阅读说明：本技术 骨软骨修复支架及其制备方法 (Osteochondral repair scaffold and preparation method thereof ) 是由 潘洋 魏欣苗 陈雪霓 谭荣伟 佘振定 于 2019-10-24 设计创作，主要内容包括：本发明涉及一种骨软骨修复支架,包括依次设置的软骨下骨层、钙化软骨层、软骨层和软骨膜层；其中,所述软骨膜层为位于所述软骨层远离所述钙化软骨层的一侧表面的致密膜层,所述致密膜层能够防止外部纤维细胞长入到软骨层。本发明还公开了一种骨软骨修复支架的制备方法。本发明中的骨软骨修复支架,在软骨层一侧表面的设置有致密的软骨膜层,该软骨膜层作为软骨层的屏障膜,可以防止外部纤维细胞长入软骨层,同时修复得到软骨组织不会超过周围正常组织,避免造成软骨增生。(The invention relates to a bone cartilage repair support, which comprises a subchondral bone layer, a calcified cartilage layer, a cartilage layer and a cartilage membrane layer which are sequentially arranged; the cartilage layer is a compact film layer located on the surface of one side, far away from the calcified cartilage layer, of the cartilage layer, and the compact film layer can prevent external fiber cells from growing into the cartilage layer. The invention also discloses a preparation method of the osteochondral repair scaffold. According to the osteochondral repair scaffold, the compact cartilage membrane layer is arranged on the surface of one side of the cartilage layer and serves as a barrier membrane of the cartilage layer, external fiber cells can be prevented from growing into the cartilage layer, meanwhile, cartilage tissues obtained through repair cannot exceed surrounding normal tissues, and cartilage hyperplasia is avoided.)

1. The osteochondral repair bracket is characterized by comprising a subchondral bone layer, a calcified cartilage layer, a cartilage layer and a cartilage membrane layer which are sequentially arranged;

the cartilage layer is a compact film layer located on the surface of one side, far away from the calcified cartilage layer, of the cartilage layer, and the compact film layer can prevent external fiber cells from growing into the cartilage layer.

2. The osteochondral repair scaffold according to claim 1, wherein the cartilage membrane layer is a collagen membrane layer;

preferably, the subchondral bone layer, the calcified cartilage layer and the cartilage layer are all porous structures, the calcified cartilage layer is provided with a barrier layer for preventing the tissue of the subchondral bone layer from growing into the cartilage layer, and the average pore diameter of the barrier layer is smaller than the average pore diameter of the subchondral bone layer and the cartilage layer;

more preferably, the pore size of the barrier layer is from 100nm to 5 μm;

more preferably, the calcified cartilage layer further comprises a first surface layer and a second surface layer, the first surface layer and the second surface layer are respectively arranged at the positions of the barrier layer adjacent to the subchondral bone layer and the cartilage layer, and the pore diameter of the first surface layer and the pore diameter of the second surface layer are mainly 50-1000 μm.

3. The osteochondral repair scaffold according to claim 1 or 2, wherein the main component of the calcified cartilage layer is polylactic acid.

4. The osteochondral repair scaffold according to claim 1, wherein the subchondral bone layer comprises a metal outer frame having pores and a subchondral bone layer body having a non-metal main component, the subchondral bone layer body being located inside the metal outer frame;

preferably, the metal outer frame comprises a plurality of metal strips which are arranged at intervals;

preferably, the metal outer frame is made of one or a compound of more of stainless steel, titanium alloy except the stainless steel and magnesium alloy except the stainless steel;

preferably, the subchondral bone layer body is a collagen-inorganic salt composite porous scaffold;

preferably, the inorganic salt is selected from one of tricalcium phosphate, hydroxyapatite, bone particles and calcium polyphosphate, or a mixture of any of the above inorganic salts;

preferably, the inorganic salt is calcium polyphosphate doped with magnesium and/or gallium.

