Chondrocytes for treating osteoarthritis and preparation method and application thereof

Abstract

The present invention discloses chondrocytes for treating osteoarthritis, and a preparation method and application thereof, which relate to the field of biomedical technology, and include the following process: taking mesenchymal stem cells and collagen microspheres for mixing, inoculating them in a culture device for culture, adding induction culture medium for induction culture, harvesting, and obtaining chondrocytes. The present invention mixes mesenchymal stem cells with collagen microspheres so that the mesenchymal stem cells are adsorbed / inoculated on the collagen microspheres, the cells are amplified, and cultured induction is performed. The mesenchymal stem cells are induced to form chondrocytes to obtain chondrocytes, and the product is collected, resuspended in 0.9% physiological saline, and injected locally into the bone and joints. It can be used for the treatment of osteoarthritis, reducing the adverse reactions caused by the use of traditional drugs and the damage caused by surgical methods. Chondrocytes fundamentally solve the cause of osteoarthritis by repairing cartilage through cells, and have a significant therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, in particular to a chondrocyte sphere for treating osteoarthritis and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA), a chronic degenerative disease, is characterized by degeneration of articular cartilage, changes in subosseous structures, and damage to the soft tissues surrounding the joints. OA is one of the most common joint diseases worldwide, with its prevalence increasing significantly with age. OA often causes joint pain, motor dysfunction, and even disability, significantly impacting patients' quality of life. Currently, traditional treatments for OA include medication, physical therapy, and, in severe cases, surgery. Medication includes nonsteroidal anti-inflammatory drugs, metalloproteinase inhibitors, intra-articular injections of glucocorticoids and hyaluronic acid, while surgery includes osteochondral transplantation and total joint replacement. However, both treatments can only provide palliative effects and are unable to promote regeneration of damaged cartilage. Medication often comes with significant adverse reactions, while surgery can cause significant pain and injury to patients, with limited effectiveness in preventing disease progression or repairing damaged articular cartilage.

[0003] With the development of tissue engineering and cell regeneration technologies, researchers have discovered that through their inherent anti-inflammatory and immunomodulatory abilities and the active factors they secrete, mesenchymal stem cells can regulate the local microenvironment of damaged cartilage, protecting it from further damage. However, depending on the route of administration, the duration of mesenchymal stem cells' residence in the body varies, and they are cleared from the body over time. Furthermore, influenced by the disease microenvironment, stem cells can undergo spontaneous migration and homing, making it difficult to maintain large numbers of stem cells around joints for extended periods.

[0004] In recent years, the development of microsphere drug delivery systems has provided a promising option for the targeted and sustained-release delivery of macromolecular drugs, including nucleic acids, proteins, cells, and cytokines. Microspheres can effectively deliver macromolecular drugs to their targets, enhancing their efficacy. Therefore, combining microspheres with mesenchymal stem cells, either systemically or through local injection, could address a significant challenge in current cell-based applications. Therefore, we propose chondrocytes for the treatment of osteoarthritis, as well as their preparation and application. Summary of the Invention

[0005] The purpose of the present invention is to provide a chondrocyte for treating osteoarthritis and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing chondrocytes for treating osteoarthritis, comprising the following process:

[0007] Mesenchymal stem cells are mixed with collagen microspheres, inoculated into a culture device for culture, induction culture medium is added for induction culture, and harvested to obtain chondrocytes.

[0008] Furthermore, mesenchymal stem cells (MSC cells) used were cells of passage 3 to 5, which were derived from umbilical cord mesenchymal stem cells prepared by laboratory personnel of our unit.

[0009] Furthermore, mesenchymal stem cells were cultured using a special culture medium for human mesenchymal stem cells. Primary cells were isolated from the umbilical cord and passaged at a ratio of 1:3 when the cells grew to 80% to 90%. Stem cells of passage 3 to 5 were selected for subsequent experiments.

[0010] Then, the cells were mixed with a culture medium containing collagen microspheres according to an appropriate seeding density; the culture medium was DMEM / F12 basal culture medium.

[0011] Furthermore, the induction medium is: DMEM / F12 basal medium and 8-12 ng / mL transforming growth factor β3 (TGFβ3), 0.09-0.11 μmol / mL dexamethasone, 90-110 μg / mL sodium pyruvate, 38-42 μg / mL proline, and 45-54 μg / mL ascorbic acid in DMEM / F12 basal medium.

[0012] Transforming Growth Factor (TGF) in the induction culture medium is a multifunctional protein polypeptide that promotes cell proliferation, regulates cell differentiation, and enhances extracellular matrix synthesis. TGFβ3, a bioactive molecule that can induce chondrogenesis, promotes chondrocyte proliferation and the synthesis of extracellular matrix (such as proteoglycans and hyaluronidase), thereby enhancing the ability of mesenchymal stem cells to differentiate into cartilage and achieve chondrogenic differentiation. Dexamethasone, a long-acting adrenal glucocorticoid, directly binds to the glucocorticoid receptor and promotes the expression of bone morphogenetic protein-6 (BMP-6). BMP-6, in turn, induces the expression of TGFβ receptor. This combination promotes the differentiation of mesenchymal stem cells into chondrocytes. Sodium pyruvate helps maintain intracellular acid-base balance, stabilizes the intracellular environment, and promotes chondrocyte growth and proliferation. Proline, an amino acid required for the production of collagen and cartilage, promotes the formation of new cartilage and the strengthening of chondrocytes. Ascorbic acid can promote the hydroxylation of lysproline during collagen formation, thereby promoting the formation of the three-dimensional structure of the chondrocytes.

[0013] The induction culture process is as follows: stop the automated culture stirring program, wait for the MSC-loaded collagen microspheres to settle, and then pump out the excess supernatant. Take a sample for cell counting and add induction medium based on the counted number of cells. Replace the induction medium with fresh one every three days until chondrocyte spheres form on day 15. Samples are then stained with Alcian blue for identification.

