Osteochondral repair scaffold and preparation method thereof

Abstract

The invention discloses an osteochondral repair scaffold and a preparation method thereof.The osteochondral repair scaffold comprises a subchondral bone layer, a calcified cartilage layer and a cartilage layer which are sequentially connected, and the calcified cartilage layer comprises an upper calcified cartilage layer, a middle calcified cartilage layer and a lower calcified cartilage layer; according to the osteochondral repair scaffold and the preparation method thereof, the layered structure is adopted to simulate the physiological cartilage of the human body, and through the layering and gradient porosity design of the calcified cartilage layer, the flexibility of the scaffold is improved; the use performance of the scaffold is improved, and it can be ensured that the scaffold can rapidly recruit autoblood of a patient after being implanted into an osteochondral defect part, so that mesenchymal stem cells are guided to be differentiated into cartilage cells in a cartilage layer, and cartilage tissue generation is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an osteochondral repair scaffold and a preparation method thereof. Background Art

[0002] In the clinical treatment of articular cartilage injury, microfracture surgery and 3D bionic scaffolds are the current main technical means. As a conventional therapy for small and medium-sized (≤4 cm²) localized cartilage defects, microfracture surgery releases mesenchymal stem cells in bone marrow blood through drilling hard bone, promotes the formation of fibrous blood clots and differentiates them into fibrous cartilage tissue to achieve defect repair. However, the fibrous cartilage generated by this method is significantly inferior to natural hyaline cartilage in terms of mechanical properties, biological functions and long-term stability. Moreover, microfracture surgery mainly focuses on the repair of simple cartilage, ignoring the repair of the calcified layer and subchondral bone injuries, resulting in poor integration efficiency at the bone-cartilage interface, which in turn affects tissue maturation and long-term stability.

[0003] Aiming at the above defects, 3D bionic scaffolds have become a research hotspot, aiming to simulate the layered structure of natural cartilage, including the surface layer, the middle layer, the deep radial layer and the calcified cartilage layer. Among them, the calcified cartilage layer is the key interface between cartilage and bone, contains mineralized matrix, and helps chondrocyte anchoring and differentiation. The calcified cartilage layer plays a key role in mechanical conduction and cartilage-bone integration. However, existing osteochondral repair scaffolds and simple cartilage repair scaffolds are difficult to achieve an osteochondral scaffold with a gradient mineralized calcified cartilage layer.

[0004] Therefore, introducing a gradient mineralized structure in the design of osteochondral repair scaffolds can not only improve the mechanical properties of the scaffolds, but also better simulate the biological and mechanical properties of natural osteochondral tissue, promote the integration of new cartilage and host bone, and avoid postoperative cartilage shedding or repair failure, which has important clinical application value. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides an osteochondral repair scaffold with mechanical adaptability, biological activity and layered integration ability and a preparation method thereof.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions.

[0007] The present application provides an osteochondral repair scaffold, comprising a subchondral bone layer, a calcified cartilage layer and a cartilage layer connected in sequence; The subchondral bone layer is internally provided with through pores and comprises a bioactive ceramic; The calcified cartilage layer is internally provided with gradient through pores and comprises a bioactive ceramic; The cartilage layer is in a porous sponge shape and includes at least one of medicinal gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester short fibers loaded with magnetic nanoparticles; Among them, the calcified cartilage layer includes an upper calcified cartilage layer, a middle calcified cartilage layer, and a lower calcified cartilage layer. The upper calcified cartilage layer and the middle calcified cartilage layer are inlaid with porous gels. The porous gels include at least one of PVA, bioactive ceramics, medicinal gelatin, hyaluronic acid, hyaluronate, or porous fragmented polyester short fibers.

[0008] Further defined, for the above-mentioned osteochondral repair scaffold, among them, the bioactive ceramics in the subchondral bone layer and the calcified cartilage layer include at least one of hydroxyapatite and α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass.

[0009] Further defined, for the above-mentioned osteochondral repair scaffold, among the bioactive ceramics in the subchondral bone layer, the ratio of hydroxyapatite to at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass is 50-70:30-50; And / or, among the bioactive ceramics in the calcified cartilage layer, the ratio of hydroxyapatite to at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass is 60-80:20-40.

[0010] Further defined, for the above-mentioned osteochondral repair scaffold, among them, the through pores of the bioactive ceramics in the calcified cartilage layer are distributed in a gradient; Among them, from the subchondral bone layer to the side of the cartilage layer, for the through pores of the bioactive ceramics in the calcified cartilage layer, the porosity increases from 20% to 90%.

[0011] Further defined, for the above-mentioned osteochondral repair scaffold, among them, the porosity of the upper calcified cartilage layer is specifically set to 60% - 90%, the porosity of the middle calcified cartilage layer is specifically set to 35% - 60%, and the porosity of the lower calcified cartilage layer is specifically set to 20% - 35%.

[0012] Further defined, for the above-mentioned osteochondral repair scaffold, among them, the cartilage layer further includes one or more of repair cells, drug carriers, bioactive substances, and antibacterial components.

[0013] This application provides a preparation method for the osteochondral repair scaffold described in any one of the above, including: Preparing printing slurry; Using 3D bioprinting technology to prepare a green body scaffold; Performing drying treatment and sintering treatment on the green body scaffold to form a scaffold main body; Wherein, a subchondral bone layer is formed on the support body, or a subchondral bone layer and a calcified cartilage layer are formed on the support body.

[0014] It is further defined that the above-mentioned preparation method further comprises: During the printing process, the inter-layer angle parameters and printing height parameters can be adjusted in real time.

