CN116421784A - Osteochondral tissue repair scaffold and preparation method thereof - Google Patents

Abstract

The invention provides an osteochondral tissue repair stent and a preparation method thereof. The osteochondral tissue repair stent is integrally manufactured by self-made photo-curing 3D printing equipment and comprises a bionic double-layer structure: the cartilage layer is provided with lotus-shaped and radial holes, the subchondral bone layer is provided with lotus-shaped holes, longitudinal holes between the cartilage layer and the subchondral bone layer are mutually communicated, the pore diameter is accurately controlled, and a calcium phosphate nano-cluster material with the particle size of 1 nanometer can be added into the subchondral bone layer. The osteochondral tissue repair scaffold prepared by the invention aims at two different tissue characteristics and regeneration requirements of cartilage and subchondral bone, designs a double-layer scaffold with different physical and biological characteristics, simulates the anisotropy of a natural osteochondral structure, promotes cell migration and tissue ingrowth, and achieves the effect of promoting synchronous regeneration of cartilage and subchondral bone, thereby having great application potential.

Description

Osteochondral tissue repair scaffold and preparation method thereof

technical field

The invention belongs to the technical field of biomedical materials, and in particular relates to an integrated osteochondral tissue repair scaffold for promoting cell migration and a preparation method thereof.

Background technique

Cartilage damage in the knee joint is mainly caused by trauma, disease or aging, which affects the normal life of patients and causes pain. Composed of chondrocytes and extracellular matrix (ECM) such as type II collagen and proteoglycans, articular cartilage is a smooth, elastic tissue without blood vessels, lymphatics, or nerves that has limited self-healing when damaged ability. According to the depth of cartilage damage, it can usually be divided into three types: partial cartilage defect, full-thickness cartilage defect and osteochondral defect. However, clinical studies have shown that cartilage damage always extends deeply into the subchondral bone, resulting in knee joint damage Osteochondral defect. If left untreated, osteochondral defects can further aggravate articular cartilage defects and lead to osteoarthritis. And based on the natural complex structure of osteochondral bone, cartilage and subchondral bone are two tissues with obvious biological specificity, which means that the clinical treatment of osteochondral injury is more challenging. Therefore, the repair of osteochondral defects is very important. However, the current traditional treatment and surgical methods can only temporarily relieve the pain of patients, and cannot truly repair osteochondral defects and regenerate hyaline cartilage with complete functions. Therefore, facing the clinical problems of osteochondral repair, it is urgent to propose new treatment methods.

Tissue engineering is an emerging field that has brought new hope for in vivo transplantation and regeneration of tissues and organs. In recent years, tissue engineering has gradually become an effective and cutting-edge method for the treatment of articular cartilage injuries. However, there are still some problems to be solved in the osteochondral scaffolds in tissue engineering, such as the non-layered scaffolds have little repair effect and the lack of effective interfacial bonding between the layers of the multilayer scaffolds. However, 3D printing technology has been studied more in the preparation of tissue engineering scaffold materials, and has shown advantages. The scaffold material printed by 3D printing technology has a shape that matches the defect tissue, as well as an internal three-dimensional porous structure that can provide cell adhesion, growth and proliferation. Through material screening and modification, 3D printing "bio-ink" can be prepared. 3D bioprinters can print scaffolds with good mechanical properties. As a new technology in optical printing, DLP has already demonstrated many advantages. For example, the printing speed is fast, the printing precision is high, and it is conducive to cell survival, etc., which have been used in many research directions in the field of tissue engineering.

Previous studies have proved that scaffolds with porous structures are conducive to promoting cell migration, and different structures can also affect the mechanical properties of scaffolds. Therefore, in order to promote tissue regeneration, it is essential to construct an ideal scaffold microenvironment that affects the fate of cells and tissues. CN101810885B provides a double-layer bionic osteochondral tissue engineering support with circular holes and vertical holes, but the formation of the circular holes and vertical holes in the support comes from the characteristics of the material itself, GelMA used in the present invention Hydrogel also has a similar porous structure, and the distribution of pores is random. It is difficult to precisely control the pore size, which seriously affects the migration and growth of chondrocytes. The degree of biomimesis is low, and the effect of cartilage repair is not good.

Therefore, DLP printing technology can be used to construct a multi-layer osteochondral scaffold structure in an integrated manner, realize personalized customization, and effectively mimic the specific physical and biological characteristics of natural osteochondral.

Contents of the invention

In order to solve the above problems, the present invention provides a novel scaffold for osteochondral tissue repair. The scaffold for osteochondral tissue repair is a hydrogel scaffold, which is integrally produced by self-made light-curing 3D printing equipment, including bionic double Layer structure: cartilage layer and subchondral bone layer, the cartilage layer has "lotus root-like" and "radial" pores, the subchondral bone layer has "lotus root-like" pores, and the cartilage layer and The longitudinal pores between the bone layers are connected to each other, and the pore size can be precisely controlled, and calcium phosphate nano-cluster materials with a particle size of 1 nanometer can also be added to the subchondral bone layer. The purpose of the present invention is to provide a hydrogel osteochondral tissue repair scaffold with good biocompatibility, good mechanical properties, high bionic degree, which is more conducive to the migration of chondrocytes and bone marrow mesenchymal stem cells and promotes the repair of osteochondral defects and its Preparation.

Although the hydrogel used to prepare the repair scaffold may have randomly distributed vertical and circular holes in its material, its cartilage repair effect is still poor. The osteochondral tissue repair scaffold provided by the present invention is integrally produced by self-made light-curing 3D printing equipment, which can precisely control the arrangement and pore size of the hydrogel micropores of the osteochondral tissue repair scaffold, thereby achieving better osteochondral tissue repair. Cartilage defect repair effect.

