Controllable degradation magnesium-based bionic biphasic stent as well as preparation method and application thereof

Abstract

The invention provides a controllable degradation magnesium-based bionic biphasic stent as well as a preparation method and application thereof. The stent comprises a porous magnesium stent, hydrogel and a ceramic coating, the porous magnesium scaffold comprises a cartilage phase scaffold and a subchondral bone phase scaffold which are arranged from top to bottom; hydrogel is arranged on pores and the outer surface of the cartilage phase scaffold, and a ceramic coating is arranged on the surface of the subchondral bone phase scaffold. From the bionic angle, the three-dimensional communicated porous magnesium stent with gradient porosity is designed by simulating the structure, mechanics and other characteristics of joint tissues (upper cartilage and lower subchondral bone); meanwhile, according to the characteristics of joint tissue components, biological properties and the like, hydrogel (an upper cartilage phase) is filled in the stent, and a bioactive ceramic coating (a lower subchondral bone phase) is prepared on the surface of the stent, so that the degradation rate of the biphasic magnesium stent is regulated and controlled, and the cytotoxicity is reduced; meanwhile, the biocompatibility and the biological activity are improved, and endogenous cartilage-subchondral bone integrated repair is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and relates to a controllable degradable magnesium-based biomimetic biphasic scaffold as well as a preparation method and application thereof, in particular to the design and preparation of a controllable degradable magnesium-based biomimetic biphasic scaffold and the application of the same to the repair of endogenous osteochondral integration injury. BACKGROUND

[0002] With the aggravation of social population aging and the increase of young and middle-aged trauma, the incidence of articular cartilage injury is rising rapidly. The number of chondrocytes in cartilage tissue is small, and the activity is low, and there is no vascular tissue, so once the cartilage is damaged, it is almost impossible to self-repair. If not treated in time, cartilage injury often expands inward, causing cartilage-subchondral bone injury. Cartilage and subchondral bone have different structures, mechanics and biological properties, and it is extremely difficult to repair osteochondral integration injury. At present, the methods for treating osteochondral injury in clinical practice mainly include microfracture, autologous bone transplantation and allogeneic bone transplantation, etc., but these methods have many limitations and are difficult to achieve ideal repair effect. Therefore, artificial osteochondral repair materials have gradually become a research hotspot.

[0003] Due to the different structures, mechanics, compositions and biological properties of cartilage and subchondral bone, it is often difficult to achieve ideal osteochondral integration repair effect using a single material and structure. Therefore, many researchers prepare multi-phase composite scaffolds through step-by-step methods such as layered freeze-drying or layered salting-out, respectively endowing the cartilage phase and the subchondral bone phase of the scaffold with specific biological properties for osteochondral integration repair. Patent CN106178126A discloses a repair osteochondral biphasic porous composite scaffold and a preparation method thereof, which respectively prepares a lower layer osteogenic phase (the composition contains nanometer hydroxyapatite, silk fibroin and chitosan) and an upper layer chondrogenic phase (the composition contains silk fibroin and chitosan) through a step-by-step freeze-drying method. The biphasic scaffold has good osteochondral repair ability, but the bonding strength between the phases of the scaffold prepared by the step-by-step freeze-drying method is often poor, which cannot guarantee the mechanical strength of the whole biphasic scaffold, which may greatly limit its application in clinical practice. In addition, in recent years, tissue engineering scaffolds and stem cell therapy have made significant development and progress, but they still face some limitations, such as low survival rate of exogenous cells, poor migration, uncontrolled differentiation of stem cells, and extremely short half-life of growth factors. Therefore, developing a new type of osteochondral repair scaffold to ensure the stability of the scaffold structure while integrally promoting the repair of endogenous cartilage and subchondral bone has become another big problem that needs to be solved.

