New use of a class of phosphorylated polymers containing syringolide structural units

Abstract

The present invention discloses a new use of a phosphorylated polymer containing a dihydroeugenol structural unit, belonging to the technical field of polymers. The preparation method of the phosphorylated polymer is as follows: First, anhydrous ethylenediamine, formaldehyde, potassium hydroxide solution and a polymer containing a dihydroeugenol structure are mixed to obtain an aminated polymer. Then, the aminated polymer is mixed with a 2-hydroxy-3-chloropropyl phosphate sodium solution. After the reaction is completed, dialysis and freeze-drying are carried out to finally obtain a phosphorylated polymer containing a dihydroeugenol structure. By phosphorylating and modifying the polymer containing a dihydroeugenol structure, the prepared phosphorylated polymer containing a dihydroeugenol structure has the ability to promote the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells and inhibit the osteoclastic differentiation of bone marrow mononuclear macrophages, and can effectively promote the repair of osteochondral unit defects.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymers, and particularly relates to a new use of a phosphorylated polymer containing a dihydroeugenol structural unit. Background Art

[0002] The osteochondral unit consists of articular cartilage and subchondral bone and plays a crucial role in joint movement and weight-bearing functions. Articular cartilage is a low-cell tissue that covers the bone ends to reduce friction and absorb stress during joint movement, while subchondral bone supports and nourishes the cartilage. This dynamic system maintains joint balance and function through biochemical and biomechanical signals and responds to changes in the joint environment. However, the osteochondral unit is easily damaged and degenerated due to aging, injury, or disease, thereby impairing joint function and leading to osteoarthritis. Therefore, protecting and restoring the osteochondral unit is crucial for joint health and mobility. The simultaneous regeneration of subchondral bone and articular cartilage is a challenging part of osteochondral unit repair and has become an urgent problem to be solved for orthopedic surgeons and patients worldwide. Currently, the method for promoting the repair of damaged osteochondral is to implant biomaterials from natural or synthetic sources to replace the damaged tissue.

[0003] Polymers containing dihydroeugenol structures (DHP) can effectively promote the repair of cartilage defects in rats, but their effect on subchondral bone repair is not clear. The coordinated action of osteoblasts that generate new bone and osteoclasts that resorb old bone leads to bone remodeling. The importance of osteoclasts in bone homeostasis and human health can be demonstrated by various forms of osteoclast formation and dysfunction. Under the stimulation of external stressors, bisphosphonates reduce the activity of osteoclasts and promote apoptosis to achieve the effect of inhibiting bone resorption. The oxygen atoms on the phosphate group form a synergistic chelation with bone minerals and show a strong affinity for hydroxyapatite. However, existing bisphosphonate drugs may cause serious adverse reactions such as osteonecrosis of the jaw, esophageal cancer, and renal failure.

[0004] Therefore, there is an urgent need to provide a phosphorylated polymer containing a dihydroeugenol structure that has no toxic side effects and is used for the repair of osteochondral defects. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the first technical problem to be solved by the present invention is to provide the application of a phosphorylated polymer containing a dihydroeugenol structural unit in the preparation of a drug for promoting the repair of osteochondral unit defects, which can promote the chondrogenic differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells, and at the same time inhibit the osteoclastic differentiation of bone marrow mononuclear macrophages, thereby promoting the repair of osteochondral defects. The second technical problem to be solved by the present invention is to provide a preparation method of a phosphorylated polymer containing a dihydroeugenol structure, which is simple and convenient, and the prepared phosphorylated polymer containing a dihydroeugenol structure has no toxic and side effects. The third technical problem to be solved by the present invention is to provide a phosphorylated polymer containing a dihydroeugenol structural unit, which has good biocompatibility and a uniform structure, can promote the chondrogenic differentiation of mesenchymal stem cells, and at the same time can effectively regulate the bone metabolism balance, inhibit osteoclastic differentiation while promoting osteogenic differentiation.

[0006] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0007] The application of a phosphorylated polymer containing a dihydroeugenol structural unit in the preparation of a drug for promoting the repair of osteochondral unit defects.

[0008] Furthermore, the phosphorylated polymer containing a dihydroeugenol structural unit promotes the chondrogenic differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells, and / or inhibits the osteoclastic differentiation of bone marrow mononuclear macrophages.

