-3.

[0194] (2) CCK-8 test

[0195] BMSCs cells in the logarithmic phase were collected, the concentration of the cell suspension was adjusted, and 100 μL was added to each well of the 96-well plate to adjust the density of the cells to be tested to 5000 / well. After the cells adhered, the culture medium was replaced with 100 μL of the co-culture medium shown in Table 1 below (wherein the co-culture medium of the blank group was blank culture medium, and the blank culture medium was α-MEM complete culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin mixture (100X) double antibody). After co-culture for 1d, 3d, and 5d, the culture medium in the 96-well plate was aspirated and replaced with 100 μL of culture medium containing 10% CCK-8 solution. The sample was then placed in an incubator in the dark and incubated for 2 hours. The absorbance at 450 nm was measured on a microplate reader, and the cell survival rate was calculated, where cell survival rate = (OD value of the experimental group - OD value of the culture medium containing 10% CCK8 solution) / (OD value of the blank group - OD value of the culture medium containing 10% CCK8 solution) × 100%. The results are shown in Figure 2. Figure 8 、 Figure 9 As shown in Table 1, all results are the average values of three parallel experiments.

[0196] Table 1

[0197]

[0198] (3) Live / Dead testing

[0199] A 14 mm glass slide was placed in a 24-well plate. The BMSC cells in the culture dish were digested with trypsin and resuspended in complete culture medium. After counting the cells, 3 × 10 5 BMSCs were cultured in blank culture medium (α-MEM complete culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin mixture (100X) and double-antibody), extract A-1 and extract A-3, respectively. The cells were removed on day 1, day 3, and day 5, and the culture medium was discarded. The cells were washed three times with PBS, and 300 μl of dead-alive staining reagent was added to each well. The cells were placed back in the cell culture incubator and incubated for 15 minutes, then removed, the staining solution was discarded, and 300 μl of PBS was added to each well. The cells were then observed and photographed under a laser confocal microscope. The results are shown in Figure 2. Figure 10 shown.

[0200] It can be seen from the CCK-8 and Live / Dead tests that the artificial periosteum material of the present invention has low cytotoxicity and good biocompatibility.

[0201] Example 3 Artificial periosteum in vitro cell experiment

[0202] 1. Cell Adhesion

[0203] SCs cells were cultured at 3×10 5 Cells were seeded in 24-well plates containing 5mm×5mm×1mmA-1 or A-3 samples in a sterilized 5mm×5mm material, so that the cells were seeded on the artificial periosteum material. The culture medium in the well plate was DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% double antibody. The cells were cultured in a 37°C CO2 incubator for 3 days (the blank group contained only the above-mentioned complete medium without artificial periosteum material). After washing with PBS 3 times and fixing with 2.5% glutaraldehyde, the cells were washed with PBS, and then dehydrated with 50%, 70%, 85%, 95%, and 100% ethanol in a gradient manner. The cells were dried using a critical point dryer, sprayed with gold powder, and the morphology was observed by SEM. The results are as follows Figure 11 As shown, it can be seen that A-1 has better cell adhesion performance.

[0204] 2. Promote neurogenesis

[0205] (1) Expression of neurotrophic factor genes in co-cultured SCs

[0206] After A-1, A-4 and A-6 were co-cultured with SCs cells according to the above steps, the expression of neurotrophic factor genes NGF, BDNF and GDNF in each sample was detected. The results are as follows: Figure 12As shown in Table 2, the values on the ordinate represent the expression folds of the corresponding genes (based on the blank group), and the results are the averages of three parallel experiments. It can be seen that A-1 has a better effect in promoting the expression of neurotrophic factors.

[0207] Table 2

[0208] sample NGF BDNF GDNF Blank group 1.000 1.000 1.000 A-4 1.465 1.609 2.380 A-6 1.481 2.201 5.285 A-1 1.732 2.897 7.073

[0209] (2) Cell repair phenotypic gene expression

[0210] SCs cells were cultured at a rate of 3 × 10 5 Each well was seeded with 5mm×5mm sterilized materials in a 6-well plate. The culture medium in the well plate was DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% double antibody. 5mm×5mm×1mm glass slide samples of A-1 or A-3 were added respectively, and the expression of Olig1 and cjun was detected after culturing in a 37°C CO2 incubator for 3 days (the blank group contained only the above-mentioned complete culture medium (i.e., the blank culture medium in the table) without artificial periosteum material). Sample RNA was collected using the RNA extraction adsorption column kit from Novozymes, and then the RNA was reverse transcribed into cDNA using a reverse transcription kit, and then gene expression was detected using a qPCR instrument. The results are as follows Figure 15 As shown in Table 3, Figure 15 The repair phenotype expression folds are based on the blank group, and the control and experimental groups have the same gene expression folds. The results are the average of three parallel experiments. This shows that A-1 can enhance the expression of the repair phenotype in cells and promote nerve repair.

