Small molecule compound for improving cell adaptability and promoting cartilage regeneration

Abstract

The invention provides a small-molecule compound for improving cell adaptability and promoting cartilage regeneration, the optimal small-molecule compound FPH2 is screened from more than 2000 small-molecule compounds through screening of a cartilage cell adaptability model, and multiple rounds of screening and verification prove that the FPH2 has the effects of improving functional phenotype and adaptability of cartilage cells in in-vitro culture and promoting cartilage regeneration. The cartilage cell culture medium has the effects of promoting cartilage cell redifferentiation, promoting cartilage precursor cell differentiation to cartilage, inhibiting fatty acid oxidase, promoting mitochondrial metabolism adaptability and the like, and can be used in in-vitro cartilage cell culture (used for cell transplantation) and in-vivo cartilage regeneration (used for in-situ medication). A safe, low-cost, convenient and easy-to-popularize strategy for regulating and controlling the adaptability of the cartilage cells is provided for cartilage regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a small molecule compound that enhances cell adaptability and promotes cartilage regeneration. Background Art

[0002] Adult articular cartilage has no spontaneous regeneration ability, and injuries and diseases often cannot heal on their own. For example, if early cartilage defects are not properly treated, they will progress to osteoarthritis, affecting hundreds of millions of people and bringing serious economic and medical burdens. There is an urgent need to find regenerative treatment strategies that can treat early and avoid joint replacement. Regenerative medicine aims to combine multidisciplinary means to help tissue regeneration. Cells are an important element among them, and the adaptability (cell fitness) of cells is crucial for tissue regeneration, referring to the characteristics of cells to adapt, survive, grow continuously and maintain functions in a new environment.

[0003] Adult chondrocytes are typical cells with low adaptability, which is the fundamental reason for the difficulty of cartilage tissue regeneration. Chondrocytes are not only scarce in number and difficult to give a regenerative response in injuries and diseases. In addition, the low adaptability of chondrocytes is mainly manifested in the following aspects: 1) Without external intervention, articular chondrocytes are difficult to survive and maintain functions in environments such as injuries and diseases for a long time; 2) If chondrocyte transplantation is used to treat cartilage injuries, chondrocytes need to be cultured in vitro first. Chondrocytes that leave the in-vivo environment will rapidly lose their functions in the in-vitro planar environment and gradually undergo growth arrest. This phenomenon is called chondrocyte dedifferentiation, which is mainly caused by the inadaptability of chondrocytes to the in-vitro growth environment. These dysfunctional chondrocytes, as grafts, often cannot maintain the functional phenotype after entering the patient's body, form fibrous cartilage, and cannot be completely repaired.

[0004] Basic research on cell adaptability focuses on the fields of development and tumor biology. For example, drugs are developed for the culture of T cells (epigenetic inhibitor JQ1, kinase inhibitor ibrutinib, etc.), but the findings in this field are not applicable to the field of cartilage regeneration.

[0005] In the field of tissue regeneration, regulating the gene regulatory network that dominates cell adaptation is a direct method for regulating cell adaptation. However, this type of method currently faces several drawbacks: 1) The understanding of gene regulation of adaptability is limited, and genes do not have universality; 2) Genes that regulate cell adaptability are often related to tumorigenesis and the cell cycle. High expression of such genes in cells has a carcinogenic risk; 3) Gene editing involves the use of vectors such as viruses, which may bring risks such as safety and immune rejection.

[0006] In the field of cartilage repair, there is no ideal treatment strategy for patients with osteoarthritis. In the early stage of osteoarthritis, it may be manifested as superficial cartilage matrix loss or injury. Weight loss, exercise regulation, etc. can be adopted, and non-steroidal anti-inflammatory drugs and glucocorticoids can be given to relieve pain and inflammation. Some patients also take glucosamine health products or inject hyaluronic acid into the joints to supplement the loss of cartilage matrix. However, none of the above methods can effectively prevent the progressive development of osteoarthritis. The remaining drugs under development include stem cell and derivative injection, growth factor injection, etc., but the results are still controversial and have not been widely promoted. In the end stage of osteoarthritis, only joint surgery replacement can be carried out. Therefore, it is very important to find methods for early prevention and treatment, and effectively block cartilage degeneration or progressive injury.

[0007] For local cartilage defects in young and middle-aged patients, methods such as chondrocyte transplantation and cartilage mosaic tissue block transplantation can help cartilage regeneration to a certain extent. To reduce allogeneic rejection, such therapies generally need to extract cartilage tissue from the non-weight-bearing area of the patient's own joint. However, the tissue size or cell number is limited, and it still needs to proliferate and grow in vitro or in situ. Considering the poor adaptability of chondrocytes and their easy dedifferentiation, three-dimensional scaffold materials can also be combined to provide a biomimetic microenvironment, which can relieve the problem of poor cartilage growth to a certain extent but cannot fundamentally solve it. At present, the failure rate of autologous chondrocyte transplantation is about 10-15%, and it is only applicable to people under 50 years old. For middle-aged and elderly patients, the lack of cell sources and poor transplantation environment are still unsolved problems. It can be seen that improving the adaptability of chondrocytes is very important.

[0008] To develop new treatment strategies, the types of drugs that can be considered include: gene / nucleic acid drugs, protein and other macromolecular drugs, cell drugs, chemical small molecule drugs, etc. Gene drugs need to have clear and reliable targets, and there are risks such as low universality, carcinogenicity, and immune rejection; protein and other macromolecular drugs are restricted from promotion due to disadvantages such as high price, short validity period in the joint cavity, and poor permeability; cell drugs, such as mesenchymal stem cells, may increase the survival of chondrocytes in the joint cavity through immunomodulation, but they are expensive, the dosage is difficult to standardize, and there is still controversy about their efficacy at present.

[0009] In recent years, the research on the regulation of cell fate by chemical small molecule drugs has increased. It can not only achieve somatic cell reprogramming and transdifferentiation, but also be used to directly regulate cell function phenotypes. Chemical small molecule drugs have good application prospects. Compared with gene drugs, the use of small molecule drugs is easier to standardize and precisely regulate, and it is safer and more convenient. It will not introduce foreign carcinogenic genes or viruses and directly acts at the protein level; compared with protein and other macromolecular drugs, small molecule drugs are easy to synthesize, store, transport, have lower costs, are easy to use, have small molecular weights and are easily taken up by cells, and have lower potential immunogenicity.

[0010] Currently, small molecule drugs reported in the fields of osteoarthritis and cartilage regeneration include Kartogenin, BNTA, etc., but there is no direct evidence indicating that their mechanisms of action, targets, and cell adaptability are related. At present, no chemical small molecule drugs that can enhance the adaptability of chondrocytes have been discovered.

[0011] Therefore, there is an urgent need to find a safe and convenient treatment method to enhance the adaptability of chondrocytes, promote cartilage regeneration, prevent the progressive development of cartilage injuries, and prevent osteoarthritis. Summary of the Invention

[0012] To solve the above problems, the present invention provides a small molecule compound that enhances cell adaptability and promotes cartilage regeneration. Through screening using a chondrocyte adaptability model, the most preferred small molecule compound FPH2 was screened from more than 2,000 small molecule compounds. After multiple rounds of screening and verification, it was proven that FPH2 has the effects of enhancing the functional phenotypes and adaptability of chondrocytes in in vitro culture, promoting the redifferentiation of chondrocytes, promoting the differentiation of chondroprogenitor cells into cartilage, inhibiting fatty acid oxidase, and promoting mitochondrial metabolic adaptability. It can be used in in vitro chondrocyte culture (for cell transplantation) and in vivo cartilage regeneration (in situ drug administration), providing a safe, low-cost, convenient, and easy-to-promote strategy for regulating chondrocyte adaptability for cartilage regeneration.

