Bone joint injection as well as preparation method and application thereof

Abstract

The invention relates to a bone joint injection as well as a preparation method and application thereof. The bone joint injection comprises dopamine modified hyaluronic acid, amino-polyethylene glycol and a dissolving solution. The amino-polyethylene glycol is introduced into the dopamine modified hyaluronic acid, so that the micro-crosslinking effect can be enhanced, the mechanical property of the bone joint injection is improved, and the problems of joint abrasion and the like caused by lack of bone joint synovial fluid are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bone joint injection, in particular to a bone joint injection and a preparation method and application thereof. BACKGROUND

[0002] Synovial fluid is a synovial fluid secreted by synovial membrane and existing in joints, which plays a crucial physiological function in joint movement. Synovial fluid plays a lubricating role, which can effectively prevent the mutual friction between articular cartilages, while absorbing impact and nourishing cartilage. However, in the case of osteoarthritis, trauma or aging, the decrease in the amount or viscosity of synovial fluid can cause direct friction between articular cartilages, aggravate wear and tear, and cause pain, stiffness and limited joint movement. Therefore, maintaining the amount and viscosity of synovial fluid at a normal level is crucial for the normal spatial movement of bone joints.

[0003] Traditional physical therapy and exercise rehabilitation treatment methods can reduce joint load through muscle strengthening to indirectly protect synovial fluid, but cannot solve the problem of existing synovial fluid loss. Hyaluronic acid is a macromolecular polysaccharide with good biocompatibility, and is also the main component of synovial fluid, which plays an important physiological function of water retention, osmotic pressure regulation and lubrication in vivo. Dopamine is an endogenous nitrogen-containing organic compound, also known as catecholamine or hydroxytyramine, which is an intermediate product produced by tyrosine in the metabolic process of dihydroxyphenylalanine, and is a kind of catecholamine, which has good wet adhesion to biological tissues. Dopamine has certain antioxidant capacity and can remove excess free radicals in the case of oxidative stress in bone joints. Hyaluronic acid modified by dopamine has a certain degree of improvement in mechanical properties, but the effect is limited.

[0004] Therefore, it is necessary to provide a bone joint injection with better mechanical properties. SUMMARY

[0005] To solve the above technical problems, the present application provides a bone joint injection and a preparation method and application thereof. By introducing amino-polyethylene glycol into dopamine-modified hyaluronic acid, the present application can enhance the micro-crosslinking effect, improve the mechanical properties of the bone joint injection, and significantly improve the joint wear caused by the lack of synovial fluid.

[0006] To achieve this purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a bone joint injection, which comprises dopamine-modified hyaluronic acid, amino-polyethylene glycol and a dissolving solution.

[0008] The amino-polyethylene glycol is a high-molecular polymer, including single-end amino-polyethylene glycol, double-end amino-polyethylene glycol or multi-arm amino-polyethylene glycol. The amino-polyethylene glycol has good water solubility, excellent lubricity and moisturizing property, and forms a hydration layer after being dissolved in water to have anti-fouling effect. The amino-polyethylene glycol is introduced into the dopamine-modified hyaluronic acid in the application, which enhances the micro-crosslinking of the dopamine-modified hyaluronic acid, effectively improves the mechanical properties of the obtained bone joint injection solution, provides good buffering effect for joint friction, and at the same time endows the joint lubricating fluid with anti-fouling effect and injectability.

[0009] Preferably, the grafting rate of dopamine in the dopamine-modified hyaluronic acid is 10-50% (for example, can be 10%, 20%, 30%, 40%, 50% and the like, preferably 20-40%).

[0010] In the application, if the grafting rate of dopamine in the hyaluronic acid is too low, the antioxidant effect is poor; if the grafting rate is too high, the viscosity is too high.

[0011] Preferably, the weight average molecular weight of the hyaluronic acid in the dopamine-modified hyaluronic acid is 200-2000 kDa (for example, can be 200 kDa, 500 kDa, 1000 kDa, 1500 kDa, 2000 kDa and the like, preferably 800-1500 kDa).

[0012] In the application, if the molecular weight of the hyaluronic acid is too low, the viscosity is too low, and it is difficult to occur micro-crosslinking to maintain the lubricating effect; if the molecular weight is too high, the viscosity is too high, and it is difficult to maintain good performance.

