A joint repair protein composition and its preparation method and application

Abstract

The present invention relates to a joint repair protein composition, and its preparation comprises the following steps: adding 20U / mL-35U / mL of any one or combination of nuclease or universal nuclease to a cell protein extract, placing it under 37°C±1°C for enzymolysis for 15min-40min, and separating and purifying the obtained enzymolysis solution. The cell protein composition obtained by the present invention has the effects of cell repair, joint repair, and cartilage repair, and is used for preventing and treating any one or complications of joint traumatic lesions, degenerative osteoarthritis, joint injuries, refractory wound lesions, knee osteoarthritis, and cartilage damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a joint repair protein composition, a preparation method thereof and an application thereof. Background Art

[0002] Arthritis is called the immortal cancer, with a very high disability rate, which seriously affects the quality of life. There are more than 150 million people in my country suffering from joint diseases caused by degenerative lesions and sports injuries. It is predicted that the domestic market size of joints and sports medicine alone will exceed 20 billion per year and grow at a rate of more than 20%. The "Survey on the Prevalence of Primary Osteoarthritis in People Over 40 Years Old in China" shows that the prevalence of primary osteoarthritis in people aged 40-70 is 62.2% and is age-related. National statistics show that the population over 65 years old in my country is expected to exceed 210 million (accounting for about 15%) in 2025, and problems such as joint injuries and degenerative lesions or cartilage damage in the elderly are increasing year by year. Joint injuries (such as traumatic lesions of joints, degenerative osteoarthritis, etc.) are common clinical diseases, including refractory wound lesions, osteoarthritis of the knee, cartilage wear of the knee, cartilage damage, etc.

[0003] Treatment or repair methods for joint injuries and articular cartilage injuries include anti-inflammation, analgesia (pain relief), physical therapy, acupuncture, joint replacement, stem cell therapy, radiofrequency ablation, chondrocyte transplantation, osteochondral transplantation, and engineered cartilage, such as physical therapy and nutrition represented by traditional Chinese medicine physiotherapy and glucosamine, lubrication relief represented by chitosan and hyaluronic acid, anti-inflammatory treatment represented by PRP, and joint replacement represented by arthroscopy and artificial joints. Except for joint replacement, all of them focus on relieving symptoms and delaying the course of the disease, and there is a lack of truly effective and safe treatment methods.

[0004] Articular cartilage has no blood vessels, nerves, or lymphatic systems, contains a small number of chondrocytes, and has limited migration and proliferation capabilities, making it difficult for articular cartilage damage to heal on its own. When the blood supply and nerve innervation of articular cartilage fail, it can easily lead to joint damage, which in turn affects the joint's ability to repair. Without timely and effective treatment and repair of the injury, the injury will continue to worsen and cause osteoarticular lesions, leading to joint pain, joint swelling, or movement disorders, and even irreversible pathological changes and joint degenerative changes, affecting the patient's mobility and disability. Repair of joint injuries is a long, gradual, and slow process. Timely repair of damaged cells can significantly improve and treat related lesions.

[0005] When cells and microorganisms are subjected to external stimuli and exogenous stressors (including cold, heat, acid, alkali, electric current, radiation, chemicals, etc.), they will induce stress proteins in cells and microorganisms due to stress response. Document 1 (Newlimonophyllines AC from the stem of Atalantia monophylla and cytotoxicityagainst cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41: 431–437) discloses that compounds 1-16 extracted from Rutaceae plants (Atalantia monophylla) have activities such as inhibiting tumor cell growth.

[0006] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, and urine. They have the characteristics of wide sources, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and convenient collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN). They participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, and immune maturation processes. They can be used for immune regulation, tissue repair, and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome. However, MSCs products need to be refrigerated in their production, storage, transportation, and application, and their cell viability is maintained at ≤12h, which limits their therapeutic application. To this end, it is necessary to develop safe and effective joint repair drugs to meet clinical needs. Summary of the invention

[0007] The object of the present invention is to provide a joint repair protein composition, the preparation of which comprises the following steps:

[0008] (1) adding 20U / mL-35U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract of the present invention, placing it at 37°C±1°C for enzymatic hydrolysis for 15min-40min to obtain an enzymatic hydrolyzate, and placing it at 2°C-4°C for standby use;

[0009] (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution portion at an ultraviolet wavelength of 280 nm is monitored and collected, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0010] In a preferred technical solution of the present invention, the preparation of the cell protein extract comprises the following steps:

[0011] S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 10h-14h, and then separated, washed and collected, wherein the stressor is selected from any one or a combination of compounds 1-16,

[0012]

[0013]

[0014] S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then ultrasonically treated at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer, or a combination thereof;

[0015] S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

[0016] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid (18AA) 0.02-0.05% and 3-8μmol / L of stressor.

[0017] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 45%, RPMI 164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid (18AA) 0.05% and 4-6μmol / L of stressor.

[0018] In the preferred technical solution of the present invention, the density of mesenchymal passage cells in step S-1 is 6.0×10 6 -2.0×10 7 / mL, preferably 8.0×10 6 -1.0×10 7 Pieces / mL.

[0019] In a preferred technical solution of the present invention, the mesenchymal stem cells in step S-1 are cultured in the culture medium for 11h-13h.

[0020] In a preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of times of washing cells is 2-5 times, preferably 3-4 times.

[0021] In the preferred technical scheme of the present invention, the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm*5-10min.

[0022] In the preferred technical solution of the present invention, the ultrasonic conditions of step S-2 are: working for 3s and then resting for 1s at 2°C-8°C, 25kHZ, 360W, and ultrasonic treatment for 1-5min.

[0023] In the preferred technical solution of the present invention, the separation in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multi-stage centrifugation, multi-stage filtration or a combination thereof, preferably 3000-7000rpm*15-25min.

[0024] In the preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min respectively.

[0025] In the preferred technical solution of the present invention, the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

[0026] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 or the protein composition obtained in step (2) is frozen, preferably at -40°C to -20°C.

[0027] In a preferred technical scheme of the present invention, a lyophilization protectant is added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), and lyophilized to obtain a cell protein extract lyophilized preparation or a protein composition lyophilized preparation, wherein the lyophilization protectant is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

[0028] In a preferred technical solution of the present invention, the freeze-dried preparation contains 0.5-8%, preferably 1-5%, of freeze-protectant by mass percentage.