[0013] On the one hand, the present invention provides a reagent for enhancing the adaptability of chondrocytes, and the reagent contains FPH2 and its solvent.

[0014] Based on the correlation between the targets of more than 2,000 small molecule drugs and the in vitro dedifferentiation pathway of chondrocytes, the present invention finally obtained the small molecule compound FPH2 with the best effect on increasing the number of chondrocytes and preventing the loss of functional phenotypes of chondrocytes in in vitro culture through a large number of repeated screenings. After a large number of verification experiments, it was proven that FPH2 can effectively enhance the adaptability of chondrocytes, thereby effectively promoting cartilage regeneration.

[0015] Furthermore, the structural formula of the FPH2 is shown in formula (1):

[0016]

[0017] Furthermore, the concentration of FPH2 in the reagent is 0.2 - 20 μM.

[0018] In some embodiments, the concentration of FPH2 in the reagent is 0.5 - 5 μM.

[0019] In some embodiments, the solvent is dimethyl sulfoxide (DMSO).

[0020] On the other hand, the present invention provides a method for improving the adaptability of chondrocytes, which uses a reagent containing FPH2 to treat chondrocytes.

[0021] Furthermore, the adaptability includes one or more of in vitro amplification adaptability, chondrogenic differentiation and redifferentiation adaptability, metabolic adaptability, and in vivo adaptability after cell transplantation.

[0022] In some ways, the method for improving the in vitro amplification adaptability of chondrocytes is (plate amplification): chondrocytes are routinely adherently cultured until the confluence reaches 20 - 40%, the medium dissolved with FPH2 is replaced, and the cells are cultured in an environment of 37°C, 3 - 8% carbon dioxide, and the rest is air. The medium dissolved with FPH2 is replaced once every 2 days until the cells are completely confluent, and then passage or cryopreservation is carried out.

[0023] In some ways, the method for improving the chondrogenic differentiation and redifferentiation adaptability of chondrocytes is: first, the number of chondrocytes is amplified (plate amplification), and then the redifferentiation of chondrocytes is carried out.

[0024] In some ways, the method for amplifying the number of chondrocytes is: chondrocytes are routinely adherently cultured until the confluence reaches 20 - 40%, the medium dissolved with FPH2 is replaced, and the cells are cultured in an environment of 37°C, 3 - 8% carbon dioxide, and the rest is air. The medium dissolved with FPH2 is replaced once every 2 days until the cells are completely confluent, and then passage is carried out. When passing the cells, the chondrocytes are digested with 0.03 - 0.08% concentration of trypsin for 2 - 3 minutes, then the digestion is terminated with DMEM / F - 12 medium containing 10% fetal bovine serum. After centrifuging at 1200 - 1500 rpm for 3 - 5 minutes, the medium is discarded, and the cells are resuspended with the medium containing FPH2 and re - inoculated at a cell confluence of 40 - 50%, and this is recorded as passage 1. The cells are continuously cultured in this way until passage 4.

[0025] In some ways, the method for redifferentiating chondrocytes is: when the confluence of chondrocytes continuously cultured to passage 4 reaches more than 90%, the chondrocytes are digested with 0.03 - 0.08% concentration of trypsin for 2 - 3 minutes, then the digestion is terminated with DMEM / F - 12 medium containing 10% fetal bovine serum. The cell suspension is transferred to a 15 ml sterile centrifuge tube, and the number of cells in each tube is 1 - 3×10 5 cells. After centrifuging at 1200 - 1500 rpm for 3 - 5 minutes. After centrifugation, it can be seen with the naked eye that the cell aggregates sink to the bottom of the tube. Slowly aspirate the medium and add the chondrogenic differentiation induction medium containing FPH2.

[0026] In some ways, the culture medium can be DMEM / F-12 medium with 10% (volume fraction) fetal bovine serum. Cultivate for 48 hours in an environment of 37 °C, 3-8% carbon dioxide, and the rest is air.

[0027] In some ways, the culture medium is preferably a chondrogenic differentiation induction medium, and its components are as follows: add 1% (volume fraction) Insulin, Transferrin, Selenium, Ethanolamine Solution to H-DMEM (High Glucose Dulbecco's Modified Eagle Medium) medium, 2.5-10 ng / ml Recombinant Human TGFβ3, 1% sodium pyruvate, 50 μg / ml L-ascorbic acid 2-phosphate hydrate, 10-7 M dexamethasone, and the pH value is natural. The chondrogenic differentiation induction medium is changed every 3-4 days and cultured continuously for 5-10 days. During this process, chondrocytes will spontaneously aggregate into spherical or disc-shaped microtissues.

[0028] In some ways, implant the chondrocyte microtissues obtained by cultivation into the body of an animal model to observe the cartilage repair effect.

[0029] In some ways, FPH2 is continuously added throughout the in vitro culture medium, and the concentration used is 0.2-20 μM, preferably 0.5-2.5 μM.

[0030] In some ways, the number of chondrocytes implanted into the rat body is about 1-2×1 5 0⁶. The number of cells actually used clinically is adjusted according to the injury size. The in vivo injury applied can be, but is not limited to, full-thickness cartilage defects.

[0031] In some ways, the defect size can be, but is not limited to, a cylindrical injury with a diameter of 1-2 mm and a depth of 1-4 mm.

[0032] In some ways, the dissolving method of FPH2 is to dissolve FPH2 powder in dimethyl sulfoxide as a stock solution, and the concentration of FPH2 is 10-50 mM. When in use, directly dissolve the FPH2 stock solution in the culture medium, and the concentration used is 0.2-20 μM.

[0033] In some ways, the concentration of FPH2 can be preferably 0.5-5 μM.

[0034] In some ways, the conventional growth medium required for chondrocytes is DMEM / F-12 (Dulbecco's Modified Eagle Medium: F-12) medium supplemented with 10% (v / v) fetal bovine serum and with a natural pH value;

[0035] In some ways, the chondrocytes can be chondrocytes of mouse or human origin.

[0036] In some ways, the culture conditions can also preferably be a hypoxic environment at 37°C, 3-8% carbon dioxide, 3-8% oxygen, and the rest nitrogen.

[0037] In some ways, the number of passages can be 1-5.

[0038] On the other hand, the present invention provides the use of a small molecule compound for preparing a reagent for improving the adaptability of chondrocytes, and the small molecule compound includes Cediranib and FPH2.

[0039] Further, the most preferred small molecule compound is FPH2.

[0040] Further, the adaptability includes one or more of in vitro amplification adaptability, chondrogenic differentiation and redifferentiation adaptability, metabolic adaptability, and in vivo adaptability after cell transplantation.

[0041] On the other hand, the present invention provides the use of FPH2 for preparing a reagent for promoting the differentiation of chondrogenic progenitor cells into chondrocytes.

[0042] On the other hand, the present invention provides the use of FPH2 for preparing a reagent for inhibiting fatty acid oxidase and promoting mitochondrial metabolism.

[0043] On the other hand, the present invention provides the use of FPH2 for preparing a reagent for improving the therapeutic effect of chondrocyte transplantation.

[0044] On the other hand, the present invention provides the use of FPH2 for preparing a reagent for preventing osteoarthritis.

