Joint repair protein compositions and methods for making and using same

Abstract

The present invention relates to a protein composition having joint repair effect, and its preparation includes the steps of adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to a cell protein extract, performing enzymatic hydrolysis for 15 to 40 minutes at 37°C ± 1°C, and isolating and purifying the prepared enzymatic hydrolysis liquid. The cell protein composition obtained by the present invention has effects of cell repair, joint repair, and cartilage repair, and is used for preventing and treating any one of traumatic joint lesions, degenerative bone joint lesions, joint damage, intractable wound lesions, bone joint lesions of the knee joint, and cartilage damage, or complications thereof.

Description

[Technical field]

[0001] The present invention is in the field of biomedical technology, specifically, to joint repair protein compositions and methods for making and using same. [Background technology]

[0002] Arthritis is called "cancer that does not lead to death" and has a very high rate of physical disability and serious impact on quality of life. In China, more than 150 million people suffer from joint diseases caused by degenerative lesions and movement damage. It is predicted that the market size of joint and movement medicine alone in China will exceed 20 billion yuan / year and is growing at a rate of more than 20%. According to the "Survey on the Prevalence of Primary Osteoarthritis in People Aged 40 and Over in China", the prevalence of primary osteoarthritis in people aged 40 to 70 years old is 62.2% and is age-related. According to national statistical data, the population of China aged 65 and over is expected to exceed 210 million (about 15%) in 2025, and problems such as joint damage, degenerative lesions, and cartilage damage in the elderly are increasing year by year. Joint damage (eg, joint traumatic lesions, degenerative osteoarticular lesions, etc.) is a common clinical condition, including intractable wound lesions, osteoarticular lesions of the knee joint, wear and tear of knee joint cartilage, cartilage damage, etc.

[0003] Treatment or repair methods for joint injury and articular cartilage injury include anti-inflammation, analgesia (painkillers), physical therapy, acupuncture, joint replacement, stem cell therapy, radiofrequency ablation, chondrocyte transplantation, osteochondral transplantation, engineered cartilage, such as physical therapy and nutrition represented by traditional Chinese medicine physical therapy and ammonia sugar, lubrication relief represented by chitosan and hyaluronic acid, anti-inflammatory treatment represented by PRP, and joint replacement represented by arthroscopy and artificial joints, etc. However, other than joint replacement, all of them are mainly aimed at relieving symptoms and delaying the progression of the disease, and there is no truly effective and safe treatment method.

[0004] Articular cartilage does not have the distribution of blood vessels, nerves, and lymphatic systems, contains a small amount of chondrocytes, and has limited migration and proliferation ability. Damage to articular cartilage is difficult to self-heal. When the blood supply and innervation of articular cartilage are impaired, it is easy to lead to joint damage, which further affects the joint's repair ability. If there is no timely and effective treatment and repair of the damage, the damage will continue to worsen, causing osteoarticular lesions, leading to joint pain, joint swelling or movement disorder, which in turn causes irreversible pathological changes and joint degenerative changes, affecting the patient's behavior and physical disability. Repair of joint damage is a long-term, gradual and slow process. Timely repair of damaged cells can significantly improve and treat related lesions.

[0005] When cells and microorganisms are subjected to stress induction by external stimuli and exogenous stress substances (including cold, heat, acid, alkali, electric current, radiation, chemical substances, etc.), the cells and microorganisms are induced to produce stress proteins through a stress response. Literature 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) discloses that compounds 1-16 extracted from a citrus plant (Atalantia monophylla) have activity to inhibit the proliferation of tumor cells, etc.

[0006] Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate in multiple directions. They are widely distributed in bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, urine and other tissues. They have characteristics such as wide supply source, no need for matching, low infection rate, strong differentiation ability, strong growth ability and easy collection. They can produce activating factors such as stem cell factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN) and other activating factors. They are involved in the regulation of processes such as cell growth, apoptosis, cell differentiation, antiviral and immune maturation, and 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 require refrigeration during production, storage, transportation, and use, and their cell activity can only be maintained for 12 hours or less, limiting their therapeutic use. Therefore, there is a need to develop a safe and effective skin repair drug to meet clinical needs. Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to provide a joint repair protein composition. [Means for solving the problem]

[0008] A joint repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting solution to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution, and storing the solution at 2°C to 4°C for use; and (2) adjusting the enzymatic degradation solution produced in step (1) to 5-15 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the eluted sample at an ultraviolet wavelength of 280 nm.