[0014] Furthermore, the inoculation ratio of collagen microspheres to mesenchymal stem cells is 96-105 mg collagen microspheres per million MSC cells.

[0015] Furthermore, the culture equipment is a 3D automated culture device, purchased from Beijing Huakan Biological Co., Ltd. Compared to two-dimensional cell culture technology, 3D cell culture can better simulate the growth and activity of cells in a real environment; it can also better promote cell-cell and cell-matrix interactions, thereby increasing intercellular communication, which is conducive to the plasticity of cell growth and differentiation.

[0016] Furthermore, the inoculation process is as follows: mesenchymal stem cells and collagen microspheres are pumped into the automated culture tank through the loading tube of the 3D automated culture equipment, the temperature is turned on, the stirring program, temperature, and pH are set, and the speed is subsequently adjusted according to the growth status of the cells.

[0017] Furthermore, the 3D automated culture equipment was pre-configured before inoculation. The pre-configuration process included connecting the sterilized sample loading line of the 3D automated culture equipment, opening the surface and deep vents, and introducing oxygen, carbon dioxide, and air to ensure the internal environment of the automated culture equipment matched that of conventional cell culture. A dissolved oxygen electrode was connected, stirring was initiated, and oxygen consumption in the automated culture equipment was monitored overnight.

[0018] Furthermore, the harvesting process is as follows: stop all programs of the 3D automated culture equipment, pump the culture medium mixture containing the chondrocytes into a centrifuge bottle, let it stand, discard the supernatant, add physiological saline or PBS to wash 2 to 4 times, resuspend the chondrocytes with physiological saline according to the dosage requirements, and divide them for use.

[0019] In the above technical solution, mesenchymal stem cells are mixed with collagen microspheres, allowing the mesenchymal stem cells to be adsorbed / seeded on the collagen microspheres. The cells are then expanded and cultured using chondrocyte induction medium, inducing the mesenchymal stem cells to form chondrocytes. The resulting product, chondrocytes, is collected and resuspended in 0.9% saline solution for local injection into joints. This can be used to treat osteoarthritis, reducing the adverse reactions associated with traditional medications and the damage caused by surgical procedures. Chondrocytes fundamentally address the cause of osteoarthritis by repairing cartilage through cell-mediated repair, demonstrating significant therapeutic efficacy.

[0020] Furthermore, collagen microspheres are prepared by the following process:

[0021] Step 1: Dissolve hydroxypropyl cellulose in dimethylacetamide, add methylsulfonyl chloride, and stir at 68-72°C for 150-200 minutes;

[0022] Add rosin acid and continue the reaction for 8 to 12 hours; precipitate, wash, and dry to obtain a rosin-based hydroxypropyl cellulose compound;

[0023] Step 2: Take the collagen solution, adjust the pH of the system to 3.5-4.5, add rosin-based hydroxypropyl cellulose and mix, and let it stand at 36-38°C for 1-6 hours; collect the colloid, freeze-dry it, and irradiate it for sterilization to obtain collagen microspheres.

[0024] Furthermore, in step 1, the mass ratio of hydroxypropyl cellulose, methylsulfonyl chloride, and rosin acid is 10: (5.6-5.9): (9.0-9.5);

[0025] The ratio of hydroxypropyl cellulose and dimethylacetamide is 10 g / 250 mL.

[0026] Furthermore, in step 1, after the reaction, petroleum ether is added to precipitate the product, which is then washed three times with petroleum ether and dried under vacuum to obtain a rosin-based hydroxypropyl cellulose compound;

[0027] The ratio of hydroxypropyl cellulose, petroleum ether for precipitation, and petroleum ether for washing is 10 g: 1000 mL: 500 mL.

[0028] Furthermore, in step 2, the mass ratio of collagen to rosin-based hydroxypropyl cellulose is (2-3): (2-3).

[0029] Furthermore, in step 2, rosin-based hydroxypropyl cellulose is added in the form of a solution with a concentration of 10 wt %; the concentration of the collagen solution is 10 wt %;

[0030] All solvents were sterilized deionized pure water.

[0031] The collagen solution and the rosin-based hydroxypropyl cellulose solution are in equal volume ratios.

[0032] Furthermore, in step 2, the reagent used to adjust the pH is 1 M hydrochloric acid (HCl).

[0033] Furthermore, in step 2, the collagen is bovine collagen.

[0034] In this technical solution, hydroxypropyl cellulose is mixed with methylsulfonyl chloride under anhydrous conditions, resulting in nucleophilic substitution of the sulfonyl chloride groups with the hydroxyl groups. This acylation reaction generates a sulfonyl ester compound, which then introduces a sulfonamide group to produce sulfonamide cellulose. Abietic acid is then added, and the hydroxyl groups in the sulfonamide cellulose structure undergo an esterification reaction with the carboxyl groups to produce rosin-based hydroxypropyl cellulose. The rosin-based hydroxypropyl cellulose is then mixed with collagen in an aqueous solution, hydrogen-bonded, and freeze-dried to form porous collagen microspheres, which serve as a carrier for chondrocytes.

[0035] As a natural biopolymer material, collagen microspheres have excellent biocompatibility, biodegradability, and low immunogenicity, and are widely used in the field of biomedical materials. Porous microspheres prepared based on collagen have good affinity with cell tissues, and feature a large specific surface area, low density, and good cell adhesion. As a cell culture and drug delivery system, by combining collagen microspheres with chondrocytes, chondrocytes are adsorbed / loaded onto the collagen microspheres, effectively preventing chondrocyte inactivation in vivo and improving cell stability to better exert their effectiveness. The porous nature of collagen microspheres facilitates chondrocyte regeneration in vivo, controlling the release rate of chondrocytes and achieving a sustained-release effect. They also have excellent targeting, reaching the lesion site, allowing the chondrocytes to fully function, thereby achieving a better therapeutic effect for osteoarthritis.