[0009] Furthermore, the concentration of the phosphorylated polymer containing a dihydroeugenol structural unit used in the repair of osteochondral unit defects does not exceed 100 μg / mL.

[0010] Furthermore, the concentration of the phosphorylated polymer containing a dihydroeugenol structural unit used in the repair of osteochondral unit defects does not exceed 1 μg / mL.

[0011] Furthermore, the specific steps of the phosphorylated polymer containing a dihydroeugenol structural unit are as follows:

[0012] 1) Weigh 1 g of the polymer containing a dihydroeugenol structure and dissolve it in 10 mL of potassium hydroxide solution. Then, add 1 mL of anhydrous ethylenediamine and 1.5 mL of formaldehyde aqueous solution in sequence, and react at 85 °C for 5 h to obtain an aminated polymer; among them, the concentration of the potassium hydroxide solution is 10%, and the concentration of the formaldehyde aqueous solution is 37%;

[0013] 2) Mix the aminated polymer prepared in step 1) with a 2-hydroxy-3-chloropropyl phosphate sodium solution, react for 5 h, then dialyze with a dialysis bag with a cut-off relative molecular mass of 500. After dialysis is completed, freeze-dry to obtain a phosphorylated polymer containing a dihydroeugenol structure.

[0014] Preparation method of phosphorylated polymer containing dihydroeugenol structure, the specific steps are as follows:

[0015] 1) Weigh 1 g of the polymer containing dihydroeugenol structure and dissolve it in 10 mL of potassium hydroxide solution. Then, add 1 mL of anhydrous ethylenediamine and 1.5 mL of aqueous formaldehyde solution in sequence. After reacting at 85 °C for 5 h, an aminated polymer is obtained; wherein, the concentration of the potassium hydroxide solution is 10%, and the concentration of the aqueous formaldehyde solution is 37%;

[0016] 2) Mix the aminated polymer prepared in step 1) with 2-hydroxy-3-chloropropyl phosphate sodium solution. After reacting for 5 h, dialysis is carried out using a dialysis bag with a cut-off relative molecular mass of 500. After dialysis is completed, freeze-drying is carried out to obtain a phosphorylated polymer containing dihydroeugenol structure.

[0017] Furthermore, in step 2), the molar ratio of the aminated polymer to phosphate ions is 2 - 4:1.

[0018] Furthermore, the molar ratio of the aminated polymer to phosphate ions is 4:1.

[0019] The phosphorylated polymer containing dihydroeugenol structural unit prepared by the method.

[0020] Application of the phosphorylated polymer containing dihydroeugenol structural unit in the preparation of a drug for promoting the repair of osteochondral unit defects.

[0021] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:

[0022] (1) The phosphorylated polymer containing dihydroeugenol structural unit prepared by the present invention has good biocompatibility and a uniform structure, can promote the chondrogenic differentiation of mesenchymal stem cells, and at the same time can effectively regulate the bone metabolism balance, inhibit osteoclast differentiation while promoting osteoblast differentiation.

[0023] (2) By phosphorylating and modifying the polymer containing dihydroeugenol structure, the phosphorylated polymer containing dihydroeugenol structure prepared by the present invention has the ability to promote the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells and inhibit the osteoclast differentiation of bone marrow mononuclear macrophages, and can effectively promote the repair of osteochondral unit defects.

[0024] (3) The preparation method of the present invention is simple and convenient, and the prepared phosphorylated polymer containing dihydroeugenol structure has no side effects and has good economic benefits. Description of the drawings

[0025] Figure 1 It is a flow chart of the phosphorylated polymer containing dihydroeugenol structure;

[0026] Figure 2 Fourier transform infrared spectra of DHP, DHP-P1 and DHP-P2;

[0027] Figure 3 Cryo-electron microscopy images of DHP-P1 and DHP-P2;

[0028] Figure 4 HSQC spectra side chain (δC / δH 50 - 90 / 2.5 - 6.0) and aromatic (δC / δH 100 - 135 / 5.5 - 8.5) region maps of DHP-P1 and DHP-P2;

[0029] Figure 5 Schematic diagram of the main substructures in the process of preparing the phosphorylated polymer containing eugenol structure in the present invention;

