Mesenchymal stem cell preparation for promoting knee joint repair, preparation method and application

Abstract

The invention provides a mesenchymal stem cell preparation for promoting knee joint repair as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: loading transforming growth factor-beta1 and interleukin-6 on chitosan microspheres, then preparing a chitosan-oxidized starch-carboxymethyl-beta-cyclodextrin bracket loaded with sustained release microspheres, loading luteolin and quercetin, carrying out mixed culture with mesenchymal stem cells, adding a traditional Chinese medicine extract, and carrying out freeze-drying to obtain the luteolin-quercetin-modified mesenchymal stem cells. The mesenchymal stem cell preparation for promoting knee joint repair is prepared. The preparation method of the carbon anode plate for fluorine preparation is simple, the generation of polarization of the carbon anode plate is prevented, and the carbon anode plate has the advantages of low porosity, low ash content, high mechanical strength, high volume density, obviously prolonged service life, low resistivity and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a mesenchymal stem cell preparation for promoting knee joint repair, a preparation method and an application thereof. Background Art

[0002] Cartilage injury is one of the most common knee diseases. Since cartilage has no blood vessels, nerves, or lymphatic tissue, its nutrients mainly come from the synovial fluid of the knee joint. These histological characteristics make the self-repair ability of cartilage injury extremely limited. Traumatic cartilage injury can cause pain and swelling in the patient's joints. If not treated in time, it will accelerate joint degeneration and cause more serious functional disorders.

[0003] At present, the clinical treatment effect is not good, mainly to relieve the patient's pain and improve the function of the knee joint. The reason is that the pathogenesis of cartilage damage is unclear. Drug treatment is mainly symptomatic treatment, temporarily relieving the patient's pain, but the long-term effect is poor, and the symptoms are prone to recurrence. While the drug achieves a certain therapeutic effect, it is also accompanied by some side effects. For elderly and late-stage patients with severe symptoms and poor conservative treatment effects, artificial knee replacement surgery is required. Not only is the cost of the operation high, it will cause a serious burden to the patient and his family, and a second operation is also required. At the same time, cartilage damage, as a long-term and chronic disease, also consumes a lot of medical resources. However, with the development of tissue engineering-related regenerative medicine, the repair of cartilage damage has been achieved.

[0004] Umbilical cord mesenchymal stem cells have stronger proliferation and differentiation capabilities, low immunogenicity, easy access, no ethical restrictions, and are easy to prepare industrially. Umbilical cord mesenchymal stem cells can develop into bone, cartilage, fat, and other types of cells. Once injected into the knee joint of a patient with knee cartilage damage, they can be directed to differentiate into chondrocytes and promote the recovery of damaged cartilage in the body. Umbilical cord mesenchymal stem cells have long-term development prospects in the repair of knee cartilage damage and can cure more patients with knee arthritis who are suffering from pain.

[0005] The existing therapeutic agents using umbilical cord mesenchymal stem cells have ensured the activity of umbilical cord mesenchymal stem cells as much as possible, so that they promote the regeneration of endogenous stem cells in the damaged part and differentiate into osteoblasts and chondrocytes, while promoting the proliferation and replication of original chondrocytes to supplement and repair damaged cartilage tissue; however, the existing preparations have relatively weak protection for articular cartilage during treatment, which prolongs the treatment time and increases the pain of patients.

[0006] Chinese invention patent CN108865986B discloses a mesenchymal stem cell preparation for repairing articular cartilage damage / defects, and its preparation method and application. Mesenchymal stem cells are indirectly co-cultured with synovial cells, and the mesenchymal stem cells after co-culture are harvested and frozen; the umbilical cord mesenchymal stem cells obtained after co-culture by inoculating synovial cells and umbilical cord mesenchymal stem cells at a density ratio of 4:1 are more likely to differentiate into cartilage under the same induction conditions. The preparation is effectively promoted to repair articular cartilage damage / defects by injection into the joint cavity, and the therapeutic effect is significantly better than that of a simple mesenchymal stem cell preparation.

[0007] Chinese invention patent CN102899287B discloses a method for inducing mesenchymal stem cells to differentiate into chondrocytes and its application in osteoarthritis, specifically providing a method for preparing umbilical cord mesenchymal stem cells differentiated into chondrocytes. The scaffold-free cartilage constructed by this method can be used to repair damaged articular cartilage.

[0008] In the above-mentioned prior art, repair of articular cartilage damage is achieved by culturing and inducing mesenchymal stem cells. However, in the above-mentioned culture process, the culture medium used is a serum-containing culture medium. While the serum provides necessary nutrients for cell growth and proliferation, it also has defects such as differences between different batches, large workload, unguaranteed composition, and a high chance of infection by viruses and mycoplasmas. Summary of the invention

[0009] The purpose of the present invention is to propose a mesenchymal stem cell preparation, preparation method and application for promoting knee joint repair. The preparation method is simple, prevents the polarization of the carbon anode plate, has the advantages of low porosity, low ash content, high mechanical strength, high volume density, etc., significantly improved life, low resistivity, etc., and has broad application prospects.