[0036] Hydroxypropyl cellulose acts as a disintegrant, causing collagen microspheres to dissociate when applied, promoting the sustained release of chondrocytes. The methylsulfonamide structure introduced by the reaction can enhance the interaction between the chondrocytes and the target protein, improving their activity and selectivity. It can also improve the sustained release and metabolic stability of the chondrocytes. The introduction of the rosin ester structure can serve as the skeleton material of the collagen microspheres and can interfere with the synthesis of bacterial cell walls, affecting bacterial metabolic processes, and has certain anti-inflammatory activity, exerting anti-inflammatory and bactericidal effects, and preventing infection.

[0037] Furthermore, the bovine collagen is surface modified, and the specific process is as follows:

[0038] Take collagen, dissolve it in phosphate buffer, adjust the pH of the system to 7.2-7.5, slowly add crotonic anhydride within 20-30 minutes, adjust the system temperature to 48-52°C, and react for 150-200 minutes; transfer it to a dialysis bag with a molecular weight cutoff of 500D, and dialyze it in water to obtain functionalized collagen.

[0039] Furthermore, the mass ratio of collagen to crotonic anhydride is 10:(2.0-2.8);

[0040] The ratio of collagen to phosphate buffer is 5 g:100 mL.

[0041] Furthermore, collagen microspheres are prepared by the following process:

[0042] Functionalized collagen, rosin-based hydroxypropyl cellulose, and thiolated chondroitin sulfate are mixed in a phosphate buffer solution with a pH of 7.2 to 7.5, a photoinitiator is added, the mixture is stirred, and ultraviolet light is irradiated for 10 to 15 minutes; the mixture is washed with ultrapure water, washed with gradient ethanol, freeze-dried, and sterilized by irradiation to obtain collagen microspheres.

[0043] Furthermore, the collagen microspheres include the following components by mass: 10 parts of functionalized collagen, 7 to 15 parts of rosin-based hydroxypropyl cellulose, 1 to 3 parts of thiolated chondroitin sulfate, and 0.2 to 1.0 parts of photoinitiator I2959;

[0044] The wavelength of ultraviolet light is 10-400nm, and the light intensity is 10-15mW / cm 2 .

[0045] Furthermore, the thiolated chondroitin sulfate is prepared by the following process:

[0046] Chondroitin sulfate is mixed with deionized water, the pH is adjusted to 5-6, EDC and NHS are added, and the mixture is stirred for activation for 150-200 minutes; 3-mercaptotyramine hydrochloride is added, and the mixture is reacted in the dark for 24 hours; ethanol is precipitated, and vacuum drying is performed to obtain thiolated chondroitin sulfate.

[0047] Furthermore, the ratio of chondroitin sulfate to deionized water was 5 g:100 mL;

[0048] The mass ratio of chondroitin sulfate and 3-mercaptotyramine hydrochloride is 10: (6.7-7.0);

[0049] The mass ratio of chondroitin sulfate, EDC, and NHS is 10:(7.6-7.8):(4.5-4.7);

[0050] EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0051] NHS is N-hydroxysuccinimide.

[0052] In the above technical solution, collagen and crotonic anhydride are mixed, and the amino group reacts with the anhydride to introduce double bonds into the collagen to form a crotonamide structure, thereby generating functionalized collagen, which can promote the osteogenic differentiation of mesenchymal stem cells, contribute to the proliferation of chondrocytes and the formation of chondrocytes.

[0053] 3-Mercaptotyramine hydrochloride is mixed with chondroitin sulfate. Under the action of EDC and NHS, the carboxyl group is activated and reacts with the amino group in 3-mercaptotyramine hydrochloride, introducing a sulfhydryl group into the chondroitin sulfate, causing it to be sulfhydrated to form thiolated chondroitin sulfate, which can promote the synthesis and repair process of chondrocytes, contribute to the growth and maintenance of cartilage tissue; inhibit the activity of cartilage-degrading enzymes, alleviate the wear and degeneration of articular cartilage, and protect the joints; help improve joint lubricity, reduce joint friction and wear; and inhibit protein adsorption and bacterial adhesion, reduce joint inflammation and pain, thereby improving joint function.

[0054] Under the action of light initiation, thiolated chondroitin sulfate is used as a cross-linking agent to covalently cross-link and assemble with functionalized collagen with double bonds and rosin-based hydroxypropyl cellulose to form collagen microspheres. The prepared collagen microspheres have better mechanical properties and biological activity, can effectively cope with the fluid shear brought by the reactor, exert their buffering effect, ensure the activity of chondrocytes, and improve their survival rate; the collagen microspheres have good stability and biocompatibility, are not easy to dissociate during culture, which helps to maintain their complete morphology, cells are more likely to grow and differentiate on the surface of the collagen microspheres, and the cell phenotype is effectively maintained. They are suitable as a regeneration scaffold for cartilage tissue, help promote the catabolism and anabolism of adjacent chondrocytes, accelerate cartilage formation, and achieve cartilage tissue repair and regeneration.

[0055] Application of cartilage balls for treating osteoarthritis, and application as osteoarthritis treatment materials.

[0056] In the above technical solution, the chondrocytes are resuspended in 0.9% normal saline, which has injectable properties and can be locally injected into bone joints for the treatment of osteoarthritis. Chondrocytes are used to repair cartilage, and the sustained release of chondrocytes and the promotion of cartilage tissue repair and regeneration are achieved, which fundamentally solves the cause of osteoarthritis and has a significant therapeutic effect.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. The method for preparing chondrocytes for treating osteoarthritis described in the present invention uses hydroxypropyl cellulose, methylsulfonyl chloride, rosin acid, and collagen as the main raw materials to prepare collagen microspheres, which adsorb / load chondrocytes and serve as carriers. This can effectively prevent chondrocyte inactivation in the body and improve cell stability so that they can better exert their effectiveness. The porous characteristics of the collagen microspheres are conducive to the regeneration of chondrocytes in the body and the control of the release rate of chondrocytes to achieve sustained release. They can also target the lesion site, fully utilize the functionality of chondrocytes, and achieve the treatment of osteoarthritis.

