阅读说明：本技术 一种组织特异性半月板细胞外基质材料及其制备方法和应用 (Tissue-specific meniscus extracellular matrix material and preparation method and application thereof ) 是由 钟刚 于 2021-10-14 设计创作，主要内容包括：本发明涉及生物医药技术领域,具体涉及一种组织特异性细胞外基质材料及其制备方法以及在保护半月板损伤后的关节损伤和退化中的应用。本发明通过对猪半月板的内侧1/3部分进行脱细胞化制备mECM,该制备方法能够彻底地清除细胞和细胞内的核酸,同时保留细胞外基质的主要成分,有利于维持细胞稳态；该方法所制备的mECM材料兼具了优异的力学性能和生物相容性,不仅能够修复半月板损伤,还能够有效地减少损伤部位纤维化,该mECM可作为支架材料与骨髓间充质干细胞复合,起到保护半月板损伤后的关节损伤和退化的作用,这对组织再生和患者后期生活质量方面具有重大意义。(The invention relates to the technical field of biomedicine, in particular to a tissue-specific extracellular matrix material, a preparation method thereof and application thereof in protecting joint injury and degeneration after meniscus injury. According to the invention, the mECM is prepared by decellularizing the inner 1/3 part of the pig meniscus, and the preparation method can thoroughly eliminate cells and nucleic acid in the cells, and simultaneously retains the main components of extracellular matrix, thereby being beneficial to maintaining the steady state of the cells; the mECM material prepared by the method has excellent mechanical property and biocompatibility, can repair meniscus injury, and can effectively reduce fibrosis of an injury part, and the mECM can be used as a scaffold material to be compounded with bone marrow mesenchymal stem cells to play a role in protecting joint injury and degeneration after meniscus injury, so that the mECM material has great significance in tissue regeneration and later life quality of patients.)

1. A method for preparing a tissue-specific meniscal extracellular matrix material, comprising: the method comprises the following steps:

(1) collecting fresh semilunar plate of 6-7 month-old pig in slaughterhouse, cleaning, taking out inner 1/3 part, carefully cutting into 5-10 mm pieces³Tissue patches of (a);

(2) placing the tissue small blocks in an acetic acid solution with the concentration of 0.02mol/L, and soaking for 48 hours at the temperature of 4 ℃;

(3) freeze-drying the tissue blocks at-80 deg.C for 24 hr, re-warming at room temperature for 4 hr to obtain freeze-dried tissue, and repeating the above steps to obtain three freeze-dried tissues;

(4) grinding the freeze-dried tissues into powder, placing the powder in a mixed system of 2% SDS and 10mmol/L tris, stirring the powder for 24 hours at 25 ℃, removing supernate, and circulating the supernate for three times to obtain a precipitate;

(5) treating the precipitate with 0.1% peracetic acid for 2 hr, centrifuging again to remove supernatant, stirring with sterile water and PBS with aprotinin, washing, and precipitating for 12 hr;

(6) placing the precipitate in PBS, adjusting the pH value of the solution to 8-8.5 by using 2mol/L NaOH, stirring for 6 hours at 4 ℃, and then adjusting the pH value of the solution to 2-2.5 by using 2mol/L HCl;

(7) centrifuging the solution with the adjusted pH value by a refrigerated centrifuge, and removing supernatant;

(8) putting the precipitate into a dialysis bag, dialyzing in distilled water, changing water every day, oscillating at low temperature until pH is close to neutral, then conducting for less than 30 μ s, centrifuging by a refrigerated centrifuge, collecting precipitate, and pouring out waste liquid;

(9) dissolving the pellet in PBS, treating with 200U/mL DNase and 50U/mL RNase solutions at 37 deg.C for 24 hours to remove nucleic acids from the meniscal extracellular matrix, and then washing with PBS repeatedly;

(10) centrifuging the washed solution by a refrigerated centrifuge, collecting precipitate, wherein the obtained precipitate is a meniscus extracellular matrix material, freeze-drying the meniscus extracellular matrix material, and storing the meniscus extracellular matrix material at-20 ℃ for later use.

