A method for increasing the glycosaminoglycan content in cartilage using a cationic polymer

Abstract

The application provides a method for improving the content of cartilage glycosaminoglycan by using cationic polymer, and the method promotes the content of glycosaminoglycan in cartilage tissue to be obviously increased by treating cartilage with a preparation containing polybrene, creates a good microenvironment for cartilage repair, and promotes the cartilage to be better repaired and regenerated; the method can improve the content of glycosaminoglycan in the differentiated tissue of mesenchymal stem cells by culturing the mesenchymal stem cells in an induction medium containing polybrene, promotes the mesenchymal stem cells to be better differentiated into cartilage, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a method for improving the content of glycosaminoglycan in cartilage by using cationic polymers. BACKGROUND

[0002] Adult articular cartilage has limited regenerative capacity and is difficult to repair spontaneously after injury, resulting in permanent damage such as osteoarthritis, pain and loss of labor, which has affected the quality of life of more than 100 million people in China. At present, the complete regeneration of the extracellular matrix of articular cartilage is still a great challenge. Irreversible loss of glycosaminoglycan is a typical pathological feature of osteoarthritis. The extracellular matrix component accounts for 95% of the total weight of hyaline cartilage and is the material basis for its support and lubrication, and there is a large amount of sulfated glycosaminoglycan in it, which creates a negative charged microenvironment for cartilage, protecting cartilage cells and maintaining tissue moisture. However, glycosaminoglycan is easily lost and difficult to regenerate in cartilage lesions. For example, under the conditions of mechanical damage, inflammation, etc., the reduction of glycosaminoglycan on the surface of the joint and the fibrous proliferation are typical signs of early osteoarthritis. Its progressive loss causes the decline of the lubrication and support capacity of the articular cartilage.

[0003] At present, there are the following treatment methods for the loss of glycosaminoglycan:

[0004] 1. Oral or injection of hyaluronic acid, chondroitin sulfate and other glycosaminoglycans to supplement the glycosaminoglycan components of cartilage tissue. Such methods can short-term relieve osteoarthritis symptoms, but the effect is controversial because the supplemented glycosaminoglycan is difficult to stay in the joint cavity for a long time and be continuously synthesized by cartilage tissue, and cannot effectively prevent the progression of osteoarthritis.

[0005] 2. Using a biomimetic hydrogel containing glycosaminoglycan components to fill the missing cartilage tissue, which can physically protect the cartilage surface and provide a microenvironment similar to the physiological environment for in situ cells; however, the biomimetic hydrogel cannot completely mimic the complex structure and composition of natural glycosaminoglycan, and once the endogenous cells cannot be effectively attracted to participate in the regeneration of glycosaminoglycan, the regeneration effect is not complete; in addition, the effect of glycosaminoglycan on cell adhesion and proliferation is still controversial, and for the repair of large area defects, there is a problem of poor tissue integration.

[0006] 3. The use of mesenchymal stem cells for the treatment of osteoarthritis or cartilage injury, wherein the method involves: intra-articular injection of mesenchymal stem cells, microfracture (surgical puncture of subchondral bone to allow migration of bone marrow mesenchymal stem cells in the subchondral bone marrow cavity to the injury for repair), mesenchymal stem cells have a certain chondrogenic ability, but it is difficult to completely regenerate hyaline (articular) chondrocytes, and the newly formed fibrous cartilage will have a lack of glycosaminoglycan and compensatory abnormal extracellular matrix. Using methods such as tissue engineering, using growth factors to regulate the chondrogenic differentiation of mesenchymal stem cells, or biologic scaffold loaded cell implantation, can improve the repair effect to a certain extent, but still accompanied by fibrous tissue formation.

[0007] In summary, the existing methods cannot effectively solve the problem that glycosaminoglycan cannot be completely regenerated in cartilage injury and degeneration. The main reasons include 1) the supplemented glycosaminoglycan is difficult to effectively reside on the joint surface; 2) the existing methods cannot accurately stimulate cells to produce new glycosaminoglycan.

[0008] It can be seen that glycosaminoglycan is very important for cartilage function, and the existing methods are difficult to achieve effective regeneration of glycosaminoglycan, so it is difficult to provide a good microenvironment for cartilage regeneration to promote the repair of cartilage. Therefore, it is urgent to find a method that can realize the formation of cartilage glycosaminoglycan. SUMMARY

[0009] To solve the above problems, the present application provides a method for improving the content of glycosaminoglycan in cartilage by using cationic polymer, which can significantly increase the content of glycosaminoglycan in cartilage tissue by using a preparation containing polybrene to treat cartilage, create a good microenvironment for cartilage repair, and promote better repair and regeneration of cartilage. By using an induction medium containing polybrene to culture mesenchymal stem cells, the content of glycosaminoglycan in the differentiated tissue of mesenchymal stem cells can be increased, and the differentiation of mesenchymal stem cells into cartilage can be promoted.