[0030] Figure 6 Cell viability data of DHP-P1 and DHP-P2 on bone marrow mesenchymal stem cells;

[0031] Figure 7 Cell viability data of DHP-P1 and DHP-P2 on bone marrow mononuclear macrophages;

[0032] Figure 8 Expression of osteoclast differentiation-related genes mRNA (such as TRAP, MMP-9 and ATP6VOD2) evaluated by RT-qPCR for DHP-P1;

[0033] Figure 9 TRAP staining images;

[0034] Figure 10 Phalloidin staining images;

[0035] Figure 11 Expression of osteogenesis-related genes mRNA evaluated by RT-qPCR for DHP-P1;

[0036] Figure 12 ALP and ARS staining results;

[0037] Figure 13 Expression of mRNA of transcription factors for chondrogenic expression (SOX9, ACAN). Detailed implementation manners

[0038] The present invention will be further described below in conjunction with specific embodiments.

[0039] The polymer containing the dihydroeugenol structural unit used in the following examples was prepared in the laboratory, and the reference is Chen Xuekuan, Zhao Houkuan, Wu Hongfei, etc. Study on the synthesis and antioxidant properties of dehydrogenation polymerization products of isoeugenol [J]. Chemistry and Industry of Forest Products, 2018, 38(01): 87-92.

[0040] Example 1

[0041] The preparation method of the phosphorylated polymer containing the dihydroeugenol structure is as follows:

[0042] 1) Weigh 1 g of the polymer containing the dihydroeugenol structural unit and dissolve it in 10 mL of potassium hydroxide solution. Then, add 1 mL of anhydrous ethylenediamine and 1.5 mL of formaldehyde aqueous solution in sequence, and react at 85 °C for 5 h to obtain an aminated polymer; among them, the concentration of the potassium hydroxide solution is 10%, and the concentration of the formaldehyde aqueous solution is 37%.

[0043] 2) Mix the aminated polymer prepared in step 1) with the 2-hydroxy-3-chloropropyl phosphate sodium solution (the molar ratio of the aminated polymer to the phosphate ion is 4:1). After reacting for 5 h, perform dialysis with a dialysis bag having a cut-off relative molecular mass of 500. After the dialysis is completed, freeze-dry at 1.33 Pa and -60 °C for 48 h to obtain the phosphorylated polymer containing the dihydroeugenol structure, denoted as DHP-P1, with a yield of 80% and a purity of 98%. The results are as Figures 1 - 5 shown in Table 1. Figure 1 It is a flow chart of the phosphorylated polymer containing the dihydroeugenol structure.

[0044] Example 2

[0045] The preparation method of the phosphorylated polymer containing the dihydroeugenol structure is as follows:

[0046] 1) Weigh 1 g of the polymer containing the dihydroeugenol structural unit and dissolve it in 10 mL of potassium hydroxide solution. Then, add 1 mL of anhydrous ethylenediamine and 1.5 mL of formaldehyde aqueous solution in sequence, and react at 85 °C for 5 h to obtain an aminated polymer; among them, the concentration of the potassium hydroxide solution is 10%, and the concentration of the formaldehyde aqueous solution is 37%.

[0047] 2) Mix the aminated polymer prepared in step 1) with the 2-hydroxy-3-chloropropyl phosphate sodium solution (the molar ratio of the aminated polymer to the phosphate ion is 2:1). After reacting for 5 h, perform dialysis with a dialysis bag having a cut-off relative molecular mass of 500. After the dialysis is completed, freeze-dry at 1.33 Pa and -60 °C for 48 h to obtain the phosphorylated polymer containing the dihydroeugenol structure, denoted as DHP-P2, with a yield of 85% and a purity of 96%. The results are as Figures 1 - 5 shown in Table 1. Figure 1Flow chart of the phosphorylated polymer containing syringolide structure

[0048] Example 3

[0049] DHP-P1 and DHP-P2 were characterized by nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). The specific test methods are as follows:

[0050] The chemical structures of DHP-P1 and DHP-P2 were analyzed by nuclear magnetic resonance (NMR). 2 Both the D-HSQC NMR spectrum and the 31P-NMR spectrum were measured using a Bruker (Germany) 600 MHz superconducting nuclear magnetic resonance spectrometer at 30 °C. Weigh 20 mg of the sample and dissolve it in deuterated dimethyl sulfoxide (DMSO-d6) for the detection of two-dimensional spectra. The conditions are as follows: 1 The spectral widths of H and 13 C are 3497 Hz and 18750 Hz respectively, 1 The H relaxation time is 1.5 s, the number of acquisition points is 1024, and the number of scans is 128 and 257 respectively. 31 Detection steps for the 31P-NMR spectrum: Accurately weigh 40 mg of the absolutely dry sample into a nuclear magnetic resonance tube, dissolve it in 0.5 mL of deuterated pyridine / deuterated chloroform (1.6:1, v / v), add 0.2 mL of the internal standard reagent N-hydroxy-5-norbornene-2,3-dicarboximide solution (e-NHI, 9.23 mg / mL) and 0.05 mL of the relaxation reagent chromium(III) acetylacetonate solution (5.6 mg / mL). After thorough mixing, add 0.1 mL of the phosphating reagent 2-chloro-1,3,2-dioxaphospholane for phosphating treatment, and then perform on-machine analysis after 15 minutes.

[0051] The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of the phosphorylated polymer containing syringolide structural units were determined using a high-performance gel permeation chromatograph. Weigh 0.004 g of the absolutely dry sample, add 2 mL of tetrahydrofuran, completely dissolve it and then filter and load it onto the machine. The analytical columns are Styragel HR-1 and Styragel HR-2, the injection volume is 50 μL, the mobile phase is tetrahydrofuran, the flow rate is 1 mL / min, and the standard sample is polystyrene.

[0052] The results are shown in Table 1 below.

[0053] Table 1 Functional group content and molecular weight of DHP-P1 and DHP-P2

[0054]

[0055] Table 1 shows the functional group contents and molecular weights of DHP-P1 and DHP-P2. As can be seen from the table, compared with the aliphatic hydroxyl content (0.7 mmol / g) and phenolic hydroxyl content (1.9 mmol / g) of the polymer containing syringolide structural units, the aliphatic hydroxyl contents of DHP-P1 and DHP-P2 are 0.7 mmol / g and 0.9 mmol / g respectively; the phenolic hydroxyl contents are 2.2 mmol / g and 2.3 mmol / g respectively. Therefore, after phosphorylation modification of the polymer containing syringolide structure, the aliphatic hydroxyl contents of DHP-P1 and DHP-P2 decrease, while the phenolic hydroxyl contents increase, which is related to the breakage of the linkage bonds during the modification process of the polymer, thus exposing more phenolic hydroxyls. In addition, the content of phosphate ions in the phosphorylated modified polymer varies with different reaction input amounts.

[0056] Figure 2 Figure 4 shows the Fourier transform infrared spectra of DHP, DHP-P1 and DHP-P2. As can be seen from the figure, the Fourier transform infrared spectra of DHP-P1 and DHP-P2 are similar to those of the dehydrogenated polymer. The absorption peaks at 3418 cm -1 and 3415 cm -1 are attributed to the stretching of -OH in alcohols and phenols. In addition, characteristic absorption peaks of phosphates appear at 420 cm -1 for DHP-P1 and DHP-P2, and the sizes of the absorption peaks vary with the addition amount of phosphates, indicating that phosphate groups are successfully incorporated into the polymer containing syringolide structural units.

[0057] Figure 3 Figure 5 shows the cryo-electron microscopy images of DHP-P1 and DHP-P2. As can be seen from the figure, the suspension aqueous solutions of DHP-P1 and DHP-P2 are photographed, and the results show that DHP-P1 and DHP-P2 can be uniformly dispersed in water.

