Construction method of in vitro articular cartilage injury model and application thereof

Abstract

The application discloses a kind of construction methods of in vitro joint cartilage injury model and its application, belong to biomedical experimental model technical field.Experiment sample is taken to adult long white pig knee joint cartilage, after sampling, washing is placed in vitro culture system and is pre-cultured, then using interleukin-1 beta (IL-1 beta) is induced to handle, and the in vitro model that simulates the pathological characteristics of human early cartilage injury and osteoarthritis is constructed.The application determines the induction concentration and induction length of IL-1 beta, and establishes a five-dimensional model verification system.Compared with the existing in vitro joint cartilage injury model, the model has high pathological simulation degree, strong standardization, good repeatability, low cost, small ethical risk, and covers a wide range of applications, such as drugs, health products, functional foods, natural product extracts and other types of cartilage protection active substances screening, and can be used for mechanism research of cartilage sub-health state and osteoarthritis, with wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental model technology, specifically to a method for constructing an isolated articular cartilage injury model and its application. Background Technology

[0002] Articular cartilage is a transparent cartilaginous tissue covering the ends of joint bones, playing a vital role in transmitting loads, cushioning impacts, and reducing joint friction. Because articular cartilage lacks blood vessels, lymphatic vessels, and nerves, its self-regenerative and repair capabilities are extremely poor, and once damaged, it is prone to continuous progression. The superficial cartilage layer (SFZ) is the first line of defense against external damage. Cartilage degeneration and early pathological changes in osteoarthritis (OA) are concentrated in the superficial layer, accompanied by a series of changes such as matrix metabolic disorders, collagen fiber and proteoglycan degradation, and chondrocyte damage. With the increasing aging population and sports injuries, the incidence of cartilage damage and osteoarthritis is rising year by year and showing a trend towards affecting younger people. Developing cartilage-protective products and elucidating the damage mechanisms have become hot research topics in the industry, and standardized, pathologically accurate damage models are the core foundation for conducting such research.

[0003] Currently, the mainstream cartilage injury models in the industry are mainly divided into four categories, each with obvious shortcomings: Enzymatic hydrolysis is a classic method for constructing superficial cartilage injury models in vitro. The principle involves using exogenous digestive enzymes such as chymotrypsin, collagenase, and agglutinase to selectively degrade matrix components such as proteoglycans and collagen fibers in the superficial cartilage layer, causing damage to the cartilage surface structure in a short time. It is characterized by its simple operation and rapid modeling, and is widely used in in vitro cartilage injury research. For example, the non-patent literature "The Influence of Removing the Superficial Cartilage Region on the Morphology and Mechanical Behavior of Cartilage Surface [J]" (Wang Yabo, Liu Jie, Gao Lilan, et al. Journal of Biomedical Engineering, 2024, 41(2):328-335. (PMC: PMC11058486)) used chymotrypsin to treat porcine articular cartilage to construct a superficial cartilage injury model, confirming that enzymatic hydrolysis can rapidly destroy the cartilage surface structure. However, this injury model is an immediate injury model and cannot simulate the progressive injury process mediated by inflammatory factors in vivo. It lacks an inflammatory microenvironment, cannot reflect chondrocyte functional disorders, and is not suitable for evaluating the sub-health status of cartilage.

[0004] Mechanical impact models apply directional mechanical forces to cartilage using drop impact devices and mechanical loading equipment to simulate acute cartilage damage caused by sports injuries and external impacts. However, due to the difficulty in accurately controlling the impact force parameters, the degree of damage varies greatly among different samples, the model has poor repeatability and cannot quantitatively evaluate the loss of matrix components.