[0013] Preferably, the amino-polyethylene glycol is selected from amino-polyethylene glycol-amino and / or O-(2-aminoethyl) polyethylene glycol, preferably amino-polyethylene glycol-amino.

[0014] Preferably, the weight average molecular weight of the amino-polyethylene glycol is 2000-8000 Da (for example, can be 2000 Da, 4000 Da, 6000 Da, 8000 Da and the like).

[0015] In the application, if the weight average molecular weight of the amino-polyethylene glycol is too low, the anti-fouling property is poor; if the molecular weight is too high, the steric hindrance is large, and the micro-crosslinking effect is weak.

[0016] Preferably, the dissolving solution comprises any one of water, phosphate buffer, phosphate buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, or sodium carbonate-sodium bicarbonate buffer, or a combination of at least two thereof.

[0017] Preferably, the use amount ratio of the dopamine-modified hyaluronic acid, the amino-polyethylene glycol, and the dissolving solution is (0.001-5) g:(0.001-3) g:100 mL, preferably (2-5) g:(0.5-2) g:100 mL.

[0018] The above numerical value (0.001-3) g may, for example, be independently 0.001 g, 0.01 g, 0.1 g, 0.5 g, 1 g, 1.5 g, 2 g, 3 g, 5 g, etc., and (0.001-3) g may, for example, be independently 0.001 g, 0.01 g, 0.1 g, 0.5 g, 1 g, 1.5 g, 2 g, 3 g, etc. In the present application, the use amount ratio of the dopamine-modified hyaluronic acid and the amino-polyethylene glycol can be 1:1-10:1. If the use amount ratio is too high, the cross-linking is too strong, and the lubricity is poor. If the use amount ratio is too low, the micro-cross-linking effect is weak, and the stain resistance is poor.

[0019] In a second aspect, the present application provides a preparation method of the bone joint injection solution according to the first aspect, the preparation method comprising: mixing dopamine-modified hyaluronic acid, amino-polyethylene glycol, and a dissolving solution to obtain the bone joint injection solution.

[0020] Preferably, the preparation method of the dopamine-modified hyaluronic acid comprises: mixing hyaluronic acid, an activating agent, dopamine hydrochloride, and a dissolving solution, and reacting to obtain the dopamine-modified hyaluronic acid.

[0021] Preferably, the hyaluronic acid, the activating agent, and the dissolving solution are first mixed for activation, and then the system is mixed with dopamine hydrochloride for modification to obtain the dopamine-modified hyaluronic acid.

[0022] Preferably, the activating agent comprises 1-ethyl-(3-dimethylaminopropyl) carbodiimide and / or N-hydroxysuccinimide.

[0023] Preferably, the dissolving solution comprises any one of water, phosphate buffer, phosphate buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer or sodium carbonate-sodium bicarbonate buffer or a combination of at least two thereof.

[0024] Preferably, the hyaluronic acid, the activating agent, the dopamine hydrochloride and the dissolving solution are used in a ratio of (0.001-1) g:(0.001-1) g:(0.001-1) g:100 mL, preferably (0.01-1) g:(0.01-0.8) g:(0.01-0.5) g:100 mL.

[0025] The above numerical value (0.001-1) g may be, for example, independently 0.001 g, 0.01 g, 0.1 g, 0.2 g, 0.5 g, 0.8 g, 1 g, etc.

[0026] Preferably, the reaction is carried out at a temperature of 20-40℃ (for example, 20℃, 22℃, 25℃, 30℃, 32℃, 35℃, 40℃, etc.) for a time of 6-15 h (for example, 6 h, 8 h, 10 h, 12 h, 15 h, etc.).

[0027] Preferably, the reaction is followed by a post-treatment step, wherein the post-treatment comprises dialysis of the reaction system, followed by freeze-drying to obtain the dopamine-modified hyaluronic acid.

[0028] Preferably, the dialysis is carried out at a pH of 4-5 (for example, 4, 4.2, 4.5, 4.8, 5, etc.) for a time of 2-4 days (for example, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, etc.).

