Multilayered biomimetic scaffolds and methods of making the same

Abstract

The application discloses a kind of multilayer biomimetic scaffolds and preparation method thereof.The preparation method of the multilayer biomimetic scaffold includes the following steps:S1, sacrificial material is layer-by-layer 3D printed, n layers are continuously printed, and grid-shaped porous inner mold is obtained;S2, biomimetic hydrogel precursor solution is poured in the grid-shaped porous template, then light curing crosslinking is carried out, and sacrificial material-hydrogel complex is formed;S3, repeat step S1 and S2, and form multilayer sacrificial material-hydrogel complex;S4, solvent dissolution method is used to carry out inner demolding treatment to the multilayer sacrificial material-hydrogel complex, and multilayer biomimetic scaffold is obtained.The preparation method realizes layered biomimetic design based on inner demolding technology, ensures that hydrogel of different biomimetic layers exists independently, avoids mixing and diffusion problem of biomimetic material between adjacent layers when pouring once, accurately realizes the design of different biomimetic layers, and also ensures effective curing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tissue engineering products and regenerative medicine technology, and particularly relates to a multilayer biomimetic scaffold and a preparation method thereof. BACKGROUND

[0002] At present, the number of patients who need treatment due to multi-layer tissue damage such as joint cartilage injury, skin tissue defect, vascular disease, etc. shows a continuous growth trend. The limitations of traditional treatment methods make the clinical demand for new repair technology increasingly urgent, and the market prospect of related tissue engineering products and regenerative medicine technology is broad.

[0003] There are a wide range of multi-layer composite structures in the human body, such as osteochondral interface, dermal-epidermal layer of skin, and multi-layer composite tissues such as blood vessel wall. The structural characteristics of these tissues are that the upper and lower layers are different, but they need to maintain the integrity of the whole. Once these tissues are damaged on a large scale, due to the complexity of their internal structure and the limitations of their self-repairing ability, clinical treatment faces great challenges.

[0004] Taking osteochondral tissue as an example, it is composed of a surface cartilage layer, a subchondral transition layer, and a bottom bone layer. The extracellular matrix (ECM) composition, cell types, pore structure, and mechanical properties of each layer are significantly different, and the articular cartilage lacks nerves and blood vessels, and its self-repairing ability after injury is extremely limited. The current clinical repair methods, including microfracture, autologous chondrocyte transplantation, and tissue engineering scaffold implantation, can alleviate symptoms to some extent, but all have their own limitations and are difficult to achieve ideal anatomical structure and functional reconstruction. The shortcomings of these treatment methods result in long-term pain, dysfunction, and other problems in patients, which seriously affect the quality of life, so it is urgent to develop new treatment programs that can meet the requirements of multi-layer tissue repair.

[0005] With the rapid development of the field of tissue engineering and regenerative medicine, 3D printing technology has opened up a new technical path for the preparation of tissue engineering scaffolds due to its precision, controllability, and personalized customization. However, the existing technology still has a series of key technical problems in constructing multi-layer, structure-function integrated tissue engineering scaffolds, which restricts its clinical translation application and industrial development.

[0006] Currently, direct 3D printing of hydrogels is one of the common strategies for fabricating tissue engineering scaffolds. Although natural bio-inks have good biocompatibility and bioactivity, they are not suitable for fine printing and cannot meet the precision requirements of complex multi-layer scaffolds. In addition, extrusion-based bio-3D printers have significant technical and economic barriers: the cost of the equipment is extremely high, usually 10-100 times that of a fused deposition modeling (FDM) polymer material printer, greatly increasing the research and production costs; at the same time, the bio-printing speed is slow, which seriously affects the production efficiency and the feasibility of industrialization. To improve the printing precision, it is usually necessary to increase the concentration of hydrogels or add non-cell extracellular matrix (ECM) components, which will inevitably sacrifice the biological function of the scaffold. Many biomimetic formulations with excellent biological activity cannot be applied directly due to their viscosity, rheological properties, and other characteristics. The combination of these technical and economic factors has severely limited the large-scale application and clinical promotion of bio-3D printing technology.

[0007] Given the limitations of direct bio-printing methods, researchers have tried other preparation strategies. To achieve the connection of multi-layer scaffolds, some studies have used adhesive methods. Although this method is simple, it introduces chemical adhesives, which can form non-bioactive regions at the interface, hindering cell migration and nutrient exchange. The adhesive area can also form closed pores, disrupting the overall interconnectedness of the scaffold. The microenvironment at the interface is significantly different from that of natural tissues, which is not conducive to tissue integration and long-term repair.

[0008] In addition to the above methods, some existing 3D printing technologies have improved the scaffold preparation process to some extent, but still have common problems: the controllability of the scaffold pore structure is poor, making it difficult to balance mechanical strength and cell permeability; most technologies are designed for a single tissue type and cannot simulate the complex microenvironment of multi-layer tissues; more importantly, existing technologies cannot achieve precise hierarchical distribution of extracellular matrix (ECM) components within the scaffold, which is crucial for simulating the multi-layer structure and function of natural tissues; in addition, the specific surface area of the scaffold is usually insufficient, limiting the efficiency of cell adhesion and nutrient exchange.

[0009] In particular, when preparing multi-layer biomimetic scaffolds containing osteogenic materials in the form of solid particles (such as bioactive glass, hydroxyapatite, etc.), existing technologies face more challenges: on the one hand, using a biomimetic hydrogel precursor solution containing solid particles directly for 3D printing can easily clog the nozzle, making it difficult to prepare multi-layer scaffolds containing particles, and the rheological properties of the hydrogel formulation are required to be high for direct printing, which requires a compromise between printability and biomimicry, resulting in optimized biomimetic hydrogel precursor solution formulations that are not suitable for direct printing; on the other hand, when preparing thick-layer scaffolds by one-time perfusion, solid particles can further affect the photocuring effect, leading to incomplete curing inside the scaffold and decreased mechanical properties.

[0010] In the face of growing clinical treatment needs, the existing technology has many deficiencies in the preparation of multi-layer biomimetic tissue engineering scaffolds, which seriously restricts the industrialization process of related products and is difficult to meet the urgent needs of complex tissue repair in clinical practice. Therefore, it is urgent to develop new technical solutions to solve the above key technical problems. SUMMARY

[0011] In order to solve the following problems in the prior art: (1) accurate control of the composition of the biomimetic layered structure: accurate implementation of the design of the layered biomimetic structure - when using the "internal demolding method" (i.e. a method of constructing the internal pore structure of the scaffold by using a soluble or removable sacrificial material as a mold, and then removing the mold to form a porous scaffold) to prepare a hydrogel scaffold, in order to simulate the complex structure of natural multi-layered tissues (such as the bone-cartilage interface, skin, etc.), it is necessary to accurately configure different extracellular matrix components, bioactive factors and functional particles in different layers, and to control the thickness ratio and composition gradient of each layer. However, when directly pouring different component solutions into a thin-layer multi-component scaffold, the different component solutions are easily mixed, resulting in a blurred interface between layers, uneven distribution of components, and the inability to achieve the ideal layered biomimetic structure; (2) interface bonding and pore regulation of multi-layer scaffolds - the existing preparation methods have technical difficulties in the following two key aspects: in terms of firm bonding between layers, multi-layer scaffolds often have insufficient bonding strength at the interface between layers, which easily leads to delamination, cannot withstand physiological loads, and affects the overall mechanical properties and long-term stability of the scaffold; in terms of accurate control of the pore connectivity structure, traditional methods cannot effectively connect the pores across layers, which leads to blocked cell migration and poor exchange of nutrients, and cannot accurately control the pore size and distribution between different layers, making it difficult to meet the specific functional requirements of different tissue levels.

[0012] In the present application, the single-layer printing layer height of the sacrificial material 3D printed refers to the thickness of the extruded sacrificial material set in the 3D printing equipment parameters. The "n" in each continuous printing of n layers of sacrificial material refers to the number of layers that are subjected to hydrogel perfusion and solidification after each continuous printing. The selection of this number of layers is mainly determined by the light transmission performance of the hydrogel and the penetration depth of the light curing.

[0013] The cycle logic of the preparation process is: printing several layers of sacrificial material → perfusing hydrogel of corresponding thickness → light curing → repeating the cycle until the total thickness of the target multi-layer biomimetic scaffold is reached.

[0014] Relationship between hydrogel light transmission and printing strategy:

[0015] (1) Transparent hydrogel: light can penetrate deeply, and a relatively thick layer of hydrogel can be perfused and completely cured at one time;

[0016] (2) translucent hydrogel: moderate light penetration ability, need moderate thickness of layered perfusion curing;

[0017] (3) hydrogel containing osteogenic material (in the form of solid particles): particles block light penetration, only thin layer can be cured by frequent perfusion.

[0018] The application provides a preparation method of a multilayer biomimetic scaffold, which comprises the following steps:

[0019] S1, layer-by-layer 3D printing of a sacrificial material, continuous printing of n layers to obtain a grid-shaped porous inner mold;

[0020] wherein the single-layer printing layer height is 0.1-0.3 mm, and n is 1-50;

[0021] S2, perfusion of a biomimetic hydrogel precursor solution in the grid-shaped porous inner mold, and then light curing crosslinking to form a sacrificial material-hydrogel composite;

[0022] S3, repeating steps S1 and S2 on the upper surface of the sacrificial material-hydrogel composite obtained in step S2 to form a multilayer sacrificial material-hydrogel composite, wherein the number of layers n continuously printed in each step S1 is the same or different, and the biomimetic hydrogel precursor solution perfused in each step S2 is the same or different;

[0023] S4, inner demolding treatment of the multilayer sacrificial material-hydrogel composite by a solvent dissolution method to obtain a multilayer biomimetic scaffold.

[0024] In the application, the value of n depends on the light transmission performance of the components in the sacrificial material and the biomimetic hydrogel precursor solution. In order to make the curing more sufficient, when the light transmission performance of the above components is not good, the value of n needs to be increased accordingly.

[0025] In some embodiments, the biomimetic hydrogel precursor solution comprises a transparent material and does not comprise a translucent material or an osteogenic material, and n is 10-50.

[0026] In some embodiments, the biomimetic hydrogel precursor solution comprises a translucent material and does not comprise an osteogenic material, and n is 3-10.

[0027] In some embodiments, the biomimetic hydrogel precursor solution comprises an osteogenic material, and n is 1-5.

[0028] In the application, the steps S1 and S2 are repeated, and the perfusion can be performed using a biomimetic hydrogel precursor solution with the same components or a biomimetic hydrogel precursor solution with different components.

