Mesenchymal stem cell composition for promoting bone joint repair and preparation method thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a mesenchymal stem cell composition for promoting bone joint repair and a preparation method thereof, wherein the mesenchymal stem cell composition comprises mesenchymal stem cells, a hydrogel framework, heparin / hyaluronic acid-VEGF microspheres, modified PLGA nanoparticles and nano-hydroxyapatite. In the inflammatory damage area, activated neutrophils secrete MPO, MPO generates HOCl and ROS through catalytic reaction, acts on the sulfide bond, makes the microspheres separate from the hydrogel framework, the hydrophilicity of the microsphere shell layer is enhanced by the oxidized sulfoxide group, the microspheres swell and depolymerize, release VEGF, start angiogenesis, VEGF recruits a large number of inflammatory cells, produces ROS, makes the sulfur ketal bond between the modified PLGA nanoparticles and the hydrogel framework break, releases TGF-beta 3 and sFlt-1, sFlt-1 inhibits the activity of the remaining VEGF, TGF-beta 3 can effectively promote cartilage formation, sFlt-1 forms a stable complex with heparin to achieve the mild and persistent release of sFlt-1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically a mesenchymal stem cell composition for promoting bone and joint repair and its preparation method. Background Technology

[0002] Osteoarthritis and other joint diseases leading to cartilage defects are common clinical problems, with extremely poor self-repair capabilities. Mesenchymal stem cell therapy is considered the most promising repair strategy due to its multi-lineage differentiation potential and paracrine effects. Current techniques often use hydrogels as cell carriers and attempt to load them with growth factors such as angiogenesis factor (VEGF) and chondrogenic differentiation factors (such as TGF-β3 or BMP-2).

[0003] However, tissue repair is a multi-stage process, each with its unique biological tasks and required signaling molecules. VEGF is primarily used in the early angiogenesis and cell recruitment phase, while TGF-β3 is mainly used in the intermediate tissue formation and differentiation phase. If both are released simultaneously and continuously, on the one hand, continuous stimulation by VEGF can lead to excessive vascularization into the cartilage defect area, which can eventually trigger endochondrial ossification, resulting in vascularization or hypertrophy of the repair tissue, forming dysfunctional fibrocartilage or heterotopic ossification, ultimately leading to repair failure. On the other hand, VEGF instructs cells to "migrate and form blood vessels," while TGF-β / BMP instructs them to "settle, differentiate, and synthesize matrix." Mesenchymal stem cells, receiving these two conflicting signals, will be confused and unable to effectively execute either program, resulting in low repair efficiency. Furthermore, growth factors are very expensive; if released at an unnecessary stage, they will be rapidly degraded and become ineffective, resulting in significant waste of the drug. Summary of the Invention

[0004] (1) Technical problems to be solved The purpose of this invention is to provide a mesenchymal stem cell composition that promotes bone and joint repair and its preparation method, in order to solve the problem that the simultaneous release of VEGF and TGF-β by hydrogels used as mesenchymal stem cell carriers in vivo leads to vascularization or hypertrophy of the repaired tissue and low repair efficiency.

[0005] (2) Technical solution To achieve the above objectives, in one respect, the present invention provides a mesenchymal stem cell composition for promoting bone and joint repair, the mesenchymal stem cell composition comprising mesenchymal stem cells and a multifunctional hydrogel; The multifunctional hydrogel comprises the following components by weight fraction: 80-100 parts of hydrogel framework, 15-20 parts of heparin / hyaluronic acid-VEGF microspheres, and 15-20 parts of modified PLGA nanoparticles. The hydrogel framework is methacrylamide hyaluronic acid; The heparin / hyaluronic acid-VEGF microspheres are linked to the hydrogel framework via thioether bonds; The modified PLGA nanoparticles are connected to the hydrogel framework via thioketal bonds, and are internally loaded with a TGF-β3 and sFlt-1-heparin complex.

[0006] Furthermore, the multifunctional hydrogel also includes nano-hydroxyapatite, with a mass of 7-10% of the mass of methacrylamide hyaluronic acid.

