Bone cartilage repair scaffold and preparation method thereof

Abstract

The present application discloses a bone cartilage repair scaffold and a preparation method thereof, wherein the bone cartilage repair scaffold includes a subchondral bone layer, a calcified cartilage layer and a cartilage layer connected in sequence, the calcified cartilage layer includes a calcified cartilage upper layer, a calcified cartilage middle layer, a calcified cartilage lower layer, and a porous gel embedded in the calcified cartilage upper layer and the calcified cartilage middle layer. In an embodiment of the present application, the above-mentioned bone cartilage repair scaffold and a preparation method thereof are adopted, and a layered structure is adopted to simulate human physiological cartilage. Through the layering and gradient porosity design of the calcified cartilage layer, not only the flexibility of the scaffold is improved and the performance of the scaffold is improved, but also it can ensure that the scaffold can quickly recruit the patient's autologous blood after being implanted into the bone cartilage defect site, thereby guiding bone marrow mesenchymal stem cells to differentiate into chondrocytes in the cartilage layer and promote the generation of cartilage tissue.

Description

Technical Field

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

[0002] Microfracture and 3D biomimetic scaffolds are currently the mainstays of clinical treatment for articular cartilage injuries. Microfracture, a conventional treatment for small to medium-sized (≤4 cm²) localized cartilage defects, involves drilling holes in the bone to release mesenchymal stem cells from the bone marrow, prompting the formation of fibrous blood clots and their differentiation into fibrocartilage tissue to repair the defect. However, the fibrocartilage generated by this method is significantly inferior to natural hyaline cartilage in terms of mechanical properties, biological function, and long-term stability. Furthermore, microfracture primarily focuses on repairing cartilage alone, neglecting the repair of the calcified layer and subchondral bone. This results in poor integration efficiency at the bone-cartilage interface, which in turn affects tissue maturation and long-term stability.

[0003] To address these deficiencies, 3D biomimetic scaffolds have become a research hotspot, aiming to mimic the layered structure of natural cartilage, including the surface layer, intermediate layer, deep radial layer, and calcified cartilage layer. The calcified cartilage layer is the key interface between cartilage and bone, containing a mineralized matrix that facilitates 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 have difficulty achieving an osteochondral scaffold with a gradient mineralized calcified cartilage layer.

[0004] Therefore, introducing a gradient mineralized structure into 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 detachment or repair failure, which has important clinical application value. Summary of the Invention

[0005] In view of the deficiencies of the existing technology, the present invention provides a bone cartilage 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 implemented 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;

[0008] The subchondral bone layer is provided with through pores therein and comprises bioactive ceramics;

[0009] The calcified cartilage layer has gradient through-pores inside and includes bioactive ceramics;

[0010] The cartilage layer is in a porous sponge shape and comprises at least one of pharmaceutical gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester staple fibers loaded with magnetic nanoparticles;

[0011] The calcified cartilage layer includes a calcified cartilage upper layer, a calcified cartilage middle layer and a calcified cartilage lower layer. The calcified cartilage upper layer and the calcified cartilage middle layer are inlaid with porous gel, and the porous gel includes at least one of PVA, bioactive ceramics, medicinal gelatin, hyaluronic acid, hyaluronate or porous fragmented polyester staple fibers.

[0012] It is further defined that in the above-mentioned osteochondral repair scaffold, 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.

[0013] It is further defined that in the above-mentioned osteochondral repair scaffold, the ratio of hydroxyapatite to at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass in the bioactive ceramic of the subchondral bone layer is 50-70:30-50;

[0014] And / or, in the bioactive ceramic 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.

[0015] It is further defined that, in the above-mentioned osteochondral repair scaffold, the through-pores of the bioactive ceramic in the calcified cartilage layer are distributed in a gradient manner;

[0016] The porosity of the through pores of the bioactive ceramic in the calcified cartilage layer increases from 20% to 90% from the subchondral bone layer to the cartilage layer.

[0017] It is further defined that in the above-mentioned osteochondral repair scaffold, the porosity of the upper layer of the calcified cartilage is specifically set to 60% to 90%, the porosity of the middle layer of the calcified cartilage is specifically set to 35% to 60%, and the porosity of the lower layer of the calcified cartilage is specifically set to 20% to 35%.

[0018] It is further defined that in the above-mentioned osteochondral repair scaffold, the cartilage layer also includes one or more of repair cells, drug carriers, bioactive substances, and antibacterial components.

[0019] The present application provides a preparation method for any of the above-mentioned osteochondral repair scaffolds, comprising:

[0020] preparing a printing paste;

[0021] 3D bioprinting technology was used to prepare embryo scaffolds;

[0022] performing drying and sintering on the green embryo scaffold to form a scaffold body;

[0023] Wherein, a subchondral bone layer is formed on the stent body, or a subchondral bone layer and a calcified cartilage layer are formed on the stent body.

[0024] It is further defined that the above-mentioned preparation method further comprises:

[0025] Adjust the inter-layer angle parameters and printing height parameters in real time during the printing process.