[0029] The preparation method of the multilayer biomimetic scaffold provided by the application has the following advantages:

[0030] Firstly, the layer-by-layer process can control the thickness of each layer, enabling sufficient curing of the bottom layer material by light, thereby effectively avoiding the problem of incomplete one-time photocuring of thick scaffolds. Considering the transmission depth limit of photocuring, the thickness of the sacrificial material and the perfused biomimetic hydrogel precursor solution cannot be too thick, otherwise the light source cannot be fully transmitted and crosslinking curing cannot be completed. The present application gradually accumulates the required thickness by layer-by-layer circulation of "printing-perfusing-curing", thereby ensuring that each layer is fully cured, ensuring the integrity and mechanical properties of the overall structure of the scaffold;

[0031] Secondly, by layer-by-layer circulation of "printing-perfusing-curing", the perfusion of the biomimetic hydrogel precursor solution of the next layer after the curing of the previous layer of hydrogel can achieve precise division of components between layers, preventing the mixing and diffusion of different components of the biomimetic hydrogel precursor solution at the interface in the traditional overall perfusion method. In this way, the selection of hydrogel for each layer is more flexible, and different formulations of hydrogel can be selected flexibly according to the biomimetic needs;

[0032] Thirdly, the perfusion of the biomimetic hydrogel precursor solution of the next layer after the curing of the previous layer of hydrogel, the surface formed after the curing of the previous layer of hydrogel still has active photosensitive groups, which can chemically crosslink with the molecules in the biomimetic hydrogel precursor solution of the next layer during the photocuring crosslinking process, forming a covalently bonded interface, thereby achieving firm chemical bonding between layers and obtaining a firm integrated scaffold, while avoiding the mixing of components between different layers;

[0033] Finally, the present application uses an "internal demolding method" to print an internal mold using a high molecular material (such as PLA, PCL, PVA, etc.) as a dissolvable sacrificial material. Compared with the preparation method of directly 3D printing with a biomimetic hydrogel precursor solution, the high molecular material has excellent printing performance and higher printing precision. After selectively dissolving and removing the sacrificial material (internal mold), a three-dimensionally interconnected and through-porous network is formed, which can achieve precise control and gradient design of porosity and pore size. The pore size can be precisely controlled according to the biomimetic needs of different layers, thereby achieving the best matching of the mechanical properties and biological functions of each layer.

[0034] In summary, this preparation method realizes layered biomimetic design based on the internal demolding technology, ensures that the hydrogel materials of different biomimetic layers exist independently, avoids the mixing and diffusion of biomimetic materials between adjacent layers during one-time perfusion, precisely realizes the composition of different biomimetic layers, and also ensures the effective curing of the hydrogel material.

[0035] In the present application, in step S2, the sacrificial material-hydrogel composite comprises a porous mold and a biomimetic hydrogel formed by photo-crosslinking of the biomimetic hydrogel precursor solution filled in the porous mold, and the biomimetic hydrogel is formed into a corresponding layer of the multilayer biomimetic scaffold after the internal demolding treatment in step S3.

[0036] In the present application, the layers of the multilayer sacrificial material-hydrogel composite prepared by repeating steps S1 and S2 are sequentially connected, and have good mechanical strength between layers.

[0037] In the present application, the sacrificial material-hydrogel composite in step S2 refers to a sacrificial material-hydrogel composite that has completed photo-crosslinking, and subsequent steps are performed on the upper surface thereof. The upper surface refers to the surface that is away from and parallel to the printing platform during 3D printing.

[0038] In some preferred embodiments, in step S1, the thickness of the grid-shaped porous internal mold is 0.1-15 mm, preferably 1-5 mm.

[0039] In some preferred embodiments, in step S1, the 3D printing is performed by extrusion grid printing. The use of extrusion grid printing can achieve more precise pore structure and facilitate the realization of the overall through structure of the multilayer biomimetic scaffold. The extrusion grid printing can also be referred to as extrusion vertical cross printing.

[0040] In some embodiments, in step S1, the 3D printing is performed by fused deposition modeling (FDM) 3D printing.

[0041] In some preferred embodiments, the parameters of the fused deposition modeling 3D printing include:

[0042] The printing temperature is 200-250°C, for example, 210°C;

[0043] The platform temperature is 50-70°C, for example, 60°C;

[0044] The printing speed is 50-80 mm / s, for example, 60 mm / s;

[0045] The height of a single printed layer is 0.1-0.3 mm, for example, 0.2 mm.

[0046] In the present application, the grid-shaped porous inner mold is obtained by 3D printing with a sacrificial material. At this time, the filament diameter of the sacrificial material extruded by the nozzle of 3D printing becomes the pore diameter of the hydrogel in the multilayer biomimetic scaffold. The pore diameter of the grid-shaped porous inner mold of the sacrificial material becomes the filament diameter of the hydrogel. Therefore, the filament diameter of the sacrificial material during printing is close to the stable filament width formed by the nozzle diameter. Once the nozzle diameter is determined, the filament diameter of the corresponding sacrificial material during printing (i.e. the pore diameter of the hydrogel) is within a controllable range. On this basis, controlling the nozzle diameter within a suitable range such as 0.25-0.40 mm has the following advantages: ① ensures continuous filament extrusion; ② ensures smooth nozzle without blockage; ③ ensures printing speed and controls size consistency; ④ ensures the interlayer bonding strength of the printed structure, avoids deformation or collapse of the inner demolding structure due to insufficient interlayer adhesion, ensures smooth pore channels, and obtains excellent permeability and actual accessible surface.

[0047] In the present application, the single-layer printing layer height in the range of 0.1-0.3 mm can ensure the size and strength of the inner mold, and the demolding without deformation. However, if the single-layer printing layer height is too large or too small, the above-mentioned excellent multilayer biomimetic scaffold cannot be obtained. Specifically, if the single-layer printing layer height is too small (<0.1 mm): the printing strip is pressed very flat, the pore opening is narrowed, and even at some positions, it is almost closed; it looks more "dense", but the actual accessible surface is reduced, and the horizontal position permeable hole is reduced; if the single-layer printing layer height is too large (>0.3 mm): the contact between the upper and lower layers is less, and the layers are easy to separate; the scaffold is more prone to collapse or deformation under immersion or slight stress. At the same time, due to the increase of the single-layer printing layer height, the number of layers per unit thickness decreases, and the specific surface area decreases accordingly.

[0048] In some specific embodiments, in step S1, the 3D printing is fused deposition modeling 3D printing, the printing temperature is 210℃, the platform temperature is 60℃, the printing speed is 60 mm / s, and the single-layer printing layer height is 0.2 mm.

[0049] In the present application, in step S1, the sacrificial material can be a conventional high molecular material in the field, preferably including one or more of polylactic acid (PLA), polycaprolactone (PCL) and polyvinyl alcohol (PVA), for example, PLA.

[0050] In some embodiments, in step S1, the sacrificial material used in each step S1 is the same or different.

[0051] In some preferred embodiments, in step S1, the type of sacrificial material used in each step S1 is the same.

[0052] In some more preferred embodiments, in step S1, the sacrificial material in the multi-layer sacrificial material-hydrogel composite is PLA.

[0053] In some preferred embodiments, in step S1, the 3D printing is fused deposition modeling 3D printing, and the printing parameter of single-layer printing layer height is set to 0.1-0.3 mm, preferably 0.2 mm. The number of layers n of each continuous printing is selected according to the light transmittance of the hydrogel, for example:

[0054] For transparent biomimetic hydrogel precursor solutions (such as gelatin-containing, hyaluronic acid-based hydrogels), 10-50 layers of sacrificial material (e.g., 2-5 mm thick) can be printed each time and then perfused;

[0055] For semi-transparent biomimetic hydrogel precursor solutions (such as elastin-containing), 3-10 layers of sacrificial material (e.g., 1-2 mm thick) can be printed each time and then perfused;

[0056] For biomimetic hydrogel precursor solutions containing particulate materials (such as hydrogels containing hydroxyapatite, bioglass particles, or cytokine release microspheres), 1-5 layers of sacrificial material (e.g., 0.2-0.6 mm thick) can be printed each time and then perfused.

[0057] In the present application, in step S2, the photo-curing crosslinking can be performed using conventional methods in the art under light irradiation.

[0058] In some embodiments, in step S2, the light source for photo-curing crosslinking is ultraviolet light, blue light, or near-infrared light; wherein the wavelength of the ultraviolet light is preferably 355-375 nm, for example 365 nm; the wavelength of the blue light is preferably 395 nm-415 nm, for example 405 nm; and the wavelength of the near-infrared light is preferably 700-2500 nm.

[0059] In some embodiments, in step S2, the photo-curing crosslinking time is 10-80 s, preferably 30-80 s, for example 45 s or 60 s.

[0060] In some embodiments, in step S2, the photo-curing crosslinking is performed under light irradiation with an intensity of 5-50 mW / cm 2 .

[0061] In some preferred embodiments, the photo-curing crosslinking is performed under light irradiation with a wavelength of 365-405 nm and an intensity of 5-50 mW / cm 2 .

[0062] In some specific embodiments, the photo-curing crosslinking is performed under light irradiation with a wavelength of 405 nm and an intensity of 10 mW / cm 2 .

[0063] In some embodiments, in step S2, the photocuring crosslinking is performed under irradiation of light with a wavelength of 405 nm and an intensity of 10 mW / cm2for 60 s.

[0064] In some embodiments, in step S1, the photocuring crosslinking is performed under irradiation of light with a wavelength of 405 nm and an intensity of 10 mW / cm2for 45 s.

[0065] In some preferred embodiments, in step S3, the repeating of steps S1 and S2 is performed for 1-5 times.

[0066] In the present application, in step S3, the solvent used in the solvent dissolution method can be an organic solvent conventionally used in the art to selectively dissolve the sacrificial material, preferably including one or both of chloroform and dichloromethane. Among them, the "selective dissolution" refers to dissolving only the sacrificial material without affecting the hydrogel after photocuring crosslinking.

[0067] In the present application, in step S2, the biomimetic hydrogel precursor solution can include a hydrogel component, a hydrogel medium and a photoinitiator.

[0068] In the present application, the hydrogel component can be selected and designed according to the characteristics of the biomimetic object (i.e. the target natural multi-layer tissue) of the multi-layer biomimetic scaffold.

[0069] In some embodiments, the hydrogel component includes one or a combination of several of methacrylated gelatin (GelMA), methacrylated collagen, methacrylated elastin (ElaMA), methacrylated keratin (KerMA), methacrylated hyaluronic acid (HAMA), methacrylated chondroitin sulfate (CSMA), methacrylated sodium alginate, methacrylated heparin, gelatin, collagen, elastin, hyaluronic acid, chondroitin sulfate, heparin and sodium alginate.

[0070] In the present application, the hydrogel medium can be conventional in the art and can be selected according to the gelable component.

[0071] In some embodiments, the hydrogel medium is physiological saline, cell culture medium, phosphate buffered solution (PBS) or carbonate buffered solution. Among them, the cell culture medium is preferably DMEM, DMEM / F12 or RPMI 1640; the pH of the phosphate buffered solution is preferably 7.4.

[0072] In some embodiments, the hydrogel medium further comprises glycerol and / or DMSO. Among them, the volume fraction of the glycerol is preferably 5%-30%, and the percentage is the volume percentage of the glycerol in the volume of the hydrogel medium; the volume fraction of the DMSO is preferably 5%-50%, and the percentage is the volume percentage of the DMSO in the volume of the hydrogel medium.