2. The method of claim 1, wherein: in step (1), the menisci need to be harvested within 12 hours after slaughter.

3. The method of claim 1, wherein: in step (7), the centrifugation conditions were 4 ℃ and 9000rpm for 45 min.

4. The method of claim 1, wherein: in the step (8), the centrifugation conditions were 4 ℃ and 9000rpm for 40 min.

5. The method of claim 1, wherein: in the step (10), the centrifugation conditions were 4 ℃ and 9000rpm for 45 min.

6. A tissue-specific meniscal extracellular matrix material, comprising: the meniscal extracellular matrix material is produced by the production method according to any one of claims 1 to 5.

7. Use of a meniscal extracellular matrix material of claim 6 in the preparation of a composite material for the protection of joint damage and degeneration following meniscal injury.

8. A composite material for protecting joints from damage and degeneration following meniscal injury, characterized by: the composite material is compounded by the meniscal extracellular matrix material of claim 6 and bone marrow mesenchymal stem cells.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a tissue-specific meniscus extracellular matrix material, a preparation method thereof and application thereof in protecting joint damage and degeneration after meniscus damage.

Background

The meniscus is a C-shaped tissue which is positioned between a femoral condyle and a tibial plateau and is formed by elastic cartilages in the knee joint, and the meniscus has the main function of buffering mechanical pressure between the tibia and the femur in the movement process and avoiding damage to the articular cartilage caused by direct mechanical impact. With increased outdoor activity, the incidence of meniscal damage is increasing. After meniscus injury, the protective function of the meniscus to joints is weakened, so that the damage of soft tissues of the knee, such as cruciate ligament injury, joint capsule injury, cartilage surface injury and the like, is frequently complicated, and clinical symptoms of severe pain of the knee joint, incapability of automatically straightening, joint swelling and the like are shown.

At present, meniscus suture is mostly adopted as a clinical treatment mode of meniscus injury, and the arthroscopic follow-up evaluation is carried out on the treatment mode by Henning, and the results show that about 40 percent of cases have the condition of no healing or incomplete healing. Meniscal resections are another important way to clinically treat meniscal injuries, but often after meniscal row resections, articular cartilage degeneration occurs, eventually leading to osteoarthritis. As can be seen, there is currently no ideal clinical treatment for meniscal damage.

With the development of tissue engineering, a new hope is brought to meniscus injury repair, and a new treatment mode is provided for meniscus repair. Three-dimensional scaffold materials have been widely regarded as important components of tissue engineering. Because mechanical stress in the process of buffer motion of the meniscus needs very strong mechanical strength and the traditional natural materials are difficult to achieve the condition, the current scaffold material for meniscus tissue engineering repair mainly adopts artificially synthesized materials, and the biocompatibility of the artificially synthesized materials is poor, so that the application of the scaffold material in meniscus repair is greatly limited. More importantly, current scaffold materials and meniscal repair strategies ignore the limitations on the overall function of the joint, particularly the degradation of articular cartilage, which is extremely important for tissue regeneration and later quality of life of the patient.

Disclosure of Invention

One of the objectives of the present invention is to provide a tissue-specific meniscus extracellular matrix material with both mechanical properties and biocompatibility of meniscus and a preparation method thereof, wherein the prepared meniscus extracellular matrix material can protect the damaged and degenerated joints of meniscus.

Another object of the present invention is to provide a use of a meniscus extracellular matrix material for the preparation of a composite material for protecting joint damage and degeneration after meniscus damage.