[0010] The technical solution of the present invention is achieved in this way: The present invention provides a method for preparing a mesenchymal stem cell preparation for promoting knee joint repair, wherein transforming growth factor-β 1 and interleukin-6 were loaded on chitosan microspheres, and then a chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was prepared, and luteolin and quercetin were loaded, mixed with mesenchymal stem cells, and traditional Chinese medicine extracts were added to prepare a mesenchymal stem cell preparation that promotes knee joint repair.

[0011] As a further improvement of the present invention, the following steps are included: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: Chitosan was dissolved in acid solution and transforming growth factor-β was added 1and interleukin-6, add an emulsifier octanol solution, stir, dropwise add sodium tripolyphosphate aqueous solution for cross-linking, centrifuge, wash, freeze-dry, and obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: Chitosan was dissolved in acid solution, oxidized starch and carboxymethyl-β-cyclodextrin were added, and transforming growth factor-β was added after stirring and mixing. 1 & interleukin-6 sustained-release chitosan microspheres, adding EDC and NHS, stirring for reaction, filtering, washing, and freeze-drying to prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: dissolving luteolin and quercetin in ethanol, adding chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres, stirring the loading reaction, filtering, washing, and freeze-drying to obtain a composite scaffold material; S4. Composite culture: dripping a mesenchymal stem cell suspension onto the composite scaffold material, culturing, then dripping a cartilage-inducing culture medium, continuing to culture, freeze-drying, and obtaining a cell-laden composite scaffold material; S5. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: dissolving the Chinese medicine extract in water, adding the cell-laden composite scaffold material for impregnation, and freeze-drying to obtain the mesenchymal stem cell preparations for promoting knee joint repair.

[0012] As a further improvement of the present invention, the acid solution in step S1 is a 1-2wt% acetic acid solution, and the chitosan, transforming growth factor-β 1 The mass ratio of interleukin-6, emulsifier and sodium tripolyphosphate is 4-6:0.1-0.15:0.05-0.1:0.5-1:0.1-0.2, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

[0013] As a further improvement of the present invention, the acid solution in step S2 is a 1-2wt% acetic acid solution, and the chitosan, oxidized starch, carboxymethyl-β-cyclodextrin, transforming growth factor-β 1The mass ratio of interleukin-6 sustained-release chitosan microspheres, EDC and NHS is 5-7:5-7:3-6:4-6:1-2:1-2, the stirring reaction time is 5-7h, and the preparation method of the oxidized starch is as follows: 10-15 parts by weight of corn starch is added to 200 parts by weight of water, the pH value of the solution is adjusted to 3-4, 20-25 parts by weight of hydrogen peroxide is continuously added, 0.1-0.3 parts by weight of copper chloride is added, 1000-1200W microwave treatment is performed for 5-10min, the reaction is kept warm for 1-3h, the pH value of the solution is adjusted to 5-6, filtering, washing, and drying to obtain the oxidized starch.

[0014] As a further improvement of the present invention, in step S3, the mass ratio of luteolin, quercetin and chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres is 2-3:1-2:20-25, and the stirring loading reaction time is 1-3h.

[0015] As a further improvement of the present invention, the cell density of the mesenchymal stem cell suspension in step S4 is 10 4 -10 5 The liquid-solid ratio of the mesenchymal stem cell suspension and the composite scaffold material is 1-2 mL: 1 g, and the culture conditions are 35-37°C, 5 v / v% CO 2 , culture for 1-2 days, and the continued culture time is 2-4 days.

[0016] As a further improvement of the present invention, in step S5, the mass ratio of the Chinese medicine extract to the cell-laden composite scaffold material is 3-5:12-15, and the immersion time is 5-7 hours.

[0017] As a further improvement of the present invention, the preparation method of the Chinese medicine extract is as follows: 10-15 parts by weight of Rehmannia glutinosa, 10-15 parts by weight of Psoralea corylifolia, 5-10 parts by weight of Eucommia ulmoides, 5-10 parts by weight of Lycium barbarum, 10-15 parts by weight of Salvia miltiorrhiza, and 3-5 parts by weight of Chuanxiong are washed, dried, and crushed respectively, added to 300-400 parts by weight of water, heated and boiled for extraction for 2-4 hours, filtered, and the extraction was repeated 1-2 times, the filtrate was combined, and dried to obtain the Chinese medicine extract.

[0018] The present invention further protects a mesenchymal stem cell preparation for promoting knee joint repair obtained by the above preparation method.

[0019] The present invention further protects the use of the above-mentioned mesenchymal stem cell preparation for promoting knee joint repair in the preparation of a medicine for treating knee joint inflammation and knee joint injury.