[0073] In the present application, by further introducing glycerol and / or DMSO into the hydrogel medium, the following two advantages can be achieved: on the one hand, a low-volatility moisturizing mixed co-solvent system can be realized, the evaporation rate is reduced, and the perfusion rheology is stabilized, and after solidification is completed, it can be washed away in the subsequent demolding process; on the other hand, it is beneficial to the control of volatilization under the platform warming condition: when using fused deposition modeling (FDM) 3D printing sacrificial material, the printing platform is usually maintained at 50-70℃ to ensure the forming accuracy and interlayer adhesion; under this temperature condition, using water-based (such as physiological saline, PBS, etc.) combined with glycerol and / or DMSO as the hydrogel medium for perfusion can inhibit local water volatilization during perfusion and solidification, thereby avoiding changes in the effective concentration of the biomimetic hydrogel precursor solution, which is beneficial to ensuring the consistency of the interfacial morphology and the pores.

[0074] In some specific embodiments, the hydrogel medium is PBS.

[0075] In some specific embodiments, the hydrogel medium is PBS, glycerol and DMSO; the volume ratio of the PBS, glycerol and DMSO is, for example, 50:25:25.

[0076] In the present application, the photoinitiator can be a photoinitiator commonly used in the art. The photoinitiator can initiate a covalent cross-linking reaction under light irradiation, thereby improving the cross-linking degree, stability and controllability of degradation of the hydrogel, so as to adapt to the needs of the tissue repair process.

[0077] In some embodiments, the photoinitiator is a blue light photoinitiator, an ultraviolet light photoinitiator or a near-infrared light photoinitiator.

[0078] Among them, the blue light photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), riboflavin, flavin mononucleotide, eosin Y or tris (2,2'-bipyridyl) ruthenium chloride / sodium persulfate, for example, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate.

[0079] Among them, the ultraviolet light photoinitiator is preferably I2959.

[0080] Among them, the near-infrared light photoinitiator is, for example, squarylium cyanine, and the model number of the squarylium cyanine is, for example, SQ-739.

[0081] In some embodiments, the biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5-20% methacrylated gelatin, 10-40% osteogenic material, and 0.1-0.5% photoinitiator; wherein the osteogenic material comprises one or more of bioactive glass, tricalcium phosphate, inorganic bone powder, and hydroxyapatite; wherein the concentrations are the mass (g) of each component per volume (mL) of hydrogel medium in the biomimetic hydrogel precursor solution.

[0082] In some embodiments, the biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5-20% methacrylated gelatin, 5-15% osteogenic material, and 0.1-0.5% photoinitiator; wherein the osteogenic material comprises one or more of bioactive glass, tricalcium phosphate, inorganic bone powder, and hydroxyapatite; wherein the concentrations are the mass (g) of each component per volume (mL) of hydrogel medium in the biomimetic hydrogel precursor solution.

[0083] In some embodiments, the biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5-20% methacrylated gelatin, 0.5-5% methacrylated hyaluronic acid, 0.5-5% methacrylated chondroitin sulfate, and 0.1-0.5% photoinitiator; wherein the concentrations are the mass (g) of each component per volume (mL) of hydrogel medium in the biomimetic hydrogel precursor solution.

[0084] In some embodiments, the biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5-20% methacrylated gelatin, 0.5-5% methacrylated elastin, and 0.1-0.5% photoinitiator; wherein the concentrations are the mass (g) of each component per volume (mL) of hydrogel medium in the biomimetic hydrogel precursor solution.

[0085] In some embodiments, the biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5-20% methacrylated gelatin, 0.5-5% methacrylated keratin, and 0.1-0.5% photoinitiator; wherein the concentrations are the mass (g) of each component per volume (mL) of hydrogel medium in the biomimetic hydrogel precursor solution.

[0086] In the present invention, the multi-layered biomimetic scaffold can be a three-layered biomimetic scaffold or a two-layered biomimetic scaffold.

[0087] Method of making osteochondral biomimetic scaffold

[0088] In some alternative embodiments, the multi-layered biomimetic scaffold is an osteochondral biomimetic scaffold.

[0089] In some preferred embodiments, the method for preparing the osteocartilage biomimetic scaffold comprises the following steps:

[0090] S1, layer-by-layer 3D printing of a sacrificial material, continuously printing n layers to obtain a grid-shaped porous inner mold; wherein the single-layer printing layer height is 0.1-0.3 mm, and n is 1-5;

[0091] S2, pouring a bone layer biomimetic hydrogel precursor solution into the grid-shaped porous inner mold, and then performing photocuring crosslinking to form part of a bone layer composite;

[0092] S3-1, repeatedly performing steps S1 and S2 on the upper surface of the part of the bone layer composite obtained in step S2 to form a bone layer composite; wherein the number of layers n continuously printed in each step S1 is the same, and the bone layer biomimetic hydrogel precursor solution poured in each step S2 is the same;

[0093] S3-2, repeatedly performing steps S1 and S2 on the upper surface of the bone layer composite to form an intermediate layer composite; wherein the biomimetic hydrogel precursor solution poured in step S2 is an intermediate layer biomimetic hydrogel precursor solution;

[0094] S3-3, repeatedly performing steps S1 and S2 on the upper surface of the intermediate layer composite to form a cartilage layer composite; wherein in step S1, n is 3-10, and the biomimetic hydrogel precursor solution poured in step S2 is a cartilage layer biomimetic hydrogel precursor solution;

[0095] After steps S1, S2, S3-1, S3-2, and S3-3, a multilayer sacrificial material-hydrogel composite is obtained, which sequentially comprises a bone layer composite, an intermediate layer composite, and a cartilage layer composite;

[0096] S4, performing inner demolding treatment on the multilayer sacrificial material-hydrogel composite by a solvent dissolution method to obtain an osteocartilage biomimetic scaffold.

[0097] In some more preferred embodiments, the bone layer biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5%-20% methacrylated gelatin (GelMA), 10%-40% osteogenic material, and 0.1%-0.5% photoinitiator;

[0098] The intermediate layer biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5%-20% methacrylated gelatin (GelMA), 5%-15% osteogenic material, and 0.1%-0.5% photoinitiator;

[0099] The cartilage layer biomimetic hydrogel precursor solution comprises the following concentrations of each component: 5%-20% methacrylated gelatin (GelMA), 0.5%-5% methacrylated hyaluronic acid (HAMA), 0.5%-5% methacrylated chondroitin sulfate (CSMA), and 0.1%-0.5% photoinitiator;

[0100] wherein the concentration is the ratio of the mass (g) of each component to the volume (mL) of the hydrogel medium in the biomimetic hydrogel precursor solution;

[0101] wherein the osteogenic material comprises one or more of bioactive glass, tricalcium phosphate, inorganic bone powder, and hydroxyapatite (HAP).

[0102] In the present application, the intermediate layer composite is a transition composite for transitioning from the bone layer composite to the cartilage layer composite.

[0103] In the prior art, on the one hand, the biomimetic hydrogel precursor solution containing osteogenic material in the form of solid particles (such as bioactive glass, hydroxyapatite, etc.) is prone to clogging the nozzle when directly 3D printed, and it is difficult to achieve multi-layer scaffold preparation containing particles, and direct printing has high requirements for the rheological properties of the hydrogel formulation, and a compromise needs to be made between printability and biomimicry, resulting in an optimized biomimetic hydrogel precursor solution formulation that is often not suitable for direct printing preparation; on the other hand, when preparing thick layer scaffolds by one-time perfusion, the hydroxyapatite and other osteogenic materials in the form of solid particles also block light, further affecting the light curing effect, resulting in incomplete curing inside the scaffold and a decrease in mechanical properties. In the above embodiments, the formulation of the biomimetic hydrogel precursor solution is further optimized, and components simulating the natural extracellular matrix (ECM) are introduced, while the biological activity of the obtained multi-layer biomimetic scaffold is improved, specifically, methacrylated hyaluronic acid (HAMA) and chondroitin sulfate (CSMA) are added to the biomimetic hydrogel precursor solution corresponding to the cartilage layer to promote the formation of cartilage matrix, and inorganic components such as bioactive glass, tricalcium phosphate, inorganic bone powder, and hydroxyapatite (HAP) are added to the biomimetic hydrogel precursor solution of the bone layer and the intermediate layer to enhance the osteogenic performance.

[0104] In some preferred embodiments, the particle size Dv50 of the bioactive glass is 30-50 μm, for example 40 μm. In the prior art, when the biomimetic hydrogel precursor solution containing bioactive glass and other osteogenic materials in the form of solid particles is directly 3D printed, it will cause clogging, and in the one-time perfusion mode, the solid particles are prone to sedimentation and are difficult to distribute uniformly, while in the present application, the "layer-by-layer perfusion-printing-curing" mode can avoid this defect.

[0105] In some more preferred embodiments, in step S1, the bone layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 5-20% methacrylated gelatin, 10-40% bioactive glass, and 0.1-0.5% photoinitiator.

[0106] In some specific embodiments, in step S1, the bone layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 15% methacrylated gelatin, 30% bioactive glass, and 0.2% photoinitiator.

[0107] In some more preferred embodiments, in step S1, the bone layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 5-20% methacrylated gelatin, 10-30% hydroxyapatite, and 0.1-0.5% photoinitiator.

[0108] In some more preferred embodiments, in step S1, the intermediate layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 5-20% methacrylated gelatin, 5-15% bioactive glass, and 0.1-0.5% photoinitiator.

[0109] In some specific embodiments, in step S1, the intermediate layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 15% methacrylated gelatin, 10% bioactive glass, and 0.2% photoinitiator.

[0110] In some more preferred embodiments, in step S1, the intermediate layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 5-20% methacrylated gelatin, 5-15% hydroxyapatite, and 0.1-0.5% photoinitiator.

[0111] In some specific embodiments, in step S1, the cartilage layer biomimetic hydrogel precursor solution comprises each component at the following concentrations: 10% methacrylated gelatin, 0.5% methacrylated hyaluronic acid, 1% methacrylated chondroitin sulfate, and 0.2% photoinitiator.

[0112] In some more preferred embodiments, in step S1, the bone layer biomimetic hydrogel precursor solution, the mass ratio of the methacrylated gelatin, bioactive glass, and photoinitiator is 1: (1-5): (0.005-0.05).

[0113] In some more preferred embodiments, in step S1, the intermediate layer biomimetic hydrogel precursor solution, the mass ratio of the methacrylated gelatin, bioactive glass, and photoinitiator is 1: (0.1-0.8): (0.05-0.5).

[0114] In some more preferred embodiments, in step S1, the mass ratio of the methacrylated gelatin, the methacrylated hyaluronic acid, the methacrylated chondroitin sulfate and the photoinitiator in the cartilage layer biomimetic hydrogel precursor solution is 1: (0.01-0.1): (0.05-0.5): (0.01-0.05).

