Construction method of in-vitro articular cartilage superficial layer injury model

Abstract

The invention belongs to the technical field of experimental model preparation, and provides a construction method of an in-vitro articular cartilage superficial layer injury model aiming at proteoglycan and collagenous fibers in a superficial cartilage layer (SFZ) in order to simulate cartilage surface component loss and structural damage in the early stage of OA morbidity, and the in-vitro articular cartilage superficial layer injury model is constructed by taking a pig knee joint cartilage as a material. A mixed solution of 0.01 mg / ml chymotrypsin and 0.5 mg / ml collagenase D is used as digestive enzyme, the surface of an in-vitro articular cartilage column is treated for 20 min, proteoglycan and collagenous fibers in SFZ are accurately destroyed, and an in-vitro articular cartilage superficial layer damage model is constructed; histological staining, microscopic observation and mechanical detection results all show that the method can cause SFZ structure damage and mechanical property reduction on the premise of not influencing deep tissues. The model has the advantages of simplicity and convenience in operation, low cost and controllable injury range, and provides a reliable experimental tool for research on early pathological mechanisms of osteoarthritis and exploration of related diagnosis and treatment means.

Description

Technical Field

[0001] This invention belongs to the field of experimental model preparation technology, specifically relating to a method for constructing an isolated articular cartilage superficial layer injury model. Background Technology

[0002] The main pathological changes in osteoarthritis (OA) are degeneration of articular cartilage and synovial hyperplasia. The pain, swelling, and functional impairment caused by OA remain among the major diseases affecting middle-aged and elderly patients. With the acceleration of aging and the increase in the incidence of sports injuries, the incidence of OA is increasing year by year, and the age of onset is trending younger. However, the early pathogenesis of OA is unclear, and often by the time a definitive diagnosis is made, irreversible damage to the cartilage tissue has already occurred, missing the optimal window for etiological treatment. Treatment is then limited to symptomatic and surgical interventions, placing a heavy burden on patients and society.

[0003] The superficial zone (SFZ) of articular cartilage is a special structure that accounts for about 5%-10% of the cartilage thickness. It is characterized by parallel-arranged collagen fibers and flattened cells. Figure 1 The superficial joint zone (SFZ) plays a crucial role in maintaining extremely low surface friction and transmitting mechanical signals, serving as the first line of defense for cartilage tissue against damage. Recent studies have found that many pathological changes in cartilage degeneration first occur in the superficial joint zone (SFZ), and SFZ damage plays a significant role in the development and progression of full-thickness degeneration of articular cartilage. Furthermore, chondrocyte progenitor cells, cells with self-renewal capabilities, have been found to be closely related to the superficial joint layer in recent years. Therefore, the SFZ may be the earliest site of articular cartilage degeneration and an indispensable part of research on early cartilage injury.

[0004] To study the pathogenesis and treatment of osteoarthritis (OA), OA models other than those used in humans are indispensable tools, especially various animal OA models, which are widely used in OA pathophysiological research. However, animal knee OA models, such as those for rat anterior cruciate ligament transection and mouse meniscus instability surgery, require a 2-3 month modeling period, and the success rate is not 100%. This leads to increased time and cost. Therefore, in vitro cartilage tissue OA models are the best choice to solve these problems. Compared with in vivo animal knee OA modeling, ex vivo cartilage tissue block OA models have the advantages of rapid modeling, short modeling cycle, significant structural changes, and smaller changes in physiological parameters.

[0005] Current research on osteoarthritis (OA) is limited to the middle and late stages of the disease, where cartilage has already suffered severe damage. To advance the window of opportunity for OA diagnosis and treatment, the development of early OA models is urgently needed. The superficial articular cartilage layer (SFZ) is the primary site for resisting mechanical stress damage to the joint, capable of withstanding shear, compressive, and tensile forces, and plays a crucial buffering role. Furthermore, the cartilage surface has extremely low surface friction, providing excellent lubrication during joint movement. Therefore, the SFZ, as the interface structure between cartilage and the joint cavity, plays a vital role in maintaining joint mechanical stability and is the initial site of damage in the development of OA. The development of in vitro cartilage tissue SFZ damage models is urgently needed to advance the window of opportunity for OA research.