[0059] 2. The present invention describes a method for preparing chondrocytes for treating osteoarthritis. The collagen microspheres are prepared using hydroxypropyl cellulose, methylsulfonyl chloride, rosin acid, and collagen as the primary raw materials. Hydroxypropyl cellulose dissociates during application, promoting sustained release of chondrocytes. The introduced methylsulfonamide groups enhance the interaction between the chondrocytes and target proteins, increasing their activity and selectivity. Furthermore, the chondrocytes' sustained release and metabolic stability are improved. The introduced rosin acid ester structure interferes with bacterial cell wall synthesis, affecting bacterial metabolism and achieving anti-inflammatory, bactericidal, and infection-preventing effects.

[0060] 3. The method for preparing chondrocytes for treating osteoarthritis described in the present invention utilizes crotonic anhydride to modify collagen to form a crotonamide structure, generating functionalized collagen that can promote the osteogenic differentiation of mesenchymal stem cells. 3-Mercaptotyramine hydrochloride is then reacted with chondroitin sulfate to form thiolated chondroitin sulfate, which promotes the synthesis and repair process of chondrocytes and improves joint function. Subsequently, under the action of light, the thiolated chondroitin sulfate, functionalized collagen, and rosin-based hydroxypropyl cellulose are cross-linked and assembled to form collagen microspheres. These microspheres have superior mechanical properties and bioactivity, can effectively withstand the fluid shear generated by the reactor, and improve chondrocyte survival rate. The collagen microspheres are not easily dissociated in the culture matrix, allowing the cells to effectively maintain their phenotype.

[0061] 4. The chondrocytes for treating osteoarthritis described in the present invention are resuspended in 0.9% physiological saline to make them injectable and can be injected locally into bone joints to treat osteoarthritis, achieve sustained release of chondrocytes, utilize chondrocytes to repair cartilage, promote cartilage tissue repair and regeneration, fundamentally solve the cause of osteoarthritis, and have significant therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a 100x staining image of a cartilage ball section in Example 1 of the present invention;

[0063] Figure 2 This is a 400x staining image of a cartilage ball section in Example 1 of the present invention;

[0064] Figure 3 This is an electron microscope image of the collagen microspheres in Example 1 of the present invention;

[0065] Figure 4 2.5x H&E section images of the forelimb and hindlimb joints of rats after administration;

[0066] A is a 2.5x H&E section of a normal left hind limb joint of a Lewis rat before the experiment of the present invention;

[0067] B is a 2.5x H&E section of the right hind limb joint of a rat after the experiment in Example 2 of the present invention;

[0068] C is a 2.5x H&E section of the right hind limb joint of a rat after the experiment in Example 1 of the present invention;

[0069] D is a 2.5x H&E section of the left hind limb joint of a rat after the experiment in Comparative Example 1 of the present invention;

[0070] E is a 2.5x H&E section of the left hind limb joint of the arthritis-like rat model induced by bovine type II collagen of the present invention;

[0071] Figure 5 This is a diagram of osteogenic differentiation of mesenchymal stem cells in Example 1 of the present invention, stained with alizarin red for osteogenic differentiation and observed at 100x magnification;

[0072] Figure 6 This is a diagram of adipogenic differentiation of mesenchymal stem cells in Example 1 of the present invention, using Oil Red O staining for adipogenic staining and observed at 200 times magnification;

[0073] Figure 7 This is a diagram of the chondrogenic differentiation of mesenchymal stem cells in Example 1 of the present invention, stained with Alcian blue for chondrogenic differentiation and observed at 100x magnification;

[0074] Figure 8 This is an electron micrograph of the collagen microspheres in Example 4 of the present application;

[0075] Figure 9 This is a fluorescent staining image of the chondrocytes in Example 4 of the present application. DETAILED DESCRIPTION

[0076] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0077] In the following specific embodiments:

[0078] Human mesenchymal stem cells (hMSCs) were isolated from the umbilical cord. Primary cells were passaged at a 1:3 ratio until the cells reached 85 ± 5% growth. Stem cells from passage 5 were used in the following experiments. 3DFloTrix serum-free mesenchymal stem cell medium was used as the culture medium, which was purchased from Beijing Huakan Biotechnology Co., Ltd.

[0079] The inoculation medium was DMEM / F12 basal medium, which was obtained from Beijing Huakan Biological Co., Ltd.

[0080] Hydroxypropyl cellulose: molecular weight 95,000, from Hercules, USA;

[0081] Collagen: bovine type II collagen, sourced from Sigma, USA;

[0082] 3D automated culture equipment, sourced from Beijing Huakan Biological Co., Ltd.;

[0083] The freeze-drying temperature is selected to be -20°C;

[0084] Incomplete Freund's adjuvant, from Sigma, USA;

[0085] In Example 3-5, the concentration of the phosphate buffer was 0.1M.

[0086] Example 1: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0087] Step 1: Dissolve 10 g of hydroxypropyl cellulose in 250 mL of dimethylacetamide, add 5.68 g of methylsulfonyl chloride, and stir at 70°C for 180 minutes; add 9 g of rosin acid and continue the reaction for 10 hours; add 1000 mL of petroleum ether for precipitation, wash three times with 500 mL of petroleum ether, and vacuum dry to obtain a rosin-based hydroxypropyl cellulose compound;

[0088] A 10 wt% collagen solution was taken, the pH of the system was adjusted to 4.0 with 1 M hydrochloric acid, and an equal volume of 10 wt% rosin-based hydroxypropyl cellulose solution was added and mixed, and the mixture was allowed to stand at 37°C for 12 hours. The colloid was collected, washed with ultrapure water, washed with gradient ethanol, freeze-dried for 12 hours, and irradiated for sterilization to obtain collagen microspheres.