[0020] The present invention has the following beneficial effects: Articular cartilage is a very durable tissue, but once damaged, it is difficult to repair naturally because the cartilage tissue lacks blood vessels and lymphatics. 1 (Transforming Growth Factor-β 1 ) and IL-6 (interleukin-6) are encapsulated in chitosan microspheres to make sustained-release microspheres dispersed in the scaffold for mesenchymal stem cells to differentiate into chondrocytes, and the microspheres have a sustained-release effect, which can protect TGF-β 1 The activity of IL-6 improves the bioavailability of cytokines and regulates the growth and differentiation of cells. When the body suffers from knee joint injury, the expression of IL-6 in plasma and knee joint homogenate is activated, inhibiting the necrosis of the knee joint injury site; the mesenchymal stem cell preparation for promoting knee joint repair of the present invention can significantly increase the IL-6 level after addition, stimulate the body to produce IL-6, and promote the repair of knee joint injury by stimulating the release of IL-6 and enriching it at the knee joint injury site. It has the effect of promoting the proliferation of articular chondrocytes, maintaining the phenotype of chondrocytes in maintaining the normal engineering of articular cartilage and the repair of cartilage injuries, and has a sustained release effect, thereby prolonging the efficacy of the drug.

[0021] The present invention prepares an oxidized starch, and the oxidized starch with high carboxyl content and low depolymerization degree can be prepared by oxidizing the starch with hydrogen peroxide. Hydrogen peroxide can easily penetrate deep into the starch granules, oxidize the starch molecules at C 2 and C 3 The chain is broken at the end, causing the starch to depolymerize and producing carboxyl content. Excessive hydrogen peroxide will eventually decompose into water, which will not affect the purity of the product or the environment. It is an ideal green process. Oxidized starch is the same as native starch, non-toxic, non-irritating, biodegradable and biocompatible. The scaffold uses starch-based materials with good degradability and compatibility. It can form hydrogels with balanced solid and liquid mechanical properties at the joints, which can improve the mechanical microenvironment of the joints. It can also promote the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs), thereby affecting the early mineralization process of new bone. In the later stage, the scaffold carrier material will slowly degrade, leaving almost no residue in the body.

[0022] In the present invention, chitosan, oxidized starch, and carboxymethyl-β-cyclodextrin are subjected to condensation between carboxyl groups and amino groups to form a polymer scaffold. On the one hand, the polymer scaffold can load transforming growth factor-β through hydrogen bonding. 1 &Interleukin-6 sustained-release chitosan microspheres, on the other hand, the cavity structure of β-cyclodextrin can fix the loading of luteolin and quercetin, greatly increasing the loading amount of such water-insoluble anti-inflammatory active components, thereby improving their efficacy, and having the effects of auxiliary synergistic anti-inflammatory and enhancing autophagy. In addition, the present invention adopts EDC and NHS for cross-linking, without the use of glutaraldehyde and genipin cross-linking agents, which reduces toxicity and sensitization, and is safer to use.

[0023] Since the scaffold prepared by chitosan has a small pore size, cell growth is restricted, resulting in low cell viability on the chitosan scaffold. The chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold prepared by the present invention has abundant pores and a large specific surface area. The present invention carries out composite culture of mesenchymal stem cells and the prepared composite scaffold material, providing abundant space and nutrition for the culture of stem cells, thereby greatly promoting cell proliferation and growth. The stem cells can differentiate into chondrocytes, directly participate in the synthesis of cartilage, and secrete a variety of growth factors and cytokines to promote the proliferation and differentiation of chondrocytes, increase the synthesis of cartilage matrix, and upregulate the expression of cartilage formation-related genes, such as type II collagen (Col-Ⅱ) and B cell lymphoma-2 gene (BCL-2), promote chondrocyte survival, thereby greatly promoting the repair of knee joints.

[0024] In addition, the preparation of the present invention, which is made of the Chinese medicine compatibility of Radix Rehmanniae Preparata, Fructus Psoraleae, Eucommia ulmoides, Fructus Lycii, Radix Salviae Miltiorrhizae, and Rhizoma Chuanxiong, has the effects of tonifying the kidney and strengthening bones, promoting blood circulation and relieving pain, and has a significant improvement effect on the treatment of knee joint injuries such as knee osteoarthritis. The Chinese medicine extract is used in combination with mesenchymal stem cells, and the various active ingredients of the Chinese medicine extract can cooperate with the biological characteristics of stem cells to promote the repair of knee joint cartilage, and the Chinese medicine ingredients can enhance the proliferation and differentiation ability of stem cells, and further promote the generation of chondrocytes. In addition, the anti-inflammatory effect of Chinese medicine can be combined with the immunomodulatory function of stem cells to jointly improve the inflammatory microenvironment of the knee joint.