[0007] Furthermore, the preparation method of the heparin / hyaluronic acid-VEGF microspheres includes the following steps: S11. Dissolve heparin and hyaluronic acid in PBS buffer, slowly add VEGF solution, gently vortex, add sodium alginate solution and mix, add 1-5% calcium chloride solution dropwise with a microsyringe, crosslinking reaction, let stand to solidify, centrifuge to collect solid, wash with PBS buffer to obtain the first compound; S12. Resuspend the first compound in MES buffer, add EDC and NHS, stir to activate, and obtain the second compound; S13. Add the CRGDS short peptide to the second compound, stir the reaction, centrifuge and wash to obtain heparin / hyaluronic acid-VEGF microspheres.

[0008] Furthermore, the mass of the VEGF is 0.5-1.5% of the mass of heparin and hyaluronic acid, and the mass of the CRGDS short peptide is 10-20% of the mass of the first compound.

[0009] Furthermore, the preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve heparin in ultrapure water to obtain a heparin solution; dissolve sFlt-1 in PBS buffer, slowly add it dropwise to the heparin solution, and incubate until the sFlt-1-heparin complex is obtained. S22. Dissolve TGF-β3 in PBS buffer, add sFlt-1-heparin complex, and then add trehalose to obtain the inner aqueous phase; dissolve PLGA polymer in dichloromethane to obtain the oil phase; S23. Under ice bath conditions, the inner aqueous phase is slowly added dropwise to the oil phase and ultrasonically emulsified. The resulting emulsion is poured into a PVA solution and ultrasonically emulsified again under ice bath conditions. The resulting double emulsion solution is stirred in an open container to evaporate the solvent and obtain a nanoparticle suspension. The solid is collected by centrifugation, washed with pre-cooled ultrapure water, resuspended in trehalose aqueous solution, and freeze-dried to obtain the third compound. S24. The third compound was dispersed in MES buffer, EDC·HCl and NHS were added, and the mixture was stirred to activate it. Ethylenediamine was dissolved in MES buffer, and the pH was adjusted to 7.2 before being slowly added dropwise. The reaction was continued at room temperature for 2 hours. The resulting reaction solution was centrifuged and purified, and washed with pre-cooled ultrapure water to obtain the fourth compound. S25. The fourth compound was dispersed in borate buffer, and methacryloyl-PEG-thioacetate-PEG-NHS ester was added. The reaction was carried out in the dark. The resulting reaction solution was centrifuged to collect the solid, washed with pre-cooled ultrapure water, and freeze-dried to obtain modified PLGA nanoparticles.

[0010] On the other hand, based on the same inventive concept, the present invention also provides a method for preparing a mesenchymal stem cell composition for promoting bone and joint repair, which is applied to the aforementioned mesenchymal stem cell composition for promoting bone and joint repair, comprising the following steps: S1. Dissolve methacrylamide hyaluronic acid in PBS buffer, add nano-hydroxyapatite, vortex disperse, then add heparin / hyaluronic acid-VEGF microspheres and modified PLGA nanoparticles in sequence, and continue vortex dispersion to obtain the first mixture; S2. Mesenchymal stem cells were digested with trypsin, the cell pellet was collected by centrifugation, and resuspended in PBS buffer to obtain a second mixture; S3. Add lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the first mixture, mix well, and then continue to add the second mixture dropwise. After shaking and mixing well, transfer to a mold, place in an ice bath, and use a blue light source with a wavelength of 405~470nm at 15~20mW / cm². 2 Mesenchymal stem cell composition was obtained by irradiating the sample with light intensity for 60-100 seconds.

[0011] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. Heparin / hyaluronic acid-VEGF microspheres are linked to the hydrogel framework via thioether bonds. In the inflammatory damage area, activated neutrophils secrete large amounts of MPO. MPO catalyzes the reaction of H2O2 with Cl-. - The reaction generates HOCl and ROS. HOCl / ROS oxidizes and breaks the thioether bonds between the heparin / hyaluronic acid-VEGF microspheres and the hydrogel backbone, releasing the microspheres from the hydrogel network. After the bonds are broken, hydrophilic sulfoxide groups are formed on the surface of the microspheres, greatly enhancing the hydrophilicity of the shell. The microspheres absorb water, swell, and depolymerize, thereby releasing the VEGF encapsulated inside into the local microenvironment, initiating angiogenesis and cell recruitment.

