A bone cartilage tissue repair scaffold and a preparation method thereof

Abstract

The application provides a kind of osteochondral tissue repair scaffold and its preparation method.The osteochondral tissue repair scaffold is made by self-made photo-curing 3D printing equipment integration, including bionic double-layer structure: cartilage layer and subchondral bone layer, cartilage layer has "lotus root-like" and "radiation-like" hole, subchondral bone layer has "lotus root-like" hole, and longitudinal interstices between cartilage layer and subchondral bone layer are mutually through, aperture is accurately controlled, and also can add calcium phosphate nanocluster material with 1 nanometer particle size in subchondral bone layer.The osteochondral tissue repair scaffold prepared by the application aims at the different tissue characteristics and regeneration requirements of cartilage and subchondral bone, designs double-layer scaffold with different physical and biological characteristics, simulates the anisotropy of natural osteochondral structure, promotes cell migration and tissue growth, and achieves the effect of promoting synchronous regeneration of cartilage and subchondral bone, so it has great application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an integrated osteochondral tissue repair scaffold that promotes cell migration and its preparation method. Background Technology

[0002] Cartilage damage in the knee joint is primarily caused by trauma, disease, or aging, which can affect a patient's daily life and cause pain. Articular cartilage, composed of chondrocytes and extracellular matrix (ECM) (such as type II collagen and proteoglycans), is a smooth, elastic tissue without blood vessels, lymphatics, or nerves, and has a limited capacity for self-healing when damaged. Based on the depth of cartilage damage, it can generally be classified into three types: partial cartilage defects, full-thickness cartilage defects, and osteochondral defects. However, clinical studies have shown that cartilage damage often extends deeply into the subchondral bone, leading to osteochondral defects in the knee joint. If left untreated, osteochondral defects can further aggravate articular cartilage damage and lead to osteoarthritis. Furthermore, due to the inherently complex structure of osteochondral, cartilage and subchondral bone are two tissues with distinct biological characteristics, meaning that the clinical treatment of osteochondral injuries is more challenging. Therefore, osteochondral defect repair is crucial. However, current traditional treatments and surgical methods can only temporarily relieve pain and cannot truly achieve osteochondral defect repair and regeneration of fully functional hyaline cartilage. Therefore, it is urgent to propose new treatment methods to address the clinical challenges of osteochondral repair.

[0003] Tissue engineering, an emerging field, has brought new hope to in vivo transplantation and the regeneration of tissues and organs. In recent years, tissue engineering has gradually become an effective and cutting-edge method for treating articular cartilage injuries. However, there are still some unresolved issues in the osteocartilage scaffolds used in tissue engineering, such as the limited effectiveness of non-layered scaffold repair and the lack of effective interfacial bonding between multilayered scaffolds. 3D printing technology has been extensively studied in the preparation of tissue engineering scaffold materials and has shown advantages. Scaffold materials printed using 3D printing technology have shapes that match the damaged tissue and internal three-dimensional porous structures that can provide cell adhesion, growth, and proliferation. Through material screening and modification, 3D printing "bio-inks" can be prepared, and 3D bioprinters can be used to print scaffold materials with good mechanical properties. DLP, as a new technology in photolithography, has already demonstrated many advantages, such as fast printing speed, high printing precision, and improved cell survival, and has been used in multiple research directions in the field of tissue engineering.

[0004] Previous studies have shown that porous scaffolds facilitate cell migration, and different structures can affect the mechanical properties of the scaffold. Therefore, constructing an ideal scaffold microenvironment that influences cell and tissue fate is essential for promoting tissue regeneration. CN101810885B provides a bilayer biomimetic osteochondral tissue engineering scaffold with circular and vertical pores. However, the formation of the circular and vertical pores in its scaffold is due to the inherent properties of the material itself. The GelMA hydrogel used in this invention also has a similar porous structure, and the pore distribution is random, making it difficult to precisely control the pore size. This severely affects the migration and growth of chondrocytes, resulting in a low degree of biomimicry and poor cartilage repair.

[0005] Therefore, DLP printing technology can be used to construct multi-layer osteocartilage scaffold structures in an integrated manner, achieving personalized customization and effectively mimicking the unique physical and biological characteristics of natural osteocartilage. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a novel osteochondral tissue repair scaffold. The scaffold is a hydrogel scaffold, integrally fabricated using a self-made photopolymerization 3D printing device. It comprises a biomimetic double-layer structure: a cartilage layer and a subchondral bone layer. The cartilage layer has "lotus root-like" and "radial" pores, while the subchondral bone layer has "lotus root-like" pores. The longitudinal pores between the cartilage and subchondral bone layers are interconnected, allowing for precise pore size control. Furthermore, calcium phosphate nanoclusters with a particle size of 1 nanometer can be added to the subchondral bone layer. The purpose of this invention is to provide a hydrogel osteochondral tissue repair scaffold with good biocompatibility, excellent mechanical properties, high biomimetic properties, and that promotes the migration of chondrocytes and bone marrow mesenchymal stem cells and facilitates the repair of osteochondral defects, along with its preparation method.

[0007] Although the hydrogel used to prepare the repair scaffold may itself contain randomly distributed vertical and circular pores, its cartilage repair effect is still relatively poor. The osteocartilage tissue repair scaffold provided by this invention is integrally fabricated using a self-made photopolymerization 3D printing device, which can precisely control the arrangement and pore size of the hydrogel micropores in the osteocartilage tissue repair scaffold, thereby achieving a better osteocartilage defect repair effect.

[0008] On one hand, the present invention provides an osteochondral tissue repair scaffold, which includes a cartilage layer and a subchondral bone layer from top to bottom. The cartilage layer has pores distributed in a "lotus root shape" and a "radial shape". The subchondral bone layer has "lotus root shape" pores that communicate with the cartilage layer.

[0009] The "lotus root-shaped" and "radial" holes described in this invention are both strip-shaped through holes.