5. A method for preparing the osteochondral repair scaffold of any one of claims 1-4, comprising the steps of:

preparing a subchondral bone layer, a calcified cartilage layer and a cartilage layer which are connected in sequence; and

forming a compact cartilage film layer on the surface of the side, away from the calcified cartilage layer, of the cartilage layer;

preferably, the forming of the dense perichondrium layer on the surface of the side of the perichondrium layer away from the calcified perichondrium layer comprises:

providing a collagen solution with the concentration of 0.5-1.5% (g/100 ml);

crosslinking the collagen solution to obtain a crosslinked collagen solution; and

and forming a compact cartilage film layer on the surface of the side, away from the calcified cartilage layer, of the cartilage layer by using the crosslinked collagen solution.

6. The method for preparing a calcium-containing cartilage according to claim 5, wherein the step of forming a dense perichondrium layer on the surface of the cartilage layer on the side far from the calcified cartilage layer comprises:

providing a collagen solution with the concentration of 0.5-1.5% (g/100 ml);

drying the surface of the side, away from the calcified cartilage layer, of the cartilage layer by using the collagen solution to form a membranous structure; and

and crosslinking the membranous structure in a crosslinking solution to form a compact cartilage membranous layer.

7. The method for preparing according to claim 5, wherein the preparing of the subchondral bone layer, the calcified cartilage layer and the cartilage layer connected in sequence comprises:

adopting polylactic acid to form a calcified cartilage layer by thermally induced phase separation;

forming a subchondral bone layer on the surface of one side of the calcified cartilage layer; and

and forming a cartilage layer on the surface of the other side of the calcified cartilage layer.

8. The method of claim 7, wherein the forming of the calcified cartilage layer from the thermally induced phase separation using polylactic acid comprises:

providing a polylactic acid solution with the concentration of 4-10% (g/100 ml);

providing a pore-foaming agent with the particle size of 50-1000 mu m;

stacking a first layer of pore-forming agent, adding a polylactic acid solution to immerse the first layer of pore-forming agent, and forming a layer of polylactic acid solution layer above the first layer of pore-forming agent;

a second layer of pore-forming agent is stacked on the polylactic acid solution layer, and then the polylactic acid solution is added to immerse the second layer of pore-forming agent, so that a polylactic acid solution precursor is obtained; and

thermally phase-separating the polylactic acid solution precursor at 4-196 ℃ for 5 min-2 h, replacing the solvent of the polylactic acid solution by a replacement solvent, and freeze-drying at-60-80 ℃ to form a calcified cartilage layer.

9. The method for preparing a calcified cartilage layer according to claim 7, wherein the forming of a subchondral bone layer on the surface of the calcified cartilage layer includes:

providing a metal outer frame;

placing the metal outer frame on the surface of one side of the calcified cartilage layer;

mixing an inorganic salt with the cross-linked collagen solution to form an inorganic salt-cross-linked collagen mixed solution; and

and pouring the inorganic salt-crosslinked collagen mixed solution into the metal outer frame, and freeze-drying to form the subchondral bone layer body.

10. The method for preparing a calcified cartilage layer according to claim 7, wherein the forming of a subchondral bone layer on the surface of the calcified cartilage layer includes:

providing a metal outer frame;

placing the metal outer frame on the surface of one side of the calcified cartilage layer;

providing a collagen-inorganic salt mixed solution;

adding the mixed solution of the collagen and the inorganic salt into the metal outer frame, and freeze-drying to form a sponge scaffold; and

and crosslinking the sponge support in a crosslinking solution, and freeze-drying to obtain the subchondral bone layer body.

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a bone cartilage repair scaffold and a preparation method thereof.

Background

Cartilage damage is osteoarthritis, is a common and frequent disease of middle-aged and elderly people, and some current clinical treatment methods cannot provide a good repairing effect on large-area cartilage defects formed in the middle and later stages of osteoarthritis. Tissue engineered osteochondral repair scaffolds are the most promising repair methods. The osteochondral repair scaffold is gradually replaced with normal tissue in vivo after being implanted in vivo, thereby completing the repair. Whereas, in order to simulate the natural osteochondral structure, osteochondral repair scaffolds are generally designed as a three-layer structure: cartilage layer, calcified cartilage layer and subchondral bone layer.