[0029] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

[0030] According to a preferred technical solution of the present invention, the cell protein extract freeze-dried preparation or the protein composition freeze-dried preparation has a pH of 6-8, preferably a pH of 7-7.5.

[0031] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is hydrolyzed by either nuclease or omnipotent nuclease and then separated and purified.

[0032] In a preferred technical solution of the present invention, the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

[0033] In the preferred technical scheme of the present invention, 25U / mL-30U / mL of any one or a combination of nuclease or universal nuclease is added to the cell protein extract prepared by the present invention, and the extract is placed at 37°C±1°C for enzymatic hydrolysis for 20min-30min to prepare an enzymatic hydrolyzate.

[0034] In a preferred technical solution of the present invention, the molecular weight of the joint repair protein composition is 25 kDa-245 kDa, preferably 50 kDa-200 kDa.

[0035] The preferred technical solution of the present invention is that the protein composition of the joint repair protein composition is as follows Figure 2 shown.

[0036] In a preferred technical solution of the present invention, the lyophilized preparation is reconstituted with an isotonic solution before use and is used by any one or a combination of smearing, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, and lumbar puncture, wherein the isotonic solution is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer.

[0037] The culture of mesenchymal stem cells or primary mesenchymal stem cells of the present invention adopts a culture method in the art.

[0038] In the preferred technical solution of the present invention, the culture of mesenchymal stem cells comprises the following steps: primary mesenchymal stem cells are cultured at an initial density of 5.0×10 5 -5.0×10 6 The culture medium is added with 100 μg / ml of DMEM / F12 medium, and then cultured at 37.0°C±0.5°C and 5%±1.0% CO2 for 10-15 days. Every 2-3 days, the culture medium is observed to turn yellow and replaced by half. The culture medium contains 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin.

[0039] In the preferred technical solution of the present invention, the culture of primary mesenchymal stem cells comprises the following steps:

[0040] A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2, and the medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells;

[0041] B: Shake, collect the lower layer cells, wash with PBS, add 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, pipette gently, centrifuge at 1200-1500rpm / min*5-8min, and collect the cells.

[0042] The object of the present invention is to provide a method for preparing a cell protein extract having joint repair efficacy, comprising the following steps:

[0043] S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0°C±0.5°C, 5%±1.0% CO2 for 10h-14h, and then separated, washed and collected, wherein the stressor is selected from any one or a combination of compounds 1-16;

[0044] S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then ultrasonically treated at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer, or a combination thereof;

[0045] S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

[0046] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid (18AA) 0.02-0.05% and 3-8μmol / L of stressor.

[0047] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 45%, RPMI 164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid (18AA) 0.05% and 4-6μmol / L of stressor.

[0048] In the preferred technical solution of the present invention, the density of mesenchymal passage cells in step S-1 is 5.0×10 6 -2.0×10 7 / mL, preferably 8.0×10 6 -1.0×10 7 Pieces / mL.

[0049] In a preferred technical solution of the present invention, the mesenchymal stem cells in step S-1 are cultured in the culture medium for 11h-13h.

[0050] In a preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of times of washing cells is 2-5 times, preferably 3-4 times.

[0051] In the preferred technical scheme of the present invention, the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm*5-10min.

[0052] In the preferred technical solution of the present invention, the ultrasonic conditions of step S-2 are: working for 3s and then resting for 1s at 2°C-8°C, 25kHZ, 360W, and ultrasonic treatment for 1-5min.

[0053] In the preferred technical solution of the present invention, the separation in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multi-stage centrifugation, multi-stage filtration or a combination thereof, preferably 3000-7000rpm*15-25min.

[0054] In the preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min respectively.

[0055] In the preferred technical solution of the present invention, the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

[0056] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is frozen, preferably at -40°C to -20°C.

[0057] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is hydrolyzed by either nuclease or omnipotent nuclease and then separated and purified.

[0058] The culture of mesenchymal stem cells or primary mesenchymal stem cells of the present invention adopts a culture method in the art.

[0059] In the preferred technical solution of the present invention, the culture of mesenchymal stem cells comprises the following steps: primary mesenchymal stem cells are cultured at an initial density of 5.0×10 5 -5.0×10 6 The culture medium is added with 100 μg / ml of DMEM / F12 medium, and then cultured at 37.0°C±0.5°C and 5%±1.0% CO2 for 10-15 days. Every 2-3 days, the culture medium is observed to turn yellow and replaced by half. The culture medium contains 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin.

[0060] In the preferred technical solution of the present invention, the culture of primary mesenchymal stem cells comprises the following steps:

[0061] A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2, and the medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells;

[0062] B: Shake, collect the lower layer cells, wash with PBS, add 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, pipette gently, centrifuge at 1200-1500rpm / min*5-8min, and collect the cells.

[0063] Another object of the present invention is to provide a method for preparing a joint repair protein composition, comprising the following steps:

[0064] (1) adding 20U / mL-35U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract of the present invention, placing it at 37°C±1°C for enzymatic hydrolysis for 15min-40min to obtain an enzymatic hydrolyzate, and placing it at 2°C-4°C for standby use;

[0065] (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution portion at an ultraviolet wavelength of 280 nm is monitored and collected, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0066] In a preferred technical solution of the present invention, the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

[0067] In the preferred technical scheme of the present invention, 25U / mL-30U / mL of any one or a combination of nuclease or universal nuclease is added to the cell protein extract prepared by the present invention, and the extract is placed at 37°C±1°C for enzymatic hydrolysis for 20min-30min to prepare an enzymatic hydrolyzate.

[0068] In a preferred technical solution of the present invention, the molecular weight of the joint repair protein composition is 25 kDa-245 kDa, preferably 50 kDa-200 kDa.

[0069] In a preferred technical solution of the present invention, the protein composition obtained in step (2) is frozen, preferably at -40°C to -20°C.

[0070] In a preferred technical scheme of the present invention, a lyophilization protective agent is added to the protein composition obtained in step (2), and lyophilization is performed to obtain the protein composition, wherein the lyophilization protective agent is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

[0071] In a preferred technical solution of the present invention, the freeze-dried preparation contains 0.5-8%, preferably 1-5%, of freeze-protectant by mass percentage.

[0072] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the protein composition obtained in step (2), wherein the protein stabilizer is selected from any one of albumin, zinc salt and aluminum salt.

[0073] According to a preferred technical solution of the present invention, the lyophilized preparation of the protein composition has a pH of 6-8, preferably a pH of 7-7.5.