[0010] Extensive research has confirmed that the shape and size of the pores in the osteochondral repair scaffold have a significant impact on the scaffold's mechanical properties and cell migration. The shape, distribution, and size of the pores must be precisely controlled to obtain suitable mechanical properties and better cell migration, thereby achieving better cartilage repair results.

[0011] The "lotus root-shaped" holes described in this invention refer to longitudinally continuous holes perpendicular to the support (see details). Figure 1 and Figure 2 "Radial" holes refer to transverse holes that penetrate the support horizontally and radiate outwards from the center of the cross-section, arranged horizontally (see details). Figure 1 and Figure 2 ).

[0012] The cartilage layer has pores distributed in a "lotus root" and "radial" pattern to facilitate the migration and distribution of chondrocytes; the subchondral bone layer has "lotus root" pores to guide bone marrow mesenchymal stem cells (BMSCs) to migrate to the upper space.

[0013] Furthermore, both the cartilage layer and the subchondral bone layer are cylindrical, and the diameter of the cylindrical base of the cartilage layer and the subchondral bone layer is the same.

[0014] Furthermore, the "lotus root-shaped" holes longitudinally penetrate the cylindrical bottom surface of the cartilage layer and the subchondral bone layer, and the "lotus root-shaped" holes of the subchondral bone layer and the cartilage layer are connected as one.

[0015] Furthermore, the "radial" holes penetrate laterally through the cartilage layer and pass through the center of the cross-section of the cartilage layer.

[0016] Furthermore, the pore diameter of the cartilage layer and the subcartilaginous layer is 100~400μm.

[0017] The interconnected porous structure within the scaffold can form a robust three-dimensional tissue, facilitating cell proliferation and differentiation, as well as nutrient / waste transport, thereby achieving efficient tissue regeneration. Furthermore, pore size can regulate cell fate; sufficiently large pores provide adequate osteogenic niches for bone formation, while sufficiently small pores maintain stem cell characteristics and prevent differentiation.

[0018] The vertical pores in the subchondral bone scaffold facilitate the migration of bone marrow mesenchymal stem cells to the upper layers, and the "lotus root-like" scaffold has stronger mechanical properties than the upper layers, which is beneficial for bone repair.

[0019] The hydrogel scaffold of the cartilage layer has pores distributed in a "lotus root" and "radial" pattern, which not only facilitates the migration of bone marrow mesenchymal stem cells, but also facilitates the migration of surrounding normal chondrocytes to the damaged area, thereby enhancing the repair of the cartilage layer.

[0020] The scaffold, which includes the cartilage layer and the subchondral bone layer, is a porous scaffold that facilitates cell migration, nutrient transport and blood vessel ingrowth. Its mechanical strength and biological properties are suitable for the regeneration and repair of osteochondrium.

[0021] Furthermore, the "radial" holes may be provided in one or more layers, with an interval of more than 100 μm between each layer, and are evenly distributed in the cartilage layer.

[0022] Furthermore, each layer is provided with 2 to 10 radial holes that traverse the cartilage layer and pass through the center of the cross-section.

[0023] Furthermore, the included angle between two adjacent "radial" holes on the same layer remains consistent.

[0024] Furthermore, multiple "lotus root-shaped" holes can be provided, with a spacing of more than 100 μm between each hole, and they are evenly distributed longitudinally in the cross-section of the cartilage layer and the subchondral bone layer.

[0025] Furthermore, the size of the osteochondral tissue repair scaffold is designed to match the actual size of the human or animal osteochondral tissue to be transplanted.

[0026] In some embodiments, the cartilage repair scaffold provided by this invention has the following dimensions: the thickness of the multilayer osteocartilage tissue repair scaffold is 4 mm, the thickness of the cartilage layer is 1 mm, the thickness of the subchondral bone layer is 3 mm, and the pore size is 200 µm micropores. This size and specification of the cartilage repair scaffold is primarily used for matching animal experimental rabbit osteocartilage modeling.

[0027] Furthermore, the cartilage layer includes GelMA, LAP, and growth factors.

[0028] Furthermore, the subchondral bone layer includes GelMA and LAP.

[0029] Furthermore, the subchondral bone layer also includes calcium phosphate nanoclusters with a particle size of 1 nanometer.

[0030] The calcium phosphate nanocluster material with a particle size of 1 nanometer described in this invention is prepared based on the calcium phosphate nanocluster in CN109718249A.

[0031] Hydroxyapatite is the main inorganic component of human and animal bones. Ordinary hydroxyapatite is mainly used to prepare functionalized composite materials. Existing nano-hydroxyapatite generally has a particle size of more than 10 nanometers and is mostly used as a scaffold material in bone tissue engineering.

[0032] The calcium phosphate nanoclusters with a particle size of 1 nanometer used in this invention were independently developed by the research team. The preparation method has been patented (CN109718249A) and is mainly used to prepare drugs for repairing osteoporosis and can effectively promote the regeneration of tooth enamel.

[0033] Extensive research has revealed that when calcium phosphate nanoclusters with a particle size of 1 nanometer are used to prepare cartilage tissue repair scaffolds, their smaller particle size not only results in better biocompatibility but also better biomimetic effects, effectively promoting osteogenic differentiation of mesenchymal stem cells and leading to better cartilage tissue repair.

[0034] Furthermore, the GelMA and LAP concentrations are the same in the cartilage layer and the subchondral bone layer.

[0035] Furthermore, the GelMA content is 15%, and the LAP content is 0.2%.

[0036] Furthermore, the growth factor is KGN, and the concentration of KGN is 200 μM.

[0037] On the other hand, the present invention provides a method for preparing a bone and cartilage tissue repair scaffold, characterized in that the preparation method includes:

[0038] (1) Prepare the first solution by adding GelMA, LAP, and KGN to single-distilled water under light-protected conditions and stirring until homogeneous. The first solution contains GelMA, LAP, and growth factors and is used to prepare the cartilage layer.