The traditional osteochondral repair scaffold with a three-layer structure can cause some problems in the process of repairing cartilage injury. For example, after being implanted into a body, wherein a cartilage layer is in contact with other tissues and cells, external fibroblasts can grow into the cartilage layer from pores of the cartilage layer or even into a scaffold, so that the normal bone regeneration process is influenced, and the healing is influenced; meanwhile, the repaired cartilage tissue is easy to exceed the surrounding normal tissue, thereby causing cartilage hyperplasia.

Disclosure of Invention

Therefore, it is necessary to provide an osteochondral repair scaffold and a preparation method thereof aiming at the technical problems of the traditional osteochondral repair scaffold with a three-layer structure.

The osteochondral repair bracket comprises a subchondral bone layer, a calcified cartilage layer, a cartilage layer and a cartilage membrane layer which are arranged in sequence; the cartilage layer is a compact film layer positioned on the surface of one side of the cartilage layer, and the compact film layer can prevent external fiber cells from growing into the cartilage layer.

In one embodiment, the cartilage membrane layer is a collagen membrane layer.

In one embodiment, the subchondral bone layer, the calcified cartilage layer and the cartilage layer are all porous structures, the calcified cartilage layer has a barrier layer preventing the growth of subchondral bone tissue into the cartilage layer, and the pore size of the barrier layer is smaller than the pore size of the subchondral bone layer and the cartilage layer.

In one embodiment, the barrier layer has a pore size of 100nm to 5 μm.

In one embodiment, the calcified cartilage layer further comprises a first surface layer and a second surface layer, the first surface layer and the second surface layer are respectively arranged at the positions of the barrier layer adjacent to the subchondral bone layer and the cartilage layer, and the pore diameters of the first surface layer and the second surface layer are both mainly 50-1000 μm.

The first surface layer and the second surface layer mainly form macropores through a pore-forming agent, but the walls of the macropores can simultaneously have small micropores, and the pore diameter of each micropore is 100nm-5 microns.

In one embodiment, the main component of the calcified cartilage layer is polylactic acid.

In one embodiment, the subchondral bone layer comprises a metal outer frame with pores and a subchondral bone layer body with a non-metal main component, wherein the subchondral bone layer body is positioned inside the metal outer frame.

In one embodiment, the metal outer frame comprises a plurality of metal strips which are arranged at intervals.

In one embodiment, the metal outer frame is made of one or a composite of several of stainless steel, titanium alloy except for stainless steel and magnesium alloy except for stainless steel.

In one embodiment, the subchondral bone layer body is a collagen-inorganic salt composite porous scaffold.

In one embodiment, the inorganic salt is selected from one of tricalcium phosphate, hydroxyapatite, natural calcined bone particles, and calcium polyphosphate, or a mixture of any of the foregoing inorganic salts.

In one embodiment, the inorganic salt is calcium polyphosphate doped with magnesium and/or gallium.

The preparation method of the osteochondral repair scaffold comprises the following steps:

preparing a subchondral bone layer, a calcified cartilage layer and a cartilage layer which are connected in sequence; and

and forming a compact cartilage film layer on the surface of one side of the cartilage layer, which is far away from the calcified cartilage layer.

In one embodiment, the forming of the dense perichondrium layer on the surface of one side of the perichondrium layer includes:

providing a collagen solution with the concentration of 0.5-1.5% (0.8%, 1%, 1.2%, 1.4% can be selected, and the concentration percentage of the collagen solution is calculated by g/100 ml);

crosslinking the collagen solution to obtain a crosslinked collagen solution; and

and forming a compact collagen film layer on the surface of one side of the cartilage layer by using the crosslinked collagen solution, wherein the compact collagen film layer is the cartilage film layer.