The purpose of the invention is realized by the following technical scheme:

the invention provides a preparation method of a tissue-specific meniscus extracellular matrix material, which comprises the following steps:

(1) collecting fresh semilunar plate of 6-7 month-old pig in slaughterhouse, cleaning, taking out inner 1/3 part, carefully cutting into 5-10 mm pieces³Tissue patches of (a);

(2) placing the tissue small blocks in an acetic acid solution with the concentration of 0.02mol/L, and soaking for 48 hours at the temperature of 4 ℃;

(3) freeze-drying the tissue blocks at-80 deg.C for 24 hr, re-warming at room temperature for 4 hr to obtain freeze-dried tissue, and repeating the above steps to obtain three freeze-dried tissues;

(4) grinding the freeze-dried tissues into powder, placing the powder in a mixed system of 2% SDS and 10mmol/L tris, stirring the powder for 24 hours at 25 ℃, removing supernate, and circulating the supernate for three times to obtain a precipitate;

(5) treating the precipitate with 0.1% peracetic acid for 2 hr, centrifuging again to remove supernatant, stirring with sterile water and PBS with aprotinin, washing, and precipitating for 12 hr;

(6) placing the precipitate in PBS, adjusting the pH value of the solution to 8-8.5 by using 2mol/L NaOH, stirring for 6 hours at 4 ℃, and then adjusting the pH value of the solution to 2-2.5 by using 2mol/L HCl;

(7) centrifuging the solution with the adjusted pH value by a refrigerated centrifuge, and removing supernatant;

(8) putting the precipitate into a dialysis bag (molecular weight 8000-;

(9) dissolving the pellet in PBS, treating with 200U/mL DNase and 50U/mL RNase solutions at 37 deg.C for 24 hours to remove nucleic acids from the meniscal extracellular matrix, and then washing with PBS repeatedly;

(10) centrifuging the washed solution by a refrigerated centrifuge, collecting precipitate, wherein the obtained precipitate is a meniscus extracellular matrix material, freeze-drying the meniscus extracellular matrix material, and storing the meniscus extracellular matrix material at-20 ℃ for later use.

Preferably, in step (1), the meniscus is acquired within 12 hours after slaughter.

Preferably, in step (7), the centrifugation conditions are 4 ℃, 9000rpm, 45 min.

Preferably, in step (8), the centrifugation conditions are 4 ℃, 9000rpm, 40 min.

Preferably, in step (10), the centrifugation conditions are 4 ℃, 9000rpm, 45 min.

The invention also provides a tissue-specific meniscus extracellular matrix material, which is prepared by the preparation method.

The meniscus is a very dense connective tissue, complete decellularization is difficult to achieve by the conventional method for removing the extracellular matrix, and the method for removing the extracellular matrix from the meniscus can completely remove cells and intracellular nucleic acids while retaining main components of the extracellular matrix, such as Collagen (Collagen), hydroxyproline (OHP) and glycosaminoglycans (GAGs), and is beneficial to maintaining cell homeostasis.

The invention also provides application of the meniscus extracellular matrix material in preparing a composite material for protecting joint damage and degeneration after meniscus damage.

The invention also provides a composite material for protecting joint injury and degeneration after meniscus injury, which is compounded by the meniscus extracellular matrix material and bone marrow mesenchymal stem cells.

The invention has the beneficial effects that:

the invention is inspired by the bionics principle, and by adopting the original method for removing the extracellular matrix from the meniscus, compared with the conventional method for removing the extracellular matrix, the preparation method can thoroughly remove cells and nucleic acid in the cells, and simultaneously retains the main components of the extracellular matrix, such as collagen, hydroxyproline and glycosaminoglycan, thereby being beneficial to maintaining the steady state of the cells; experiments prove that the meniscus extracellular matrix (mECM) material prepared by the method has excellent mechanical property and biocompatibility, can repair meniscus injury and can effectively reduce fibrosis of the injured part. Therefore, the mECM can be used as a scaffold material to be compounded with bone marrow mesenchymal stem cells (BMSCs) to play a role in protecting joint damage and degeneration after meniscus damage, and has great significance on tissue regeneration and later life quality of patients.

Drawings

FIG. 1 is a graph showing the experimental effect of decellularization of mECM in example 1.

FIG. 2 is a graph showing the experimental effect of mECM of example 2 on the ability of BMSCs to differentiate into fibrocartilage.

FIG. 3 is a graph showing the experimental effect of mECM in the dorsal rat of example 3 on the ability of BMSCs to differentiate into cartilage.

Fig. 4 is a graph of the experimental effect of the ecm of example 4 in protecting joint degeneration following meniscal injury.