[0026] It is further defined that the above-mentioned preparation method further comprises:

[0027] preparing a precursor solution;

[0028] introducing the precursor solution into a silicone soft mold;

[0029] 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, and then frozen and subjected to a first vacuum freeze-drying process;

[0030] 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.

[0031] It is further defined that the above-mentioned preparation method further comprises:

[0032] The cartilage layer is integrally formed on the calcified cartilage layer using a negative pressure impregnation process;

[0033] Alternatively, a cartilage layer is prepared on the calcified cartilage layer using 3D printing technology.

[0034] The present invention has at least the following beneficial effects:

[0035] 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 into the osteochondral defect site, it can quickly recruit the patient's autologous blood, allowing the autologous blood to enter the calcified cartilage layer and cartilage layer along the scaffold, thereby guiding the bone marrow mesenchymal stem cells to differentiate into chondrocytes in the cartilage layer and promote the generation 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 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, and realizing the integrated repair of osteochondral defects.

[0036] 2. The calcified cartilage layer is configured as a bioactive ceramic with pores throughout and a gradient distribution, including an upper calcified cartilage layer, a middle calcified cartilage layer, and a lower calcified cartilage layer. The upper and middle calcified cartilage layers are inlaid with porous gel (porous sponge-like). Since the porosity of the calcified cartilage layer is designed to be a gradient of 90% to 20%, when the osteochondral repair scaffold is implanted in the osteochondral defect, this structure can smoothly "drain" bone marrow mesenchymal stem cells from the subchondral bone defect to the calcified cartilage layer and the cartilage layer under capillary action. In particular, the inlaying of porous gel in the calcified cartilage layer accelerates this process.

[0037] 3. The calcified subcartilage layer of the calcified cartilage layer has a low porosity of 20% to 35%. The porosity of the calcified subcartilage layer can be controlled in real time through 3D printing parameters. This low porosity can inhibit the new bone tissue in the subchondral bone layer from adhering to the calcified cartilage layer and crawling into the cartilage layer to grow, causing calcification of the cartilage area.

[0038] 4. The calcified cartilage upper layer and calcified cartilage middle layer of the calcified cartilage layer have a large porosity, of which the middle layer has a porosity of 35% to 60% and the upper layer has a porosity of 60% to 90%. Through the control of printing parameters (model parameters, printing needle inner diameter, printing spacing, interlayer angle and layer height, etc.), this increasing porosity structure combined with the porous sponge-like gel embedded in it can buffer the pressure in the intermediate interface structure. Compared with traditional rigid scaffolds, the calcified cartilage upper layer and calcified cartilage middle layer of the calcified cartilage layer help the osteochondral repair scaffold to transmit and disperse stress and resist shear force when bearing pressure, thereby 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;

[0039] 5. The upper layer of calcified cartilage and the middle layer of calcified cartilage are inlaid with porous sponge-like gel, 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, improves the problems of traditional composite scaffolds such as brittleness and poor interlayer adhesion, and improves the overall performance of the scaffold.

[0040] 6. The subchondral bone layer and calcified cartilage layer utilize a combination of slowly degrading HA and a rapidly degrading inorganic phase powder (at least one of α-TCP, β-TCP, calcium carbonate, calcium sulfate, calcium silicate, and bioactive glass). The degradation rates of the subchondral bone layer and 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 no higher than that of the calcified cartilage layer, resulting in an overall faster degradation rate of the subchondral bone layer than that of the calcified cartilage layer. As the subchondral bone layer of the scaffold gradually degrades and new bone is generated, the calcified cartilage layer can still be present at a relatively large amount at the middle interface of the osteochondral repair scaffold, thereby inhibiting the penetration of new bone tissue below through the interfacial calcified cartilage layer and reducing the risk of cartilage repair failure of the osteochondral scaffold.

[0041] 7. The cartilage layer uses medicinal 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 absorb sufficient BMSCs, nutrients and growth factors, etc., forming an in situ "bioreactor" to provide conditions for differentiation into chondrocytes;

[0042] 8. The cartilage layer can act as a biological barrier. The hydrophilic groups of the medicinal gelatin (such as amino, carboxyl, and hydroxyl groups) it contains can interact with tissue fluid / blood and expand in situ, which is conducive to its fixation in the cartilage defect. The strong water absorption can keep the blood and recruited BMSCs in place, effectively preventing cells and nutrients from flowing into the joint cavity and causing the loss of effective ingredients.

