Bone-cartilage integrated scaffold with directional pore structure and preparation method thereof

Abstract

The present invention discloses an integrated bone and cartilage scaffold with a directional pore structure and a preparation method thereof, which belongs to the fields of tissue engineering, biomedicine and biomanufacturing technology. The method simulates the natural material and structure of bone cartilage, uses the principle of directional crystallization to prepare a bone and cartilage scaffold with a directional structure, and uses a low-temperature deposition manufacturing process based on rapid prototyping technology to prepare a horizontally oriented cartilage layer scaffold and a vertically oriented subchondral bone layer scaffold. Between the cartilage layer and the subchondral bone layer, methacrylated gelatin capable of UV cross-linking is combined with other materials with chemical grafting and bonding effects as a transition layer, thereby preparing a tissue engineering bone and cartilage integrated composite scaffold. The present invention is aimed at the treatment of articular bone and cartilage defects, and uses a bioactive material to manufacture a bone and cartilage composite scaffold with a directional pore structure. It can promote the coordinated repair of bone and cartilage tissue according to the characteristics of different functional areas of bone and cartilage, thereby improving the repair effect of bone and cartilage defects.

Description

Technical Field

[0001] The present invention belongs to the technical fields of tissue engineering, biomedicine and biomanufacturing, and particularly relates to a bone-cartilage integrated scaffold with a directional pore structure and a preparation method thereof. Background Art

[0002] Osteochondral defects are usually caused by trauma, disease or aging, and manifest as simultaneous damage to the articular cartilage and subchondral bone. Current treatments for osteochondral defects include autologous osteochondral transplantation, allogeneic transplantation and engineered biomaterial implantation. Although the above treatments have been widely used in clinical practice, they all have their limitations and shortcomings, such as damage to osteochondral tissue, damage to the donor site, mismatch in mechanical properties, etc. Due to the lack of an effective strategy to reconstruct the multi-layered functional tissue structure of the damaged articular cartilage, the above methods cannot simultaneously achieve the reconstruction of cartilage and subchondral bone. Therefore, the repair of osteochondral damage remains a very challenging topic.

[0003] In recent years, research on repairing articular cartilage damage has gradually shifted toward utilizing tissue engineering methods to promote tissue regeneration and reconstruction. Because tissue engineering is based on the properties of seed cells, scaffold materials, and growth factors, it has become a promising approach for repairing osteochondral defects larger than a critical size. Simultaneously, the rapid development of 3D printing technology has also advanced research in tissue engineering repair. This layer-by-layer addition technique makes it possible to manufacture functionally graded porous materials for osteochondral tissue engineering. Scaffolds manufactured using bio-3D printing technology possess a macroporous structure that is interconnected, facilitating the transport of nutrients and cell migration into the interior, thereby enabling tissue induction or regeneration. Furthermore, to a certain extent, it can effectively simulate the complexity of tissues and create multi-layered gradient scaffold structures.

[0004] Tissue regeneration places higher demands on biomaterials, requiring them to have suitable chemical properties and physical structures. Hydrogels are three-dimensional polymer networks with high water content, cross-linking capabilities under mild conditions, good biocompatibility, and adjustable biochemical and physical properties. Because the structure and properties of hydrogels are similar to the microenvironment of many human tissues, especially the extracellular matrix (ECM) has a more similar structure and physical and chemical properties that are closer to normal living tissues, hydrogels can be directly used in tissue engineering or formulated into scaffolds after processing. Another major feature of hydrogels as scaffolds is that they can maintain a fluid state under normal conditions and cross-link into a solid-like shape with a certain strength under specific external stimuli. Therefore, hydrogels have unparalleled advantages as tissue engineering scaffolds.

[0005] According to the strategy of layer-by-layer regeneration structure, based on the difference in mechanical and biological structure of natural osteocartilage, it has been developed from single-layer scaffold to double-layer or multi-layer scaffold. The scaffold has a certain mechanical strength, can mimic the natural joint structure, and the implanted scaffold can be well integrated with the host tissue to maintain biological function. Although the research on osteocartilage scaffold has experienced the development process from single to multi-layer biomimetic structure, there are still many problems in the current research process: (1) The prepared osteocartilage scaffold has poor binding force with cells and tissue integration, making it difficult to induce scaffolds for integrated and coordinated repair of osteocartilage; (2) The osteocartilage scaffold prepared in layers only mimics the layered structure of natural osteocartilage, but not its anisotropy; (3) The osteocartilage layer and the bone layer of the osteocartilage scaffold prepared in layers are not tightly bonded, and the interface bonding strength is generally poor, which makes it easy to separate after long-term culture. Summary of the Invention

[0006] In view of the current demand for repairing osteochondral damage and the defects of existing osteochondral damage repair scaffolds and materials, the present invention provides an integrated osteochondral scaffold material with high interfacial bonding strength and directional pore structure, as well as its preparation method and application. The specific technical solutions are as follows:

[0007] The first aspect of the present invention provides an integrated osteochondrocyte scaffold with a directional pore structure, wherein the integrated osteochondrocyte scaffold is a double-layer composite hydrogel scaffold, wherein the double-layer composite hydrogel sequentially comprises an upper composite hydrogel layer, a transition layer, and a lower composite hydrogel layer, wherein:

[0008] The upper composite hydrogel has a horizontally oriented pore structure, is used to simulate the cartilage tissue environment, and contains components such as methacryloylated gelatin (GelMA), chitosan (CS), and silk fibroin (SF);

[0009] The lower layer composite hydrogel has a vertically oriented pore structure, is used to simulate the subchondral bone tissue environment, and comprises components such as methacryloylated gelatin (GelMA), sodium alginate (SA), and hydroxyapatite (HA);

[0010] The transition layer is a bonding interface layer between the two layers, and contains components such as methacrylated gelatin (GelMA) and dopamine (DA) that can generate chemical grafting with the upper and lower composite hydrogel layers.