[0005] Animal in vivo models are the traditional mainstream models for basic research on osteoarthritis. Commonly used modeling methods include rat / mouse anterior cruciate ligament transection (ACLT), Hulth meniscus instability surgery, and rabbit intra-articular drug injection, which can simulate the complex humoral, immune, and tissue microenvironment in vivo. For example, the non-patent literature "Tougu Xiaotong Capsules Regulate Nav1.7 to Reduce Chondrocyte Degeneration in Mice with Knee Osteoarthritis [J]" (Fu Changlong, Lin Yanming, et al. Journal of Southern Medical University, 2024, 44(11):2074-2081) uses the mouse Hulth method to construct an OA model. Animal models can simulate the cartilage degeneration process under complex in vivo environments, but they have the following problems: the modeling period is long, usually requiring 2-3 months or even longer; the modeling rate is unstable, affected by factors such as surgical skills and individual animal differences; surgery involving live animals requires animal ethics review, which does not meet the requirements of the 3R principle (reduce, optimize, replace animal experiments); and a large amount of money is required for animal purchase, feeding, surgery, anesthesia, and postoperative care, resulting in high time and economic costs.

[0006] Chondrocyte models are in vitro monolayer cell culture systems that stimulate chondrocytes with inflammatory factors such as IL-1β and TNF-α to simulate inflammatory damage. They are often used for molecular mechanism analysis and drug screening, offering advantages such as ease of operation, high throughput, and short cycle time. However, chondrocytes undergo dedifferentiation during in vitro culture, losing their original phenotypic characteristics; monolayer culture cannot simulate the unique three-dimensional microenvironment and cell-matrix interactions of cartilage tissue; it cannot reflect the hierarchical structure of cartilage tissue (superficial, intermediate, and deep layers) and its differentiated responses to external stimuli; and it is difficult to evaluate bioactive substances that need to penetrate the matrix to exert their effects.

[0007] In addition to the inherent defects of the aforementioned models, existing technologies also have common problems: First, there is a lack of unified and standardized modeling schemes, and the experimental conditions of different laboratories vary greatly, resulting in poor data comparability; second, the application scenarios are narrow, with most models only targeting the development of osteoarthritis drugs and rarely used for the evaluation of cartilage sub-health, efficacy verification of health products and functional foods; third, although there is relevant academic research on IL-1β-induced in vitro models, the parameters of induction concentration and culture time vary greatly, there is no optimal parameter combination, and no systematic protection scheme has been formed.

[0008] Meanwhile, existing cell evaluation models are two-dimensional evaluation systems, which cannot accurately reflect the actual pathological process of human cartilage damage, resulting in low verification dimensions for drug efficacy evaluation.

[0009] Therefore, developing an ex vivo articular cartilage injury model with high pathological simulation, standardization, repeatability, low cost, ethical friendliness, and wide applicability is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0010] To address the numerous shortcomings of existing technologies, this invention provides a method for constructing an isolated articular cartilage injury model and its application. Using adult Landrace pig knee cartilage as a standardized sample, this invention employs interleukin-1β (IL-1β) inflammatory factors to induce a progressive injury model, identifies optimal modeling parameters, establishes a multi-dimensional validation system, and ensures the model closely reflects the actual pathological process of human cartilage injury. Furthermore, it expands application scenarios, meeting the evaluation needs of various fields such as mechanism research, pharmaceuticals, health products, functional foods, and natural product extracts.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] The term used in this article refers to the superficial cartilage zone (SFZ), the outermost layer of articular cartilage. It serves as the first line of defense against external mechanical damage and inflammatory stimuli, playing a dominant role in surface lubrication and mechanical signal transduction, and is the initial site of early cartilage degeneration. Interleukin-1β (IL-1β) is a classic pro-inflammatory cytokine and a core mediator of osteoarthritis. It can induce chondrocyte apoptosis, increase matrix metalloproteinase expression, and accelerate the degradation of chondrocyte proteoglycans and collagen.