[0074] The preferred technical solution of the present invention is that the protein composition of the joint repair protein composition is as follows Figure 2 shown.

[0075] In a preferred technical solution of the present invention, the lyophilized preparation is reconstituted with an isotonic solution before use and is used by any one or a combination of smearing, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, and lumbar puncture, wherein the isotonic solution is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer.

[0076] Another object of the present invention is to provide a joint repair composition, which is composed of any one or a combination of the cell protein extract or joint repair protein composition with joint repair efficacy of the present invention and a pharmaceutically acceptable carrier.

[0077] The dosage or type of the pharmaceutically acceptable carrier of the present invention is determined according to factors such as the physicochemical properties and content of the active ingredients in the composition, the type of preparation, the dissolution and bioavailability of the preparation, and the like.

[0078] The composition of the present invention can be in the dosage form of the art and can be prepared using the formulation technology of the art.

[0079] In a preferred technical solution of the present invention, the composition is selected from any one of a lyophilized agent, a gel, a nasal spray, a liquid dressing, an injection, a patch, an ointment, a cream, an emulsion, and a suppository.

[0080] In a preferred technical solution of the present invention, the administration method of the composition is selected from any one or a combination of smearing, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, and lumbar puncture.

[0081] Another object of the present invention is to provide the use of the cell protein extract with joint repair efficacy, the joint repair protein composition or the composition thereof for preparing products for cell repair, joint repair and cartilage repair.

[0082] In the preferred technical scheme of the present invention, the joint repair or cartilage repair is selected from any one of traumatic joint lesions, degenerative osteoarthritis, joint injury, refractory wound lesions, osteoarthritis of the knee joint, cartilage injury or their complications.

[0083] In the preferred technical scheme of the present invention, the product is used for any one of or a combination of people who need to care for their bones and joints, people with bone degenerative diseases in the middle-aged and elderly, people with sports injuries, people who need to maintain their health through sports, and people who need to recover after surgery for bone and joint diseases.

[0084] Another object of the present invention is to provide the use of compound 1-16 for stress-induced stem cells to produce functional proteins with repair efficacy.

[0085] In the preferred technical solution of the present invention, the repair is any one of cell repair, hair follicle repair, joint repair, and repair.

[0086] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0087] Unless otherwise specified, the identification of mesenchymal stem cells MSCs in the present invention refers to "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neuroestorative clinical application".

[0088] Compared with the prior art, the present invention has the following beneficial effects:

[0089] 1. The present invention scientifically screens a culture medium containing stressors to induce mesenchymal stem cells to produce cell proteins with cell repair efficacy. The obtained cell protein extract, cell protein composition or its composition has the effects of cell repair, joint repair and cartilage repair, activates wound chondrocyte regeneration, promotes injury repair, and is used for preventing and treating any of traumatic joint lesions, degenerative osteoarticular lesions, joint injuries, refractory wound lesions, knee osteoarticular lesions, cartilage injuries or their complications, and has the advantages of high purity, good stability, safety and effectiveness, and easy storage and transportation.

[0090] 2. The preparation method of the present invention has the advantages of simple operation, environmental protection, better cost, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The electrophoretic separation results of the joint repair protein composition of the present invention;

[0092] Figure 2 The results of high performance liquid chromatography (HPLC) testing of the joint repair protein composition of the present invention;

[0093] Figure 3 In Experimental Example 1, the repair effect of left medial condylar joint injury of the experimental sheep on day 0 and day 49 was studied;

[0094] Figure 4 In Experimental Example 1, the repair effect of right lateral plateau joint injury on the 0th day and the 49th day of the experimental sheep was studied.

[0095] Figure 5 Clinical scoring results of the joint repair protein composition of the present invention used to repair joint damage or cartilage damage in patients;

[0096] Figure 6 The joint repair protein composition of the present invention is used to repair the functional scoring result of the patient's joint damage or cartilage damage. DETAILED DESCRIPTION

[0097] The details of the present invention are further explained and described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0098] 1. Culture of primary mesenchymal stem cells

[0099] The culture of primary mesenchymal stem cells includes the following steps:

[0100] 1) After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected. They were placed in a culture bottle, and DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin was added. They were then cultured at 37°C and 5% CO2 to promote their adhesion. After the medium turned yellow every 2-3 days, half of the medium was replaced. The culture was cultured for 10-12 days until cells were seen crawling out of the edge of the tissue block.

[0101] 2) Gently shake the tissue blocks to make them fall off, collect the tissue blocks and the lower layer cells respectively, and culture the collected tissue blocks again on the wall;

[0102] 3) After washing the collected lower layer cells with PBS, add an appropriate amount of 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, gently blow the bottom of the bottle with a pipette, centrifuge at 1500rpm / min*5min, and collect the cells.

[0103] 2. Subculture of primary mesenchymal stem cells

[0104] Subculture of primary mesenchymal stem cells: Primary mesenchymal stem cells were cultured at an initial density of 1.0×10 5 -6.0×10 5Add 100U / ml of the culture medium to DMEM / F12 containing 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin, and then culture it at 37.0℃±0.5℃, 5%±1.0% CO2 for 10-15 days. Replace half of the culture medium after observing it turn yellow every 2-3 days.

[0105] 3. The preparation of compounds 1-16 refers to document 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41: 431-437).

[0106] Example 1 Preparation of cell protein extract with joint repair efficacy of the present invention

[0107] The method for preparing the cell protein extract having joint repairing effect of the present invention comprises the following steps:

[0108] (1) Mesenchymal cells were cultured at a density of 5.0×10 6 10 μmol / mL of the cells were added to a medium containing 45% DMEM / F12, 45% RPMI 1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% compound amino acid (18AA) and 5μmol / L compound 16, and then cultured at 37°C and 5% CO2 for 12h, centrifuged at 1200rpm*5min, washed 3 times with PBS, and then the cells were collected;

[0109] (2) The cells collected in step (1) were cultured at a density of 1.0×10 7 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0110] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0111] Example 2 Preparation of the joint repair protein composition of the present invention

[0112] The preparation of the joint repair protein composition of the present invention comprises the following steps:

[0113] (1) Add 25 U / mL of UCF.ME UltraNuclease to the cell protein extract prepared in Example 1, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0114] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared into 10 mg / ml with an elution solvent, and sequentially passed through a high-purity silica gel liquid chromatography protection column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.1-1 ml / min, and the elution portion with an ultraviolet wavelength of 280 nm is monitored and collected, thereby obtaining the enzymatic hydrolyzate, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0115] Example 3 Preparation of freeze-dried preparation of joint repair protein composition of the present invention

[0116] A required amount of mannitol was added to the cell protein composition prepared in Example 2, stirred, mixed evenly, and then freeze-dried. The resulting freeze-dried preparation contained 2% mannitol (m / m).