[0004] Metallic magnesium and magnesium alloys have attracted much attention due to their biodegradability, good mechanical properties, excellent biosafety and bioactivity. The latest research shows that magnesium ions generated by magnesium alloy degradation have excellent properties of promoting blood vessels and bone formation, promoting stem cell chondrogenesis, inhibiting chondrocyte calcification and protecting chondrocytes. These characteristics of magnesium alloy make it have unique advantages as a bone-cartilage integrated repair material. However, there is no report on the application of magnesium alloy to cartilage repair and bone-cartilage integrated repair scaffold materials at home and abroad. This may be because the standard electrode potential of magnesium is low, the degradation rate in the body is too fast, and it is difficult to maintain sufficient mechanical strength before the function of tissue repair and regeneration is restored, which is a major challenge for the application of magnesium-based bone-cartilage integrated repair. SUMMARY

[0005] The present application provides a controllable degradation magnesium-based biomimetic dual-phase scaffold, its preparation method and application, aiming at the problems of different structures, mechanical and biological properties of cartilage and subchondral bone, and the too fast degradation rate of magnesium metal. From the perspective of biomimicry, the gradient porosity is designed by simulating the structure and mechanical properties of joint tissue (upper cartilage and lower subchondral bone). A magnesium alloy three-dimensional interconnected gradient scaffold with high precision, high density and mechanical properties meeting the requirements of clinical use is prepared by one-step 3D printing technology, solving the problem of poor bonding strength between cartilage phase scaffold and subchondral bone phase scaffold. Then, according to the characteristics of joint tissue composition and biological properties, bioactive hydrogel is filled in the cartilage phase of the scaffold, and ceramic coating is prepared in the subchondral bone phase, to control the Mg 2+ degradation rate, reduce cytotoxicity and improve the biosafety of magnesium alloy scaffold. At the same time, the dual-phase scaffold is endowed with better bioactivity, which can significantly induce the regeneration of cartilage and subchondral bone without the need of loading exogenous cells, drugs, growth factors, etc., and can be applied to bone-cartilage integrated injury repair.

[0006] The object of the present application can be achieved by the following scheme:

[0007] In the first aspect, the present application provides a controllable degradation magnesium-based biomimetic dual-phase scaffold, which comprises a porous magnesium scaffold, a hydrogel and a ceramic coating. The porous magnesium scaffold comprises a cartilage phase scaffold and a subchondral bone phase scaffold arranged from top to bottom. The cartilage phase scaffold is provided with a hydrogel on the pores and the outer surface, and the surface of the subchondral bone phase scaffold is provided with a ceramic coating.

[0008] The present application utilizes 3D printing technology to process and form in one step. The cartilage phase scaffold and the subchondral bone phase scaffold are connected through a gradient transition scaffold to form a biomimetic gradient porous magnesium scaffold.

[0009] As an embodiment of the present application, the porosity of the cartilage phase scaffold is 86%-98%, and the compressive modulus is 40-150 MPa. If the porosity is too low and the compressive modulus is too large, the mechanical properties of the cartilage phase scaffold will be too strong.

[0010] As an embodiment of the present application, the porosity of the subchondral bone phase scaffold is 60%-85%, and the compressive modulus is 350-1000 MPa. If the porosity is too high and the compressive modulus is too small, the mechanical properties of the cartilage phase scaffold will be too weak.

[0011] According to the principle of mechanical simulation, the present application designs a cartilage phase scaffold with weak mechanical properties and a subchondral bone phase scaffold with strong mechanical properties, which is beneficial to subsequent bone and cartilage repair. At the same time, the cartilage phase scaffold with a relatively high porosity produces a relatively high Mg 2+ release concentration, which is beneficial to promoting chondrogenic differentiation; the subchondral bone phase scaffold with a relatively low porosity produces a relatively low Mg 2+ release concentration, which is beneficial to promoting osteogenic differentiation, but both concentrations are far lower than the Mg 2+ release concentration of the bare magnesium scaffold, otherwise it will produce high cytotoxicity.