In one embodiment, the forming of the dense perichondrium layer on the surface of the cartilage layer on the side far away from the calcified cartilage layer comprises:

providing a collagen solution with the concentration of 0.5-1.5% (0.8%, 1%, 1.2%, 1.4% can be selected, and the concentration percentage of the collagen solution is calculated by g/100 ml);

drying the surface of the side, away from the calcified cartilage layer, of the cartilage layer by using the collagen solution to form a membranous structure; and

and crosslinking the membranous structure in a crosslinking solution to form a compact cartilage membranous layer.

In one embodiment, the preparing of the subchondral bone layer, the calcified cartilage layer and the cartilage layer connected in sequence comprises:

adopting polylactic acid to form a calcified cartilage layer by thermally induced phase separation;

forming a subchondral bone layer on the surface of one side of the calcified cartilage layer; and

and forming a cartilage layer on the surface of the other side of the calcified cartilage layer.

In one embodiment, the forming of the calcified cartilage layer by thermally induced phase separation using polylactic acid comprises:

providing a polylactic acid solution with the concentration of 4-10%;

providing a pore-foaming agent with the particle size of 50-1000 mu m;

stacking a first layer of pore-forming agent, adding a polylactic acid solution to immerse the first layer of pore-forming agent, and forming a layer of polylactic acid solution layer above the first layer of pore-forming agent;

a second layer of pore-forming agent is stacked on the polylactic acid solution layer, and then the polylactic acid solution is added to immerse the second layer of pore-forming agent, so that a polylactic acid solution precursor is obtained; and

thermally phase-separating the polylactic acid solution precursor at 4-196 ℃ for 5 min-2 h, replacing the solvent of the polylactic acid solution by a replacement solvent, and freeze-drying at-60-80 ℃ to form a calcified cartilage layer.

In one embodiment, the forming of the subchondral bone layer on the surface on one side of the calcified cartilage layer comprises:

providing a metal outer frame;

placing the metal outer frame on the surface of one side of the calcified cartilage layer;

mixing an inorganic salt with the cross-linked collagen solution to form an inorganic salt-cross-linked collagen mixed solution; and

and pouring the inorganic salt-crosslinked collagen mixed solution into the metal outer frame, and freeze-drying to form the subchondral bone layer body.

In one embodiment, the forming of the subchondral bone layer on the surface on one side of the calcified cartilage layer comprises:

providing a metal outer frame;

placing the metal outer frame on the surface of one side of the calcified cartilage layer;

providing a collagen-inorganic salt mixed solution;

adding the mixed solution of the collagen and the inorganic salt into the metal outer frame, and freeze-drying to form a sponge scaffold;

and crosslinking the sponge support in a crosslinking solution, and freeze-drying to obtain the subchondral bone layer body.

According to the osteochondral repair scaffold, the compact cartilage membrane layer is arranged on the surface of one side of the cartilage layer and serves as a barrier membrane of the cartilage layer, external fiber cells can be prevented from growing into the cartilage layer, meanwhile, cartilage tissues obtained through repair cannot exceed surrounding normal tissues, and cartilage hyperplasia is avoided.

Drawings

FIG. 1 is a schematic cross-sectional view of an osteochondral repair scaffold according to an embodiment of the present invention;

FIG. 2 is a schematic cross-sectional view of a calcified cartilage layer in an osteochondral repair scaffold according to an embodiment of the present invention;

fig. 3-5 are schematic diagrams showing the structure of the metal frame of the subchondral bone layer in the osteochondral repair frame according to the embodiment of the present invention, wherein the upper row is a top view of the metal frame, and the lower row is a front view corresponding to the upper row;

FIG. 6 is an electron microscope image of the barrier layer in the calcified cartilage layer in the osteochondral repair scaffold according to example 2 of the present invention;