[0015] It is further defined that the above-mentioned preparation method further comprises: preparing a precursor solution; introducing the precursor solution into the silicone soft mold; After the calcified cartilage upper layer and the calcified cartilage middle layer of the calcified cartilage layer are immersed in the precursor solution under negative pressure, they are frozen and subjected to a first vacuum freeze-drying treatment; After the first vacuum freeze-drying treatment, the calcified cartilage layer is subjected to a gradient alcohol dehydration treatment, a cross-linking agent cross-linking treatment, and then a second vacuum freeze-drying treatment is performed to generate a stable mosaic porous gel.

[0016] It is further defined that the above-mentioned preparation method further comprises: The cartilage layer is formed integrally on the calcified cartilage layer by using a negative pressure impregnation process; Alternatively, a cartilage layer is prepared on the calcified cartilage layer using 3D printing technology.

[0017] The present invention has at least the following beneficial effects: 1. The structure and material design of the osteochondral repair scaffold, which includes the subchondral bone layer, calcified cartilage layer and cartilage layer, simulates the physiological cartilage of the human body to the greatest extent. The layered design of the calcified cartilage layer improves the flexibility of the scaffold and improves the performance of the scaffold. At the same time, the layered structure combined with the gradient porosity of the calcified cartilage layer can ensure that after the scaffold is implanted in the osteochondral defect, it can quickly recruit the patient's autologous blood, so that the autologous blood enters the calcified cartilage layer and the cartilage layer along the scaffold, thereby guiding the bone marrow mesenchymal stem cells to differentiate into chondrocytes in the cartilage layer and promote the formation of cartilage tissue. Since the patient's autologous bone remodeling rate is faster than the repair rate of cartilage tissue, the subchondral bone layer will first combine with the patient's own hard bone tissue, thereby achieving the fixation of the scaffold at the defect site, providing a stable environment for the differentiation of chondrocytes and the formation of cartilage tissue on the cartilage layer, and realizing the integrated repair of osteochondral defects.

[0018] 2. The calcified cartilage layer is set as a bioactive ceramic with through pores and a gradient distribution, including the upper calcified cartilage layer, the middle calcified cartilage layer, and the lower calcified cartilage layer. The upper calcified cartilage layer and the middle calcified cartilage layer are inlaid with porous gels (in a porous sponge-like shape). Due to the gradient design of the porosity of the calcified cartilage layer ranging from 90% to 20%, when the osteochondral repair scaffold is implanted at the osteochondral defect site, this structure can smoothly "drain" the bone marrow mesenchymal stem cells in the subchondral bone defect site to the calcified cartilage layer and the cartilage layer under capillary action. In particular, the porous gels inlaid in the calcified cartilage layer will accelerate this process; 3. The lower calcified cartilage layer of the calcified cartilage layer has a relatively low porosity of 20% - 35%. The porosity of the lower calcified cartilage layer can be controlled in real time through 3D printing parameters. This relatively low porosity can inhibit the adhesion and crawling of the newly formed bone tissue in the subchondral bone layer through the calcified cartilage layer and grow into the cartilage layer, resulting in cartilage area calcification; 4. The upper calcified cartilage layer and the middle calcified cartilage layer of the calcified cartilage layer have relatively large porosities. Among them, the porosity of the middle layer is 35% - 60%, and the porosity of the upper layer is 60% - 90%. Through the control of printing parameters (model parameters, inner diameter of the printing needle, printing spacing, layer angle, layer height, etc.), this increasing porosity structure combined with the porous sponge-like gels inlaid in it can play a role in buffering pressure at the intermediate interface structure. Compared with traditional rigid scaffolds, the upper calcified cartilage layer and the middle calcified cartilage layer of the calcified cartilage layer help the osteochondral repair scaffold to conduct and disperse stress and resist shear force when bearing pressure, reducing the risk of postoperative cartilage repair failure in patients due to excessive stress difference between the implanted scaffold material and the local cartilage defect; 5. The upper calcified cartilage layer and the middle calcified cartilage layer of the calcified cartilage layer are inlaid with porous sponge-like gels, and the cartilage layer is generated in situ during the preparation process. This not only improves the elasticity, toughness, and compressive strength of the scaffold, but also increases the adhesion of the interface, improving problems such as the brittleness of traditional composite scaffolds and poor interlayer adhesion, and improving the overall performance of the scaffold.

[0019] 6. The subchondral bone layer and the calcified cartilage layer are combined with HA with a slower degradation rate and inorganic phase powders with a faster degradation rate (at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, bioactive glass). The degradation rates of the subchondral bone layer and the calcified cartilage layer are controlled by adjusting the ratio of the two. The proportion of hydroxyapatite in the bioactive ceramic of the subchondral bone layer is not higher than that of the calcified cartilage layer, so that the overall degradation rate of the subchondral bone layer is faster than that of the calcified cartilage layer. When the subchondral bone layer of the scaffold gradually degrades and corresponding new bone is generated, the calcified cartilage layer can still exist in a large amount between the intermediate interfaces of the osteochondral repair scaffold, playing a role in inhibiting the new bone tissue below from calcifying through the interface calcified cartilage layer and reducing the risk of cartilage repair failure of the osteochondral scaffold; 7. The cartilage layer uses pharmaceutical gelatin as the framework material and has a porous sponge-like structure. When implanted into the body, under the "drainage" effect of the calcified cartilage layer, it can greatly recruit and adsorb sufficient BMSCs, nutrients, growth factors, etc., and form a "bioreactor" in situ, providing conditions for differentiating into chondrocytes. 8. The cartilage layer can act as a biological barrier. The hydrophilic groups (such as amino, carboxyl, and hydroxyl groups) of the pharmaceutical gelatin it contains can interact with tissue fluid / blood, swell in situ, which is beneficial for its fixation at the cartilage defect. Its strong water absorption can keep blood and recruited BMSCs in place, effectively preventing cells and nutrients from flowing into the joint cavity and causing loss of active ingredients. 9. Hyaluronic acid (sodium) in the cartilage layer can play a lubricating role, which is beneficial for inducing the differentiation of BMSCs into chondrocytes. At the same time, the repair cells, drugs, and carriers in the cartilage layer can play the role of drug loading (antibacterial, analgesic, promoting cartilage differentiation). 10. The main body of the scaffold uses hydroxyapatite, β-tricalcium phosphate, bioactive glass, etc. as the main materials. Using an aqueous PVA solution as the binder for inorganic phase ceramic powder to prepare a printing slurry, a hard bone scaffold with a porosity gradient can be prepared by using three-dimensional printing technology and designing different interlayer porosities. The porous gel inlaid with the cartilage layer and the calcified cartilage layer uses pharmaceutical gelatin as the main material. The whole preparation method requires fewer types of raw materials, the preparation process is less affected by the environment, and the raw materials have good biocompatibility, good osteogenic inductivity, and compressive strength comparable to that of human cancellous bone. Description of the Drawings