[0039] (2) To prepare the second solution, GelMA, LAP, or GelMA, LAP, and calcium phosphate nanoclusters with a particle size of 1 nanometer are added to single-distilled water under light-protected conditions and stirred evenly to obtain the second solution. The second solution includes GelMA, LAP, and 1 nanometer hydroxyapatite, and is used to prepare the subchondral bone layer;

[0040] (3) Pour the first liquid into the liquid tank of the photopolymerization 3D printing equipment and print it. After the upper cartilage layer scaffold is completely printed, the upper cartilage layer hydrogel scaffold is obtained.

[0041] (4) After cleaning the printer liquid tank, pour the second liquid into the printer liquid tank and continue printing. After the lower cartilage subchondral bone layer scaffold is completely printed, a double-layer osteochondral tissue repair scaffold is obtained.

[0042] Based on the principles of bionics and the size and structure of normal osteochondral defects, a CAD model of a double-layer osteochondral tissue integrated repair scaffold with a specific shape and bionic spatial structure is established using computer-aided design software. The designed double-layer osteochondral tissue repair scaffold CAD model is converted into a Task Scheduler Task Object (.job) file format suitable for a self-designed and developed UV-curing 3D printing device, and imported into the 3D printing device. Using the UV curing printing system, the first and second liquid materials are printed according to the preset parameters using the self-made UV curing 3D printing device, thus obtaining an osteochondral tissue repair scaffold structure with precise pore arrangement and pore size. The scaffold structure is divided into a cartilage layer and a subchondral bone layer from top to bottom. The cartilage layer has pores distributed in a "lotus root" and "radial" pattern, that is, the pore structure is arranged laterally and longitudinally to facilitate the migration and distribution of chondrocytes. The subchondral bone layer has "lotus root" shaped pores, that is, the pore structure is arranged longitudinally to guide the migration of bone marrow mesenchymal stem cells (BMSCs) to the upper space. The longitudinal "lotus root" shaped pores between the cartilage layer and the subchondral bone layer are interconnected.

[0043] The invention utilizes a self-made photopolymerization 3D printing device, which is prepared according to the method of the patent application CN110228193A. This device can achieve simultaneous photopolymerization and printing, and can precisely control the pore size and pore arrangement of the osteocartilage tissue repair scaffold, thereby achieving better cartilage tissue repair results.

[0044] The bilayer osteochondral tissue repair scaffold involved in this invention is constructed by mimicking the physiological structure and composition of natural osteochondral tissue. Different growth factors, inorganic ions or organic components, such as KGN, BMP, TGF-β1, etc., can be added to both the upper and lower layers based on the original components.

[0045] Although GelMA hydrogel itself has randomly distributed vertical and circular pores, its cell migration effect is still poor, making it difficult to achieve sufficient defect repair. This invention uses a photopolymerization 3D printing device to print gels with specific 200µm pore sizes in the form of "lotus root" and "radial" pores, which can significantly improve the cell migration promotion effect, thereby greatly improving the defect repair effect.

[0046] The osteochondral tissue repair scaffold provided by this invention has the following beneficial effects:

[0047] (1) The osteochondral tissue repair scaffold is precisely printed by a self-made photopolymerization 3D printing equipment. It has precise pore size and pore arrangement, which has a good effect on cell migration.

[0048] (2) The calcium phosphate nanoclusters with a particle size of 1 nanometer were used to prepare osteochondral repair scaffolds and had better osteogenic induction properties than ordinary hydroxyapatite.

[0049] (3) It has adjustable mechanical properties and good biocompatibility;

[0050] (4) It has a better ability to promote the migration of chondrocytes and bone marrow mesenchymal stem cells;

[0051] (5) It has the ability to transport nutrients / transfer waste;

[0052] (6) It has the ability to promote the ingrowth of new blood vessels and better promote the repair of osteochondral defects. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the osteochondral tissue repair scaffold in Example 1.

[0054] Figure 2 This is a cross-sectional view of the osteochondral tissue repair scaffold in Example 1.

[0055] Figure 3 This is a design drawing of the osteochondral tissue repair scaffold in Example 1.

[0056] Figure 4 This is a 3D model and a schematic diagram of the preparation of the osteochondral tissue repair scaffold in Example 1.

[0057] Figure 5 This is a general diagram of the osteochondral tissue repair scaffold in Example 1.

[0058] Figure 6 Fluorescence and electron micrographs of the pores in the osteochondral tissue repair scaffold in Example 1.

[0059] Figure 7 This is a microscopic structural diagram of the upper cartilage layer scaffold with different pore sizes in the osteochondral tissue repair scaffold of Example 2.

[0060] Figure 8 Mechanical properties of the osteochondral tissue repair scaffold with 15% GelMA in Example 2 with different pore sizes.

[0061] Figure 9 Mechanical properties of different pore sizes of the osteochondral tissue repair scaffold in Example 2 with 10% GelMA.

[0062] Figure 10 The image shows scanning electron microscope (SEM) images of 15% GelMA and 10% GelMA in the osteochondral tissue repair scaffold of Example 2.

[0063] Figure 11The mechanical properties of the upper and lower hydrogel scaffolds in the osteochondral tissue repair scaffold of Example 3 are shown.

[0064] Figure 12 The results are from the biocompatibility test of the GelMA hydrogel used as a bone and cartilage tissue repair scaffold in Example 4.

[0065] Figure 13 The results of alkaline phosphatase staining are from the osteogenic induction performance experiment of the GelMA hydrogel used as a bone and cartilage tissue repair scaffold in Example 5.

[0066] Figure 14 The results of the osteogenic induction performance experiment of the GelMA hydrogel of the osteochondral tissue repair scaffold in Example 5 are the quantitative results of alkaline phosphatase activity.

[0067] Figure 15 The results of in vitro cell migration promotion of the osteochondral tissue repair scaffold in Example 6 are shown.

[0068] Figure 16 The in vivo cell infiltration results are shown for the osteochondral tissue repair scaffold in Example 7.

[0069] Figure 17 , 18 Figures 1 and 19 show the in vivo cell infiltration results of osteochondral tissue repair scaffolds with different micropore distributions in Case 8.

[0070] Figure 20 These are gross images of joint samples taken 8 and 16 weeks after stent implantation in Example 9.