[0025] The mesenchymal stem cell preparation for promoting knee joint repair prepared by the present invention can promote the differentiation of mesenchymal stem cells into chondrocytes, promote the generation and proliferation of chondrocytes, and maintain the chondrocyte phenotype. It is safe and non-toxic, has a significant improvement effect on the treatment of knee osteoarthritis, knee joint injuries and other diseases, and has broad application prospects. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] NHS, N-hydroxysuccinimide, EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0028] Mesenchymal stem cells were porcine adipose-derived mesenchymal stem cells purchased from Shanghai Lianzu Biotechnology Co., Ltd.

[0029] The preparation method of mesenchymal stem cell suspension is as follows: add porcine adipose mesenchymal stem cells into α-MEM complete culture medium containing 10wt% fetal bovine serum, and adjust the cell density to 10 4 -10 5 / mL to prepare mesenchymal stem cell suspension.

[0030] Chondrogenic culture medium, CTCC-Y002, for in vitro induction of MSCs differentiation into chondrocytes, was purchased from Wuxi Puhe Biopharmaceutical Technology Co., Ltd.

[0031] Preparation Example 1 Preparation of oxidized starch The method is as follows: add 10g corn starch to 200wt water, adjust the pH value of the solution to 3, continue to add 20g hydrogen peroxide, add 0.1g copper chloride, microwave at 1000W for 5min, keep warm for 1h, adjust the pH value of the solution to 5, filter, wash, and dry to obtain oxidized starch.

[0032] Preparation Example 2 Preparation of oxidized starch The method is as follows: add 15g corn starch to 200wt water, adjust the pH value of the solution to 4, continue to add 25g hydrogen peroxide, add 0.3g copper chloride, microwave at 1200W for 10min, keep warm for 3h, adjust the pH value of the solution to 6, filter, wash, and dry to obtain oxidized starch.

[0033] Preparation Example 3 Preparation of oxidized starch The method is as follows: add 12g corn starch to 200wt water, adjust the solution pH to 3.5, continue to drop 22g hydrogen peroxide, add 0.2g copper chloride, microwave at 1100W for 7min, keep warm for 2h, adjust the solution pH to 5.5, filter, wash, and dry to obtain oxidized starch.

[0034] Preparation Example 4 Preparation of Chinese herbal medicine extract The method is as follows: wash, dry, and crush 10g of Rehmannia glutinosa, 10g of Psoralea corylifolia, 5g of Eucommia ulmoides, 5g of Lycium barbarum, 10g of Salvia miltiorrhiza, and 3g of Chuanxiong respectively, add them into 300g of water, heat and boil to extract for 2h, filter, repeat the extraction once, combine the filtrate, and dry to obtain the Chinese medicine extract.

[0035] Preparation Example 5 Preparation of Chinese herbal medicine extract The method is as follows: wash, dry, and crush 15g of Rehmannia glutinosa, 15g of Psoralea corylifolia, 10g of Eucommia ulmoides, 10g of Lycium barbarum, 15g of Salvia miltiorrhiza, and 5g of Chuanxiong respectively, add them into 400g of water, heat and boil to extract for 4h, filter, repeat the extraction twice, combine the filtrate, and dry to obtain the Chinese medicine extract.

[0036] Preparation Example 6 Preparation of Chinese herbal medicine extract The method is as follows: wash, dry, and crush 12g of Rehmannia glutinosa, 12g of Psoralea corylifolia, 7g of Eucommia ulmoides, 7g of Lycium barbarum, 12g of Salvia miltiorrhiza, and 4g of Chuanxiong respectively, add them into 350g of water, heat and boil to extract for 3h, filter, repeat the extraction twice, combine the filtrate, and dry to obtain the Chinese medicine extract. Example 1

[0037] This embodiment provides a method for preparing a mesenchymal stem cell preparation for promoting knee joint repair, comprising the following steps: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 4 g chitosan was dissolved in 200 mL 1 wt% acetic acid solution, and 0.1 g transforming growth factor-β was added. 1 and 0.05 g interleukin-6, add 50 mL of octanol solution containing 0.5 g Tween-20, stir, add 10 mL of 0.1 g sodium tripolyphosphate aqueous solution, cross-link for 1 h, centrifuge, wash, and freeze-dry to obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 5 g of chitosan was dissolved in 200 mL of 1 wt% acetic acid solution, 5 g of oxidized starch prepared in Preparation Example 1 and 3 g of carboxymethyl-β-cyclodextrin were added, and 4 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 1g EDC and 1g NHS, stir and react for 5h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: 2 g luteolin and 1 g quercetin were dissolved in 200 mL ethanol, and 20 g chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added, and the loading reaction was stirred for 1 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S4. Composite culture: 10 mL of mesenchymal stem cell suspension was added dropwise to 10 g of composite scaffold material at 35°C and 5 v / v% CO 2 , cultured for 1 day, then added 10 mL of chondrogenic culture medium, cultured for another 2 days, and freeze-dried to obtain a cell-laden composite scaffold material; S5. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: 3 g of the Chinese medicine extract prepared in Preparation Example 4 was dissolved in 200 ml of water, 12 g of the cell-laden composite scaffold material was added and immersed for 5 hours, and freeze-dried to obtain a mesenchymal stem cell preparation for promoting knee joint repair. Example 2