[0006] The pathogenesis of osteoarthritis (OA) includes aging-related degeneration, changes in mechanical factors, and inflammatory damage. Existing OA modeling methods include aged animal models, stress impact models, and in vitro cartilage tissue block culture inflammatory factor stimulation models. While these methods can effectively model the in vivo environment and pathogenesis of OA, they have certain drawbacks. For example, aged animal models are costly in terms of time and money, and cannot accurately determine the timing of OA formation. Stress impact models can only cause structural changes in cartilage and cannot precisely control the loss of important components (collagen fibers and proteoglycans) during OA cartilage damage. Inflammatory stimulation models simulate the inflammatory environment of articular cartilage in vivo, but the degree of cartilage damage is unpredictable, and in vitro cultured cartilage samples need to be as fresh as possible. This places extremely high demands on the process from cartilage collection to laboratory culture; the interval between collection and culture cannot be too long, and microbial contamination must be avoided. Therefore, for the early stage of OA cartilage damage, namely SFZ (form-free fracture zone) damage, there is an urgent need to find a simple, cost-effective, and precisely controllable method for modeling. The main components of cartilage SFZ are type I collagen (COL I) and proteoglycans (GAG). Summary of the Invention

[0007] To simulate the loss of cartilage surface components and structural damage in the early stages of osteoarthritis (OA), this invention provides a method for constructing an isolated articular cartilage superficial layer injury model targeting proteoglycans and collagen fibers in the superficial cartilage layer. The method utilizes enzymatic digestion to construct SFZ injury of isolated cartilage tissue under non-sterile conditions, selecting chymotrypsin and collagenase D as digestive enzymes to construct the isolated articular cartilage superficial layer injury model. It is hoped that this model will provide a rapid and convenient research model for OA cartilage injury studies.

[0008] The present invention is achieved by the following technical solution: a method for constructing a superficial injury model of isolated articular cartilage, using a mixture of 0.01 mg / ml chymotrypsin and 0.5 mg / ml collagenase D as digestive enzymes, treating the surface of isolated articular cartilage for 20 min, precisely destroying the proteoglycans and collagen fibers in SFZ, and constructing a superficial injury model of isolated articular cartilage. The specific steps are as follows: (1) Sample collection: Select isolated knee joint cartilage. Before extracting the cartilage tissue block, ensure that the knee joint capsule is intact and undamaged so that the cartilage is in a normal physiological environment before the experiment. Open the skin, fascia and joint capsule in sequence according to the tissue layers to expose the cartilage. Select specimens with bright color, smooth surface and no damage marks on the cartilage surface. Use a bone cartilage hollow drill to remove the cartilage column from the trochlea of ​​the femur and place it in PBS solution for later use. (2) Enzymatic hydrolysis of cartilage column: The latex tube is placed on the outside of the sample cartilage column, with the lower edge of the cartilage column flush with the lower edge of the latex tube, and the upper edge of the cartilage column 10 mm lower than the upper edge of the latex tube; to ensure good sealing between the outside of the cartilage column and the latex tube: add colored dye from the top of the cartilage column and let it stand for 20 minutes, then observe the cartilage and the latex tube. If there is residual dye on the surface of the cartilage and no dye on the side, then the sealing between the cartilage column and the latex tube is good. The cartilage column fitted with a latex tube was placed in a 12-well plate. A mixture of chymotrypsin and collagenase D was dropped onto the cartilage surface from above the latex tube, with the amount of liquid added enough to fill the latex tube. The concentration of chymotrypsin after mixing was 0.01 mg / ml, and the concentration of collagenase D was 0.5 mg / ml. The reaction time was 20 minutes. (3) Indicator detection of the model: Histological staining of the cartilage column showed that the superficial surface tissue structure of the cartilage was destroyed by safranin and HE staining, presenting a rough-like change, and the damage range did not exceed the parallel SFZ structure. Sirius red staining showed that the originally complete and continuous collagen fibers had a moth-eaten change, and the collagen fibers in the SFZ were partially interrupted; Indian ink stained the cartilage surface. Microscopic observation revealed obvious traces of cartilage damage. In three-dimensional images, the surface of normal cartilage was smooth, while the surface of enzymatically hydrolyzed cartilage gradually became more undulating, forming an appearance similar to "corrosion". The tissue structure became blurred, and its roughness information also increased. Mechanical property testing: Nanoindentation testing showed that the Young's modulus of the cartilage surface in the isolated articular cartilage superficial layer injury model was 232.56±88.24 kPa.