[0115] A method for preparing a skin biomimetic scaffold

[0116] In some alternative embodiments, the multi-layer biomimetic scaffold is a skin biomimetic scaffold.

[0117] In some preferred embodiments, the method for preparing the skin biomimetic scaffold comprises the following steps:

[0118] S1, layer-by-layer 3D printing of a sacrificial material, continuously printing n layers to obtain a grid-shaped porous inner mold; wherein the single-layer printing layer height is 0.1-0.3 mm, and n is 10-50;

[0119] S2, pouring a dermis layer biomimetic hydrogel precursor solution into the grid-shaped porous inner mold, and then performing photocuring crosslinking to form a dermis layer composite;

[0120] S3, repeating steps S1 and S2 on the upper surface of the dermis layer composite obtained in step S2 to form an epidermis layer composite; wherein the biomimetic hydrogel precursor solution poured in step S2 is an epidermis layer biomimetic hydrogel precursor solution;

[0121] Through steps S1, S2 and S3, a multi-layer sacrificial material-hydrogel composite is obtained, which comprises a dermis layer composite and an epidermis layer composite in sequence;

[0122] S4, performing inner demolding treatment on the multi-layer sacrificial material-hydrogel composite by a solvent dissolution method to obtain a skin biomimetic scaffold;

[0123] In some preferred embodiments, the dermis layer biomimetic hydrogel precursor solution comprises the following concentrations of components: 5%-20% methacrylated gelatin, 0.5%-5% methacrylated elastin and 0.1%-0.5% photoinitiator.

[0124] In some preferred embodiments, the epidermis layer biomimetic hydrogel precursor solution comprises the following concentrations of components: 5%-20% methacrylated gelatin, 0.5%-5% methacrylated keratin and 0.1%-0.5% photoinitiator.

[0125] In some preferred embodiments, the concentration is the ratio of the mass (g) of each component to the volume (mL) of the hydrogel medium in the biomimetic hydrogel precursor solution.

[0126] In some embodiments, in step S2, the dermal layer biomimetic hydrogel precursor solution comprises the following concentrations of each component: 10% methacrylated gelatin, 1% methacrylated elastin, and 0.25% photoinitiator.

[0127] In some embodiments, in step S2, the epidermal layer biomimetic hydrogel precursor solution comprises the following concentrations of each component: 10% methacrylated gelatin, 1% methacrylated keratin, and 0.25% photoinitiator.

[0128] The present application provides a multi-layer biomimetic scaffold prepared by the method for preparing a multi-layer biomimetic scaffold as described above.

[0129] In the present application, the multi-layer biomimetic scaffold can be a three-layer biomimetic scaffold or a two-layer biomimetic scaffold, such as an osteochondral biomimetic scaffold, a skin biomimetic scaffold, a gastrointestinal tract biomimetic scaffold, a bladder biomimetic scaffold, or a skull biomimetic scaffold.

[0130] Osteochondral biomimetic scaffold

[0131] In the present application, the multi-layer biomimetic scaffold can be an osteochondral biomimetic scaffold.

[0132] In some preferred embodiments, the multi-layer biomimetic scaffold is an osteochondral biomimetic scaffold comprising a bone layer, an intermediate layer, and a cartilage layer covalently connected in sequence; each of the bone layer, the intermediate layer, and the cartilage layer is a through-porous structure.

[0133] In the present application, the intermediate layer is a transition layer for transitioning from the bone layer to the cartilage layer.

[0134] In some preferred embodiments, the bone layer, the intermediate layer, and the cartilage layer are arranged in a vertical and horizontal cross stereo grid pattern.

[0135] In some preferred embodiments, the bone layer has a pore size of 300-500 μm, a porosity of 30%-50%, and a thickness of 1-5 mm;

[0136] The intermediate layer has a pore size of 200-400 μm, a porosity of 50%-70%, and a thickness of 0.2-0.8 mm;

[0137] The cartilage layer has a pore size of 100-300 μm, a porosity of 50%-90%, and a thickness of 1-3 mm.

[0138] In some preferred embodiments, the intermediate layer does not cover or partially covers the pores of the cartilage layer and / or the bone layer.

[0139] In some preferred embodiments, the osteochondral biomimetic scaffold is a through-porous structure.

[0140] In some embodiments, the bone layer has a pore size of 500 μm, a porosity of 50%, and a thickness of 4 mm.

[0141] In some embodiments, the intermediate layer has a pore size of 300 μm, a porosity of 60%, and a thickness of 0.6 mm.

[0142] In some embodiments, the cartilage layer has a pore size of 250 μm, a porosity of 80%, and a thickness of 2 mm.

[0143] In some embodiments, the bone layer has a pore size of 500 μm, a porosity of 50%, and a thickness of 4 mm; the intermediate layer has a pore size of 300 μm, a porosity of 60%, and a thickness of 0.6 mm; and the cartilage layer has a pore size of 250 μm, a porosity of 80%, and a thickness of 2 mm.

[0144] In the present application, the mechanical strength between the layers of the osteochondral biomimetic scaffold is comparable to the mechanical strength within the layers of the bone layer, the intermediate layer, and the cartilage layer.

[0145] Skin biomimetic scaffold

[0146] In the present application, the multi-layer biomimetic scaffold can be a skin biomimetic scaffold.

[0147] In some preferred embodiments, the multi-layer biomimetic scaffold is a skin biomimetic scaffold, which comprises a covalently linked dermis layer and an epidermis layer, both of which are through-porous structures.

[0148] In some preferred embodiments, the pores in the dermis layer and the epidermis layer are arranged in a vertical cross pattern.

[0149] In some preferred embodiments, the dermis layer has a pore size of 100-300 μm, for example 250 μm, a porosity of 40%-60%, for example 50%, and a thickness of 1-4 mm, for example 2 mm.

[0150] In some preferred embodiments, the epidermis layer has a pore size of 50-100 μm, for example 60 μm, a porosity of 70%-90%, for example 80%, and a thickness of 0.2-2 mm, for example 1.5 mm.

[0151] In some preferred embodiments, the dermis layer has a pore size of 250 μm, a porosity of 50%, and a thickness of 2 mm; and the epidermis layer has a pore size of 60 μm, a porosity of 80%, and a thickness of 1.5 mm.

[0152] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred examples of the present application.

[0153] The reagents and raw materials used in the present application are commercially available.

[0154] The positive progress effect of the present application is that:

[0155] (1) The present application realizes the true sense of hierarchical biomimetic design. Through the process design of "printing-perfusion-curing" in each layer, further combined with the optimization design of the formula, the independent existence of the hydrogel materials of different biomimetic layers is ensured, the mixing and diffusion problem of the materials between adjacent layers during one-time perfusion is avoided, and the precise realization of different component compositions of each biomimetic layer is ensured. The method of the present application can prepare a multi-layer biomimetic scaffold with stable structure, controllable porosity and good biological activity. The interconnected porous network is formed inside the obtained scaffold, and the structures of each layer are closely combined, which simulates the gradient transition characteristics of natural multi-layer tissues, has good pore structure and interlayer bonding strength, and can more accurately simulate the heterogeneous structure and functional partition characteristics of natural tissues, and provides a specific microenvironment for the growth of different types of cells.

[0156] (2) In view of the problem of weak combination caused by the material or structural difference of different functional layers, the method of printing-curing in each layer of the present application effectively prevents the interface separation, and ensures the structural integrity and excellent mechanical properties of the whole multi-layer scaffold. In the preparation method of the present application, stable covalent bond is formed between adjacent functional layers, which ensures that the layers are closely connected and will not fall off. At the same time, due to the use of high-precision 3D printing sacrificial material mold and selective dissolution method, the obtained multi-layer biomimetic scaffold still retains the through-pore structure while maintaining the firm combination between layers, and does not appear the phenomenon of sealing the pores, which is beneficial to cell growth, nutrient substance transmission and tissue regeneration.

[0157] (3) The strategy of printing sacrificial material mold in each layer, perfusing biological ink in each layer and curing in each layer is particularly suitable for biological ink containing solid-phase particles (such as hydroxyapatite HAP particles). Such ink often faces the risk of nozzle blockage and the challenge of insufficient light curing in 3D printing due to the presence of particles. The method of "printing-perfusion-curing" in each layer of the present application not only effectively overcomes the limitation of light penetration depth, ensures that each part can still be fully light cross-linked in the presence of high-concentration light-blocking materials, thereby obtaining excellent and uniform mechanical properties, but also provides a more reliable and effective technical approach for stably integrating such functional particle-containing and difficult-to-print materials into complex multi-layer composite scaffold structures.

[0158] Further preferably, by employing a low-volatility moisturizing mixed co-solvent system as the hydrogel medium, such as PBS supplemented with glycerol and dimethyl sulfoxide (DMSO) to reduce evaporation rate and stabilize perfusion rheology, volatilization control under platform warming conditions can be achieved. Glycerol and DMSO can be washed away after solidification is completed during subsequent demolding. BRIEF DESCRIPTION OF DRAWINGS

[0159] Figure 1 A comparison chart of the mechanical properties of the scaffolds based on different concentrations of HAP hydrogel precursor solutions in Example 2 (layer-by-layer solidification) and Comparative Example 1 (one-time solidification).

[0160] Figure 2 A scanning electron microscope image of the inside of the osteochondral biomimetic scaffold obtained in Example 1.

[0161] Figure 3 A micro-CT three-dimensional reconstruction image of the osteochondral biomimetic scaffold obtained in Example 1.

[0162] Figure 4 A cell culture section image of the layered biomimetic scaffold obtained in Example 3 (each layer showing the cell growth condition of the corresponding pore size and porosity structure).

[0163] Figure 5 A comparison chart of the interlayer and intralayer shear strength of the osteochondral biomimetic scaffold obtained in Example 1.

[0164] Figure 6 A comparison chart of the mechanical strength of the osteochondral biomimetic scaffold obtained in Example 1 before and after mineralization of the layered structure.

[0165] Figure 7 A comparison chart of the mechanical properties of the osteochondral biomimetic scaffold obtained in Example 1 before and after mineralization of the layered structure.

[0166] Figure 8 A comparison chart of the mechanical properties of the osteochondral biomimetic scaffold obtained in Example 1 before and after mineralization of the layered structure.

[0167] Figure 9 A micro-CT image of the osteochondral biomimetic scaffold obtained in Example 1 implanted into the joint defect of a rabbit.

[0168] Figure 10 A histological staining image of the osteochondral biomimetic scaffold obtained in Example 1 implanted into the joint defect of a rabbit.

[0169] Figure 11 A fluorescence microscope observation image of the dermal cells cultured on the dermal layer of the skin biomimetic scaffold obtained in Example 5.

[0170] Figure 12Fig. 2 shows the fluorescence microscope observation of the epidermal cells cultured on the epidermal layer of the skin biomimetic scaffold obtained in Example 5. DETAILED DESCRIPTION

[0171] The present application is further illustrated by the following examples without thereby limiting the present application to the examples described. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to the conventional methods and conditions, or according to the instructions of the commercial products.