[0211] Table 3

[0212]

[0213] (3) Fluorescent immunostaining test

[0214] A-1 and A-3 were made into 5 mm × 5 mm × 1 mm samples and placed in 24-well plates after disinfection. The blank group did not add artificial periosteum samples, washed 3 times with PBS, and immersed in culture medium to inoculate SCs cells or PC12 cells. Each cell was 2 × 10 5 After 3 days of culture, the cells were fixed with 4% paraformaldehyde.

[0215] Subsequently, each sample was treated with S100 antibody ( Figure 13 ) or C-jun( Figure 14) was diluted in PBS at a ratio of 1:200 and incubated with primary antibody at 4°C overnight. The next day, after washing with PBS three times, Alexa-488 was diluted in PBS at a ratio of 1:200 and incubated at room temperature for 1 hour. After that, the cells were washed with PBS three times, each for 5 minutes. Then, the cells were stained with Phalloidin-iFluor 594 reagent (ab176757) for 30 minutes, washed with PBS three times, each for 5 minutes. After DAPI staining, the cells were mounted and observed with a laser confocal microscope. The results are shown in the figure. Figure 13 and Figure 14 shown. Figure 13 and Figure 14 Phalloidin indicates cytoskeleton staining, C-jun or S100-β indicates antibody expression of repair phenotypes, Dapi indicates cell nuclei, and Merge indicates a merged layer. This indicates that A-1 has a significant effect in promoting neurogenesis.

[0216] 3. Promote bone formation

[0217] Add 2 mL of rat dorsal root ganglion cell culture medium to a six-well plate and co-culture with 5 mm × 5 mm × 1 mm samples of A-1, A-2, or A-3 (blank group without artificial periosteum). Each of the above co-culture solutions or the blank group was mixed with α-MEM complete medium at a volume ratio of 1:1 and then co-cultured with rat BMSCs. Co-culture with BMSCs was performed in the same manner as in step 3 above for 7 days. A-1, A-2, A-3, and the blank group formed one experiment, for a total of 3 experiments.

[0218] (1) Cell immunofluorescence staining

[0219] Take a group of experimental samples and wash them 3 times with PBS, discard PBS, add an appropriate amount of 4% paraformaldehyde, and fix them at room temperature for 15 minutes; discard paraformaldehyde, wash them 3 times with PBS, add Triton X-100, let them stand at room temperature for 15 minutes, and punch the cells; discard Triton X-100, wash them 3 times with PBS, add the target primary antibody (RUNX-2, Col 1), and incubate them at 4°C overnight; wash them 3 times with PBS, add the corresponding fluorescent-labeled secondary antibody, and incubate them at 37°C for 1 hour; wash them 3 times with PBS, add DAPI staining solution; wash them 3 times with PBS, and observe them with a laser confocal microscope. The results are as follows Figure 16 As shown in Figure 1, where col 1 represents the expression of type I collagen antibody and merge is a composite image of different layers. It can be seen that A-1 has a good effect in promoting bone formation.

[0220] (2) Expression of osteogenic differentiation-related proteins

[0221] Protein immunoblotting (WB) was used to detect the expression levels of osteogenic differentiation-related proteins (ALP, COL-1, OCN, OPN, RUNX2).

[0222] Take a set of experimental samples and add RIPA protein lysis buffer to each sample. Pipette and mix well. Shake on ice at 4°C for 30 minutes. Collect the lysate and transfer it to a centrifuge tube. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Collect the supernatant. Prepare a standard protein concentration sample and determine the protein concentration using the BCA assay. Prepare a separating gel and a stacking gel. Denature the protein at 95°C-100°C for 5 minutes before loading the sample for electrophoresis. After electrophoresis, transfer the sample to the membrane using a semi-dry method. Block with blocking buffer, incubate with primary and secondary antibodies, and analyze using a gel imaging system.

[0223] (3) Expression of genes related to osteogenic differentiation

[0224] qRT-PCR was used to detect the expression levels of osteogenic differentiation-related genes (ALP, COL-1, OCN, OPN, and RUNX2).