Fig. 5 is a graph of the experimental effect of the ecm of example 4 in protecting osteoarthritis after meniscal injury.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1. preparation of mescm.

1. The meccm was prepared by decellularising the medial 1/3 portion of the fresh porcine meniscus as follows:

(1) collecting fresh half-moon plate of 6-7 month-old pig in slaughterhouse (within 12 hr after slaughtering), cleaning, collecting inner 1/3 part, carefully cutting into 5-10 mm size³Tissue patches of (a);

(2) placing the tissue small blocks in an acetic acid solution with the concentration of 0.02mol/L, and soaking for 48 hours at the temperature of 4 ℃;

(3) freeze-drying the tissue blocks at-80 deg.C for 24 hr, re-warming at room temperature for 4 hr to obtain freeze-dried tissue, and repeating the above steps to obtain three freeze-dried tissues;

(4) grinding the freeze-dried tissues into powder, placing the powder in a mixed system of 2% SDS and 10mmol/l tris, stirring the powder for 24 hours at 25 ℃, removing supernate, and circulating the supernate for three times to obtain a precipitate;

(5) treating the precipitate with 0.1% peracetic acid for 2 hr, centrifuging again to remove supernatant, stirring with sterile water and PBS with aprotinin, washing, and precipitating for 12 hr;

(6) placing the precipitate in PBS, adjusting the pH value of the solution to 8-8.5 by using 2mol/L NaOH, stirring for 6 hours at 4 ℃, and then adjusting the pH value of the solution to 2-2.5 by using 2mol/L HCl;

(7) centrifuging the pH-adjusted solution with a refrigerated centrifuge (9000 rpm, 45min, 4 deg.C), and removing supernatant;

(8) loading the precipitate into dialysis bag (molecular weight 8000-;

(9) dissolving the pellet in PBS, treating with 200U/mL DNase and 50U/mL RNase solutions at 37 ℃ for 24 hours to remove nucleic acids from the mECM, and then repeatedly washing with PBS;

(10) centrifuging the washed solution with a refrigerated centrifuge, collecting precipitate (9000 rpm, 45min, 4 deg.C), and freeze-drying the precipitate to obtain meniscal extracellular matrix (mECM) and storing at-20 deg.C.

2. Verification of decellularization of ecm:

2.1 Experimental methods:

(1) and (3) detecting the DNA content: double stranded DNA content in the ecm and fresh meniscal tissue was measured using PicoGreen dsDNA quantification kit as per the instructions to reflect the effectiveness of the decellularization and DNA clearance of the invention.

(2) Detecting the content of Collagen (Collagen), hydroxyproline (OHP) and glycosaminoglycan (GAGs): the collagen and GAGs content in mECM and fresh menisci tissue is detected by collagen quantitative detection kit, hydroxyproline detection kit and GAGs kit.

(3) Scanning electron microscope: after the mECM and the Cowhide collagen are lyophilized at-80 ℃ for 24 hours, gold spraying is carried out for two minutes, and then the morphology structure of the tissue is photographed by a scanning electron microscope.

2.2 Experimental results:

as shown in fig. 1a, the significant decrease in dsDNA content relative to normal meniscal tissue for the mmecm prepared by the unique decellularization method of the invention, as a nuclear feature, reflects the effectiveness of the decellularization treatment of the invention.

As shown in fig. 1b-d, the mmecm is very close to the Collagen (Collagen), hydroxyproline (OHP) and glycosaminoglycans (GAGs) content of the meniscal tissue, indicating that the decellularization method employed in the present invention has little effect on the main components of the meniscal extracellular matrix while removing cells in the meniscus.

As shown in fig. 1e, by swelling the mesms in acetic acid, with a loose porous structure, interconnected pores ranged from 10 μm to 40 μm, which is thought to facilitate cell infiltration and proliferation.