[0071] Figure 21 HE staining results of joint samples after stent implantation in Example 9.

[0072] Figure 22 The SO staining results are shown for the joint sample after stent implantation in Example 9.

[0073] Figure 23 The results are the mechanical test results of the joint sample after repair 16 weeks after stent implantation in Example 9. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The advantages and features of the present invention will become clearer from the following description and claims. It should be noted that the drawings are all in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the purpose of illustrating the embodiments of the present invention.

[0075] Example 1: The osteochondral tissue repair scaffold provided by the present invention

[0076] The osteocartilage tissue repair scaffold structure provided in this embodiment is as follows: Figures 1-3 As shown, where Figure 1 This is a schematic diagram of the structure of a bone and cartilage tissue repair scaffold. Figure 2 This is a cross-sectional view of the osteochondral tissue repair scaffold. Figure 3 Design drawing for a scaffold for osteochondral tissue repair.

[0077] Depend on Figure 1 , Figure 2 and Figure 3 As can be seen, the osteochondral tissue repair scaffold provided in this embodiment includes an upper cartilage layer 1 and a lower subchondral bone layer 2. The cartilage layer 1 has "lotus root-shaped" pores 3 and "radial" pores 4. The subchondral bone layer 2 has "lotus root-shaped" pores 3 that penetrate the cartilage layer. The "lotus root-shaped" pores between the cartilage layer 1 and the subchondral bone layer 2 are interconnected. The "lotus root-shaped" pores 3 refer to longitudinally continuous pores perpendicular to the scaffold, and the "radial" pores 4 refer to transverse pores that penetrate the scaffold and radiate outward from the center of the cross-section. In this embodiment, both the cartilage layer 1 and the subchondral bone layer 2 are cylindrical, and the diameter of the cylindrical bottom surface of the cartilage layer 1 and the subchondral bone layer 2 is the same. The "lotus root-shaped" pores 3 penetrate the cylindrical bottom surface of the cartilage layer 1 and the subchondral bone layer 2. The "radial" pores 4 are located on the cylindrical sidewall of the cartilage layer 1 and penetrate the cartilage layer 1 transversely.

[0078] Preferably, the pore diameter of the "lotus root-like" and "radial" pores in the cartilage layer 1 and the subchondral bone layer 2 is 200 μm.

[0079] Preferably, the radial pores 4 can be provided in one or more layers, with a spacing of more than 100 μm between each layer, and are evenly distributed in the cartilage layer 1. In this embodiment, there are three layers of radial pores 4, which are evenly arranged from top to bottom. If it is necessary to prepare a larger osteocartilage tissue repair scaffold, or if the cartilage layer 1 is thick, more layers of radial pores 4 can be provided.

[0080] Preferably, each layer has 2 to 10 radial holes 4 that traverse the cartilage layer 1 laterally and pass through the center of the cross-section. In this embodiment, each layer has a total of 5 radial holes 4. If a larger osteocartilage tissue repair scaffold needs to be prepared, or if the cross-sectional diameter of the cartilage layer 1 is large, more radial holes 4 can be provided in each layer.

[0081] Preferably, the included angle between two adjacent "radial" holes 4 on the same layer is consistent. In this embodiment, since five "radial" holes 4 are provided, the included angle between any two adjacent "radial" holes 4 is 36 degrees.

[0082] Preferably, multiple "lotus root-shaped" holes 3 can be provided, with a spacing of more than 100 μm between each hole, and they are evenly distributed longitudinally in the cross-section of the cartilage layer 1 and the subchondral bone layer 2. In this embodiment, a total of 30 "lotus root-shaped" holes 3 are provided, evenly distributed.

[0083] Preferably, the cartilage layer 1 includes GelMA, LAP, and growth factors, and the subchondral bone layer 2 contains GelMA, LAP, and calcium phosphate nanoclusters with a particle size of 1 nanometer. The preparation method of the calcium phosphate nanoclusters with a particle size of 1 nanometer is described in CN201910068395.1.

[0084] Preferably, the cartilage layer 1 and the subchondral bone layer 2 have the same concentration of GelMA and LAP, with the GelMA concentration being 10-15% and the LAP concentration being 0.2%.

[0085] Preferably, the growth factor of the cartilage layer 1 is KGN, and the concentration of KGN is 200 μM.

[0086] The upper cartilage layer 1 and the lower subchondral bone layer 2 were printed using a self-made photopolymerization 3D printing device to create a pre-designed double-layer osteochondral tissue repair scaffold.

[0087] The method for preparing the osteochondral tissue repair scaffold provided in this embodiment specifically includes:

[0088] (1) Prepare the first solution, which includes GelMA, LAP and KGN. Specifically, GelMA, LAP and KGN are added to single-distilled water in a 42°C water bath under light-protected conditions and stirred evenly to obtain the first solution. The concentration of GelMA is 15%, the concentration of LAP is 0.2% and the concentration of KGN is 200 μM.

[0089] (2) Prepare the second solution, which includes GelMA, LAP, and 1-nanometer calcium phosphate nanoclusters. Specifically, GelMA, LAP, and 1-nanometer calcium phosphate nanoclusters are added to single-distilled water in a 42°C water bath and stirred until homogeneous to obtain the second solution. The concentration of GelMA is 15%, and the concentration of LAP is 0.2%.

[0090] (3) Pour 6 mL of the first liquid into the 42°C heated liquid tank of the self-made photopolymerization 3D printing equipment to obtain the upper cartilage layer scaffold after printing; transfer the remaining first liquid in the liquid tank, clean the liquid tank with single distilled water, pour 6 mL of the second liquid into the 42°C heated liquid tank, and continue printing to obtain an integrated double-layer osteocartilage tissue repair scaffold.