[0043] 9. Hyaluronic acid (sodium) in the cartilage layer can act as a lubricant, which is conducive to inducing BMSCs to differentiate into chondrocytes. At the same time, the repair cells or drugs and carriers in the cartilage layer can play the role of drug delivery (antibacterial, analgesic, and promoting cartilage differentiation);

[0044] 10. The main body of the scaffold is made of hydroxyapatite, β-tricalcium phosphate, bioglass, etc., and a PVA aqueous solution is used as a binder for the inorganic ceramic powder to prepare the printing slurry. The use of 3D printing technology and the design of the porosity between different layers can prepare a hard bone scaffold with a porosity gradient. The porous gel inlaid with the cartilage layer and the calcified cartilage layer is made of medicinal gelatin as the main material. The entire preparation method requires a small number of raw materials, the preparation process is less affected by the environment, and the raw materials have good biocompatibility and good osteogenic induction, and have a compressive strength comparable to that of human cancellous bone. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic structural diagram of the "stent body" in the osteochondral repair stent of the embodiment of the present application;

[0046] Figure 2 This is a schematic structural diagram of the osteochondral repair scaffold according to an embodiment of the present application;

[0047] Figure 3 This is a schematic diagram of the microstructure of the osteochondral repair scaffold according to an embodiment of the present application;

[0048] Figure 4 This is a schematic diagram of the microstructure of the osteochondral repair scaffold according to an embodiment of the present application;

[0049] Figure 5 Schematic diagram of the microstructure of the "calcified cartilage layer 300" in the osteochondral repair scaffold of the embodiment of the present application;

[0050] Figure 6 This is a stress-strain curve diagram of the "scaffold body" in the osteochondral repair scaffold of the embodiment of the present application;

[0051] Figure 7 This is a stress-strain curve diagram of the osteochondral repair scaffold according to an embodiment of the present application.

[0052] Reference numerals

[0053] The porous ceramic scaffold upper layer is 110, the porous ceramic scaffold lower layer is 120, the subchondral bone layer is 200, the calcified cartilage layer is 300, and the cartilage layer is 400. DETAILED DESCRIPTION

[0054] The following will be combined with the accompanying drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of this application.

[0055] The terms "first," "second," and the like in the specification and claims of this application are used to distinguish similar objects, and are not used to describe a specific order or precedence. It should be understood that the terms used in this manner are interchangeable where appropriate, so that the embodiments of this application can be implemented in an order other than that illustrated or described herein, and that the objects distinguished by "first," "second," and the like are generally of the same type, and do not limit the number of objects; for example, the first object can be one or more. In addition, the term "and / or" in the specification and claims refers to at least one of the connected objects, and the character " / " generally indicates that the objects connected are in an "or" relationship.

[0056] The following describes 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 in conjunction with the accompanying drawings.

[0057] Example 1

[0058] like Figures 1 to 7 As shown, an embodiment of the present application provides a bone cartilage repair scaffold, comprising a cartilage layer 400, a subchondral bone layer 200 and a calcified cartilage layer 300 therebetween.

[0059] Among them, the main body of the osteochondral repair scaffold is a bioactive ceramic scaffold;

[0060] The calcified cartilage layer 300 includes a calcified cartilage upper layer, a calcified cartilage middle layer, and a calcified cartilage lower layer, and has through pores therein, and the porosity decreases from 90% to 20% from the calcified cartilage upper layer to the calcified cartilage lower layer;

[0061] The main structure of the calcified cartilage layer is also a bioactive ceramic scaffold. The calcified cartilage upper layer and the calcified cartilage middle layer of the calcified cartilage layer 300 are inlaid with porous gel. The porous gel includes at least one of PVA (polyvinyl alcohol), bioactive ceramics, pharmaceutical gelatin, hyaluronic acid, hyaluronate, or porous fragmented polyester staple fibers.

[0062] The cartilage layer 400 is a porous gel in the form of a porous sponge and includes at least one of pharmaceutical gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester staple fibers loaded with magnetic nanoparticles.

[0063] In the embodiment of the present application, the above-mentioned osteochondral repair scaffold is used, which adopts a layered three-layer structure. Among them, the designed calcified cartilage layer 300 has a special structure, which not only improves the flexibility of the osteochondral repair scaffold, but also, when the cartilage repair scaffold is implanted in the integrated osteochondral defect, the calcified cartilage layer 300 has a gradually decreasing porosity from top to bottom. Under capillary action, it can smoothly "drain" the patient's bone marrow mesenchymal stem cells and nutrients from the subchondral bone defect to the cartilage layer 400, accelerating the recruitment of the patient's autologous blood, allowing the autologous blood to flow along the scaffold body into the calcified cartilage layer 300 and the cartilage layer 400. The special biochemical composition and structure of the cartilage layer also guide the bone marrow mesenchymal stem cells stored in the cartilage layer to differentiate into chondrocytes, promoting the formation of cartilage tissue. Since the patient's autologous bone remodeling rate is faster than the repair rate of cartilage tissue, the design of the present invention allows the subchondral bone layer of the osteochondral repair scaffold to first combine with the patient's own hard bone tissue, thereby achieving fixation of the scaffold at the defect site, providing a stable environment for chondrocyte differentiation and cartilage tissue generation on the cartilage layer 400, and realizing integrated repair of osteochondral defects.

[0064] In a preferred embodiment, the cartilage layer 400 includes pharmaceutical gelatin and hyaluronate.

[0065] It is understood that the hyaluronate may specifically be sodium hyaluronate, or other hyaluronates such as calcium hyaluronate and potassium hyaluronate.