[0011] Furthermore, in the double-layer composite hydrogel, in the methacrylated gelatin / chitosan / silk fibroin (GelMA / CS / SF) composite hydrogel layer, i.e., the upper composite hydrogel, the mass percentages of methacrylated gelatin, chitosan, and silk fibroin in the hydrogel are each independently 3 wt%-10 wt%.

[0012] Furthermore, in the double-layer composite hydrogel scaffold, in the methacrylated gelatin / chitosan / silk fibroin (GelMA / CS / SF) composite hydrogel layer, i.e., the upper composite hydrogel, the mass ratio of methacrylated gelatin, chitosan, and silk fibroin is 5:5:7.

[0013] Furthermore, in the methacrylated gelatin / sodium alginate / hydroxyapatite (GelMA / SA / HA) composite hydrogel layer, ie, the lower composite hydrogel, the mass percentages of methacrylated gelatin, sodium alginate, and hydroxyapatite in the hydrogel are each independently 3 wt % to 10 wt %.

[0014] Furthermore, in the methacrylated gelatin / sodium alginate / hydroxyapatite (GelMA / SA / HA) composite hydrogel layer, ie, the lower composite hydrogel, the mass ratio of methacrylated gelatin, sodium alginate, and hydroxyapatite is 2:2:3.

[0015] Furthermore, in the double-layer composite hydrogel scaffold, the methacrylated gelatin / chitosan / silk fibroin (GelMA / CS / SF) composite hydrogel layer can simulate the cartilage tissue environment; the methacrylated gelatin / sodium alginate / hydroxyapatite (GelMA / SA / HA) composite hydrogel layer can simulate the subchondral bone tissue environment.

[0016] Furthermore, the transition layer interface of the double-layer composite hydrogel can be methacrylated gelatin / dopamine / chitosan (GelMA / PDA / CS), and a polymer network organic connection is generated between the transition layer and the cartilage layer and the bone layer, which can simulate the normal bone and cartilage interface.

[0017] Furthermore, in the transition layer interface of the double-layer composite hydrogel, the mass percentages of methacrylated gelatin, polydopamine, and chitosan in the transition layer hydrogel are independently 15wt%-25wt%, 0.5wt%-2wt%, and 2wt%-10wt%.

[0018] Furthermore, the mass percentages of methacrylated gelatin, polydopamine, and chitosan in the transition layer hydrogel are independently 20 wt %, 0.5 wt %, and 5 wt %, respectively.

[0019] Furthermore, the thickness of the upper layer composite hydrogel is 0 mm-5 mm, and the pore size is 0.2 mm-0.5 mm.

[0020] Furthermore, the thickness of the lower layer composite hydrogel is 0 mm-5 mm, and the pore size is 0.2 mm-0.5 mm.

[0021] Furthermore, the transition layer is a solid layer with a thickness of 0mm-1mm.

[0022] Optionally, factors that promote the regeneration of osteochondral tissue, preferably stem cells, human fibroblasts, and bioactive factors, can be added to the double-layer composite hydrogel. These factors can be added to both the upper and lower layers of the composite hydrogel.

[0023] A third aspect of the present invention provides a method for preparing the integrated osteochondral scaffold with a directional pore structure, the method comprising 3D bioprinting, directional freezing, and photocuring cross-linking, and comprising the following steps:

[0024] S1. Printing the subchondral bone layer: dissolving methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and a photoinitiator in water to prepare a first mixed solution, pouring the solution into a barrel, and loading the 3D bioprinter nozzle to print the subchondral bone layer;

[0025] S2, printing a transition layer: preparing aqueous solutions of methacrylated gelatin GelMA, chitosan CS, dopamine DA, a photoinitiator, and sodium hydroxide, respectively, and then mixing them to form a second mixed solution, which is poured into a barrel and loaded into a 3D bioprinter nozzle to print a transition layer on the subchondral bone layer obtained in step S1;

[0026] S3, printing the cartilage layer: dissolving methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and a photoinitiator in water to prepare a third mixed solution, pouring the solution into a barrel, and loading the solution into a 3D bioprinter nozzle to print the cartilage layer on the transition layer of step S2, forming an osteochondral scaffold consisting of a cartilage layer and a subchondral bone layer bonded by the transition layer;

[0027] S4, freezing the directional osteochondral scaffold: placing the osteochondral scaffold printed in step S3 in a directional freezing box, and placing the osteochondral scaffold and the directional freezing box together in a liquid nitrogen tank for directional freezing, wherein the cartilage layer is horizontally directional frozen, and the subchondral bone layer is vertically directional frozen;

[0028] S5. Cross-linking of osteochondral scaffold: The osteochondral scaffold after directionally freezing is first irradiated with ultraviolet light, and then immersed in sodium citrate solution and calcium chloride solution respectively, to finally form a double-layered directional osteochondral scaffold.