[0091] (4) According to the designed CAD model, the printed double-layer osteochondral tissue repair scaffold consists of two parts. The upper cartilage layer has transversely penetrating micropores with a diameter of 200µm, used to induce and promote the migration and distribution of chondrocytes; and longitudinally penetrating micropores with a diameter of 200µm, used to induce the migration of chondrocytes and bone marrow mesenchymal stem cells. The lower subchondral bone layer has micropores with a diameter of 200µm that are longitudinally interpenetrating with the upper cartilage layer, used to guide the migration of bone marrow mesenchymal stem cells to the upper space. The thickness of the double-layer osteochondral tissue repair scaffold is 4mm, the thickness of the upper cartilage layer is 1mm, and the thickness of the lower subchondral bone layer is 3mm.

[0092] Figure 4 The 3D model and preparation schematic diagram of the double-layer osteochondral tissue repair scaffold provided by the present invention. Figure 5 General views of the double-layer osteochondral tissue repair scaffold provided by the present invention. (a) Top view of the scaffold; (b) Side view of the upper scaffold; (c) Side view of the lower scaffold. Figure 6 Fluorescence and electron microscopy images of the pores in the double-layer osteochondral tissue repair scaffold provided by this invention. The electron microscopy images also show that the gel itself has a porous structure. (a) Fluorescence images of the "lotus root-like" and "radial" structures of the upper scaffold; (b) Scanning electron microscopy image of the side of the upper scaffold. Figure 4-6 This demonstrates that the double-layer osteochondral tissue repair scaffold of the present invention can be printed using DLP to produce a precise double-layer structure with a diameter of 4 mm, an upper scaffold thickness of 1 mm, a lower scaffold thickness of 3 mm, and a pore size of 200 μm.

[0093] Example 2: Effects of different GelMA concentrations and pore sizes on the mechanical properties of bilayer osteochondral tissue repair scaffolds

[0094] This embodiment describes the mechanical testing of hydrogel scaffolds with different pore sizes formed at 10% and 15% GelMA concentrations using a tensile-compression mechanical testing instrument (Instron-5543 with a 1kN sensor). The specific implementation steps are as follows:

[0095] (1) Use a DLP printer to print lotus root-shaped subchondral bone layer GelMA hydrogel scaffolds with two concentrations of 10% and 15%, with scaffold pore diameters of 0µm, 100µm, 200µm and 400µm respectively.

[0096] (2) Incubate the printed GelMA hydrogel scaffold in 1xPBS for 4 hours;

[0097] (3) Place the hydrogel scaffold to be tested on the stage of the mechanical testing instrument, and set the probe to press the scaffold down at a speed of 1 mm / min until the height of the scaffold becomes 65% of the initial height. The computer automatically records the compression deformation and pressure data.

[0098] (4) Calculate the compression modulus by linear fitting based on the strain range of 40%-60%.

[0099] Figure 7 The diagram shows the microstructure of the upper cartilage layer GelMA hydrogel scaffold of the present invention. The pore diameters of the scaffold are 0µm, 100µm, 200µm and 400µm (a, b and c). Figure 8-10 The optimal GelMA concentration and structure were screened for the double-layer osteochondral tissue repair scaffold of the present invention. Figure 8 and Figure 9 Load-compression deformation curves and compressive modulus are shown for printed stents with concentrations of 15% and 10% at stent pore diameters of 0µm, 100µm, 200µm, and 400µm. Figure 10 Scanning electron microscopy (SEM) images of GelMA hydrogels with concentrations of 15% and 10% are shown. By measuring the relationship between load and compressive deformation in mechanical tests of the 10% and 15% GelMA concentrations at different pore sizes, it was demonstrated that different pore sizes affect the scaffold's mechanical properties, and under the same conditions, larger pore sizes result in relatively poorer overall mechanical performance. Furthermore, the 15% GelMA hydrogel scaffold exhibits better overall mechanical properties than the 10% GelMA hydrogel. We also observed the microstructure of the 10% and 15% GelMA hydrogels using SEM, revealing that both hydrogels possess a porous structure. In addition, the specific surface area of ​​the hydrogel scaffolds with different pore sizes was calculated, as shown in Table 1.

[0100] Table 1. Specific surface area of ​​hydrogel scaffolds with different pore sizes

[0101]

[0102] Ultimately, through mechanical screening of material structures and comparison of the overall surface area of ​​the scaffold, it was found that larger pore sizes result in a larger specific surface area for the cartilage repair scaffold. However, increased pore size can easily lead to a decrease in mechanical support performance. This invention selected a 200µm pore size 15% GelMA hydrogel as the scaffold to be used. This hydrogel scaffold possesses both good mechanical properties and a large specific surface area. (*p<0.05, **p<0.01)

[0103] Example 3 Mechanical testing of upper and lower hydrogel scaffolds

[0104] This embodiment uses a tensile-compression mechanical testing instrument (Instron-5543 with a 1kN sensor) to conduct mechanical tests on the upper and lower hydrogel scaffolds respectively. The specific implementation steps are as follows:

[0105] (1) Incubate the printed upper and lower hydrogel scaffolds in 1xPBS for 4 hours;

[0106] (2) Place the hydrogel scaffold to be tested on the stage of the mechanical testing instrument, and set the probe to press the scaffold down at a speed of 1 mm / min until the height of the scaffold becomes 45% of the initial height. The computer automatically records the compression deformation and pressure data.

[0107] (3) Calculate the compression modulus by linear fitting according to the strain range of 20%-40%.

[0108] Figure 11 The mechanical properties of the upper and lower hydrogel scaffolds of the double-layer osteochondral tissue repair scaffold show that the mechanical properties of the lower subchondral bone layer are significantly stronger than those of the upper cartilage layer. This is consistent with the biological differences between cartilage and subchondral bone in natural osteochondral tissue, indicating that the double-layer osteochondral tissue repair scaffold of the present invention has excellent biomimetic mechanical properties.

[0109] Example 4: Cell viability assay using CCK-8 in GelMA hydrogel

[0110] This embodiment uses CCK-8 assay to determine cell viability in GelMA hydrogels. The GelMA hydrogels are divided into two groups: the first group contains only GelMA and LAP, and the second group is a blank control. The specific implementation steps are as follows:

[0111] (1) Prepare the second solution, in which the concentration of GelMA is 15% and the concentration of LAP is 0.2%.