[0117] Example 4 Preparation of cell protein extract with joint repair efficacy of the present invention

[0118] The method for preparing the cell protein extract having joint repairing effect of the present invention comprises the following steps:

[0119] (1) Mesenchymal cells were cultured at a density of 5.0×10 6 10 μmol / mL of the cells were added to a medium containing 45% DMEM / F12, 45% RPMI 1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% compound amino acid (18AA) and 8μmol / L compound 13, and then cultured at 37°C and 5% CO2 for 12h, centrifuged at 1200rpm*5min, washed 3 times with PBS, and then the cells were collected;

[0120] (2) The cells collected in step (1) were cultured at a density of 1.0×10 7Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0121] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0122] Example 5 Preparation of the joint repair protein composition of the present invention

[0123] The preparation of the joint repair protein composition of the present invention comprises the following steps:

[0124] (1) Add 30 U / mL of UCF.ME UltraNuclease to the cell protein extract prepared in Example 4, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0125] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared into 10 mg / ml with an elution solvent, and sequentially passed through a high-purity silica gel liquid chromatography protection column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.1-1 ml / min, and the elution portion with an ultraviolet wavelength of 280 nm is monitored and collected, thereby obtaining the enzymatic hydrolyzate, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0126] Example 6 Preparation of freeze-dried preparation of joint repair protein composition of the present invention

[0127] Dextran was added to the cell protein composition prepared in Example 5, stirred, mixed evenly, and then freeze-dried to obtain a freeze-dried preparation containing 5% dextran (m / m).

[0128] Example 7 Preparation of cell protein extract with joint repair efficacy of the present invention

[0129] The method for preparing the cell protein extract having joint repairing effect of the present invention comprises the following steps:

[0130] (1) Mesenchymal cells were cultured at a density of 6.0×10 610 μg / mL of 5% 5-nitropropene was added to a medium containing 45% DMEM / F12, 45% RPMI 1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% compound amino acid (18AA) and 6μmol / L compound 14, and then cultured at 37°C and 5% CO2 for 12h, centrifuged at 1200rpm*5min, washed 3 times with PBS, and then the cells were collected;

[0131] (2) The cells collected in step (1) were cultured at a density of 9.0×10 6 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0132] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0133] Example 8 Preparation of the joint repair protein composition of the present invention

[0134] The preparation of the joint repair protein composition of the present invention comprises the following steps:

[0135] (1) Add 20 U / mL of UCF.ME UltraNuclease to the cell protein extract obtained in Example 7, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0136] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared into 10 mg / ml with an elution solvent, and sequentially passed through a high-purity silica gel liquid chromatography protection column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.8 ml / min, and the elution portion with an ultraviolet wavelength of 280 nm is monitored and collected, thereby obtaining the enzymatic hydrolyzate, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0137] Example 9 Preparation of freeze-dried preparation of joint repair protein composition of the present invention

[0138] Sorbitol was added to the cell protein composition prepared in Example 8, stirred, mixed evenly, and then freeze-dried to obtain a freeze-dried preparation containing 3% sorbitol (m / m).

[0139] Example 10 Preparation of cell protein extract with joint repair efficacy of the present invention

[0140] The method for preparing the cell protein extract having joint repairing effect of the present invention comprises the following steps:

[0141] (1) Mesenchymal cells were cultured at a density of 1.0×10 7 10 μmol / mL of the cells were added to a medium containing 45% DMEM / F12, 45% RPMI 1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% compound amino acid (18AA) and 7μmol / L compound 15, and then cultured at 37°C and 5% CO2 for 12h, centrifuged at 1200rpm*5min, washed 3 times with PBS, and then the cells were collected;

[0142] (2) The cells collected in step (1) were cultured at a density of 5.0×10 7 Cells were dispersed in physiological saline at 2-8°C, 25kHz, and 360W, and ultrasonicated for 3s, 1s at rest, and 2min to obtain cell lysate.

[0143] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0144] Embodiment 11 Preparation of the joint repair protein composition of the present invention

[0145] The preparation of the joint repair protein composition of the present invention comprises the following steps:

[0146] (1) Add 30 U / mL of UCF.ME UltraNuclease to the cell protein extract obtained in Example 10, and place the extract at 37° C. for 30 min to obtain an enzymatic solution;

[0147] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared into 10 mg / ml with an elution solvent, and sequentially passed through a high-purity silica gel liquid chromatography protection column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.1-1 ml / min, and the elution portion with an ultraviolet wavelength of 280 nm is monitored and collected, thereby obtaining the enzymatic hydrolyzate, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0148] Example 12 Preparation of freeze-dried preparation of joint repair protein composition of the present invention

[0149] Mannitol was added to the cell protein composition obtained in Example 11, stirred, mixed evenly, and then freeze-dried to obtain a freeze-dried preparation containing 5% mannitol (m / m).

[0150] Example 13 Preparation of freeze-dried preparation of cell protein extract of the present invention

[0151] A required amount of mannitol was added to the cell protein extract obtained in Example 1, stirred, mixed evenly, and then freeze-dried. The resulting freeze-dried preparation contained 2% mannitol (m / m).

[0152] Embodiment 14 Molecular weight distribution detection of the joint repair protein composition of the present invention

[0153] The molecular weight distribution of the nerve repair protein composition of the present invention is detected by standard molecular weight polyacrylamide gel electrophoresis, comprising the following steps:

[0154] 1. Select a glass plate with a thickness of 1.5 mm and place it horizontally. Spread the glue solution (15% lower glue, 4% separation glue) prepared according to Table 1 on the glass plate in sequence. Insert the comb vertically into the lower glue.

[0155] Table 1

[0156]

[0157]

[0158] 2. Thaw the Marker (20KDa-245KDa) frozen at -40°C, and use PBS to prepare the Marker and the lyophilized powder of the joint repair protein composition prepared in Example 3, Example 6, Example 9, and Example 12 into 10ug / ul of the test sample, add 20ul of the test sample to the loading well, and perform electrophoresis at 60-80V until clear bands appear. Adjust the voltage to 100-120V until the Marker is completely separated, place the PAGE gel in Coomassie Brilliant Blue dye solution, and stop dyeing when clear bands appear on the gel. After washing with pure water, decolorize with 10% acetic acid solution, and stop decolorizing when the gel becomes transparent. See the results. Figure 1 , wherein strip A is embodiment 3, strip B is embodiment 6, strip C is embodiment 9, and strip D is embodiment 12.