[0020] Figure 1 It is a schematic structural diagram of the "main body of the scaffold" in the osteochondral repair scaffold of the embodiment of the present application; Figure 2 It is a schematic structural diagram of the osteochondral repair scaffold of the embodiment of the present application; Figure 3 It is a schematic microstructural diagram of the osteochondral repair scaffold of the embodiment of the present application; Figure 4 It is a schematic microstructural diagram of the osteochondral repair scaffold of the embodiment of the present application; Figure 5 It is a schematic microstructural diagram of the "calcified cartilage layer 300" part in the osteochondral repair scaffold of the embodiment of the present application; Figure 6 It is a stress-strain curve diagram of the "main body of the scaffold" in the osteochondral repair scaffold of the embodiment of the present application; Figure 7 It is a stress-strain curve diagram of the osteochondral repair scaffold of the embodiment of the present application.

[0021] Reference Signs Upper layer of the porous ceramic scaffold - 110, lower layer of the porous ceramic scaffold - 120, subchondral bone layer - 200, calcified cartilage layer - 300, cartilage layer - 400. Detailed implementation manners

[0022] The following will clearly describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application belong to the scope of protection of the present application.

[0023] The terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, rather than to describe a specific order or sequence. It should be understood that such used data can be interchanged under appropriate circumstances so that the embodiments of the present application can be implemented in an order other than those illustrated or described herein, and the objects distinguished by "first", "second", etc. are generally of the same category, and the number of objects is not limited. For example, the first object can be one or multiple. In addition, "and / or" in the specification and claims means at least one of the connected objects, and the character " / " generally represents an "or" relationship between the related objects before and after.

[0024] The following will, with reference to the accompanying drawings, describe in detail the osteochondral repair scaffold and its preparation method provided by the embodiments of the present application through specific embodiments and their application scenarios.

[0025] Embodiment 1 As Figures 1 to 7 shown, the embodiment of the present application provides an osteochondral repair scaffold, including a cartilage layer 400, a subchondral bone layer 200, and a calcified cartilage layer 300 therebetween.

[0026] Among them, the main body of the osteochondral repair scaffold is a bioactive ceramic scaffold; The calcified cartilage layer 300 includes an upper calcified cartilage layer, a middle calcified cartilage layer, and a lower calcified cartilage layer, with through pores inside, and the porosity decreases successively from the upper calcified cartilage layer to the lower calcified cartilage layer along 90% - 20%; The main structure of the calcified cartilage layer is also a bioactive ceramic scaffold. The upper calcified cartilage layer and the middle calcified cartilage layer of the calcified cartilage layer 300 are inlaid with porous gels, and the porous gels include at least one of PVA (polyvinyl alcohol), bioactive ceramics, pharmaceutical gelatin, hyaluronic acid, hyaluronate, or porous fragmented polyester short fibers; The cartilage layer 400 is a porous gel, in a porous sponge shape, and includes at least one of pharmaceutical gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester short fibers loaded with magnetic nanoparticles.

[0027] In the embodiment of the present application, the above-mentioned osteochondral repair scaffold is adopted, which has a three-layer structure with a hierarchical design. Among them, the designed calcified cartilage layer 300 has a special structure, which not only improves the flexibility of the osteochondral repair scaffold. When the cartilage repair scaffold is implanted into the osteochondral defect site, the porosity of the calcified cartilage layer 300 decreasing gradually from top to bottom can smoothly "drain" the bone marrow mesenchymal stem cells and nutrients in the subchondral bone defect site of the patient to the cartilage layer 400 under capillary action, accelerating the recruitment of the patient's autologous blood, so that the autologous blood enters the calcified cartilage layer 300 and the cartilage layer 400 along the main body of the scaffold. The special biochemical composition and structure of the cartilage layer will also guide the bone marrow mesenchymal stem cells stored in the cartilage layer to differentiate into chondrocytes, promoting the generation of cartilage tissue. Since the rate of autologous osteogenesis remodeling of the patient is faster than the repair rate of cartilage tissue, the present invention is designed so that the subchondral bone layer of the osteochondral repair scaffold will first combine with the patient's own hard bone tissue, thereby realizing the fixation of the scaffold at the defect site, providing a stable environment for the differentiation of chondrocytes and the generation of cartilage tissue on the cartilage layer 400, and realizing the integrated repair of osteochondral defects.

[0028] In a preferred embodiment, the cartilage layer 400 includes medicinal gelatin and hyaluronate.

[0029] It can be understood that the hyaluronate can specifically be sodium hyaluronate, and other hyaluronates such as calcium hyaluronate and potassium hyaluronate can also be used.

[0030] In the embodiment of the present application, the above-mentioned osteochondral repair scaffold is adopted. The cartilage layer 400 has a porous sponge structure and uses medicinal gelatin as the framework material. After being implanted into the body, it can recruit and adsorb sufficient BMSCs (bone marrow mesenchymal stem cells) and nutrients, providing conditions for differentiating chondrocytes. At the same time, the cartilage layer 400 contains hyaluronate, which can play a lubricating role and is beneficial to inducing BMSCs (bone marrow mesenchymal stem cells) to differentiate into chondrocytes.