[0038] This embodiment provides a method for preparing a mesenchymal stem cell preparation for promoting knee joint repair, comprising the following steps: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 6 g chitosan was dissolved in 200 mL 2 wt% acetic acid solution, and 0.15 g transforming growth factor-β was added. 1 and 0.1 g interleukin-6, add 50 mL of octanol solution containing 1 g Tween-40, stir, add 10 mL of 0.2 g sodium tripolyphosphate aqueous solution, cross-link for 1 h, centrifuge, wash, and freeze-dry to obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 7 g of chitosan was dissolved in 200 mL of 2 wt% acetic acid solution, 7 g of oxidized starch prepared in Preparation Example 2 and 6 g of carboxymethyl-β-cyclodextrin were added, and 6 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 2g EDC and 2g NHS, stir and react for 7h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: 3 g of luteolin and 2 g of quercetin were dissolved in 200 mL of ethanol, and 25 g of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added, and the loading reaction was stirred for 3 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S4. Composite culture: 12 mL of mesenchymal stem cell suspension was added dropwise to 10 g of composite scaffold material at 37°C and 5 v / v% CO 2 , cultured for 2 days, then added 10 mL of chondrogenic culture medium, cultured for another 4 days, and freeze-dried to obtain a cell-laden composite scaffold material; S5. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: 5 g of the Chinese medicine extract prepared in Preparation Example 5 was dissolved in 200 ml of water, 15 g of the cell-laden composite scaffold material was added and immersed for 7 hours, and freeze-dried to obtain a mesenchymal stem cell preparation for promoting knee joint repair. Example 3

[0039] This embodiment provides a method for preparing a mesenchymal stem cell preparation for promoting knee joint repair, comprising the following steps: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, and 0.12 g transforming growth factor-β was added. 1and 0.07g interleukin-6, add 50mL of octanol solution containing 0.7g Tween-80, stir, add 10mL of 0.15g sodium tripolyphosphate aqueous solution, cross-link for 1h, centrifuge, wash, freeze-dry, and obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 6 g of oxidized starch prepared in Preparation Example 3 and 4.5 g of carboxymethyl-β-cyclodextrin were added, and 5 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 1.5g EDC and 1.5g NHS, stir and react for 6h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: 2.5 g luteolin and 1.5 g quercetin were dissolved in 200 mL ethanol, and 22 g chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added, and the loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S4. Composite culture: 11 mL of mesenchymal stem cell suspension was added dropwise to 10 g of composite scaffold material at 36°C and 5 v / v% CO 2 , cultured for 1.5 days, then added 10 mL of chondrogenic culture medium, cultured for another 3 days, and freeze-dried to obtain a cell-laden composite scaffold material; S5. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: 4 g of the Chinese medicine extract prepared in Preparation Example 6 was dissolved in 200 ml of water, 13 g of the cell-laden composite scaffold material was added, immersed for 6 hours, and freeze-dried to obtain a mesenchymal stem cell preparation for promoting knee joint repair.

[0040] Comparative Example 1 Compared with Example 3, the difference is that transforming growth factor-β is not added in step S1 1 .

[0041] The details are as follows: S1. Preparation of interleukin-6 sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, 19 g interleukin-6 was added, 50 mL octanol solution containing 0.7 g Tween-80 was added, stirred, 10 mL 0.15 g sodium tripolyphosphate aqueous solution was added dropwise, cross-linked for 1 h, centrifuged, washed, and freeze-dried to obtain interleukin-6 sustained-release chitosan microspheres.

[0042] Comparative Example 2 Compared with Example 3, the difference is that interleukin-6 is not added in step S1.

[0043] The details are as follows: S1. Transforming Growth Factor-β 1 Preparation of sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, and 0.19 g transforming growth factor-β was added. 1 , add 50 mL of octanol solution containing 0.7 g of Tween-80, stir, add 10 mL of 0.15 g of sodium tripolyphosphate aqueous solution, cross-link for 1 h, centrifuge, wash, and freeze-dry to obtain transforming growth factor-β 1 Sustained release chitosan microspheres.

[0044] Comparative Example 3 Compared with Example 3, the difference is that step S1 is not performed.