[0112] (2) Add the prepared second solution to the 96-well plate, with 50 µL added to each well. After cross-linking with ultraviolet light, soak it in low-sugar culture medium.

[0113] (3) After soaking for 24 hours, the cells were seeded at 1000 cells / well. After 24 hours of adhesion, the original culture medium was discarded and new culture medium was added at 100 µL per well. Fresh culture medium was added to the blank control group and cell suspension was added to the positive control group. There were 6 replicates for each experimental group.

[0114] (4) Detection of cell proliferation rate at 1, 3, and 5 days. Before detection, add 10 µL of CCK-8 reagent to each well and incubate the 96-well plate in a 37°C, 5% CO2 humidity incubator for 1 hour. After incubation, aspirate the culture medium from each well and place it in a separate 96-well plate. Measure the OD value at 450 nm using a microplate reader, and record and process the cell proliferation data.

[0115] Figure 12The biocompatibility of the GelMA hydrogel used as a bone and cartilage tissue repair scaffold indicates that the cells are in a state of significant proliferation. GelMA has good biocompatibility, which is beneficial to cell proliferation, indicating that the bone and cartilage tissue repair scaffold of the present invention has good biocompatibility.

[0116] Example 5: Qualitative and quantitative determination of the osteogenic induction capacity of GelMA hydrogel using ALP.

[0117] This embodiment uses alkaline phosphatase (ALP) to qualitatively and quantitatively determine the osteogenic induction capacity of GelMA hydrogel. The GelMA hydrogel is divided into three groups: the first group contains only GelMA and LAP; the second group contains GelMA, LAP, and calcium phosphate nanoclusters with a particle size of 1 nm (referred to as CaP in the figure); and the third group contains GelMA, LAP, and commercial hydroxyapatite with a particle size of 20 nm (referred to as HANP in the figure). The specific implementation steps are as follows:

[0118] (1) Prepare the second solution. The concentration of GelMA in the second solution is 15% and the concentration of LAP is 0.2%. The second and third groups also need to add 1 nanometer calcium phosphate nanoclusters or commercial hydroxyapatite with a particle size of 20 nanometers.

[0119] (2) Add the prepared second solution to the 48-well plate, with 150µL added to each well. After cross-linking with ultraviolet light, soak in low-sugar culture medium.

[0120] (3) After soaking for 24 hours, the cells were divided into groups of 1×10⁻⁶ cells. 5 Cells were seeded per well. Once the cells adhered and reached 80% confluence, the original culture medium was discarded, and fresh osteogenic induction medium was added at 250 µL per well. Each experimental group was replicated four times.

[0121] (4) On the seventh day after the start of osteogenic induction, alkaline phosphatase colorimetric and alkaline phosphatase quantitative experiments were performed on the cells, and the alkaline phosphatase data of the samples were recorded and processed.

[0122] Figure 13-14 The qualitative and quantitative results of alkaline phosphatase activity in samples from day 7 indicate that the combination of GelMA with calcium phosphate nanoclusters with a particle size of 1 nanometer has better osteogenic induction properties than GelMA with commercial hydroxyapatite with a particle size of 20 nanometers, which is beneficial to osteogenic differentiation of cells. This shows that the osteochondral tissue repair scaffold of the present invention has good osteogenic induction properties.

[0123] Example 6: The effect of scaffold micropores on in vitro cell migration experiments

[0124] This embodiment describes an in vitro cell migration experiment using a hydrogel scaffold. The specific implementation steps are as follows:

[0125] (1) DiI staining of cells: DiI stock solution was diluted to 2 µM / mL working solution with PBS or serum-free medium, cells were digested with trypsin, centrifuged, and then resuspended in working solution. Cells were then placed in a 37°C, 5% CO2 humidity incubator for 20 min. After the incubation, the supernatant was discarded by centrifugation, and the preheated 37°C medium was slowly added by pipette. The cells were then centrifuged again, and the cells were washed twice in total.

[0126] (2) DiI-stained C3H cells were sorted at 5×10 5 Cells / mL were seeded on 3.5cm culture dishes and the medium was changed after 24 hours.

[0127] (3) Gently place the printed subchondral bone layer scaffold with “lotus root-like” and “radial” pores with a pore size of 200µm, and the printed scaffold without micropores (but with random porous structure in the gel itself) in the middle of the culture dish, ensuring that the culture medium liquid submerges the scaffold.

[0128] (4) Two groups of scaffold samples were collected on the first day after the experiment, and scaffold samples with micropores were collected on the 3rd and 5th days. The collected scaffold samples were observed and photographed using an Olympus upright two-photon confocal microscope, and three-dimensional image reconstruction and data processing were performed using Imaris.

[0129] Figure 15 This study provides a quantitative analysis of cell numbers after migration onto scaffold samples with and without micropores. The results show that the 200µm pore size "lotus root-like" and "radial" pore structures in the bilayer osteochondral tissue repair scaffold of this invention effectively promote cell migration into the scaffold, thereby promoting defect repair in in vivo repair experiments. However, if the gel does not use a photopolymerization 3D printing device to print specific 200µm pore size "lotus root-like" and "radial" pore structures, although it also has vertical and circular pores, its cell migration effect is still poor, making it difficult to achieve sufficient defect repair.

[0130] Example 7: The effect of scaffold micropores on in vivo cell migration experiments

[0131] This embodiment describes an in vivo cell migration experiment using a hydrogel scaffold. The specific implementation steps are as follows:

[0132] (1) Under sterile conditions, a subchondral bone scaffold with “lotus root-like” and “radial” pores with a pore size of 200µm and a scaffold without micropores (but with random porous structures in the gel itself) were printed.

[0133] (2) Pentobarbital was used to anesthetize SPF-grade SD rats (approximately 250g, male rats), and pentobarbital was injected into the peritoneal cavity of the rats at a dose of 50mg / kg.

[0134] (3) Under sterile conditions, make an incision of about 2 cm in length on the inner side of the rat's back, place the two types of GelMA hydrogel scaffolds into it respectively, and then suture.