[0029] The core principle of the high-strength transition layer of the double-layer bone-cartilage integrated scaffold of the present invention is:

[0030] In the transition layer, the amino group of dopamine DA is modified on the carboxyl group of GelMA by a covalent bond reaction. Subsequently, DA self-polymerizes into polydopamine PDA under alkaline conditions. CS is then added to increase the viscosity of the material and further promote the oxidative polymerization of DA. At the same time, hydrogen bonding exists between CS and DA, and CS reacts with PDA to form Schiff base bonds. Finally, the transition layer itself is cross-linked and stabilized, forming a GelMA-DA polymerization network between the cartilage layer and the subchondral bone layer. The multiple cross-linking makes the transition layer of the osteochondral scaffold have a higher strength of bonding, which can organically integrate the cartilage layer and the subchondral bone layer more firmly.

[0031] Furthermore, in step S1, the total mass percentage of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution is 15 wt % to 25 wt %.

[0032] Furthermore, the mass percentages of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution are 3wt%-8wt%, 3wt%-8wt%, 5wt%-10wt%, and 0.5wt%-2wt%, respectively.

[0033] Furthermore, the mass percentages of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution are 5%, 5%, 7.5%, and 1%, respectively.

[0034] Furthermore, in step S2, the second mixed solution is composed of 5wt%-10wt% chitosan solution and a pre-gel mixture in a volume ratio of 0.3:1, and the pre-gel mixture is composed of methacrylated gelatin GelMA, dopamine DA, a photoinitiator, and sodium hydroxide in mass percentages of 15wt%-25wt%, 0.5wt%-2wt%, 0.1wt%-1wt%, and 0.05wt%-0.2wt%.

[0035] Furthermore, in step S2, the pre-gel mixture consists of methacrylated gelatin GelMA, dopamine DA, a photoinitiator, and sodium hydroxide in the mass percentages of 20 wt%, 0.5 wt%, 0.5 wt%, and 0.1 wt%.

[0036] Furthermore, in step S2, the concentration of the chitosan solution is 5 wt%.

[0037] Furthermore, in step S2, the second mixed solution is prepared by combining a 30-50 wt% methacrylated gelatin solution, a 2 wt% dopamine solution, a 2.5 wt% photoinitiator solution, and a 2 wt% sodium hydroxide solution in a volume ratio of 1:0.5:0.4:0.1 to form a pregel mixture, which is then heated in a water bath at 35-40°C, preferably 37°C. A 5-10 wt% chitosan solution, preferably 5 wt%, is then added, with the volume ratio of the chitosan solution to the pregel mixture being 0.3:1. Chitosan improves the viscosity, crosslinking, and printability of the hydrogel.

[0038] Furthermore, in step S3, the total amount of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and photoinitiator in the third mixed solution is 15 wt % to 25 wt %.

[0039] Furthermore, the mass ratios of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and photoinitiator in the third mixed solution are 2wt%-8wt%, 2wt%-8wt%, 5wt%-10wt%, 1wt%-5wt%, and 0.5wt%-2wt%, respectively;

[0040] Furthermore, the mass ratios of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, and photoinitiator in the third mixed solution are 5wt%, 5wt%, 7wt%, 3.5wt%, and 1wt%, respectively.

[0041] Furthermore, the photoinitiator is a conventional photoinitiator in the art, preferably 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959).

[0042] Furthermore, the mass percentage of the photoinitiator is 0.5 wt%-2 wt%, preferably 1 wt%.

[0043] Furthermore, the methacrylated gelatin can be conventional in the art and commercially available, or can be obtained by methacrylating gelatin using conventional methods in the art.

[0044] Furthermore, in step S1, the thickness of the subchondral bone layer is 0 mm-5 mm, the wire diameter is 0.4 mm-0.5 mm, the filling spacing is 0.6 mm-1 mm, the printing speed is 4-5 mm / s, the air pressure is 0.2-0.3 MPa, and the platform temperature is 0°C-5°C.

[0045] Furthermore, in step S2, the transition layer is a solid layer, the thickness of the transition layer is 0mm-1mm, the wire diameter is 0.2mm-0.3mm, the filling spacing is equal to the wire diameter, the printing speed is 2-4mm / s, the air pressure is 0.05-0.15Mpa, and the platform temperature is 0℃-5℃.

[0046] Furthermore, in step S3, the thickness of the cartilage layer is 0 mm-5 mm, the wire diameter is 0.2 mm-0.3 mm, the filling spacing is 0.4 mm-0.8 mm, the printing speed is 3-4 mm / s, the air pressure is 0.1-0.2 MPa, and the platform temperature is 0°C-5°C.

[0047] The aperture is the width of the printed hole, and the fill pitch refers to the distance between the printed filaments. Therefore, the fill pitch, aperture, and filament diameter all satisfy the following equation: fill pitch = aperture + filament diameter.

[0048] Furthermore, in step S4, the directional freezing box is composed of copper plates and ABS plates, the top surface is made of ABS plates, and the left and right upper plates are made of copper plates, so that the upper ice crystals grow horizontally, the left and right lower plates are made of ABS plates, and the bottom is made of copper plates, so that the lower ice crystals grow vertically.

[0049] Furthermore, in step S5, the ultraviolet light irradiation time is 10 min-30 min, preferably 20 min; the ultraviolet light wavelength is 365 nm-405 nm, preferably 365 nm; the ultraviolet light intensity is 5 mW / cm 2 -50mW / cm 2 , preferably 10 mW / cm 2 .