[0016] This invention is achieved through the following technical solutions: The first aspect of this invention relates to a method for constructing an isolated articular cartilage injury model, comprising the following steps: (1) Cartilage sample collection and pretreatment: Select cartilage blocks from the knee joints of adult pigs, rinse them with buffer solution and set them aside for use; (2) In vitro pre-culture: Cartilage blocks were placed in DMEM high-glucose medium with added compound additives and pre-cultured under constant temperature, constant humidity and constant carbon dioxide concentration conditions; (3) Induced culture of inflammatory factors: Interleukin-1β recombinant protein was added to the pre-cultured system in step (2) for induced culture to obtain an isolated articular cartilage injury model; wherein, the final concentration of interleukin-1β in the culture medium was 15-40 ng / mL, and the induction culture time was 5-10 days. A schematic diagram of the isolated cartilage culture plate is shown below. Figure 1 As shown.

[0017] In some embodiments of the present invention, the final concentration of interleukin-1β in the culture medium in step (3) is 15-30 ng / mL, preferably 15 ng / mL.

[0018] In some embodiments of the present invention, the induction culture time in step (3) is 5-8 days, preferably 7 days.

[0019] In some embodiments of the present invention, the adult pig in step (1) is an adult Landrace pig, and the sampling site is the femoral trochlea of ​​the knee joint.

[0020] In some embodiments of the present invention, the rinsing with buffer in step (1) specifically involves rinsing twice with 4°C HBSS buffer and three times with PBS buffer.

[0021] In some embodiments of the present invention, the culture medium in step (2) contains insulin-transferrin-sodium selenate (ITS, 100×), penicillin, streptomycin, gentamicin sulfate, and amphotericin B.

[0022] In some embodiments of the present invention, the ratio of the mass of the cartilage block to the volume of the culture medium in step (2) is 0.05-0.15 g: 1 mL.

[0023] In some embodiments of the present invention, the culture conditions for the pre-culture in step (2) are: temperature 37°C, carbon dioxide volume fraction 5%, ambient humidity 90%, and the culture medium is changed every 3 days.

[0024] In some embodiments of the present invention, the pre-culture time in step (2) is 5-8 days, preferably 7 days.

[0025] In some embodiments of the present invention, after the inflammatory factor induction culture in step (3) is completed, step (4) model verification is also included.

[0026] In some embodiments of the present invention, the model validation employs a five-dimensional validation system to test the constructed model. The five-dimensional validation system includes: LDH relative release detection, S-GAG relative release detection, Safranin O-Fix Green staining detection, H&E staining morphological observation, and scanning electron microscopy ultrastructure observation.

[0027] A second aspect of the invention relates to an isolated articular cartilage injury model constructed by the above method.

[0028] A third aspect of the present invention relates to the application of the isolated articular cartilage injury model constructed by the above method in screening cartilage-protective active substances.

[0029] In some embodiments of the present invention, the cartilage-protecting active substance is selected from at least one of pharmaceutical active ingredients, health product efficacy ingredients, functional food ingredients, and natural product extracts.

[0030] In some embodiments of the present invention, the natural product extract is selected from at least one of plant extracts, marine bioactive substances, and fungal extracts.

[0031] Compared with the prior art, the beneficial effects of the present invention are: (1) High pathological simulation: The present invention uses IL-1β inflammatory factor induction to simulate the progressive and functional damage of cartilage in the in vivo inflammatory microenvironment, which is different from the instantaneous damage of the enzymatic method. It highly restores the real pathological process of human cartilage damage and osteoarthritis, and can simulate the functional decline of cartilage in the sub-healthy state. (2) High standardization and good repeatability: The present invention has screened the optimal parameter combination of 15ng / mL IL-1β and 7 days of induction time through gradient experiments, established a unified modeling process and a five-dimensional verification system, solved the problem of chaotic parameters and incomparable data in traditional induction models, and the experimental results of different batches and different laboratories are highly consistent. (3) Stable sample source, ethically friendly and low cost: The knee joint cartilage of adult Landrace pigs from slaughterhouses is selected, the sample supply is sufficient and the cost is low; there is no need to carry out live animal surgery, avoid animal ethics review, and fully comply with the 3R principle of animal experiments. It has significant advantages over animal in vivo models. (4) Comprehensive and reliable verification system: The first five-dimensional verification system of LDH+S-GAG+Safranin O staining+H&E staining+Scanning electron microscopy is used to evaluate the model from four levels: cell activity, matrix composition, tissue morphology and ultrastructure. The test results are objective and highly reliable. (5) Wide range of applications: Breaking through the limitation of traditional models that are only used for drug development, this model can be applied to disease mechanism research, cartilage sub-health evaluation, activity screening of various products such as drugs, health products, functional foods and natural product extracts. It has rich application scenarios and high market and scientific research value. (6) Simple operation and moderate cycle: The overall experimental cycle is about 14 days (7 days of pre-culture and 7 days of induction period). The equipment requirements are conventional and the operation process is simple. It can achieve high-throughput sample screening and is suitable for routine laboratory operations and industrial screening. Attached Figure Description