[0031] In a preferred embodiment, the cartilage layer 400 further includes repair cells, and / or drug carriers.

[0032] Among them, the repair cells include but are not limited to chondrocytes, bone marrow mesenchymal stem cells, and extended potential stem cells, and the drug carriers include but are not limited to biomimetic nanoparticles, nano-hydrogel microspheres, and targeted peptide complexes.

[0033] In a preferred embodiment, the cartilage layer 400 further includes bioactive substances for promoting cartilage generation, and / or antibacterial components.

[0034] In a preferred embodiment, the bioactive ceramic scaffold in the calcified cartilage layer 300 and the subchondral bone layer 200 includes at least one of hydroxyapatite, α-TCP (α-tricalcium phosphate), β-TCP (β-tricalcium phosphate), calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass.

[0035] In a preferred embodiment, the bioactive ceramic scaffold is specifically configured as a HA / β-TCP scaffold.

[0036] Among them, the bioactive ceramics of bioactive ceramic scaffolds include HA (hydroxyapatite) and β-TCP (β-tricalcium phosphate).

[0037] In a preferred embodiment, two ends of the calcified cartilage layer 300 are fixedly connected to the bioactive ceramic scaffold and the cartilage layer 400 respectively.

[0038] In a preferred embodiment, the cartilage layer 400 is specifically configured as a porous gel, and the bioactive ceramic scaffold and the cartilage layer 400 have a fusion portion.

[0039] The part of the bioactive ceramic scaffold that is not fused with the cartilage layer 400 forms the subchondral bone layer 200 , and the calcified cartilage layer 300 is specifically configured as the fused part of the bioactive ceramic scaffold and the cartilage layer 400 .

[0040] In a preferred embodiment, the thickness of the upper layer of the calcified cartilage of the calcified cartilage layer 300 is set to 0.1-2.0 mm, and the porosity is set to 60% to 90%.

[0041] In a preferred embodiment, the thickness of the calcified cartilage middle layer of the calcified cartilage layer 300 is set to 0.1-2.0 mm, and the porosity is set to 35% to 60%.

[0042] In a preferred embodiment, the thickness of the calcified subcartilage layer of the calcified cartilage layer 300 is set to 0.1-1.0 mm, and the porosity is set to 20% to 35%.

[0043] In an embodiment of the present application, the above-mentioned bone cartilage repair scaffold is used, and the calcified cartilage layer 300 is configured as a bioactive ceramic scaffold and a porous gel filled therein. The porous gel has a strong ability to recruit blood, and can keep a large amount of blood in the calcified cartilage layer 300, and continuously transport / provide the patient's bone marrow mesenchymal stem cells and nutrients required for cell proliferation and differentiation to the cartilage layer 400. At the same time, the porous gel has a certain elasticity when absorbing water and swelling, which can improve the mechanical strength and toughness of the scaffold, increase the adhesion of the interface, improve the defects of traditional composite scaffolds such as brittleness and poor adhesion, and improve the performance of the scaffold.

[0044] In a preferred embodiment, the osteochondral repair scaffold as a whole has a pore gradient that increases from bottom to top along one axial side.

[0045] Among them, the cartilage layer 400 merges with the end of the bioactive ceramic scaffold body with larger pores to form a calcified cartilage layer 300.

[0046] It can be understood that the bioactive ceramic scaffold on the side of the calcified cartilage layer 300 close to the cartilage layer has a higher porosity, which is beneficial to the delivery of autologous blood.

[0047] In a preferred embodiment, the compressive strength of the bioactive ceramic scaffold is set to 4 MPa to 10 MPa.

[0048] In a preferred embodiment, as Figure 1 shown, the bioactive ceramic scaffold includes a scaffold upper layer 110 and a scaffold lower layer 120 fixedly connected to the scaffold upper layer 110.

[0049] Among them, the porosity of the scaffold upper layer 110 is greater than that of the scaffold lower layer 120, and the cartilage layer 400 merges with the scaffold upper layer 110 to form a calcified cartilage layer 300.

[0050] In a preferred embodiment, the porosity of the scaffold upper layer 110 is specifically set to 65% - 75%, and / or the porosity of the scaffold lower layer 120 is specifically set to 25% - 55%.

[0051] In a preferred embodiment, the scaffold body is specifically set as an integrally formed structure by 3D printing and has at least two interlayer angles.

[0052] It can be understood that the interlayer angle is an execution parameter of 3D printing, that is, the angle between the bidirectional printing trajectories, and different interlayer angles correspond to different pore patterns.

[0053] In a preferred embodiment, the interlayer angle of the scaffold lower layer 120 is specifically set to 0 - 60°, and the interlayer angle of the scaffold upper layer 110 is specifically set to 60 - 90°.

[0054] It can be understood that the interlayer angle of the scaffold upper layer 110 is set to 60 - 90°, which has a higher porosity compared to the scaffold lower layer 120, and can recruit more bone marrow mesenchymal stem cells and accommodate more nutrients, which is beneficial to the differentiation and regeneration of cartilage.

[0055] Example 2 The embodiment of the present application provides a preparation method for the osteochondral repair scaffold in the above embodiment, including: Preparing a precursor solution; Introducing the precursor solution into a silica gel soft mold; After the precursor solution is vacuum-cured, a first vacuum freeze-drying treatment is performed on it to generate a cartilage germ layer; The cartilage germ layer is first subjected to gradient alcohol dehydration treatment and cross-linking agent cross-linking treatment, and then a second vacuum freeze-drying treatment is performed to generate a cartilage layer 400.