[0009] In a preferred technical solution of the present invention, the preparation of the cell protein extract comprises: Density is 5.0×10 6 pieces / mL~1.0×10 7 Step S-1: placing mesenchymal passaged cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium at 37.0℃±0.5℃ and 5%±1.0% CO2 for 10h-14h, followed by separating and washing the cells to collect them; [ka] [ka] The collected cells were 5.0 x 10 6 pieces / mL~1.0×10 7 Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0010] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0011] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0012] In a preferred technical solution of the present invention, the mesenchymal passage cell density in step S-1 is 6.0×10 6 ~2.0×10 7 5.0×10 6 ~1.0×10 7 pieces / mL.

[0013] In a preferred technical solution of the present invention, the mesenchymal passaged stem cells in step S-1 are cultured in the medium for 11 to 13 hours.

[0014] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of washing the cells is 2 to 5 times, preferably 3 to 4 times.

[0015] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0016] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0017] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0018] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0019] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0020] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0021] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is added with a freeze-drying protection agent selected from any one or combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch, and then freeze-dried to prepare a freeze-dried cell protein extract preparation or a freeze-dried protein composition preparation.

[0022] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass.

[0023] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cellular protein extract prepared in step S-3 or the protein composition prepared in step (2), which is any one selected from albumin, zinc salts, and aluminum salts.

[0024] In a preferred technical solution of the present invention, the lyophilized preparation of the cell protein extract or the lyophilized preparation of the protein composition has a pH of 6 to 8, preferably 7 to 7.5.

[0025] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically digested using either nuclease or ultranuclease, and then separated and purified.

[0026] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0027] In a preferred technical solution of the present invention, 25 U / mL to 30 U / mL of either one or a combination of nuclease or ultranuclease is added to the cell protein extract prepared by the present invention, and the mixture is subjected to enzymatic hydrolysis at 37°C±1°C for 20 to 30 minutes to prepare an enzymatic hydrolysis solution.

[0028] 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.

[0029] In a preferred technical solution of the present invention, the protein composition in the joint repair protein composition is as shown in FIG.

[0030] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted immediately before use with an isotonic solution selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof, and then used by one or a combination of smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture.

[0031] For the culture of the passaged mesenchymal stem cells or primary mesenchymal stem cells of the present invention, a culture method known in the art is employed.

[0032] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The cells are added to a subculture medium containing a step of DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin so as to achieve an initial density of cells / ml, and then the medium is cultured for 10 to 15 days at 37.0°C ± 0.5°C and 5% ± 1.0% CO2. After observing the yellowing of the subculture medium every 2 to 3 days, half of the subculture medium is replaced.

[0033] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Step A is to cut the tissue into small pieces, centrifuge and wash the tissue pieces to collect the tissue pieces, add them to DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, changing half of the medium every 2 to 3 days, and culturing until cells emerge from the tissue pieces; and step B, shaking the mixture to collect the lower layer cells and washing them with PBS, adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min* for 5-8 minutes, and then collecting and obtaining the cells.

[0034] The object of the present invention is to provide a method for producing a cellular protein extract having a joint repair effect, the method comprising the steps of: Density is 5.0×10 6 pieces / mL~1.0×10 7 Step S-1: placing mesenchymal passaged cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium at 37.0℃±0.5℃ and 5%±1.0% CO2 for 10h-14h, followed by separating and washing the cells to collect them; The collected cells were 5.0 x 10 6 pieces / mL~1.0×10 7Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0035] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0036] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0037] In the preferred technical solution of the present invention, the mesenchymal passage cell density in step S-1 is 5.0 × 10 6 ~2.0×10 7 5.0×10 6 ~1.0×10 7 pieces / mL.

[0038] In a preferred technical solution of the present invention, the mesenchymal passaged stem cells in step S-1 are cultured in the medium for 11 to 13 hours.

[0039] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of washing the cells is 2 to 5 times, preferably 3 to 4 times.

[0040] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0041] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0042] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0043] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0044] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0045] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is frozen and stored, preferably at -40°C to -20°C.

[0046] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically digested using either nuclease or ultranuclease, and then separated and purified.

[0047] For the culture of the passaged mesenchymal stem cells or primary mesenchymal stem cells of the present invention, a culture method known in the art is employed.