[0050] Furthermore, in step S5, the soaking time of the sodium citrate solution is 30 min-120 min, preferably 60 min; the soaking time of the calcium chloride solution is 30 min-120 min, preferably 60 min.

[0051] Furthermore, in step S5, the concentration of the sodium citrate solution is 0.3 mol / L, and the concentration of the calcium chloride solution is 3 wt %.

[0052] A double-layer directional bone and cartilage integrated scaffold is prepared by the above method.

[0053] Compared with the prior art, this invention has the following advantages and beneficial effects:

[0054] (1) The double-layer directional osteochondral integrated scaffold simulates the fiber arrangement and distribution of the natural osteochondral structure to a certain extent, and the different directional structures of the upper and lower layers are conducive to the growth and penetration of cells along the pore direction. On the one hand, the horizontally arranged tubular pores of the cartilage layer can induce the directional lateral growth of cells. On the other hand, the osteochondral defect starts to heal from the bottom, so the vertically oriented pore structure of the subchondral bone layer can guide the active cells at the bottom to migrate and diffuse upward rapidly. At the same time, the highly interconnected porous structure of the upper and lower layers can support the mutual flow between natural tissue and new tissue, leading to the induced regeneration of different tissues, that is, the cartilage and subchondral bone layers regenerate at the same time at the spatial level.

[0055] (2) The solid structure of the transition layer can, on the one hand, prevent the migration of cells between the cartilage and subchondral bone layers and the growth of blood vessels into the cartilage layer, and on the other hand, as a hydrogel, it can allow the transport of oxygen and waste;

[0056] (3) The transition layer can be organically combined with the upper and lower layers, and the interface bonding strength is high, which to a certain extent prevents the upper and lower layers from falling off after long-term culture of the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the directional freezing box.

[0058] Figure 2 Schematic diagram of the preparation process of integrated bone and cartilage scaffold.

[0059] Figure 3 Schematic diagram of the integrated bone and cartilage scaffold and SEM image of the internal directional structure.

[0060] Figure 4 Figure 1 shows the combined force of the bone and cartilage integrated scaffold.

[0061] Figure 5 This is the cartilage gene expression map of the integrated osteochondral scaffold.

[0062] Figure 6 This is the osteogenic gene expression map of the integrated osteochondral scaffold. DETAILED DESCRIPTION

[0063] The present invention discloses an integrated bone and cartilage scaffold with a directional pore structure and a preparation method thereof, belonging to the field of 3D printing technology. The method simulates the natural material and structure of bone cartilage, utilizes the principle of directional crystallization to prepare a bone and cartilage scaffold with a directional structure, utilizes a low-temperature deposition manufacturing process based on rapid prototyping technology to prepare a horizontally oriented cartilage layer scaffold and a vertically oriented subchondral bone layer scaffold, and utilizes methacrylated gelatin capable of UV cross-linking in combination with other materials with chemical grafting and bonding effects as a transition layer between the cartilage layer and the subchondral bone layer, thereby preparing a tissue engineering bone and cartilage integrated composite scaffold. The present invention is aimed at the treatment of articular bone and cartilage defects, and uses a bioactive material to manufacture a bone and cartilage composite scaffold with a directional pore structure, which can promote the coordinated repair of bone and cartilage tissue according to the characteristics of different functional areas of bone and cartilage, thereby improving the repair effect of bone and cartilage defects.

[0064] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0065] Example

[0066] like Figure 2 As shown, the present invention provides a method for preparing the integrated osteochondral scaffold with a directional pore structure, the method comprising 3D bioprinting, directional freezing and photocuring cross-linking, and comprising the following steps:

[0067] S1. Printing the subchondral bone layer: dissolving methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and a photoinitiator in water to prepare a first mixed solution, pouring the solution into a barrel, and loading the 3D bioprinter nozzle to print the subchondral bone layer;

[0068] S2, printing a transition layer: preparing aqueous solutions of methacrylated gelatin GelMA, chitosan CS, dopamine DA, a photoinitiator, and sodium hydroxide, respectively, and then mixing them to form a second mixed solution, which is poured into a barrel and loaded into a 3D bioprinter nozzle to print a transition layer on the subchondral bone layer obtained in step S1;

[0069] S3, printing the cartilage layer: dissolving methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and a photoinitiator in water to prepare a third mixed solution, pouring the solution into a barrel, and loading the solution into a 3D bioprinter nozzle to print the cartilage layer on the transition layer of step S2, forming an osteochondral scaffold consisting of a cartilage layer and a subchondral bone layer bonded by the transition layer;

[0070] S4, freezing the directional osteochondral scaffold: placing the osteochondral scaffold printed in step S3 in a directional freezing box, and placing the osteochondral scaffold and the directional freezing box together in a liquid nitrogen tank for directional freezing, wherein the cartilage layer is horizontally directional frozen, and the subchondral bone layer is vertically directional frozen;

[0071] S5. Cross-linking of osteochondral scaffold: The osteochondral scaffold after directionally freezing is first irradiated with ultraviolet light, and then immersed in sodium citrate solution and calcium chloride solution respectively, to finally form a double-layered directional osteochondral scaffold.

[0072] Furthermore, in step S1, the total mass percentage of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution is 15 wt % to 25 wt %.