On the one hand, the present invention provides a scaffold for repairing osteochondral tissue, which includes a cartilage layer and a subchondral bone layer from top to bottom, and the cartilage layer has holes distributed in "lotus root shape" and "radial shape"; the subchondral bone layer The bone layer has "lotus root-shaped" holes that communicate with the cartilage layer.

Both the "lotus root-shaped" and "radial" holes in the present invention are strip-shaped through holes.

The present invention has been proved by a large number of studies that the shape and diameter of the pores in the osteochondral repair scaffold have a significant impact on the mechanical properties and cell migration of the scaffold, and the shape, distribution and diameter of the pores must be precisely controlled to obtain suitable mechanical properties. And have a better cell migration effect, so as to have a better cartilage repair effect.

The "lotus root-shaped" hole in the present invention refers to the longitudinal through arrangement of holes perpendicular to the bracket (see Figure 1 and Figure 2 for details); As the center, the holes radiating outward are arranged in a horizontal direction (see Figure 1 and Figure 2 for details).

The cartilage layer has "lotus root-shaped" and "radial" pores to facilitate the migration and distribution of chondrocytes; the subchondral bone layer has "lotus root-shaped" pores to guide bone marrow mesenchymal stem cells (BMSCs) ) to the upper space.

Further, both the cartilage layer and the subchondral bone layer are cylindrical, and the diameters of the cylindrical bottom surfaces of the cartilage layer and the subchondral bone layer are consistent.

Further, the "lotus root-shaped" holes longitudinally penetrate the cylindrical bottom surfaces 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 into one body.

Further, the "radial" holes transversely penetrate the cartilage layer and pass through the center of the cross-section of the cartilage layer.

Further, the pore diameter of the cartilage layer and the subchondral layer is 100-400 μm.

The interconnected pore structure in the scaffold can form a strong three-dimensional organization, which is conducive to cell proliferation and differentiation and nutrient/waste transfer, thereby achieving efficient tissue regeneration. And pore size can regulate cell fate, large enough pores provide a sufficient osteogenic niche for bone formation, while small enough pores will maintain stemness and prevent differentiation.

The vertical holes in the subchondral bone layer scaffold are conducive to the migration of bone marrow mesenchymal stem cells to the upper layer, and the "lotus root-shaped" scaffold has stronger mechanical properties than the upper layer, which is conducive to bone repair.

The cartilage layer hydrogel scaffold has "lotus root-shaped" and "radial" distribution of holes, 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, strengthening the repair of the cartilage layer .

The scaffold including the cartilage layer and the subchondral bone layer is a porous scaffold that facilitates the migration of cells, the transportation of nutrients and the ingrowth of blood vessels, and its mechanical strength and biological properties are suitable for the regeneration and repair of osteochondral.

Furthermore, the "radial" holes can be provided in one or more layers, with the interval between each layer being greater than 100 μm, and evenly distributed in the cartilage layer.

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

Furthermore, the included angles between two adjacent "radial" holes on the same layer are kept consistent.

Further, the "lotus root-shaped" holes can be arranged in multiples, and the distance between each hole is greater than 100 μm, and they are evenly distributed in the cross-section of the cartilage layer and the subchondral bone layer in the longitudinal direction.

Further, the size of the osteochondral tissue repair scaffold is designed to match the size of the actual human or animal osteochondral to be transplanted.

In some ways, the size of the cartilage repair scaffold provided by the present invention is that the thickness of the multilayer osteochondral 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 aperture is Micropores of 200 μm. The cartilage repair scaffold of this size is mainly used to match the animal experiment rabbit osteochondral modeling.

Further, the cartilage layer includes GelMA, LAP and growth factors.

Further, the subchondral bone layer includes GelMA and LAP.

Further, the subchondral bone layer also includes calcium phosphate nano-cluster materials with a particle size of 1 nanometer.

The calcium phosphate nano-cluster material with a particle size of 1 nanometer in the present invention is prepared according to the calcium phosphate nano-cluster in CN109718249A.

Hydroxyapatite is the main inorganic component of human and animal bones, and ordinary hydroxyapatite is mainly used to prepare functional composite materials. The existing nano-hydroxyapatite has a particle size of more than 10 nanometers and is mostly used as a scaffold material for bone tissue engineering.

The calcium phosphate nanocluster material with a particle size of 1 nanometer used in the present invention is independently developed by the research team, and its preparation method has applied for patent CN109718249A. It is mainly used to prepare drugs for repairing osteoporosis, and can effectively promote the regeneration of tooth enamel.

After a lot of research, the present invention finds that when the calcium phosphate nanocluster material with a particle size of 1 nanometer is used to prepare a scaffold for cartilage tissue repair, it not only has better biocompatibility because of its smaller particle size, but also has Better bionic effect can effectively promote the osteogenic differentiation of mesenchymal stem cells, and the cartilage tissue repair effect is better.

Further, the GelMA and LAP concentrations of the cartilage layer and the subchondral bone layer are the same.

Further, the GelMA content is 15%, and the LAP content is 0.2%.

Further, the growth factor is KGN, and the concentration of KGN is 200 μM.