[0009] Furthermore, in step (1), the diameter of the cartilage hollow drill is 8.5 mm, the length of the cartilage column is 5 mm, and the inner diameter of the latex tube is 8 mm and the length is 15 mm.

[0010] Furthermore, in step (2), the colored dyes are safranin, HE staining, Sirius red, and Indian ink. The isolated knee cartilage is knee cartilage from an adult Landrace pig, an adult cow, or a goat.

[0011] The model was tested for indicators: Histological staining of the cartilage column showed that the superficial surface tissue structure of the cartilage was destroyed and roughened by safranin and HE staining. The damage range did not exceed the parallel SFZ structure. Sirius red staining showed that the originally intact and continuous collagen fibers had worm-eaten changes and the collagen fibers in the SFZ were partially interrupted. Indian ink stained the cartilage surface. Microscopic observation revealed that, when the damaged area was magnified 400 times, compared with the control group, the enzymatically hydrolyzed cartilage showed obvious signs of damage. Three-dimensional images showed that the surface of normal cartilage was smooth, while the surface of the enzymatically hydrolyzed cartilage gradually became more undulating, forming an appearance similar to "corrosion," and the tissue structure became blurred. Simultaneously, the roughness information also increased after enzymatic hydrolysis. Mechanical property testing: Nanoindentation testing showed that the average Young's modulus of normal cartilage was 586.61±41.77 kPa; the Young's modulus of the cartilage surface after enzymatic hydrolysis was 232.56±88.24 kPa, and the difference was statistically significant.

[0012] The method described in this invention precisely disrupts the proteoglycans and collagen fibers in SFZ (fiber fibrous tissue). Histological staining, microscopic observation, and mechanical testing results all show that this method can cause structural damage and decreased mechanical properties of SFZ without affecting deeper tissues. This model has the advantages of simple operation, low cost, and controllable damage range, providing a reliable experimental tool for the study of early pathological mechanisms of osteoarthritis and the exploration of related diagnostic and treatment methods. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of articular cartilage and the superficial septum (SFZ). In the diagram: the left image shows a schematic diagram of articular cartilage and the SFZ; the right image shows porcine cartilage stained with hematoxylin and eosin (HE). Figure 2 It is an osteochondral column; in the figure: A is an osteochondral hollow drill, B is an osteochondral column; Figure 3 To seal around the cartilage, in the diagram: A is a rubber tube, B is a rubber tube fitted onto the osteocartilage column, with staining reagent dripped in from above, and C is the dye that exists only on the surface of the cartilage in contact and does not seep into the surrounding area. Figure 4 This is an enzymatic digestion process; Figure 5 For histological staining, in the figure: A is safranin-fix green, B is hematoxylin and eosin (HE) staining, and C is Sirius red staining; Figure 6 The image shows the surface morphology of cartilage; in the figure: A is stained with Indian ink, B is taken with super depth of field, and C is roughness statistics. Figure 7 For the mechanical characterization of cartilage surface, in the figure: A is nanoindentation, B is Young's modulus; Figure 8The results of the enzymatic hydrolysis experiment on porcine knee cartilage using chymotrypsin alone are shown in the figure. In the figure: A represents a chymotrypsin concentration of 0.001 mg / ml; B represents a chymotrypsin concentration of 0.01 mg / ml; C represents a chymotrypsin concentration of 0.1 mg / ml. Figure 9 The results of an enzymatic hydrolysis experiment using collagenase D alone on porcine knee cartilage are shown in the figure. In the figure: A represents a collagenase D concentration of 0.1 mg / ml; B represents a collagenase D concentration of 0.5 mg / ml. Figure 10 The image shows the results of safranin staining of cartilage after enzymatic hydrolysis of the cartilage surface using a compound enzyme. Figure 11 The results of safranin staining after enzymatic hydrolysis of porcine knee joint cartilage using collagenase III; Figure 12 The results of safranin staining after enzymatic hydrolysis of porcine knee joint cartilage using chondroitinase; Figure 13 The results of the enzymatic hydrolysis of cartilage were obtained by comparing adult cattle and goats with pig cartilage, using chymotrypsin at a concentration of 0.01 mg / ml and collagenase D at a concentration of 0.5 mg / ml. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise defined, 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 pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0016] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0018] I. Materials: Hollow cartilage drill: Smith & Nephew; Latex tubing: CCID Consulting; Red dye: Solarbio; 12-well plate: Solarbio; HE staining kit: Solarbio; Safranin-Fix-Green staining kit: Solarbio; Sirius Red staining kit: Solarbio; India ink staining kit: Beyotime; Chymotrypsin: GLPBIO; Collagenase D: EpiZyme; High depth-of-field microscope: Keyence; Nanoindentation instrument: Leica.