[0012] As an embodiment of the present application, the hydrogel includes at least one of sodium alginate hydrogel, amino acid-based supramolecular hydrogel, methacrylated gelatin hydrogel, and chitosan hydrogel.

[0013] As an embodiment of the present application, the thickness of the hydrogel on the outer surface of the cartilage phase scaffold is 1-100 μm.

[0014] As an embodiment of the present application, the ceramic coating includes one of calcium phosphate coating, HA coating, and Sr-P coating. The thickness of the ceramic coating is 21-80 μm. If the coating is too thin (the reaction time for preparing the coating is short), the coating cannot completely cover the surface of the magnesium alloy scaffold, and the protection effect is poor.

[0015] As an embodiment of the present application, the hydrogel is doped with active substances. The extracellular matrix of the cartilage is simulated to further improve the biological activity of the cartilage phase scaffold.

[0016] Further, the active substances include at least one of polypeptides and nanofibers.

[0017] In some preferred embodiments, the active substances include phenylalanine-based nanofibers and protein N-terminal peptides.

[0018] In a second aspect, the present application provides a preparation method of a controllable degradable magnesium-based bionic biphasic scaffold, including the following steps:

[0019] S1, preparing a porous magnesium scaffold by 3D printing technology under a protective atmosphere;

[0020] S2, chemically depositing and / or hydrothermally converting the subchondral bone scaffold in the porous magnesium scaffold to form a ceramic coating;

[0021] S2, immersing the cartilage scaffold in a hydrogel solution to solidify and form a cartilage phase.

[0022] As an embodiment of the present application, in step S1, the oxygen content of the protective atmosphere is less than 10 ppm.

[0023] In step S1 of the present application, a modeling software is used to design a gradient scaffold structure in which the cartilage scaffold and the subchondral bone scaffold are connected in a gradient transition, and the sliced structure is imported into a 3D printing device.

[0024] As an embodiment of the present application, in step S1, the 3D printing method includes any one of powder selective melting method, electric arc method, and paste extrusion deposition method; and the material used for 3D printing is any one of pure magnesium and magnesium alloy. In some embodiments, the magnesium alloy is Mg-Nd-Zn-Zr alloy.

[0025] As an embodiment of the present application, in step S2, the subchondral bone scaffold in the porous magnesium scaffold is chemically deposited to form a calcium phosphate coating;

[0026] Or, the subchondral bone scaffold in the porous magnesium scaffold is chemically deposited and hydrothermally converted to form a HA coating; the hydrothermal conversion uses a mixed solution of 200-250 mM sodium phosphate salt and 200-250 mM calcium ethylenediaminetetraacetate;

[0027] Or, the subchondral bone scaffold in the porous magnesium scaffold is chemically deposited and hydrothermally converted to form a Sr-P coating; the hydrothermal conversion uses a mixed solution of 200-250 mM sodium phosphate salt, 200-250 mM calcium ethylenediaminetetraacetate, 20-25 mM sodium ethylenediaminetetraacetate, and 20-25 mM strontium nitrate.

[0028] Further, the chemical deposition uses a mixed solution of 0.25-0.4 mol / L calcium phosphate salt and 1-1.6 mol / L sodium nitrate.

[0029] Further, the chemical deposition time is 4-12 hours; the hydrothermal conversion temperature is 80-100℃, and the time is 6-12h.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1. The application is designed from the perspective of bionics, simulating the structure and mechanics of joint tissue (upper cartilage, lower subchondral bone) and designing gradient porosity, and through 3D printing technology, a magnesium-based three-dimensional interconnected gradient scaffold with high precision, high density and mechanical properties meeting the requirements of clinical use is prepared by one-step method, effectively solving the problem of poor interface bonding force of the dual-phase scaffold, and the magnesium alloy scaffold matches the mechanical properties and structural characteristics of human cartilage and bone.