[0089] Step 2: Pre-set the 3D automated culture equipment. The process is as follows: connect the pre-sterilized 3D automated culture equipment's sample loading line, open the surface and deep vents, and introduce oxygen, carbon dioxide, and air to ensure the automated culture equipment's internal environment meets the standard cell culture environment of 5% CO2 and 37°C. Connect a dissolved oxygen electrode, start the stirring program, and monitor the automated culture equipment's oxygen consumption overnight. (The piping connection and usage instructions for this equipment should be carried out according to the manufacturer's training instructions.)

[0090] Inoculation: The process is as follows: mesenchymal stem cells and collagen microspheres are pumped into the automated culture tank through the loading tube of the 3D automated culture equipment, the temperature is turned on, the stirring program, temperature, and pH are set, and the speed is subsequently adjusted according to the growth status of the cells;

[0091] The induction culture process is as follows: stop the automated culture stirring program, wait for the MSC-loaded collagen microspheres to settle, and then pump out the excess supernatant; take a sample for cell counting, and add induction medium based on the cell count; replace with fresh induction medium every 3 days until chondrocyte spheres form on the 15th day; the induction medium contains: DMEM / F12 basal medium, 10 ng / mL transforming growth factor β3, 0.10 μmol / mL dexamethasone, 100 μg / mL sodium pyruvate, 40 μg / mL proline, and 50 μg / mL ascorbic acid;

[0092] The harvesting process is as follows: stop all programs of the 3D automated culture equipment, pump the culture medium containing the chondrocytes into a centrifuge bottle, let it stand, discard the supernatant, rinse three times with saline, resuspend the chondrocytes in saline according to the dosage requirements, and divide them into groups to obtain chondrocytes; the inoculation ratio of collagen microspheres to mesenchymal stem cells is: 100 mg collagen microspheres per million MSC cells;

[0093] The chondrocytes were resuspended in 0.9% saline to prepare the injection, with a dose of 8 × 10 5 The drug was administered at a dose of 100 mg / mL, once every two weeks for 3 times.

[0094] Example 2: The process steps are the same as those in Example 1. The chondrocytes are resuspended in 0.9% saline to prepare an injection. The dosage is 1×10 6 The drug was administered at a dose of 100 mg / mL, once every two weeks for 3 times.

[0095] Example 3: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0096] Step 1: Dissolve 10 g of collagen in 200 mL of phosphate buffer, adjust the pH of the system to 7.2, slowly add 2.0 g of crotonic anhydride within 20 minutes, and adjust the temperature of the system to 48°C; transfer to a dialysis bag with a molecular weight cutoff of 500D, and dialyze in water to obtain functionalized collagen;

[0097] 10 g of chondroitin sulfate was mixed in 200 mL of deionized water, the pH was adjusted to 5, 7.6 g of EDC and 4.5 g of NHS were added, and the mixture was stirred and activated for 150 min; 6.7 g of 3-mercaptotyramine hydrochloride was added, and the mixture was reacted in the dark for 24 h; ethanol was precipitated, and vacuum dried to obtain thiolated chondroitin sulfate;

[0098] Take 10g of hydroxypropyl cellulose, dissolve it in 250mL of dimethylacetamide, add 5.6g of methylsulfonyl chloride, and stir the reaction at 68°C for 150min; add 9g of rosin acid and continue the reaction for 8h; add 1000mL of petroleum ether to precipitate, wash it with 500mL of petroleum ether three times, and vacuum dry it to obtain a rosin-based hydroxypropyl cellulose compound;

[0099] 10 g of functionalized collagen, 10 g of rosin-based hydroxypropyl cellulose, and 1 g of thiolated chondroitin sulfate were mixed in 200 mL of pH 7.2 phosphate buffer, 0.2 g of photoinitiator I2959 was added, and the mixture was stirred at 150 rpm and exposed to 365 nm ultraviolet light for 10 min at an intensity of 10 mW / cm 2 ; Wash with ultrapure water, wash with gradient ethanol, freeze-dry for 12 hours, and sterilize by irradiation to obtain collagen microspheres;

[0100] Step 2 is the same as in Example 2, and cartilage balls and injections are obtained.

[0101] Example 4: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0102] Step 1: Dissolve 10 g of collagen in 200 mL of phosphate buffer, adjust the pH of the system to 7.3, slowly add 2.4 g of crotonic anhydride within 25 minutes, adjust the system temperature to 50°C, and react for 180 minutes; transfer to a dialysis bag with a molecular weight cutoff of 500D, and dialyze in water to obtain functionalized collagen;

[0103] 10 g of chondroitin sulfate was mixed in 200 mL of deionized water, the pH was adjusted to 5.5, 7.7 g of EDC and 4.6 g of NHS were added, and the mixture was stirred and activated for 180 min; 6.8 g of 3-mercaptotyramine hydrochloride was added, and the mixture was reacted in the dark for 24 h; ethanol was precipitated, and vacuum dried to obtain thiolated chondroitin sulfate;

[0104] Take 10g of hydroxypropyl cellulose, dissolve it in 250mL of dimethylacetamide, add 5.7g of methylsulfonyl chloride, and stir the reaction at 70°C for 180min; add 9g of rosin acid and continue the reaction for 10h; add 1000mL of petroleum ether to precipitate, wash it with 500mL of petroleum ether three times, and vacuum dry it to obtain a rosin-based hydroxypropyl cellulose compound;

[0105] 10 g of functionalized collagen, 10 g of rosin-based hydroxypropyl cellulose, and 2 g of thiolated chondroitin sulfate were mixed in 200 mL of pH 7.4 phosphate buffer, 0.6 g of photoinitiator I2959 was added, and the mixture was stirred at 160 rpm and exposed to 365 nm ultraviolet light for 12 min at an intensity of 12 mW / cm 2; Wash with ultrapure water, wash with gradient ethanol, freeze-dry for 12 hours, and sterilize by irradiation to obtain collagen microspheres;

[0106] Step 2 is the same as in Example 2, and cartilage balls and injections are obtained.