[0225] RNA was extracted from a set of experimental samples. After extraction, the RNA concentration and purity were determined by UV absorption, followed by reverse transcription into sample cDNA. qRT-PCR of the housekeeping gene (GAPDH) was performed with a serial dilution standard and the test samples. DNA template was prepared for the serial dilution standard curve, and qRT-PCR of the test gene was performed on the test samples.

[0226] Example 4 Animal Experiments on Artificial Periosteum

[0227] 1. Animal Modeling and Sample Administration

[0228] Thirty-two 8-week-old male Sprague-Dawley (SD) rats with an average weight of approximately 300 g were selected. After intraperitoneal injection of sodium pentobarbital (3.5 mg / 100 g) for anesthesia, the rat skull was prepared and disinfected. In a sterile environment, a midline incision was made on the skull, and the scalp tissue was exposed by blunt separation. After locating the midline of the skull, two 5 mm defects were made at the midpoints of the midline. An electric drill was used to slowly drill holes in the two centers. After slowly drilling through the skull with a 5 mm diameter drill, free bone fragments were removed with microtweezers. The rat skull was then infiltrated and rinsed with ice-cold saline while drilling. The rat skull defect model was established as follows: Figure 17 shown.

[0229] The mice were divided into four groups (blank group, A-1 group, A-2 group, and A-3 group), with 8 mice in each group. 5mm×5mm×1mm irradiated and sterilized A-1, A-2, or A-3 samples were attached to the bone defect surface of the corresponding group of rats (the blank group did not have artificial periosteum); then the fascia and skin were closed layer by layer, the incision was disinfected, and the mice were returned to the cage and continued to be raised after waking up.

[0230] 2. Micro-CT testing

[0231] Four SD rats in each group were killed at 4 and 8 weeks after surgery. The skulls of the rats were isolated, wrapped with gauze soaked in saline, and stored on ice. Micro-CT scanning and three-dimensional reconstruction were performed immediately to analyze and evaluate the bone regeneration. The results are shown in the figure. Figure 18 and Figure 19 shown.

[0232] The AX-2000 mirco ct machine was used to quantitatively analyze the relevant data. A 5×5 mm region of interest was selected and the bone mineral density (BMD) BV / TV (bone volume / total volume), trabecular number (Tb.N), and trabecular thickness (Tb.Th) within the region of interest were analyzed. The results are shown in the figure below. Figure 20 and Figure 21 , and shown in Tables 4 and 5, where all results are the average values of 3 sets of parallel experiments.

[0233] Table 4

[0234] Sample (4 weeks) BV / TV Tb.N Tb.Th Blank group 5.225 0.419 0.1 A-3 9.019 0.687 0.131 A-2 11.525 0.778 0.14 A-1 18.422 1.096 0.182

[0235] Table 5

[0236] Sample (8 weeks) BV / TV Tb.N Tb.Th Blank group 10.168 0.602 0.214 A-3 15.036 0.605 0.247 A-2 18.105 0.644 0.295 A-1 26.101 1.410 0.324

[0237] 3. Bone Repair Histology

[0238] Preparation of paraffin sections: After Micro-CT scanning, skulls were removed 4 weeks after surgery and immediately immersed in 4% paraformaldehyde for 48 hours. The fixed specimens were removed and decalcified in 10% EDTA decalcification solution, with the decalcification solution changed every two days for four weeks. After decalcification, the specimens were rinsed under running water overnight to remove residual EDTA before subsequent embedding. The rinsed tissue specimens were removed and dehydrated in 70% ethanol for 5 minutes, 80% ethanol for 2-4 hours, 95% ethanol for 2-4 hours, 95% ethanol for 2-4 hours, 100% ethanol for 2-4 hours, and 100% ethanol for 2-4 hours. The dehydrated specimens were removed and transparentized in an ethanol / xylene (1:1) mixture for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes. The transparent tissue blocks were removed, immersed in wax, and embedded. The embedded wax blocks were stored at -20°C.