[0066] In an embodiment of the present application, the above-mentioned osteochondral repair scaffold is used, and the cartilage layer 400 has a porous sponge structure and uses medicinal gelatin as a frame material. After implantation into the body, it can recruit and absorb sufficient BMSCs (bone marrow mesenchymal stem cells) and nutrients to provide conditions for the differentiation of chondrocytes. At the same time, the cartilage layer 400 contains hyaluronate, which can play a lubricating role and is conducive to inducing BMSCs (bone marrow mesenchymal stem cells) to differentiate into chondrocytes.

[0067] In a preferred embodiment, the cartilage layer 400 further includes repair cells and / or drug carriers.

[0068] Among them, repair cells include but are not limited to chondrocytes, bone marrow mesenchymal stem cells, and expanded potential stem cells, and drug carriers include but are not limited to biomimetic nanoparticles, nanohydrogel microspheres, and targeted peptide complexes.

[0069] In a preferred embodiment, the cartilage layer 400 further includes bioactive substances for promoting cartilage formation, and / or antibacterial components.

[0070] 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.

[0071] In a preferred embodiment, the bioactive ceramic scaffold is specifically configured as a HA / β-TCP scaffold.

[0072] Among them, the bioactive ceramics of bioactive ceramic scaffolds include HA (hydroxyapatite) and β-TCP (β-tricalcium phosphate).

[0073] In a preferred embodiment, both ends of the calcified cartilage layer 300 are fixedly connected to the bioactive ceramic scaffold and the cartilage layer 400 respectively.

[0074] 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.

[0075] The portion 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 portion of the bioactive ceramic scaffold and the cartilage layer 400 .

[0076] 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%.

[0077] 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%.

[0078] In a preferred embodiment, the thickness of the calcified subchondral layer of the calcified cartilage layer 300 is set to 0.1-1.0 mm, and the porosity is set to 20% to 35%.

[0079] In an embodiment of the present application, the above-mentioned bone cartilage repair scaffold is used, and the calcified cartilage layer 300 is set 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.

[0080] 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.

[0081] The cartilage layer 400 is fused with the end of the bioactive ceramic scaffold body with larger pores to form the calcified cartilage layer 300 .

[0082] It is understandable 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 conducive to the transportation of autologous blood.

[0083] In a preferred embodiment, the compressive strength of the bioactive ceramic scaffold is set to 4 MPa-10 MPa.

[0084] In a preferred embodiment, as Figure 1 As shown, the bioactive ceramic stent includes a stent upper layer 110 and a stent lower layer 120 fixedly connected to the stent upper layer 110 .

[0085] The porosity of the upper layer 110 of the scaffold is greater than the porosity of the lower layer 120 of the scaffold, and the cartilage layer 400 blends with the upper layer 110 of the scaffold to form a calcified cartilage layer 300 .

[0086] In a preferred embodiment, the porosity of the upper layer 110 of the support is specifically set to 65% to 75%, and / or the porosity of the lower layer 120 of the support is specifically set to 25% to 55%.

[0087] In a preferred embodiment, the bracket body is specifically configured as an integrated structure formed by 3D printing and has at least two inter-layer angles.

[0088] It can be understood that the interlayer angle is an execution parameter of 3D printing, that is, the angle between the bidirectional printing tracks, and different interlayer angles correspond to different hole types.

[0089] In a preferred embodiment, the interlayer angle of the support lower layer 120 is specifically set to 0-60°, and the interlayer angle of the support upper layer 110 is specifically set to 60-90°.

[0090] It is understandable that the interlayer angle of the upper layer 110 of the scaffold is set to 60-90°, which has a higher porosity than the lower layer 120 of the scaffold, can recruit more bone marrow mesenchymal stem cells and accommodate more nutrients to facilitate cartilage differentiation and regeneration.

[0091] Example 2

[0092] The present application provides a preparation method for the osteochondral repair scaffold in the above embodiment, comprising:

[0093] preparing a precursor solution;

[0094] introducing the precursor solution into a silicone soft mold;

[0095] After the precursor solution is vacuum solidified, a first vacuum freeze-drying process is performed on the precursor solution to generate a cartilage germ layer;

[0096] The cartilage germ layer is first subjected to a gradient alcohol dehydration treatment, a cross-linking agent cross-linking treatment, and then subjected to a second vacuum freeze-drying treatment to generate a cartilage layer 400 .

[0097] In a preferred embodiment, the mass concentration of the precursor solution is set to 1% to 5%wt.

[0098] In a preferred embodiment, the precursor solution is prepared as follows:

[0099] At a predetermined temperature, at least one of medicinal gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester staple fibers loaded with magnetic nanoparticles is dispersed in deionized water and stirred evenly.

[0100] In a preferred embodiment, the predetermined temperature is specifically set to 38-45°C.

[0101] In a preferred embodiment, it also includes:

[0102] preparing a printing paste;

[0103] Use 3D printing technology to prepare embryo scaffolds;

[0104] The green scaffold is dried and sintered to form a scaffold body.

[0105] In a preferred embodiment, preparing the printing paste includes:

[0106] preparing a liquid adhesive solution and a dual-phase ceramic powder respectively;

[0107] Preparation of solid phase mixed powder based on dual phase ceramic powder;

[0108] A liquid binder solution is mixed with a solid mixed powder to create a printing paste.