[0045] The present invention has been proven by research that FPH2 can not only slow down the loss of chondrocyte function during chondrocyte amplification and improve the effect of chondrocyte transplantation, but also directly enhance the adaptability of chondrocytes in the joint cavity through in situ action, promote cartilage regeneration, and prevent osteoarthritis.

[0046] In some ways, the method for using FPH2 to prevent chondroarthritis is to directly apply FPH2 to the damaged surface of articular cartilage.

[0047] In some ways, the concentration of FPH2 used is 100 - 500 μM, and the drug dose in the adult rat model is 0.02 - 5 nmol / joint.

[0048] In some ways, the cartilage surface damage includes, but is not limited to, superficial cartilage defects.

[0049] In some ways, the size of the superficial defect in the rat joint model can be a square damage with a side length of 1 - 3 mm and a depth of 0.1 - 1 mm.

[0050] Furthermore, it is preferred to encapsulate FPH2 in a material to maintain the slow in-situ release of FPH2.

[0051] In some ways, FPH2 is encapsulated in microspheres for sustained release for the prevention of osteoarthritis.

[0052] In some ways, the microspheres for sustained release include, but are not limited to, those prepared from gelatin.

[0053] In some ways, the microspheres for sustained release are prepared as follows: FPH2 and gelatin are mixed and dissolved in deionized water at a mass ratio of 1:10; the gelatin solution is dropped into liquid paraffin containing sorbitan oleate (0.1 - 0.2% by volume fraction) and stirred at 600 revolutions per minute for 13 minutes; cooled on ice and stirred for 30 minutes, then 1 ml of glutaraldehyde is added for crosslinking; further stirred for 2 hours, 2 ml of acetone is added, the supernatant is taken and 1 ml of acetone is added, and it is allowed to solidify overnight at 4°C; washed three times with acetone and deionized water respectively, and stored at -20°C after freeze-drying for later use.

[0054] Furthermore, it is preferred to encapsulate the microspheres loaded with FPH2 in an adhesive hydrogel, apply and attach it to the joint surface, which is beneficial for the continuous in-situ action of FPH2.

[0055] In some ways, the adhesive hydrogel can be selected from, but is not limited to, GelMA / HA-NB hydrogel. GelMA (Gelatin-Methacryloyl) and HA-NB (Hyaluronic Acid-N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butanamide) are dissolved in phosphate buffer (pH 7.2) and mixed evenly. The dosage of GelMA is 2 - 10% (mass fraction), and the dosage of HA-NB is 0.2 - 2% (mass fraction). Sterilization is carried out at 120°C for 8 minutes.

[0056] In some methods, after mixing the hydrogel with the FPH2-microsphere solution, 0.25% (mass fraction) of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) needs to be mixed in as a photoinitiator. Gelation can occur after irradiating with an ultraviolet lamp for 10 - 30 seconds. The wavelength of ultraviolet irradiation is 405 nm.

[0057] The technical solutions provided by the present invention have the following beneficial effects:

[0058] 1. Discover a novel small molecule compound FPH2 that can enhance cell adaptability and promote cartilage regeneration; ]>

[0059] 2. Discover that FPH2 has the effects of enhancing the functional phenotype and adaptability of chondrocytes in in vitro culture, promoting the redifferentiation of chondrocytes, promoting the differentiation of chondroprogenitor cells into cartilage, inhibiting fatty acid oxidase, and promoting mitochondrial metabolic adaptability;

[0060] 3. Discover that FPH2 can be used in in vitro chondrocyte culture (for cell transplantation) and in vivo cartilage regeneration (in situ drug administration);

[0061] 4. Provide a safe, low-cost, convenient, and easy-to-popularize strategy for regulating chondrocyte adaptability for cartilage regeneration. Description of the Drawings

[0062] Figure 1 Schematic diagram of the results of the effect of 288 drugs on the number of chondrocytes and Col2-pd2EGFP fluorescence in Example 1 (primary screening);

[0063] Figure 2 Schematic diagram of the results of the effect of 2 candidate drugs on the proliferation viability of chondrocytes and Col2-pd2EGFP fluorescence in Example 1;

[0064] Figure 3 Chemical structural formulas of 2 candidate drugs in Example 1;

[0065] Figure 4 White light and green fluorescence images of Col2-pd2EGFP mouse chondrocytes after being treated with FPH2 for 48 hours in Example 1;

[0066] Figure 5 Green fluorescence expression rate of Col2-pd2EGFP mouse chondrocytes after being treated with different concentrations of FPH2 for 48 hours in Example 1;

[0067] Figure 6 Relative cell viability of mouse chondrocytes after being treated with different concentrations of FPH2 for 48 hours in Example 1;

[0068] Figure 7 Schematic diagram of the immunofluorescence staining results of COL2 and ACAN after treating human infant chondrocytes (passage 4) with FPH2 under normoxic conditions for 48 hours in Example 2;

[0069] Figure 8 Schematic diagram of the statistical results of the immunofluorescence staining intensity of COL2 and ACAN after treating human infant chondrocytes (passage 4) with FPH2 under normoxic conditions for 48 hours in Example 2;

[0070] Figure 9 Representative pictures of the co-immunostaining of COL2 and ACAN after treating human infant chondrocytes (passage 3) with FPH2 under hypoxic conditions for 48 hours in Example 2;

[0071] Figure 10 Schematic diagram of the statistical results of the immunofluorescence staining intensity of COL2 and ACAN after treating human infant chondrocytes (passage 3) with FPH2 under hypoxic conditions for 48 hours in Example 2;

[0072] Figure 11 Flow chart of the in vitro expansion and redifferentiation of chondrocytes in Example 3;

[0073] Figure 12 Representative pictures of alcian blue (extracellular matrix) staining after treating mouse and human chondrocytes (passage 4) with FPH2 at high density for 48 hours in Example 3;

[0074] Figure 13 Representative pictures of alcian blue staining after treating the ATDC5 chondrogenic differentiation model with FPH2 for 8 days in Example 4;

[0075] Figure 14 Result diagram of predicting potential targets of FPH2 by the Similarity ensemble approach tool in Example 5;

[0076] Figure 15 Amino acid sequence alignment analysis of carnitine palmitoyl transferase I (CPT1A, liver subtype; CPT1B, muscle subtype) in different species in Example 5;

[0077] Figure 16 Detection results of carnitine palmitoyl transferase I activity after treating cells with FPH2 in Example 5, indicating that FPH2 has an inhibitory effect on the target;

[0078] Figure 17Schematic diagram of the detection results of ATP energy production, relative mitochondrial membrane potential level, and reactive oxygen species production level after treating chondrocytes with FPH2 in Example 5;

[0079] Figure 18 Photographs of implanting chondrocytes continuously cultured with FPH2 into the joints of rats to repair full-thickness cartilage defects in Example 6;

[0080] Figure 19 Schematic diagram of the analysis of the probability of forming hyaline cartilage in the joints of rats (12 weeks after surgery) with different treatment methods in Example 6;

[0081] Figure 20 Schematic diagram of the analysis of the area ratio of transplanted cells participating in interface healing calculated based on human cell antibody labeling in Example 6;

[0082] Figure 21 Flow chart of preventing osteoarthritis by directly adding FPH2 at the in-situ cartilage defect in Example 7;

[0083] Figure 22 Immunohistochemical pictures of COL2 and MMP13 after repairing superficial cartilage defects in rats with different treatment methods for 8 weeks in Example 7;

[0084] Figure 23 Schematic diagram of the ICRS-II score results after repairing superficial cartilage defects in rats with different treatment methods for 8 weeks in Example 7;

[0085] Figure 24 Schematic diagram of the OARSI score results after repairing superficial cartilage defects in rats with different treatment methods for 8 weeks in Example 7. Detailed implementation manners

[0086] The present invention will be further described in detail below in conjunction with the drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not impose any limitations on it. According to the following description and the claims, the advantages and features of the present invention will be clearer. It should be noted that the drawings are all in a very simplified form and use non-precise scales, only for conveniently and clearly assisting in explaining the purpose of the embodiments of the present invention.