[0048] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The cells are added to a subculture medium containing a step, which is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, so that the initial density of the cells is adjusted to 10 cells / ml, and then the medium is cultured at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days. After observing the yellowing of the subculture medium every 2 to 3 days, half of the subculture medium is replaced.

[0049] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Step A is to cut the tissue into small pieces, centrifuge and wash the tissue pieces to collect the tissue pieces, add them to DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, changing half of the medium every 2 to 3 days, and culturing until cells emerge from the tissue pieces; and step B, shaking the mixture to collect the lower layer cells and washing them with PBS, adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min* for 5-8 minutes, and then collecting and obtaining the cells.

[0050] Another object of the present invention is to provide a method for making a joint repair protein composition, the method comprising: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting solution to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution, and storing the solution at 2°C to 4°C for use; and (2) adjusting the enzymatic degradation solution produced in step (1) to 5-15 mg / ml at 2°C-8°C with an elution solvent comprising 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride, passing the solution through a chromatography column at an elution flow rate of 0.1-1 ml / min, and monitoring and collecting the eluted sample at an ultraviolet wavelength of 280 nm.

[0051] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0052] In a preferred technical solution of the present invention, 25 U / mL to 30 U / mL of either one or a combination of nuclease or ultranuclease is added to the cell protein extract prepared by the present invention, and the mixture is subjected to enzymatic hydrolysis at 37°C±1°C for 20 to 30 minutes to prepare an enzymatic hydrolysis solution.

[0053] 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.

[0054] In a preferred technical solution of the present invention, the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0055] In a preferred technical solution of the present invention, the protein composition prepared in step (2) is added with a freeze-drying protection agent, which is any one or combination of selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch, and then freeze-dried to obtain the protein composition.

[0056] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass.

[0057] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the protein composition prepared in step (2), which is any one selected from albumin, zinc salts, and aluminum salts.

[0058] In a preferred technical solution of the present invention, the pH of the lyophilized protein composition preparation is 6-8, preferably 7-7.5.

[0059] In a preferred technical solution of the present invention, the protein composition in the joint repair protein composition is as shown in FIG.

[0060] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted immediately before use with an isotonic solution selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof, and then used by one or a combination of smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture.

[0061] Another object of the present invention is to provide a joint repair composition comprising any one or combination of the cellular protein extract or joint repair protein composition having the joint repair effect of the present invention and a pharma- ceutically acceptable carrier.

[0062] The amount or type of the pharma- ceutically acceptable carrier of the present invention is determined by factors such as the physicochemical properties and content of the active ingredient in the composition, the type of formulation, the dissolution and bioavailability of the formulation, and the like.

[0063] The compositions of the present invention may be in any dosage form known in the art and may be prepared using formulation techniques known in the art.

[0064] In a preferred technical solution of the present invention, the composition is any one selected from the group consisting of freeze-dried agents, gelling agents, nasal sprays, liquid dressings, injections, patches, ointments, creams, emulsions, and suppositories.

[0065] In a preferred technical solution of the present invention, the method of administering the composition is selected from the following: smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture, or any combination thereof.

[0066] Another object of the present invention is to provide the use of a cell protein extract, a joint repair protein composition or a composition thereof having the joint repair effect described in the present invention in the preparation of products for cell repair, joint repair, and cartilage repair.

[0067] In a preferred technical solution of the present invention, the joint repair or cartilage repair is selected from the group consisting of traumatic joint lesions, degenerative osteoarticular lesions, joint damage, intractable wound lesions, osteoarticular lesions of the knee joint, and cartilage damage, or any complication thereof.

[0068] In a preferred technical solution of the present invention, the product is used for bone and joint care people, elderly people with bone degenerative diseases, people injured by exercise, people in exercise care, and people in post-surgery rehabilitation for bone and joint diseases, or any combination thereof.

[0069] Another object of the present invention is to provide a use of compounds 1-16 in stress-inducing stem cells to produce functional proteins with repair effects.

[0070] In a preferred technical solution of the present invention, the repair is one of cell repair, hair follicle repair, joint repair, and repair.

[0071] Unless otherwise stated, when the invention relates to percentages between liquids and liquids, said percentages are volume / volume percentages, when the invention relates to percentages between liquids and solids, said percentages are volume / weight percentages, when the invention relates to percentages between solids and liquids, said percentages are weight / volume percentages and the remainder are weight / weight percentages.