[0032] 2. The application prepares an active ceramic coating on the lower layer of the magnesium alloy gradient scaffold by chemical deposition or chemical deposition and hydrothermal conversion method, and a subchondral bone phase is prepared. The coating is dense, which not only effectively improves the corrosion resistance of the magnesium alloy scaffold, regulates the degradation rate of Mg 2+ , but also improves the bioactivity of the magnesium alloy, and can realize endogenous bone regeneration.

[0033] 3. The application fills the upper layer of the magnesium-based metal gradient scaffold with hydrogel, and a cartilage phase is prepared. On the one hand, the hydrogel forms an interpenetrating network structure, simulates the extracellular matrix of cartilage, is conducive to cell adhesion, migration and growth, and promotes cell proliferation and chondrogenic differentiation; on the other hand, the hydrogel can regulate the degradation rate of the cartilage phase magnesium-based scaffold, reduce the Mg 2+ release concentration and cytotoxicity, improve the biological safety of the scaffold, so as to ensure the realization of cell proliferation and chondrogenic differentiation effect, and then realize endogenous cartilage regeneration. In addition, active substances such as polypeptides or nanofibers can be added to the hydrogel to further improve the cell proliferation effect.

[0034] 4. The application has wide application range and can be applied to bone and cartilage damage in various physiological and pathological environments (osteoarthritis, etc.), and has the advantages of not needing to load exogenous cells, drugs, growth factors, etc., and is suitable for all magnesium and magnesium alloy materials and any human body part cartilage, bone and cartilage integrated damage repair plant tissue regeneration and repair implant devices. BRIEF DESCRIPTION OF DRAWINGS

[0035] Other features, objects and advantages of the application will become more apparent through reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0036] Figure 1 The design drawing of the magnesium alloy bionic gradient scaffold in Example 1 and the Micro-CT scanning result of the 3D printed magnesium alloy bionic gradient scaffold;

[0037] Figure 2 The macroscopic photograph of the controllable degradation magnesium-based bionic dual-phase scaffold after coating modification in Example 1;

[0038] Figure 3 The digital photograph of the subchondral bone phase scaffold after coating modification in Example 2;

[0039] Figure 4 Digital photo of the scaffold filled with hydrogel for the cartilage phase in Example 2;

[0040] Figure 5 Stress-strain curve of the scaffold for the cartilage phase and subchondral bone phase in Example 2;

[0041] Figure 6 Cell proliferation viability test results of the magnesium-based scaffold leaching liquor in Example 4, a) for the cartilage phase, b) for the subchondral bone phase;

[0042] Figure 7 Photos of the surgery implantation of the magnesium-based biomimetic gradient scaffold in the New Zealand white rabbit osteochondral defect model in Example 5;

[0043] Figure 8 Mg2+ release concentration of the hydrogel-wrapped magnesium alloy scaffold and the hydrogel-filled magnesium alloy scaffold in Comparative Example 1 after in-vitro soaking for 3d; 2+

[0044] Figure 9 Surface live cell density of the hydrogel-wrapped magnesium alloy scaffold and the hydrogel-filled magnesium alloy scaffold in Comparative Example 1;

[0045] Figure 10 Photos of the immunofluorescence staining of hBMSCs after 3d culture on the surface of the hydrogel without nanofiber (a) and the hydrogel with nanofiber (b) in Example 6;

[0046] Figure 11 Alcian blue staining results of hBMSCs after 21d culture of the blank control and the hydrogel-filled magnesium alloy scaffold sample in Comparative Example 1; wherein a is the blank control sample, and b is the hydrogel-filled magnesium alloy scaffold sample;

[0047] Figure 12 Structural schematic diagram of the controllable degradation magnesium-based biomimetic biphasic scaffold. DETAILED DESCRIPTION

[0048] The present application will be described in detail below with reference to the drawings and specific examples. The following examples are implemented on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are provided, which will help those skilled in the art to further understand the present application. It should be pointed out that the protection scope of the present application is not limited to the following examples, and several adjustments and improvements made on the premise of the concept of the present application all belong to the protection scope of the present application.