[0012] 2. The released VEGF recruits a large number of inflammatory cells, which produce a large amount of ROS during the respiratory burst. The high level of ROS causes the thioacetate connecting the modified PLGA nanoparticles and the hydrogel backbone to be cleaved by high levels of hydrogen peroxide and other ROS, releasing TGF-β3 and sFlt-1. sFlt-1 binds to heparin and is released slowly, inhibiting the activity of the remaining VEGF in the later stage of repair, thus avoiding the long-term continuous release of VEGF that leads to vascularization or hypertrophy of the repaired tissue. TGF-β3 can effectively promote cartilage formation.

[0013] 3. Heparin / hyaluronic acid-VEGF microspheres and modified PLGA nanoparticles achieve the sequential release of VEGF, TGF-β3 and sFlt-1 by utilizing changes in MPO and ROS in the environment at different times. This avoids the low repair efficiency caused by the simultaneous release of both, and the release of VEGF and TGF-β3 only at the required stage also saves costs.

[0014] 4. Modified PLGA nanoparticles produce acidic byproducts after degradation. Adding nano-hydroxyapatite to the multifunctional hydrogel can act as a pH buffer, and the calcium and phosphorus ions released by nano-hydroxyapatite can also work together with TGF-β3 to promote osteogenic / chondrogenic differentiation. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: This example discloses a mesenchymal stem cell composition that promotes bone and joint repair, the mesenchymal stem cell composition comprising mesenchymal stem cells and a multifunctional hydrogel; The multifunctional hydrogel comprises the following components by weight fraction: 100 parts hydrogel framework, 20 parts heparin / hyaluronic acid-VEGF microspheres, and 20 parts modified PLGA nanoparticles. The hydrogel framework is methacrylamide hyaluronic acid; The heparin / hyaluronic acid-VEGF microspheres are linked to the hydrogel framework via thioether bonds; The modified PLGA nanoparticles are connected to the hydrogel framework via thioketal bonds, and are internally loaded with a TGF-β3 and sFlt-1-heparin complex.

[0017] The multifunctional hydrogel also includes nano-hydroxyapatite, which accounts for 10% of the mass of methacrylamide hyaluronic acid.

[0018] The preparation method of the heparin / hyaluronic acid-VEGF microspheres includes the following steps: S11. Heparin and hyaluronic acid were mixed and dissolved in PBS buffer at a 1:1 ratio. VEGF solution was slowly added dropwise, and the mixture was gently vortexed. Sodium alginate solution (final concentration 1%, w / v) was added and mixed. 1-5% calcium chloride solution was added dropwise using a microsyringe. The cross-linking reaction was carried out for 15-30 min. The mixture was allowed to stand and solidify for 30 min. The solid was collected by centrifugation and washed with PBS buffer to obtain the first compound. S12. Resuspend the first compound in MES buffer, add EDC and NHS, stir and activate for 15-30 min to obtain the second compound; S13. Add the CRGDS short peptide to the second compound, adjust the pH of the system to 5.5~6.0, stir the reaction at 4℃ for 2~4h, centrifuge and wash to obtain heparin / hyaluronic acid-VEGF microspheres.

[0019] The mass of the VEGF is 1.5% of the mass of heparin and hyaluronic acid, and the mass of the CRGDS short peptide is 20% of the mass of the first compound.

[0020] It should be noted that the amino group at one end of the CRGDS short peptide forms an amide bond with the activated carboxyl group on the surface of the second compound, thereby covalently grafting the thiol group at the other end onto the microsphere surface. The methacryloyl group on the hydrogel precursor molecule undergoes a Michael addition reaction with the thiol group on the microsphere surface, pre-forming some thioether bonds. Then, photocrosslinking is used for shaping, stably anchoring the microspheres within the hydrogel network. Furthermore, the RGD sequence in the CRGDS short peptide is an integrin ligand, which can significantly promote the adhesion and colonization of mesenchymal stem cells on the microsphere surface and in the hydrogel matrix, helping to improve the survival rate and local retention rate of transplanted cells, further enhancing the bone and joint repair effect.