[0019] II. Methods 1. Sample Collection: Knee joints of adult Landrace pigs (from a local slaughterhouse) were selected. Before extracting cartilage tissue blocks, the knee joint capsule must be ensured to be intact and undamaged, so that the cartilage is in a normal physiological environment before the experiment. The skin, fascia, and joint capsule were opened sequentially according to tissue layers to expose the cartilage. The condition of the cartilage was observed, and specimens with a bright color, smooth surface, and no signs of damage were selected. An 8.5mm diameter bone-cartilage hollow drill was used. Figure 2 A) Remove the cartilage column from the femoral trochlea site ( Figure 2 B), 5 mm in length and 2 mm in cartilage thickness, and placed in PBS solution for later use.

[0020] 2. Enzymatic hydrolysis method: Prepare a latex tube with an inner diameter of 8 mm and a length of 15 mm and attach it to the outside of the osteocartilage column. Figure 3 A), the lower edge of the cartilage column is flush with the lower edge of the latex tubing, leaving a 10mm height space above the cartilage surface. Add red dye from above and let stand for 20 minutes. Figure 3 B), then observe whether the dye leaks into the gap between the cartilage and the latex tubules. After 20 minutes, discard the dye. If only dye remains on the surface of the cartilage and not on the sides, then the seal between the osteocartilage and the latex tubules is good. Figure 3 C).

[0021] The osteocartilage column fitted with a latex tube was placed in a 12-well plate, and different enzymes were added from above the latex tube to ensure that they only digested the surface tissue of the cartilage.

[0022] (1) Comparative experiment a. Enzymatic hydrolysis of porcine knee cartilage using chymotrypsin alone: ​​The cartilage surface was hydrolyzed using chymotrypsin at concentrations of 0.001 mg / ml, 0.01 mg / ml, and 0.1 mg / ml, respectively, for 10 min, 20 min, and 30 min, respectively. The hydrolyzed cartilage was then sectioned in paraffin and stained with safranin.

[0023] The results are as follows Figure 8As shown, the results indicated that when the chymotrypsin concentration was 0.001 mg / ml, there was no significant change in safranin staining of the cartilage at the three time points. That is, at this concentration, chymotrypsin cannot enzymatically digest the superficial layer of cartilage (…). Figure 8 A). The enzymatic hydrolysis time will not be increased further, as increased cost is not the objective of this invention.