[0049] Example 1

[0050] ​A Mg alloy gradient scaffold with 95% porosity and 120 MPa compressive modulus in the upper layer and 75% porosity and 750 MPa compressive modulus in the lower layer is prepared by laser selective melting 3D printing technology in an argon atmosphere with an oxygen content of less than 10 ppm, as shown in Figure 1 . After electrochemical polishing and fluorination of the Mg alloy gradient scaffold, the lower layer of the fluorinated Mg-Nd-Zn-Zr Mg alloy gradient scaffold (i.e., the porous Mg scaffold) is immersed in a mixed solution of calcium phosphate (0.28 mol / L) and sodium nitrate (1.2 mol / L) for chemical deposition reaction for 4 hours to prepare a calcium phosphate coating (21 μm in thickness) on the surface of the scaffold, thereby preparing the subchondral bone phase. Then, the upper layer of the Mg alloy gradient scaffold is immersed in a sodium alginate (1.5 mg / mL) and amino acid-based supramolecular (1 mg / mL) composite hydrogel, which is doped with bioactive Sr ions (also as a crosslinking agent), to fill the hydrogel (5 μm in thickness) on the upper layer of the Mg alloy gradient scaffold, thereby preparing the cartilage phase. Through the above steps, a controllable degradable Mg alloy biphase scaffold is prepared, as shown in Figure 2 . The structural schematic diagram is shown in Figure 12 .

[0051] Example 2

[0052] A Mg-Nd-Zn-Zr Mg alloy scaffold with 92% porosity and 90 MPa compressive modulus in the cartilage layer and 73% porosity and 700 MPa compressive modulus in the subchondral bone layer is prepared by laser selective melting 3D printing technology in an argon atmosphere with an oxygen content of less than 10 ppm for mechanical test and research. After electrochemical polishing and fluorination of the Mg alloy scaffold, the fluorinated Mg alloy subchondral bone layer scaffold is immersed in a mixed solution of calcium phosphate (0.3 mol / L) and sodium nitrate (1.6 mol / L) for chemical deposition reaction for 4 hours to prepare a brushite coating (21 μm in thickness) on the surface of the scaffold, and then the brushite coating is immersed in a mixed solution of sodium phosphate (200 mM) and calcium ethylenediaminetetraacetate (200 mM) for hydrothermal conversion reaction for 6 hours in a 90°C reaction kettle to prepare a HA coating (as shown in Figure 3 ) on the surface of the scaffold. The Mg alloy cartilage layer scaffold is immersed in a methacrylated gelatin hydrogel, and the hydrogel is filled on the Mg alloy cartilage layer scaffold after photocrosslinking (10 μm in thickness on the outer surface), as shown in Figure 4 . Through the above steps, a controllable degradable Mg-based bionic scaffold is prepared.

[0053] The mechanical properties of the cartilage phase and the subchondral bone phase of the scaffold are tested, respectively. The biphase scaffold matches the mechanical properties of human cartilage and bone, respectively, as shown in Figure 5 .

[0054] Example 3

[0055] Mg-Nd-Zn-Zr magnesium alloy scaffolds with 90% porosity and 70 MPa compressive modulus for cartilage layer and 68% porosity and 450 MPa compressive modulus for subchondral bone layer were prepared by arc 3D printing technology in argon atmosphere with oxygen content less than 10 ppm for in vitro degradation experiment. The magnesium alloy scaffolds were electrochemically polished and then fluorinated. The fluorinated magnesium alloy scaffolds for subchondral bone layer were immersed in a mixed solution of calcium phosphate (0.25 mol / L) and sodium nitrate (1 mol / L) to prepare a brushite coating (surface thickness of 50 μm) on the surface of the scaffolds by chemical deposition reaction for 8 hours. The magnesium alloy scaffolds for cartilage layer were immersed in a methacrylated hyaluronic acid photocured hydrogel, and the hydrogel was filled on the magnesium alloy scaffolds for cartilage layer after photocrosslinking (outer surface thickness of 10 μm). The controllable degradation magnesium-based bionic scaffolds were prepared by the above steps.