[0159] Embodiment 15 High performance liquid chromatography detection of the joint repair protein composition of the present invention

[0160] The lyophilized powder of the joint repair protein composition of Example 3 was dissolved in deionized water to prepare a 10 mg / ml test solution.

[0161] Chromatographic column: SHIMSEN Ankylo (300mm*4.6mm.D.,3um; P / N:380-01215-05)

[0162] Mobile phase: 50mmol / L phosphate buffer (pH=6.8), containing 300mmol / L sodium chloride; flow rate: 0.3mL / min; injection volume: 10ul; column temperature: 25℃; detection wavelength: 280nm; isocratic elution, collection time: 30min. Results are shown in Figure 2 .

[0163] Test Example 1 The cell protein extract with joint repair efficacy of the present invention is used for research on joint repair effect

[0164] 120 mg of the cell protein extract freeze-dried powder prepared in Example 13 was dissolved in 5 ml of physiological saline to prepare a cell protein extract solution with a concentration of 2.4%. The solution was prepared and used immediately before each injection.

[0165] 1. Experimental Methods

[0166] A 12-month-old male goat weighing 47 kg was placed under general anesthesia. An 8-cm midline incision was made on the left medial condyle. The medial edge of the patellar tendon was separated and incised to the joint to expose the medial femoral condyle and the medial tibial plateau. The surface of the articular cartilage was smooth. A 0.3×0.3×0.6 cm (length×width×depth) cartilage injury wound was made on the medial femoral condyle with a 3 mm ball drill. The incision was sutured (see Figure 2). Figure 3). An 8-cm midline incision was made on the right lateral platform, and the layers were separated layer by layer. The joint capsule was entered on the lateral side of the patellar tendon to expose the lateral femoral condyle and the lateral tibial platform. The cartilage was found to be intact and smooth. A 3-mm ball drill was used to make a 0.6×0.3×0.3-cm (length×width×depth) cartilage injury wound on the lateral side of the platform, and the incision was sutured (see Figure 4 ).

[0167] 2.5 ml of the cell protein extract freeze-dried preparation solution prepared in Example 13 was injected into the goat's knee joint cavity unilaterally every week for three consecutive weeks. The goats were observed once in the morning and once in the evening every day. The results are shown in Table 2.

[0168] Table 2

[0169]

[0170]

[0171] After 49 days of observation, the sheep were anesthetized and killed, and cartilage slices were obtained (see Figure 3-4 ).

[0172] This project has completed seven safety tests, including pyrogen detection, acute systemic toxicity test, cytotoxicity test, skin sensitization test, intradermal reaction test, local tissue reaction and degradation test after implantation, and subacute systemic toxicity test, and no adverse reactions were observed.

[0173] The cell protein extract of the invention has excellent effect of repairing joint damage and is safe and effective.

[0174] Experimental Example 2 Study on the effect of the joint repair protein composition of the present invention on repairing joint damage or cartilage damage

[0175] Twenty-one volunteer patients with joint diseases aged 50-80 years were selected as subjects (see Table 3) to investigate and evaluate the effect of the joint repair protein composition of the present invention on repairing joint damage or cartilage damage.

[0176] Inclusion criteria for subjects: ① Patients diagnosed with knee osteoarthritis based on the American College of Rheumatology (ACR) classification criteria; ② Patients with knee pain caused by various factors lasting for more than half a year, or patients who have improved after routine clinical treatment but have relapsed or worsened osteoarthritis after stopping medication; patients have knee pain of more than 4 points when walking on flat ground, and the pain lasts for at least 4 weeks; ③ Through X-ray observation, the Kellgren-Lawrence grade of the target study side knee joint is grade II-III, and the Kellgren-Lawrence grade of the other side knee joint is lower than grade II; ④ 30-80 years old, regardless of gender; able to complete four outpatient follow-up visits within half a year after the first treatment (2 weeks, 4 weeks, 3 months, and 6 months after treatment); ⑤ Able to understand and voluntarily sign the informed consent form, and can voluntarily complete the trial procedures and follow-up examinations.

[0177] Exclusion criteria for subjects: 1) secondary osteoarthritis, history and / or any evidence of the following diseases in the observed target joint: suppurative arthritis, inflammatory joint disease, gout, recurrent pseudogout, Paget's bone disease, joint fractures, chloasma, acromegaly, hemochromatosis, Wilson's disease, primary osteochondroma, genetic diseases (such as ADHD) and collagen gene mutations; 2) patients with other rheumatic diseases, including (but not limited to) systemic lupus erythematosus, inflammatory bowel disease, Felty's syndrome, scleroderma, inflammatory myopathy or other connective tissue diseases, overlap syndrome, etc.); 3) patients who have received intra-articular drug injections within 12 weeks; 4) patients who have undergone arthroscopy, correction or total joint replacement within 6 months; 5) patients who need arthroplasty; 6) patients who use anticoagulants (such as warfarin, low molecular weight heparin) and antiplatelet aggregation drugs; 7) patients with or with a history of tumor diseases. 8) Patients with serious poorly controlled diseases, such as diabetes, hypertension, kidney disease, liver disease or severe heart disease (such as moderate to severe congestive heart failure (New York Heart Association heart function grade III / IV)), and are judged by the researchers to be unsuitable for joining this study. 9) Abnormal laboratory test results: blood routine: WBC <3×10^9 / L; HGB <90g / L; PLT <100×10^9 / L; liver function: TBIL >1.5 times the upper limit of normal; ALT or AST >2.5 times the upper limit of normal; kidney function: creatinine >1.5 times the upper limit of normal, accompanied by creatinine clearance <50ml / min (measured value, or Cockcroft-Gault formula calculated value); HIVAb positive, HBsAg positive, HBcAb positive, HCVAb positive, syphilis antibody positive (any one of the test results is positive and the patient is excluded). 10) Those who have participated in other drug clinical trials within 3 months before screening; 11) Those who have received intra-articular or intramuscular injections or intravenous injections of corticosteroids within 4 weeks before screening; 12) Women who are pregnant, breastfeeding, or preparing for pregnancy. 13) Those who have used stem cell treatment within six months; 14) Those with mental disorders or uncontrolled or poorly controlled mental illnesses; 15) Those who are in the process of medical litigation; 16) Those with a history of alcoholism and drug abuse, as well as those with allergic constitution or a history of allergies; 17) Patients who are considered by the researchers to be unsuitable for participating in this clinical trial.