On the other hand, the present invention provides a preparation method of osteochondral tissue repair scaffold, it is characterized in that, described preparation method comprises:

(1) To prepare the first feed liquid, add GelMA, LAP and KGN into single distilled water under the condition of avoiding light, and stir evenly to obtain the first feed liquid. The first feed solution includes GelMA, LAP and growth factors for preparing the cartilage layer;

(2) Prepare the second feed liquid, GelMA, LAP, or GelMA, LAP, calcium phosphate nano-cluster material with a particle size of 1 nanometer are added to single distilled water under the condition of avoiding light, and after stirring evenly, the second Liquid. The second feed solution includes GelMA, LAP and 1 nanometer hydroxyapatite for preparing subchondral bone layer;

(3) Pour the first material liquid into the liquid tank of the light-curing 3D printing device for printing, and after the upper cartilage layer support is completely printed, the upper cartilage layer hydrogel support is obtained;

(4) After cleaning the liquid tank of the printer, pour the second material liquid into the liquid tank of the printer, and continue printing. After all the lower subchondral bone layer brackets are printed, a double-layer bone cartilage tissue repair bracket is obtained.

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 space structure was established by using computer-aided design software; the designed double-layer osteochondral tissue The repair bracket CAD model is converted into the Task Scheduler Task Object (.job) file format suitable for the self-designed and developed UV light projection-based light-curing 3D printing equipment through software, and imported into the 3D printing equipment, using ultraviolet light The curing printing system prints the first material liquid and the second material liquid through self-made photo-curing 3D printing equipment according to the preset parameters, and then obtains the osteochondral tissue repair scaffold with precise hole arrangement and pore size structure. 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 shape" and a "radial shape", that is, the pore structure is arranged horizontally and vertically to facilitate chondrocytes. Migration and distribution; and the subchondral bone layer has "lotus root-shaped" pores, that is, the pore structure is arranged longitudinally to guide bone marrow mesenchymal stem cells (BMSCs) to migrate to the upper space; and the cartilage layer and subchondral bone The longitudinal "lotus root-shaped" pores between the layers are interconnected.

The self-made light-curing 3D printing equipment used in the present invention is prepared according to the method of the patent CN110228193A, which can realize printing while light-curing, and can precisely control the aperture and hole arrangement of the osteochondral tissue repair bracket, thereby Can achieve better cartilage tissue repair effect.

The double-layer osteochondral tissue repair scaffold involved in the present invention is constructed by simulating the physiological structure and composition of natural osteochondral, wherein the upper and lower layers can be added with different growth factors, inorganic ions or organic components on the basis of the original components, such as KGN , BMP, TGF-β1, etc.

Although the GelMA hydrogel itself also has randomly distributed vertical holes and circular holes, its cell migration effect is still poor, and it is difficult to achieve a sufficient defect repair effect; The gel with "lotus root shape" and "radial" pore structure can significantly improve the effect of promoting cell migration, thereby greatly improving the effect of defect repair.

The osteochondral tissue repair scaffold provided by the present invention has the following beneficial effects:

(1) Precisely printed by self-made light-curing 3D printing equipment, the prepared osteochondral tissue repair scaffold has precise pore size and pore arrangement, and has a good effect on cell migration;

(2) The calcium phosphate nanocluster material with a particle size of 1 nanometer is used in the preparation of osteochondral repair scaffolds, which has better osteoinductive properties than ordinary hydroxyapatite;

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

(4) have a better ability to promote the migration of chondrocytes and bone marrow mesenchymal stem cells;

(5) Capable of nutrient transport/waste transfer;

(6) It has the ability to grow new blood vessels and better promote the repair of osteochondral defects.

Description of drawings

FIG. 1 is a schematic structural view of the osteochondral tissue repair scaffold in Example 1.

FIG. 2 is a cross-sectional view of the osteochondral tissue repair scaffold in Example 1. FIG.

FIG. 3 is a design diagram of the osteochondral tissue repair scaffold in Example 1. FIG.

FIG. 4 is a 3D model and a schematic diagram of preparation of the osteochondral tissue repair scaffold in Example 1. FIG.

FIG. 5 is a general view of the scaffold for repairing osteochondral tissue in Example 1. FIG.

FIG. 6 is a fluorescence image and an electron microscope image of pores in the scaffold for repairing osteochondral tissue in Example 1. FIG.

Fig. 7 is a microstructure diagram of the upper cartilage layer scaffold with different pore sizes in the scaffold for repairing osteochondral tissue in Example 2.

FIG. 8 shows the mechanical properties of the osteochondral tissue repair scaffold in Example 2 with 15% GelMA and different pore sizes.

FIG. 9 shows the mechanical properties of the osteochondral tissue repair scaffold in Example 2 with 10% GelMA and different pore sizes.

10 is a scanning electron micrograph of 15% GelMA and 10% GelMA of the bone cartilage tissue repair scaffold in Example 2.

Fig. 11 shows the mechanical properties of the upper and lower layers of the hydrogel scaffold in the osteochondral tissue repair scaffold in Example 3.

Fig. 12 is the biocompatibility test result of the GelMA hydrogel of the osteochondral tissue repair scaffold in Example 4.

Fig. 13 is the alkaline phosphatase staining results of the osteoinductive performance experiment of the GelMA hydrogel of the osteochondral tissue repair scaffold in Example 5.

Fig. 14 is the quantitative result of the alkaline phosphatase activity of the osteoinductive performance experiment of the GelMA hydrogel of the osteochondral tissue repair scaffold in Example 5.

Fig. 15 is the result of promoting cell migration in vitro of the osteochondral tissue repair scaffold in Example 6.

Fig. 16 is the in vivo cell infiltration result of the osteochondral tissue repair scaffold in Example 7.