[0172] Example 1 Preparation of osteochondral biomimetic scaffold

[0173] 1. Design of inner mold and setting of printing parameters

[0174] In this example, polylactic acid (PLA) material was selected as the sacrificial material. In this example, a fused deposition modeling (FDM) 3D printer was used for layer-by-layer printing of the sacrificial material, with the printing nozzle temperature set to 210°C, the platform temperature set to 60°C, the printing speed set to 60 mm / s, and the single-layer printing layer height set to 0.2 mm.

[0175] According to the osteochondral 3D model, a grid with different pore structures in different regions was designed: the porosity of the bone layer region was 50%, and the grid line diameter was set to 500 μm; the porosity of the middle layer region was 60%, and the grid line diameter was set to 300 μm; the porosity of the cartilage layer region was 80%, and the grid line diameter was set to 250 μm, to simulate the structural characteristics of the natural osteochondral tissue.

[0176] 2. Preparation of biomimetic hydrogel precursor solution

[0177] The following three kinds of biomimetic hydrogel precursor solutions were prepared respectively (the concentration of each component was calculated from the mass of each component / PBS volume):

[0178] (1) Bone layer biomimetic hydrogel precursor solution: GelMA was dissolved in PBS to prepare a solution with a concentration of 15% (w / v), and then 30% (w / v) of bioactive glass (BG) was added.

[0179] (2) Middle layer biomimetic hydrogel precursor solution: GelMA was dissolved in PBS to prepare a solution with a concentration of 15% (w / v), and then 10% (w / v) of bioactive glass was added.

[0180] (3) Cartilage layer biomimetic hydrogel precursor solution: GelMA, HAMA and CSMA were dissolved in PBS to prepare a mixed solution with a GelMA concentration of 10% (w / v), a HAMA concentration of 0.5% (w / v), and a CSMA concentration of 1% (w / v).

[0181] Wherein, the particle size Dv50 of BG is 40 μm;

[0182] The light initiator LAP is added in all solutions with a concentration of 0.2% (w / v), and the pH of PBS is 7.4. After the above solutions are prepared, they are preheated in a 50°C water bath for 10 min to reduce the viscosity and facilitate perfusion.

[0183] 3. Preparation of osteochondral biomimetic scaffold

[0184] In this embodiment, the integrated osteochondral biomimetic scaffold (diameter 7 mm) with mechanical gradient is prepared by using the "layer-by-layer printing-perfusion-photocuring" cycle combined with internal demolding dissolution process. The specific steps are as follows:

[0185] (1) Preparation of bone layer complex:

[0186] Based on the design parameters of the bone layer, the PLA sacrificial material is printed layer by layer on the 3D printer platform, and 5 layers are continuously printed, with a total printing thickness of about 1 mm.

[0187] The bone layer biomimetic hydrogel precursor solution (GelMA + 30% bioactive glass) is preheated and perfused into the printed PLA grid structure to ensure that the solution fills the pores of the grid.

[0188] Using a 405 nm blue light (LED light source, light intensity 10 mW / cm²) for 60 seconds, the bone layer biomimetic hydrogel precursor solution is photocrosslinked to form the first part of the bone layer.

[0189] The above "layer-by-layer printing-perfusion-photocuring" process is repeated 3 times to construct a bone layer complex with a thickness of about 4 mm.

[0190] (2) Preparation of intermediate layer complex:

[0191] On the surface of the prepared bone layer complex, the PLA sacrificial material is continuously printed for 3 layers, with a total printing thickness of about 0.6 mm.

[0192] The intermediate layer biomimetic hydrogel precursor solution (GelMA + 10% bioactive glass) is preheated and perfused into the newly printed PLA grid structure.

[0193] Using the same light source for 60 seconds, the hydrogel precursor solution is photocrosslinked to form the intermediate layer, and an intermediate layer complex with a thickness of about 0.6 mm is constructed.

[0194] (3) Preparation of cartilage layer complex:

[0195] On the surface of the prepared intermediate layer complex, based on the design parameters of the cartilage layer, the PLA sacrificial material is continuously printed for 10 layers, with a total printing thickness of about 2 mm.

[0196] The chondral layer of the biomimetic hydrogel precursor solution (GelMA+HAMA+CSMA) was preheated and perfused into the newly printed PLA lattice structure.

[0197] The chondral layer of the biomimetic hydrogel precursor solution was photo-crosslinked using the same light source for 45 seconds to form a 2mm-thick chondral layer composite.

[0198] (4) Internal demolding treatment:

[0199] After completing the printing-perfusion-photo-curing process of the entire bone layer composite, the intermediate layer composite, and the chondral layer composite, the constructed multilayer sacrificial material-hydrogel composite was immersed in dichloromethane to dissolve the PLA sacrificial material, and finally the scaffold was thoroughly washed with ultrapure water to ensure complete removal of residual solvents, and a bone-cartilage biomimetic scaffold with a fine pore structure was obtained after freeze-drying.

[0200] After the above preparation process, the bone-cartilage biomimetic scaffold formed has the following characteristics:

[0201] Bone layer: composed of GelMA+30% bioactive glass, porosity 50%, pore size 500 μm, thickness about 4mm;

[0202] Intermediate layer: composed of GelMA+10% bioactive glass, porosity 60%, pore size 300 μm, thickness about 0.6mm;

[0203] Chondral layer: composed of GelMA+HAMA+CSMA, porosity 80%, pore size 250 μm, thickness about 2mm;

[0204] The three layers are stably covalently bonded through the photo-curing process, ensuring that there is no interlayer peeling. The overall scaffold retains a through-pore structure and does not have a sealing phenomenon, which is conducive to cell growth and nutrient transport. At the same time, the layer-by-layer solidification process ensures that the components of the biomaterials in each layer remain independently distributed without intermixing, achieving the layered biomimetic characteristics of bone-cartilage tissue.

[0205] Example 2:

[0206] 1. Preparation of biomimetic hydrogel precursor solution

[0207] (1) Prepare the basic biomimetic hydrogel precursor solution: 15%(w / v) GelMA and 0.2%(w / v) photo-initiator LAP in PBS solution.

[0208] (2) Add different concentrations of hydroxyapatite (HAP) as osteogenic material to the above basic biomimetic hydrogel precursor solution to obtain four kinds of biomimetic hydrogel precursor solutions, respectively:

[0209] 0% HAP group: no HAP was added;

[0210] 10% HAP group: 10% (w / v) HAP was added;

[0211] 30% HAP group: 30% (w / v) HAP was added;

[0212] 50% HAP group: 50% (w / v) HAP was added;

[0213] 70% HAP group: 70% (w / v) HAP was added.

[0214] 2. Design of inner mold and setting of printing parameters

[0215] In this embodiment, polylactic acid (PLA) material was selected as the sacrificial material. In this embodiment, a fused deposition modeling (FDM) 3D printer was used for layer-by-layer printing of the sacrificial material, the printing nozzle temperature was set to 210°C, the platform temperature was set to 60°C, the printing speed was set to 60 mm / s, and the single layer printing layer height was 0.2 mm.

[0216] According to the osteochondral 3D model, the porosity was 60%, and the grid line diameter was set to 350 μm.

[0217] 3. Printing-Perfusion-Photocuring

[0218] Print one layer, perfuse the prepared biomimetic hydrogel precursor solution, and cure under a 405 nm light source for 10 s, repeat the cycle until the whole printing perfusion is completed, and then cure the whole for 5 min; then perform inner demolding treatment. 25 layers were continuously printed, and the total printing thickness was 5 mm.

[0219] Among them, the above four kinds of biomimetic hydrogels were used for perfusion respectively, and four different HAP content multilayer biomimetic scaffolds were obtained respectively.

[0220] Among them, the inner demolding treatment method is described in Example 1.

[0221] Example 3: Integrated preparation of layered scaffold

[0222] The osteochondral biomimetic scaffold with gradient pore structure was prepared by the method of Example 1.

[0223] Prepare the biomimetic hydrogel precursor solution: 15% (w / v) GelMA and 0.2% (w / v) photoinitiator LAP in PBS solution. Using the layer-by-layer printing-perfusion-curing process, four different pore structure layers were designed from top to bottom in the same scaffold:

[0224] First layer: pore size 300 μm, porosity 30%, grid line diameter set to 300 μm; 5 layers were continuously printed, and the total printing thickness was 1 mm;

[0225] Second layer: Pore size 250 pm, porosity 50%, grid line diameter set to 250 pm; 4 layers printed consecutively, total printed thickness 0.8 mm;

[0226] Third layer: Pore size 200 pm, porosity 70%, grid line diameter set to 200 pm; 3 layers printed consecutively, total printed thickness 0.6 mm;

[0227] Fourth layer: Pore size 150 pm, porosity 90%, grid line diameter set to 150 pm; 2 layers printed consecutively, total printed thickness 0.4 mm.

[0228] Example 4

[0229] 1. Design of inner mold and setting of printing parameters

[0230] In this example, polylactic acid (PLA) was chosen as the sacrificial material, and a fused deposition modeling (FDM) 3D printer was used for layer-by-layer printing of the sacrificial material. The printing nozzle temperature was set to 210°C, the platform temperature was set to 60°C, the printing speed was set to 60 mm / s, and the single-layer printing height was set to 0.2 mm.

[0231] According to the osteochondral 3D model, the bone layer region was designed with a porosity of 50% and a grid line diameter of 500 pm; the middle layer region was designed with a porosity of 60% and a grid line diameter of 300 pm; and the cartilage layer region was designed with a porosity of 80% and a grid line diameter of 250 pm.

[0232] 2. Preparation of biomimetic hydrogel precursor solution (moisturizing co-solvent medium)

[0233] The following three kinds of biomimetic hydrogel precursor solutions were prepared respectively (the concentration of each component was calculated based on the mass of each component per volume of hydrogel medium):

[0234] (1) Bone layer biomimetic hydrogel precursor solution: 15% (w / v) GelMA, 30% (w / v) bioactive glass, 0.2% (w / v) LAP; the hydrogel medium was PBS, glycerol and DMSO, PBS / glycerol / DMSO = 50 / 25 / 25 (v / v / v);

[0235] (2) Middle layer biomimetic hydrogel precursor solution: 15% (w / v) GelMA, 10% (w / v) bioactive glass, 0.2% (w / v) LAP; the hydrogel medium was PBS, glycerol and DMSO, PBS / glycerol / DMSO = 50 / 25 / 25 (v / v / v);

[0236] (3) Cartilage layer biomimetic hydrogel precursor solution: 10% (w / v) GelMA, 0.5% (w / v) HAMA, 1% (w / v) CSMA, 0.2% (w / v) LAP; hydrogel is PBS, glycerol and DMSO, PBS / glycerol / DMSO=50 / 25 / 25 (v / v / v);

[0237] The pH of PBS in all solutions is 7.4. After the above solutions are prepared, they are preheated at 50°C for 10 min to facilitate perfusion.