[0021] The preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve 40 μg heparin in 0.2 mL of ultrapure water to obtain a heparin solution; dissolve 20 μg sFlt-1 in 0.2 mL of PBS buffer and slowly add it dropwise to the heparin solution. Incubate at 4 °C for 60 min to obtain the sFlt-1-heparin complex. S22. Dissolve 20 µg TGF-β3 in 0.1 mL PBS buffer, add 0.4 mL sFlt-1-heparin complex, and then add 1% (w / v) trehalose to obtain the inner aqueous phase; dissolve 50 mg PLGA polymer in dichloromethane and bring the volume to 2 mL to obtain the oil phase; S23. Under ice bath conditions, the inner aqueous phase was slowly added dropwise to the oil phase at a volume ratio of 1:4. The mixture was ultrasonically emulsified for 30 seconds. The resulting emulsion was poured into 20 mL of 2% (w / v) PVA solution and ultrasonically emulsified again for 60 seconds under ice bath conditions. The resulting double emulsion solution was stirred in an open container and the solvent was evaporated for 3-4 hours to obtain a nanoparticle suspension. The solid was collected by centrifugation at 4°C, washed with pre-cooled ultrapure water, resuspended in 5% (w / v) trehalose aqueous solution, and freeze-dried to obtain the third compound. S24. Disperse 10 mg of the third compound in 10 mL of MES buffer, add 40 µL of LEDC·HCl (10 mg / mL) and 40 µL of NHS (10 mg / mL), stir and activate for 30 min. Dissolve ethylenediamine in MES buffer at 10 molar equivalents (relative to EDC volume), adjust the pH to 7.2 and slowly add it dropwise. Continue to react at room temperature for 2 h. Centrifuge and purify the resulting reaction solution, wash with pre-cooled ultrapure water to obtain the fourth compound. S25. The fourth compound was dispersed in borate buffer, 5 mg of methacryloyl-PEG-thioacetate-PEG-NHS ester was added, and the reaction was carried out in the dark for 4 h. The resulting reaction solution was centrifuged to collect the solid, washed with pre-cooled ultrapure water, and freeze-dried to obtain modified PLGA nanoparticles.

[0022] The method for preparing the mesenchymal stem cell composition for promoting bone and joint repair includes the following steps: S1. Dissolve methacrylamide hyaluronic acid in PBS buffer, add nano-hydroxyapatite, vortex disperse, then add heparin / hyaluronic acid-VEGF microspheres and modified PLGA nanoparticles in sequence, continue vortex dispersion, and let stand at room temperature for 30~60 min to obtain the first mixture; S2. Mesenchymal stem cells were digested with trypsin, the cell pellet was collected by centrifugation, and resuspended in PBS buffer to obtain a second mixture; S3. Add lithium phenyl-2,4,6-trimethylbenzoylphosphonate to the first mixture, mix well, and then continue to add the second mixture dropwise. After shaking and mixing well, transfer to a mold, place in an ice bath, and use a blue light source with a wavelength of 405~470nm at 15~20mW / cm². 2 Mesenchymal stem cell composition was obtained by irradiating the sample with light intensity for 60-100 seconds.

[0023] It should be noted that mesenchymal stem cells were passaged using trypsin digestion to collect P3-P5 generation cells, and the second mixture contained... cell.

[0024] It should be noted that the concentration of lithium phenyl-2,4,6-trimethylbenzoylphosphonate is 0.05%~0.1%, and the mixture needs to be placed in an ice bath for irradiation to ensure that the cell survival rate is >90%.

[0025] Example 2: This example is based on Example 1, but differs from Example 1 in that the multifunctional hydrogel in this example includes the following components by weight fraction: 90 parts of hydrogel skeleton, 17 parts of heparin / hyaluronic acid-VEGF microspheres, and 17 parts of modified PLGA nanoparticles; The other components and preparation methods are the same as in Example 1.

[0026] Example 3: This example is based on Example 1, but differs from Example 1 in that the multifunctional hydrogel in this example includes the following components by weight fraction: 80 parts of hydrogel skeleton, 15 parts of heparin / hyaluronic acid-VEGF microspheres, and 15 parts of modified PLGA nanoparticles; The other components and preparation methods are the same as in Example 1.

[0027] Example 4: This example differs from Example 1 in that the multifunctional hydrogel in this example also includes nano-hydroxyapatite, with a mass of 7% of methacrylamide hyaluronic acid. The other components and preparation methods are the same as in Example 1.

[0028] Example 5: This example is based on Example 1, but differs from Example 1 in that the mass of VEGF in this example is 0.5% of the mass of heparin and hyaluronic acid, and the mass of CRGDS short peptide is 10% of the mass of the first compound.

[0029] The other components and preparation methods are the same as in Example 1.

[0030] Comparative Example 1: This comparative example is based on Example 1, but differs from Example 1 in that the mass of VEGF in this comparative example is 0.1% of the mass of heparin and hyaluronic acid.