Figures 17, 18, and 19 show the in vivo cell infiltration results of osteochondral tissue repair scaffolds with different micropore distributions in Example 8.

Fig. 20 is a general view of the joint samples at 8 and 16 weeks after implantation of the stent in Example 9.

Fig. 21 is the HE staining result of the joint sample after implanting the stent in Example 9.

Fig. 22 is the SO staining result of the joint sample after implanting the stent in Example 9.

Fig. 23 is the result of the mechanical test of the repaired joint sample 16 weeks after implantation of the stent in Example 9.

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention, but do not limit it in any way. Advantages and features of the present invention will be apparent from the following description and claims. It should be noted that all the drawings are in very simplified form and use inaccurate scales, and are only used to facilitate and clearly assist the purpose of illustrating the embodiments of the present invention.

Embodiment 1 Osteochondral tissue repair scaffold provided by the present invention

The osteochondral tissue repair scaffold structure provided in this embodiment is shown in Figures 1-3, wherein Figure 1 is a schematic structural view of the osteochondral tissue repair scaffold, Figure 2 is a cross-sectional view of the osteochondral tissue repair scaffold, and Figure 3 is the osteochondral tissue repair scaffold Design drawing of tissue repair scaffold.

It can be seen from Fig. 1, Fig. 2 and Fig. 3 that the osteochondral tissue repair scaffold provided by this embodiment includes an upper cartilage layer 1 and a lower subchondral bone layer 2, and the cartilage layer 1 has "lotus root-shaped" holes 3 and " The subchondral bone layer 2 has a "lotus root-shaped" hole 3 that penetrates the cartilage layer; the "lotus root-shaped" pores between the cartilage layer 1 and the subchondral bone layer 2 communicate with each other. The "lotus root-shaped" hole 3 refers to the vertical through arrangement of holes perpendicular to the bracket, and the "radial" hole 4 refers to the horizontal hole that runs through the bracket horizontally, and is centered on the center of the cross section, radiating outwards and arranged horizontally. In this embodiment, both the cartilage layer 1 and the subchondral bone layer 2 are cylindrical, and the cylindrical bottom surfaces of the cartilage layer 1 and the subchondral bone layer 2 have the same diameter; the "lotus root-shaped" hole 3 runs through the cartilage layer 1 and the subchondral bone layer. The cylindrical bottom surface of the bone layer 2; the "radial" holes 4 are located on the cylindrical side wall of the cartilage layer 1, penetrating the cartilage layer 1 transversely.

Preferably, the "lotus root-shaped" and "radial" pores of the cartilage layer 1 and subchondral bone layer 2 have a pore diameter of 200 μm.

Preferably, "radial" holes 3 may be provided in one or more layers, and the interval between each layer is greater than 100 μm, evenly distributed in the cartilage layer 1 . In this embodiment, there are three layers of "radial" holes 3, which are evenly arranged from top to bottom. If it is necessary to prepare a larger-sized bone cartilage tissue repair scaffold, or when the cartilage layer 1 is thicker, more layers can be set. The "radial" holes3.

Preferably, each layer is provided with 2 to 10 "radial" holes 3 transversely penetrating the cartilage layer 1 and passing through the center of the cross-section. In this embodiment, five "radial" holes 3 are arranged on each layer. If it is necessary to prepare a scaffold for osteochondral tissue repair with a larger size, or when the cross-sectional diameter of the cartilage layer 1 is larger, more "radial" holes 3 can be provided in each layer.

Preferably, the included angles between two adjacent "radial" holes 3 on the same layer are consistent. In this embodiment, since five "radial" holes 3 are provided, the angle between every two adjacent "radial" holes 3 is 36 degrees.

Preferably, a plurality of "lotus root-shaped" holes 4 can be provided, and the distance between each hole is greater than 100 μm, and they are evenly distributed longitudinally in the cross-section of the cartilage layer 1 and the subchondral bone layer 2 . In this embodiment, there are altogether 30 "lotus root-shaped" holes 4 evenly distributed.

Preferably, the cartilage layer 1 includes GelMA, LAP and growth factors, and the subchondral bone layer 2 contains GelMA and LAP, as well as calcium phosphate nano-cluster materials with a particle size of 1 nm. For the preparation method of calcium phosphate nanocluster materials with a particle size of 1 nanometer, see CN201910068395.1.

Preferably, the GelMA and LAP concentrations of the cartilage layer 1 and the subchondral bone layer 2 are the same, the GelMA concentration is 10-15%, and the LAP concentration is 0.2%.

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

The upper layer of cartilage layer 1 and the lower layer of subchondral bone layer 2 were printed with a self-made light-curing 3D printing device to print a pre-designed model structure of the double-layer osteochondral tissue repair scaffold.

The preparation method of the osteochondral tissue repair scaffold provided in this embodiment specifically includes:

(1) Prepare the first feed liquid, the first feed liquid includes GelMA, LAP and KGN. Specifically, GelMA, LAP and KGN were added into single distilled water in a water bath at 42° C. under the condition of avoiding light, and stirred evenly to obtain the first feed liquid. Among them, the concentration of GelMA is 15%, the concentration of LAP is 0.2%, and the concentration of KGN is 200 μM.

(2) Prepare the second feed liquid, the second feed liquid includes GelMA, LAP and 1 nanometer calcium phosphate nanocluster material. Specifically, GelMA, LAP and 1 nanometer calcium phosphate nanocluster material were added to single distilled water in a water bath at 42° C., and stirred evenly to obtain a second feed solution. Among them, the concentration of GelMA is 15%, and the concentration of LAP is 0.2%.