[0072] Unless otherwise specified, the identification of the mesenchymal stem cells (MSCs) of the present invention refers to "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application." Effect of the Invention

[0073] Compared with the conventional technology, the present invention has the following advantageous effects:

[0074] 1. In the present invention, a culture medium containing a stress substance that induces stress so that mesenchymal stem cells produce a cell protein with a cell repair effect is scientifically screened. The obtained cell protein extract, cell protein composition or composition thereof has the effect of cell repair, joint repair, and cartilage repair, and can be used to activate the regeneration of wounded chondrocytes and promote the repair of damage, and can be used to prevent and treat any one of traumatic joint lesions, degenerative osteoarticular lesions, joint damage, intractable wound lesions, osteoarticular lesions of the knee joint, and cartilage damage, or complications thereof, and has the advantages of high purity, good stability, safety and effectiveness, and easy storage and transportation.

[0075] 2. The preparation method of the present invention has the advantages of being simple in operation, environmentally friendly, cost-effective, and suitable for industrial production. [Brief description of the drawings]

[0076] [Figure 1] Electrophoretic separation results of the joint repair protein composition of the present invention. [Diagram 2] 1 shows the results of high performance liquid chromatography detection of the joint repair protein composition of the present invention. [Diagram 3] Test Example 1 is a study of the repair effect of left medial condyle joint damage on days 0 and 49 in test sheep. [Figure 4] Test Example 1 is a study of the repair effect of right lateral plateau joint damage on days 0 and 49 of test sheep. [Diagram 5] 1 shows the clinical score results when the joint repair protein composition of the present invention is used to repair joint or cartilage damage in patients. [Figure 6] 1 shows the functional score results when the joint repair protein composition of the present invention is used to repair joint or cartilage damage in patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The present invention will be further explained and described in detail with reference to the following specific examples, but the scope of protection of the present invention is not limited thereto.

[0078] 1. Cultivation of primary mesenchymal stem cells Primary mesenchymal stem cell culture After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Then, cut the tissue into small pieces, centrifuge and wash to collect the tissue pieces, place them in a culture flask, add DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture it under 37℃ and 5% CO2 conditions to promote adhesion to the wall. After observing the yellowing of the medium every 2-3 days, replace half of the medium and culture for 10-12 days until cells are seen emerging from the edge of the tissue pieces. 2) gently shaking the plate to drop the tissue fragments, collecting the tissue fragments and the lower layer cells, and then reattaching and culturing the collected tissue fragments; The collected lower layer cells are washed with PBS, and then an appropriate amount of 0.25% trypsin is added to digest the cells for 2 to 3 minutes. The digestion is stopped by adding an equal volume of trypsin stop solution. The bottom of the flask is gently blown with a pipette, and the cells are centrifuged at 1500 rpm / min for 5 minutes. Then, the cells are collected and obtained.

[0079] 2. Subculture of primary mesenchymal stem cells Subculture of primary mesenchymal stem cells: 1.0×10 primary mesenchymal stem cells 5 ~6.0×10 5 Add the cells to DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin to an initial density of cells / ml, then culture it at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days. After observing the yellowing of the medium every 2 to 3 days, replace half of the medium.

[0080] 3. For the preparation of compounds 1 to 16, refer to Reference 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).

[0081] Example 1: Preparation of cell protein extract having joint repair effect according to the present invention The method for producing a cell protein extract having a joint repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6Step (1) of adding the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 5μmol / L compound 16 so as to achieve a density of 100μmol / mL cells; and then culturing the medium at 37℃ and 5% CO2 for 12h, centrifuging the medium at 1200rpm*5min, washing the medium with PBS three times, and then collecting the cells. The cells collected in step (1) were diluted to 1.0 × 10 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0082] Example 2: Preparation of joint repair protein composition of the present invention The joint repair protein composition of the present invention can be prepared by: (1) adding 25 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 1, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0083] Example 3: Preparation of a freeze-dried preparation of the joint repair protein composition of the present invention The cell protein composition prepared in Example 2 was added with a required amount of mannitol, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 2% mannitol (m / m).