[0058] Figure 4HSQC spectra side chain (δC / δH 50 - 90 / 2.5 - 6.0) and aromatic (δC / δH 100 - 135 / 5.5 - 8.5) region maps of DHP - P1 and DHP - P2. As can be seen from the figure, the spectra of DHP - P1 and DHP - P2 both show prominent signals corresponding to methoxy (δC / δH 55.6 / 3.73), the β - position signal (A) of the β - O - 4 ether bond structure connected to the syringyl group (S) of the lignin carbonyl, and the signal peak (B) related to the β - β structure position of resinol, located at 86.0 / 4.11, 84.9 / 4.69 and 53.7 / 3.05 ppm respectively. In addition, the chemical shifts of α and β of the β - 5 (C) structure are 86.8 / 5.49 and 53.1 / 3.49 ppm respectively. The signals of the 2, 5 and 6 - position units of the guaiacyl (G) structure in the aromatic ring region are at 111.0 / 7.01, 114.4 / 6.73 - 115.3 / 6.98 ppm and 119.0 / 6.82 ppm respectively. At the same time, the side - chain position of G - type lignin is oxidized to a keto group (G'), and the signal is located at 112.5 / 7.32 ppm. In addition, the cinnamyl alcohol end - group (I) structure is found in both DHP - Ps, and the related signal peaks at its α and β positions are 128.5 / 6.47 ppm and 128.5 / 6.25 ppm respectively.

[0059] Figure 5 Schematic diagram of the main sub - structures in the process of preparing the phosphorylated polymer containing dihydroeugenol structure in the present invention; in the figure, A and A ， is the β - O - 4 aryl ether bond structure, the difference is that the γ - position of A is a hydroxyl group, and the γ - position of A' is an esterified p - coumaric acid ester; B is the resinol structure; C is the phenylcoumaran structure; G is the guaiacyl structure; I is the p - hydroxycinnamyl alcohol unit.

[0060] Example 4

[0061] Cytotoxicity determination of the phosphorylated polymer containing dihydroeugenol structural units:

[0062] The cytotoxicity tests of DHP-P1 and DHP-P2 on bone marrow mesenchymal stem cells and bone marrow mononuclear macrophages were determined by co-culturing the cells with different concentrations (1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL) of DHP-P1 and DHP-P2 for 24 h. The cell viability was analyzed using the Cell Counting Kit-8 (CCK-8). Cells in the logarithmic growth phase were digested with trypsin, gently pipetted and mixed, and then inoculated into a 96-well cell culture plate with a multi-channel pipette and cultured in a constant temperature incubator at 37 °C for 24 h. Different concentrations of DHP-P1 and DHP-P2 were added to the well plates to make their final concentrations reach 0, 10 μg / mL, 50 μg / mL, and 100 μg / mL. Each group of experiments was repeated 6 times. After the culture, the old medium was discarded, and the cells were washed 3 times with PBS buffer. 100 μL of CCK-8 detection solution was added to each well, and finally, the well plates were sealed with plastic wrap and cultured in a constant temperature incubator at 37 °C for 60 min. The absorbance of each well of cells was measured at 450 nm using a microplate reader for cytotoxicity analysis. The results are as Figure 5 and 6 shown.

[0063] Figure 6 The data of the cell viability of DHP-P1 and DHP-P2 affecting bone marrow mesenchymal stem cells are shown in the figure. It can be seen from the figure that at low concentrations (below 1 μg / mL), DHP-P1 showed a certain promoting effect on the proliferation of bone marrow mesenchymal stem cells. DHP-P2 had no obvious promoting effect on the proliferation of bone marrow mesenchymal stem cells. When the concentration was in the range of 1 - 100 μg / mL, the proliferation effect of DHP-P1 on bone marrow mesenchymal stem cells gradually weakened. DHP-P2 had a certain toxicity to bone marrow mesenchymal stem cells.

[0064] Figure 7 The data of the cell viability of DHP-P1 and DHP-P2 affecting bone marrow mononuclear macrophages are shown in the figure. It can be seen from the figure that at low concentrations (below 1 μg / mL), neither DHP-P1 nor DHP-P2 had toxic effects on bone marrow mesenchymal stem cells and bone marrow mononuclear macrophages. In the concentration range of 1 - 100 μg / mL, the promoting effect of DHP-P1 on the proliferation of bone marrow mononuclear macrophages increased with the increase of concentration. However, the toxicity of DHP-P2 to bone marrow mononuclear macrophages increased with the increase of concentration. Based on the proliferation promoting effects and toxic effects of DHP-P1 and DHP-P2 on the two types of cells, 1 μg / mL DHP-P1 was finally selected as the optimal concentration for subsequent experiments.