[0024] When the concentration of chymotrypsin was 0.01 mg / ml, after 30 minutes of safranin staining, the lighter-colored areas were in the superficial layer of cartilage. However, this only indicates that the polysaccharide components in the superficial layer of cartilage had been enzymatically degraded. The surface was still relatively smooth compared to normal cartilage, and the structure was not damaged, which does not conform to the development pattern of OA. Figure 8 B).

[0025] When the concentration of chymotrypsin was 0.1 mg / ml, after 10 minutes of safranin staining, the area that became lighter in color extended beyond the superficial layer of the cartilage. Figure 8 C). The depth of enzymatic hydrolysis cannot be controlled by shortening the time, because the shorter the time, the more difficult it is to control, which is not conducive to large-scale cartilage modeling.

[0026] b. Enzymatic hydrolysis of porcine knee cartilage using collagenase D alone: ​​Collagenase D at concentrations of 0.1 mg / ml and 0.5 mg / ml was used to hydrolyze the cartilage surface, with hydrolysis times of 10 min, 20 min, and 30 min for each concentration. The hydrolyzed cartilage was then sectioned in paraffin and stained with safranin.

[0027] The results are as follows Figure 9 As shown, the results indicated that when the collagenase D concentration was 0.1 mg / ml, safranin staining revealed that the cartilage surface became rougher and the continuity was interrupted compared to normal cartilage. At a concentration of 0.5 mg / ml, the roughness of the cartilage surface was even greater, indicating more severe damage; however, no polysaccharide degradation was observed when collagenase D was used alone.

[0028] Considering the simultaneous loss of glycosaminoglycans and collagen degradation during the development of osteoarthritis (OA), and based on the above findings, a combined application of chymotrypsin and collagenase D was chosen. Specifically, the concentration of chymotrypsin was selected as 0.01 mg / ml, and the concentration of collagenase D as 0.5 mg / ml (the concentration after mixing the two enzymes). The combined enzyme was used to enzymatically hydrolyze the cartilage surface for 1 min, 10 min, 20 min, and 30 min.

[0029] The cartilage was then stained with safranin. The results are as follows: Figure 10As shown, the results indicated that safranin staining revealed the loss of polysaccharides on the cartilage surface at 20 min, and the surface became rougher rather than smooth. Furthermore, the damage was limited to the superficial layer of the cartilage. At 1 min and 10 min, the damage was less severe, while at 30 min, the damage extended beyond the superficial layer of the cartilage.

[0030] c. Collagenase III was used to enzymatically hydrolyze porcine knee joint cartilage at a concentration of 0.5 g / ml for 20 minutes. After hydrolysis, safranin staining was performed, and the results are as follows: Figure 11 As shown, the results indicate that superficial cartilage damage is not obvious. Compared to normal, undigested cartilage, no structural damage was observed on its surface; only the staining was slightly lighter than that of normal cartilage. Figure 11 ).

[0031] d. Chondroitinase was used to enzymatically hydrolyze porcine knee joint cartilage at a concentration of 1.0 U / ml for 20 min. Safranin staining was performed after hydrolysis, and the results are shown below. Figure 12 As shown, the superficial glycosaminoglycans of the cartilage were significantly lost. Compared to normal, undigested cartilage, its surface was relatively smooth, and no structural damage was observed. Figure 12 ).

[0032] e. Select a mixture of chymotrypsin (GLPBIO, California, USA) and collagenase D (EpiZyme, Shanghai, China). After mixing, the concentration of chymotrypsin is 0.01 mg / ml and the concentration of collagenase D is 0.5 mg / ml. Then, add it dropwise to the cartilage surface. Figure 4 The effect lasts for 20 minutes.

[0033] 3. Modeling criteria for cartilage SFZ damage: Cartilage SFZ is characterized by parallel arrangement of collagen and cells. Therefore, in this invention, the modeling criteria are that structural damage and component loss are limited to the SFZ.