(3) Pour 6mL of the first material liquid into the liquid tank heated at 42°C in the self-made light-curing 3D printing equipment, and obtain the upper cartilage layer scaffold after printing; transfer the remaining first material liquid in the liquid tank, and use a single After cleaning the liquid tank with distilled water, pour 6mL of the second material liquid into the liquid tank heated at 42°C, and continue printing to obtain an integrated double-layer osteochondral tissue repair scaffold.

(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 200 μm for inducing and promoting the migration and distribution of chondrocytes; and has longitudinally penetrating micropores with a diameter of 200 μm for inducing the migration of chondrocytes and bone marrow mesenchymal stem cells. The lower subchondral bone layer has micropores with a diameter of 200 μm longitudinally interpenetrating with the upper cartilage layer for guiding bone marrow mesenchymal stem cells to migrate to the upper space. And the thickness of the double-layer osteochondral tissue repair scaffold is 4 mm, the thickness of the upper cartilage layer is 1 mm, and the thickness of the lower subchondral bone layer is 3 mm.

Fig. 4 is a 3D model and a schematic diagram of preparation of the double-layer osteochondral tissue repair scaffold provided by the present invention. Fig. 5 is a general view of the double-layer osteochondral tissue repair scaffold provided by the present invention. (a) Top view of the bracket; (b) Side view of the upper bracket; (c) Side view of the lower bracket. Fig. 6 is a fluorescence image and an electron microscope image of the pores in the double-layer osteochondral tissue repair scaffold provided by the present invention. It can also be seen from the electron microscope images that the gel itself has a porous structure. (a) Fluorescence image of the “lotus root-like” and “radial” structures of the upper scaffold; (b) SEM image of the side of the upper scaffold. Figures 4-6 show that the double-layer osteochondral tissue repair scaffold of the present invention can print a precise double-layer structure with a diameter of 4 mm, a thickness of the upper layer of 1 mm, a thickness of the lower layer of 3 mm, and a pore diameter of 200 μm through DLP.

Example 2 Effects of Different GelMA Concentrations and Different Aperture Scaffolds on the Mechanical Properties of Double-Layer Osteochondral Tissue Repair Scaffolds

In this embodiment, mechanical tests are carried out on hydrogel scaffolds with different pore sizes formed by two GelMA concentrations of 10% and 15% using a tensile-compression mechanical tester (Instron-5543 with a 1kN sensor), and the specific implementation steps are as follows:

(1) Use a DLP printer to print lotus root-shaped GelMA hydrogel scaffolds with two concentrations of 10% and 15% in the lower subchondral bone layer. The diameters of the scaffold holes are 0 μm, 100 μm, 200 μm and 400 μm, respectively.

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

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

(4) Calculate the compressive modulus by linear fitting according to the strain in the range of 40%-60%.

Fig. 7 is a microstructure diagram of the GelMA hydrogel scaffold of the upper cartilage layer of the present invention, and the diameters of the scaffold holes are 0 μm, 100 μm, 200 μm and 400 μm (a, b and c). 8-10 are the optimal GelMA concentration and structure screening of the double-layer osteochondral tissue repair scaffold of the present invention. Figure 8 and Figure 9 are the load-compression deformation curves and compression modulus of the printed scaffolds with concentrations of 15% and 10% respectively when the diameters of the scaffold holes are 0 μm, 100 μm, 200 μm and 400 μm, and Figure 10 is the concentration of 15% respectively and SEM images of 10% GelMA hydrogels. By measuring the relationship between 10% and 15% GelMA concentrations in mechanical tests of different pore sizes, the relationship between load and compression deformation shows that scaffolds with different pore sizes will affect the mechanics of the stent, and under the same conditions, the larger the pore size, the better the overall mechanical properties. The performance is relatively poor. In addition, the overall mechanical properties of the 15% GelMA hydrogel scaffold are better than those of the 10% GelMA hydrogel. We also observed the microstructure of hydrogels with scanning electron microscope for 10% GelMA and 15% GelMA, which showed that the microstructures of hydrogels were all porous. In addition, the specific surface area of hydrogel scaffolds with different pore sizes was calculated, as shown in Table 1.

Table 1 The specific surface area of hydrogel scaffolds with different pore sizes

Printed micropore diameter (μm) 0 100 200 400 Specific surface area (m ² /m ³ ) 2.5 3.62 4.71 6.85

Finally, through the mechanical screening of the material structure and the comparison of the overall surface area of the scaffold, the larger the pore size, the larger the specific surface area of the cartilage repair scaffold, but the increase in the pore size will easily lead to a decrease in the mechanical support performance. The present invention decides to select 15% GelMA hydrogel with a pore size of 200 μm as the scaffold used, and the hydrogel scaffold not only has better mechanical properties, but also has a larger specific surface area of the scaffold. (*p<0.05, **p<0.01)

The mechanical test of embodiment 3 upper and lower layer hydrogel support

In this embodiment, a tensile-compression mechanical tester (Instron-5543 with a 1kN sensor) is used to perform mechanical tests on the upper and lower hydrogel supports respectively. The specific implementation steps are as follows:

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

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

(3) Calculate the compressive modulus by linear fitting according to the strain in the range of 20%-40%.

Figure 11 shows the mechanical properties of the upper and lower hydrogel scaffolds of the double-layer osteochondral tissue repair scaffold, indicating that the mechanical properties of the lower subchondral bone layer are significantly stronger than the upper cartilage layer, which is in line with the biological relationship between cartilage and subchondral bone in natural osteochondral tissue The differences indicate that the double-layer osteochondral tissue repair scaffold of the present invention has excellent bionic mechanical properties.