[0056] In a preferred embodiment, the mass concentration of the precursor solution is set to 1% - 5% wt.

[0057] In a preferred embodiment, the preparation of the precursor solution is specifically as follows: At a predetermined temperature, at least one of medicinal gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester short fibers loaded with magnetic nanoparticles is dispersed in deionized water and stirred evenly.

[0058] In a preferred embodiment, the predetermined temperature is specifically set to 38 - 45 °C.

[0059] In a preferred embodiment, it further includes: Preparing a printing slurry; Using three-dimensional printing technology to prepare a green body scaffold; Performing a drying treatment and a sintering treatment on the green body scaffold to form a scaffold main body.

[0060] In a preferred embodiment, the preparation of the printing slurry includes: Separately preparing a liquid-phase adhesive solution and a biphasic ceramic powder; Preparing a solid-phase mixed powder based on the biphasic ceramic powder; Mixing the liquid-phase adhesive solution with the solid-phase mixed powder to generate a printing slurry.

[0061] In a preferred embodiment, the liquid-phase adhesive solution includes at least one of an aqueous PVA solution, glycerol, and ammonium polyacrylate.

[0062] Among them, the mass fraction of PVA is set to 8% - 11%.

[0063] The mass ratio of the aqueous PVA solution to glycerol, or ammonium polyacrylate, is 10:(0.4 - 0.8).

[0064] In a preferred embodiment, the preparation method of the biphasic ceramic powder is: Mixing nano-HA and nano-β-TCP evenly according to a mass ratio of (2:8) - (8:2).

[0065] In a preferred embodiment, the preparation method of the solid-phase mixed powder is: The biphasic ceramic powder is mixed with at least one of calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass in any proportion.

[0066] In a preferred embodiment, mixing the liquid-phase adhesive solution and the solid-phase mixed powder to generate a printing slurry specifically includes: Mixing the solid-phase mixed powder and the liquid-phase adhesive solution according to a mass ratio of (5 - 8.5):10; During the mixing process of the solid-phase mixed powder and the liquid-phase adhesive solution, slowly add the solid-phase mixed powder and stir evenly.

[0067] In a preferred embodiment, preparing a green body scaffold using three-dimensional printing technology includes: Filling the printing slurry into a three-dimensional printing cartridge; Configuring three-dimensional printing parameters; Among them, the bottom diameter of the model is set to 10 - 12 mm, the layer height is set to 0.5 mm, the line spacing is set to 1 mm, and the printing path is set to a Z shape; The height of the green body scaffold is set to 8 - 9 mm, and the pore structure is set to be filled at 0 - 60 - 120°; The printing discharge speed is set to 0.5 mm 3 / s, the wire walking speed is set to 2.8 mm / s, the temperatures of the syringe and the table are set to a constant 25°C, and the printing needle has a diameter of 0.5 mm.

[0068] In a preferred embodiment, preparing a green body scaffold using three-dimensional printing technology further includes: During the printing process, the interlayer angle parameter and the printing height parameter are adjusted in real time.

[0069] It can be understood that by adjusting the interlayer angle parameter and the printing height parameter, a green body scaffold with uniform or non-uniform pore size distribution and uniform or step-varying overall porosity distribution can be obtained.

[0070] In the embodiments of the present application, using the above-mentioned preparation method, hydroxyapatite and β-tricalcium phosphate are used as the main materials of the scaffold, and a PVA aqueous solution is used as the binder for nano-hydroxyapatite powder to prepare a printing slurry. By using three-dimensional printing technology and designing the porosity of different layers, a hard scaffold body with a porosity gradient can be prepared. At the same time, the types of raw materials required for preparation are few, the influence of the environment is small, and the raw materials have good biocompatibility, osteogenic inductivity, and fixation, so that the scaffold has a compressive strength equivalent to that of human cancellous bone.

[0071] In a preferred embodiment, the drying treatment specifically is to place the green body scaffold in a drying environment at 25°C and let it stand for 4 - 24 h; And / or, the sintering treatment specifically is to heat the green body scaffold after drying treatment to 1100°C - 1200°C at a rate of 2.5°C / h and keep it warm for 2 - 4 h.

[0072] In a preferred embodiment, it further includes: The stent body is negatively pressure impregnated into the precursor solution, and a calcified cartilage layer 300 is formed on the immersed part of the stent body during the formation of the cartilage layer 400.

[0073] It can be understood that the subchondral bone layer and the calcified cartilage layer are formed on the stent body. When the stent body is negatively pressure impregnated into the precursor solution, the infiltrated part is the corresponding part of the upper calcified cartilage layer and the middle calcified cartilage layer of the stent body with respect to the calcified cartilage layer.

[0074] In a preferred embodiment, negatively pressure impregnating the stent body into the precursor solution and forming the calcified cartilage layer 300 on the immersed part of the stent body during the formation of the cartilage layer 400 specifically includes: The upper layer 110 of the stent of the stent body is negatively pressure impregnated into the precursor solution, the immersion height is set to 0.5 - 1.5 mm, and the vacuum degree of the negative pressure impregnation is set to be greater than 0.2.

[0075] In the embodiment of the present application, by adopting the above preparation method, a three-dimensional printing technology is used to prepare a cartilage layer 400 with an elastic structure.

[0076] In a preferred embodiment, preparing the cartilage layer 400 on the stent body by using a three-dimensional printing technology includes: Mix a 15% - 40% wt pharmaceutical gelatin solution with hyaluronic acid (salt) or solid phase powder in a ratio of (10:6) - (10:0.5) to prepare a paste-like extrudate; Control the room temperature to be 29 - 35 °C, use a ceramic stent as the substrate, and use a 3D printing technology to print an elastic stent with a thickness of 0.5 - 7 mm.