[0056] The scaffolds before and after coating modification were immersed in cell culture medium (aMEM) to evaluate the degradation behavior of the scaffolds. The results showed that the coating and filled hydrogel on the surface of the scaffolds could significantly improve the corrosion resistance of the scaffolds; the degradation rate of the bare scaffolds was ≥1.0 mm / year, and the degradation rate of the scaffolds after coating modification was ≤0.2 mm / year.

[0057] Example 4

[0058] Mg-Nd-Zn-Zr magnesium alloy scaffolds with 93% porosity and 100 MPa compressive modulus for cartilage layer and 70% porosity and 500 MPa compressive modulus for subchondral bone layer were prepared by paste extrusion deposition printing technology in argon atmosphere with oxygen content less than 10 ppm for in vitro cell experiment. The magnesium alloy scaffolds were electrochemically polished and then fluorinated. The fluorinated magnesium alloy scaffolds for subchondral bone layer were immersed in a mixed solution of calcium phosphate (0.4 mol / L) and sodium nitrate (1 mol / L) to prepare a calcium phosphate coating on the surface of the scaffolds by chemical deposition reaction for 10 hours, and then the calcium phosphate coating was immersed in a mixed solution of sodium phosphate (250 mM), calcium ethylenediaminetetraacetate (200 mM), sodium ethylenediaminetetraacetate (20 mM) and strontium nitrate (20 mM) for hydrothermal conversion reaction for 10 hours in a 90°C reaction kettle to prepare a Sr-P coating (surface thickness of 65 μm) on the surface of the scaffolds. The magnesium alloy scaffolds for cartilage layer were immersed in a composite hydrogel of chitosan (10 mg / mL) and amino acid-based supramolecules coupled with N-terminal peptides of connective proteins (2 mg / mL) to fill the hydrogel on the scaffolds (outer surface thickness of 30 μm). The controllable degradation magnesium-based bionic scaffolds were prepared by the above steps.

[0059] The cell activity of the cartilage phase scaffold and the subchondral bone phase scaffold prepared in this embodiment was evaluated (cck8 detection) using human bone marrow mesenchymal stem cells (hBMSCs) Figure 6 ), and the results showed that the cartilage phase scaffold and the subchondral bone phase scaffold had good cell compatibility and could promote cell proliferation. Compared with the bare magnesium scaffold, the cytotoxicity was improved from level 2 to level 0; in addition, the cartilage phase scaffold promoted the chondrogenic differentiation of hBMSCs, and the subchondral bone phase scaffold promoted the osteogenic differentiation of hBMSCs.

[0060] Example 5

[0061] Under an argon atmosphere with an oxygen content of less than 10 ppm, a Mg-Nd-Zn-Zr magnesium-based biomimetic gradient scaffold was prepared by laser selective melting 3D printing technology, in which the porosity of the upper cartilage layer was 94%, and the compressive modulus was 110 MPa, and the porosity of the subchondral bone layer was 80%, and the compressive modulus was 750 MPa. After electrochemical polishing of the magnesium-based gradient scaffold, fluorination treatment was performed. Then, the lower layer of the fluorinated magnesium alloy gradient scaffold was soaked in a mixed solution of calcium phosphate salt (0.3 mol / L) and sodium nitrate (1 mol / L), and a calcium phosphate coating was prepared on the surface of the scaffold by chemical deposition reaction for 12 h. Then, the calcium phosphate coating was soaked in a mixed solution of sodium phosphate salt (250 mM) and calcium ethylenediaminetetraacetate (250 mM), and a hydrothermal conversion reaction was performed in a 90°C reaction kettle for 12 hours, to prepare a HA coating (surface thickness of 80 μm) on the surface of the lower layer of the magnesium alloy gradient scaffold. Then, the upper layer of the magnesium alloy gradient scaffold was soaked in a methyl methacrylate gelatin hydrogel, and after photo-crosslinking, the hydrogel was filled on the upper layer of the magnesium alloy gradient scaffold (outer surface thickness of 10 μm). Through the above steps, a controllable degradation magnesium-based biomimetic biphasic scaffold was prepared.