[0073] Furthermore, in step S2, the mass percentages of the methacrylated gelatin GelMA, chitosan CS, dopamine DA, photoinitiator, and sodium hydroxide in the second mixed solution are 15wt%-25wt%, 2wt%-10wt%, 0.5wt%-2wt%, 0.1wt%-1wt%, and 0.05wt%-0.2wt%, respectively.

[0074] Furthermore, in step S3, the total amount of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and photoinitiator in the third mixed solution is 15 wt % to 25 wt %.

[0075] Furthermore, in step S1, the thickness of the subchondral bone layer is 0 mm-5 mm, the wire diameter is 0.4 mm-0.5 mm, the filling spacing is 0.6 mm-1 mm, the printing speed is 4-5 mm / s, the air pressure is 0.2-0.3 MPa, and the platform temperature is 0°C-5°C.

[0076] Furthermore, in step S2, the transition layer is a solid layer, the thickness of the transition layer is 0mm-1mm, the wire diameter is 0.2mm-0.3mm, the filling spacing is equal to the wire diameter, the printing speed is 2-4mm / s, the air pressure is 0.05-0.15Mpa, and the platform temperature is 0℃-5℃.

[0077] Furthermore, in step S3, the thickness of the cartilage layer is 0 mm-5 mm, the wire diameter is 0.2 mm-0.3 mm, the aperture is 0.4 mm-0.8 mm, the printing speed is 3-4 mm / s, the air pressure is 0.1-0.2 MPa, and the platform temperature is 0°C-5°C.

[0078] Furthermore, in step S4, the directional freezing box is composed of a copper plate and an ABS plate, such as Figure 1As shown in the right picture, the top surface is made of ABS board, and the left and right upper half plates are made of copper board, so that the upper ice crystals grow horizontally. The left and right lower half plates are made of ABS board, and the bottom is made of copper board, so that the lower ice crystals grow vertically (the dark blocks in the picture are copper boards, and the light blocks are ABS boards). Ordinary directional freezing boxes are also made of copper and ABS boards, but they can only meet the ice crystal growth in one direction, such as Figure 1 As shown, a single-layer horizontally oriented freezing box is designed as follows: copper plates are used only on the left and right sides, while ABS plates are used on the remaining four sides, allowing ice crystals to grow horizontally. A single-layer vertically oriented freezing box is designed as follows: copper plates are used only on the top and bottom sides, while ABS plates are used on the remaining four sides, allowing ice crystals to grow vertically in the vertical direction. The freezing box used in the present invention can simultaneously meet the requirements for ice crystal growth in different directions at the top and bottom.

[0079] Furthermore, in step S5, the ultraviolet light irradiation time is 10 min-30 min, preferably 20 min; the ultraviolet light wavelength is 365 nm-405 nm, preferably 365 nm; the ultraviolet light intensity is 5 mW / cm 2 -50mW / cm 2 , preferably 10 mW / cm 2 .

[0080] Furthermore, in step S5, the soaking time of the sodium citrate solution is 30 min-120 min, preferably 60 min; the soaking time of the calcium chloride solution is 30 min-120 min, preferably 60 min.

[0081] The following are specific embodiments to further illustrate the technical solutions and technical effects of the present invention.

[0082] Example 1

[0083] A double-layer directional bone and cartilage integrated scaffold and a preparation method thereof, comprising the following steps:

[0084] (1) Synthesis of methacryloylated gelatin (GelMA): A 10 wt% gelatin solution was prepared in a phosphate buffer at 50°C. After 1 hour, 8 mL of methacrylic anhydride was added dropwise to the gelatin solution at a rate of 0.5 mL / min and reacted at 50°C for 3 hours. The reaction was stopped by diluting the solution 5-fold with phosphate buffer. To remove any unreacted methacrylic anhydride, the solution was dialyzed in distilled water using an 80 kDa dialysis membrane for 1 week, and the solution was freeze-dried for 7 days and stored at -80°C. The chemical structure and degree of methacryloylation of GelMA were studied by nuclear magnetic resonance hydrogen spectroscopy (1H-NMR). The degree of substitution of GelMA was calculated to be about 40% using the formula (1-integral area ratio of the lysine methylene proton peak of GelMA and pure gelatin).

[0085] (2) Preparation of 5GMA / 5CS / 7SF hydrogel ink for cartilage layer: First, 2.5 g of 5% methacrylated gelatin, 2.5 g of 5% chitosan, 3.5 g of 7% silk fibroin, 1.75 g of 3.5% acetic acid solution, and 0.5 g of 1% photoinitiator were weighed according to their mass fractions. The photoinitiator powder was then added to a beaker containing 39.25 g of deionized water and magnetically stirred at a constant temperature of 60°C for 1 h at a speed of 200 r / min to dissolve the photoinitiator in the water. The weighed chitosan and acetic acid solution was then poured into the beaker and magnetically stirred at 50°C for 1 h (at a speed of 200 r / min) to fully dissolve the chitosan in the weak acid environment. Finally, the weighed GelMA and SF were blended in a beaker and magnetically stirred at 50°C for 5 h at a stirring speed of 200 r / min. After GelMA and SF were uniformly dissolved and mixed, the mixed gel of GelMA and SF was transferred to the printing cylinder, ultrasonicated for 30 min to remove bubbles, and stored for later use.