[0084] (Example 4) Preparation of cell protein extract having joint repair effect of the present invention The method for producing a cell protein extract having a joint repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6 To obtain a density of 1000 cells / mL, add the medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 8μmol / L compound 13, then culture it at 37℃, 5% CO2 for 12h, then centrifuge it at 1200rpm*5min, wash it with PBS three times, and collect the cells (1); The cells collected in step (1) were diluted to 1.0 × 10 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0085] Example 5: Preparation of joint repair protein composition of the present invention The joint repair protein composition of the present invention can be prepared by: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 4, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0086] Example 6: Preparation of a lyophilized preparation of the joint repair protein composition of the present invention Dextran was added to the cell protein composition prepared in Example 5, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 5% dextran (m / m).

[0087] (Example 7) Preparation of cell protein extract having joint repair effect of the present invention The method for producing a cell protein extract having a joint repair effect of the present invention comprises the steps of: Mesenchymal cell lineages 6.0 x 10 6 To obtain a density of 1000 cells / mL, add the medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 6μmol / L compound 14, then culture it at 37℃, 5% CO2 for 12h, then centrifuge it at 1200rpm*5min, wash it with PBS three times, and collect the cells (1); The cells collected in step (1) were 9.0 × 10 6(2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0088] Example 8: Preparation of joint repair protein composition of the present invention The joint repair protein composition of the present invention can be prepared by: (1) adding 20 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 7, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic digestion solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in sequence at an elution flow rate of 0.8 ml / min and monitoring and collecting the elution site at an ultraviolet wavelength of 280 nm.

[0089] (Example 9) Preparation of a lyophilized preparation of the joint repair protein composition of the present invention Sorbitol was added to the cell protein composition prepared in Example 8, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 3% sorbitol (m / m).

[0090] (Example 10) Preparation of cell protein extract having joint repair effect of the present invention The method for producing a cell protein extract having a joint repair effect of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7 To obtain a density of 1000 cells / mL, add the medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 7μmol / L compound 15, then culture it at 37℃, 5% CO2 for 12h, then centrifuge it at 1200rpm*5min, wash it with PBS three times, and collect the cells (1); 5.0 × 10 cells collected in step (1) 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0091] Example 11: Preparation of joint repair protein composition of the present invention The joint repair protein composition of the present invention can be prepared by: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 10, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride under conditions of 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the elution site at an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0092] (Example 12) Preparation of a lyophilized preparation of the joint repair protein composition of the present invention Mannitol was added to the cell protein composition prepared in Example 11, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 5% mannitol (m / m).

[0093] (Example 13) Preparation of freeze-dried preparation of cell protein extract of the present invention The cell protein extract prepared in Example 1 was added with a required amount of mannitol, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 2% mannitol (m / m).

[0094] Example 14: Measurement of molecular weight distribution of the joint repair protein composition of the present invention The molecular weight distribution of the nerve repair protein composition of the present invention was measured using standard molecular weight polyacrylamide gel electrophoresis, Step 1: select a glass plate with a thickness of 1.5 mm, place it horizontally, and apply the prepared gel solutions (15% lower gel, 4% separating gel) on the glass plate in sequence according to Table 1, and insert the comb vertically into the lower gel; [Table 1] After thawing the marker (20KDa-245KDa) frozen at -40°C, the marker and the freeze-dried powder of the joint repair protein composition prepared in Example 3, Example 6, Example 9, and Example 12 are mixed with PBS to prepare a test sample of 10ug / ul, and 20ul of the test sample is added to the loading well, and then electrophoresis is performed under 60-80V conditions until a relatively clear band appears, and the voltage is adjusted to 100-120V until the marker separation is completed, and the PAGE gel is placed in Coomassie Brilliant Blue staining solution, and when a clear band appears on the gel, the staining is stopped, and after washing with pure water, it is destained with a 10% acetic acid solution, and destaining is stopped when the gel becomes transparent. The results are shown in Figure 1, where band A is Example 3, band B is Example 6, band C is Example 9, and band D is Example 12.

[0095] Example 15: High performance liquid chromatography detection of the joint repair protein composition of the present invention The lyophilized powder of the joint repair protein composition of Example 3 is dissolved in deionized water and then made into a test solution of 10 mg / ml. Chromatography column: SHIMSEN Ankylo (300mm*4.6mm.D., 3um; P / N: 380-01215-05) Shimadzu Mobile phase: 50 mmol / L phosphate buffer (pH=6.8) containing 300 mmol / L sodium chloride, flow rate is 0.3 mL / min, injection volume is 10 ul, column temperature is 25° C., measurement wavelength is 280 nm, isocratic elution, 30 min collection. The results are shown in FIG. 2.