[0238] 3. Preparation of osteochondral biomimetic scaffold

[0239] This example uses a "layer-by-layer printing-perfusion-photocuring" cycle to prepare an integrated osteochondral biomimetic scaffold (diameter 7 mm) with mechanical gradient combined with internal demolding dissolution process. The specific steps are as follows:

[0240] (1) Osteal layer complex preparation:

[0241] Based on the design parameters of the bone layer, the PLA sacrificial material is printed layer by layer on the printing platform, and 5 layers are continuously printed, with a total printing thickness of about 1 mm.

[0242] The bone layer biomimetic hydrogel precursor solution is perfused into the printed PLA grid structure to ensure that the solution fills the pores of the grid.

[0243] Use 405 nm blue light (LED light source, light intensity 10 mW / cm²) to irradiate for 60 s to solidify, so that the bone layer biomimetic hydrogel precursor solution is photocrosslinked to form the first part of the bone layer.

[0244] Continue to repeat the above "layer-by-layer printing-perfusion-photocuring" process 3 times to construct a bone layer complex with a thickness of about 4 mm.

[0245] (2) Preparation of intermediate layer complex:

[0246] On the surface of the prepared bone layer complex, continue to print the PLA sacrificial material, and continuously print 3 layers with a total printing thickness of about 0.6 mm.

[0247] The intermediate layer biomimetic hydrogel precursor solution is preheated and perfused into the newly printed PLA grid structure.

[0248] Use the same light source to irradiate for 60 seconds to photocrosslink the hydrogel precursor solution to form the intermediate layer. Construct an intermediate layer complex with a thickness of about 0.6 mm.

[0249] (3) Preparation of cartilage layer complex:

[0250] On the surface of the prepared intermediate layer composite, based on the design parameters of the cartilage layer, continue to print the PLA sacrificial material, print 10 layers in succession, and the total thickness is about 2 mm.

[0251] After preheating the cartilage layer biomimetic hydrogel precursor solution, it is infused into the newly printed PLA grid structure.

[0252] The cartilage layer biomimetic hydrogel precursor solution is irradiated for 45 seconds using the same light source to cause photo-crosslinking, forming a 2 mm thick cartilage layer composite.

[0253] (4) Internal demolding treatment:

[0254] After completing the printing-infusion-photocuring process of the entire bone layer composite, the intermediate layer composite, and the cartilage layer composite, the constructed multi-layer sacrificial material-hydrogel composite is soaked in dichloromethane to dissolve the PLA sacrificial material, and finally the scaffold is thoroughly washed with ultrapure water to ensure complete removal of residual solvents, and a bone-cartilage biomimetic scaffold with fine pore structure is obtained after freeze-drying.

[0255] In this embodiment, by using a low-volatility moisturizing glycerol and DMSO co-solvent system as the hydrogel medium, volatilization control under platform warming conditions can be achieved to reduce the evaporation rate and stabilize the infusion rheology. After solidification, the glycerol and DMSO co-solvent can be removed by water washing during the subsequent demolding process. In addition, it is also beneficial to ensure the consistency of the interfacial morphology and the pores.

[0256] Example 5 Preparation of skin biomimetic scaffold

[0257] 1. Design of internal mold and setting of printing parameters

[0258] In this embodiment, polylactic acid (PLA) material is selected as the sacrificial material. In this embodiment, a fused deposition modeling (FDM) 3D printer is used for layer-by-layer printing of the sacrificial material, the printing nozzle temperature is set to 210°C, the platform temperature is set to 60°C, the printing speed is set to 60 mm / s, and the single-layer printing layer height is 0.1 mm.

[0259] According to the 3D model of the skin, a grid with different pore structures in different regions is designed: the dermis layer region has a porosity of 50% and the grid line diameter is set to 250 μm; the epidermis layer region has a porosity of 80% and the grid line diameter is set to 60 μm, to simulate the structural characteristics of natural skin.

[0260] 2. Preparation of biomimetic hydrogel precursor solution

[0261] (1) Dermal layer biomimetic hydrogel precursor solution for preparing dermis layer composite: 10% GelMA and 1% methacrylated elastin (ElaMA) are dissolved in PBS, and 0.25% photoinitiator LAP is added;

[0262] (2) Epidermis layer biomimetic hydrogel precursor solution for preparing epidermis layer composite: 10% GelMA and 1% methacrylated keratin protein (KerMA) were dissolved in PBS, and 0.25% photoinitiator was added;

[0263] Wherein, the pH of PBS is 7.4.

[0264] 3. Preparation of skin biomimetic scaffold

[0265] This embodiment adopts the process of "layer-by-layer printing-perfusion-photocuring" cycle combined with internal demolding dissolution to prepare the skin biomimetic scaffold, and the specific steps are as follows:

[0266] (1) Preparation of dermis layer composite

[0267] Based on the design parameters of the dermis layer, PLA sacrificial material was printed layer by layer on the platform of the 3D printer, and 20 layers were continuously printed, with a total printing thickness of about 2.0 mm.

[0268] The prepared dermis layer biomimetic hydrogel precursor solution (GelMA+ElaMA) was perfused into the PLA inner mold, and the pores were fully filled, followed by 365 nm ultraviolet curing to form the dermis layer composite.

[0269] (2) Preparation of epidermis layer composite

[0270] Based on the design parameters of the epidermis layer, PLA sacrificial material was continuously printed on the surface of the solidified dermis layer composite, and 15 layers were continuously printed, with a total printing thickness of about 1.5 mm.

[0271] The prepared epidermis layer biomimetic hydrogel precursor solution (GelMA+KerMA) was perfused into the PLA inner mold, and the second ultraviolet curing was performed to ensure firm bonding between the layers, forming the epidermis layer composite.

[0272] (3) Internal demolding treatment

[0273] After completing the printing-perfusion-curing process of the entire dermis layer composite and epidermis layer composite, the constructed multilayer sacrificial material-hydrogel composite was immersed in dichloromethane to dissolve the PLA sacrificial material, and then washed with water. After freeze-drying, the skin biomimetic scaffold was obtained.

[0274] Comparative Example 1:

[0275] On the basis of Example 2, one-time printing-curing instead of layer-by-layer printing-curing was adopted in the printing-perfusion-photocuring, and the rest of the conditions and steps were the same as those of Example 2. The one-time printing-curing specifically includes the following steps:

[0276] The inner mold was once printed and perfusion completed, the perfusion prepared biomimetic hydrogel precursor solution, the whole was cured under 405 nm light source for 10 min, and then the inner demolding treatment was carried out. 25 layers were continuously printed, and the total thickness was 5 mm.

[0277] Comparative Example 2

[0278] On the basis of Example 1, only the single-layer printing layer height was adjusted from 0.2 mm to 0.05 mm, the thickness of the bone layer complex, the intermediate layer complex and the cartilage layer complex remained unchanged, and the rest of the conditions and steps were the same.

[0279] Comparative Example 3

[0280] On the basis of Example 1, only the single-layer printing layer height was adjusted from 0.2 mm to 0.35 mm, the thickness of the bone layer complex, the intermediate layer complex and the cartilage layer complex remained unchanged, and the rest of the conditions and steps were the same.

[0281] Effect Example 1: Test of physical and chemical properties of the scaffold and verification of composition optimization

[0282] 1. Morphology characterization

[0283] The microstructure of the osteochondral biomimetic scaffold obtained in Example 1 was observed by scanning electron microscope and micro-CT, as shown in Figure 2 and Figure 3 respectively. The results show that each layer forms a three-dimensional porous structure that is interconnected, and the pore size and porosity are basically consistent with the design values. The bone layer region has a rough surface due to the addition of a higher concentration of bioactive glass particles; the intermediate layer contains less bioactive glass, and the roughness is moderate; the cartilage layer region is relatively smooth. The interface between the three layers transitions naturally and is tightly bonded.

[0284] 2. Interlayer and intralayer shear strength

[0285] To evaluate the uniform consistency of the connection strength of the osteochondral biomimetic scaffold prepared by the "layer-by-layer printing-perfusion-light curing" method of the application, whether there is a strength difference between the interlayer interface and the intralayer region, the following shear strength test was carried out:

[0286] (1) The osteochondral biomimetic scaffold (diameter 7 mm) prepared in Example 1 was placed in the cylindrical sample groove at the lower part of the specially designed test fixture, so that the measured surface (interface layer or any region inside) was flush with the edge of the cylinder, ensuring that the measured surface was in the exact test position.

[0287] (2) Fix the cylinder clamp on the bottom platform of the universal material testing machine. Install a precision shear cutter with a square cross section and a side length greater than 7 mm on the upper clamp of the testing machine, and accurately position it above the edge of the cylinder. Drive the shear cutter downward at a constant rate of 0.5 mm / min to produce a precise shear on the measured surface of the osteochondral biomimetic scaffold, until it is completely broken, forming a shear surface at the measured surface.

[0288] (3) Record the force-displacement curve of the entire shear process through the testing system, and extract the maximum shear force (F_max, unit: N).

[0289] (4) Calculate the shear strength according to the following formula: τ = F_max / A

[0290] Where τ is the shear strength (kPa), F_max is the maximum shear force (N), and A is the shear area (π×r²=π×3.5²mm²).

[0291] The above shear strength test is carried out on the cross section perpendicular to the thickness direction of the cartilage layer, the interface between the cartilage layer and the intermediate layer, the intermediate layer, the interface between the intermediate layer and the bone layer, and the bone layer of the osteochondral biomimetic scaffold, respectively, to obtain the shear strength of different measured surfaces. The shear strength of the cartilage layer, the interface between the cartilage layer and the intermediate layer, the intermediate layer, the interface between the intermediate layer and the bone layer, and the bone layer is shown in Table 1-1.

[0292] Further calculation gives the average value of the interlayer shear strength and the average value of the interlayer shear strength, and the calculation results are shown in Table 1-2 and Figure 5 Where the average value of the interlayer shear strength = (the shear strength of the cartilage layer + the shear strength of the intermediate layer + the shear strength of the bone layer) / 3; the average value of the interlayer shear strength = (the shear strength of the interface between the cartilage layer and the intermediate layer + the shear strength of the interface between the intermediate layer and the bone layer) / 2.

[0293] Table 1-1

[0294]

[0295] Table 1-2

[0296]

[0297] The scaffold internal strength test shows that the average value of the interlayer shear strength (225.6±16.7kPa) and the average value of the interlayer shear strength (230.1±14.9kPa) prepared by the layer-by-layer solidification method of the present application are comparable, and the average value results are shown in Figure 5As shown, it is proved that the method of the present application can ensure firm interlayer bonding while guaranteeing the multilayer structure, thereby providing an effective solution for the preparation of the biomimetic multilayer tissue engineering scaffold.

[0298] Effect Example 2: Verification of the Mineralization Performance of the Layered Structure

[0299] The osteochondral biomimetic scaffold prepared in Example 1 was placed in SBF simulated body fluid (purchased from Beijing Solaybao Technology Co., Ltd., model G0390), and taken out after 21 days of 37°C immersion. The scaffold was cut into a bone layer, an intermediate layer and a cartilage layer along the designed interface, and the elastic modulus of each layer was tested before immersion (i.e. on day 0) and after 21 days of immersion (i.e. on day 21), respectively. The elastic modulus of the bone layer, the intermediate layer and the cartilage layer is shown in Table 2 and Figure 6 .