[0010] In one aspect, the present application provides a method for improving the content of glycosaminoglycan in a tissue, which comprises treating the tissue with a preparation containing polybrene.

[0011] Glycosaminoglycan, also known as mucopolysaccharide, is a general term for the glycan part of proteoglycan macromolecules, which exists in many substances in nature, such as animal skin, animal cartilage and even the surface mucosa of the gastrointestinal tract or respiratory tract of the human body. Glycosaminoglycan is composed of disaccharide repeating units of glycosamine, one of which is an amino-containing sugar, and the other is usually uronic acid, and the hydroxyl group of the sugar group is usually sulfated. Glycosaminoglycan is an important component of cartilage tissue, which plays a role in lubrication, tissue support and buffering. Glycosaminoglycan is easily lost in cartilage lesions, which affects the function of cartilage tissue and brings serious social impact. Therefore, new treatment methods need to be developed to promote the regeneration of cartilage glycosaminoglycan.

[0012] GAGs can be divided into chondroitin sulfate, keratin sulfate, hyaluronic acid, heparin and heparan sulfate. GAGs have protective support and other functions in tissues, and provide a steady-state microenvironment for cells, and are important substances in regenerative medicine. The main components of cartilage GAGs are chondroitin sulfate and hyaluronic acid, and chondroitin sulfate is a component of glycoprotein (proteoglycan, aggrecan (ACAN)).

[0013] Polybrene is also known as bromohexamethyleneimmonium, which is a cationic polymer. It is most commonly used in virus-mediated gene transfection. The mechanism of action may be to neutralize the electrostatic repulsion on the surface of cells and viruses. In cell culture, it is mainly used to improve the efficiency of retrovirus infection of cells by neutralizing the charge repulsion between sialic acid on the cell surface and viral particles. Polybrene also has a certain effect in protein sequencing, and can be autoclaved at high temperature and high pressure, which is safe, controllable, cheap and convenient to use.

[0014] Since the cationic polymer has a positive charge and the GAG has a negative charge, the application attempts to use a cationic polymer to treat damaged cartilage tissue, hoping to effectively improve the concentration of GAG in the cartilage tissue.

[0015] After a large number of research experiments, the application found that by using a preparation containing polybrene to treat cartilage tissue, the content of GAG in the cartilage tissue can be effectively improved, and the cartilage repair and regeneration can be promoted. The reason may be that polybrene has a positive charge, GAG and cell membrane have a negative charge, and polybrene may use the charge attraction to adsorb GAG in the tissue, promote the adhesion and integration of cells and GAG, and maintain the residence of GAG. It is also possible that polybrene can effectively increase the content of GAG in the cartilage tissue differentiated from mesenchymal stem cells, which will promote the increase of GAG metabolism. This needs to be further studied and proved.

[0016] Further, the structural formula of the polybrene is shown as formula (1):

[0017]

[0018] Wherein, n = 2000-8000.

[0019] In some ways, preferably, n = 4000-6000.

[0020] Further, the concentration of polybrene in the preparation is 5-100 ug / mL; the tissue is cartilage tissue or cartilage-like tissue. The GAG includes chondroitin sulfate, PRG4, ACAN glycoprotein, etc.

[0021] In some ways, the preparation containing polybrene is a photo-crosslinked hydrogel, which can be applied to the surface of cartilage injury and photo-crosslinked after ultraviolet lamp irradiation.

[0022] In some embodiments, the method of dissolving the polybrene is to weigh the corresponding powder, pre-dissolve in double distilled water as a stock solution, the concentration of the polybrene in the stock solution is 10-100 mg / ml. Filter sterilization using a sterile filter with a pore size of 0.22 μm, then add the polybrene stock solution to the hydrogel solution that has not been photo-crosslinked, add dropwise, mix evenly while adding, until the final concentration of polybrene in the hydrogel solution is 5-100 μg / ml (preferably 10-50 μg / ml).

[0023] In some embodiments, the composition of the hydrogel solution is preferably but not limited to the following: phosphate buffer as the solvent (pH 7.2), 20% (mass fraction) of gelatin-methacryloyl (Gelatin-Methacryloyl, hereinafter referred to as GelMA), 10% (mass fraction) of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide modified chondroitin sulfate (Chondroitin Sulfate-N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide, abbreviated as CS-NB), and 0.25% (mass fraction) of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide modified hyaluronic acid (Hyaluronic Acid-N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide, abbreviated as HA-NB). The solution is subjected to sterilization treatment at 120°C for 8 minutes before use.