[0062] The magnesium-based biomimetic biphasic scaffold was implanted into a rabbit articular osteochondral defect model, as shown in Figure 7 , to evaluate the in vivo biological activity of the biphasic scaffold. The results showed that the biphasic scaffold could promote osteogenesis and chondrogenesis, and the osteochondral defect was basically repaired within 2-3 months, and the scaffold was basically degraded.

[0063] Example 6

[0064] The preparation method of this embodiment is basically the same as that of Example 5, except that phenylalanine-based molecules (1 mg / mL) were dissolved in the methyl methacrylate gelatin hydrogel, and phenylalanine-based nanofibers were introduced into the hydrogel framework. Figure 10 The immunofluorescence staining photographs of hBMSCs cultured on the surface of the hydrogel without nanofibers (a) and the surface of the hydrogel containing nanofibers (b) for 3 days showed that the introduction of phenylalanine-based nanofibers promoted cell adhesion and proliferation, and had better cell compatibility and cell proliferation activity.

[0065] Comparative Example 1

[0066] A magnesium-based bionic gradient scaffold with a porosity of 94% and a compressive modulus of 110 MPa in the upper cartilage layer and a porosity of 80% and a compressive modulus of 750 MPa in the lower subchondral bone layer was prepared by a laser selective melting 3D printing technology in an argon atmosphere with an oxygen content of less than 10 ppm. The magnesium-based gradient scaffold was electrochemically polished and then fluorinated. The lower layer of the fluorinated magnesium alloy gradient scaffold was immersed in a mixed solution of calcium phosphate (0.3 mol / L) and sodium nitrate (1 mol / L) to prepare a calcium phosphate coating on the surface of the scaffold by a chemical deposition reaction for 12 h. Then, the calcium phosphate coating was immersed in a mixed solution of sodium phosphate (250 mM) and calcium ethylenediaminetetraacetate (250 mM) to prepare a HA coating (a surface thickness of 80 μm) on the surface of the lower layer of the magnesium alloy gradient scaffold in a reaction kettle at 90°C for 12 h. After the methacrylated gelatin hydrogel was photo-crosslinked, the hydrogel (a thickness of 10 μm) was connected to the magnesium alloy gradient scaffold to obtain a magnesium alloy gradient scaffold sample wrapped with the hydrogel on the outer layer.

[0067] The in vitro degradation experiment results show that the magnesium alloy scaffold sample of Comparative Example 1 wrapped with the hydrogel releases Mg 2+ at a higher concentration than the magnesium alloy scaffold sample of Example 5 filled with the hydrogel Figure 8 , indicating that the hydrogel wrapped on the outer layer has a poor protective effect on the magnesium scaffold. The two scaffolds were co-cultured with cells, and the cytotoxicity of the scaffolds was evaluated by cell live / dead staining. The results prove that the cytotoxicity of the magnesium alloy scaffold sample of Example 5 filled with the hydrogel is significantly lower than that of the magnesium scaffold sample of Comparative Example 1 wrapped with the hydrogel Figure 9 , significantly improving the cell proliferation activity, while the magnesium scaffold sample of Comparative Example 1 wrapped with the hydrogel has a high cytotoxicity, resulting in poor cell proliferation and chondrogenic differentiation.