[0032] Figure 1 Schematic diagram of a culture plate for isolated cartilage; Figure 2 Bar chart of relative LDH release induced by different concentrations of IL-1β; Figure 3 Bar chart of relative S-GAG release under different concentrations of IL-1β; Figure 4 Bar chart of relative LDH release at different induction durations; Figure 5 : Bar chart of relative intensity of safranin O-fast green staining; Figure 6 : Morphological images of cartilage tissue stained with H&E; Figure 7 Scanning electron microscope images of cartilage surface; Figure 8 The model is used for screening the activity of ingredients in health supplements. Figure 9 The model is used for screening different active monomer components. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0034] The experimental materials used in the following experiments are shown in Table 1.

[0035] Table 1: Experimental Materials

[0036] Example 1: Concentration selection of modeling agent IL-1β A method for constructing an isolated articular cartilage injury model was performed in each experimental group according to the following steps: (1) Cartilage sample collection: The femoral trochlea was fully exposed, and a cartilage block was taken from the femoral trochlea of ​​the knee joint. The wet weight was weighed and recorded as 0.1g. The cartilage block was first soaked in HBSS at 4℃ and rinsed twice, then rinsed three times with PBS and placed in a 24-well plate for culture. (2) In vitro culture: The cartilage was incubated at 37℃ and 5% CO2 for 7 days, with 2mL of culture medium per well and the medium was changed every 3 days. (3) Induction of inflammatory factors: DMEM culture medium containing 2, 5, 10, 15, 20, 30, and 40 ng / mL of IL-1β was added to each model group, and DMEM culture medium without IL-1β was added to the negative control group (NC group). The culture was continued for 7 days. The experiment lasted for 14 days. (4) Sample collection: After the culture is completed, the supernatant of each group is collected for LDH and S-GAG detection; cartilage tissue is collected for histological staining and scanning electron microscopy observation.

[0037] LDH test results are as follows Figure 2 As shown, compared with the NC group, the relative LDH release in the model group (M group) was significantly increased after treatment with IL-1β at concentrations of 15, 20, 30, and 40 ng / mL (P<0.05), indicating that IL-1β successfully induced chondrocyte damage. Among these, the induction effect was better at an IL-1β concentration of 15 ng / mL, with a relatively low standard deviation, suggesting that the induction effect at this concentration was relatively stable. However, there was no statistically significant difference in the relative LDH release between the NC group and the treatment groups with IL-1β concentrations of 2, 5, and 10 ng / mL.

[0038] S-GAG assay was performed according to the S-GAGs kit instructions, measuring absorbance at a wavelength of 656 nm. Results are as follows: Figure 3 As shown in the figure, compared with the NC group, the relative release of S-GAG in the model group (M group) was significantly increased after treatment with IL-1β at concentrations of 15, 20, and 30 ng / mL (P<0.05), indicating that IL-1β successfully induced the degradation of proteoglycans in the cartilage matrix. Among them, the induction effect was best at an IL-1β concentration of 15 ng / mL, suggesting that the induction effect at this concentration was relatively stable.

[0039] Based on the combined detection results of LDH and S-GAGs, this invention determined the treatment concentration of IL-1β to be 15-30 ng / mL. Furthermore, 15 ng / mL was selected as the induction concentration of IL-1β in subsequent experiments.