[0096] (Test Example 1) Study of the effect of the cell protein extract having joint repair effect of the present invention on joint repair 120 mg of the freeze-dried powder of the cell protein extract 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.

[0097] 1. Experimental Method A male goat, aged 12 months and weighing 47 kg, was generally bred under general anesthesia, and an 8 cm midline incision was made on the left medial condyle, and the medial edge of the patellar tendon was incised to the joint to expose the medial femoral condyle and the medial tibial plateau. The surface of the articular cartilage was found to be smooth, and a cartilage injury wound measuring 0.3×0.3×0.6 cm (length×width×depth) was made on the medial femoral condyle with a 3 mm ball drill, and the incision was sutured (see Figure 3). An 8 cm incision was made in the middle of the right lateral plateau, and the layer was separated, and the joint capsule was entered from the outside of the patellar tendon to expose the lateral femoral condyle and the lateral tibial plateau. The cartilage was found to be intact and smooth, and a cartilage injury wound measuring 0.6×0.3×0.3 cm (length×width×depth) was made on the outside of the plateau with a 3 mm ball drill, and the incision was sutured (see Figure 4).

[0098] Goats were injected with 2.5 ml of the freeze-dried cell protein extract solution prepared in Example 6 into the knee joint cavity of one side every week for three consecutive weeks. Observations were performed once every morning and evening. The results are shown in Table 2.

[0099] [Table 2]

[0100] After 49 days of observation, the experimental goats were anesthetized and sacrificed, and materials were taken to prepare cartilage slices (see Figures 3-4).

[0101] For this project, seven safety tests have already been completed, including heat source measurements, acute systemic toxicity tests, cytotoxicity tests, skin sensitization tests, intradermal reaction tests, local tissue reactions and decomposition tests after transplantation, and subacute systemic toxicity tests, and no side effects were observed.

[0102] The cell protein extract of the present invention has an excellent effect of repairing joint damage, and is safe and effective.

[0103] (Test Example 2) Study of the effect of the joint repair protein composition of the present invention in repairing joint or cartilage damage The effect of the joint repair protein composition of the present invention in repairing joint damage or cartilage damage was examined and evaluated using 21 patients with joint lesions aged 50 to 80 years old (see Table 3).

[0104] Subject selection criteria: (i) Patients diagnosed with osteoarthritis of the knee based on the American College of Rheumatology (ACR) classification criteria; (ii) Patients who have knee pain due to various causes that continues for more than six months, or who have improved after conventional clinical treatment but whose osteoarthritis has recurred or worsened after discontinuing administration, and who have pain in the knee joint for more than 4 minutes when walking on a flat road, and this phenomenon continues for at least 4 weeks; (iii) Patients whose knee joint on the study side had a Kellgren-Lawrence grade of II to III and whose knee joint on the other side had a Kellgren-Lawrence grade of less than II, as observed by radiographic examination; (iv) Patients aged 30–80 years, regardless of gender, who were able to complete four follow-up visits within six months of initial treatment (2 weeks, 4 weeks, 3 months, and 6 months after treatment); (v) Patients who are able to understand and willingly sign the informed consent form and willingly complete the study procedures and follow-up surveys.