[0300] The results show that:

[0301] The elastic modulus of the bone layer increased from 200 kPa to 600 kPa;

[0302] The elastic modulus of the intermediate layer increased from 170 kPa to 280 kPa;

[0303] The elastic modulus of the cartilage layer remained basically unchanged at 150 kPa;

[0304] which indicates that the composition of each layer is clear, the bone layer and the intermediate layer containing the bioactive glass are mineralized and deposited, and the cartilage layer maintains the original characteristics, thereby verifying the effectiveness of the layered biomimetic design.

[0305] Table 2

[0306]

[0307] Effect Example 3: Verification of the In Vivo Repair Effect

[0308] A bone and cartilage defect with a diameter of 4 mm and a height of 4 mm was made in the trochlea of the knee joint of a rabbit, and the osteochondral biomimetic scaffold prepared in Example 1 was implanted. The micro-CT images on the first day of implantation and one month after implantation are shown in Figure 9 A and B, respectively. The results show that the scaffold is not mineralized and has low density and is not visible on the first day of implantation. However, one month after implantation, the bone layer and the intermediate layer are mineralized and deposited, the density is increased and begins to be visible, and the cartilage layer is still not visible, which proves that the bone layer is mineralized in vivo and the compositions of the layers are not mixed, thereby meeting the layered biomimetic effect.

[0309] The histological ponceau-fuchsin staining image is shown in Figure 10 . According to Figure 10 , the scaffold outline is visible one month after implantation, the cartilage layer begins to form cartilage tissue, and the bone layer begins to ossify, as shown in Figure 10The scaffold is degraded at 3 months, the cartilage layer forms mature cartilage tissue connected with the adjacent cartilage, and the bone layer forms new bone tissue, as shown in part B of FIG. 6. Figure 10

[0310] The effect embodiment verifies that the scaffold prepared by the layer-by-layer solidification process realizes composition stratification without mixing, and each layer independently plays the expected function.

[0311] Effect embodiment 4: Elastic modulus test

[0312] The elastic modulus of the scaffolds obtained in example 2 (layer-by-layer solidification) and comparative example 1 (one-time solidification) was tested, and the specific test method was as follows: a universal material testing machine was used to determine the elastic modulus of the scaffold. The scaffold sample was subjected to uniaxial compression test at room temperature at a constant rate of 1 mm / min, and the stress-strain curve was recorded. The linear section in the strain range of 10%-15% was selected for linear regression analysis, and the curve slope was the elastic modulus. Six parallel samples were tested in each group, and the results were expressed as mean ± standard deviation.

[0313] The elastic modulus of the biomimetic scaffolds obtained by different concentrations of HAP hydrogel precursor solution through example 2 (layer-by-layer solidification) and comparative example 1 (one-time solidification) is as follows: Figure 1 and Table 3, in the presence of osteogenic material (HAP), the layer-by-layer printing and solidification method in example 2 can obtain higher mechanical strength than the multi-layer biomimetic scaffold obtained by the one-time solidification method in comparative example 1. Especially at high concentrations of HAP (50% and 70%), the difference between the two solidification methods is more significant, indicating that layer-by-layer solidification can effectively solve the influence of light-blocking material on the light solidification effect, ensure that the material can be fully cross-linked, and thus ensure the mechanical properties of the scaffold.

[0314] Table 3

[0315]

[0316] Effect embodiment 5: cell distribution verification

[0317] The scaffold obtained in example 3 was subjected to cell distribution verification, specifically:

[0318] Bone marrow mesenchymal stem cells (BMSCs) were inoculated on the prepared integrated stratified scaffold at a density of 2×10 6 cells / mL, and cultured at 37°C in a 5% CO2 environment for 7 days. After the culture ended, the scaffold was cut along the designed stratification interface, and each layer was taken for Calcein-AM live cell fluorescent dye (green) staining, and the cell distribution of each layer was observed by fluorescence microscopy, and the cell culture section of the stratified scaffold was obtained as shown in FIG. 8. Figure 4 ​As shown in part A-D of the figure, the cell growth of each layer presents the corresponding pore size and porosity structure. The results show that the porosity structure of each layer in a single scaffold can be precisely controlled by the layer-by-layer solidification process of the present application, realizing the gradient cell microenvironment, and providing an ideal biomimetic scaffold platform for the layered repair of osteochondral tissue.

[0319] The corresponding relationship between the porosity and the specific surface area increase multiple of the layers with different porosity structures in the scaffold obtained in Example 3 is shown in Table 4 (for example, a scaffold with a size of 10*10*10 mm and a wire diameter of 0.25 mm).

[0320] Table 4

[0321]

[0322] Effect Example 6: Optimization of the porosity of the cartilage layer and study of the mechanical properties

[0323] In order to obtain the optimal porosity structure with the biomimetic mechanical properties of cartilage, the porosity optimization of the cartilage layer is studied in this effect example. A biomimetic hydrogel precursor solution is prepared: 15% (w / v) GelMA, 2% HAMA, 2% CSMA and 0.2% (w / v) light initiator LAP in PBS solution. The scaffold samples of the cartilage layer with different porosities are prepared by the method of Example 1 (10 layers are continuously printed, and the total printing thickness is about 2 mm):

[0324] Group A: porosity 50%, grid line diameter set to 250 μm;

[0325] Group B: porosity 60%, grid line diameter set to 250 μm;

[0326] Group C: porosity 70%, grid line diameter set to 250 μm;

[0327] Group D: porosity 80%, grid line diameter set to 250 μm;

[0328] Mechanical property test:

[0329] (1) Static compression test: uniaxial compression test is performed by using an electronic universal testing machine, and the compression rate is 1 mm / min. The obtained static compression test graph is as shown in Figure 7 As shown in part A of the figure, with the increase of the porosity, the elastic modulus of the scaffold gradually decreases, and the elastic modulus of the group with a porosity of 60% is about 350 kPa, which is close to the mechanical range of natural cartilage.

[0330] (2) Dynamic compression cycle test: in order to evaluate the rebound characteristics of the cartilage layer, 10 cycles of dynamic compression-release test (strain range 0-70%) are performed. The obtained dynamic compression cycle test graph is as shown in Figure 7As shown in the middle B part, the scaffolds with 60% and 70% porosity showed good resilience performance, and the stress-strain curve remained stable in multiple cycles, with similar viscoelastic characteristics to natural cartilage.

[0331] Effect Example 7: Optimization of Cartilage Layer Formulation

[0332] In order to meet the cartilage layer formulation of the component biocompatibility of natural cartilage, the components of the cartilage layer biocompatible hydrogel precursor solution were optimized and designed, and the specific formulation design was as follows:

[0333] Control group (15G): 15% GelMA;

[0334] Optimized group (15G-2H-2C): 15% GelMA + 2% HAMA + 2% CSMA;

[0335] Among them, the introduction of HAMA (methyl acrylate hyaluronic acid) and CSMA (methyl acrylate chondroitin sulfate) aims to better simulate the component composition of the natural cartilage matrix.

[0336] Gene expression analysis:

[0337] Rabbit bone marrow mesenchymal stem cells were inoculated into the scaffolds of the above formulations, and RNA was extracted after 7 days, 14 days and 21 days of culture for qRT-PCR analysis. The expression of cartilage-related marker genes (Col2a1, Col1a1, Col2a1 / Col1a1, Col10a1, Acan and Sox 9) was detected, and the relative expression of genes was calculated by 2^(-ΔΔCt) method. GAPDH was used as an internal reference gene for standardization. The results are shown in Figure 8 As shown in the middle G-L part, the three-component formulation (15G-2H-2C) is significantly better than the single GelMA formulation in terms of cartilage-specific gene expression.

[0338] Among them, the related data was statistically analyzed by GraphPad Prism 6.0 software. Two-way ANOVA was used for comparison between groups. When the overall test P < 0.05, further Bonferroni method was used for pairwise comparison. P < 0.05 was considered statistically significant, in which * represented P < 0.05, ** represented P < 0.01, *** represented P < 0.001, and **** represented P < 0.0001.

[0339] Effect Example 8: Optimization of Single Layer Printing Layer Height

[0340] In the 3D printing of the sacrificial material in Examples 1-5, the single-layer printing layer height is in the range of 0.1-0.3 mm, the specific surface area of the obtained multilayer biomimetic scaffold is moderate, and excellent mechanical strength is also considered.

[0341] In the 3D printing of the sacrificial material in Comparative Example 2, the single-layer printing layer height is too small, only 0.05 mm, and when 3D printing is performed, the printing strips are obviously flattened, the gap between adjacent strips is narrowed, the orifice is narrowed or even blocked; the throughness is obviously reduced after internal demolding. In the obtained multilayer biomimetic scaffold, the orifice is narrowed or blocked, the actual contact surface is reduced, and the through hole is reduced, which is not conducive to the subsequent cell entry and nutrient exchange.

[0342] In the 3D printing of the sacrificial material in Comparative Example 3, the single-layer printing layer height is too large, 0.35 mm, and when 3D printing is performed, the contact between upper and lower layers is less, and the layers are easy to separate; the interlayer shear force is weak, and the whole is more prone to collapse / deformation; due to the increase of the single-layer printing layer height and the decrease of the number of layers per unit thickness, the exposed pore wall length is reduced, and the specific surface area is reduced (the actual contact surface is reduced under the same volume). In the obtained multilayer biomimetic scaffold, the layers are separated, collapsed, and the specific surface area is reduced, and the comprehensive performance is worse than that of Example 1.

[0343] In the 3D printing of the sacrificial material in the present application, the single-layer printing layer height is in the range of 0.1-0.5 mm, and the specific values are 0.1 mm, 0.15 mm, 0.25 mm, 0.3 mm, 0.4 mm and 0.5 mm, respectively. The optimization results of the single-layer printing layer height are shown in Table 5. It is found that in the present application, the single-layer printing layer height is controlled in the range of 0.10-0.30 mm to ensure the stability and specific surface area of the scaffold.

[0344] Table 5

[0345]

[0346] Effect Example 9: Verification of Layered Cell Distribution of Double-layer Skin Biomimetic Scaffold

[0347] In order to verify the layered function of the biomimetic dermis-epidermis double-layer skin biomimetic scaffold prepared in Example 5, a cell culture and distribution observation experiment was carried out.