[0077] After cooling to room temperature for shaping, the elastic cartilage layer is frozen, vacuum freeze-dried, dehydrated with gradient alcohol, washed and then freeze-dried again to obtain an osteochondral repair stent. Observe the osteochondral repair stent. The 3D printed pharmaceutical gelatin layer penetrates into the interior of the ceramic stent to form a porous gel. The part of the uppermost layer that does not blend with the ceramic stent is the cartilage layer 400; the part that blends with the ceramic stent is the calcified cartilage layer 300, and the part of the ceramic stent that does not blend with the cartilage layer 400 is the subchondral bone layer 200.

[0078] Wherein, the cartilage layer blends with the side of the bioactive ceramic stent body with larger pores to form a calcified cartilage layer.

[0079] In the embodiment of the present application, by adopting the above preparation method, the overall flexibility of the osteochondral repair scaffold is improved. Due to the use of medicinal gelatin with a relatively high mass concentration, when the subsequent freeze-drying treatment generates an integrated scaffold, a calcified cartilage layer 300 is formed at the joint with the bioactive ceramic scaffold. The dense bottom structure in the calcified cartilage layer 300 combines with the absorption and swelling of blood by the middle and upper layers of medicinal gelatin, enabling high-efficiency accommodation of bone marrow mesenchymal stem cells, growth factors, nutrients, etc. The entire calcified cartilage layer 300 can also achieve the effect of a filter / semipermeable membrane.

[0080] In the embodiment of the present application, by adopting the above preparation method, the precursor solution is impregnated under negative pressure on the side with a relatively high porosity of the bioactive ceramic body. After freeze-drying and forming, the calcified cartilage layer 300 includes a scaffold main body part with a high porosity and a porous gel distributed in the scaffold main body, effectively improving the mechanical strength and toughness of the calcified cartilage layer 300.

[0081] Example 3 The embodiment of the present application provides a preparation method for preparing the scaffold main body of the osteochondral repair scaffold in the above Example 1, including: (1) Preparation of the liquid-phase adhesive solution Weigh 0.8 g of PVA powder according to the PVA mass fraction of 8%. Put the PVA powder and 10 ml of deionized water into a beaker and seal it with plastic wrap. Set the oil bath temperature to 95 °C and the magnetic stirring speed to 120 r / min, and stir for 2 h 10 min; Then lower the oil bath temperature to 60 °C, keep the speed unchanged, stir for 1 h, and add 0.4 mL of glycerol and stir for 9 min after returning to room temperature to finally obtain a colorless, uniform, and transparent liquid-phase adhesive solution.

[0082] (2) Preparation of the printing slurry Weigh 8 g of the mixed powder of nHA (nano-hydroxyapatite) and β-TCP, gradually and slowly add it to the liquid-phase adhesive solution while stirring. After complete addition and uniform stirring, perform ultrasonic degassing treatment to obtain a uniform printing slurry with good extrudability and formability.

[0083] (3) Establishment of the printing model Import a cylindrical printing model on a three-dimensional printing device, adjust the bottom diameter of the mold to 10 mm, copy the model, keep the bottom diameters of the 3 models equal, adjust the height to 7 mm, and the filling route to 0 - 60 - 120°; continue to adjust the height to 1 mm, the filling route to 0 - 70°, and the remaining height to 2 mm, the filling route to 0 - 90° to obtain the final printing model.

[0084] (4) Setting of the printing parameters Set the discharging speed to 0.5 mm 3 / s, the wire speed is 1.8 mm / s, the temperature of the syringe and the table is set to a constant 25 °C, the diameter of the printing needle is 0.5 mm, the extrusion method of the printer is an electric drive screw propeller, the workbench moves in a combined motion along the x-y axis, the print head moves along the z axis, and prints layer by layer in sequence, and finally completes the printing according to the specified model.

[0085] (5) The green body scaffold is subjected to a drying treatment Place the just-printed green body scaffold in a drying environment at 25 °C for 12 h.

[0086] (6) The green body scaffold is subjected to a sintering treatment Put the completely dried green body scaffold into a corundum crucible, heat it to 1150 °C at a heating rate of 2.5 °C / h and hold for 2 h, then cool it to room temperature at the same speed. After sintering, an HA / β-TCP scaffold with different porosities is obtained.

[0087] Example 4 The embodiment of the present application provides a preparation method for preparing the scaffold body of the osteochondral repair scaffold in the above Example 1, including: (1) Preparation of the liquid-phase adhesive solution Weigh 1 g of PVA powder according to a PVA mass fraction of 10%. Put the PVA powder and 10 ml of deionized water into a beaker and seal it with plastic wrap. Set the oil bath temperature to 95 °C, the magnetic stirring speed to 120 r / min, and stir for 2 h 10 min; Then lower the oil bath temperature to 60 °C, keep the speed unchanged, stir for 1 h, add 0.6 ml of glycerol after returning to room temperature and stir for 9 min, and finally obtain a colorless, uniform and transparent liquid-phase adhesive solution.

[0088] (2) Preparation of the printing slurry Weigh 7 g of the mixed powder of nHA (nano-hydroxyapatite) and β-TCP, gradually and slowly add it to the liquid-phase adhesive solution while stirring. After complete addition and stirring evenly, perform ultrasonic degassing treatment to obtain a uniform printing slurry with good extrusion and formability.

[0089] (3) Establishment of the printing model Import a cylindrical printing model on a three-dimensional printing device, adjust the bottom diameter of the mold to 12 mm, copy the model, keep the bottom diameters of the two models equal, adjust the height to 9 mm, the filling route to 0-60-120°, and the remaining height to 3 mm, the filling route to 0-90° to obtain the final printing model.