In conclusion, since the meniscus is a very dense connective tissue, complete decellularization is difficult to achieve by the conventional method for removing the extracellular matrix, the method for removing the extracellular matrix from the meniscus can completely remove cells and nucleic acids in the cells, and simultaneously main components of the extracellular matrix, such as Collagen (Collagen), proline (OHP) and glycosaminoglycan (GAGs), are retained, thereby being beneficial to maintaining cell homeostasis.

Example 2. experiment of the ability of ecm to promote differentiation of BMSCs into fibrocartilage.

In vitro experiments, in this example, BMSCs were mixed with ecm and conventional bovine-derived collagen hydrogel (the most commonly used natural scaffold material, cow collagen) for three-dimensional culture, and BMSCs were examined for proliferation in the ecm and cow collagen, and their ability to induce BMSCs to differentiate into fibrocartilage was further evaluated by tissue staining, qPCR, and Western blotting.

1. The experimental method comprises the following steps:

(1) isolation and culture of BMSCs: three-day-old SD rats were euthanized, soaked in alcohol for two minutes, the hind limbs of the suckling rats were harvested and placed in a solution of penicillin streptomycin for two minutes, and the femoral shaft was harvested by peeling off the muscle tissue in a sterile environment. A one-milliliter syringe needle (22 gauge needle) containing media was inserted into the femoral shaft, and BMSCs in the femur were flushed and collected. BMSCs were cultured in complete medium. The medium was changed every three days and subcultured once every five days. All subsequent experiments were performed using passage 3 BMSCs.

(2) Three-dimensional culture of BMSCs; mECM and Cowhide collagen are dissolved in 1mol/l acetic acid, and the pH value is adjusted to about 7 by 1mol/l sodium hydroxide solution; collecting BMSCs (1 × 105 per tube), centrifuging to the bottom of a 15 ml centrifuge tube, respectively dripping 0.1ml of mECM with PH =7 or Cowhide collagen into centrifuge tubes corresponding to experiment groups, fully mixing, standing at 37 ℃ for 20 minutes, slowly adding a culture medium after the compound is solidified, and culturing in an incubator for 7 days, 14 days or 21 days to collect samples for further experiments.

(3) And (3) GAGs detection: the three-dimensional cultured sample was washed with PBS, followed by grinding into a homogenate with liquid nitrogen, and adding 60. mu.g/ml proteinase K to the homogenate in a water bath at 56 ℃ for 10 hours. The homogenate was incubated for 15 minutes using 33258 reagent and then absorbance was measured at 460nm wavelength with a fluorescent microplate reader to reflect the DNA content and cell number in the three-dimensional tissue. The homogenate was incubated with DMMB reagent for 30 minutes in the dark, followed by absorbance at wavelength 525nm with a fluorescent microplate reader to reflect the total GAGs content in the three-dimensional tissue. Finally, the level of secretion of GAGs in the cell is reflected by the total GAGs content divided by the total DNA content.

(4) And (3) qPCR detection: the three-dimensional cultured samples were washed with PBS, then ground to homogenate with liquid nitrogen, mRNA in the samples was extracted with mRNA extraction kit, and cartilage-related gene (Collagen I, Collagen II and aggrecan) expression in the three-dimensional samples was detected according to standard qPCR protocol.

(5) Western blotting detection:

PBS washes the three-dimensional cultured sample, followed by grinding into homogenate with liquid nitrogen, extracting total protein using a cell protein extraction kit, determining the concentration of the protein solution, electrophoresis and inversion using a sample loading of 50ug, blocking with blocking solution, the module was used to incubate primary antibodies (Collagen I, Collagen II and aggrecan) for 24 hours, and after incubating fluorescent secondary antibodies for two hours, the ODYSSEY bicolor infrared laser imaging system scanned the membrane.

(6) Histological staining: the three-dimensionally cultured tissue was sucrose-dehydrated and paraffin-embedded, and then tissue sections having a thickness of 5 μm were taken. Dewaxing was followed by HE staining, Alisin Blue staining (Alcian Blue) and Toluidine Blue staining (Toluidine Blue).