[0348] Experimental method:

[0349] Dermatofibroblasts and keratinocytes were inoculated on the dermis layer and epidermis layer, respectively, and after 7 days of culture, the living cells were labeled with Calcein-AM fluorescent dye, and the cell distribution was observed by fluorescence microscope. The test figures are respectively as follows: Figure 11 andFigure 12 As shown in the figure, the double-layer skin biomimetic scaffold presents obvious layered cell distribution characteristics:

[0350] Dermis layer: cells are distributed in loose network, which meets the design requirements of the large pore structure (pore size 250 μm, porosity 50%) of the dermis layer, providing good three-dimensional growth space for fibroblasts, as shown in the figure. Figure 11

[0351] Epidermis layer: cells are densely distributed and arranged in layers, which embodies the characteristics of the small pore structure (pore size 60 μm, porosity 80%) of the epidermis layer, which is conducive to the formation of a tight epidermal barrier structure by keratinocytes, as shown in the figure. Figure 12

[0352] The effect embodiment proves that the layer-by-layer printing-perfusion-curing process can successfully prepare ultrathin layered scaffolds with different pore structures, realize the layered specific distribution of cells, and verify the effectiveness of the design of the biomimetic double-layer skin biomimetic scaffold.

[0353] Although the specific embodiments of the present application are described above, those skilled in the art should understand that this is only an example, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present application, and these changes and modifications all fall within the protection scope of the present application.​​

Claims

1. A method for fabricating a multi-layer biomimetic scaffold, characterized in that, It includes the following steps: S1. Perform 3D printing on the sacrificial material layer by layer, continuously printing n layers to obtain a mesh-like porous inner mold; The single-layer printing height is 0.1-0.3 mm, and n is 1-50; S2. Inject the biomimetic hydrogel precursor solution into the mesh-like porous inner mold, and then perform photocuring and cross-linking to form a sacrificial material-hydrogel composite. S3. On the upper surface of the sacrificial material-hydrogel composite obtained in step S2, repeat steps S1 and S2 to form a multilayer sacrificial material-hydrogel composite; wherein, the number of layers n printed continuously in each step S1 is the same or different, and the biomimetic hydrogel precursor solution poured in each step S2 is the same or different. S4. The multilayer sacrificial material-hydrogel composite is subjected to internal demolding treatment using a solvent dissolution method to obtain a multilayer biomimetic scaffold.

2. The method for preparing the multi-layer biomimetic scaffold as described in claim 1, characterized in that, The n satisfies any one of the following conditions: (1) The biomimetic hydrogel precursor solution includes transparent materials, but does not include translucent materials or osteogenic materials, and n is 10-50; (2) The biomimetic hydrogel precursor solution includes a translucent material but does not include osteogenic materials, and n is 3-10; (3) The biomimetic hydrogel precursor solution includes osteogenic materials, and n is 1-5.

3. The method for preparing the multi-layer biomimetic scaffold as described in claim 1, characterized in that, Steps S1-S3 satisfy one or more of the following conditions (1)-(8): (1) In step S1, the thickness of the mesh-like porous inner mold is 0.1-15mm; (2) In step S1, the 3D printing method is extrusion mesh printing; (3) In step S1, the 3D printing is fused deposition modeling 3D printing; (4) In step S1, the sacrificial material is a molten polymer material; (5) In step S2, the light source for photocuring crosslinking is ultraviolet light, blue light or near-infrared light; wherein the wavelength of the ultraviolet light is 355-375 nm; the wavelength of the blue light is 395 nm-415 nm; and the wavelength of the near-infrared light is 700-2500 nm. (6) In step S2, the photocuring crosslinking time is 10-80 s; (7) In step S2, the photocuring crosslinking is performed at a light intensity of 5-50 mW / cm. 2 The process was carried out under the illumination of light; (8) In step S3, steps S1 and S2 are repeated 1 to 5 times.

4. The method for preparing the multi-layer biomimetic scaffold as described in claim 1, characterized in that, In step S2, the biomimetic hydrogel precursor solution includes hydrogel components, a hydrogel medium, and a photoinitiator; The biomimetic hydrogel precursor solution satisfies one or more of the following conditions (1)-(4): (1) The hydrogel components include one or more of the following: methacrylamide gelatin, methacrylamide collagen, methacrylamide elastin, methacrylamide keratin, methacrylamide hyaluronic acid, methacrylamide chondroitin sulfate, methacrylamide sodium alginate, methacrylamide heparin, gelatin, collagen, elastin, hyaluronic acid, chondroitin sulfate, heparin and sodium alginate; (2) The hydrogel medium is physiological saline, cell culture medium, phosphate buffer solution or carbonate buffer solution; (3) The hydrogel medium further includes glycerol and / or DMSO; (4) The photoinitiator is a blue light photoinitiator, an ultraviolet light photoinitiator or a near-infrared light photoinitiator.

5. The method for preparing the multi-layer biomimetic scaffold as described in claim 4, characterized in that, The biomimetic hydrogel precursor solution satisfies any one of the following conditions (1)-(5): (1) The biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 10%-40% osteogenic material and 0.1%-0.5% photoinitiator; (2) The biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 5%-15% osteogenic material and 0.1%-0.5% photoinitiator; (3) The biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide hyaluronic acid, 0.5%-5% methacrylamide chondroitin sulfate and 0.1%-0.5% photoinitiator; (4) The biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide elastin and 0.1%-0.5% photoinitiator; (5) The biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide keratin and 0.1%-0.5% photoinitiator; The osteogenic material includes one or more of bioactive glass, tricalcium phosphate, inorganic bone meal, and hydroxyapatite; Wherein, the concentration is the ratio of the mass of each component to the volume of the hydrogel medium in the biomimetic hydrogel precursor solution, with mass expressed in g and volume expressed in mL.

6. The method for preparing a multi-layer biomimetic scaffold as described in any one of claims 1-5, characterized in that, The multi-layer biomimetic scaffold is a osteochondral biomimetic scaffold, and its preparation method includes the following steps: S1. Perform 3D printing on the sacrificial material layer by layer, continuously printing n layers to obtain a mesh-like porous inner mold; wherein, the height of a single printed layer is 0.1-0.3 mm, and n is 1-5; S2. Inject the bone layer biomimetic hydrogel precursor solution into the mesh-like porous inner mold, and then perform photocuring crosslinking to form part of the bone layer composite. S3-1. Repeat steps S1 and S2 multiple times on the upper surface of a portion of the bone layer composite obtained in step S2 to form a bone layer composite; wherein, the number of layers n printed continuously in each step S1 is the same, and the bone layer biomimetic hydrogel precursor solution injected in each step S2 is the same. S3-2. Repeat steps S1 and S2 on the upper surface of the bone layer composite to form an intermediate layer composite; wherein, the biomimetic hydrogel precursor solution injected in step S2 is the intermediate layer biomimetic hydrogel precursor solution. S3-3. Repeat steps S1 and S2 on the upper surface of the intermediate layer composite to form a cartilage layer composite; wherein, in step S1, n is 3-10, and the biomimetic hydrogel precursor solution injected in step S2 is a cartilage layer biomimetic hydrogel precursor solution. Through steps S1, S2, S3-1, S3-2 and S3-3, a multilayer sacrificial material-hydrogel composite is obtained, which successively includes a bone layer composite, an intermediate layer composite and a cartilage layer composite. S4. The multilayer sacrificial material-hydrogel composite is subjected to internal demolding treatment using a solvent dissolution method to obtain a osteochondral biomimetic scaffold.

7. The method for preparing the multi-layer biomimetic scaffold as described in claim 6, characterized in that, The bone-bearing biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 10%-40% osteogenic material, and 0.1%-0.5% photoinitiator; The intermediate layer biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 5%-15% osteogenic material, and 0.1%-0.5% photoinitiator; The biomimetic hydrogel precursor solution for the cartilage layer comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide hyaluronic acid, 0.5%-5% methacrylamide chondroitin sulfate, and 0.1%-0.5% photoinitiator; Wherein, the concentration is the ratio of the mass of each component to the volume of the hydrogel medium in the biomimetic hydrogel precursor solution of each layer, with mass in g and volume in mL. The osteogenic material includes one or more of bioactive glass, tricalcium phosphate, inorganic bone meal, and hydroxyapatite.

8. The method for preparing a multi-layer biomimetic scaffold as described in any one of claims 1-5, characterized in that, The multilayer biomimetic scaffold is a skin biomimetic scaffold, and its preparation method includes the following steps: S1. The sacrificial material is 3D printed layer by layer, and n layers are printed continuously to obtain a mesh-like porous inner mold; wherein the height of a single printed layer is 0.1-0.3 mm, and n is 10-50. S2. A biomimetic hydrogel precursor solution for the dermal layer is injected into the mesh-like porous inner mold, and then photocured and cross-linked to form a dermal layer composite. S3. Repeat steps S1 and S2 on the upper surface of the dermal complex obtained in step S2 to form an epidermal complex; wherein, the biomimetic hydrogel precursor solution injected in step S2 is an epidermal biomimetic hydrogel precursor solution. Through steps S1, S2 and S3, a multilayer sacrificial material-hydrogel composite is obtained, which successively includes a dermal composite and an epidermal composite. S4. The multilayer sacrificial material-hydrogel composite is subjected to internal demolding treatment using a solvent dissolution method to obtain a skin biomimetic scaffold. The dermal biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide elastin, and 0.1%-0.5% photoinitiator; The epidermal biomimetic hydrogel precursor solution comprises the following components at the following concentrations: 5%-20% methacrylamide gelatin, 0.5%-5% methacrylamide keratin, and 0.1%-0.5% photoinitiator; The concentration is the ratio of the mass of each component to the volume of the hydrogel medium in the precursor solution of each layer of biomimetic hydrogel, with mass expressed in g and volume expressed in mL.

9. A multi-layer biomimetic scaffold, characterized in that, It is prepared by the method of any one of claims 1-8 for the preparation of a multilayer biomimetic scaffold.

10. The multi-layer bionic scaffold as described in claim 9, characterized in that, The multi-layer bionic scaffold satisfies the following condition (1) or (2): (1) The multi-layer biomimetic scaffold is an osteochondral biomimetic scaffold, which includes a bone layer, an intermediate layer and a cartilage layer covalently connected in sequence; the bone layer, the intermediate layer and the cartilage layer are all through porous structures; The pores in the bone layer, intermediate layer, and cartilage layer are arranged in a three-dimensional grid pattern with vertical and horizontal intersections. The bone layer has a pore size of 300-500 μm, a porosity of 30%-50%, and a thickness of 1-5 mm; The intermediate layer has a pore size of 200-400 μm, a porosity of 50%-70%, and a thickness of 0.2-0.8 mm; The cartilage layer has a pore size of 100-300 μm, a porosity of 50%-90%, and a thickness of 1-3 mm; The intermediate layer does not cover or partially covers the pores of the cartilage layer and / or the bone layer; (2) The multilayer biomimetic scaffold is a skin biomimetic scaffold, which includes a covalently connected dermis and epidermis, both of which are through-porous structures; The pores in the dermis and epidermis are arranged in a vertically crisscross pattern. The dermis layer has a pore size of 100-300 μm, a porosity of 40%-60%, and a thickness of 1-4 mm; The pore size of the epidermal layer is 50-100 μm, the porosity is 70%-90%, and the thickness is 0.2-2 mm.

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