FIG. 7 is an electron microscope photograph of the surface layer in the calcified cartilage layer in the osteochondral repair scaffold according to example 2 of the present invention;

FIG. 8 is a graph showing the results of cell culture on the surface of the perichondrium layer on the side away from the perichondrium layer after cell culture on the perichondrium layer in the osteochondrium repair scaffold in example 2 according to the present invention in Experimental example 2;

FIG. 9 is a graph showing the results of cell culture on the surface of the perichondrium layer on the side close to the perichondrium layer after cell culture on the perichondrium layer in the osteochondrial repair scaffold of example 2 in Experimental example 2 of the present invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Fig. 1 shows an osteochondral repair scaffold according to an embodiment of the present invention, which includes a subchondral bone layer 1, a calcified cartilage layer 2, a cartilage layer 3, and a cartilage membrane layer 4; wherein, the cartilage membrane layer 4 is a compact membrane layer positioned on the surface of one side of the cartilage layer 3 far away from the calcified cartilage layer 2, and the compact membrane layer can prevent external fiber cells from growing into the cartilage layer.

The osteochondral repair support in this embodiment keeps away from the calcified cartilage layer 2 in the cartilage layer and is provided with compact cartilage membrane layer on one side surface, and this cartilage membrane layer can prevent that outside fibre cell from growing into the cartilage layer as the barrier membrane on cartilage layer, and the while is restoreed and is obtained cartilage tissue and can not surpass normal tissue on every side, avoids causing cartilage hyperplasia. Wherein the periosteum layer 4 has a higher density, which is such that cells cannot pass through the periosteum layer.

In one embodiment, the perichondrium layer 4 is a collagen film layer. Further, the cartilage membrane layer 4 is a compact membrane layer consisting of crosslinked collagen, and the thickness of the cartilage membrane layer 4 is 0.1 mm-1 mm. In the actual operation, the crosslinking is carried out by using collagen with concentration of 0.5% -1.5% (0.8%, 1%, 1.2%, 1.4% can be selected, the concentration percentage of the collagen solution is calculated by g/100ml, the solvent can be selected from the common solvent in the field, for example, about 0.05mol/L acetic acid solution).

Of course, the perichondrium layer may be formed using other materials and methods. For example, 10% PLGA is dissolved by acetone (mass to volume ratio, e.g., 1g PLGA dissolved in 10mL acetone), then the solution is poured into a beaker to form a height of 1mm, and then the prepared subchondral bone/calcified cartilage/cartilage layer scaffold is deposited into the solution in the beaker with the side surface of the cartilage layer 3 away from the calcified cartilage layer 2, and then air-dried to form the cartilage film layer 4.

In one embodiment, the subchondral bone layer 1, the calcified cartilage layer 2 and the cartilage layer 3 are porous, and the calcified cartilage layer 2 has a barrier layer for preventing the growth of subchondral bone tissue into the cartilage layer, and the pore size of the barrier layer is smaller than the pore size of the subchondral bone layer 1 and the cartilage layer 3.

In the osteochondral repair scaffold in the embodiment, the subchondral bone layer, the calcified cartilage layer and the cartilage layer are all of porous structures, so that nutrient transmission of the osteochondral repair scaffold is facilitated, and smooth proceeding of a osteochondral repair scaffold repair process is guaranteed. The calcified cartilage layer has a barrier layer for preventing the bone tissue of the subchondral bone layer from growing into the cartilage layer, and the barrier layer has a relatively small aperture, so that the calcified cartilage layer can be used as a barrier to prevent the bone tissue of the subchondral bone layer from growing into the cartilage layer and can transmit nutrients through the relatively small aperture.

As shown in fig. 2, the calcified cartilage layer 2 in the present embodiment includes a first surface layer 21, a barrier layer 22 and a second surface layer 23, wherein the pore size of the first surface layer 21 and the pore size of the second surface layer 23 are 50 μm to 1000 μm, and the pore size of the barrier layer 22 is 100nm to 5 μm.