[0034] 4. Histological staining after enzymatic digestion confirmed the model damage in SFZ (historical staining is the gold standard, providing a direct visual assessment of the degree and extent of cartilage damage): Safranin staining, HE staining, and Sirius red staining were used. Safranin staining primarily stains glycosaminoglycans in cartilage, with staining depth representing glycosaminoglycan content. HE staining primarily stains collagen in cartilage, with staining depth representing collagen content. Sirius red staining of collagen allows for a direct visual observation of collagen continuity. Normal cartilage (soaked in PBS) showed a smooth surface in Safranin and HE staining, with relatively consistent staining levels in the superficial and intermediate-deep layers. Cartilage cells arranged parallel to the surface could also be observed. Enzymatic digestion of cartilage revealed destruction of the superficial cartilage surface structure in Safranin and HE staining; the surface was no longer smooth but exhibited a rough appearance, indicating structural damage to the superficial cartilage. Furthermore, the superficial cartilage stained lighter than the intermediate-deep layers after enzymatic digestion, indicating the loss of polysaccharide components in the superficial cartilage after digestion. Most importantly, the damage did not extend beyond the parallel SFZ structures. Figure 5 A, B); Sirius red staining of normal cartilage shows good collagen continuity in the superficial layer, but after enzymatic hydrolysis, the originally intact and continuous collagen fibers show worm-eaten changes, and the collagen fibers in SFZ are partially interrupted ( Figure 5 C).

[0035] 5. Macroscopic observation after enzymatic hydrolysis: Indian ink staining: Indian ink cannot stain on smooth, undamaged cartilage surfaces. However, when the cartilage is damaged, Indian ink can stain, and the larger and deeper the damage, the deeper the staining. Compared to the control group of normal cartilage, Indian ink stained the cartilage surface after enzymatic hydrolysis (…). Figure 6 A).

[0036] 6. Submicroscopic Surface Morphology of the SFZ Damage Model: The surface morphology of normal and enzymatically hydrolyzed cartilage was scanned using the unique embossing mode of a super-depth-of-field microscope. This scanning mode clearly displays the smoothness of the cartilage surface. After magnifying the enzymatically hydrolyzed cartilage surface to 400x, compared with the smooth and uniform surface of the control group, the enzymatically hydrolyzed cartilage showed obvious damage marks, exhibiting a groove-like appearance. Three-dimensional reconstruction of the scanned cartilage surface was performed. The more blue the color of the reconstructed image, the smoother it is; the more yellow, the greater the roughness. The three-dimensional image shows that the normal cartilage surface is smooth and mostly blue, while the reconstructed surface of the enzymatically hydrolyzed cartilage is yellow, indicating a gradual increase in surface undulation. Figure 6 B), forming an appearance similar to "corrosion," with blurred tissue structure. Simultaneously, statistical analysis was performed on the cartilage surface roughness, selecting at least 15 points on the cartilage surface and calculating their height differences (the height difference of the cartilage surface represents its roughness). The roughness information for each point was then compiled into a bar chart. Figure 6C), statistical difference analysis revealed that the roughness information of the cartilage surface also increased after enzymatic hydrolysis treatment. P < 0.05.

[0037] 7. Changes in the mechanical properties of cartilage SFZ after enzymatic hydrolysis: To assess the role of the superficial cartilage layer in maintaining the mechanical properties of cartilage tissue, we used nanoindentation to detect the Young's modulus of the cartilage surface. Nanoindentation showed that the average Young's modulus of normal cartilage was 586.61 ± 41.77 kPa; the Young's modulus of the enzymatically hydrolyzed cartilage surface was 232.56 ± 88.24 kPa. Statistical analysis of the nanoindentation data revealed that the mechanical properties of the cartilage surface decreased after enzymatic hydrolysis, and the difference was statistically significant. Figure 7 A, B) P < 0.05.