[0086] (3) Preparation of 5GMA / 5SA / 7.55HA hydrogel ink for subchondral bone layer: First, weigh 2.5g of modified gelatin with a mass fraction of 5%, 2.5g of sodium alginate with a mass fraction of 5%, 3.75g of nanohydroxyapatite with a mass fraction of 7.5%, and 0.5g of photoinitiator with a mass fraction of 1%. Then, add the photoinitiator powder into a beaker containing 40.75g of deionized water, and stir magnetically at a constant temperature of 60℃ for 1h, with the speed set at 200r / min, so that the photoinitiator is fully dissolved in the water. Then, pour HA into the beaker and stir evenly. Finally, mix GelMA and SA in a beaker, place it in a constant temperature water bath at 50℃ and stir magnetically for 5h, with the speed set at 200r / min. After GelMA and SA are evenly dissolved and mixed, transfer the mixed gel of GelMA and SA into the 3D printing barrel, remove bubbles by ultrasonication for 30min, and store for later use. The subchondral bone layer hydrogel ink obtained at this time was named 5GMA / 5SA / 7.5HA.

[0087] (4) Preparation of transition layer GelMA / PDA / CS hydrogel ink: First, a 40 wt% GelMA stock solution was prepared (the final concentration of GelMA after mixing with other components was 20 wt%), and the pH value was set to 5. The photoinitiator (Irgacure 2959) solution was prepared at a stock concentration of 2.5%, and the concentration of photoinitiator in the final sample was 0.5 wt%. A 2 wt% dopamine (DA) stock solution (corresponding to a final DA concentration of 0.5 wt%) and a 2 wt% NaOH solution (corresponding to a final NaOH concentration of 0.1 wt%) were prepared. To achieve the desired final concentration, GelMA, photoinitiator, DA, and NaOH were mixed in a volume ratio of 1:0.4:0.5:0.1 to prepare a pre-mixed solution, i.e., a pre-gel mixture. The pre-gel mixture was kept at 37°C for 1 hour before the experiment to allow DA to oxidize. A chitosan solution with a concentration of 5 wt% was added to the pre-gel mixture, and the volume ratio of the chitosan solution to the pre-gel mixture was 0.3:1 to improve the viscosity and cross-linking degree of the ink to obtain GelMA / PDA / CS hydrogel ink.

[0088] (5) Preparation of a double-layer directional bone-cartilage integrated scaffold: The three inks of cartilage layer, transition layer, and subchondral bone layer were poured into the 3D printing barrel, and then the temperature of the printing barrel and the printing platform were adjusted. The subchondral bone layer, transition layer, and cartilage layer were printed in sequence through the imported STL model.

[0089] The printing parameters of the cartilage layer are: needle 0.31 mm, pressure 0.12 MPa, speed 4 mm / s, platform temperature 0 °C, thickness 2 mm, and filling spacing 0.7 mm.

[0090] The printing parameters of the transition layer are: needle tip 0.26 mm, pressure 0.08 MPa, speed 3 mm / s, platform temperature 0 °C, thickness 0.2 mm, and filling spacing 0.3 mm.

[0091] The printing parameters of the subchondral bone layer are: needle tip 0.51 mm, pressure 0.22 MPa, speed 4 mm / s, platform temperature 0°C, thickness 2 mm, and filling spacing 0.7 mm.

[0092] The scaffold is initially solidified and formed on a low-temperature platform, and then the printed scaffold is quickly placed in a directional freezing box (the upper layer is horizontally oriented and the lower layer is vertically oriented), and placed in a liquid nitrogen tank and frozen at -160°C for 12 hours to allow the scaffold to obtain a preliminary internal orientation structure. After the scaffold is taken out of the liquid nitrogen tank, it is placed under ultraviolet light for preliminary curing and photocrosslinking for 20 minutes to maintain the internal orientation structure of the scaffold. The cartilage layer is then immersed in a 0.3 mol / L sodium citrate solution for 60 minutes, and the subchondral bone layer is immersed in a 3wt% CaCl2 solution for 60 minutes to further complete ionic crosslinking, and finally the construction of the bone / cartilage integrated scaffold is completed. The preparation process and scaffold structure are shown in the figure. Figure 2 and Figure 3 .

[0093] like Figure 3 As shown, the double-layer composite hydrogel scaffold prepared in this embodiment includes an upper composite hydrogel layer (cartilage layer), a transition layer and a lower composite hydrogel layer (subchondral bone layer) in sequence. The cartilage layer has a thickness of 2 mm, a horizontally oriented pore structure, and a pore size of 0.4 mm. The transition layer is a solid layer with a thickness of 0.2 mm. The subchondral bone layer has a thickness of 2 mm, a vertically oriented pore structure, and a pore size of 0.2 mm.

[0094] Example 2

[0095] A double-layer directional bone and cartilage integrated scaffold was prepared according to the method of Example 1, except that the light curing time in step (5) was 30 minutes.

[0096] Comparative Example 1

[0097] A double-layer directional bone and cartilage integrated scaffold was prepared according to the method of Example 1, except that the transition layer hydrogel ink in step (4) was composed of GelMA and DA, and no chitosan was added.

[0098] Comparative Example 2

[0099] A double-layer non-directional bone and cartilage integrated scaffold was prepared according to the method of Example 1, except that, in the non-directional group, the scaffold was not placed in a directional freezing box for orientation, that is, step S4 was omitted and S5 was directly performed.