The calcified cartilage layer 2 has a three-layer structure, and as can be seen from fig. 1 and 2, in the present embodiment, the pore size of the barrier layer 22 is much smaller than the pore size of the first surface layer 21 and the second surface layer 23. The calcified cartilage layer 2 can be used as a barrier membrane to prevent the lower bone tissue from growing into the upper cartilage layer, and can also transmit nutrients through micropores; the first surface layer and the second surface layer in the calcified cartilage layer contain macropores, and the first surface layer and the second surface layer in the calcified cartilage layer can be used as transition layers between the subchondral bone layer and the calcified cartilage layer and between the calcified cartilage layer and the cartilage layer so as to enhance the connection strength between the subchondral bone and the cartilage layer.

In one embodiment, the subchondral bone layer 1 includes a metal outer frame having pores and a subchondral bone layer body whose main component is a non-metal, the subchondral bone layer body being located inside the metal outer frame.

In the traditional osteochondral repair scaffold, the subchondral bone layer has more problems. Some subchondral bone layers adopt composite scaffolds of polymers, macromolecules and inorganic salts, and although the scaffolds can be completely degraded, the mechanical strength of the scaffolds is low, and the brittleness of the scaffolds is increased after the inorganic salts are added, so that the clinical application of the scaffolds is also limited. Some methods for enhancing the strength of the subchondral bone layer use metal materials as a matrix to prepare the subchondral bone layer, namely the obtained subchondral bone layer has a metal scaffold, the mechanical strength of the metal scaffold is high, but the whole prepared metal scaffold is of a porous structure, the area into which bone tissues can grow is limited, and the elastic modulus of metal is high, so that the finally repaired subchondral bone layer has a great difference from the normal bone tissues.

The subchondral bone layer 1 in the embodiment includes a metal outer frame with pores and a subchondral bone layer body with a non-metal main component, wherein the metal outer frame has an effect of protecting the subchondral bone layer body with lower internal mechanical strength; when the subchondral bone layer 1 keeps high strength, the metal bracket is only used as an outer frame, so that the ratio of the metal bracket to the subchondral bone layer 1 is low, and the difference between the osteochondral tissue and the surrounding normal bone tissue after repair is reduced.

In one embodiment, the metal outer frame comprises a plurality of metal strips arranged at intervals. Fig. 3 to 5 are schematic structural diagrams of three different structures of the metal outer frame in the embodiment of the present invention, and the invention is not limited thereto, for example, the bottom surface of the metal outer frame may be set to be triangular, square or customized according to the specific shape of the cartilage defect of the patient. In a specific embodiment, in which the metal casing is of a cylindrical configuration, the upper row is a top view of the metal casing in fig. 3-5, and the lower row is a front view corresponding to the upper row. The metal casing shown in fig. 3 is composed of a plurality of metal strips arranged in parallel. The metal casing shown in fig. 4 is a casing constructed by a plurality of metal strips intersecting perpendicularly in a grid arrangement. The metal casing shown in fig. 5 is a frame constructed by a plurality of obliquely intersecting metal strips in a grid arrangement. Preferably, the metal strips are spaced at intervals of 0.5mm to 5mm, and the metal strips have a diameter of 0.5mm to 1 mm. The metal outer frame may be made of stainless steel, titanium alloy other than stainless steel, or magnesium alloy other than stainless steel. Of course, a composite structure composed of the above metals may be used, and for example, the plurality of metal strips may be made of different metal materials.

Preferably, the structure of the metal outer frame can be designed according to the actual requirement. For example, software can be used to design the metal outer frame, and then the metal outer frame is printed by using a 3D printing technology.

In one embodiment, the subchondral bone layer body is a collagen-inorganic salt composite porous scaffold. Preferably, the subchondral bone body is a porous scaffold formed by mixing the crosslinked collagen and inorganic salt particles. Wherein the mass ratio of the collagen to the inorganic salt is 10: 90-90: 10. The aperture of the obtained collagen-inorganic salt composite porous scaffold is 50-500 mu m.