[0239] HE staining: immerse the paraffin sections of the four groups in xylene I and xylene II for 30 minutes each; then immerse the sections in pure alcohol I for 2 minutes, pure alcohol II for 2 minutes, 95% alcohol I for 2 minutes, 95% alcohol II for 2 minutes, 80% alcohol for 2 minutes, 70% alcohol for 2 minutes, and rinse with distilled water; immerse the sections in hematoxylin stain for 2 minutes, rinse with running water, immerse in 1% hydrochloric acid alcohol solution for 3 seconds, rinse with running water for 5 minutes until fully blue, and examine the cell nucleus for appropriateness; immerse the sections in alcoholic eosin solution for 15 seconds, and then immerse in 85%, 95%, 95%, 100%, and 100% alcohol for 1 minute each; immerse the sections in xylene I and xylene II for 1 minute each; take out the transparent sections, wipe off the xylene outside the specimen, use neutral gum to seal the sections, and observe the seals under a fluorescence microscope. The results are as follows Figure 22 shown.

[0240] Masson trichrome staining: Paraffin sections from the four groups were immersed in xylene I and xylene II for 30 minutes each. The sections were then immersed in pure alcohol I for 2 minutes, pure alcohol II for 2 minutes, 95% alcohol I for 2 minutes, 95% alcohol II for 2 minutes, 80% alcohol for 2 minutes, and 70% alcohol for 2 minutes, followed by a rinse in distilled water. Stain with Weigert's iron hematoxylin solution for 3 minutes. Differentiate with hydrochloric acid and alcohol for 15 seconds (if the iron hematoxylin stain is too dark, the differentiation time can be increased appropriately). Wash with distilled water three times for 1 minute each. Bluing was performed with Masson's bluing solution, followed by a wash with water and a 1-minute wash in distilled water. Stain with Ponceau fuchsin solution for 7-8 seconds, followed by a 1-minute wash with 0.2% weak acid. Wash with phosphomolybdic acid solution for 2 minutes, followed by a 1-minute wash with 0.2% weak acid. Stain with aniline blue solution for 1 minute 30 seconds (staining time can be adjusted based on tissue density), followed by a 1-minute wash with 0.2% weak acid. Dehydrate quickly with 95% ethanol for 2-3 seconds, dehydrate with anhydrous ethanol 3 times, 5-10 seconds each time. Transparent with xylene 3 times, 1-2 minutes each time, seal the slides and observe under a fluorescence microscope. Figure 23 shown.

[0241] Immunofluorescence staining: ① Make a note on the slide containing the paraffin sections, insert it into the slide rack and place it in an oven at 60-65℃ for 1-1.5 hours. ② Dewax and hydrate the slide containing the paraffin sections:

[0242] Xylene 1, 10 min → Xylene 2, 10 min → Xylene 3, 5 min → Anhydrous ethanol 1, 5 min → Anhydrous ethanol 2, 5 min → 95% ethanol, 5 min → 85% ethanol, 5 min - Wash with distilled water. ③ Place the slides containing paraffin sections in a repair box filled with EDTA antigen retrieval buffer (pH 9.0) in a microwave oven for antigen retrieval. After boiling over medium heat, turn off the power and wait for 10 minutes before boiling over medium-low heat. During this process, prevent excessive evaporation of the buffer and do not let the slides dry. After cooling naturally, place the slides in PBS (pH 7.4) and shake on a decolorizing shaker to wash three times, each time for 5 minutes. ④ After BSA blocking, use a tissue pen to draw a circle around the tissue (to prevent the antibody from flowing away) and add 3% BSA in the circle to evenly cover the tissue. Block at room temperature for 30 minutes. ⑤ Primary Antibody Incubation: Gently shake off the blocking solution and apply 1:200 dilution of NF200 and RUNX-2 primary antibodies to the sections. Place the sections flat in a humidified chamber and incubate overnight at 4°C. ⑥ Secondary Antibody Incubation: Wash the slides three times in PBS (pH 7.4) for 5 minutes each. Allow the sections to dry slightly before adding a secondary antibody of the same species as the primary antibody to the circle and covering the tissue. Incubate for 2 hours at room temperature, protected from light. ⑦ DAPI Nuclei Counterstaining: Wash the slides three times in PBS (pH 7.4) for 5 minutes each. Allow the sections to dry slightly before adding a DAPI staining solution to the circle and incubate for 5 minutes at room temperature, protected from light.

[0243] ⑧Wash the slides in PBS (pH 7.4) three times for 5 minutes each time. After the sections are slightly dried, seal them with anti-fluorescence quenching sealant and observe them under a laser confocal microscope. Figure 24 shown.

[0244] It can be seen that A-1 has a good effect in promoting bone tissue repair.

[0245] In summary, the artificial periosteum of the present invention has a good effect in promoting bone tissue and nerve repair.

[0246] The invention is suitable for preparing medicines or products for treating bone defects.