Embodiment 4 measures the cell viability test of cell in GelMA hydrogel with CCK-8

In this example, CCK-8 is used to measure the cell activity of cells in GelMA hydrogel. The GelMA hydrogel is 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 :

(1) Prepare the second feed liquid, the concentration of GelMA in the second feed liquid is 15%, and the concentration of LAP is 0.2%.

(2) The prepared second feed solution was added to a 96-well plate, wherein 50 μL was added to each well, cross-linked with ultraviolet light, and soaked in a low-sugar medium.

(3) After soaking for 24 hours, inoculate the cells at 1000 cells/well. After 24 hours of attachment, discard the original medium and add new medium, 100 μL per well; add fresh medium to the blank control group; add fresh medium to the positive control group Add only the cell suspension. Each experimental group has 6 repetitions.

(4) Detect the cell proliferation rate at 1, 3, and 5 days. Before detection, add 10 μL of CCK-8 reagent to each well, and place the 96-well plate in a 37°C, 5% _CO2 humidified incubator for 1 hour. After incubation, the culture medium in each well was aspirated and put into a 96-well plate separately, and the OD value was measured at 450nm with a microplate reader, and the cell proliferation data was recorded and processed.

Figure 12 is the biocompatibility of the GelMA hydrogel of the osteochondral tissue repair scaffold, indicating that the cells are in a state of obvious proliferation, and GelMA has good biocompatibility, which is conducive to cell proliferation, indicating that the osteochondral tissue repair scaffold of the present invention has Good biocompatibility.

Example 5 Qualitative and Quantitative Determination of Osteogenic Inductive Ability of GelMA Hydrogel with ALP

This embodiment uses alkaline phosphatase (ALP) to qualitatively and quantitatively determine the osteogenic induction ability of GelMA hydrogels. The GelMA hydrogels are divided into three groups, the first group contains only GelMA and LAP, and the second group contains GelMA , LAP and calcium phosphate nanocluster materials with a particle size of 1 nm (abbreviated as CaP in the figure), the third group contains GelMA, LAP and commercial hydroxyapatite with a particle size of 20 nm (abbreviated as HANP in the figure), specifically The implementation steps are as follows:

(1) prepare the second feed liquid, the concentration of GelMA in the second feed liquid is 15%, the concentration of LAP is 0.2%, wherein the second and third groups also need to add 1 nanometer calcium phosphate nano-cluster material or particle diameter is 20 Nanoscale commercial hydroxyapatite.

(2) The prepared second feed solution was added to a 48-well plate, wherein 150 μL was added to each well, cross-linked with ultraviolet light, and soaked in a low-sugar medium.

(3) After soaking for 24 hours, inoculate the cells at 1×10 ⁵ cells/well. After the cells adhere to the wall and grow to 80%, the original medium is discarded, and new osteogenic induction medium is added to each well. 250 μL. Each experimental group has 4 repetitions.

(4) The alkaline phosphatase chromogenic experiment and the alkaline phosphatase quantitative experiment were carried out on the seventh day after the cells started osteogenic induction, and the alkaline phosphatase data recording and processing of the samples were carried out.

Figures 13-14 are the qualitative staining and quantitative results of the alkaline phosphatase activity of the samples on the 7th day, indicating that the combination of GelMA and calcium phosphate nanocluster materials with a particle size of 1 nm has a higher ratio than GelMA and commercial hydroxyl phosphorus with a particle size of 20 nm. The better osteogenic inductivity of the limestone is beneficial to the osteogenic differentiation of cells, indicating that the osteochondral tissue repair scaffold of the present invention has good osteogenic inductivity.

The influence of the micropore of embodiment 6 support on in vitro cell migration experiment

This embodiment is an in vitro cell migration experiment of a hydrogel scaffold, and the specific implementation steps are as follows:

(1) Stain the cells with DiI, that is, dilute the DiI stock solution with PBS or serum-free medium to 2 μM/mL working solution, digest the cells with trypsin, centrifuge, resuspend the cells with the working solution, and place them at 37°C for 5 %CO ₂ humidity incubator for 20min. After the end, the supernatant was discarded by centrifugation, and the 37°C preheated medium was slowly added with a pipette gun. Centrifuge again for a total of two washes.

(2) The DiI-stained C3H cells were planted on a 3.5 cm culture dish at 5×10 ⁵ cells/mL, and the medium was changed after 24 hours of attachment.

(3) The printed lower subchondral bone scaffold with "lotus root-shaped" and "radial" pores with a pore size of 200 μm, and the printed scaffold without micropores (but there is a random porous structure in the gel itself) Gently place in the middle of the Petri dish, making sure the medium liquid submerges the holder.

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

Figure 15 is a quantitative analysis of cell numbers after migration of cells on scaffold samples with and without micropores. It shows that the "lotus root-shaped" and "radial" pore structures with a pore size of 200 μm in the double-layer osteochondral tissue repair scaffold of the present invention can effectively promote cell migration into the scaffold, thereby achieving the effect of promoting defect repair in vivo repair experiments, and if The gel with a specific 200 μm pore size "lotus root-like" and "radiant-like" pore structure has not been printed by photocuring 3D printing equipment. Although it also has vertical and circular holes, its cell migration effect is still poor and it is difficult to Sufficient defect repair effect is achieved.

Influence of the micropores of the support in embodiment 7 on the cell migration experiment in vivo

This embodiment is an in vivo cell migration experiment of a hydrogel scaffold, and the specific implementation steps are as follows:

(1) In a sterile environment, the scaffolds of the lower subchondral bone layer with "lotus root-shaped" and "radial" pores with a pore size of 200 μm and scaffolds without micropores (but there are random porous structures in the gel itself) were printed .