[0107] Example 5: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0108] Step 1: Dissolve 10 g of collagen in 200 mL of phosphate buffer, adjust the pH of the system to 7.5, slowly add 2.8 g of crotonic anhydride within 30 minutes, adjust the system temperature to 52°C, and react for 200 minutes; transfer to a dialysis bag with a molecular weight cutoff of 500D, and dialyze in water to obtain functionalized collagen;

[0109] 10 g of chondroitin sulfate was mixed in 200 mL of deionized water, the pH was adjusted to 6, 7.8 g of EDC and 4.7 g of NHS were added, and the mixture was stirred and activated for 200 min; 7.0 g of 3-mercaptotyramine hydrochloride was added, and the mixture was reacted in the dark for 24 h; ethanol was added for precipitation, and the mixture was vacuum dried to obtain thiolated chondroitin sulfate;

[0110] Take 10g of hydroxypropyl cellulose, dissolve it in 250mL of dimethylacetamide, add 5.9g of methylsulfonyl chloride, and stir the reaction at 72°C for 200min; add 9.5g of rosin acid and continue the reaction for 12h; add 1000mL of petroleum ether for precipitation, wash it three times with 500mL of petroleum ether, and vacuum dry it to obtain a rosin-based hydroxypropyl cellulose compound;

[0111] 10 g of functionalized collagen, 10 g of rosin-based hydroxypropyl cellulose, and 3 g of thiolated chondroitin sulfate were mixed in 200 mL of pH 7.5 phosphate buffer, 1.0 g of photoinitiator I2959 was added, and the mixture was stirred at 180 rpm and exposed to 365 nm ultraviolet light for 15 min at an intensity of 15 mW / cm 2 ; Wash with ultrapure water, wash with gradient ethanol, freeze-dry for 12 hours, and sterilize by irradiation to obtain collagen microspheres;

[0112] Step 2 is the same as in Example 2, and cartilage balls and injections are obtained.

[0113] Comparative Example 1: The immunosuppressant methotrexate was administered orally at a rate of 10 mg / kg once a week for 7 consecutive weeks.

[0114] Comparative Example 2: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0115] Step 1: 10 g of chondroitin sulfate was mixed in 200 mL of deionized water, the pH was adjusted to 5, 7.6 g of EDC and 4.5 g of NHS were added, and the mixture was stirred and activated for 150 min; 6.7 g of 3-mercaptotyramine hydrochloride was added, and the mixture was reacted in the dark for 24 h; ethanol was precipitated, and vacuum dried to obtain thiolated chondroitin sulfate;

[0116] Take 10g of hydroxypropyl cellulose, dissolve it in 250mL of dimethylacetamide, add 5.6g of methylsulfonyl chloride, and stir the reaction at 68°C for 150min; add 9g of rosin acid and continue the reaction for 8h; add 1000mL of petroleum ether to precipitate, wash it with 500mL of petroleum ether three times, and vacuum dry it to obtain a rosin-based hydroxypropyl cellulose compound;

[0117] 10 g collagen, 10 g rosin-based hydroxypropyl cellulose, and 1 g thiolated chondroitin sulfate were mixed in 200 mL of pH 7.2 phosphate buffer, 0.2 g photoinitiator I2959 was added, and the mixture was stirred at 150 rpm and exposed to 365 nm ultraviolet light for 10 min at an intensity of 10 mW / cm 2 ; Wash with ultrapure water, wash with gradient ethanol, freeze-dry for 12 hours, and sterilize by irradiation to obtain collagen microspheres;

[0118] Step 2 is the same as in Example 2, and cartilage balls and injections are obtained.

[0119] Comparative Example 3: A method for preparing chondrocytes for treating osteoarthritis, comprising the following steps:

[0120] Step 1: Take a 10 wt% collagen solution, adjust the pH of the system to 4.0 with 1 M hydrochloric acid, add an equal volume of 10 wt% hydroxypropyl cellulose solution, mix, and let stand at 37°C for 12 hours; collect the colloid, wash with ultrapure water, wash with ethanol gradient, freeze-dry for 12 hours, and irradiate for sterilization to obtain collagen microspheres;

[0121] Step 2 is the same as in Example 2, and cartilage balls and injections are obtained.

[0122] Experiment: The collagen microspheres, cartilage spheres and injections obtained in Examples 1-5 and Comparative Examples 2-3 were used to prepare samples, and their properties were tested and the test results were recorded:

[0123] During the osteogenic differentiation of mesenchymal stem cells, calcium deposits secreted by stem cells form calcium nodules, which is an important sign of osteogenic differentiation. Calcium ions can chelate with alizarin red to show red, which can be used for staining to show the osteogenic differentiation of stem cells after 5, 10, and 15 generations of culture. Figure 5 It can be seen that mesenchymal stem cells loaded on the surface of collagen microspheres can be induced to differentiate into osteoblasts in vitro, and cells cultured for 5 generations are suitable for subsequent experiments.

[0124] During the adipogenic differentiation process of mesenchymal stem cells, adipocytes differentiate and form, producing fat droplets. The fat droplets appear red when stained with Oil Red O, while undifferentiated cells have no obvious color. Staining is performed to show the adipogenic differentiation of stem cells after culturing for 5, 10, and 15 generations. Figure 6 It can be seen that mesenchymal stem cells loaded on the surface of collagen microspheres can be induced to differentiate into adipocytes in vitro, and cells cultured for 5 generations are suitable for subsequent experiments.