[0087] Example 1. Screening of small molecule compounds

[0088] In this example, an in vitro dedifferentiation model of Col2-pd2EGFP mouse chondrocytes was selected for drug screening. The Col2-pd2EGFP chondrocytes were isolated from transgenic mice. When the cells transcribe Col2a1, enhanced green fluorescence is emitted. The protein encoded by the Col2a1 gene is collagen type II, a cartilage marker. The fluorescence intensity of this system can be used to monitor the expression of Col2a1 in real time and characterize the function of chondrocytes.

[0089] In this example, 288 small molecule drugs were selected from the Selleck small molecule drug library containing more than 2,000 compounds. The selection criterion was that their targets were related to the in vitro dedifferentiation pathway of chondrocytes. Then, in vitro culture of Col2-pd2EGFP mouse chondrocytes was carried out for these 288 small molecule drugs, with a concentration of 1 μM added, and 0.01% (volume fraction) of the drug solvent dimethyl sulfoxide (DMSO) was used as a control.

[0090] According to previous studies, chondrocytes have low adaptability, manifested as the loss of functional phenotypes during in vitro culture, that is, dedifferentiation, and the Col2-pd2EGFP fluorescence will weaken with culture. For cells at an earlier passage (before the 4th passage), the functional phenotype may be restored by stimulating with certain factors; chondrocytes with a longer passage time are difficult to restore the phenotype. At the same time, chondrocytes experience growth arrest and slower proliferation during passage. Therefore, in this screening system, the fluorescence intensity of green fluorescence and the number of proliferating cells are used as screening indicators. It is hoped to screen out small molecule drugs from 288 small molecule drugs that can increase the cell number and green fluorescence intensity.

[0091] The specific method for in vitro culture of chondrocytes is as follows:

[0092] The 2nd - 3rd passage Col2-pd2EGFP mouse chondrocytes were seeded in a 96-well plate at a seeding density of 4,000 - 6,000 cells / well and cultured for 16 - 24 hours in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest air. The added medium was DMEM / F-12 (Dulbecco's Modified Eagle Medium:F-12) medium supplemented with 10% (volume fraction) fetal bovine serum, and the pH was natural.

[0093] The next day, the medium was discarded, and the 96-well plate was respectively added with DMEM / F-12 medium (10% fetal bovine serum) containing different small molecule drugs and cultured for another 48 hours.

[0094] After 48 hours of treatment, the CQ1 high-precision cell quantitative imaging analysis system detected that more than 80 drugs may promote the proliferation of chondrocytes and the transcription of Col2a1 during culture (see details inFigure 1 )。Among them, the DMSO group served as a control.

[0095] After the culture was terminated, the cells were fixed with 4% (mass fraction) paraformaldehyde in the dark for 20 minutes. Using the CQ1 high-precision cell quantitative imaging analysis system (Yokogawa), the cells were photographed with high throughput and high content, and the cell number and green fluorescence intensity were calculated. Finally, two small molecule drugs that could increase the cell number and green fluorescence intensity were screened and obtained, namely: Cediranib and FPH2( Figure 3 ).

[0096] After obtaining the candidate drugs, the experiment was continued and repeated, and the number of cell replicates was increased, hoping to screen the most optimal small molecule drug from them.

[0097] After multiple rounds of screening and further verification, we found that compared with the blank control, Cediranib and FPH2 could significantly promote the expression of green fluorescence. Among them, FPH2 could effectively promote the expression of Col2-pd2EGFP green fluorescence and proliferation activity( Figure 2 , Figure 4 , Figure 5 ), that is to say, it could promote the expression of green fluorescence without significantly reducing cell viability, while Cediranib could only promote the expression of green fluorescence( Figure 2 ), but could not promote the proliferation activity of cell transplantation. Therefore, the most optimal small molecule drug was FPH2. Statistical difference *p<0.05, **p<0.01, ***p<0.001. It was suggested that FPH2 could effectively improve the functional phenotype and adaptability of chondrocytes in vitro culture, and would not cause serious cytotoxicity( Figure 6 ), and the cell viability was significantly improved after the addition of FPH2, and still maintained a high cell viability when the concentration of FPH2 was 10 μM.

[0098] Example 2. FPH2 maintains the functional phenotype of chondrocytes in in vitro planar culture

[0099] Obtain human infantile polydactyl chondrocytes and perform routine in vitro culture in a culture dish. The required culture medium is DMEM / F-12 medium supplemented with 10% (volume fraction) fetal bovine serum, and the pH value is natural. When the cells are adherently cultured to a confluence of 20-40%, replace the culture medium dissolved with FPH2 (the dissolution method of FPH2 is to dissolve FPH2 powder in dimethyl sulfoxide (DMSO) as a stock solution, and the concentration of FPH2 is 10 mM), with a concentration of 0.5-5 μM (preferably 1 μM in this example). Culture in an environment of 37 °C, 3-8% carbon dioxide, and the rest air, and replace the fresh culture medium dissolved with FPH2 every 2 days until the cells are completely confluent, and then passage or cryopreserve. The number of cell passages does not exceed the 5th generation.

[0100] Culture for 48-72 hours, fix the cells being cultured with 4% (mass fraction) paraformaldehyde in the dark for 20 minutes, and perform immunofluorescence staining on chondrocyte function markers, take pictures with a confocal microscope, use the treatment with the same concentration of DMSO as a control, and compare the differences between FPH2 and the control group. Examine the expression of chondrocyte function markers COL2 (collagen type II) and ACAN (aggrecan) under normoxic conditions (oxygen concentration is 21%) or hypoxic conditions (oxygen concentration is 5%) (chondrocytes are not adapted in vitro (new environment), manifested as a decrease in the two function markers COL2 and ACAN, so an increase in COL2 and ACAN indicates that the cells are not unhealthy in the new environment, or the degree of unhealthiness is reduced. See the previously published literature by the inventor, A high-resolution route map reveals distinct stages of chondrocyte dedifferentiation for cartilage regeneration), and the detection results are shown in Figures 7 - 10 , in which Figure 7 and Figure 8 are respectively the representative pictures of COL2 and ACAN immunofluorescence staining and the statistical results of light staining intensity after treating human infantile chondrocytes (passage 4) with FPH2 for 48 hours under normoxic conditions; Figure 9 and Figure 10 are respectively the representative pictures of COL2 and ACAN immunofluorescence co-staining and the statistical results of light staining intensity after treating human infantile chondrocytes (passage 3) with FPH2 for 48 hours under hypoxic conditions.

[0101] According to Figures 7 - 10 it can be seen that human chondrocytes treated with FPH2 express higher-intensity chondrocyte function markers COL2 and ACAN. Under normoxic conditions ( Figure 7 and Figure 8) and hypoxic conditions ( Figure 9 and Figure 10 ) had significant effects. Statistical differences were *p < 0.05, **p < 0.01, ***p < 0.001. This indicates that FPH2 can enhance the adaptability of chondrocytes in in vitro culture and in petri dish culture, and can effectively maintain the functional phenotype of chondrocytes.