[0109] In a preferred embodiment, the liquid adhesive solution includes at least one of a PVA aqueous solution, glycerol, and ammonium polyacrylate.

[0110] Among them, the mass fraction of PVA is set to 8%~11%.

[0111] The mass ratio of PVA aqueous solution to glycerol or ammonium polyacrylate is 10: (0.4~0.8).

[0112] In a preferred embodiment, the preparation method of the dual-phase ceramic powder is:

[0113] Mix nano-HA and nano-β-TCP in a mass ratio of (2:8) to (8:2).

[0114] In a preferred embodiment, the preparation method of the solid phase mixed powder is:

[0115] The dual-phase ceramic powder is mixed with at least one of calcium carbonate, calcium sulfate, calcium silicate and bioactive glass in any proportion.

[0116] In a preferred embodiment, the liquid adhesive solution is mixed with the solid mixed powder to generate the printing slurry as follows:

[0117] The solid phase mixed powder and the liquid phase adhesive solution are mixed in a mass ratio of (5-8.5):10;

[0118] 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 thoroughly.

[0119] In a preferred embodiment, the preparation of embryo scaffolds using three-dimensional printing technology includes:

[0120] Filling the printing slurry into the 3D printing cylinder;

[0121] Configure 3D printing parameters;

[0122] The model bottom diameter is set to 10-12mm, the layer height is set to 0.5mm, the line spacing is set to 1mm, and the printing path is set to Z-type;

[0123] The embryo scaffold height is set to 8-9mm, and the pore structure is set to 0-60-120° filling;

[0124] The printing speed is set to 0.5mm 3 / s, the line speed is set to 2.8mm / s, the temperature of the syringe and table is set to a constant temperature of 25℃, and the printing needle adopts a 0.5mm diameter.

[0125] In a preferred embodiment, the process of preparing the embryo scaffold using three-dimensional printing technology further comprises:

[0126] Adjust the inter-layer angle parameters and printing height parameters in real time during the printing process.

[0127] It is understandable that by adjusting the interlayer angle parameters and the printing height parameters, a green embryo scaffold with uniform or uneven pore size distribution and uniform or step-varied overall porosity distribution can be obtained.

[0128] In an embodiment of the present application, the above-mentioned preparation method is adopted, hydroxyapatite and β-tricalcium phosphate are used as the main materials of the scaffold, and PVA aqueous solution is used as a binder for nano-hydroxyapatite powder to prepare a printing slurry. Three-dimensional printing technology is used and the porosity between different layers is designed, so that a hard scaffold body with a porosity gradient can be prepared. At the same time, the preparation requires fewer types of raw materials and is less affected by the environment. The raw materials all have good biocompatibility, osteoinduction and fixation, so that the scaffold has a compressive strength equivalent to that of human cancellous bone.

[0129] In a preferred embodiment, the drying process is specifically to place the embryo scaffold in a dry environment at 25° C. for 4-24 hours;

[0130] And / or, the sintering process specifically comprises heating the dried green embryo scaffold to 1100° C. to 1200° C. at a rate of 2.5° C. / h and keeping the temperature for 2 to 4 hours.

[0131] In a preferred embodiment, it also includes:

[0132] The stent body is immersed in the precursor solution under negative pressure, and the calcified cartilage layer 300 is formed on the immersed portion of the stent body during the generation of the cartilage layer 400 .

[0133] 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 immersed in the precursor solution under negative pressure, the infiltrated part is the corresponding part of the calcified cartilage upper layer and the calcified cartilage middle layer of the stent body relative to the calcified cartilage layer.

[0134] In a preferred embodiment, the stent body is immersed in the precursor solution under negative pressure, and the calcified cartilage layer 300 is formed on the immersed portion of the stent body during the formation of the cartilage layer 400. Specifically,

[0135] The upper layer 110 of the support body is immersed in the precursor solution under negative pressure, the immersion height is set to 0.5-1.5 mm, and the vacuum degree of the negative pressure immersion is set to be greater than 0.2.

[0136] In an embodiment of the present application, the above-mentioned preparation method is adopted to prepare the cartilage layer 400 with an elastic structure using three-dimensional printing technology.

[0137] In a preferred embodiment, the process of preparing the cartilage layer 400 on the scaffold body using three-dimensional printing technology includes:

[0138] 15% to 40% wt of a pharmaceutical gelatin solution is mixed with hyaluronic acid (salt) or solid phase powder in a ratio of (10:6) to (10:0.5) to prepare a paste extrudate;

[0139] The room temperature is controlled at 29-35℃, and an elastic bracket with a thickness of 0.5-7mm is printed using 3D printing technology with a ceramic bracket as the base.

[0140] After cooling to room temperature to set, the elastic cartilage layer was frozen, freeze-dried in a vacuum, dehydrated with graded alcohol, washed, and freeze-dried again to produce an osteochondral repair scaffold. Observation of the osteochondral repair scaffold revealed that the 3D-printed medicinal gelatin layer had infiltrated the interior of the ceramic scaffold, forming a porous gel. The topmost layer, unattached to the ceramic scaffold, represented the cartilage layer 400; the layer that had integrated with the ceramic scaffold represented the calcified cartilage layer 300; and the portion of the ceramic scaffold not integrated with the cartilage layer 400 represented the subchondral bone layer 200.