[0125] During the chondrogenic differentiation of mesenchymal stem cells, chondrocytes are differentiated and formed, which contain a large amount of acidic mucopolysaccharides that can bind to the acidic mucopolysaccharides of Alcian blue and appear blue. Staining is performed to show the chondrogenic differentiation of stem cells after culturing for 5, 10, and 15 generations. Figure 7 It can be seen that mesenchymal stem cells loaded on the surface of collagen microspheres can be induced to differentiate into chondrocytes in vitro, and cells cultured for 5 generations are suitable for subsequent experiments.

[0126] Combine Figure 1 、 Figure 2 This is a stained section of the chondrocytes in Example 1. The microsphere state of the collagen microspheres can be observed; and the chondrocytes appear blue and are loaded on the surface of the collagen microspheres. Figure 3 The porous nature of the collagen microspheres in Example 1 can be observed.

[0127] Figure 8 The microsphere state and porous characteristics of the collagen microspheres in Example 4 can be observed. Figure 9 After fluorescent staining, the chondrocytes on the surface of the chondrocytes appeared blue, which can indicate the loading of chondrocytes on the surface of the collagen microspheres.

[0128] The collagen microspheres were dispersed in deionized water to form a 1.5 g / L suspension. The particle size of the collagen microspheres was measured using a laser particle size analyzer. The specific surface area of the collagen microspheres was measured using a specific surface area analyzer.

[0129] The collagen microspheres were loaded into a 20×1.6 cm PS / DVB column. After stabilization, the flow rate was gradually increased at intervals to observe the pressure changes. When the flow rate increased to a certain level, the pressure at this flow rate point was no longer constant but continued to rise, and the measurement was terminated. The pressure before the continuous rise was taken as the pressure resistance value of the collagen microspheres (unit: MPa). The test data are shown in Table 1:

[0130]

[0131] The experimental subjects were a bovine type II collagen-induced arthritis rat model. To establish the model, 8-week-old syngeneic Lewis rats were selected, anesthetized, and injected with a 4 mg / mL bovine type II collagen emulsion (dissolved in 0.05 mol / L acetic acid and mixed with an equal volume of incomplete Freund's adjuvant) via the tail vein. 200 μL was administered initially, followed by 100 μL each time one week later, for three consecutive times. The model was successfully established when the rats' limbs became swollen and their movements became difficult. Compared with the normal left hind limb joints of Lewis rats, the left hind limb joints of the bovine type II collagen-induced arthritis rat model showed mild joint cavity stenosis, mild synovial tissue hyperplasia, and mild articular cartilage necrosis (see ). Figure 4 A and E).

[0132] 0.5 mL of the injections obtained in Examples 1-5 and Comparative Examples 2-3 were injected into the knee joint cavity of rats; 10 mg / kg / w methotrexate obtained in Comparative Example 1 was administered orally. The specific dosage, volume, and frequency of administration are shown in Table 2.

[0133]

[0134] After administration, the rats in Example 1 (low-dose stem cell group) showed slight joint stenosis in their right hind limb joints (see pathological sections). Figure 4 C). After the experiment in Example 2 (high-dose stem cell group), the right hind limb joints of the rats showed good condition, and the joint cavity, synovial tissue, and articular cartilage were similar to those of the normal group (see pathological sections). Figure 4 B). After the experiment in comparative example 1 (drug group), the left hind limb joints of the rats showed slight joint cavity stenosis, mild synovial tissue hyperplasia and mild articular cartilage necrosis (pathological sections see Figure 4 D).

[0135] According to the clinical scoring table of rat arthritis inflammation in Table 3, the manifestations of arthritis inflammation in rats at different time points were detected, as shown in Table 4;

[0136] Table 3. Clinical scoring table of arthritis inflammation degree in rats:

[0137]

[0138] Table 4. Clinical signs scores of rats during the experiment:

[0139]

[0140] Note: a, arthritis group compared with normal group, P < 0.01; b, drug group compared with arthritis group, P < 0.05; c, low-dose stem cell group compared with arthritis group, P < 0.05; d, high-dose stem cell group compared with arthritis group, P < 0.05; e, low-dose stem cell group compared with drug group, P < 0.05; g: Example 3-5 compared with arthritis group, P < 0.05; h: Comparative Example 2-3 compared with arthritis group, P < 0.05;

[0141] Table 5. Measurement of rat foot mass during the experiment:

[0142]

[0143] Note: *P<0.05, **P<0.01, ***P<0.001, compared with the normal group; #P<0.05, ##P<0.01, ###P<0.001, compared with the arthritis group; ①P<0.05, ①①P<0.01, compared with the drug group.

[0144] Table 6. Organ wet weight and organ index of rats in each group:

[0145]

[0146] Note: *P<0.05, **P<0.01, ***P<0.001, compared with the normal group; #P<0.05, ##P<0.01, ###P<0.001, compared with the arthritis group; ①P<0.05, ①①P<0.01, compared with the drug group.

[0147] 0.5 mL of the injections obtained in Examples 1-5 and Comparative Examples 2-3 were injected into the knee joint cavity of rats. 10 mg / kg / w of methotrexate obtained in Comparative Example 1 was administered orally. The levels of TGF-β, TNF-α, IL-1β, and IL-6 were measured at the end of the observation period (mean ± standard deviation, unit: pg / mL). The test data are shown in Table 7:

[0148] Table 7. Test results of serum immune indexes of rats at the end of the observation period:

[0149]

[0150] Note: *P<0.05, **P<0.01, ***P<0.001, compared with the normal group; #P<0.05, ##P<0.01, ###P<0.001, compared with the arthritis group; ①P<0.05, ①①P<0.01, compared with the drug group.