[0040] Example 2: Screening of Modeling Time A method for constructing an isolated articular cartilage injury model, each experimental group followed these steps: (1) Cartilage sample collection: The femoral trochlea was fully exposed, and a cartilage block was taken from the femoral trochlea of ​​the knee joint. The wet weight was weighed and recorded as 0.1g. The cartilage block was first soaked in HBSS at 4℃ and rinsed twice, then rinsed three times with PBS and placed in a 24-well plate for culture. (2) In vitro culture: Incubated at 37℃ and 5% CO2 for 7 days, with 2mL of culture medium per well, and the medium was changed every 3 days. (3) Induction of inflammatory factors: Each model group was added to DMEM culture medium containing 15ng / mL IL-1β and cultured for 3, 5, 7, 10 and 14 days respectively. The negative control group (NC group) was added to DMEM culture medium without IL-1β and cultured for 7 days. (4) Sample collection: After the culture was completed, the supernatant of each group was collected for LDH detection.

[0041] The results are as follows Figure 4 As shown in the figure, compared with the NC group, there was no significant difference in the relative release of LDH on day 3 of modeling (P>0.05); however, the relative release of LDH was significantly increased on days 5, 7, 10, and 14 of modeling (P<0.05), indicating that IL-1β successfully induced chondrocyte damage. Among them, the relative release of LDH was the highest on day 7 of modeling, and the difference was statistically significant (P<0.001). Therefore, day 7 was selected as the optimal modeling time for subsequent experiments.

[0042] Example 3: Safranin O-Fixed Green Staining (Detection of Matrix Proteoglycans) (Validation of Optimal Conditions) Methods: The model group was cultured in DMEM medium containing 15 ng / mL IL-1β, while the negative control group was cultured in DMEM medium without IL-1β. Cultures were continued for 7 days. Staining was performed according to the instructions of the Safranin O-Fixed Green Cartilage Staining Kit, and the staining intensity was analyzed using ImageJ software.

[0043] The results are as follows Figure 5 As shown in the figure, safranin O staining results indicated that the cartilage matrix in the NC group was uniformly stained, appearing as a deep red color; the superficial cartilage layer in the M group showed significantly lighter staining, with its relative staining intensity decreasing to 54% of that in the NC group, indicating a significant decrease in proteoglycan content. In summary, this suggests that the osteoarthritis cartilage matrix degradation model was successfully established.

[0044] Example 4: H&E staining (morphological observation) (validation of optimal conditions) Methods: The model group was cultured in DMEM medium containing 15 ng / mL IL-1β, while the negative control group was cultured in DMEM medium without IL-1β. Cultures were continued for 7 days. Cartilage tissue was fixed, dehydrated, embedded, sectioned, and then stained with H&E.

[0045] The results are as follows Figure 6 As shown in the figure. Histological observation revealed that the cartilage surface in the NC group was smooth and intact, with superficial cells arranged in a spindle shape and parallel to each other, and the matrix was uniformly stained. In contrast, the cartilage surface in the M group was rough and uneven, with a reduced number and irregular shape of superficial cells, and localized fissures were visible, but the damage was mainly limited to the superficial layer and did not involve the middle and deep layers. These results indicate that IL-1β successfully induced typical damaging changes in the superficial layer of cartilage.

[0046] Example 5: Scanning electron microscopy observation (surface ultrastructure) (optimal condition verification) Methods: The model group was cultured in DMEM medium containing 15 ng / mL IL-1β, while the negative control group was cultured in DMEM medium without IL-1β. Cultures were continued for 7 days. Cartilage tissue was fixed, dehydrated, critically dried, and sputter-coated with gold before being observed under a scanning electron microscope.

[0047] The results are as follows Figure 7 As shown, scanning electron microscopy revealed that the cartilage surface in group NC was smooth, with a tightly packed and continuous collagen fiber network. In contrast, the cartilage surface in group M was uneven, with exposed and broken collagen fiber networks, exhibiting a typical "worm-eaten" appearance. These results indicate that IL-1β induction can lead to the destruction of the ultrastructure of the cartilage surface, consistent with the early pathological characteristics of cartilage injury.