[0102] Example 3. FPH2 promotes the redifferentiation of chondrocytes in high-density in vitro culture

[0103] Before autologous chondrocyte transplantation, chondrocytes need to be first amplified in vitro and then redifferentiated (the flow chart is as Figure 11 shown), where amplification is because the number of human chondrocytes obtained clinically is limited and needs to be cultured in vitro to increase the cell number; redifferentiation aims to enable the cells that have lost chondrocyte function markers during the amplification process to regain their functional phenotype to ensure the function of the cells in vivo.

[0104] According to the physiological characteristics of chondrocytes, dedifferentiated chondrocytes can regain their phenotype in three-dimensional or high-density cell culture. This process is called the redifferentiation of chondrocytes. This model can be used to detect the ability of chondrocytes to adapt to a new environment and regain their phenotype, that is, adaptability.

[0105] First, perform the amplification of chondrocyte number: Obtain primary chondrocytes from mice or humans (during biopsy or surgery), and routinely culture them adherently until the confluence reaches 20 - 40%. Replace the culture medium dissolved with FPH2 (the dissolution method of FPH2 is to dissolve FPH2 powder in dimethyl sulfoxide (DMSO) as a stock solution, with an FPH2 concentration of 10 mM), with a concentration of 0.5 - 2.5 μM (preferably 1 μM in this example), and culture in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest is air. Replace the culture medium dissolved with FPH2 once every 2 days until the cells are completely confluent and then passage. During passage, digest the chondrocytes with 0.03 - 0.08% concentration trypsin for 2 - 3 minutes, then terminate the digestion with DMEM / F-12 medium containing 10% fetal bovine serum, centrifuge at 1200 - 1500 rpm for 3 - 5 minutes, discard the culture medium, resuspend with fresh culture medium containing FPH2, and re-inoculate the cells at a cell confluence of 40 - 50%, and record this as passage 1. Continuously culture the cells in this way until passage 4.

[0106] Redifferentiation of chondrocytes: When the confluence of chondrocytes cultured continuously to the 4th passage reaches more than 90%, digest the chondrocytes with 0.03 - 0.08% trypsin for 2 - 3 minutes, then terminate the digestion with DMEM / F-12 medium containing 10% fetal bovine serum, centrifuge, discard the supernatant, resuspend the cells, and the cell concentration is 1 - 3×10 7 / ml. Pipette 10 μl of the cell suspension in the center of the wells of a 24-well plate and culture it in an environment of 37°C, 3 - 8% carbon dioxide, and the rest is air for 3 - 4 hours. Wait until the cells adhere to form a concentrated and high-density chondrocyte micromass, and slowly add the medium containing 0.5 - 5 μM FPH2, with the volume of the medium in each well being 400 - 600 μl. The control group is treated with the same concentration of DMSO. The medium components are: add 1% (volume fraction) Insulin, Transferrin, Selenium, Ethanolamine Solution, 2.5 - 10 ng / ml Recombinant Human TGFβ3, 1% sodium pyruvate, 50 μg / ml L-ascorbic acid 2-phosphate hydrate, 10 -7 μM dexamethasone to H-DMEM (HighGlucose Dulbecco's Modified Eagle Medium), and the pH value is natural. Culture in an environment of 37°C, 3 - 8% carbon dioxide, and the rest is air for 2 - 10 days, and replace the fresh medium containing FPH2 once every 3 - 4 days. After the culture is completed, fix the cells with 4% (mass fraction) paraformaldehyde in the dark for 20 minutes. Stain the extracellular matrix of chondrocytes with Alcian blue staining solution, and the staining conditions are at room temperature for 12 - 16 hours. After staining, wash with double-distilled water and take pictures for observation. Figure 12 Representative pictures of Alcian blue (extracellular matrix) staining of mouse and human chondrocytes (4th passage) treated with FPH2 after high-density culture for 48 hours.

[0107] According to Figure 12 the results show that the cell micromasses treated with FPH2 have larger sizes and stronger staining of chondrocyte matrix. It shows that FPH2 can promote the adaptation of chondrocytes under high-density culture conditions, including growth and redifferentiation phenotypes.

[0108] Example 4: FPH2 promotes the differentiation of chondrogenic progenitor cell line ATDC5 into cartilage

[0109] ATDC5 is a murine prechondrocyte cell line and can be used to establish a chondrogenic differentiation model. The ATDC5 chondrocyte cell line was obtained and cultured in DMEM / F-12 supplemented with 5% (v / v) fetal bovine serum in an environment placed at 37 °C, 3 - 8% carbon dioxide, and the rest air. When the cell confluence reached 60% - 80%, the chondrogenic induction medium containing FPH2 was replaced. The composition of the medium was: DMEM / F-12 as the medium, supplemented with 5% (v / v) fetal bovine serum and 1% (v / v) Insulin, Transferrin, Selenium, Ethanolamine Solution, and the pH was natural. In the experimental group, FPH2 at a concentration of 0.5 - 5 μM (preferably 1 μM in this example) was added, and in the control group, an equal concentration of the solvent DMSO was added. The fresh chondrogenic induction medium containing FPH2 was replaced once every 3 - 4 days, and the culture was terminated on the 7th - 14th day.

[0110] After the culture was completed, the cells were fixed with 4% (w / v) paraformaldehyde in the dark for 20 minutes. The extracellular matrix of chondrocytes was stained with Alcian blue staining solution, and the staining conditions were at room temperature for 12 - 16 hours. After staining, they were washed with double-distilled water and photographed for observation. Representative pictures of Alcian blue staining after FPH2 treatment of the ATDC5 chondrogenic differentiation model for 8 days are shown as Figure 13 shown, where the cells with deep-stained aggregates are chondronodules, which are the characteristics of chondrogenic differentiation.

[0111] Figure 13 The results show that the addition of FPH2 promotes the aggregation of ATDC5 cells and the formation of Alcian blue-deeply stained chondronodules. The aggregation of cells positive for Alcian blue staining / chondronodule formation is a characteristic of chondrogenic differentiation. This indicates that in addition to acting on chondrocytes, FPH2 can also promote the differentiation of prechondrocytes into chondrocytes.

[0112] Example 5: FPH2 inhibits fatty acid oxidase and promotes mitochondrial metabolic adaptation

[0113] In this example, the action target of FPH2 was predicted and its function in cellular metabolic adaptation was verified.

[0114] 1. Prediction of FPH2 target:

[0115] Based on the SEA drug prediction tool (Similarity ensemble approach), we input the FPH2 molecule according to the Simplified molecular input line entry system (SMILES) into the website SEA (sea16.docking.org). This prediction tool can predict potential drug targets by comparing the molecular chemical structure similarities of ligand-receptor in a large number of databases. After running, the SEA tool gives a candidate list. According to the candidate list, we use the Sequence alignments tool in the Uniprot database (www.uniprot.org) to view the similarities of the target protein amino acid sequences in different species, in order to verify the phenomenon that FPH2 has functions on both human and mouse cells.