[0141] The cartilage layer is integrated with the side of the bioactive ceramic scaffold body with larger pores to form a calcified cartilage layer.

[0142] In an embodiment of the present application, the above-mentioned preparation method is adopted to improve the overall flexibility of the osteochondral repair scaffold. Since a large mass concentration of medicinal gelatin is used, when the integrated scaffold is subsequently freeze-dried to form a scaffold, a calcified cartilage layer 300 is formed at the junction with the bioactive ceramic scaffold. The dense underlying structure in the calcified cartilage layer 300, combined with the swelling effect of the middle and upper layers of medicinal gelatin absorbing blood, can efficiently accommodate bone marrow mesenchymal stem cells, growth factors and nutrients, etc. The entire calcified cartilage layer 300 can also act as a filter / semi-permeable membrane.

[0143] In an embodiment of the present application, one of the above-mentioned preparation methods is adopted, and a precursor solution is impregnated under negative pressure on a side of the bioactive ceramic body with a higher porosity. After freeze-drying and forming, the calcified cartilage layer 300 includes a high-porosity scaffold body part and a porous gel distributed in the scaffold body, which effectively improves the mechanical strength and toughness of the calcified cartilage layer 300.

[0144] Example 3

[0145] The present embodiment provides a preparation method for preparing the scaffold body of the osteochondral repair scaffold in the above embodiment 1, comprising:

[0146] (1) Liquid adhesive solution preparation

[0147] Weigh 0.8 g of PVA powder with a PVA mass fraction of 8%, place the PVA powder and 10 ml of deionized water into a beaker, seal the beaker with plastic wrap, set the oil bath temperature to 95°C, and the magnetic stirring speed to 120 rpm for 2 h 10 min.

[0148] The oil bath temperature was then lowered to 60°C, the rotation speed was kept constant, and the mixture was stirred for 1 hour. After returning to room temperature, 0.4 mL of glycerol was added and stirred for 9 minutes to finally obtain a colorless, uniform, and transparent liquid adhesive solution.

[0149] (2) Printing slurry configuration

[0150] Weigh 8 g of mixed powder of nHA (nanohydroxyapatite) and β-TCP, gradually and slowly add it to the liquid adhesive solution while stirring. After complete addition and stirring, ultrasonic bubbling treatment is performed to obtain a uniform printing slurry with good extrudability and formability.

[0151] (3) Create a printing model

[0152] Import the cylindrical printing model on the 3D printing device, adjust the bottom diameter of the mold to 10 mm, copy the model, keep the bottom diameters of the three models equal, adjust the height to 7 mm, and the filling route to 0-60-120°; continue to adjust the height to 1 mm and the filling route to 0-70°, leaving the remaining height to 2 mm and the filling route to 0-90° to obtain the final printing model.

[0153] (4) Set printing parameters

[0154] Set the discharge speed to 0.5mm 3 / s, the line speed is 1.8mm / s, the temperature of the syringe and table is set to a constant temperature of 25℃, the diameter of the printing needle is 0.5mm, the printer extrusion method is an electric-driven screw propeller, the workbench performs a composite motion along the xy axis, and the print head moves along the z axis, printing layer by layer in sequence, and finally completing the printing according to the specified model.

[0155] (5) Drying of the embryo scaffold

[0156] Place the freshly printed embryo scaffold in a dry environment at 25°C for 12 hours.

[0157] (6) Sintering of the embryonic support

[0158] The completely dried green embryo scaffold was placed in a corundum crucible, and the temperature was raised to 1150°C at a rate of 2.5°C / h and kept warm for 2 hours, and then lowered to room temperature at the same rate. After sintering, HA / β-TCP scaffolds with different porosities were obtained.

[0159] Example 4

[0160] The present embodiment provides a preparation method for preparing the scaffold body of the osteochondral repair scaffold in the above embodiment 1, comprising:

[0161] (1) Liquid adhesive solution preparation

[0162] Weigh 1 g of PVA powder with a PVA mass fraction of 10%, place the PVA powder and 10 ml of deionized water into a beaker, seal the beaker with plastic wrap, set the oil bath temperature to 95°C, and stir at a magnetic stirring speed of 120 rpm for 2 h 10 min.

[0163] Then lower the oil bath temperature to 60°C, keep the speed unchanged, stir for 1 hour, return to room temperature, add 0.6 ml of glycerol and stir for 9 minutes, and finally obtain a colorless, uniform and transparent liquid adhesive solution.

[0164] (2) Printing slurry configuration

[0165] Weigh 7 g of mixed powder of nHA (nanohydroxyapatite) and β-TCP and slowly add it to the liquid adhesive solution while stirring. After complete addition and stirring, ultrasonic bubbling treatment is performed to obtain a uniform printing slurry with good extrudability and formability.

[0166] (3) Create a printing model

[0167] Import the cylindrical printing model on the 3D 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°, the remaining height to 3 mm, and the filling route to 0-90° to obtain the final printing model.