[0100] Test Example 1: Interface bonding performance test of osteochondral scaffold

[0101] A laboratory-made shear force testing device was used to test the bonding strength. The test system consists of a test platform and a dynamometer fixed on a mobile platform. The sample was fixed to the test platform with double-sided tape. The dynamometer probe was circular. The height of the bottom of the circular probe of the dynamometer was adjusted to be located at the junction of the two layers of the bracket. At this time, the circular probe should face the upper layer of the bracket. The instrument was started and the dynamometer probe was slowly moved toward the bracket. When the probe contacted the upper layer of the bracket, the bracket was subjected to the shear thrust of the probe. At this time, the dynamometer reading was constantly changing. When the dynamometer probe destroyed the double-layer bond of the bracket, causing the upper and lower layers of the bracket to separate, the dynamometer reading F at this time was recorded. By calculating the ratio of F to the cross-sectional area of ​​the bracket, the shear force data of the bracket can be obtained.

[0102] The results of the test are as follows Figure 4 As shown in the figure, the control group refers to the scaffold without the addition of a transition layer, the GelMA / PDA group refers to the scaffold prepared in Example 1, and the GelMA / PDA / CS group refers to the scaffold prepared in Example 1. As can be seen from the figure, the double-layer directional bone and cartilage integrated scaffold prepared in Example 1 of the present invention has a high interfacial bonding strength, and the maximum shear force can reach 10KPa.

[0103] Test Example 2: In vitro repair ability of osteochondral scaffolds

[0104] Take the osteochondral scaffolds of Example 1 and Comparative Example 1 as examples.

[0105] Osteochondral scaffolds (Example 1: directional group, Comparative Example 2: non-directional group) were co-cultured with ADTC5 and MC3T3E1 cells in vitro. Cultures were removed after 1, 2, 3, and 4 weeks, respectively. RNA was then extracted using a total RNA extraction kit, and reverse transcription and RT-PCR reactions were performed to detect the expression of cartilage- and osteogenesis-related genes.

[0106] like Figure 5 、 Figure 6 As shown, the double-layer oriented osteochondral scaffold obtained in Example 1 can secrete specific extracellular matrix that promotes chondrogenic differentiation and osteogenic differentiation. The content of cartilage-specific genes (Col-X, Col-Ⅱ, Sox-9, Aggrecan) and osteogenic-specific genes (RUNX2, ALP, OCN, Col-Ⅰ) reached the highest level at the third and fourth weeks, respectively, and were higher than those of the non-oriented group scaffold in Comparative Example 2. The osteochondral scaffold of the present invention has good cell compatibility and the ability to induce osteochondral tissue differentiation in vitro.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bone and cartilage integrated scaffold with a directional pore structure, characterized in that: The bone and cartilage integrated scaffold is a double-layer composite hydrogel scaffold, which includes an upper composite hydrogel layer, a transition layer, and a lower composite hydrogel layer in sequence. The upper composite hydrogel has a horizontally oriented pore structure and is used to simulate the cartilage tissue environment, and includes methacryloylated gelatin GelMA, chitosan CS and silk fibroin SF in an independent weight percentage of 3wt%-10wt%; The lower layer composite hydrogel has a vertically oriented pore structure and is used to simulate the subchondral bone tissue environment, and includes methacrylated gelatin GelMA, sodium alginate SA and hydroxyapatite HA in an independent weight percentage of 3wt%-10wt%; The transition layer is a component that can produce chemical grafting with the upper composite hydrogel and the lower composite hydrogel, and includes methacrylated gelatin GelMA, polydopamine PDA and chitosan CS in mass percentages of 15wt%-25wt%, 0.5wt%-2wt% and 2wt%-10wt%.

2. The integrated osteochondral scaffold according to claim 1, characterized in that: In the upper layer composite hydrogel, the mass ratio of methacrylated gelatin, chitosan and silk fibroin is 5:5:

7.

3. The bone and cartilage integrated scaffold according to claim 1, characterized in that: In the lower layer composite hydrogel, the mass ratio of methacrylated gelatin, sodium alginate and hydroxyapatite is 2:2:

3.

4. The integrated bone and cartilage scaffold according to claim 1, characterized in that: The transition layer is methacrylated gelatin / polydopamine / chitosan, denoted as GelMA / PDA / CS, and the mass percentages of methacrylated gelatin, polydopamine, and chitosan in the transition layer hydrogel are 20 wt%, 0.5 wt%, and 5 wt%.

5. The integrated osteochondral scaffold according to claim 1, characterized in that: The thickness of the upper composite hydrogel is 0 mm to 5 mm, and the pore size is 0.2 mm to 0.5 mm; The thickness of the lower layer composite hydrogel is 0 mm to 5 mm, and the pore size is 0.2 mm to 0.5 mm; The transition layer is a solid layer with a thickness of 0mm-1mm.

6. The integrated osteochondral scaffold according to claim 1, characterized in that: The double-layer composite hydrogel also contains factors that promote the regeneration of osteochondral tissue, including at least one of stem cells, human fibroblasts and bioactive factors.