[0178] Table 3

[0179]

[0180]

[0181] The knee joint function score table KSS (Keen Society Score) (see Table 4) was used to observe and evaluate the therapeutic effect of the drug. 65 mg of the joint repair protein composition freeze-dried powder of Example 3 was dissolved in 5 ml of normal saline and injected into the unilateral knee joint cavity of the subject once a week, with three injections per course of treatment. The results are shown in Figure 5-6 The joint repair protein composition of the present invention can significantly repair joint damage or cartilage damage in patients with joint damage or cartilage damage.

[0182] Table 4

[0183]

[0184] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A joint repair protein composition, the preparation of which comprises the following steps: (1) Add 20U / mL-35U / mL of nuclease to the cell protein extract, place it at 37℃±1℃ for 15min-40min to obtain the enzymatic solution, and place it at 2℃-4℃ for later use; (2) At 2°C-8°C, the enzymatic hydrolysate prepared in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min. The elution portion at an ultraviolet wavelength of 280 nm is monitored and collected to obtain: The elution solvent consisted of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer, pH 6.8; The preparation of the cell protein extract comprises the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid 18AA 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 10h-14h, and then separated, washed and collected, wherein the stressor is selected from compound 16: ； S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then ultrasonically treated at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffer, TBPS buffer, TBST buffer, and Tris buffer; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separated solution is filtered through 0.45 um and 0.22 um filter membranes in sequence to obtain; The mesenchymal stem cells are derived from the umbilical cord.

2. The protein composition according to claim 1, wherein the culture medium in step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid 18AA 0.02-0.05% and 3-8μmol / L of stressor.

3. The protein composition as claimed in claim 2, wherein the culture medium in step S-1 contains DMEM / F12 45%, RPMI164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid 18AA 0.05% and 4-6μmol / L of stressor.

4. The protein composition according to any one of claims 1 to 3, wherein the density of the mesenchymal passage cells in step S-1 is 6.0×10 6 -2.0×10 7 Pieces / mL.

5. The protein composition according to claim 4, wherein the density of the mesenchymal passage cells in step S-1 is 8.0×10 6 -1.0×10 7 Pieces / mL. 6 . The protein composition according to claim 4 , wherein the mesenchymal stem cells in step S-1 are cultured in the culture medium for 11 h to 13 h.

7. The protein composition as claimed in claim 4, wherein the solvent for washing the cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffer, TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of times of washing the cells is 2-5 times.

8. The protein composition according to claim 7, wherein the cells are washed 3-4 times in step S-1.

9. The protein composition according to claim 4, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1000-2000 rpm×3-15 min.

10. The protein composition according to claim 9, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1200rpm-1500rpm×5-10min.

11. The protein composition according to claim 4, wherein the ultrasonic conditions of step S-2 are: working for 3 seconds and then resting for 1 second at 2°C-8°C, 25kHZ, 360W, and ultrasonic treatment for 1-5 minutes.

12. The protein composition according to claim 4, wherein the separation in step S-3 is selected from any one of centrifugation at 2000-8000 rpm×10-30 min, multi-stage centrifugation, and multi-stage filtration, or a combination thereof.

13. The protein composition according to claim 12, wherein the separation in step S-3 is selected from any one of centrifugation at 3000-7000 rpm×15-25 min, multi-stage centrifugation, and multi-stage filtration, or a combination thereof.

14. The protein composition according to claim 12, wherein the multi-stage centrifugation in step S-3 is 3000-4000 rpm×3-5 min, 5000-6000 rpm×3-5 min and 7000 rpm×5-8 min, respectively.

15. The protein composition according to claim 4, wherein the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

16. The protein composition according to claim 4, wherein the cell protein extract obtained in step S-3 or the protein composition obtained in step (2) is frozen.

17. The protein composition according to claim 16, wherein the cell protein extract obtained in step S-3 or the protein composition obtained in step (2) is frozen at -40°C to -20°C.

18. The protein composition according to claim 4, wherein a lyophilization protective agent is added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), and the mixture is lyophilized to obtain a cell protein extract lyophilized preparation or a protein composition lyophilized preparation, wherein: The lyoprotectant is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin, polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

19. The protein composition according to claim 18, wherein the freeze-dried preparation contains 0.5-8% of freeze-protectant by mass.

20. The protein composition according to claim 19, wherein the freeze-dried preparation contains 1-5% of a freeze-protectant by mass.

21. The protein composition according to claim 4, wherein a protein stabilizer is optionally added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), wherein: The protein stabilizer is selected from any one of albumin, zinc salt and aluminum salt.

22. The protein composition according to claim 18, wherein the pH of the cell protein extract freeze-dried preparation or the protein composition freeze-dried preparation is 6-8.

23. The protein composition according to claim 22, wherein the pH of the lyophilized preparation of cell protein extract or the lyophilized preparation of protein composition is 7-7.

5.

24. The protein composition according to claim 4, wherein the cell protein extract obtained in step S-3 is hydrolyzed with nuclease and then separated and purified.

25. The protein composition of claim 24, wherein the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

26. The protein composition according to claim 4, wherein 25U / mL-30U / mL of nuclease is added to the cell protein extract, and the extract is subjected to enzymatic hydrolysis at 37°C±1°C for 20min-30min to obtain an enzymatic hydrolyzate.

27. The protein composition according to claim 4, wherein the molecular weight of the joint repair protein composition is 25 kDa-245 kDa.

28. The protein composition according to claim 27, wherein the molecular weight of the joint repair protein composition is 50 kDa-200 kDa.

29. The protein composition according to claim 4, wherein the lyophilized preparation is reconstituted with an isotonic solution before use and then used by applying, rolling needle, microneedling, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, lumbar puncture or any combination thereof, wherein: The isotonic solution is selected from any one of physiological saline, 5% glucose solution, phosphate buffer, TBPS buffer, TBST buffer, Tris buffer or a combination thereof.

30. The protein composition according to claim 4, wherein the mesenchymal stem cells are cultured using a culture method known in the art.