[0045] The details are as follows: S1. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 6 g of oxidized starch prepared in Preparation Example 3 and 4.5 g of carboxymethyl-β-cyclodextrin were added, and after stirring and mixing, 1.5 g of EDC and 1.5 g of NHS were added, and the mixture was stirred for 6 h, filtered, washed, and freeze-dried to obtain a chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold; S2. Loading luteolin and quercetin: 2.5 g luteolin and 1.5 g quercetin were dissolved in 200 mL ethanol, 22 g chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold was added, the loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S3. Composite culture: 11 mL of mesenchymal stem cell suspension was added dropwise to 10 g of composite scaffold material at 36°C and 5 v / v% CO 2 , cultured for 1.5 days, then added 10 mL of chondrogenic culture medium, cultured for another 3 days, and freeze-dried to obtain a cell-laden composite scaffold material; S4. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: 4 g of the Chinese medicine extract prepared in Preparation Example 6 was dissolved in 200 ml of water, 13 g of the cell-laden composite scaffold material was added, immersed for 6 hours, and freeze-dried to obtain a mesenchymal stem cell preparation for promoting knee joint repair.

[0046] Comparative Example 4 Compared with Example 3, the difference is that no oxidized starch is added in step S2.

[0047] The details are as follows: S2. Preparation of chitosan-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 6 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, 10.5 g carboxymethyl-β-cyclodextrin was added, and after stirring and mixing, 5 g transforming growth factor-β was added. 1 & Interleukin-6 sustained-release chitosan microspheres were added with 1.5 g EDC and 1.5 g NHS, stirred for 6 h, filtered, washed, and freeze-dried to obtain chitosan-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres.

[0048] Comparative Example 5 Compared with Example 3, the difference is that carboxymethyl-β-cyclodextrin is not added in step S2.

[0049] The details are as follows: S2. Preparation of chitosan-oxidized starch scaffold loaded with sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 10.5 g of oxidized starch prepared in Preparation Example 3 was added, and 5 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres were added with 1.5 g EDC and 1.5 g NHS, stirred for 6 h, filtered, washed, and freeze-dried to obtain chitosan-oxidized starch scaffolds loaded with sustained-release microspheres.

[0050] Comparative Example 6 Compared with Example 3, the difference is that luteolin is not added in step S3.

[0051] The details are as follows: S3. Loading quercetin: 4 g of quercetin was dissolved in 200 mL of ethanol, and 22 g of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added. The loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material.

[0052] Comparative Example 7 Compared with Example 3, the difference is that quercetin is not added in step S3.

[0053] The details are as follows: S3. Loading luteolin: 4 g of luteolin was dissolved in 200 mL of ethanol, and 22 g of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added. The loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material.

[0054] Comparative Example 8 Compared with Example 3, the difference is that step S3 is not performed.

[0055] The details are as follows: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, and 0.12 g transforming growth factor-β was added. 1 and 0.07g interleukin-6, add 50mL of octanol solution containing 0.7g Tween-80, stir, add 10mL of 0.15g sodium tripolyphosphate aqueous solution, cross-link for 1h, centrifuge, wash, freeze-dry, and obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 6 g of oxidized starch prepared in Preparation Example 3 and 4.5 g of carboxymethyl-β-cyclodextrin were added, and 5 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 1.5g EDC and 1.5g NHS, stir and react for 6h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Composite culture: 11 mL of mesenchymal stem cell suspension was added dropwise to 10 g of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres at 36°C and 5 v / v% CO 2 , cultured for 1.5 days, then added 10 mL of chondrogenic culture medium, cultured for another 3 days, and freeze-dried to obtain a cell-laden composite scaffold material; S4. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: 4 g of the Chinese medicine extract prepared in Preparation Example 6 was dissolved in 200 ml of water, 13 g of the cell-laden composite scaffold material was added, immersed for 6 hours, and freeze-dried to obtain a mesenchymal stem cell preparation for promoting knee joint repair.

[0056] Comparative Example 9 Compared with Example 3, the difference is that step S4 is not performed.

[0057] The details are as follows: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, and 0.12 g transforming growth factor-β was added. 1 and 0.07g interleukin-6, add 50mL of octanol solution containing 0.7g Tween-80, stir, add 10mL of 0.15g sodium tripolyphosphate aqueous solution, cross-link for 1h, centrifuge, wash, freeze-dry, and obtain transforming growth factor-β 1&Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 6 g of oxidized starch prepared in Preparation Example 3 and 4.5 g of carboxymethyl-β-cyclodextrin were added, and 5 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 1.5g EDC and 1.5g NHS, stir and react for 6h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: 2.5 g luteolin and 1.5 g quercetin were dissolved in 200 mL ethanol, and 22 g chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added, and the loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S4. Preparation of a preparation for promoting knee joint repair: 4 g of the Chinese herbal extract prepared in Preparation Example 6 was dissolved in 200 ml of water, 13 g of the composite scaffold material was added, immersed for 6 hours, and freeze-dried to prepare a preparation for promoting knee joint repair.