[0031] When the VEGF ratio is too low, the absolute value of VEGF encapsulated in each microsphere is small. After implantation, the local release concentration cannot reach the effective therapeutic threshold, cannot initiate angiogenesis signals, and cannot recruit inflammatory cells. Consequently, ROS cannot be increased, and ultimately the thioketal bonds of the modified PLGA nanoparticles cannot be broken, and TGF-β3 and sFlt-1 cannot be released.

[0032] The other components and preparation methods are the same as in Example 1.

[0033] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that the heparin / hyaluronic acid-VEGF microspheres described in this comparative example do not contain heparin.

[0034] The preparation method of the hyaluronic acid-VEGF microspheres includes the following steps: S11. Dissolve hyaluronic acid in PBS buffer, slowly add VEGF solution, gently vortex, add sodium alginate solution (final concentration 1%, w / v) and mix, add 1-5% calcium chloride solution dropwise with a microsyringe, crosslink reaction for 15-30 min, let stand to solidify for 30 min, centrifuge to collect solid, wash with PBS buffer to obtain compound 5. S12. Resuspend the fifth compound in MES buffer, add EDC and NHS, stir and activate for 15-30 min to obtain the sixth compound; S13. Add the CRGDS short peptide to the sixth compound, adjust the pH of the system to 5.5~6.0, stir the reaction at 4℃ for 2~4h, centrifuge and wash to obtain hyaluronic acid-VEGF microspheres.

[0035] The other components and preparation methods are the same as in Example 1.

[0036] Comparative Example 3: This comparative example is based on Example 1, but differs from Example 1 in that the heparin / hyaluronic acid-VEGF microspheres described in this comparative example do not contain CRGDS short peptides.

[0037] The preparation method of the heparin / hyaluronic acid-VEGF microspheres includes the following steps: S11. Mix heparin and hyaluronic acid in a 1:1 ratio and dissolve in PBS buffer. Slowly add VEGF solution, gently vortex, add sodium alginate solution (final concentration 1%, w / v) and mix. Add 1-5% calcium chloride solution dropwise using a microsyringe. Allow the cross-linking reaction to proceed for 15-30 min, let stand to solidify for 30 min, centrifuge to collect the solid, wash with PBS buffer, and obtain heparin / hyaluronic acid-VEGF microspheres.

[0038] The other components and preparation methods are the same as in Example 1.

[0039] Comparative Example 4: This comparative example is based on Example 1, but differs from Example 1 in that the modified PLGA nanoparticles in this comparative example do not contain sFlt-1.

[0040] The preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve 20 µg TGF-β3 in 0.1 mL PBS buffer, add 1% (w / v) trehalose to obtain the inner aqueous phase; dissolve 50 mg PLGA polymer in dichloromethane, and bring the volume to 2 mL to obtain the oil phase; S22. Under ice bath conditions, the inner aqueous phase was slowly added dropwise to the oil phase at a volume ratio of 1:4. The mixture was ultrasonically emulsified for 30 seconds. The resulting emulsion was poured into 20 mL of 2% (w / v) PVA solution and ultrasonically emulsified again for 60 seconds under ice bath conditions. The resulting double emulsion solution was stirred in an open container and the solvent was evaporated for 3-4 hours to obtain a nanoparticle suspension. The solid was collected by centrifugation at 4°C, washed with pre-cooled ultrapure water, resuspended in 5% (w / v) trehalose aqueous solution, and freeze-dried to obtain the seventh compound. S23. Disperse 10 mg of the seventh compound in 10 mL of MES buffer, add 40 µL of LEDC·HCl (10 mg / mL) and 40 µL of NHS (10 mg / mL), stir and activate for 30 min, dissolve ethylenediamine in 10 molar equivalents (relative to EDC volume) in MES buffer, adjust pH to 7.2 and slowly add it dropwise, continue to react at room temperature for 2 h, centrifuge and purify the resulting reaction solution, wash with pre-cooled ultrapure water to obtain the eighth compound; S24. The eighth compound was dispersed in borate buffer, 5 mg of methacryloyl-PEG-thioacetate-PEG-NHS ester was added, and the reaction was carried out in the dark for 4 h. The resulting reaction solution was centrifuged to collect the solid, washed with pre-cooled ultrapure water, and freeze-dried to obtain modified PLGA nanoparticles.