[0135] (4) Samples were taken on the 3rd and 5th days after surgery and fixed with 4% formaldehyde for more than 24 hours. After the tissue was fixed, the tissue was dehydrated in a 4°C freezer with a 20% and 30% sucrose gradient. After the tissue block settled to the bottom, it was taken out for frozen sectioning.

[0136] (5) After staining the sections with DAPI at a ratio of 1:1000 for 15 minutes, wash them three times with PBS;

[0137] (6) The Olympus upright two-photon confocal microscope was used for observation, and Imaris was used for three-dimensional image reconstruction and data processing.

[0138] Figure 16 To assess in vivo cell infiltration by subcutaneously implanting printed and unprinted GelMA hydrogel scaffolds into rats, on day 5 after implantation, the number of cells infiltrated into the subchondral bone layer scaffold with printed "lotus root-like" and "radial" pores (200µm diameter) was significantly higher than that of the scaffold without micropores. This indicates that the microporous structure in the bilayer osteochondral tissue repair scaffold of this invention can effectively promote cell migration into the scaffold, thereby promoting defect repair in in vivo repair experiments.

[0139] Example 8: Effects of hydrogel scaffolds with different micropore distributions on in vivo cell migration experiments

[0140] This embodiment is an in vivo cell migration experiment using hydrogel scaffolds. Four different scaffolds with varying micropore distributions were used in the experiment. The first type was a bone and cartilage tissue repair scaffold with printed "lotus root-shaped" and "radial" pores with a diameter of 200µm. The second type was a printed "lotus root-shaped" bone and cartilage tissue repair scaffold with a diameter of 200µm. The third type was a printed "radial" bone and cartilage tissue repair scaffold with a diameter of 200µm. The fourth type was a bone and cartilage tissue repair scaffold without printed micropores.

[0141] The specific implementation steps are as follows:

[0142] (1) Under sterile conditions, hydrogel scaffolds with “lotus root-like” and “radial” pores with a pore size of 200µm and scaffolds without micropores (but with random porous structures in the gel itself) were printed.

[0143] (2) Pentobarbital was used to anesthetize SPF-grade SD rats (approximately 250g, male rats), and pentobarbital was injected into the peritoneal cavity of the rats at a dose of 50mg / kg.

[0144] (3) Under sterile conditions, a 2cm incision was made on the inner back of the rat, and four different GelMA hydrogel scaffolds were placed in it and then sutured.

[0145] (4) Samples were taken on the 5th day after surgery and fixed in 4% formaldehyde for more than 24 hours. After the tissue was fixed, it was dehydrated in a 4°C freezer with a 20% and 30% sucrose gradient. After the tissue block settled to the bottom, it was taken out for frozen sectioning.

[0146] (5) After staining the sections with DAPI at a ratio of 1:1000 for 15 minutes, wash them three times with PBS;

[0147] (6) The Olympus upright two-photon confocal microscope was used for observation, and Imaris was used for three-dimensional image reconstruction and data processing. The results are shown in Table 2. Figure 17 , Figure 18 As shown.

[0148] (7) The sections were stained with hematoxylin in the dark for 10 minutes, followed by rapid separation with 1% hydrochloric acid-ethanol, eosin staining for 20 seconds, then dehydration with graded alcohols, clearing with xylene, and mounting with neutral resin. The staining results were scanned using an Olympus VS200 digital slide scanner. The results are as follows: Figure 19 As shown.

[0149] Table 2. Effects of hydrogel scaffolds with different micropore distributions on in vivo cell migration experiments after 5 days.

[0150]

[0151] Figure 17 This is a fluorescence staining grayscale image taken on day 5 after stent implantation. Figure 18 These are the results of fluorescence staining analysis; Figure 19 HE staining results for scaffold sections, where a represents "radial" and b represents "lotus root-like" pores.

[0152] Depend on Figure 19 As can be seen from the schematic diagram, on day 5, where there are no 200μm micropores on the scaffold, cells can only adhere to the scaffold surface and have limited ability to migrate into the scaffold. However, where there are printed micropores, cells can migrate into the scaffold through the pores. Both the "lotus root-shaped" and "radial" pores play a significant role in their migration and distribution.

[0153] From Table 2 and Figure 17 , 18It can be seen that the osteochondral tissue repair scaffold containing both 200µm "lotus root-shaped" and "radial" pores significantly increased the cell migration number on day 5 compared to osteochondral tissue repair scaffolds without micropores or containing only "lotus root-shaped" or "radial" pores. This is because when the osteochondral tissue repair scaffold has both "lotus root-shaped" and "radial" pores, it is very conducive to the migration and distribution of chondrocytes, and can also guide bone marrow mesenchymal stem cells (BMSCs) to migrate to the upper space, thereby greatly increasing the cell migration number.

[0154] This embodiment further compares the cases where the "radial" pores are located in the cartilage layer, the subchondral bone layer, or both. The experiment shows that when the "radial" pores are only located in the subchondral bone layer, the cell migration effect is similar to that of the second type of "lotus root-like" osteochondral tissue repair scaffold. When the "radial" pores are distributed in both the cartilage layer and the subchondral bone layer, the cell migration effect is still not good. Only when the "radial" pores are located in the cartilage layer and the subchondral bone layer does not contain "radial" pores can the cell migration number reach 354.5±12.19 and the cell migration depth reach 279.0±56.90 micrometers be obtained.

[0155] Example 9: Repair and regeneration experiment of osteochondral scaffold in cartilage defect sites in rabbits

[0156] This embodiment is an experiment on the repair and regeneration of cartilage defects in rabbits using hydrogel scaffolds. The specific implementation steps are as follows:

[0157] (1) Under sterile conditions, a double-layer osteochondral tissue repair scaffold (B) with “lotus root-like” and “radial” pores with a diameter of 200µm was printed, a double-layer osteochondral tissue repair scaffold (NB) without micropores, and a double-layer osteochondral GelMA hydrogel scaffold with micropores containing growth factor KGN on the upper layer (B+KGN).

[0158] (2) New Zealand white rabbits were anesthetized by ear vein injection with 1% sodium pentobarbital at a dose of 40 mg / kg.