(2) SPF grade SD rats (about 250 g, male) were anesthetized with pentobarbital, that is, pentobarbital was injected into the abdominal cavity of the rats, and the injection dose was 50 mg/kg.

(3) Under a sterile environment, an incision about 2 cm in length was made in the inner skin of the back of the rat, and the two GelMA hydrogel scaffolds were respectively placed in it, and then sutured.

(4) Samples were taken on the 3rd and 5th day after operation, and fixed with 4% formaldehyde for more than 24 hours. After the tissue fixation was completed, the tissue was dehydrated in a 4°C refrigerator with a gradient of 20% and 30% sucrose, and the tissue block was taken out for frozen section after sinking to the bottom.

(5) DAPI stained the sections at a ratio of 1:1000 for 15 minutes, then washed 3 times with PBS;

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

Figure 16 shows the subcutaneous implantation of printed and unprinted GelMA hydrogel scaffolds in rats to evaluate in vivo cell infiltration. On day 5 of implantation, the printed "lotus root" and "radial" holes with a pore size of 200 μm Subchondral bone layer scaffolds impregnated with the number of cells was significantly higher than that of scaffolds without micropores. It shows that the microporous structure in the double-layer osteochondral tissue repair scaffold of the present invention can effectively promote cell migration into the scaffold, thereby achieving the effect of promoting defect repair in vivo repair experiments.

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

This example is the in vivo cell migration experiment of the hydrogel scaffold. Four kinds of scaffolds with different micropore distribution were used for the experiment, the first one is: the printed bone with 200 μm aperture of "lotus root" and "radial" pores Cartilage tissue repair scaffold; the second type is: printed "lotus root-shaped" osteochondral tissue repair scaffold with 200μm pore size; the third type is: printed osteochondral tissue repair scaffold with 200μm "radial" holes; The fourth type is: Osteochondral tissue repair scaffold without printing micropores.

The specific implementation steps are as follows:

(1) In a sterile environment, hydrogel scaffolds with "lotus root-shaped" and "radial" pores with a pore size of 200 μm and scaffolds without micropores (but there are random porous structures in the gel itself) were printed.

(2) SPF grade SD rats (about 250 g, male) were anesthetized with pentobarbital, that is, pentobarbital was injected into the abdominal cavity of the rats, and the injection dose was 50 mg/kg.

(3) Under a sterile environment, an incision about 2 cm in length was made in the inner skin of the back of the rat, and four kinds of GelMA hydrogel scaffolds were placed in it, and then sutured.

(4) Samples were taken on the 5th day after operation and fixed with 4% formaldehyde for more than 24 hours. After the tissue fixation was completed, the tissue was dehydrated in a 4°C refrigerator with a gradient of 20% and 30% sucrose, and the tissue block was taken out for frozen section after sinking to the bottom.

(5) DAPI stained the sections at a ratio of 1:1000 for 15 minutes, then washed 3 times with PBS;

(6) 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, and Figure 18.

(7) Stain the slices with HE, that is, after hematoxylin staining in the dark for 10 minutes, quickly separate the color with 1% hydrochloric acid alcohol, and stain with eosin for 20 seconds, then dehydrate with gradient alcohol, transparent with xylene, and seal the slide with neutral resin . Staining results were scanned with an Olympus VS200 digital slide scanner. The result is shown in Figure 19.

Table 2 Effects of hydrogel scaffolds with different pore distributions on in vivo cell migration experiments after 5 days

Type of bracket number of cells migrated Cell Migration Depth (microns) "Lotus Root" and "Radial" 354.5±12.19 279.0±56.90 "lotus root shape" 223.5±63.97 222.3±63.37 "Radial" 120.5±43.88 141.3±68.04 Nonporous 65.60±23.93 25.61±14.61

Figure 17 is the grayscale image of fluorescence staining on day 5 after implantation of the stent; Figure 18 is the result of fluorescence staining analysis; Figure 19 is the result of HE staining of the stent section, where a is "radial" and b is "lotus root-shaped" holes .

It can be seen from Figure 19 that, referring to the schematic diagram, on day 5, at the scaffold without 200 μm micropores, the cells can only adhere to the surface of the scaffold, and the ability to migrate to the inside of the scaffold is limited. But at the printed micropores, cells can migrate to the inside through the holes, whether it is "lotus root-shaped" holes or "radial" holes, all play a huge role in their migration and distribution.

It can be seen from Table 2 and Figures 17 and 18 that the osteochondral tissue repair scaffolds that contain both "lotus root-shaped" and "radial" holes of 200 μm, compared with those that do not contain micropores, or only contain "lotus root-shaped" or " Compared with the osteochondral tissue repair scaffold with "radial" holes, the number of cell migration on the 5th day was significantly increased; this is because when the osteochondral tissue repair scaffold has both "lotus root" 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 number of cell migration.

This embodiment further compares the situation that the "radial" holes are located in the cartilage layer, or in the subchondral bone layer, or are distributed in both the cartilage layer and the subchondral bone layer. The experiment proves that when the "radial" holes are only located in the In the subchondral bone layer, the cell migration effect is similar to that of the second "lotus root-shaped" osteochondral tissue repair scaffold; when the "radial shape" is distributed in both the cartilage layer and the subchondral bone layer, the cell migration effect is still poor; only When the "radial" holes are located in the cartilage layer and the subchondral bone layer does not contain "radial", the number of cell migration can reach 354.5±12.19, and the cell migration depth can reach 279.0±56.90 microns.