In the present embodiment, the collagen may be type I collagen, type II collagen, type III collagen, or type IM collagen, or a mixture thereof.

In the embodiment of the invention, two ways of crosslinking the collagen are adopted, one way is that the crosslinking agent is directly added into the collagen solution (0.2-1 percent), the collagen solution is uniformly mixed for 1-24 hours, then the mixture is pre-frozen and freeze-dried, and then the cleaning solution is used for soaking the bracket to remove the redundant crosslinking agent which is not crosslinked; the other method is that the collagen solution (0.2-1%) is firstly mixed with inorganic salt particles and then is lyophilized or the collagen solution is directly dried (lyophilized, air-dried at normal temperature and vacuum-dried), then the lyophilized or dried sample is put into the crosslinking solution to be soaked for 1-24h, and then pure water is used for washing and removing the redundant non-crosslinked crosslinking agent.

In the embodiment of the invention, the cross-linking agent is one or a mixture of more of formaldehyde, glutaraldehyde and EDC (1-ethyl- (3-3-dimethylhelium propyl) -carbodiimide) -NHS (N-hydroxysuccinimide). The different layers may be cross-linked using different cross-linking agents.

In some embodiments, the added mass of ED is between 0.3% and 0.5% (m/m), preferably 0.4% (m/m), of the dry weight of the collagen.

In the embodiment of the present invention, the inorganic salt is selected from one of tricalcium phosphate, hydroxyapatite, natural calcined bone particles and calcium polyphosphate, or a mixture of any of the above inorganic salts. Further, the inorganic salt is calcium polyphosphate doped with magnesium and/or gallium. The particle size of the inorganic salt particles is 100nm-2 mm.

The embodiment of the invention also discloses a preparation method of the osteochondral repair bracket, which comprises the following steps:

s10: preparing a subchondral bone layer, a calcified cartilage layer and a cartilage layer which are connected in sequence;

s20: and forming a compact cartilage film layer on the surface of one side of the cartilage layer, which is far away from the calcified cartilage layer.

In step S10, the subchondral bone layer, the calcified cartilage layer and the cartilage layer connected in sequence may be prepared by different preparation methods and in different orders.

In one embodiment of the present invention, a calcified cartilage layer is first formed by thermally induced phase separation using polylactic acid (PLLA). And then subchondral bone layers and cartilage layers are formed on the surfaces of both sides of the calcified cartilage layers, respectively.

In this example, the concentration of polylactic acid (PLLA) solution used in forming the calcified cartilage layer by thermal phase separation was 4% to 10% (g/100ml, and 5%, 6%, 7%, 8% or 9% may be selected), and the solvent for dissolving PLLA was tetrahydrofuran. The particle diameter of the pore-foaming agent is 50-1000 μm, such as 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm or 900 μm.

The calcified cartilage layer in this embodiment includes a first surface layer, a barrier layer and a second surface layer. The pore diameter of the barrier layer is different from the pore diameters of the other two surface layers, and the method is realized by adopting the following method in the embodiment of the invention: firstly, stacking a first layer of pore-forming agent, adding a polylactic acid solution to immerse the first layer of pore-forming agent, and forming a layer of polylactic acid solution above the first layer of pore-forming agent; then, a second layer of pore-forming agent is stacked on the polylactic acid solution layer, and then the polylactic acid solution is added to immerse the second layer of pore-forming agent, so that a polylactic acid solution precursor is obtained; finally, thermally inducing the polylactic acid solution precursor to phase separate for 2h at-20 ℃, replacing tetrahydrofuran three times by adopting pure water as a replacement solvent, and freeze-drying at-20-196 ℃ to form a calcified cartilage layer. Further, after the completion of the lyophilization step, the resulting structure may be placed in an ultraviolet or ozone machine for 10min to enhance the hydrophilicity of the calcified cartilage layer. In the obtained calcified cartilage layer, the first surface layer and the second surface layer have large pore diameters of 50-1000 μm, and the barrier layer has micro pore diameters of 100nm-5 μm. The preparation method is simple and convenient to operate, and meanwhile, although the formed calcified cartilage layer has three layers with different pore diameters, the calcified cartilage layer has better integrity and good stability.