[0024] In some embodiments, a photoinitiator needs to be added to the hydrogel solution before photo-crosslinking, the photoinitiator is preferably but not limited to 0.25% (mass fraction) of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, abbreviated as LAP). The wavelength of ultraviolet irradiation is 405 nm, and the irradiation time is 20-30 seconds.

[0025] In some embodiments, for in vitro culture of cartilage, osteoarthritis patient cartilage tissue is cut into small cartilage pieces (5mm x 1cm x 5mm) under sterile conditions, and cultured in Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (Gibico 11320033) containing polybrene, at 37°C, 5% carbon dioxide, and the rest air, for 14-28 days, with fresh medium containing polybrene being replaced every 3 days.

[0026] In another aspect, the present application provides a preparation for cartilage injury repair, which contains polybrene.

[0027] In yet another aspect, the present application provides a method for promoting mesenchymal stem cell chondrogenesis, which comprises culturing mesenchymal stem cells in a medium containing polybrene.

[0028] Mesenchymal stem cells are adult stem cells derived from mesoderm, which have self-renewal and multi-directional differentiation potential. Because they can maintain biological characteristics after large-scale expansion in vitro, mesenchymal stem cells are widely used in tissue engineering and regenerative medicine. They are also often used in the treatment of osteoarthritis and cartilage injury. Mesenchymal stem cells have chondrogenic potential, but the differentiated cartilage phenotype is different from the hyaline chondrocytes on the joint surface, so the clinical treatment effect is controversial, often accompanied by fibrocartilage formation. The main influencing factors include microenvironment factors and the differentiation potential of mesenchymal stem cells.

[0029] The medium containing polybrene provided by the present application can provide a better microenvironment for mesenchymal stem cells, thereby helping mesenchymal stem cells to better differentiate into cartilage suitable for clinical treatment, and eliminating the difference from natural chondrocytes.

[0030] Further, the concentration of polybrene in the medium is 0.5-5ug / mL.

[0031] In some embodiments, the mesenchymal stem cells are induced in a planar manner, and the human mesenchymal stem cells are conventionally adherent cultured to a confluence of 60-80%, and the chondrogenic induction medium containing polybrene is replaced, and cultured at 37°C, 3-8% carbon dioxide, and the rest air, for 7-14 days, with the chondrogenic induction medium containing polybrene being replaced every 3 days.

[0032] In some embodiments, the mesenchymal stem cells are subjected to three-dimensional organoid induction, the mesenchymal stem cell suspension is collected in a 15 ml sterile centrifuge tube (1-3 x 105cells / tube), centrifuged at 1200-1500 rpm for 3-5 minutes, the cells are aggregated at the bottom, the supernatant is discarded, and the chondrogenic differentiation induction medium containing polybrene is replaced, the cells are gently blown and contacted with the medium, and then incubated at room temperature for 5-15 minutes. Then centrifuge again at 1200-1500 rpm for 3-5 minutes to make the cells settle at the bottom of the centrifuge tube. Loosen the cap of the sterile centrifuge tube, and place it in a 37°C, 5% carbon dioxide, and the rest is air environment for 21-28 days, and replace the fresh chondrogenic differentiation induction medium containing polybrene once every 3 days.

[0033] In some embodiments, the method for dissolving the polybrene is to weigh the corresponding amount of powder, and pre-dissolve it in double distilled water as a stock solution. The concentration of polybrene in the stock solution is 10-100 mg / ml. Use a sterile filter with a pore size of 0.22 μm to filter and sterilize, and then dilute the polybrene stock solution with the chondrogenic differentiation induction medium to a final concentration of 0.5-5 μg / ml.

[0034] In some embodiments, the chondrogenic differentiation induction medium is composed of H-DMEM medium (High Glucose Dulbecco's Modified Eagle Medium, Gibico 11965118) as a solvent, and the remaining components are added as follows: 1% insulin, transferrin, selenium, and ethanolamine solution (ITS for short, Gibico 51500056) is added alone, and the pH value is natural; 2.5-10 ng / ml recombinant human TGFβ3 (TGFβ3 for short, Peprotech 100-36E) is added alone, and the pH value is natural; 1% ITS and 2.5-10 ng / ml TGFβ3 are added together, and the pH value is natural; preferably, 1% sodium pyruvate (Sodium Pyruvate 100 mM Solution, Gibico 11360070), 50 μg / ml L-ascorbic acid 2-phosphate hydrate (Sigma A8960), and 10-7M dexamethasone (Sigma D4902) are added; 1% ITS and 10 ng / ml TGFβ3 are added, which helps to promote the effect of chondrogenic differentiation, and the pH value is natural.