31. The protein composition according to claim 4, wherein the culture of the mesenchymal stem cells comprises the following steps: culturing the primary mesenchymal stem cells at an initial density of 5.0×10 5 -5.0×10 6 pcs / ml was added to the subculture medium, and then cultured at 37.0℃±0.5℃, 5%±1.0%CO2 for 10-15 days. Every 2-3 days, the subculture medium was observed to turn yellow and half of the amount was replaced. The subculture medium contains DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and 100 ug / ml streptomycin.

32. The protein composition of claim 31, wherein the culturing of primary mesenchymal stem cells comprises the following steps: A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃ and 5%±1.0% CO2. The medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells. B: Shake, collect the lower layer cells and wash with PBS, add 0.25% trypsin to digest for 2 min-3 min, add an equal volume of trypsin stop solution to stop digestion, pipette gently, centrifuge at 1200-1500 rpm×5-8 min, and collect the cells.

33. A method for preparing a joint repair protein composition, comprising the following steps: (1) Add 20U / mL-35U / mL of nuclease to the cell protein extract, place it at 37℃±1℃ for 15min-40min to obtain the enzymatic solution, and place it at 2℃-4℃ for later use; (2) At 2°C-8°C, the enzymatic hydrolysate prepared in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min. The elution portion at an ultraviolet wavelength of 280 nm is monitored and collected to obtain: The elution solvent consisted of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer, pH 6.8; The preparation of the cell protein extract comprises the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid 18AA 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 10h-14h, and then separated, washed and collected, wherein the stressor is selected from compound 16: ； S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffer, TBPS buffer, TBST buffer, Tris buffer or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45 um and 0.22 um filter membranes in sequence.

34. The preparation method according to claim 33, wherein the culture medium in step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid 18AA 0.02-0.05% and 3-8μmol / L of stressor.

35. The preparation method according to claim 33, wherein the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

36. The preparation method according to claim 33, wherein 25U / mL-30U / mL of nuclease is added to the cell protein extract, and the extract is subjected to enzymatic hydrolysis at 37°C±1°C for 20min-30min to obtain an enzymatic hydrolyzate.

37. The preparation method according to any one of claims 33 to 36, wherein the molecular weight of the joint repair protein composition is 25 kDa to 245 kDa.

38. The preparation method according to claim 37, wherein the molecular weight of the joint repair protein composition is 50 kDa-200 kDa.

39. The preparation method according to any one of claims 33 to 36, wherein the protein composition obtained in step (2) is frozen.

40. The preparation method according to claim 39, wherein the protein composition obtained in step (2) is frozen at -40°C to -20°C.

41. The preparation method according to any one of claims 33 to 36, wherein a freeze-drying protective agent is added to the protein composition obtained in step (2), and freeze-dried to obtain: The lyoprotectant is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

42. The preparation method according to claim 41, wherein the freeze-dried preparation contains 0.5-8% of freeze-protectant by mass.

43. The preparation method according to claim 42, wherein the freeze-dried preparation contains 1-5% of freeze-protectant in terms of mass percentage.

44. The preparation method according to any one of claims 33 to 36, wherein a protein stabilizer is optionally added to the protein composition obtained in step (2), wherein: The protein stabilizer is selected from any one of albumin, zinc salt and aluminum salt. The preparation method according to claim 41 , wherein the lyophilized preparation of the protein composition has a pH of 6-8. The preparation method according to claim 45 , wherein the lyophilized preparation of the protein composition has a pH of 7-7.

5.

47. The preparation method according to claim 41, wherein the lyophilized preparation is reconstituted with an isotonic solution before use and then used by applying, rolling needle, microneedling, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, lumbar puncture or any combination thereof, wherein: The isotonic solution is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof.

48. A cell protein extract having joint repair efficacy, the preparation method of which comprises the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL were placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid 18AA 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0%CO2 for 10h-14h, and then separated, washed and collected. The stressor is selected from compound 16 ； S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then ultrasonically treated at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45 um and 0.22 um filter membranes in sequence.

49. The cell protein extract as described in claim 48, wherein the culture medium in step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid 18AA 0.02-0.05% and 3-8μmol / L of stressor.

50. The cell protein extract as described in claim 49, wherein the culture medium in step S-1 contains DMEM / F12 45%, RPMI1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid 18AA 0.05% and 4-6μmol / L stressor.

51. The cell protein extract according to any one of claims 48 to 50, wherein the density of mesenchymal passage cells in step S-1 is 5.0×10 6 -2.0×10 7 Pieces / mL.

52. The cell protein extract according to claim 51, wherein the density of mesenchymal passage cells in step S-1 is 8.0×10 6 -1.0×10 7 Pieces / mL. The cell protein extract according to claim 51 , wherein the mesenchymal stem cells in step S-1 are cultured in the culture medium for 11 h to 13 h.

54. The cell protein extract as described in claim 51, wherein the solvent for washing the cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of times of washing the cells is 2-5 times.

55. The cell protein extract as described in claim 54, wherein the number of times of washing the cells in step S-1 is 3-4 times.

56. The cell protein extract of claim 51, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1000-2000 rpm×3-15 min.

57. The cell protein extract of claim 56, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1200rpm-1500rpm×5-10min.

58. The cell protein extract as described in claim 51, wherein the ultrasonic conditions of step S-2 are: working at 2°C-8°C, 25kHZ, 360W for 3s and then resting for 1s, and ultrasonic treatment for 1-5min.

59. The cell protein extract as described in claim 51, wherein the separation in step S-3 is selected from any one of 2000-8000 rpm×10-30 min centrifugation, multi-stage centrifugation, multi-stage filtration, or a combination thereof.

60. The cell protein extract as described in claim 59, wherein the separation in step S-3 is selected from any one of 3000-7000 rpm×15-25 min centrifugation, multi-stage centrifugation, and multi-stage filtration, or a combination thereof.

61. The cell protein extract as described in claim 51, wherein the multi-stage centrifugation in step S-3 is 3000-4000 rpm×3-5 min, 5000-6000 rpm×3-5 min and 7000 rpm×5-8 min, respectively.

62. The cell protein extract as described in claim 51, wherein the pore size of the filter membrane of the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

63. The cell protein extract as described in claim 51, wherein the cell protein extract obtained in step S-3 is frozen.

64. The cell protein extract as claimed in claim 63, wherein the cell protein extract obtained in step S-3 is frozen at -40°C to -20°C.

65. The cell protein extract as described in claim 51, wherein the cell protein extract obtained in step S-3 is hydrolyzed with nuclease and then separated and purified. The cell protein extract of claim 51 , wherein the mesenchymal stem cells are cultured using a culture method known in the art.