[0041] The other components and preparation methods are the same as in Example 1.

[0042] Comparative Example 5: This comparative example is based on Example 1, but differs from Example 1 in that the modified PLGA nanoparticles in this comparative example do not contain thioacetate-PEG-NHS ester.

[0043] The preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve 40 μg heparin in 0.2 mL of ultrapure water to obtain a heparin solution; dissolve 20 μg sFlt-1 in 0.2 mL of PBS buffer and slowly add it dropwise to the heparin solution. Incubate at 4 °C for 60 min to obtain the sFlt-1-heparin complex. S22. Dissolve 20 µg TGF-β3 in 0.1 mL PBS buffer, add 0.4 mL sFlt-1-heparin complex, and then add 1% (w / v) trehalose to obtain the inner aqueous phase; dissolve 50 mg PLGA polymer in dichloromethane and bring the volume to 2 mL to obtain the oil phase; S23. Under ice bath conditions, the aqueous phase was slowly added dropwise to the oil phase at a volume ratio of 1:4. The mixture was ultrasonically emulsified for 30 seconds. The resulting emulsion was poured into 20 mL of 2% (w / v) PVA solution and ultrasonically emulsified again for 60 seconds under ice bath conditions. The resulting double emulsion solution was stirred in an open container and the solvent was evaporated for 3-4 hours to obtain a nanoparticle suspension. The solid was collected by centrifugation at 4°C, washed with pre-cooled ultrapure water, resuspended in 5% (w / v) trehalose aqueous solution, and freeze-dried to obtain modified PLGA nanoparticles.

[0044] The other components and preparation methods are the same as in Example 1.

[0045] Comparative Example 6: This comparative example is based on Example 1, but differs from Example 1 in that the modified PLGA nanoparticles described in this comparative example do not contain heparin.

[0046] The preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve 10 µg TGF-β3 and 10 µg sFlt-1 in 0.5 mL PBS buffer, add 1% (w / v) trehalose aqueous solution to obtain the inner aqueous phase; dissolve 50 mg PLGA polymer in dichloromethane, and bring the volume to 2 mL to obtain the oil phase; S22. Under ice bath conditions, the inner aqueous phase was slowly added dropwise to the oil phase at a volume ratio of 1:4. The mixture was ultrasonically emulsified for 30 seconds. The resulting emulsion was poured into 20 mL of 2% (w / v) PVA solution and ultrasonically emulsified again for 60 seconds under ice bath conditions. The resulting double emulsion solution was stirred in an open container and the solvent was evaporated for 3-4 hours to obtain a nanoparticle suspension. The solid was collected by centrifugation at 4°C, washed with pre-cooled ultrapure water, resuspended in 5% (w / v) trehalose aqueous solution, and freeze-dried to obtain the third compound. S23. Disperse 10 mg of the third compound in 10 mL of MES buffer, add 40 µL of LEDC·HCl (10 mg / mL) and 40 µL of NHS (10 mg / mL), stir and activate for 30 min. Dissolve ethylenediamine in MES buffer at 10 molar equivalents (relative to EDC volume), adjust the pH to 7.2 and slowly add it dropwise. Continue to react at room temperature for 2 h. Centrifuge and purify the resulting reaction solution, wash with pre-cooled ultrapure water to obtain the fourth compound. S24. The fourth compound was dispersed in borate buffer, 5 mg of methacryloyl-PEG-thioacetal-PEG-NHS ester was added, and the reaction was carried out in the dark for 4 h. The resulting reaction solution was centrifuged to collect the solid, washed with pre-cooled ultrapure water, and freeze-dried to obtain modified PLGA nanoparticles.

[0047] The other components and preparation methods are the same as in Example 1.

[0048] Comparative Example 7: This comparative example is based on Example 1, but unlike Example 1, this comparative example does not contain nano-hydroxyapatite.

[0049] The other components and preparation methods are the same as in Example 1.

[0050] Comparative Example 8: This comparative example is a blank control group.

[0051] The method for preparing the mesenchymal stem cell composition for promoting bone and joint repair includes the following steps: S1. Dissolve methacrylamide hyaluronic acid in PBS buffer, add lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and obtain a third mixture; S2. Mesenchymal stem cells were digested with trypsin, the cell pellet was collected by centrifugation, and resuspended in PBS buffer to obtain the fourth mixture; S3. Add the fourth mixture dropwise to the third mixture, shake to mix, transfer to a mold, place in an ice bath, and heat with a blue light source of 405~470nm wavelength at 15~20mW / cm². 2 Mesenchymal stem cell composition was obtained by irradiating the sample with light intensity for 60-100 seconds.