[0038] III. To extend the method of constructing superficial cartilage lesions using enzymes to other species, knee cartilage from adult cattle and goats was used as a sample for comparison with porcine cartilage. The cartilage was enzymatically hydrolyzed at a concentration of 0.01 mg / ml for chymotrypsin and 0.5 mg / ml for collagenase D.

[0039] The results are as follows Figure 13 As shown, the results indicated that after enzymatic hydrolysis, the superficial layer of cartilage in cattle and sheep showed lighter staining, loss of glycosaminoglycans, and a rougher surface, indicating damage to the cartilage structure. This suggests that this enzymatic hydrolysis method is suitable for multiple species, including large animals. Figure 13 ).

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an isolated articular cartilage superficial layer injury model, characterized in that: A mixture of 0.01 mg / ml chymotrypsin and 0.5 mg / ml collagenase D was used as a digestive enzyme to treat the surface of isolated articular cartilage for 20 min, precisely destroying the proteoglycans and collagen fibers in SFZ and constructing a superficial injury model of isolated articular cartilage. The specific steps are as follows: (1) Sample collection: Select isolated knee joint cartilage. Before extracting the cartilage tissue block, ensure that the knee joint capsule is intact and undamaged so that the cartilage is in a normal physiological environment before the experiment. Open the skin, fascia and joint capsule in sequence according to the tissue layers to expose the cartilage. Select specimens with bright color, smooth surface and no damage marks on the cartilage surface. Use a bone cartilage hollow drill to remove the cartilage column from the trochlea of ​​the femur and place it in PBS solution for later use. (2) Enzymatic hydrolysis of cartilage column: The latex tube is placed on the outside of the sample cartilage column, with the lower edge of the cartilage column flush with the lower edge of the latex tube, and the upper edge of the cartilage column 10 mm lower than the upper edge of the latex tube; to ensure good sealing between the outside of the cartilage column and the latex tube: add colored dye from the top of the cartilage column and let it stand for 20 minutes, then observe the cartilage and the latex tube. If there is residual dye on the surface of the cartilage and no dye on the side, then the sealing between the cartilage column and the latex tube is good. The cartilage column fitted with a latex tube was placed in a 12-well plate. A mixture of chymotrypsin and collagenase D was dropped onto the cartilage surface from above the latex tube, with the amount of liquid added enough to fill the latex tube. The concentration of chymotrypsin after mixing was 0.01 mg / ml, and the concentration of collagenase D was 0.5 mg / ml. The reaction time was 20 minutes. (3) Indicator detection of the model: Histological staining of the cartilage column showed that the superficial surface tissue structure of the cartilage was destroyed by safranin and HE staining, presenting a rough-like change, and the damage range did not exceed the parallel SFZ structure. Sirius red staining showed that the originally complete and continuous collagen fibers had a moth-eaten change, and the collagen fibers in the SFZ were partially interrupted; Indian ink stained the cartilage surface. Microscopic observation revealed obvious traces of cartilage damage. In three-dimensional images, the surface of normal cartilage was smooth, while the surface of enzymatically hydrolyzed cartilage gradually became more undulating, forming an appearance similar to "corrosion". The tissue structure became blurred, and its roughness information also increased. Mechanical property testing: Nanoindentation testing showed that the Young's modulus of the cartilage surface in the isolated articular cartilage superficial layer injury model was 232.56±88.24 kPa.

2. The method for constructing an isolated articular cartilage superficial layer injury model according to claim 1, characterized in that: In step (1), the diameter of the cartilage hollow drill is 8.5 mm, the length of the cartilage column is 5 mm, and the inner diameter of the latex tube is 8 mm and the length is 15 mm.

3. The method for constructing an isolated articular cartilage superficial layer injury model according to claim 1, characterized in that: In step (2), the colored dyes are safranin dye, HE dye, Sirius red, and Indian ink.

4. The method for constructing an isolated articular cartilage superficial layer injury model according to claim 1, characterized in that: The isolated knee cartilage is knee cartilage from an adult Landrace pig, or knee cartilage from an adult cow or goat.

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