[0065] Example 5

[0066] In vitro inhibitory effect of phosphorylated polymers containing syringolide structural units on osteoclast differentiation and promoting osteogenic differentiation experimental determination:

[0067] The effect of DHP-P1 on the osteoclast differentiation of osteoclast progenitors was evaluated using BMMs (bone marrow-derived macrophages). Osteoclastogenesis was evaluated by the formation of multinucleated (containing more than 3) positive cells. Cells cultured with M-CSF combined with RANKL without any drugs were used as negative controls. Osteoclast progenitors were cultured for 5 days under the action of M-CSF and RANKL, and 1 μg / mL DHP and DHP-P1 were added for co-incubation respectively. The expression of mRNA of osteoclast differentiation-related genes (such as TRAP, MMP-9, and ATP6V0D2) by DHP-P1 was evaluated by real-time PCR. The results are as Figures 8 - 10 shown.

[0068] Figure 8 For RT-qPCR to evaluate the expression of mRNA of osteoclast differentiation-related genes (such as TRAP, MMP-9, and ATP6V0D2) by DHP-P1, as can be seen from the figure, DHP-P1 can significantly inhibit the relative expression of mRNA of osteoclast differentiation genes MMP-9 and ATP6V0D2.

[0069] Figure 9 It is a TRAP staining map; Figure 10 It is a Phalloidin staining map. As can be seen from the figure, DHP-P1 significantly inhibits the fusion of multinucleated trap-positive osteoclasts. Therefore, DHP-P1 effectively inhibits the osteoclast differentiation of bone marrow mononuclear macrophages.

[0070] Example 6

[0071] In vitro promotion of osteogenic and chondrogenic differentiation experimental test of phosphorylated polymers containing syringolide structural units:

[0072] The effects of DHP-P1 on osteogenesis and chondrogenesis of BMSCs were detected using p3 BMSCs (bone marrow mesenchymal stem cells), and the results are as Figures 11 - 13 shown.

[0073] Figure 11 For RT-qPCR to evaluate the effect of DHP-P1 on the expression of mRNA of osteogenesis-related genes, as can be seen from the figure, compared with the control group, the DHP treatment group had no effect on the mRNA expression of ALP and COL1 genes. However, the mRNA (ALP and COL1) levels of transcription factors regulating osteogenic expression in the DHP-P1 treatment group were significantly increased.

[0074] Figure 12 It is the ALP and ARS staining results; as can be seen from the figure, DHP-P1 has a certain promoting effect on osteogenesis of bone marrow mesenchymal stem cells.

[0075] Figure 13 Regarding the expression of mRNAs of transcription factors for chondrogenic expression (SOX9, ACAN), as can be seen from the figure, compared with the control group, the levels of mRNAs of transcription factors for chondrogenic expression (SOX9, ACAN) in the DHP-P1 treatment group were significantly increased.

[0076] In summary, DHP-P1 promotes osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro and inhibits osteoclastic differentiation of bone marrow mononuclear macrophages. It is proved that the phosphorylated polymer containing eugenol structural units has excellent effects on the repair of osteochondral unit defects.

Claims

1. Preparation method of phosphorylated polymer containing dihydroeugenol structure, characterized in that, The specific steps are as follows: 1) Weigh 1 g of the polymer containing the dihydroeugenol structure and dissolve it in 10 mL of potassium hydroxide solution. Then, add 1 mL of anhydrous ethylenediamine and 1.5 mL of aqueous formaldehyde solution in sequence. After reacting at 85 °C for 5 h, an aminated polymer is obtained. Among them, the concentration of the potassium hydroxide solution is 10%, and the concentration of the aqueous formaldehyde solution is 37%; 2) Mix the aminated polymer prepared in step 1) with the sodium 2-hydroxy-3-chloropropyl phosphate solution. After reacting for 5 h, perform dialysis using a dialysis bag with a cut-off relative molecular mass of 500. After dialysis is completed, freeze-dry to obtain the phosphorylated polymer DHP-P1 containing the dihydroeugenol structure; Among them, the molar ratio of the aminated polymer to the phosphate ion is 2 - 4:1; The polymer containing the dihydroeugenol structure is a lignin dehydrogenation polymer DHP synthesized by laccase-catalyzed reaction using isoeugenol as a raw material.

2. The phosphorylated polymer containing the dihydroeugenol structural unit prepared by the method described in claim 1.

3. Use of the phosphorylated polymer containing the dihydroeugenol structural unit described in claim 2 in the preparation of a drug for promoting the repair of osteochondral unit defects.

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