[0105] Subject Exclusion Criteria: 1) Patients with secondary osteoarthritis (history and / or any evidence that the observed joint is affected by septic arthritis, inflammatory joint disease, gout, recurrent pseudogout, Paget's bone disease, joint fracture, histological malformation, acromegaly, hemochromatosis, Wilson's disease, primary osteochondroma, genetic diseases (e.g., hypermobility disorder), and mutations in the collagen gene), 2) Patients with other rheumatic diseases, including but not limited to systemic lupus erythematosus, inflammatory bowel disease, Felty's syndrome, scleroderma, inflammatory myopathies or other connective tissue diseases, overlap syndromes, etc. 3) Patients who have received intra-articular drug injections within the past 12 weeks. 4) Patients who have undergone arthroscopy, orthopedic surgery, or total joint replacement within the past 6 months 5) Patients requiring arthroplasty, 6) Patients who used anticoagulants (e.g., warfarin, low molecular weight heparin) and antiplatelet aggregation agents. 7) Patients with a tumor disease or a history of tumor disease. 8) Patients with severe, poorly controlled diseases such as diabetes, hypertension, kidney disease, liver disease, or severe heart disease (e.g., moderate or severe congestive heart failure (New York Heart Association cardiac function class III / IV)) who are judged by the investigator to be inappropriate for inclusion in this study. 9) Patients with abnormal laboratory test results (routine blood tests: WBC<3x10^9 / L, HGB<90g / L, PLT<100x10^9 / L, liver function: TBIL>1.5x upper limit of normal, ALT or AST>2.5x upper limit of normal, renal function: creatinine>1.5x upper limit of normal, with creatinine retention rate <50ml / min (actual measurement or calculated value using the Cockcroft-Gault formula), HIVAb positive, HBsAg positive, HBcAb positive, HCVAb positive, syphilis antibody positive (excluded if any one test result is positive)). 10) Patients who have participated in other drug clinical trials within the previous 3 months were screened. 11) Screening patients who had received intraarticular, intramuscular, or intravenous injections of corticosteroids within the previous 4 weeks. 12) Pregnant, breastfeeding, or preparing to become pregnant women; 13) Patients who have used stem cell therapy within the past six months 14) Patients with mental disorders or uncontrolled or poorly controlled mental illnesses 15) Patients undergoing medical litigation 16) Alcoholics or patients with a history of drug abuse, and patients with an allergic constitution or a history of allergies. 17) Patients who the investigators consider to be unsuitable for participation in this clinical trial.

[0106] [Table 3-1] [Table 3-2]

[0107] The therapeutic effect of the evaluation drug was observed using the knee joint function score KSS (Keen Society Score) (see Table 4). 65 mg of the lyophilized powder of the joint repair protein composition of Example 3 was dissolved in 5 ml of physiological saline, and the solution was injected into the knee joint cavity of one side of the subject once a week, and three times per treatment course. The results are shown in Figures 5 to 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.

[0108] [Table 4]

[0109] The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various modifications or variations based on the present invention, and any such modifications or variations should fall within the scope of protection of the claims of the present invention as long as they do not deviate from the spirit of the present invention.

Claims

1. A joint repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of either one of nuclease or ultranuclease or a combination thereof to the cell protein extract, and subjecting the resulting solution to enzymatic degradation at 37°C ± 1°C for 15 to 40 minutes to prepare an enzymatic degradation solution, which is then stored at 2°C to 4°C for use; and (2) preparing the enzymatic decomposition solution prepared in step (1) at 5 to 15 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a chromatography column at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the eluted portion at an ultraviolet wavelength of 280 nm. Protein composition.

2. The molecular weight of the joint repair protein composition is between 25 kDa and 245 kDa, preferably between 50 kDa and 200 kDa; The protein composition of claim 1.

3. The preparation of the cell protein extract comprises: Density is 5.0 x 10 ６ pieces / mL ~ 1.0×10 ７ The mesenchymal passaged cells (cells / mL) were placed in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15 ug / mL epidermal growth factor (EGF), 1-15 ug / mL fibroblast growth factor (FGF), 1-15 ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10 μmol / L of any one or combination of compounds 1-16, and then incubated at 37.0° C.±0.5° C., 5%±1.0% CO ２ Step S-1: culturing the cells under the above conditions for 10 to 14 hours, then isolating, washing, and collecting the cells; 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 The collected cells were 5.0 × 10 ６ pieces / mL ~ 1.0×10 ７ Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... Step S-3: separating the cell lysate prepared in step S-2, and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate. A protein composition according to any one of claims 1 to 2.

4. The mesenchymal stem cells of step S-1 are cultured in the medium for 11 to 13 hours. A protein composition according to any one of claims 1 to 3.

5. The separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000 to 2000 rpm * 3 to 15 min, preferably 1200 rpm to 1500 rpm * 5 to 10 min; A protein composition according to any one of claims 1 to 4.

6. The ultrasonic conditions for step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds. A protein composition according to any one of claims 1 to 5.

7. Add a freeze-drying protection agent selected from any one or combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch to the elution site collected in step (2), and freeze-dry to obtain a product. A protein composition according to any one of claims 1 to 6.

8. The lyophilized formulation has a pH of 6 to 8, preferably a pH of 7 to 7.

5. A protein composition according to any one of claims 1 to 7.

9. A joint repair composition comprising the joint repair protein composition of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

10. Use of a joint repair protein composition according to any one of claims 1 to 8 or a joint repair composition according to claim 9 in the production of a cell repair, joint repair, or cartilage repair product.

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