[0035] In some embodiments, the chondrogenic induction medium comprises H-DMEM medium (High Glucose Dulbecco's Modified Eagle Medium, Gibico 11965118) as a solvent, 1% sodium pyruvate (Sodium Pyruvate 100 mM Solution, Gibico 11360070), 50 μg / ml L-ascorbic acid 2-phosphate hydrate (Sigma A8960), 10-7M dexamethasone (Sigma D4902), 1% ITS, and 10 ng / ml TGFβ3, at a natural pH.

[0036] The mesenchymal stem cells can be bone marrow-derived or adipose-derived. The mesenchymal stem cells can also be replaced by chondrocytes derived from the cartilage tissue of the osteoarthritic patient.

[0037] In another aspect, the present application provides a medium for inducing chondrogenic differentiation of mesenchymal stem cells, wherein the medium comprises polybrene.

[0038] In another aspect, the present application provides the use of polybrene for the preparation of a preparation for promoting the increase of glycosaminoglycan content in a tissue.

[0039] In another aspect, the present application provides the use of polybrene for the preparation of a preparation for promoting cartilage regeneration.

[0040] The preparation comprising polybrene provided by the present application can promote complete regeneration of cartilage and induce differentiation of mesenchymal stem cells into chondrocytes more suitable for clinical treatment, thereby helping to achieve complete regeneration of cartilage.

[0041] Complete regeneration refers to the complete recovery of the structure and function of the original tissue / cells through the repair process, and is opposite to incomplete regeneration, which cannot completely reproduce the original tissue / cells and often produces scar fibrous tissue. Other existing methods cannot maintain the glycosaminoglycan content in the cartilage tissue, and thus cannot achieve complete regeneration of cartilage. By the method provided by the present application, the glycosaminoglycan content in the cartilage tissue can be significantly increased, thereby effectively maintaining the microenvironment required for cartilage repair and helping to achieve complete regeneration of cartilage.

[0042] In another aspect, the present application provides the use of polybrene for the preparation of a preparation for promoting chondrogenic differentiation of mesenchymal stem cells.

[0043] The technical solution provided by the present application has the following beneficial effects:

[0044] 1. It is found that polybrene can effectively increase the glycosaminoglycan content in the cartilage tissue, thereby providing a microenvironment suitable for cartilage regeneration.

[0045] 2. Using the induction medium containing polybrene to culture mesenchymal stem cells can increase the content of glycosaminoglycan in the differentiated tissue of mesenchymal stem cells, and promote the chondrogenic differentiation of mesenchymal stem cells better;

[0046] 3. Using the preparation containing polybrene to treat cartilage can promote the better repair and regeneration of cartilage. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Structural formulas of the six cationic polymers in Example 1;

[0048] Figure 2 Pictures of alcian blue staining of mesenchymal stem cells treated with the six cationic polymers in Example 1 after 12 days of induction after chondrogenic induction;

[0049] Figure 3 Schematic diagram of adherent culture of mesenchymal stem cells using the chondrogenic induction medium containing polybrene in Example 2;

[0050] Figure 4 Picture of glycosaminoglycan staining after polybrene promotes chondrogenic differentiation of mesenchymal stem cells (flat induction) in Example 2;

[0051] Figure 5 Schematic diagram of quantification of staining depth of eosin solution and alcian blue staining solution in Example 2;

[0052] Figure 6 Schematic diagram of polybrene promoting chondrogenic differentiation of mesenchymal stem cells in a sterile centrifuge tube in Example 3;

[0053] Figure 7 Picture of eosin and alcian blue staining results after polybrene promotes chondrogenic differentiation of mesenchymal stem cells (three-dimensional culture) in Example 3;

[0054] Figure 8 Picture of quantification of eosin and alcian blue staining depth after polybrene promotes chondrogenic differentiation of mesenchymal stem cells (three-dimensional culture) in Example 3;

[0055] Figure 9 Picture of PRG4 and ACAN immunofluorescence staining results after polybrene promotes chondrogenic differentiation of mesenchymal stem cells (three-dimensional culture) in Example 3;

[0056] Figure 10 Picture of quantification of PRG4 and ACAN immunofluorescence staining depth after polybrene promotes chondrogenic differentiation of mesenchymal stem cells (three-dimensional culture) in Example 3;

[0057] Figure 11 Picture of polybrene treatment increasing the glycosaminoglycan content of the surface layer of articular cartilage tissue (cartilage block explant culture) in Example 4;

[0058] Figure 12 Figure 4 is a photograph of safranin O staining of a bone defect treated with polybrene to promote in vivo repair of the bone cartilage defect. DETAILED DESCRIPTION

[0059] The application will be described in further detail below with reference to the drawings and embodiments, it being noted that the embodiments described below are intended to facilitate an understanding of the application and do not in any way limit the same. The advantages and features of the application will be more apparent from the following description and claims. It is noted that the drawings are in a very simplified form and use non-precise proportions, only for the purpose of facilitating, clarifying and aiding the explanation of the embodiments of the application.