[0052] Experimental verification: Materials required: mesenchymal stem cells. Cells. Umbilical cord mesenchymal stem cells purchased from Zhongqiao Xinzhou were passaged and cultured, with P3 generation selected as the experimental sample.

[0053] 1. Time-series release assay: Mesenchymal stem cell compositions (3 replicates per group) were placed in PBS buffer containing 100 ng / mL MPO and cultured with shaking at 37°C. After 3 days of MPO treatment, the mesenchymal stem cell compositions were transferred to PBS buffer containing 100 μM H2O2 and cultured for another 3 days. Release media were collected on days 3, 7, and 14, and the concentrations of VEGF, TGF-β3, and sFlt-1 were detected using an ELISA kit.

[0054] Table 1: Concentrations of VEGF, TGF-β3, and sFlt-1 (ng / mL):

[0055] Note: ND indicates not detected (below the detection limit; the detection limit for VEGF ELISA is 2.0 pg / mL, for TGF-β3 ELISA it is 4.0 pg / mL, and for sFlt-1 ELISA it is 3.0 pg / mL).

[0056] Table 1 shows the concentrations of VEGF, TGF-β3, and sFlt-1. The data in the table show that VEGF in Example 1 reached a release concentration of (52.3±7.8) ng / mL on day 3, while TGF-β3 and sFlt-1 were released at only basal levels (0.8±0.2 ng / mL and 0.3±0.1 ng / mL) on day 7. However, they significantly increased to (18.7±4.2) ng / mL and (20.5±4.6) ng / mL on day 14, indicating that VEGF was released before TGF-β3 / sFlt-1, achieving a time-sequential release.

[0057] 2. Verification of Animal Model Repair Effect: Healthy adult male rats, weighing 200-250g, were purchased and acclimatized for seven days. They were then randomly divided into 13 groups of 8 rats each. Before the experiment, rats were fasted but allowed free access to water for 12 hours. Anesthesia was administered via intraperitoneal injection of sodium pentobarbital. A longitudinal incision was made on the medial side of the right hind limb knee joint to expose the joint. The femoral condyle articular surface was located, and a full-thickness cartilage defect of 2mm in diameter and 1mm in depth was created using a micro-drill. The defect was rinsed with saline to remove debris. Mesenchymal stem cell composites were implanted into each experimental group, and the incision was sutured layer by layer. Postoperatively, penicillin was administered for three consecutive days to combat infection, and recovery was observed. Twelve weeks post-surgery, patients were euthanized under excessive anesthesia. The entire knee joint was harvested and fixed with 4% paraformaldehyde for 24 hours. The fixed specimen was decalcified with EDTA decalcification solution for 4 weeks, routinely embedded in paraffin, and serially sectioned along the sagittal plane. The tissue sections were stained with hematoxylin-eosin, safranin-fast green, and immunohistochemically according to the prescribed procedure to observe the tissue morphology and structure, and to assess cartilage thickness, proteoglycan area, type II collagen area, and mast chondrocytes.

[0058] Table 2: Evaluation of Repair Results

[0059] Table 2 shows the evaluation of the repair effect. The data in the table shows that Example 1 is the best solution of the present invention. Observing its tissue morphology and structure, it shows that the cartilage surface is smooth, the chondrocytes are arranged in an orderly manner, there is a typical cartilage lacunar structure, and it is well integrated with the surrounding normal cartilage. This indicates that the added VEGF, TGF-β3 and sFlt-1 are released sequentially in the hydrogel, which can effectively promote the repair of bone and joint by mesenchymal stem cells.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell composition for promoting bone joint repair, characterized in that, The mesenchymal stem cell composition includes mesenchymal stem cells and a multifunctional hydrogel; The multifunctional hydrogel comprises the following components by weight fraction: 80-100 parts of hydrogel framework, 15-20 parts of heparin / hyaluronic acid-VEGF microspheres, and 15-20 parts of modified PLGA nanoparticles. The hydrogel framework is methacrylamide hyaluronic acid; The heparin / hyaluronic acid-VEGF microspheres are linked to the hydrogel framework via thioether bonds; The modified PLGA nanoparticles are connected to the hydrogel framework via thioketal bonds, and are internally loaded with a TGF-β3 and sFlt-1-heparin complex.