[0159] (3) Under sterile conditions, the skin was disinfected with povidone-iodine, and an incision of approximately 1 cm was made along the medial side of the patellar ligament. The incision was continued until the knee joint was exposed. A 4 mm diameter and 4 mm deep hole was made at the femoral trochlea using a graduated manual drill to complete the osteochondral defect modeling. All 48 New Zealand white rabbits underwent modeling. After modeling, they were randomly divided into 4 groups: blank group, unprinted group, printed double-layer scaffold group, and printed double-layer scaffold group with the upper layer containing growth factor KGN. The modeling was performed at two time points: 8 weeks (n=4) and 16 weeks (n=8, of which 4 samples were used for mechanical testing). After the scaffold was placed into the defect site according to the orientation, it was immediately disinfected with povidone-iodine and sutured. After modeling, the blank group did not need to be filled with scaffold and was directly disinfected and sutured.

[0160] Figure 20 These are gross images of joint samples taken at 8 and 16 weeks after stent implantation. Figure 21 HE staining results for joint samples; Figure 22 SO staining results for joint samples; Figure 23 The results are from mechanical testing of the joint sample after repair at 16 weeks.

[0161] Figure 21-23 The results, obtained at 8 and 16 weeks, confirmed that the osteochondral tissue repair scaffold of this invention exhibits better repair effects on both the cartilage layer and the subchondral bone layer. SO staining revealed poor repair in the blank control group, with most of the bone layer not fully grown and fibrous tissue filling the gaps, resulting in minimal reduction in the area of ​​the cartilage defect. In contrast, the osteochondral tissue repair group containing KGN showed smooth repair surfaces, near-complete repair, normal cartilage layer thickness, and neatly arranged chondrocytes with tight connections to surrounding tissues. This indicates that the double-layered osteochondral tissue repair scaffold of this invention can promote both subchondral bone regeneration and cartilage regeneration, effectively promoting the repair of osteochondral tissue damage.

[0162] Matters not covered in this invention are common knowledge.

[0163] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. The use of a bone and cartilage tissue repair scaffold in the preparation of bone and cartilage defect repair products, characterized in that, The osteochondral tissue repair scaffold is composed of a cartilage layer and a subchondral bone layer from top to bottom. The cartilage layer has "lotus root-shaped" and "radial" pores, and the subchondral bone layer has "lotus root-shaped" pores that penetrate the cartilage layer. There are a total of 30 "lotus root-shaped" pores, with a spacing of more than 100 μm between each pore, and they are evenly distributed. There are three layers of "radial" pores, with 5 "radial" pores in each layer, and the included angle between any two adjacent "radial" pores is 36 degrees. Both the cartilage layer and the subchondral bone layer are cylindrical, and the diameter of the cylindrical base of the cartilage layer and the subchondral bone layer is the same, which is 4 mm. The upper scaffold is 1 mm thick, and the lower scaffold is 3 mm thick. The "lotus root-shaped" pores penetrate the cylindrical base of the cartilage layer and the subchondral bone layer longitudinally. The "radial" pores are located on the cylindrical sidewalls of the cartilage layer and penetrate the cartilage layer laterally. The "lotus root-like" and "radial" pores in the cartilage layer and subchondral bone layer have a pore diameter of 200 μm; the cartilage layer includes GelMA, LAP and growth factors; the subchondral bone layer includes GelMA, LAP and calcium phosphate nanoclusters with a particle size of 1 nanometer. The GelMA content is 15%; the GelMA and LAP concentrations in the cartilage layer and subchondral bone layer are the same; the LAP content is 0.2% by mass fraction; the growth factor is KGN, and the concentration of KGN is 200 μM.

2. The osteochondral tissue repair scaffold as described in claim 1, characterized in that, From top to bottom, it consists of a cartilage layer and a subchondral bone layer. The cartilage layer has "lotus root-shaped" and "radial" pores, and the subchondral bone layer has "lotus root-shaped" pores that communicate with the cartilage layer. There are a total of 30 "lotus root-shaped" pores, with a spacing of more than 100 μm between each pore, and they are evenly distributed. There are three layers of "radial" pores, with 5 "radial" pores in each layer. The included angle between any two adjacent "radial" pores is 36 degrees. Both the cartilage layer and the subchondral bone layer are cylindrical, and the diameter of the cylindrical base of the cartilage layer and the subchondral bone layer is the same. The structure has a diameter of 4 mm, an upper support layer with a thickness of 1 mm, and a lower support layer with a thickness of 3 mm. The "lotus root-shaped" pores longitudinally penetrate the cylindrical bottom surface of the cartilage layer and the subchondral bone layer. The "radial" pores are located on the cylindrical sidewalls of the cartilage layer and transversely penetrate the cartilage layer. The pore diameter of the "lotus root-shaped" and "radial" pores in the cartilage layer and the subchondral bone layer is 200 μm. The cartilage layer includes GelMA, LAP, and growth factors. The subchondral bone layer includes GelMA, LAP, and calcium phosphate nanoclusters with a particle size of 1 nanometer. The GelMA content is 15%; the GelMA and LAP concentrations in the cartilage layer and subchondral bone layer are the same; the LAP content is 0.2% by mass fraction; the growth factor is KGN, and the concentration of KGN is 200 μM.

3. A method for preparing a bone and cartilage tissue repair scaffold as described in claim 2, characterized in that, The preparation method includes: (1) To prepare the first solution, GelMA, LAP and KGN were added to single-distilled water under light-protected conditions and stirred evenly to obtain the first solution; (2) To prepare the second solution, GelMA, LAP and calcium phosphate nanoclusters with a particle size of 1 nanometer were added to single-distilled water under light-protected conditions and stirred evenly to obtain the second solution. (3) Pour the first liquid material into the liquid tank of the photopolymerization 3D printing equipment and print the upper cartilage layer scaffold; (4) Pour the second liquid into the printer liquid tank and continue printing the lower cartilage scaffold to obtain a double-layer osteocartilage tissue repair scaffold.

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