[0058] Comparative Example 10 Compared with Embodiment 3, the difference is that step S5 is not performed.

[0059] The details are as follows: S1. Transforming Growth Factor-β 1 Preparation of interleukin-6 sustained-release chitosan microspheres: 5 g chitosan was dissolved in 200 mL 1.5 wt% acetic acid solution, and 0.12 g transforming growth factor-β was added. 1 and 0.07g interleukin-6, add 50mL of octanol solution containing 0.7g Tween-80, stir, add 10mL of 0.15g sodium tripolyphosphate aqueous solution, cross-link for 1h, centrifuge, wash, freeze-dry, and obtain transforming growth factor-β 1 &Interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: 6 g of chitosan was dissolved in 200 mL of 1.5 wt% acetic acid solution, 6 g of oxidized starch prepared in Preparation Example 3 and 4.5 g of carboxymethyl-β-cyclodextrin were added, and 5 g of transforming growth factor-β was added after stirring and mixing. 1 & Interleukin-6 sustained-release chitosan microspheres, add 1.5g EDC and 1.5g NHS, stir and react for 6h, filter, wash, freeze-dry, and prepare chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: 2.5 g luteolin and 1.5 g quercetin were dissolved in 200 mL ethanol, and 22 g chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was added, and the loading reaction was stirred for 2 h, filtered, washed, and freeze-dried to obtain a composite scaffold material; S4. Composite culture: 11 mL of mesenchymal stem cell suspension was added dropwise to 10 g of composite scaffold material at 36°C and 5 v / v% CO 2 , culture for 1.5 days, then add 10 mL of chondrogenic culture medium, continue to culture for 3 days, and freeze-dry to obtain a cell-laden composite scaffold material, which is a mesenchymal stem cell preparation for promoting knee joint repair.

[0060] Test Example 1 New Zealand white rabbits were randomly divided into a model group, an embodiment 1-3 group, and a comparative example 1-10 group, with 6 rabbits in each group; all rabbits were subjected to knee cartilage defect modeling. Anesthesia, shaving, routine disinfection of both lower limbs; aseptic operation was performed to make an incision on the medial side of the knee joint, the subcutaneous tissue was cut and separated, the medial joint capsule was cut, the joint cavity was exposed, the patella was guided to the lateral dislocation, and the articular surface of the medial femoral condyle was exposed; an electric hand drill was used to drill a hole in the center of the medial femoral condyle with a diameter of 4 mm and a depth of 3 mm to reach the subchondral bone, causing full-thickness cartilage damage, flushing the joint cavity, resetting the patella, and suturing the knee joint layer by layer. After surgery, the rabbits were returned to the cage and were free to eat and move. Each rabbit was injected with 800,000 units of penicillin sodium intramuscularly from the day of surgery, once a day, for 3 consecutive days to prevent infection.

[0061] After successful modeling, the corresponding mesenchymal stem cell preparations for promoting knee joint repair (1 mg / mL, 2 mL / animal / time, once a week) were injected into the knee joint cavities of the groups of Examples 1-3 and Comparative Examples 1-10, and sodium hyaluronate gel (0.5 mL / animal / time, once a week) was injected into the knee joint cavities of the model group and the blank group. The Chinese medicine decoction of groups A and B was administered by gavage, and the equivalent dose converted according to the animal body surface area was 3 mL / (kg·d), and the treatment was continued for five weeks.

[0062] After 12 weeks, 0.5 mL of normal saline was injected without opening the knee joint cavity. After fully flexing and extending the joint, the synovial fluid was aspirated and centrifuged to remove residual cells. The contents of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-13 (MMP-13), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were detected according to the instructions of the ELISA kit.

[0063] Subsequently, two rabbits in each group were killed by air embolism. The medial femoral condyles of both knees of the rabbits were removed at the same time, and their general condition was observed with the naked eye. The specimens were then fixed, decalcified, embedded, and sliced, and then HE staining and immunohistochemical examination were performed: referring to the scoring standard for cartilage defect repair (Wakitani scoring method), the morphology of the repaired tissue after HE staining was semi-quantitatively scored.

[0064] The results are shown in Table 1.

[0065] Table 1

[0066] Notes: Compared with the model group, P<0.05.