[0048] Example 6: Model used for screening the activity of health supplement ingredients The injury model constructed using this invention was as follows: the model group was cultured in DMEM medium containing 15 ng / mL IL-1β, while the negative control group was cultured in DMEM medium without IL-1β, and cultured for 7 days. The chondroitin protective effect of the glucosamine + chondroitin (mass ratio 2:1) compound health product (referred to as glucosamine in the figure) was evaluated. The results are as follows: Figure 8 As shown.

[0049] Gradual concentrations of the health supplement sample were added to the IL-1β induction system, and LDH release was measured after culture. The results showed that the health supplement significantly reduced cell damage and matrix degradation, demonstrating its chondroprotective activity.

[0050] Example 7: Model used for screening different active substances Puerarin (40 μg / ml), N-chlorotaurine (100 μg / ml), and caffeine (100 μg / ml) were selected as test samples and co-cultured in the in vitro injury model of this invention to detect the effects of different active substances on the LDH release level of the model. The results are as follows: Figure 9 As shown.

[0051] The results showed that puerarin significantly inhibited LDH release in the isolated articular cartilage injury model, with a statistically significant difference compared to the model group. N-chlorotaurine and caffeine, however, had no significant effect on LDH release in the isolated articular cartilage injury model. Therefore, the isolated articular cartilage injury model prepared in this application can be used for screening active substances with therapeutic effects on cartilage injury.

[0052] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for constructing an isolated articular cartilage injury model, characterized in that, Includes the following steps: (1) Cartilage sample collection and pretreatment: Select cartilage blocks from the knee joints of adult pigs, rinse them with buffer solution and set them aside for use; (2) In vitro pre-culture: Cartilage blocks were placed in DMEM high-glucose medium with added compound additives and pre-cultured in the presence of carbon dioxide; (3) Inflammatory factor induction culture: Add recombinant interleukin-1β protein to the system after pre-culture in step (2) for induction culture to obtain an isolated articular cartilage injury model; wherein, the final concentration of interleukin-1β in the culture medium is 15-40 ng / mL, and the induction culture time is 5-10 days.

2. The construction method according to claim 1, characterized in that, The final concentration of interleukin-1β in the culture medium is 15-30 ng / mL, and the induction culture time is 5-8 days.

3. The construction method according to claim 1, characterized in that, In step (1), the adult pig is an adult Landrace pig, and the sampling site is the femoral trochlea of ​​the knee joint.

4. The construction method according to claim 1, characterized in that, In step (1), the rinsing with buffer solution specifically involves rinsing twice with 4℃ HBSS buffer and three times with PBS buffer.

5. The construction method according to claim 1, characterized in that, In step (2), the amount of culture medium added to each well is 2 mL, and the culture medium contains 100× insulin-transferrin-sodium selenate, penicillin, streptomycin, gentamicin sulfate, and amphotericin B; the pre-culture conditions are: temperature 37℃, carbon dioxide volume fraction 5%, ambient humidity 90%, and the culture medium is changed every 3 days.

6. The construction method according to claim 1, characterized in that, The pre-culture time in step (2) is 5-8 days.

7. The construction method according to claim 1, characterized in that, It also includes a model validation step, which uses a five-dimensional validation system to test the constructed model. The five-dimensional validation system includes: LDH relative release detection, S-GAG relative release detection, Safranin O-Fix Green staining detection, H&E staining morphological observation, and scanning electron microscopy ultrastructure observation.

8. The application of the isolated articular cartilage injury model constructed by the construction method according to any one of claims 1-7 in screening cartilage-protective active substances.

9. The application according to claim 8, characterized in that, The cartilage-protecting active substance is selected from at least one of pharmaceutical active ingredients, health product efficacy ingredients, functional food raw materials, and natural product extracts.

10. The application according to claim 9, characterized in that, The natural product extract is selected from at least one of plant extracts, marine bioactive substances, and fungal extracts.

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