In an embodiment of the invention, the subchondral bone layer is formed on the surface on the side of the calcified cartilage layer (which may be, for example, the surface of the first skin layer). The subchondral bone layer can be a two-part structure comprising a metal outer frame and a subchondral bone layer body. The structure ensures that the subchondral bone layer has a basic structure, and simultaneously, the metal outer frame is added, so that the subchondral bone layer has higher strength.

In one embodiment, the subchondral bone layer may be prepared by:

providing a metal outer frame: the metal outer frame can be prepared in different modes, for example, different metal strips can be assembled according to the required structure; or designing a metal outer frame by adopting software according to needs and printing the metal outer frame by using a three-dimensional printer to obtain the metal outer frame.

Providing a material for forming a subchondral bone layer body: the material forming the subchondral bone layer body may be an inorganic salt-crosslinked collagen mixed solution formed by mixing an inorganic salt with a crosslinked collagen solution.

And placing the metal outer frame on the surface of one side of the calcified cartilage layer.

And pouring the inorganic salt-crosslinked collagen mixed solution into the metal outer frame, and freeze-drying to form a subchondral bone layer body, wherein the subchondral bone layer body and the metal outer frame jointly form the subchondral bone layer.

In another embodiment, the material forming the subchondral bone layer body can also be a mixed solution of collagen and an inorganic salt, wherein the collagen is not crosslinked. Adding the mixed solution of the collagen and the inorganic salt into the metal outer frame, and freeze-drying to form a sponge scaffold; and then crosslinking the sponge support in crosslinking liquid, and freeze-drying to obtain a subchondral bone layer body, wherein the subchondral bone layer body and the metal outer frame jointly form the subchondral bone layer.

In an embodiment of the invention, the cartilage layer is formed on the surface of the other side of the calcified cartilage layer (which may be the surface of the first skin layer, for example). The cartilage layer may be formed using a cross-linked collagen solution or a composite solution of a cross-linked collagen solution and inorganic salt particles. Of course, it is also possible to form an uncrosslinked cartilage layer on the surface of the other side of the calcified cartilage layer using a composite solution of an uncrosslinked collagen solution and inorganic salt particles, and then to perform crosslinking in a crosslinking solution.

In the embodiment of the invention, the collagen solution or the uncrosslinked collagen solution for forming the subchondral cartilage layer and the cartilage layer can enter the large apertures in the first surface layer or the second surface layer of the calcified cartilage layer, so that the connection strength between the subchondral cartilage layer and the calcified cartilage layer is enhanced.

In step S20, a dense cartilage film layer is formed on the surface of the cartilage layer on the side away from the calcified cartilage layer. Further, the cartilaginous membrane layer in the embodiment of the invention is a non-porous collagen-free membrane layer.

In the examples of the present invention, the dense osteochondral film layer was prepared as follows:

providing a collagen solution with the concentration of 0.5-1.5% (g/100 ml);

crosslinking the collagen solution to obtain a crosslinked collagen solution; and

and forming a compact cartilage film layer on the surface of the side, away from the calcified cartilage layer, of the cartilage layer by using the crosslinked collagen solution.

In the examples of the present invention, the dense osteochondral film layer was prepared as follows:

providing a collagen solution with the concentration of 0.5-1.5% (g/100 ml);

drying the surface of the side, away from the calcified cartilage layer, of the cartilage layer by using the collagen solution to form a membranous structure; and

and crosslinking the membranous structure in a crosslinking solution to form a compact cartilage membranous layer.

In order to describe the embodiments of the present invention in more detail, the following more preferred embodiments are described.