Example 9 Repair and regeneration experiment of osteochondral scaffold in rabbit cartilage defect

This embodiment is an experiment on the repair and regeneration of a hydrogel scaffold in a rabbit osteochondral defect, and the specific implementation steps are as follows:

(1) In a sterile environment, the double-layer osteochondral tissue repair scaffold with 200 μm pore size "lotus root" and "radial" holes (B), the double-layer osteochondral tissue repair scaffold without micropores (NB) And the upper layer contains a microporous double-layer osteochondral GelMA hydrogel scaffold (B+KGN) containing growth factor KGN.

(2) New Zealand white rabbits were anesthetized with 1% sodium pentobarbital in the ear vein, and the injection dose was 40 mg/kg.

(3) Under a sterile environment, disinfect the skin with povidone iodine, make a roughly 1cm incision along the medial side of the patellar ligament, continue to cut until the knee joint is exposed, and take a 4mm diameter and 4mm depth at the femoral trochlea with a graduated manual drill Hole in the hole to complete the modeling of osteochondral soft defect. All 48 New Zealand white rabbits were modeled. After the modeling was completed, they were randomly divided into 4 groups: the blank group, the unprinted group, the printed double-layer scaffold group, and the printed double-layer scaffold group containing growth factor KGN on the upper layer, for a total of 8 weeks ( n=4) and 16 weeks (n=8, of which 4 samples were used for mechanical testing) two time points. After the stent was placed in the defect according to the direction, it was immediately disinfected with povidone iodine and sutured. After modeling in the blank group, there was no need to fill the stent, and they were directly sterilized and sutured.

Figure 20 is the general picture of the joint samples at 8 and 16 weeks after implantation of the stent; Figure 21 is the HE staining results of the joint samples; Figure 22 is the SO staining results of the joint samples; Figure 23 is the mechanical test results of the joint samples at 16 weeks after repair .

Figures 21-23 show that the evaluation of samples received at 8 weeks and 16 weeks proves that the osteochondral tissue repair scaffold of the present invention has a better repair effect whether it is the cartilage layer or the subchondral bone layer. In SO staining, it can be observed that the repair effect of the blank control group is not good, most of the bone layer has not yet fully grown, and there is fibrous tissue filling, and the area of cartilage defect is less reduced. However, for the osteochondral tissue repair group of the present invention containing KGN, the repair surface is smooth, basically completely repaired, the thickness of the cartilage layer is normal, and the chondrocytes are arranged neatly and closely connected with the surrounding tissues. It shows that the double-layer osteochondral tissue repair scaffold of the present invention has the ability to promote cartilage regeneration while promoting subchondral bone regeneration, and can effectively promote the repair of osteochondral tissue damage.

Matters not covered in the present invention are known technologies.

Although the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, so the protection scope of the present invention should be based on the scope defined in the claims.

Claims (10)

1. The cartilage tissue repair bracket is characterized by sequentially comprising a cartilage layer and a subchondral bone layer from top to bottom, wherein the cartilage layer is provided with lotus-shaped and radial holes, and the subchondral bone layer is provided with lotus-shaped holes communicated with the cartilage layer.

2. The osteochondral tissue repair scaffold of claim 1, wherein the cartilage layers and the subchondral layers are both cylindrical and the cylindrical bottom surfaces of the cartilage layers and the subchondral layers have the same diameter; the lotus-shaped holes longitudinally penetrate through the cylindrical bottom surfaces of the cartilage layers and the subchondral bone layers; the radial holes are positioned on the cylindrical side wall of the cartilage layer and transversely penetrate through the cartilage layer.

3. The osteochondral tissue repair scaffold of claim 2, wherein the "lotus-like" and "radial" pores of the cartilage layers are 100 to 400 μm in pore diameter.

4. The osteochondral tissue repair scaffold of claim 3, wherein the cartilage layer comprises GelMA, LAP and growth factors.

5. The osteochondral tissue repair scaffold of claim 4, wherein the subchondral bone layer comprises GelMA and LAP.

6. The osteochondral tissue repair scaffold of claim 5, wherein the subchondral bone layer further comprises calcium phosphate nanocluster material having a particle size of 1 nanometer.

7. The osteochondral tissue repair scaffold of claim 5, wherein the GelMA and LAP concentrations of the cartilage and subchondral bone layers are the same; the GelMA content is 10-15% and the LAP content is 0.2% by mass fraction.

8. The osteochondral tissue repair scaffold of claim 7, wherein the growth factor is KGN, and the concentration of KGN is 200 μm.

9. A method of preparing an osteochondral tissue repair scaffold, the method comprising:

(1) Preparing a first feed liquid, adding GelMA, LAP and KGN into single distilled water under the condition of avoiding light, and uniformly stirring to obtain the first feed liquid;

(2) Preparing a second feed liquid, adding GelMA and LAP or GelMA and LAP calcium phosphate nano-cluster materials with the particle size of 1 nanometer into single distilled water under the condition of avoiding light, and uniformly stirring to obtain the second feed liquid;

(3) Pouring the first feed liquid into a liquid tank of a photo-curing 3D printing device, and printing an upper cartilage layer bracket;

(4) And pouring the second feed liquid into a liquid tank of a printer, and continuing to print the lower cartilage scaffold to obtain the double-layer cartilage tissue repair scaffold.

10. The application of the calcium phosphate nanocluster material with the particle size of 1 nanometer in preparing an osteochondral tissue repair scaffold.

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