[0060] In vitro screening of cationic polymers in Example 1

[0061] Since the cationic polymers have positive charges and the glycosaminoglycans have negative charges, this example considers the use of cationic polymers to treat damaged cartilage tissue, with the expectation of effectively improving the concentration of glycosaminoglycans in the cartilage tissue.

[0062] Human bone marrow-derived mesenchymal stem cells (3-5 passages) were seeded on culture dishes at a confluence of 60-80%, and after the cells adhered, a chondrogenic induction medium containing dissolved cationic polymers was used to culture the cells at 37°C in a 5% carbon dioxide environment for 12 days, with fresh medium being replaced every 3 days.

[0063] A large number of cationic polymers were selected in the previous stage of this example, and six typical cationic polymers were selected for illustration in this example. The reason for using these six cationic polymers is that they have been reported in the fields of cell culture, gene transfection, drug delivery, etc., and have potential application prospects. They have different molecular weights and structural characteristics, and under the condition of a uniform mass concentration, they exhibit different electric potentials. They have a certain representativeness in the screening.

[0064] The six cationic polymers used are polyethyleneimine (Polyethyleneimine, abbreviated as PEI), polyamidoamine-G3 (Polyamidoamine-G3, abbreviated as PAMAM-G3), polybrene (Hexadimethrine bromide, abbreviated as HDMBr), poly-L-lysine (Poly-L-lysine, abbreviated as PLL) and poly-D-lysine (Poly-D-lysine, abbreviated as PDL), and poly-L-ornithine (Poly-L-ornithine, abbreviated as PLO), the structural formulas of which are shown in Figure 1 , the molecular weights and electric potentials of which are shown in Table 1.

[0065] Table 1. Molecular weights and electric potentials of six cationic polymers

[0066]

[0067] The composition of the chondrogenic induction medium is H-DMEM medium (High Glucose Dulbecco's Modified Eagle Medium, Gibico 11965118) as a solvent, adding 1% insulin, transferrin, selenium, ethanolamine solution (ITS, Gibico 51500056), and 5 ng / ml recombinant human TGFβ3 (TGFβ3, Peprotech 100-36E), pH natural. The final concentration of cationic polymer in the medium is 1 μg / ml.

[0068] After 12 days of induction, the Alcian blue staining pictures of mesenchymal stem cells after chondrogenic induction treated with six kinds of cationic polymers are shown in Figure 2 The control group is the group without adding cationic polymer.

[0069] Alcian blue is an alkaline dye that can stain the acidic proteoglycans in cartilage. It specifically binds to long chains of sulfated glycosaminoglycans present in chondrocyte matrix, staining blue. It can distinguish typical cartilage glycosaminoglycan groups, but cannot be exactly to the specific species. The glycosaminoglycans in cartilage are mainly chondroitin sulfate and hyaluronic acid.

[0070] From Figure 2 It can be seen that, compared with the control group and the rest of the cationic polymer, the mesenchymal stem cells treated with polybrene show deeper Alcian blue staining, suggesting that more glycosaminoglycans are formed, and polybrene has a unique promoting effect on the chondrogenic differentiation of mesenchymal stem cells.

[0071] Example 2 Polybrene promotes chondrogenic differentiation of human mesenchymal stem cells in vitro (flat induction)

[0072] Human bone marrow-derived mesenchymal stem cells (passage 3-5) were seeded on culture dishes at a confluence of 60-80%, and after the cells adhered, chondrogenic induction medium containing polybrene ( Figure 3 ) was used, and the cells were cultured at 37°C in a 5% carbon dioxide environment for 12 days, and the fresh medium was replaced every 3 days.

[0073] The method for dissolving the polybrene is as follows: a corresponding amount of powder is weighed, and then pre-dissolved in double distilled water as a storage solution, the concentration of the polybrene in the storage solution is 10-100 mg / ml. The storage solution is filtered and sterilized by using a sterile filter with a pore size of 0.22 μm, and then diluted with the chondrogenic induction medium. Two groups of chondrogenic induction medium with a final concentration of polybrene of 1 and 3 μg / ml are prepared, respectively. A blank group without the addition of polybrene is set as a control.