2. The mesenchymal stem cell composition for promoting bone and joint repair according to claim 1, characterized in that, The multifunctional hydrogel also includes nano-hydroxyapatite, which accounts for 7-10% of the mass of methacrylamide hyaluronic acid.

3. The mesenchymal stem cell composition for promoting bone and joint repair according to claim 1, characterized in that, The preparation method of the heparin / hyaluronic acid-VEGF microspheres includes the following steps: S11. Dissolve heparin and hyaluronic acid in PBS buffer, slowly add VEGF solution, gently vortex, add sodium alginate solution and mix, add 1-5% calcium chloride solution dropwise with a microsyringe, crosslinking reaction, let stand to solidify, centrifuge to collect solid, wash with PBS buffer to obtain the first compound; S12. Resuspend the first compound in MES buffer, add EDC and NHS, stir to activate, and obtain the second compound; S13. Add the CRGDS short peptide to the second compound, stir the reaction, centrifuge and wash to obtain heparin / hyaluronic acid-VEGF microspheres.

4. The mesenchymal stem cell composition for promoting bone and joint repair according to claim 1, characterized in that, The mass of VEGF is 0.5-1.5% of the mass of heparin and hyaluronic acid, and the mass of CRGDS short peptide is 10-20% of the mass of the first compound.

5. The mesenchymal stem cell composition for promoting bone and joint repair according to claim 1, characterized in that, The preparation method of the modified PLGA nanoparticles includes the following steps: S21. Dissolve heparin in ultrapure water to obtain a heparin solution; dissolve sFlt-1 in PBS buffer, slowly add it dropwise to the heparin solution, and incubate until the sFlt-1-heparin complex is obtained. S22. Dissolve TGF-β3 in PBS buffer, add sFlt-1-heparin complex, and then add trehalose to obtain the inner aqueous phase; dissolve PLGA polymer in dichloromethane to obtain the oil phase; S23. Under ice bath conditions, the inner aqueous phase is slowly added dropwise to the oil phase and ultrasonically emulsified. The resulting emulsion is poured into a PVA solution and ultrasonically emulsified again under ice bath conditions. The resulting double emulsion solution is stirred in an open container to evaporate the solvent and obtain a nanoparticle suspension. The solid is collected by centrifugation, washed with pre-cooled ultrapure water, resuspended in trehalose aqueous solution, and freeze-dried to obtain the third compound. S24. The third compound was dispersed in MES buffer, EDC·HCl and NHS were added, and the mixture was stirred to activate it. Ethylenediamine was dissolved in MES buffer, and the pH was adjusted to 7.2 before being slowly added dropwise. The reaction was continued at room temperature for 2 hours. The resulting reaction solution was centrifuged and purified, and washed with pre-cooled ultrapure water to obtain the fourth compound. S25. The fourth compound was dispersed in borate buffer, and methacryloyl-PEG-thioacetate-PEG-NHS ester was added. The reaction was carried out in the dark. The resulting reaction solution was centrifuged to collect the solid, washed with pre-cooled ultrapure water, and freeze-dried to obtain modified PLGA nanoparticles.

6. A method for preparing a mesenchymal stem cell composition for promoting bone and joint repair, applied to the preparation of a mesenchymal stem cell composition for promoting bone and joint repair as described in any one of claims 1 to 5, characterized in that, The method includes the following steps: S1. Dissolve methacrylamide hyaluronic acid in PBS buffer, add nano-hydroxyapatite, vortex disperse, then add heparin / hyaluronic acid-VEGF microspheres and modified PLGA nanoparticles in sequence, and continue vortex dispersion to obtain the first mixture; S2. Mesenchymal stem cells were digested with trypsin, the cell pellet was collected by centrifugation, and resuspended in PBS buffer to obtain a second mixture; S3. Add lithium phenyl-2,4,6-trimethylbenzoyl phosphinate to the first mixture, mix well, continue to add the second mixture dropwise, mix well after oscillation, and transfer to a mold, place in an ice bath, irradiate with a blue light source with a wavelength of 405-470 nm at a light intensity of 15-20 mW / cm 2 for 60-100 s to obtain a mesenchymal stem cell composition.

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