[0090] (4) Setting of the printing parameters Set the discharge speed to 0.5 mm 3 / s, the wire running speed is 1.8 mm / s, the temperatures of the syringe and the table are set at a constant 25 °C, the diameter of the printing needle is 0.4 mm, the extrusion method of the printer is an electric-driven screw propeller, the workbench makes a combined movement along the x-y axis, the print head moves along the z axis, and printing is carried out layer by layer in sequence, and finally the printing is completed according to the specified model.

[0091] (5) The green body scaffold is subjected to a drying treatment Place the just-printed green body scaffold in a drying environment at 25 °C for 12 h.

[0092] (6) The green body scaffold is subjected to a sintering treatment Put the completely dried green body scaffold into a corundum crucible, heat it to 1200 °C at a heating rate of 2.5 °C / h and hold for 3 h, then cool it to room temperature at the same rate. After sintering, an HA / β-TCP scaffold with different porosities is obtained.

[0093] (7) Test the performance of the HA / β-TCP scaffold Take the HA / β-TCP scaffold samples prepared in Example 4 to test their porosities, compressive strengths, and the dimensional shrinkage rates before and after sintering. The specific test methods are as follows: The porosity test uses the liquid phase replacement method. Inject anhydrous ethanol with a volume of V1 into a graduated cylinder, slowly put the HA / β-TCP scaffold sample into it and soak for 10 min. After soaking, record the ethanol volume V2 at this time, then take out the soaked HA / β-TCP scaffold sample and record the ethanol volume V3 at this time. The porosity calculation formula of the HA / β-TCP scaffold sample is:

[0094] To facilitate the measurement of the porosity of the HA / β-TCP scaffold samples, use the process in Example 4 to print 5 HA / β-TCP scaffold samples with different interlayer angle structures respectively. After testing and calculation, the average porosity of the filling structure of 0-60-120° is 50.7%, and the average porosity of the filling structure of 0-90° is 69.7%.

[0095] To evaluate the compressive strength of the HA / β-TCP scaffold samples, a universal testing machine is used for testing. Take 5 HA / β-TCP scaffold samples prepared by the process in Example 4 for compressive strength testing. The average compressive strengths are 9.532 MPa respectively, indicating that the HA / β-TCP scaffold samples prepared by this process have a stable structure, sufficient mechanical properties, and a compressive strength similar to that of human cancellous bone, which can ensure the stability during implantation.

[0096] Table 1 Average values of the dimensions of the HA / β-TCP scaffold samples before and after sintering

[0097]

[0098] To evaluate the collapse rate of the HA / β-TCP scaffold samples before and after sintering, the dimensions of the HA / β-TCP scaffold samples before and after sintering were measured and averaged. As shown in Table 1 above, the collapse rates of the bottom diameter and height of the HA / β-TCP scaffold samples were calculated to be approximately 15% - 25%.

[0099] Example 5 The embodiment of the present application provides a preparation method for preparing the osteochondral repair scaffold in Embodiment 1 above, including: (1) Prepare a low-concentration precursor solution, such as 1%. (2) Negative pressure impregnation and freeze-drying treatment Inject the low-concentration precursor solution into the mold; Immerse the side with a higher porosity of the HA / β-TCP scaffold prepared in Embodiment 3 or 4 into the precursor solution under negative pressure, with the immersion height of 0.5 - 1.5 mm, control the vacuum degree at 0.5 Mpa, after curing, freeze overnight at -20 °C, and then vacuum freeze-dry; After freeze-drying, dehydrate the composite scaffold with gradient alcohol. According to the gradient of 50% ethanol, 75% ethanol, 95% ethanol, and 100% ethanol, immerse the composite scaffold in the 50% ethanol solution for 10 - 20 minutes, pick it up with tweezers, transfer the composite scaffold to the 75% ethanol solution and immerse it for 10 - 20 minutes, and so on. During this process, the composite scaffold can be kept in a moist state and avoid drying. After dehydration, inject the 95% ethanol solution of 50 mmol / L carbodiimide into the mold, immerse the cartilage layer 400 of the composite scaffold in the cross-linking agent, and cross-link at room temperature for 24 h; Freeze the composite scaffold at -80 °C, quickly transfer it to a freeze-dryer, and perform vacuum freeze-drying at a temperature of -50 °C to -40 °C, control the vacuum degree at 90 - 300, and finally obtain the osteochondral repair scaffold.

[0100] It can be understood that the base material of the osteochondral repair scaffold is the HA / β-TCP scaffold prepared in Embodiment 3 or 4. The part of the HA / β-TCP scaffold that is not impregnated with the precursor solution is the subchondral bone layer 200, the part of the HA / β-TCP scaffold impregnated with the precursor solution is the calcified cartilage layer 300, and the part at the end of the HA / β-TCP scaffold is the cartilage layer 400 formed by the precursor solution.

[0101] As Figure 2 shown, in the prepared osteochondral repair scaffold, the cartilage layer 400 presents a porous and fluffy foam-like shape.

[0102] To further evaluate the microscopic morphology of the cartilage layer 400, a field emission scanning electron microscope was used to observe the cartilage layer 400, as Figure 3As shown, the figure is the microstructure diagram of the synthetic cartilage layer 400 (after two freeze-dryings). It can be seen that the cartilage layer 400 presents a radially porous structure, and the pore size ranges from 50 to 400 μm.

[0103] To further evaluate the microtopography of the cartilage layer 400 - calcified cartilage layer 300 - subchondral bone layer 200, a field emission scanning electron microscope was used to observe the cross-section of the osteochondral repair scaffold. As Figure 4 、 Figure 5 shown, Figure 4 、 Figure 5 are the microstructure diagrams at the junctions of different layers of the osteochondral repair scaffold. It can be seen from Figure 4 that the cartilage layer 400 presents a porous structure, which is consistent with the structure of the outer surface of the cartilage layer 400 in Figure 3 . The calcified cartilage layer 300 is the junction part containing the scaffold main body and the cartilage layer 400. There are also porous structures in the calcified cartilage layer 300 (scaffold main body and cartilage layer 400). It can be seen from Figure 5 that the connection between the cartilage layer 400 and the scaffold main body in the calcified cartilage layer 300 is dense, and there are micropores with staggered sizes in the part of the scaffold main body perfused with the precursor solution.