[0247] In the above embodiments, all technical solutions that fall within the scope of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, several improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A cross-linked polymer material, which is a cross-linked product of a mixture comprising the following components: (A1) conductive polymer A1 0.1-5 wt%; (A2) 0.5-30 wt% of a biocompatible polymer A2 containing at least one cross-linkable functional group; (A3) solvent A3: 50-99.4 wt%; The cross-linked polymer material comprises at least two layers: (L1) layer l1 having orientation; (L2) A porous layer l2. 2 . The cross-linked polymer material according to claim 1 , wherein a density ρ1 of the oriented layer 11 is greater than a density ρ2 of the porous layer 12 .

3. The cross-linked polymer material according to claim 1, wherein the cross-linked polymer material is prepared by a method comprising the following steps: (S1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group, and then performing ice crystal recrystallization to obtain an ice crystal polymer material A3; (S2) subjecting the ice crystal polymer material A3 to a cross-linking reaction to obtain the cross-linked polymer material; Preferably, the step (S1) comprises: (s1-1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group and freezing the mixture to form an oriented layer l1; (s1-2) applying a mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group on the surface of the oriented layer L1 to form a porous layer L2, thereby obtaining a double-layer polymer material; (s1-3) performing ice crystal recrystallization on the double-layer polymer material to obtain ice crystal polymer material A3; More preferably, the freezing temperature is -30°C to -200°C, and the ice crystal recrystallization is: heating the frozen double-layer polymer material to the temperature of ice crystal recrystallization and recrystallizing it at this temperature, and the ice crystal recrystallization temperature is -30°C to 0.5°C.

4. The cross-linked polymer material according to any one of claims 1 to 3, wherein the conductive polymer A1 is PEDOT:PSS; Preferably, the biocompatible polymer is a biometabolizable and / or degradable polymer, more preferably, at least one selected from gelatin, silk fibroin, and hyaluronic acid; Preferably, the crosslinkable functional group can be crosslinked by light; more preferably, the light crosslinking is ultraviolet light crosslinking; most preferably, the crosslinkable functional group comprises an ethylenically unsaturated functional group.

5. A method for preparing the cross-linked polymer material according to any one of claims 1 to 4, comprising: (S1) mixing a conductive polymer A1 and a biocompatible polymer A2 containing at least one cross-linkable functional group, and then performing ice crystal recrystallization to obtain an ice crystal polymer material A3; (S2) subjecting the ice crystal polymer material A3 to a cross-linking reaction to obtain the cross-linked polymer material; Preferably, the step (S1) comprises: (s1-1) mixing the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group and freezing the mixture to form an oriented layer l1; (s1-2) applying a mixture of the conductive polymer A1 and the biocompatible polymer A2 containing at least one cross-linkable functional group on the surface of the oriented layer L1 to form a porous layer L2 to obtain a double-layer polymer material; (s1-3) performing ice crystal recrystallization on the double-layer polymer material to obtain ice crystal polymer material A3; Preferably, the freezing temperature is -30°C to -200°C, and the ice crystal recrystallization is: heating the frozen double-layer polymer material to the temperature of ice crystal recrystallization and performing recrystallization at this temperature, and the ice crystal recrystallization temperature is -30°C to 0.5°C.

6. An artificial periosteum comprising the cross-linked polymer material according to any one of claims 1 to 4.

7. The artificial periosteum according to claim 6, wherein the artificial periosteum comprises: The length is 0.005-50 cm; and / or the width is 0.002-50 cm; and / or the thickness is 0.001-50 mm.

8. A bone regeneration material comprising: (A) the artificial periosteum according to any one of claims 6 to 7; and (B) Bioactive cells B.

9. The bone regeneration material according to claim 8, wherein the bioactive cells B are at least one selected from the group consisting of epithelial cells, nerve cells, astrocytes, podocytes, pancreatic islet cells, endothelial cells, mesenchymal cells, stem cells, osteoblasts, myocytes, fibroblasts, hepatocytes, and chondrocytes; Preferably, the bioactive cells B are at least one selected from epithelial cells, nerve cells, astrocytes, endothelial cells, mesenchymal cells, stem cells, osteoblasts, fibroblasts, and chondrocytes.

10. Use of the artificial periosteum according to any one of claims 6 to 7 or the bone regeneration material according to any one of claims 8 to 9 in the preparation of a medicament or product for treating bone defects, wherein the bone defect is at least one of a tibial defect, a femoral defect, or a skull defect.

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