(7) Histological staining: the three-dimensionally cultured tissue was sucrose-dehydrated and paraffin-embedded, and then tissue sections having a thickness of 5 μm were taken. Antigen retrieval by high scoring followed by deparaffinization followed by immunohistochemical staining (Collagen I and Collagen II). Finally, the expression level was quantitatively analyzed by Image J after photographing through an inverted microscope.

2. The experimental results are as follows:

as shown in fig. 2a, HE staining results can see that the number of cells in the ecm proliferated greatly over time and was greater than that in the cow collegen at the same time point. More importantly, some cytoplasmic-rich vacuolated cell chondroid cells appeared in 21-day ecm, which are considered to be characteristic cell morphology during differentiation of bmscs into cartilage.

As shown in fig. 2b, in the results of characteristic staining of cartilage (Alcian and tolulidine blue staining), large amount of significant positive expression of ecm occurred, and slightly higher expression of Collagen (Collagen II) in the mrecm + BMSCs group compared to the cow Collagen + BMSCs group type II in immunohistochemical staining, whereas expression of Collagen (Collagen i), the dominant gene of fibrocartilage, was significantly increased, which was also confirmed by qPCR results (see fig. 2 c), thus indicating that mmecm is beneficial for promoting differentiation of BMSCs into fibrocartilage.

As shown in fig. 2e, the morphological characteristics of the normal meniscus tissue and BMSCs in the ecm and cobalt collagen three-dimensional culture tissues were observed under a scanning electron microscope, and the results showed that the morphological characteristics of the three-dimensional tissues in the ecm + BMSCs were closer to the structure of the normal meniscus.

Example 3 experiment of rat dorsal mECM ability to promote differentiation of BMSCs into cartilage.

This example complexes ecm with BMSCs, cultures for one week in vitro in three dimensions, transplants into rat backs by microsurgery, removes tissue from rat backs one month later, and examines the tissue morphology and cartilage differentiation by HE, safranin and toluidine blue staining.

1. The experimental method comprises the following steps:

(1) transplanting the three-dimensional culture complex to the back of a rat: performing three-dimensional culture on BMSCs in mECM according to the experimental method (2) in the example 2, and taking out the three-dimensional culture compound for standby after one week; seven-week-old male rats were skinned near the midline of the back, sterilized, longitudinally cut approximately 1cm in length along the midline of the back, and a one-week old of the three-dimensional culture complex of mECM + BMSCs was carefully transplanted under the skin of the rats, the skin was sutured, and one month later the rats were euthanized and the grafts carefully removed for use.

(2) Tissue staining: the graft was subjected to histological staining (HE staining, safranin staining and toluidine blue staining) according to the experimental method (6) in example 2.

2. The experimental results are as follows:

as shown in fig. 3, HE staining revealed that the cells in the graft were numerous, and a large number of cells exhibited a vacuolated morphology rich in cytoplasm, and found a structure resembling the caveolar-like cartilage (characteristic structure of cartilage). The above results indicate that mECM in the heterotopic transplantation environment can promote the differentiation of BMSCs to chondrocytes.

Example 4. experiments in which mnecm protected against joint degeneration and osteoarthritis following meniscal injury.

1. The experimental method comprises the following steps:

(1) preparing a meniscus injury model: 18 male SD rats of seven weeks of age were obtained and divided into three groups: a model group; mECM + BMSCs group and Cowhide collagen + BMSCs. All rats were prepared by skin preparation, sterilized, anesthetized with 3 ml of 2% sodium pentobarbital solution, microscopically incised about 1.5cm medially at the medial knee joint, the joint cavity carefully opened to expose the meniscus, and a full-thickness defect of 0.6mm diameter was made at the medial anterior horn of the meniscus. Complexes of the scaffold material and BMSCs were prepared for storage at 4 ℃ as described in experimental method (2) in example 2, 30 μ L of the complex was injected into a meniscus defect, and after standing for five minutes, the joint capsule was closed, and the muscle layer and the skin were sutured. Rats were placed on a treadmill for two hours of exercise each day starting at the second week after surgery. After eight weeks of operation, taking the joint fluid of rats as an enzyme-linked immunosorbent assay (ELISA) of inflammation media (IL-6, IL-1 beta and TNF-alpha); all rats were euthanized and joints were scored for OARSI; joint cavity volume and bone loss were assessed by micct; histological staining of meniscus and articular cartilage tissue, including HE staining, safranin fast green staining and immunohistochemical staining (MMP-13), assessed for meniscus repair and cartilage degradation and inflammatory expression levels.