[0074] The remaining components of the chondrogenic induction medium are as follows: H-DMEM medium (High Glucose Dulbecco's Modified Eagle Medium, Gibico 11965118) as a solvent; 1% insulin, transferrin, selenium, ethanolamine solution (ITS for short, Gibico 51500056), natural pH; 10 ng / ml recombinant human TGFβ3 (TGFβ3 for short, Peprotech 100-36E), natural pH; 1% sodium pyruvate (100 mM solution, Gibico 11360070), 50 μg / ml L-ascorbic acid 2-phosphate hydrate (Sigma A8960), 10-7 M dexamethasone (Sigma D4902).

[0075] After 12 days of culture, the supernatant is removed, the cells are fixed with 4% paraformaldehyde solution for 20 minutes, and then stained with Safranin O solution (Sigma, S2255-25G) and Alcian blue staining solution (ScyTek, AF-2) at room temperature for 12-16 hours. After staining, the cells are washed with double distilled water, and then observed and photographed under an optical microscope. The photographs of the glycosaminoglycan staining of the human mesenchymal stem cells promoted to differentiate into chondrocytes by the polybrene (planar induction) are shown in Figure 4 The staining intensities of the Safranin O solution (Sigma, S2255-25G) and the Alcian blue staining solution (ScyTek, AF-2) are quantitatively calculated by using Photoshop software, and the quantitative results are shown in Figure 5

[0076] As can be seen from Figure 4 It can be seen that after 12 days of induction in the chondrogenic induction medium, the mesenchymal stem cells all exhibit a chondrocyte-like phenotype and cell aggregation. The Safranin O and Alcian blue staining both show that after induction by the medium containing polybrene, the staining intensity of the mesenchymal stem cells is significantly increased, which indicates that the addition of polybrene promotes the chondrogenic differentiation of the mesenchymal stem cells and makes them produce more chondrocyte-like glycosaminoglycan matrix.​

[0077] The principle of Safranin O staining is that the alkali cartilage combines with the basic dye Safranin O to present red color. Safranin O is a cationic dye that binds to polyanions, and it shows cartilage based on the binding of cationic dye to anionic groups in polysaccharides (chondroitin sulfate or keratan sulfate). Safranin O staining is approximately proportional to the concentration of anions, which indirectly reflects the content and distribution of proteoglycans in the matrix. Alcian blue can stain the acidic proteoglycans in the cartilage. It specifically binds to long chains of sulfated glycosaminoglycans present in the chondrocyte matrix, and stains blue. The deeper the color of Safranin O and Alcian blue staining indicates the more glycosaminoglycans. Fast red is used to label the nucleus.

[0078] According to Figure 5 , the staining intensity of Safranin O staining is higher when the concentration of polybrene is 1 μg / ml, and the staining intensity of Alcian blue staining is higher when the concentration of polybrene is 3 μg / ml. The reason may be that different staining agents have different detection sensitivity and specificity for different types of glycoproteins or glycosaminoglycans.

[0079] Example 3 Polybrene promotes the differentiation of human mesenchymal stem cells into cartilage in vitro (three-dimensional organoid induction)

[0080] The human bone marrow-derived mesenchymal stem cell suspension was collected in a 15 ml sterile centrifuge tube (2.5 x 10 5 cells / tube) Figure 6 ), and after centrifugation at 1500 rpm for 3 minutes, the cells were aggregated at the bottom Figure 6 ), the supernatant was discarded, and the cartilage differentiation induction medium containing dissolved polybrene was replaced. The method of dissolving polybrene was consistent with that of Example 2, and the final concentration was 3 μg / ml. The cells were gently blown and contacted with the medium, and then incubated at room temperature for 10 minutes. Then centrifuged at 1500 rpm for 3 minutes again to make the cells settle at the bottom of the centrifuge tube. Loosen the cap of the sterile centrifuge tube and place it in a 37°C, 5% carbon dioxide environment for 28 days. Replace the fresh cartilage differentiation induction medium containing polybrene once every 3 days. Within the first 3 days of culture, the cells will automatically aggregate into spheres Figure 6 ).

[0081] The composition of the chondrogenic induction medium is H-DMEM medium (High Glucose Dulbecco's Modified Eagle Medium, Gibico 11965118) as solvent, adding 1% sodium pyruvate (Sodium Pyruvate 100 mM Solution, Gibico 11360070), 50 μg / ml L-ascorbic acid 2-phosphate hydrate (Sigma A8960), 10-7M dexamethasone (Sigma D4902); 1% ITS, and 10 ng / ml TGFβ3, pH natural.