[0151] The bipedal balance analgesia was used to measure the difference in the supporting force of the two hind feet of rats. When the animals were unrestrained, there would be a difference in the strength of the inflamed and painful foot and the normal foot to support the body weight. The greater the difference, the more painful the inflamed foot. The bipedal balance analgesia can be used to identify the effectiveness of the treatment. The test data are shown in Table 8:

[0152]

[0153] Note: *P<0.05, **P<0.01, compared with week 0.

[0154] According to the data in the above table, we can clearly draw the following conclusions:

[0155] The cartilage balls obtained in Examples 1-5 were compared with those obtained in Comparative Examples 1-3. The test results showed that

[0156] 1. Comparing Examples 1-2 with Comparative Example 1, the high-dose stem cell group (Example 1) demonstrated superior therapeutic efficacy in a bovine type II collagen-induced arthritis rat model compared to the low-dose stem cell group (Example 2) and the drug group (Comparative Example 1), but it also resulted in elevated serum cytokines TNF-α, IL-1β, and IL-6. The low-dose injection improved arthritis symptoms in a bovine type II collagen-induced arthritis rat model to a certain extent, with serum immune markers showing superior efficacy compared to methotrexate. Methotrexate, administered orally at 10 mg / kg / w, was effective in treating bovine type II collagen-induced arthritis in rats, temporarily alleviating foot pain. Administration increased serum TGF-β expression and decreased TNF-α, IL-1β, and IL-6.

[0157] 2. Comparing Example 1 with Examples 3-5, the chondrocytes obtained in Examples 3-5 functionalized the collagen and introduced thiolated chondroitin sulfate to covalently crosslink with rosin-based hydroxypropyl cellulose. The resulting chondrocytes had a better therapeutic effect on the bovine type II collagen-induced arthritis rat model, with better improvement in arthritis symptoms and pain levels. Compared with Example 3, the preparation process of the collagen microspheres in Comparative Examples 2-3 was significantly different, and the therapeutic effect of the resulting chondrocytes was relatively deteriorated. It can be seen that the present invention's preparation process for collagen microspheres and the settings of the components used can effectively improve the therapeutic effect of chondrocytes on osteoarthritis.

[0158] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A method for preparing a chondrocyte spheroid for treating osteoarthritis, characterized in that: The method comprises the following steps: mixing mesenchymal stem cells with collagen microspheres, inoculating the cells in a culture device for culture, adding an induction culture medium for induction culture, and harvesting the cells to obtain chondrocytes; The collagen microspheres are prepared by the following process: Step 1: Dissolve hydroxypropyl cellulose in dimethylacetamide, add methylsulfonyl chloride, and stir at 68-72°C for 150-200 minutes; Add rosin acid and continue the reaction for 8 to 12 hours; precipitate, wash, and dry to obtain a rosin-based hydroxypropyl cellulose compound; Step 2: Take the collagen solution, adjust the pH of the system to 3.5-4.5, add rosin-based hydroxypropyl cellulose and mix, and let it stand at 36-38°C for 1-6 hours; collect the colloid, freeze-dry, and sterilize to obtain collagen microspheres.

2. The method for preparing chondrocytes for treating osteoarthritis according to claim 1, wherein: The inoculation ratio of the collagen microspheres to the mesenchymal stem cells is: 96-105 mg collagen microspheres per one million mesenchymal stem cells.

3. The method for preparing chondrocytes for treating osteoarthritis according to claim 1, wherein: The induction medium comprises: DMEM / F12 basal medium and 8-12 ng / mL transforming growth factor β3, 0.09-0.11 μmol / mL dexamethasone, 90-110 μg / mL sodium pyruvate, 38-42 μg / mL proline, and 45-54 μg / mL ascorbic acid in the DMEM / F12 basal medium.

4. The method for preparing chondrocytes for treating osteoarthritis according to claim 1, wherein: In step 1, the mass ratio of hydroxypropyl cellulose, methylsulfonyl chloride, and rosin acid is 10: (5.6-5.9): (9.0-9.5).

5. The method for preparing chondrocytes for treating osteoarthritis according to claim 1, wherein: In step 2, the mass ratio of collagen to rosin-based hydroxypropyl cellulose is (2-3): (2-3).

6. The method for preparing chondrocytes for treating osteoarthritis according to claim 1, wherein: The collagen is surface modified, and the specific process is as follows: Take collagen, dissolve it in phosphate buffer, adjust the pH of the system to 7.2-7.5, slowly add crotonic anhydride within 20-30 minutes, adjust the system temperature to 48-52°C, react for 150-200 minutes, dialyze to obtain functionalized collagen.

7. The method for preparing chondrocytes for treating osteoarthritis according to claim 6, characterized in that: The collagen microspheres are prepared by the following process: Functionalized collagen, rosin-based hydroxypropyl cellulose, and thiolated chondroitin sulfate are mixed in a phosphate buffer solution with a pH of 7.2 to 7.5, a photoinitiator is added, the mixture is stirred and irradiated with ultraviolet light for 10 to 15 minutes, freeze-dried, and sterilized to obtain collagen microspheres.

8. The method for preparing chondrocytes for treating osteoarthritis according to claim 7, characterized in that: The thiolated chondroitin sulfate is prepared by the following process: Chondroitin sulfate is mixed with deionized water, the pH is adjusted to 5-6, EDC and NHS are added, and the mixture is stirred and activated for 150-200 minutes; 3-mercaptotyramine hydrochloride is added, and the mixture is reacted in the dark for 24 hours to obtain thiolated chondroitin sulfate.

9. Use of the chondrocytes prepared by the preparation method according to any one of claims 1 to 8, characterized in that: Used for preparing materials for treating osteoarthritis.

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