[0168] (4) Set printing parameters

[0169] Set the discharge speed to 0.5mm 3 / s, the line speed is 1.8mm / s, the temperature of the syringe and table is set to a constant temperature of 25℃, the diameter of the printing needle is 0.4mm, the printer extrusion method is an electric-driven screw propeller, the workbench performs a composite motion along the xy axis, and the print head moves along the z axis, printing layer by layer in sequence, and finally completing the printing according to the specified model.

[0170] (5) Drying of the embryo scaffold

[0171] Place the freshly printed embryo scaffold in a dry environment at 25°C for 12 hours.

[0172] (6) Sintering of the embryonic support

[0173] The completely dried embryonic scaffold was placed in a corundum crucible, heated to 1200°C at a rate of 2.5°C / h and kept warm for 3 hours, and then cooled to room temperature at the same rate. After sintering, HA / β-TCP scaffolds with different porosities were obtained.

[0174] (7) Testing the performance of HA / β-TCP scaffolds

[0175] The HA / β-TCP scaffold sample prepared in Example 4 was tested for porosity, compressive strength, and dimensional collapse rate before and after sintering. The specific testing methods are as follows:

[0176] The porosity test was performed using the liquid phase displacement method. Anhydrous ethanol with a volume of V1 was injected into a measuring cylinder. The HA / β-TCP scaffold sample was slowly placed in the cylinder and soaked for 10 minutes. The volume of ethanol V2 was recorded after soaking. The soaked HA / β-TCP scaffold sample was then removed and the volume of ethanol V3 was recorded. The porosity of the HA / β-TCP scaffold sample was calculated as follows:

[0177]

[0178] To facilitate the measurement of the porosity of the HA / β-TCP scaffold samples, 5 HA / β-TCP scaffold samples with different interlayer angle structures were printed using the process in Example 4. After testing and calculation, the average porosity of the filling structure of 0-60-120° was 50.7%, and the average porosity of the filling structure of 0-90° was 69.7%.

[0179] To evaluate the compressive strength of the HA / β-TCP scaffold samples, a universal testing machine was used for testing. Five HA / β-TCP scaffold samples prepared using the process of Example 4 were tested for compressive strength. The average compressive strength was 9.532 MPa, indicating that the HA / β-TCP scaffold samples prepared using this process had a stable structure and sufficient mechanical properties. They had a compressive strength similar to that of human cancellous bone, and could ensure stability during implantation.

[0180] Table 1 Average dimensions of HA / β-TCP scaffold samples before and after sintering

[0181]

[0182] In order 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 the average values were taken. As shown in Table 1 above, it was calculated that the collapse rate of the bottom diameter and height of the HA / β-TCP scaffold samples was approximately 15% to 25%.

[0183] Example 5

[0184] This embodiment of the present application provides a preparation method for preparing the osteochondral repair scaffold in the above embodiment 1, comprising:

[0185] (1) Prepare a low concentration, such as 1% precursor solution

[0186] (2) Negative pressure impregnation and freeze-drying

[0187] Injecting a low concentration precursor solution into the mold;

[0188] The side of the HA / β-TCP scaffold prepared in Example 3 or 4 with higher porosity was immersed in the precursor solution under negative pressure at a immersion height of 0.5-1.5 mm and a vacuum degree of 0.5 MPa. After curing, the scaffold was frozen at -20°C overnight and then freeze-dried under vacuum.

[0189] After freeze-drying, the composite scaffold was dehydrated with gradient alcohol, following the gradient of 50% ethanol, 75% ethanol, 95% ethanol, and 100% ethanol. The composite scaffold was immersed in 50% ethanol solution for 10-20 minutes, picked up with tweezers, and moved to 75% ethanol solution for 10-20 minutes. And so on. During this process, the composite scaffold can be kept moist to avoid drying. After dehydration, a 95% ethanol solution of 50mmol / L carbodiimide is injected into the mold, and the cartilage layer of the composite scaffold is immersed in the crosslinker at 400°C for crosslinking at room temperature for 24 hours.

[0190] The composite scaffold was frozen at -80°C, quickly transferred to a freeze dryer, and vacuum freeze-dried at a temperature of -50°C to -40°C, with the vacuum degree controlled at 90-300, to finally obtain a bone cartilage repair scaffold.

[0191] It can be understood that the base material of the osteocartilage repair scaffold is the HA / β-TCP scaffold prepared in Example 3 or 4, wherein the portion of the HA / β-TCP scaffold not soaked in the precursor solution is the subchondral bone layer 200, the portion of the HA / β-TCP scaffold soaked in the precursor solution is the calcified cartilage layer 300, and the portion located at the end of the HA / β-TCP scaffold is the cartilage layer 400 formed by the precursor solution.

[0192] like Figure 2 As shown, in the prepared osteochondral repair scaffold, the cartilage layer 400 is in a porous and fluffy foam shape.

[0193] In order 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. Figure 3 As shown in the figure, the microstructure of the synthetic cartilage layer 400 (after two freeze-dryings) is shown. It can be seen that the cartilage layer 400 presents a radial porous structure with a pore size range of 50-400 μm.