[0082] After 28 days, the culture medium was discarded, the cell clusters were collected, and the cell cluster tissues were fixed with 4% paraformaldehyde solution at room temperature for 48 hours. The cell clusters were dehydrated, transparentized, and paraffin-embedded and paraffin sections were prepared. The 5 μm thick paraffin sections were hydrated, and stained with Safranin O, Alcian blue, PRG4 and ACAN immunofluorescence. Photographs were taken by optical microscope and laser confocal microscope, and the staining intensity of Safranin O, Alcian blue, PRG4 and ACAN immunofluorescence staining was quantitatively calculated by photoshop software. The results are shown in Figures 7-10 , wherein Figure 7 is a Safranin O and Alcian blue staining result graph, Figure 8 is a Safranin O and Alcian blue staining intensity quantification result graph, Figure 9 is a PRG4 and ACAN immunofluorescence staining result graph, Figure 10 is a PRG4 and ACAN immunofluorescence staining intensity quantification result graph.

[0083] Safranin O staining and Alcian blue staining are used to identify cartilage glycosaminoglycans. The darker the color, the more glycosaminoglycans there are relatively. Safranin O is used to mark the cell nucleus. PRG4 and ACAN are representative glycoproteins of articular cartilage, and PRG4 and ACAN immunofluorescence staining can accurately reflect the content of PRG4 and ACAN protein. The higher the fluorescence staining intensity, the higher the protein expression or the wider the distribution. Glycosaminoglycans are the main components of glycoproteins, and the increase of glycosaminoglycans indirectly promotes the expression of glycoproteins; on the contrary, the increase of glycoprotein expression indirectly indicates the increase of glycosaminoglycan content. The control group refers to the organoids cultured in chondrogenic induction medium without adding polybrene.

[0084] from Figure 6 It can be seen that mesenchymal stem cells can aggregate to form cell spheres at the bottom of the centrifugal tube, and gradually show chondrogenic differentiation characteristics.

[0085] According to Figure 8 and Figure 9, Safranin O and Alcian Blue staining both showed that after hyaluronan treatment, the staining intensity of the center of the cell mass was significantly deepened, and the staining intensity was enhanced by about 1 times, indicating that after adding hyaluronan to the culture medium, mesenchymal stem cells accumulated more glycosaminoglycans during the process of chondrogenic differentiation (three-dimensional organoid induction).

[0086] According to Figure 10 and Figure 11 , the content of PRG4 and ACAN protein was significantly increased after hyaluronan treatment, the staining intensity of PRG4 protein was enhanced by 2 times, and the staining intensity of ACAN protein was enhanced by more than 50%, indicating that after adding hyaluronan to the culture medium, mesenchymal stem cells accumulated more glycosaminoglycans during the process of chondrogenic differentiation (three-dimensional organoid induction).

[0087] Example 4: Hyaluronan maintains the content of glycosaminoglycans in the surface of articular cartilage (chondral explant culture)

[0088] After repeatedly washing the cartilage tissue of osteoarthritis patients with sterile phosphate buffer solution, it was cut into small cartilage blocks (5mm x 1cm x 5mm) and cultured in serum-free DMEM / F12 medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12, Gibico11320033) containing dissolved hyaluronan. It was cultured at 37℃ in an environment of 5% carbon dioxide and the rest of air for 21 days, and the fresh medium containing hyaluronan was replaced every 3 days. The method of dissolving hyaluronan in the chondrogenic induction medium was consistent with Example 2, and the final concentration was 2μg / ml.

[0089] After 21 days of culture, the cartilage block tissue was fixed with 4% paraformaldehyde solution at room temperature for 72 hours, and the cartilage block was dehydrated, transparent, and paraffin-embedded and sectioned. The 7μM thick paraffin sections were hydrated, and Safranin O staining and chondroitin sulfate immunohistochemical staining were performed. Optical microscope and digital scanner were used to take photos, and hyaluronan treatment increased the content of glycosaminoglycans in the surface of articular cartilage (chondral explant culture) as shown in Figure 11 .

[0090] Safranin O staining was used to identify cartilage glycosaminoglycans, and the deeper the staining, the higher the relative content. Chondroitin sulfate immunohistochemical staining was used to identify the representative glycosaminoglycan chondroitin sulfate in cartilage, and the deeper the staining, the higher the relative content.

[0091] As can be seen from Figure 11 , Safranin O staining (top) and chondroitin sulfate immunohistochemical staining (bottom) both showed that the cartilage tissue treated with hyaluronan had more abundant glycosaminoglycans. Especially in the position of the cartilage surface layer (10% of the whole layer), the staining intensity of glycosaminoglycans was significantly higher than that of the control group.