[0116] 2. Verification of target activity:

[0117] Combined with the SEA prediction results, further verify the effect of FPH2 on potential targets. Carnitine palmitoyl transferase I (CPT1A, liver subtype; CPT1B, muscle subtype) is a rate-limiting enzyme for fatty acid oxidation. According to the standard steps of the carnitine palmitoyl transferase (CPT-1) kit (Suzhou Keming, CPT1-1-Y), detect the effect of FPH2 treatment on the activity of the target protein. Inoculate the SW1353 human chondrosarcoma cell line in a 10 cm diameter culture dish, use DMEM / F-12 supplemented with 10% (volume fraction) fetal bovine serum as the culture medium, and culture it in an environment of 37 °C, 3-8% carbon dioxide, and the rest is air. When the cell confluence reaches 30%-40%, replace it with fresh medium containing 0.5-5 μM concentration of FPH2 (also DMEM / F-12 supplemented with 10% volume fraction fetal bovine serum), and the control group adds the same concentration of solvent DMSO. After 48 hours, digest the chondrocytes with 0.03-0.08% concentration of trypsin for 2-3 minutes, then terminate the digestion with DMEM / F-12 medium containing 10% fetal bovine serum, centrifuge, remove the supernatant, resuspend the cells, and adjust the cell concentration to 3-5×10 6 / Tube, centrifuge to remove the supernatant. According to the kit instructions, add 1 ml of Reagent 1 (the reagent in the kit) and 10 μl of Reagent 3 to each tube of the sample, and homogenize with an ice bath homogenizer; centrifuge the homogenate at 600 g for 5 minutes at 4 °C; discard the precipitate, transfer the supernatant to another centrifuge tube, and centrifuge at 11,000 g for 10 minutes at 4 °C; the supernatant is the cytoplasmic extract. Add 1 ml of absolute ethanol to Reagent 5 and mix well, then add 22 ml of Reagent 4 and mix well; add 1 mL of distilled water to Reagent 6 and mix well; incubate both at 37 °C for 5 minutes. Adjust the wavelength of the microplate reader to 412 nm. Add 10 μl of the sample, 220 μl of Reagent 5, and 10 μl of Reagent 6 to a 96-well plate, mix well, record the initial absorbance A1 at 412 nm at 20 seconds and the absorbance A2 at 2 minutes and 20 seconds, and calculate ΔA = A2 - A1. The enzyme activity calculation formula is: CPT-1 (nmol / min / mg prot) = [ΔA × Vtotal reaction ÷ (ε × d) × 109] ÷ (Vsample × Cpr) ÷ T = 1760 × ΔA ÷ Cpr. Where Vtotal reaction: the total volume of the reaction system, 2.4 × 10-4 L; ε: 1.36 × 104 L / mol / cm; d: 0.5 cm; Vsample: the volume of the added sample, 0.01 ml; Vtotal extract: the volume of the added extract, 0.202 ml; T: the reaction time, 2 minutes; Cpr: the sample protein concentration, mg / ml.

[0118] 3. Characterization of cellular mitochondrial function:

[0119] ATP production detection: Mouse or human chondrocytes (passages 3 - 5) are seeded into 24- or 12-well plates, and DMEM / F-12 with 10% (v / v) fetal bovine serum is used as the culture medium. The cells are cultured until they reach a confluence of 20 - 40%, and then the medium is replaced with a medium containing dissolved FPH2 at a concentration of 0.5 - 5 μM. The cells are cultured for 48 hours in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest is air. An equal concentration of DMSO solvent is used as a control treatment. According to the standard procedure of the Enhanced ATP Detection Kit (Beyotime), the total ATP produced by the cells is detected. Aspirate the culture medium, add lysis buffer at a ratio of 1 / 10 of the medium, and pipette repeatedly to ensure sufficient lysis. Centrifuge at 12,000 g for 5 minutes at 4 °C, and take the supernatant. Add 100 μl of ATP detection working solution to the detection wells and let it stand at room temperature for 3 - 5 minutes. Add 20 μl of the sample or standard to the detection wells, quickly mix well with a pipette, and after at least 2 seconds, measure the relative light units with a chemiluminescence analyzer. Finally, calculate the concentration of ATP in the sample according to the standard curve.

[0120] 4. Mitochondrial membrane potential detection: Mouse or human chondrocytes (passages 3 - 5) were seeded into 48 - well or 24 - well plates, using DMEM / F - 12 supplemented with 10% (v / v) fetal bovine serum as the culture medium. The cells were cultured until they reached 20 - 40% confluence, and then the medium was replaced with the medium containing dissolved FPH2 at a concentration of 0.5 - 5 μM. The cells were cultured for 48 hours in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest being air. An equal - concentration DMSO solvent was used as a control treatment. According to the standard protocol of the Enhanced Mitochondrial Membrane Potential Detection Kit (JC - 1) (Beyotime), the relative value of mitochondrial membrane potential was detected. The culture medium was aspirated, 1 ml of cell culture medium was added, 1 ml of JC - 1 staining working solution was added and mixed well, and then incubated at 37 °C for 20 minutes; the supernatant was aspirated, and the cells were washed twice with JC - 1 staining buffer; 2 ml of cell culture medium was added, and photographs were taken using a laser confocal microscope. The relative value of membrane potential was calculated based on the intensity ratio of red fluorescence (excitation wavelength 5250 nm, emission wavelength 590 nm) and green fluorescence (excitation wavelength 490 nm, emission wavelength 530 nm) of the cells.

[0121] 5. Reactive Oxygen Species (ROS) detection: Mouse or human chondrocytes (passages 3 - 5) were seeded into 48 - well or 24 - well plates, using DMEM / F - 12 supplemented with 10% (v / v) fetal bovine serum as the culture medium. The cells were cultured until they reached 20 - 40% confluence, and then the medium was replaced with the medium containing dissolved FPH2 at a concentration of 0.5 - 5 μM. The cells were cultured for 48 hours in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest being air. An equal - concentration DMSO solvent was used as a control treatment. According to the standard protocol of the ROS Detection Kit (Beyotime), the relative value of ROS production by the cells was detected. DCFH - DA was diluted 1:1000 with DMEM / F - 12 culture medium without fetal bovine serum; the cell culture medium was removed, and the diluted DCFH - DA was added, usually in a volume sufficient to completely cover the cells; the cells were incubated at 37 °C for 20 minutes; the cells were washed three times with serum - free medium, and then directly observed using a confocal microscope to calculate the green fluorescence intensity (excitation wavelength 488 nm, emission wavelength 525 nm).

[0122] 6. Result analysis

[0123] According to the prediction results of the SEA prediction tool, we found that the top 3 candidate targets were carnitine palmitoyl transferase I of human or rat (carnitine palmitoyl transferase I; CPT1A, liver subtype; CPT1B, muscle subtype) (see Figure 14 ). The higher the Z - score value, the more similar FPH2 and carnitine palmitoyl transferase I are, indicating that the ligand of FPH2 and the target protein are more similar.

[0124] According to the amino acid sequence alignment in the Uniprot database, the protein amino acid sequences of carnitine palmitoyltransferase I in humans and other species are highly similar (see Figure 15 ), which supports the result that we found FPH2 to be effective in both human and mouse cells.

[0125] Based on the activity detection of carnitine palmitoyltransferase I, we found that treating cells with FPH2 led to a significant decrease in the activity of carnitine palmitoyltransferase I in a concentration-dependent manner (see Figure 16 , statistical differences *p<0.05, **p<0.01, ***p<0.001), suggesting that the target prediction result is reasonable. Given the literature hint, a decrease in the activity of carnitine palmitoyltransferase I inhibits the fatty acid oxidation metabolism of cells, thereby compensatorily increasing the level of glucose metabolism. This can potentially enhance the energy supply of chondrocytes, protect mitochondrial homeostasis, and enhance the adaptability and regenerative potential of cells.