(2) ELISA detection of synovial fluid: after three groups of SD rats are treated for eight weeks continuously, 0.1ml of physiological saline is injected into the joint cavity, joint fluid is extracted after 1 minute, and the secretion of IL-6, IL-1 beta and TNF-alpha in the joint fluid is detected through an ELISA kit and a standard operation flow.

(3) Gross observation and scoring: after animals received four weeks of continuous treatment, all rats were euthanized, articular tissue was cut, tibial plateau and femoral condyle were isolated for photographing, and articular surface damage and tissue scores were analyzed.

(4) Immunohistochemical staining: the isolated joint tissues were decalcified with EDTA for 1 month, dehydrated and sectioned by paraffin embedding. Antigen retrieval was performed by high resolution after deparaffinization, and immunohistochemical staining for osteoarthritis specific markers (MMP-13) was performed. Finally, the expression level was quantitatively analyzed by Image J after photographing through an inverted microscope.

(5) Separating the whole joint tissues of three groups of SD rats, properly stripping the peripheral skin and muscle tissues, keeping 2cm of each of tibia and femur, performing micro-CT scanning, and analyzing the obtained data by a computer on the hyperosteogeny, the bone density, the trabecular bone density and the joint cavity volume.

2. The experimental results are as follows:

(1) protection of meniscal damage by ecm-BMSCs:

as shown in fig. 4a, from a general view of the meniscus, severe meniscus degeneration and damage occurred eight weeks after meniscus injury in control group rats without treatment; the menisci can be repaired to a certain extent by mECM and Cowhide collagen (bovine dermal collagen, the scaffold material which is most commonly used at present) composite BMSCs, and the repairing effect of the mECM on the menisci is more excellent. This conclusion was also confirmed by HE staining (see fig. 4 b), and more importantly, ecm was not only able to repair meniscal damage, but also was effective in reducing fibrosis at the site of the damage. As shown in fig. 4c, for safranin staining of meniscus tissue, glycosaminoglycan of extracellular matrix can be stained red by safranin staining solution, positive area (red) is less in control group, positive staining range can be greatly increased through tissue engineering material repair, loss of glycosaminoglycan is prevented, and effect of mlcm is more excellent than that of cow collegen.

(2) Protection of meniscal damage from mnemonic complexed BMSCs osteoarthritis:

as shown in fig. 5a, from a general view, severe injury to the cartilage surface can be caused if the meniscus is damaged and treated untimely, and the treatment of meniscus damage by the mecc and the cow collagen complex BMSCs can effectively reduce cartilage damage, particularly, the cartilage surface of the mecc + BMSCs group is greatly protected, and the result is also verified by the OASRI osteoarthritis international score.

Further immunohistochemical staining of the articular surface with the osteoarthritis-specific marker MMP-13 was performed as shown in FIGS. 5c-d, and the results showed that the model group (control) exhibited a large amount of positive expression, while mECM significantly reduced MMP-13 expression.

As shown in FIG. 5e, ELISA detected the expression of synovial inflammatory mediators (IL-1. beta., IL-6 and TNF-. alpha.) and showed that mECM significantly reduced the secretion of inflammation due to meniscal injury.

As shown in fig. 5f-h, micro-CT results show that a large number of osteophytes were present near the joint in the control group, the volume of the joint cavity was significantly increased, while the ecm was effective in less osteophyte formation, and reducing the joint cavity volume was more stable in the joint.

In addition, meniscus injury can also cause bone loss and decreased bone density, and the mECM can greatly reduce the bone loss after meniscus injury, increase trabecular bone density, and further avoid further joint degeneration.

The above description is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the invention to the precise form disclosed, and all modifications and equivalents may be resorted to, falling within the scope of the invention.