[0068] Figure 11 a is the alcian blue staining result of the blank control hBMSCs cultured for 21 d, Figure 11 b is the alcian blue staining result of hBMSCs co-cultured with the magnesium alloy scaffold sample of Example 5 filled with the hydrogel for 21 d. The alcian blue staining dye is proteoglycan in the cells, and a high expression represents chondrogenic differentiation of the cells. It can be seen that the magnesium alloy scaffold sample of Example 5 filled with the hydrogel can better promote chondrogenic differentiation than the blank control.

[0069] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the above specific embodiments, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. A controllable degradable magnesium-based biomimetic biphase scaffold, characterized in that, The scaffold comprises a porous magnesium scaffold, hydrogel, and a ceramic coating; the porous magnesium scaffold comprises a cartilage phase scaffold and a subchondral bone phase scaffold arranged from top to bottom; the pores and outer surface of the cartilage phase scaffold are coated with hydrogel, and the surface of the subchondral bone phase scaffold is coated with a ceramic coating.

2. The controllable degradable magnesium-based biomimetic biphase scaffold according to claim 1, characterized in that, The porosity of the cartilage phase scaffold is 86%-98%, and the compressive modulus is 40MPa-150 MPa; the porosity of the subchondral bone phase scaffold is 60%-85%, and the compressive modulus is 350MPa-1000 MPa.

3. The controllable degradable magnesium-based biomimetic biphase scaffold according to claim 1, characterized in that, The hydrogel includes at least one of sodium alginate hydrogel, amino acid-based supramolecular hydrogel, methacrylated gelatin hydrogel, and chitosan hydrogel; the thickness of the hydrogel on the outer surface of the cartilage scaffold is 1-100 μm.

4. The controllable degradable magnesium-based biomimetic biphase scaffold according to claim 1, characterized in that, The ceramic coating includes one of calcium phosphate coating, HA coating, and Sr-P coating; the thickness of the ceramic coating is 21-80 μm.

5. The controllable degradable magnesium-based biomimetic biphase scaffold according to claim 1, characterized in that, The hydrogel is incorporating active substances; the active substances include at least one of polypeptides and nanofibers.

6. A method for preparing a controllable degradable magnesium-based biomimetic biphase scaffold as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Under a protective atmosphere, porous magnesium scaffolds are prepared using 3D printing technology; S2. Chemical deposition and / or hydrothermal conversion of the subchondral bone scaffold in the porous magnesium scaffold to form a ceramic coating; S2. Immerse the cartilage phase scaffold in the hydrogel solution and solidify it to form the cartilage phase.

7. The preparation method according to claim 6, characterized in that, In step S1, the oxygen content of the protective atmosphere is less than 10 ppm; the 3D printing technology includes any one of the following: powder selective melting, electric arc method, and paste extrusion deposition method; the material used for 3D printing is any one of pure magnesium or magnesium alloy.

8. The preparation method according to claim 6, characterized in that, In step S2, the subchondral bone scaffold in the porous magnesium scaffold is chemically deposited to form a calcium phosphate coating. Alternatively, the subchondral bone scaffold in the porous magnesium scaffold can be chemically deposited and hydrothermally converted to form an HA coating; The hydrothermal conversion uses a mixed solution of 200-250 mM sodium phosphate and 200-250 mM calcium ethylenediaminetetraacetate; Alternatively, the subchondral bone scaffold in the porous magnesium scaffold can be chemically deposited and hydrothermally converted to form an Sr-P coating; the hydrothermal conversion uses a mixed solution of 200-250 mM sodium phosphate, 200-250 mM calcium ethylenediaminetetraacetate, 20-25 mM sodium ethylenediaminetetraacetate and 20-25 mM strontium nitrate.

9. The preparation method according to claim 8, characterized in that, Chemical deposition uses a mixed solution of 0.25-0.4 mol / L calcium phosphate and 1-1.6 mol / L sodium nitrate.

10. The preparation method according to claim 8, characterized in that, The chemical deposition time is 4-12 hours; the hydrothermal conversion temperature is 80-100℃ and the time is 6-12 hours.

Images