[0194] In order to further evaluate the microscopic morphology 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. Figure 4 、 Figure 5 As shown, Figure 4 、 Figure 5 This is the microscopic structure of the junction of different layers of the osteochondral repair scaffold. Figure 4 It can be seen that the cartilage layer 400 presents a porous structure, which is consistent with Figure 3 The structure of the outer surface of the middle cartilage layer 400 is consistent. The calcified cartilage layer 300 is the boundary portion of the scaffold body and the cartilage layer 400. The calcified cartilage layer 300 also has a porous structure (the scaffold body and the cartilage layer 400). Figure 5 It can be seen that the cartilage layer 400 in the calcified cartilage layer 300 is closely connected to the scaffold body, and micropores of varying sizes exist in the portion of the scaffold body that is perfused with the precursor solution.

[0195] In order to further evaluate the compressive strength of the osteochondral repair scaffold, the osteochondral repair scaffold was tested using a universal material testing machine. Figure 6 、 Figure 7 As shown, Figure 6 、 Figure 7They are the stress-strain curves of HA / β-TCP scaffold and osteochondral repair scaffold. The test results show that the compressive strengths of HA / β-TCP scaffold and osteochondral repair scaffold are 9.646MPa and 17.45MPa respectively. The compressive strength of the osteochondral repair scaffold after molding is significantly improved.

[0196] The reasons for the increased compressive strength of the osteochondral repair scaffold include:

[0197] (1) Adding a cartilage layer of 400 makes the osteochondral repair scaffold have a certain toughness and increased damage resistance. (The HA / β-TCP scaffold reaches its extreme value in the deformation range of 0.4-0.8mm, but the osteochondral repair scaffold reaches its extreme value at 2.5mm deformation)

[0198] (2) After secondary freeze-drying, the cartilage layer 400 is tightly integrated with the scaffold body, thereby enhancing the overall mechanical properties of the osteochondral repair scaffold.

[0199] It can be seen from this that the osteochondral repair scaffold prepared in this embodiment can improve mechanical strength, toughness and performance.

[0200] It should be noted that, in this article, the terms "comprise", "include" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprises a ..." does not exclude the presence of other identical elements in the process, method, article or device comprising the element. In addition, it should be noted 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 opposite order according to the functions involved. For example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.

[0201] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of this application, ordinary technicians in this field can also make many forms without departing from the purpose of this application and the scope of protection of the claims, all of which are within the protection of this application.

Claims

1. A bone cartilage repair scaffold, characterized in that: It includes the subchondral bone layer, calcified cartilage layer and cartilage layer connected in sequence; The subchondral bone layer is provided with through pores therein and comprises bioactive ceramics; The calcified cartilage layer has gradient through-pores inside and includes bioactive ceramics; The cartilage layer is in a porous sponge shape and comprises at least one of pharmaceutical gelatin, hyaluronic acid, hyaluronate, and porous fragmented polyester staple fibers loaded with magnetic nanoparticles; The calcified cartilage layer includes a calcified cartilage upper layer, a calcified cartilage middle layer, and a calcified cartilage lower layer. The calcified cartilage upper layer and the calcified cartilage middle layer are inlaid with porous gel, and the porous gel includes at least one of PVA, bioactive ceramics, pharmaceutical gelatin, hyaluronic acid, hyaluronate, or porous fragmented polyester staple fibers. The porosity of the gradient through-pores inside the calcified cartilage layer decreases successively from the calcified cartilage upper layer to the calcified cartilage lower layer, and the main structure of the calcified cartilage layer is configured as a bioactive ceramic scaffold.

2. The osteochondral repair scaffold according to claim 1, characterized in that: 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 ceramic 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 ceramic 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 ceramic in the calcified cartilage layer are distributed in a gradient manner; The porosity of the through pores of the bioactive ceramic in the calcified cartilage layer increases from 20% to 90% from the subchondral bone layer to the cartilage layer.

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

6. The osteochondral repair scaffold according to claim 1, characterized in that: The cartilage layer also includes one or more of repair cells, drug carriers, and bioactive substances.

7. A preparation method for preparing the osteochondral repair scaffold according to any one of claims 1 to 6, characterized in that: include: preparing a printing paste; 3D bioprinting technology was used to prepare embryo scaffolds; performing drying and sintering on the green embryo scaffold to form a scaffold body; Wherein, a subchondral bone layer is formed on the stent body, or a subchondral bone layer and a calcified cartilage layer are formed on the stent body.

8. A preparation method according to claim 7, characterized in that, Also includes: Adjust the inter-layer angle parameters and printing height parameters in real time during the printing process.

9. A preparation method according to claim 7, characterized in that, Also includes: preparing a precursor solution; introducing the precursor solution into a silicone soft mold; 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, and then frozen and subjected to a first vacuum freeze-drying process; 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.

10. A preparation method according to claim 9, characterized in that: Also includes: The cartilage layer is integrally formed on the calcified cartilage layer using a negative pressure impregnation process; Alternatively, a cartilage layer is prepared on the calcified cartilage layer using 3D printing technology.

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