[0104] To further evaluate the compressive strength of the osteochondral repair scaffold, a universal material testing machine was used to test the osteochondral repair scaffold. As Figure 6 、 Figure 7 shown, Figure 6 、 Figure 7 are the stress-strain curves of the HA / β-TCP scaffold and the osteochondral repair scaffold respectively. The measured compressive strengths of the HA / β-TCP scaffold and the osteochondral repair scaffold are 9.646 MPa and 17.45 MPa respectively. The compressive strength of the formed osteochondral repair scaffold has been significantly improved.

[0105] The reasons for the improvement of the compressive strength of the osteochondral repair scaffold include: (1) Adding the cartilage layer 400 makes the osteochondral repair scaffold have certain toughness and increased anti-destruction ability. (The HA / β-TCP scaffold reaches the extreme value within the deformation range of 0.4 - 0.8 mm, but the osteochondral repair scaffold reaches the extreme value at 2.5 mm deformation) (2) After the cartilage layer 400 is freeze-dried twice, the part where it blends with the scaffold main body is tightly combined, making the overall mechanical properties of the osteochondral repair scaffold enhanced.

[0106] It can be seen from this that the osteochondral repair scaffold prepared in this embodiment can improve the mechanical strength, toughness and service performance.

[0107] It should be noted that in this text, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the existence of additional identical elements in the process, method, article or device comprising that element. In addition, it should be pointed out that the scope of the methods and devices in the embodiments of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in the reverse order according to the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0108] The embodiments of the present application have been described above in conjunction with the accompanying drawings. However, the present application is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Those of ordinary skill in the art, under the inspiration of the present application and without departing from the spirit and scope protected by the claims of the present application, can still make many forms, all of which fall within the protection scope of the present application.

Claims

1. An osteochondral repair scaffold, characterized in that, It includes a subchondral bone layer, a calcified cartilage layer, and a cartilage layer that are connected in sequence; The subchondral bone layer is internally provided with through pores and includes bioactive ceramics; The calcified cartilage layer is internally provided with gradient through pores and includes bioactive ceramics; The cartilage layer is in a porous sponge shape and includes at least one of medicinal gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester short fibers loaded with magnetic nanoparticles; Among them, the calcified cartilage layer includes an upper calcified cartilage layer, a middle calcified cartilage layer, and a lower calcified cartilage layer. The upper calcified cartilage layer and the middle calcified cartilage layer are inlaid with porous gels. The porous gels include at least one of PVA, bioactive ceramics, medicinal gelatin, hyaluronic acid, hyaluronate, or porous fragmented polyester short fibers; The porosity of the gradient through pores inside the calcified cartilage layer decreases sequentially from the upper calcified cartilage layer to the lower calcified cartilage layer, and the main structure of the calcified cartilage layer is set as a bioactive ceramic scaffold.

2. The osteochondral repair scaffold according to claim 1, wherein The bioactive ceramics in the subchondral bone layer and the calcified cartilage layer include hydroxyapatite and at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass; 3. The osteochondral repair scaffold according to claim 2, characterized in that, In the bioactive ceramics of the subchondral bone layer, the ratio of hydroxyapatite to at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass is 50-70:30-50; And / or, in the bioactive ceramics of the calcified cartilage layer, the ratio of hydroxyapatite to at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass is 60-80:20-40.

4. The osteochondral repair scaffold according to claim 1, characterized in that, The through pores of the bioactive ceramics in the calcified cartilage layer are distributed in a gradient; Among them, from the subchondral bone layer to one side of the cartilage layer, the porosity of the through pores of the bioactive ceramics in the calcified cartilage layer increases from 20% to 90%.

5. The osteochondral repair scaffold according to claim 1 or 4, characterized in that, The porosity of the upper calcified cartilage layer is specifically set to 60% to 90%, the porosity of the middle calcified cartilage layer is specifically set to 35% to 60%, and the porosity of the lower calcified cartilage layer is specifically set to 20% to 35%.

6. The osteochondral repair scaffold according to claim 1, wherein, The cartilage layer further includes one or more of repair cells, drug carriers, bioactive substances, and antibacterial components.

7. A preparation method for preparing the osteochondral repair scaffold according to any one of claims 1 to 6 above, characterized in that, It includes: Preparing printing slurry; Preparing a green body scaffold by using 3D bioprinting technology; Performing drying treatment and sintering treatment on the green body scaffold to form a scaffold main body; Among them, the subchondral bone layer is formed on the scaffold main body, or the subchondral bone layer and the calcified cartilage layer are formed on the scaffold main body.

8. A preparation method according to claim 7, characterized in that, It further includes: Adjusting the interlayer angle parameter and the printing height parameter in real time during the printing process.

9. A preparation method according to claim 7, characterized in that, It further includes: Preparing a precursor solution; Introducing the precursor solution into a silica gel soft mold; After the upper calcified cartilage layer and the middle calcified cartilage layer of the calcified cartilage layer are impregnated under negative pressure with the precursor solution, they are frozen and subjected to the first vacuum freeze-drying treatment; After the first vacuum freeze-drying treatment, the calcified cartilage layer is subjected to gradient alcohol dehydration treatment and cross-linking agent cross-linking treatment, and then the second vacuum freeze-drying treatment is performed to generate a stable inlaid porous gel.

10. A preparation method according to claim 9, characterized in that, It further includes: On the calcified cartilage layer, a cartilage layer is integrally formed by a negative pressure impregnation process; Or, a cartilage layer is prepared on the calcified cartilage layer by 3D printing technology.

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