[0126] Based on the detection of the mitochondrial function of chondrocytes, we found that FPH2 increased the overall energy metabolism level of cells, enabling cells to produce more ATP (see Figure 17 left figure); FPH2 increased the relative value of mitochondrial membrane potential. Given that a decrease in mitochondrial membrane potential is an indication of abnormal mitochondrial function, the results show that FPH2 can enhance the function of mitochondria to maintain homeostasis (see Figure 17 middle figure); FPH2 slightly inhibited the production of reactive oxygen species, but no significant difference was observed, which may be related to FPH2 promoting mitochondrial energy production and increased workload, suggesting that FPH2 did not cause more accumulation of reactive oxygen species in cells due to increased mitochondrial activity (see Figure 17 right figure). Statistical differences *p<0.05, **p<0.01, ***p<0.001.

[0127] In summary, the results show that FPH2 can inhibit the activity of carnitine palmitoyltransferase I, activate cell metabolism, protect mitochondrial homeostasis, and enhance the metabolic adaptability of chondrocytes.

[0128] Example 6. Amplification of chondrocytes treated with FPH2 to promote the efficacy of in vivo cell transplantation

[0129] This example simulated the clinical chondrocyte transplantation process to detect the effect of culturing chondrocytes with FPH2 on in vivo cell transplantation.

[0130] 1. Cell passage and amplification:

[0131] Human primary chondrocytes were obtained. The culture medium used was DMEM / F-12 supplemented with 10% (v / v) fetal bovine serum, and the pH was natural. FPH2 was continuously added to the medium at a concentration of 0.5 - 5 μM. The cells were cultured for 48 hours in an environment of 37 °C, 3 - 8% carbon dioxide, and the rest being air. An equal concentration of DMSO solvent was used as a control treatment. The medium containing FPH2 was replaced every 2 days until the cells were completely confluent and passaged. When passaging, the chondrocytes were digested with trypsin at a concentration of 0.03 - 0.08% for 2 - 3 minutes, then the digestion was terminated with DMEM / F-12 medium containing 10% fetal bovine serum. After centrifugation at 1200 - 1500 rpm for 3 - 5 minutes, the medium was discarded, and the cells were resuspended with fresh medium containing FPH2 and re-inoculated at a cell confluence of 40 - 50%, which was recorded as passage 1. The cells were continuously cultured in this way until passage 4.

[0132] 2. Redifferentiation of chondrocytes:

[0133] When the confluence of chondrocytes continuously cultured to passage 4 reached more than 90%, the chondrocytes were digested with trypsin at a concentration of 0.03 - 0.08% for 2 - 3 minutes, then the digestion was terminated with DMEM / F-12 medium containing 10% fetal bovine serum. The cell suspension was transferred to a 15 ml sterile centrifuge tube, and the number of cells in each tube was 1 - 3×10 5 cells. After centrifugation at 1200 - 1500 rpm for 3 - 5 minutes. After centrifugation ended, cell aggregates could be seen settling at the bottom of the tube with the naked eye. The medium was slowly aspirated, and a chondrogenic differentiation induction medium containing 0.5 - 5 μM FPH2 was added. An equal concentration of DMSO solvent was used as a control treatment. The components of the chondrogenic differentiation induction medium were: H-DMEM (High Glucose Dulbecco's Modified Eagle Medium) medium supplemented with 1% (v / v) Insulin, Transferrin, Selenium, Ethanolamine Solution, 2.5 - 10 ng / ml Recombinant Human TGFβ3, 1% sodium pyruvate, 50 μg / ml L-ascorbic acid 2-phosphate hydrate, 10-7 M dexamethasone, and the pH was natural. FPH2 at 0.5 - 5 μM was continuously added during the culture process, and the medium was changed every 3 - 4 days for 5 - 10 days. During this process, the chondrocytes would spontaneously aggregate into spherical or disc-shaped microtissues.

[0134] 3. Full-thickness cartilage defect model in rats: Male SD rats at about 10 weeks of age were anesthetized by intraperitoneal injection of chloral hydrate (chloral hydrate concentration 7% mass fraction, 500 μl injected per 100 g body weight). The lower limbs were shaved, disinfected, and the knee joint cavity of the lower limbs was opened. A cylindrical injury with a diameter of 1 - 2 mm and a depth of 1 - 4 mm was created at the center of the femoral trochlear groove. After creating the model, for the blank group, the joint cavity was directly reduced and sutured without any other treatment; for the control group, cartilage microtissues cultured with DMSO as a control were transplanted; for the experimental group, cartilage microtissues continuously cultured with FPH2 were transplanted. The number of cells implanted into the defect was about 1 - 2×105, and the microtissues could be trimmed according to the actual size of the defect. When implanting the microtissues, 1 - 5% mass fraction of methacrylated gelatin hydrogel was dropped to prevent the transplanted microtissues from falling off the defect (the blank group also added hydrogel). After the operation, the rats were given routine care and were fed with the immunosuppressant cyclophosphamide (2 - 4 mg / kg / day, dissolved in drinking water, and the water was changed every 2 - 3 days) on the second day after the operation to prevent host-graft immune rejection caused by xenotransplantation. At 12 weeks after the operation, the rats were euthanized, and the joint tissues were fixed by static placement in 4% mass fraction paraformaldehyde solution at room temperature for 2 - 5 days, and then the tissues were decalcified, dehydrated, cleared, embedded in paraffin, and sectioned into paraffin sections. The 7 μM thick paraffin sections were hydrated and stained with safranin O-fast green, and photos were taken with an optical microscope and a digital scanner. Immunofluorescence staining was performed with LaminA / C antibody (Abcam, ab108595) to label the transplanted human cells, and the tissue integration area formed by the graft was calculated.

[0135] 4. Result analysis

[0136] Figure 18 Photographs of cartilage cells continuously cultured with FPH2 implanted into the joints of rats to repair full-thickness cartilage defects, where the arrow points to the tissue interface; there are tissue voids in the DMSO control group, and it is difficult for the implant and host tissues to heal; there is excessive migration at the tissue interface in the FPH2 group, indicating better integration. Figure 18 The results show that the defects without transplanted cells are difficult to spontaneously repair and cannot form hyaline cartilage; for the tissues transplanted with control or FPH2-treated cells, the formation of hyaline cartilage can be observed.

[0137] Figure 19The probability of forming hyaline cartilage in the joints of rats by different treatment methods (12 weeks after surgery). The calculation method of the probability of forming hyaline cartilage is as follows: All samples are sectioned, and 1 - 3 sections with the largest tissue defect (or graft) area in each sample among consecutive sections are selected under the microscope and marked as representative sections; after safranin O - fast green staining, the sample is qualitatively analyzed based on the histological characteristics presented by the section; if the safranin O - fast green staining shows red and the cell morphology is oval or triangular / polygonal, it is recorded as hyaline cartilage formation; if the safranin O - fast green staining shows green, or the cell morphology is spindle - shaped or fibroblastic, or there is no new tissue formation at the defect site, it is recorded as no hyaline cartilage formation; after qualitatively analyzing each joint sample in each group, the probability is statistically calculated. According to Figure 18 and 19 , the formation of hyaline cartilage indicates the existence of tissue regeneration and repair. Hyaline cartilage: Safranin O - fast green staining is red; fibrous cartilage or bone: Safranin O - fast green staining is green. Thus, the probability of forming hyaline cartilage (i.e., tissue repair) is: 50% (4 / 8 samples) in the FPH2 experimental group; 33% (3 / 9 samples) in the DMSO control group; 0% in the blank group. This shows that FPH2 can improve the regeneration efficiency of chondrocyte transplantation.