[0092] Example 5: Polybrene promotes in vivo hyaline cartilage regeneration (animal experiment)

[0093] New Zealand white rabbits (6 months old, weight 2.5-3 kg) were anesthetized with sodium pentobarbital (20 mg / kg body weight) and Xylazine (10 mg / kg body weight), and the knee joint cavity of the hind limb was opened. A cylindrical defect (5 mm in diameter and 4 mm in depth) was made in the center of the femoral trochlear groove using a drill bit.

[0094] The "sham operation" group refers to the group in which the joint cavity was opened during the operation of modeling the cartilage defect, but no operation was performed on the articular cartilage, i.e., the healthy control group; the "blank" group refers to the group in which the articular cartilage surface was modeled with a defect, and no treatment was performed, i.e., the negative control group; the "hydrogel" group refers to the group in which the articular cartilage surface was modeled with a defect, and hydrogel without polybrene was added to the surface, for comparison with the effect of hydrogel containing polybrene; the "polybrene-hydrogel" group refers to the group in which the articular cartilage surface was modeled with a defect, and hydrogel containing polybrene was added to the surface, i.e., the experimental group.

[0095] The composition of the hydrogel solution: phosphate buffer as solvent (pH 7.2), 20% (mass fraction) of gelatin-methacryloyl (GelMA), 10% (mass fraction) of chondroitin sulfate-N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide (CS-NB), and 0.25% (mass fraction) of hyaluronic acid-N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy butanamide (HA-NB). The solution was sterilized at 120°C for 8 minutes before use. The final concentration of polybrene in the hydrogel was 25 μg / ml.

[0096] The hydrogel solution needs to add 0.25% (mass fraction) of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator before being added to the defect. The wavelength of ultraviolet irradiation is 405 nm.

[0097] At 6 and 12 weeks postoperatively, New Zealand white rabbits were sacrificed with an overdose of sodium pentobarbital, and joint samples were collected. The cartilage blocks were fixed in 4% paraformaldehyde solution at room temperature for 5 days. The osteochondral tissues were decalcified, dehydrated, transparentized, and embedded in paraffin and sectioned. The 7 μM-thick paraffin sections were hydrated and stained with Safranin O. Photographs were taken with an optical microscope and a digital scanner. Representative Safranin O staining images of the in vivo osteochondral defect repair promoted by polybrene are shown in Figures 1-4. Figure 12 .

[0098] Safranin O staining was used to identify cartilage glycosaminoglycans. The darker the staining, the higher the relative content, which indirectly indicates better cartilage repair. Round chondrocytes are a physiological structure of cartilage tissue. The appearance of round chondrocytes in the sham operation group and the polybrene-hydrogel group is one of the signs of hyaline cartilage regeneration. Fibers arranged in parallel or in a network are a feature of fibrous cartilage, which mainly appears in the blank group and the hydrogel group, and is one of the signs of incomplete repair.

[0099] At 6 and 12 weeks postoperatively, the joint cartilage samples showed obvious fibrous tissue accumulation in the blank group Figure 12 , indicating that the defect could not be spontaneously healed by endogenous mesenchymal stem cells. The Safranin O staining of the hydrogel group was improved, but the tissue healing was still incomplete. In the polybrene-hydrogel group, oval chondrocytes in the hyaline-like cartilage tissue Figure 12 grew at 6 weeks, indicating faster repair of cartilage damage. In the 12-week postoperative observation, the polybrene-hydrogel group had better bone-cartilage interface integration. The animal experiment results suggest that polybrene is very helpful for in vivo cartilage repair.

[0100] The details of the present application are well known in the art. Although the present application is disclosed as above, the present application is not limited thereto. Any person skilled in the art, without departing from the spirit and scope of the present application, can make various changes and modifications, and therefore the scope of protection of the present application should be limited by the scope defined in the claims.

Claims

1. Use of polybrene for preparing a preparation for promoting cartilage regeneration.

2. Use according to claim 1, characterized in that, The polybrene increases the content of glycosaminoglycan in cartilage tissue or cartilage-like tissue.

3. Use according to claim 2, characterized in that, The tissue is treated with a preparation containing polybrene; the tissue is cartilage tissue or cartilage-like tissue.

4. Use according to claim 3, characterized in that, The structural formula of the polybrene is shown in formula (1): Formula (1); In which, n=2000-8000.

5. Use according to claim 4, characterized in that, The concentration of polybrene in the preparation is 5-100 ug / mL.

6. The use according to claim 1, characterized in that, The polybrene promotes the differentiation of mesenchymal stem cells into cartilage.

7. Use according to claim 6, characterized in that, Mesenchymal stem cells are cultured in a medium containing polybrene.

8. Use according to claim 7, characterized in that, The concentration of polybrene in the medium is 0.5-5 ug / mL.

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