67. The cell protein extract of claim 51, wherein the culture of the mesenchymal stem cells comprises the following steps: culturing the primary mesenchymal stem cells at an initial density of 5.0×10 5 -5.0×10 6 pcs / ml was added to the subculture medium, and then cultured at 37.0℃±0.5℃, 5%±1.0%CO2 for 10-15 days. Every 2-3 days, the subculture medium was observed to turn yellow and half of the amount was replaced. The subculture medium contains DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and 100 ug / ml streptomycin.

68. The cell protein extract of claim 67, wherein the culturing of the primary mesenchymal stem cells comprises the following steps: A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃ and 5%±1.0% CO2. The medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells. B: Shake, collect the lower layer cells and wash with PBS, add 0.25% trypsin to digest for 2 min-3 min, add an equal volume of trypsin stop solution to stop digestion, pipette gently, centrifuge at 1200-1500 rpm×5-8 min, and collect the cells.

69. A method for preparing a cell protein extract having joint repair efficacy, comprising the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal passage cells of 100 cells / mL were placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid 18AA 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0%CO2 for 10h-14h, and then separated, washed and collected. The stressor is selected from compound 16 ； S-2: Collect cells at a density of 5.0×10 6 / mL-1.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffer, TBPS buffer, TBST buffer, Tris buffer or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45 um and 0.22 um filter membranes in sequence.

70. The preparation method as described in claim 69, wherein the culture medium in step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid 18AA 0.02-0.05% and 3-8μmol / L of stressor.

71. The preparation method as described in claim 70, wherein the culture medium in step S-1 contains DMEM / F12 45%, RPMI164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid 18AA 0.05% and 4-6μmol / L of stressor.

72. The preparation method according to any one of claims 69 to 71, wherein the density of mesenchymal passage cells in step S-1 is 5.0×10 6 -2.0×10 7 Pieces / mL.

73. The preparation method according to claim 72, wherein the density of mesenchymal passage cells in step S-1 is 8.0×10 6 -1.0×10 7 Pieces / mL.

74. The preparation method according to claim 72, wherein the mesenchymal stem cells in step S-1 are cultured in the culture medium for 11 h to 13 h.

75. The preparation method as described in claim 72, wherein the solvent for washing the cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of times of washing the cells is 2-5 times.

76. The preparation method as described in claim 75, wherein the number of times of washing the cells in step S-1 is 3-4 times.

77. The preparation method according to claim 72, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1000-2000 rpm×3-15 min.

78. The preparation method according to claim 77, wherein the separation in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein: The centrifugal condition is 1200rpm-1500rpm×5-10min.

79. The preparation method as described in claim 72, wherein the ultrasonic conditions of step S-2 are: working at 2°C-8°C, 25kHZ, 360W for 3s and then resting for 1s, and ultrasonic treatment for 1-5min.

80. The preparation method as described in claim 72, wherein the separation in step S-3 is selected from any one of 2000-8000 rpm×10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, or a combination thereof.

81. The preparation method as described in claim 80, wherein the separation in step S-3 is selected from any one of centrifugation at 3000-7000 rpm×15-25 min, multi-stage centrifugation, and multi-stage filtration, or a combination thereof.

82. The preparation method as described in claim 72, wherein the multi-stage centrifugation in step S-3 is 3000-4000 rpm×3-5 min, 5000-6000 rpm×3-5 min and 7000 rpm×5-8 min, respectively.

83. The preparation method as described in claim 72, wherein the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

84. The preparation method as described in claim 72, wherein the cell protein extract obtained in step S-3 is frozen.

85. The preparation method as described in claim 84, wherein the cell protein extract obtained in step S-3 is frozen at -40°C to -20°C.

86. The preparation method as described in claim 72, wherein the cell protein extract obtained in step S-3 is hydrolyzed with nuclease and then separated and purified.

87. The preparation method according to claim 72, wherein the mesenchymal stem cells are cultured using a culture method known in the art.

88. The preparation method according to claim 72, wherein the culture of the mesenchymal stem cells comprises the following steps: culturing the primary mesenchymal stem cells at an initial density of 5.0×10 5 -5.0×10 6 pcs / ml was added to the subculture medium, and then cultured at 37.0℃±0.5℃, 5%±1.0%CO2 for 10-15 days. Every 2-3 days, the subculture medium was observed to turn yellow and half of the amount was replaced. The subculture medium contains DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and 100 ug / ml streptomycin.

89. The preparation method according to claim 88, wherein the culturing of primary mesenchymal stem cells comprises the following steps: A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃ and 5%±1.0% CO2. The medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells. B: Shake, collect the lower layer cells and wash with PBS, add 0.25% trypsin to digest for 2 min-3 min, add an equal volume of trypsin stop solution to stop digestion, pipette gently, centrifuge at 1200-1500 rpm×5-8 min, and collect the cells.

90. A joint repair composition, which consists of any one of the cell protein extract with joint repair efficacy as described in any one of claims 48 to 68 or the joint repair protein composition as described in any one of claims 1 to 32 or a combination thereof and a pharmaceutically acceptable carrier.

91. The composition of claim 90, wherein the composition is selected from any one of a lyophilized agent, a gel, a nasal spray, a liquid dressing, an injection, a patch, an ointment, a cream, an emulsion, and a suppository.

92. A composition as described in any one of claims 90-91, wherein the administration method of the composition is selected from any one or a combination of smearing, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, and lumbar puncture.

93. Use of the cell protein extract with joint repair efficacy as described in any one of claims 48 to 68 or the joint repair protein composition as described in any one of claims 1 to 32 or a combination thereof in the preparation of a product for joint repair.

94. The use according to claim 93, wherein the joint repair is joint injury.

95. The use as described in claim 93, wherein the joint repair is selected from any one of traumatic joint lesions, degenerative osteoarthritis, refractory wound lesions, osteoarthritis of the knee, and cartilage damage.

96. Use of the cell protein extract with joint repair efficacy as described in any one of claims 48 to 68 or the joint repair protein composition as described in any one of claims 1 to 32 or a combination thereof in the preparation of a product for cartilage repair.

97. The use as described in any one of claims 93-96, wherein the product is used for any one of or a combination of people who need to care for their bones and joints, people with bone degenerative diseases in the middle-aged and elderly, people with sports injuries, people who need to maintain their health through sports, and people who need to recover after surgery for bone and joint diseases.

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