[0138] Figure 20 Based on human - cell antibody labeling, the proportion of the area of transplanted cells participating in interface healing is calculated. Compared with the DMSO control group, the implanted cells in the FPH2 group have better integration with the surrounding bone - cartilage tissue and do not form tissue gaps ( Figure 18 ). By labeling human cells, we found that the cells in the FPH2 - treated group migrated more to the tissue healing interface ( Figure 20 , with statistical differences *p < 0.05, **p < 0.01, ***p < 0.001). This indicates that the cells treated with FPH2 have stronger regeneration potential and adaptability and can effectively grow into the tissues of the surrounding environment.

[0139] In summary, the results show that chondrocytes cultured with FPH2 for a long time have stronger regeneration potential and adaptability in in - vivo repair.

[0140] Example 7: In - situ action of FPH2 on the surface of articular cartilage to prevent osteoarthritis

[0141] This example simulates the treatment of superficial cartilage defects. Superficial cartilage defects refer to injuries that do not involve the subchondral bone, which are often in the early stage of the disease and there is no ideal treatment method yet. Improper treatment will lead to secondary osteoarthritis.

[0142] In this example, FPH2 is directly added in - situ at the cartilage defect site, combined with carriers such as hydrogels, to promote regeneration and prevent osteoarthritis. The flow chart is as Figure 21 shown.

[0143] 1. Preparation of FPH2 - microspheres: Mix and dissolve FPH2 and gelatin in deionized water at a mass ratio of FPH2:gelatin = 1:10; Drop the gelatin solution into liquid paraffin containing sorbitan oleate (0.1 - 0.2% volume fraction), and stir at a speed of 600 revolutions per minute for 13 minutes; Cool on ice, stir for 30 minutes and add 1 ml of glutaraldehyde for cross - linking; Further stir for 2 hours, add 2 ml of acetone, take the supernatant and then add 1 ml of acetone, and solidify overnight at 4°C; Wash three times with acetone and deionized water respectively, and store at - 20°C after freeze - drying for later use.

[0144] 2. Preparation of articular surface hydrogel: Dissolve GelMA (full name: Gelatin - Methacryloyl) and HA - NB (full name: Hyaluronic Acid - N-(2 - aminoethyl)-4-(4-(hydroxymethyl)-2 - methoxy - 5 - nitrosophenoxy)butanamide) in phosphate - buffered saline (pH 7.2) and mix well. The dosage of GelMA is 2 - 10% (mass fraction), and the dosage of HA - NB is 0.2 - 2% (mass fraction). Sterilize at 120°C for 8 minutes. After mixing the hydrogel with the FPH2 - microsphere solution, 0.25% (mass fraction) of lithium phenyl - 2,4,6 - trimethylbenzoylphosphinate (LAP) needs to be mixed in as a photo - initiator. The hydrogel can gel after being irradiated with an ultraviolet lamp for 10 - 30 seconds. The wavelength of ultraviolet irradiation is 405 nm.

[0145] 3. Superficial cartilage defect model in rats: Anesthetize male SD rats about 10 weeks old by intraperitoneal injection of chloral hydrate (chloral hydrate concentration 7% mass fraction, 500 μl injected per 100 g body weight). Shave the hair on the lower limbs, disinfect, open the knee joint cavity of the lower limbs, and create a square injury with a side length of 1 - 3 mm and a depth of 0.1 - 1 mm at the center of the femoral trochlear groove. After creating the model, for the blank group, directly suture the joint cavity back in place without any other treatment; For the control group, add 2 μl of microsphere - hydrogel mixture without encapsulating FPH2 to form a gel in situ; For the experimental group, add the microsphere - hydrogel mixture encapsulating FPH2 to form a gel in situ. The final concentration of FPH2 used is 100 - 500 μM, and the drug dose in the adult rat model is 0.02 - 5 nmol / joint. For the sham - operation group, only open the joint cavity and suture it back in place without performing the modeling step, serving as a healthy cartilage control.

[0146] At 8 weeks after surgery, the rats were euthanized, and the joint tissues were fixed with 4% paraformaldehyde solution at room temperature for 2 - 5 days. The tissues were then decalcified, dehydrated, cleared, embedded in paraffin, and sectioned. The 7 - μm thick paraffin sections were hydrated and subjected to immunohistochemical staining. Photographs were taken with an optical microscope and a digital scanner and scored.

[0147] 4. Results Analysis

[0148] Figure 22 These are immunohistochemical pictures of COL2 and MMP13 after 8 weeks of repairing superficial cartilage defects in rats with different treatment methods. According to Figure 22 It can be seen that in the FPH2 microsphere - hydrogel group, the expression of the cartilage function marker COL2 was higher, while the expression of the cartilage matrix degradation marker MMP13 (matrix metalloproteinase) was lower. The results indicate that the microsphere - hydrogel loaded with FPH2 can effectively regenerate cartilage tissue on the joint surface, and the results are better than those of the blank control and the blank microsphere - hydrogel group.

[0149] Figure 23 These are the ICRS - II scores after 8 weeks of repairing superficial cartilage defects in rats with different treatment methods. Among them, ICRS - II is used for scoring the effect of cartilage repair, with a full score of 100 points. The higher the score, the better the repair effect. According to the overall parameter score of the ICRS - II score for cartilage repair, the score of the FPH2 microsphere - hydrogel group was significantly higher than that of the blank group, and it was significantly better than the scoring effect of the microsphere - hydrogel system without FPH2 and the blank group.

[0150] Figure 24 These are the OARSI scores after 8 weeks of repairing superficial cartilage defects in rats with different treatment methods. Among them, OARSI grade is used for scoring the progression of osteoarthritis, with a full score of 6 points. The higher the score, the more severe the osteoarthritis. According to the OARSI score, the score of the FPH2 microsphere - hydrogel group was significantly lower than that of the blank group, and it was significantly lower than that of the microsphere - hydrogel system without FPH2 and the blank group. Statistical differences: *p < 0.05, **p < 0.01, ***p < 0.001.

[0151] In summary, the FPH2 - based sustained - release system can significantly improve the repair effect of superficial cartilage defects and play a preventive role in osteoarthritis.

[0152] Matters not covered by this invention are well - known technologies. Although this invention is disclosed as above, it is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of this invention. Therefore, the protection scope of this invention should be determined by the scope defined by the claims.

Claims

1. A reagent for improving the adaptability of chondrocytes, characterized in that, The reagent contains FPH2 and its solvent.

2. The reagent according to claim 1, wherein The structural formula of the FPH2 is shown in Formula (1):

3. The reagent according to claim 2, wherein The concentration of FPH2 in the reagent is 0.2 - 20 μM.

4. A method for improving the adaptability of chondrocytes, characterized in that, Chondrocytes are treated with the reagent containing FPH2.

5. Use of a small molecule compound for preparing a reagent for enhancing the adaptability of chondrocytes, wherein the small molecule compound comprises Cediranib and FPH2.

6. The use according to claim 5, characterized in that, The adaptability includes one or more of in vitro amplification adaptability, chondrogenic differentiation and redifferentiation adaptability, metabolic adaptability, and in vivo adaptability after cell transplantation.

7. Use of FPH2 for preparing a reagent for promoting the differentiation of chondrogenic progenitor cells into chondrocytes.

8. Use of FPH2 for preparing a reagent for inhibiting fatty acid oxidase and promoting mitochondrial metabolism.

9. Use of FPH2 for preparing a reagent for enhancing the therapeutic effect of chondrocyte transplantation.

10. Use of FPH2 for preparing a reagent for preventing osteoarthritis.

Images