[0067] After cartilage damage, the homeostasis of native cartilage is broken, and the destruction of the dynamic balance between MMP and TIMP is an important cause of the occurrence and development of knee osteoarthritis. MMP is an endopeptidase that can degrade the extracellular matrix and plays an important role in the degradation of cartilage matrix, while TIMP is a secreted glycoprotein that blocks the degradation of the extracellular matrix and inhibits the activity of MMP, and it participates in the establishment and maintenance of tissue structure. As can be seen from the above table, the mesenchymal stem cell preparations for promoting knee joint repair prepared in Examples 1-3 of the present invention can significantly reduce the Wakitani score, reduce the content of MMP-9 and MMP-13, and increase the content of TIMP-1.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell preparation for promoting knee joint repair, characterized in that: Transforming growth factor-β1 and interleukin-6 were loaded on chitosan microspheres, and then a chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres was prepared, and luteolin and quercetin were loaded, mixed with mesenchymal stem cells, and traditional Chinese medicine extracts were added to prepare a mesenchymal stem cell preparation that promotes knee joint repair.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Preparation of transforming growth factor-β1 & interleukin-6 sustained-release chitosan microspheres: dissolving chitosan in acid solution, adding transforming growth factor-β1 and interleukin-6, adding emulsifier octanol solution, stirring, adding sodium tripolyphosphate aqueous solution for cross-linking, centrifuging, washing, freeze-drying, and preparing transforming growth factor-β1 & interleukin-6 sustained-release chitosan microspheres; S2. Preparation of chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres: dissolving chitosan in acid solution, adding oxidized starch and carboxymethyl-β-cyclodextrin, stirring and mixing evenly, adding transforming growth factor-β1 & interleukin-6 sustained-release chitosan microspheres, adding EDC and NHS, stirring and reacting, filtering, washing, and freeze-drying to obtain chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres; S3. Loading luteolin and quercetin: dissolving luteolin and quercetin in ethanol, adding chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres, stirring the loading reaction, filtering, washing, and freeze-drying to obtain a composite scaffold material; S4. Composite culture: dripping a mesenchymal stem cell suspension onto the composite scaffold material, culturing, then dripping a cartilage-inducing culture medium, continuing to culture, freeze-drying, and obtaining a cell-laden composite scaffold material; S5. Preparation of mesenchymal stem cell preparations for promoting knee joint repair: dissolving the Chinese medicine extract in water, adding the cell-laden composite scaffold material for impregnation, and freeze-drying to obtain the mesenchymal stem cell preparations for promoting knee joint repair.

3. The preparation method according to claim 2, characterized in that: The acid solution in step S1 is a 1-2wt% acetic acid solution, the mass ratio of the chitosan, transforming growth factor-β1, interleukin-6, emulsifier and sodium tripolyphosphate is 4-6:0.1-0.15:0.05-0.1:0.5-1:0.1-0.2, and the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

4. The preparation method according to claim 2, characterized in that: The acid solution in step S2 is a 1-2wt% acetic acid solution, the mass ratio of the chitosan, oxidized starch, carboxymethyl-β-cyclodextrin, transforming growth factor-β1 & interleukin-6 sustained-release chitosan microspheres, EDC and NHS is 5-7:5-7:3-6:4-6:1-2:1-2, the stirring reaction time is 5-7h, and the preparation method of the oxidized starch is as follows: 10-15 parts by weight of corn starch is added to 200 parts by weight of water, the pH value of the solution is adjusted to 3-4, 20-25 parts by weight of hydrogen peroxide is continued to be added, 0.1-0.3 parts by weight of copper chloride is added, 1000-1200W microwave treatment is performed for 5-10min, the reaction is kept warm for 1-3h, the pH value of the solution is adjusted to 5-6, filtering, washing, and drying to obtain oxidized starch.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass ratio of luteolin, quercetin and chitosan-oxidized starch-carboxymethyl-β-cyclodextrin scaffold loaded with sustained-release microspheres is 2-3:1-2:20-25, and the time of the stirring loading reaction is 1-3h.

6. The preparation method according to claim 2, characterized in that: The cell density of the mesenchymal stem cell suspension in step S4 is 10 4 -10 5 / mL, the liquid-to-solid ratio of the mesenchymal stem cell suspension and the composite scaffold material is 1-2mL:1g, the culture conditions are 35-37°C, 5v / v%CO2, culture for 1-2d, and the continued culture time is 2-4d.

7. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of the Chinese medicine extract to the cell-laden composite scaffold material is 3-5:12-15, and the immersion time is 5-7 hours.

8. The preparation method according to claim 7, characterized in that: The preparation method of the Chinese medicine extract is as follows: 10-15 parts by weight of Rehmannia glutinosa, 10-15 parts by weight of Psoralea corylifolia, 5-10 parts by weight of Eucommia ulmoides, 5-10 parts by weight of Lycium barbarum, 10-15 parts by weight of Salvia miltiorrhiza, and 3-5 parts by weight of Chuanxiong are washed, dried, and crushed respectively, added into 300-400 parts by weight of water, heated and boiled for extraction for 2-4 hours, filtered, and the extraction is repeated 1-2 times, the filtrate is combined, and dried to obtain the Chinese medicine extract.

9. A mesenchymal stem cell preparation for promoting knee joint repair obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the mesenchymal stem cell preparation for promoting knee joint repair as claimed in claim 9 in the preparation of a drug for treating knee joint inflammation and knee joint injury.

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