7. A method for preparing an integrated bone and cartilage scaffold with a directional pore structure, characterized in that: The method comprises 3D bioprinting, directional freezing and photocuring cross-linking, and includes the following steps: S1. Printing the subchondral bone layer: dissolving methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and a photoinitiator in water to prepare a first mixed solution, pouring the solution into a barrel, and loading the 3D bioprinter nozzle to print the subchondral bone layer; The total mass percentage of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution is 15wt%-25wt%; The mass percentages of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution are 3wt%-8wt%, 3wt%-8wt%, 5wt%-10wt%, and 0.5wt%-2wt%, respectively; S2, printing a transition layer: preparing aqueous solutions of methacrylated gelatin GelMA, chitosan CS, dopamine DA, a photoinitiator, and sodium hydroxide, respectively, and then mixing them to form a second mixed solution, which is poured into a barrel and loaded into a 3D bioprinter nozzle to print a transition layer on the subchondral bone layer obtained in step S1; The second mixed solution is composed of a 5wt%-10wt% chitosan solution and a pre-gel mixture in a volume ratio of 0.3:1, and the pre-gel mixture is composed of methacrylated gelatin GelMA, dopamine DA, a photoinitiator, and sodium hydroxide in mass percentages of 15wt%-25wt%, 0.5wt%-2wt%, 0.1wt%-1wt%, and 0.05wt%-0.2wt%; S3, printing the cartilage layer: dissolving methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and a photoinitiator in water to prepare a third mixed solution, pouring the solution into a barrel, and loading the solution into a 3D bioprinter nozzle to print the cartilage layer on the transition layer of step S2, forming an osteochondral scaffold consisting of a cartilage layer and a subchondral bone layer bonded by the transition layer; The total mass percentage of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and photoinitiator in the third mixed solution is 15wt%-25wt%; The mass ratios of the methacryloylated gelatin GelMA, chitosan CS, silk fibroin SF, acetic acid, and photoinitiator in the third mixed solution are 2wt%-8wt%, 2wt%-8wt%, 5wt%-10wt%, 1wt%-5wt%, and 0.5wt%-2wt%, respectively; S4, freezing the directional osteochondral scaffold: placing the osteochondral scaffold printed in step S3 in a directional freezing box, and placing the osteochondral scaffold and the directional freezing box together in a liquid nitrogen tank for directional freezing, wherein the cartilage layer is horizontally directional frozen, and the subchondral bone layer is vertically directional frozen; S5. Cross-linking of osteochondral scaffold: The osteochondral scaffold after directionally freezing is first irradiated with ultraviolet light, and then immersed in sodium citrate solution and calcium chloride solution respectively, to finally form a double-layered directional osteochondral scaffold.

8. The method according to claim 7, characterized in that In step S1, the mass percentages of the methacrylated gelatin GelMA, sodium alginate SA, hydroxyapatite HA, and photoinitiator in the first mixed solution are 5%, 5%, 7.5%, and 1%, respectively.

9. The method according to claim 7, characterized in that In step S2, the pre-gel mixture consists of methacrylated gelatin GelMA, dopamine DA, a photoinitiator, and sodium hydroxide in the mass percentages of 20 wt%, 0.5 wt%, 0.5 wt%, and 0.1 wt%.

10. The method according to claim 7, characterized in that In step S3, the mass ratios of the methacrylated gelatin GelMA, chitosan CS, silk fibroin SF, and photoinitiator in the third mixed solution are 5 wt%, 5 wt%, 7 wt%, 3.5 wt%, and 1 wt%, respectively.

11. The method according to claim 7, characterized in that The parameters of the 3D bioprinting are as follows: In step S1, the wire diameter is 0.4mm-0.5mm, the filling spacing is 0.6mm-1mm, the thickness is 0mm-5mm, the printing speed is 4-5mm / s, the air pressure is 0.2-0.3MPa, and the platform temperature is 0℃-5℃; In step S2, the wire diameter is 0.2mm-0.3mm, the filling spacing is equal to the wire diameter, the thickness is 0mm-1mm, the printing speed is 2-4mm / s, the air pressure is 0.05-0.15Mpa, and the platform temperature is 0℃-5℃; In step S3, the wire diameter is 0.2mm-0.3mm, the filling spacing is 0.4mm-0.8mm, the thickness is 0mm-5mm, the printing speed is 3-4mm / s, the air pressure is 0.1-0.2MPa, and the platform temperature is 0℃-5℃.

12. The method according to claim 7, characterized in that In step S4, the directional freezing box is composed of copper plates and ABS plates. The top surface is made of ABS plates, and the left and right upper plates are made of copper plates, so that the upper ice crystals grow horizontally. The left and right lower plates are made of ABS plates, and the bottom is made of copper plates, so that the lower ice crystals grow vertically.

13. The method according to claim 7, characterized in that When preparing the solution in steps S1-S3, heating is performed in a water bath at 40°C-60°C.

14. The method according to claim 7, wherein: In step S5, the ultraviolet light irradiation time is 10 min-30 min, the ultraviolet light wavelength is 365 nm-405 nm, and the ultraviolet light intensity is 5 mW / cm 2 -50mW / cm 2 .

15. The method according to claim 14, characterized in that In step S5, the ultraviolet light irradiation time is 20 minutes.

16. The method according to claim 14, characterized in that In step S5, the wavelength of the ultraviolet light is 365 nm.

17. The method according to claim 14, characterized in that In step S5, the UV light intensity is 10 mW / cm 2 .

18. The method according to claim 7, characterized in that In step S6, the soaking time of the sodium citrate solution is 30 min-120 min.

19. The method according to claim 18, characterized in that In step S6, the soaking time of the sodium citrate solution is 60 minutes.

20. The method according to claim 7, wherein In step S6, the soaking time of the calcium chloride solution is 30 min-120 min.

21. The method according to claim 20, characterized in that In step S6, the soaking time of the calcium chloride solution is 60 minutes.

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