[0029] In a third aspect, the application provides a use of the bone joint injection solution according to the first aspect in the preparation of a bone repair material.

[0030] Compared with the prior art, the application has at least the following beneficial effects:

[0031] The application introduces amino-polyethylene glycol into the dopamine-modified hyaluronic acid, which enhances the micro-crosslinking of the dopamine-modified hyaluronic acid, effectively improves the mechanical properties of the obtained bone joint injection solution, provides good buffering effect for joint friction, and at the same time endows the joint lubricating fluid with anti-fouling effect and injectability. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1is a nuclear magnetic hydrogen spectrum of the dopamine-modified hyaluronic acid, dopamine hydrochloride and hyaluronic acid in Example 1.

[0033] Figure 2 is a modulus and viscosity change graph of the bone joint injection solution in Example 1.

[0034] Figure 3 is a shear thinning experiment test graph of the bone joint injection solution in Example 1.

[0035] Figure 4 is a cck-8 experiment graph of the bone joint injection solution and dopamine-hyaluronic acid in Example 1.

[0036] Figure 5 is a rat osteoarthritis model micro-CT graph of the bone joint injection solution in Example 1.

[0037] Figure 6 is a rat osteoarthritis model micro-CT graph of the dopamine-hyaluronic acid in Example 1. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0039] Example 1

[0040] The present embodiment provides a preparation method of a bone joint injection solution, comprising the following steps:

[0041] (1) 4.5 g of hyaluronic acid (molecular weight = 80-150 million Da) and 450 mL of pH≈5.5 double distilled water are dissolved, 2.5 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimide and 1.5 g of N-hydroxysuccinimide are added, and stirring is activated for 0.5 h, then 2.25 g of dopamine hydrochloride is added, and reaction is carried out at 25℃ for 12 h, after dialysis and freeze-drying, dopamine-modified hyaluronic acid is obtained (hydrogen spectrum test and analysis of dopamine hydrochloride, hyaluronic acid and dopamine-modified hyaluronic acid are carried out by using a 400M Bruker nuclear magnetic resonance spectrometer, and the dopamine grafting rate is calculated to be 35% by peak integration, Figure 1 );

[0042] (2) 3 g of dopamine-modified hyaluronic acid, 1 g of amino-polyethylene glycol-amino (molecular weight = 4000 Da) and 100 mL of sterile enzyme-free water are mixed to obtain a bone joint injection solution.

[0043] Example 2

[0044] The present embodiment provides a preparation method of a bone joint injection solution, comprising the following steps:

[0045] (1) 4.5 g of hyaluronic acid (molecular weight = 200,000 Da) and 450 mL of double distilled water with pH ≈ 5.5 are dissolved, 2.5 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole and 1.5 g of N-hydroxysuccinimide are added, stirring and activating for 0.5 h, then 2.25 g of dopamine hydrochloride is added, reacting at 25°C for 12 h, after dialysis and freeze-drying, dopamine-modified hyaluronic acid (calculated dopamine grafting rate is 40%) is obtained;

[0046] (2) 2 g of dopamine-modified hyaluronic acid, 2 g of amino-polyethylene glycol-amino (molecular weight = 4000 Da) and 100 mL of sterile enzyme-free water are mixed to obtain a bone joint injection solution.

[0047] Example 3

[0048] The present embodiment provides a preparation method of a bone joint injection solution, comprising the following steps:

[0049] (1) 4.5 g of hyaluronic acid (molecular weight = 200,000 Da) and 450 mL of double distilled water with pH ≈ 5.5 are dissolved, 2.5 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole and 1.5 g of N-hydroxysuccinimide are added, stirring and activating for 0.5 h, then 2.25 g of dopamine hydrochloride is added, reacting at 25°C for 12 h, after dialysis and freeze-drying, dopamine-modified hyaluronic acid (calculated dopamine grafting rate is 40%) is obtained;

[0050] (2) 2 g of dopamine-modified hyaluronic acid, 2 g of amino-polyethylene glycol-amino (molecular weight = 4000 Da) and 100 mL of sterile enzyme-free water are mixed to obtain a bone joint injection solution.

[0051] Example 4

[0052] The present embodiment provides a preparation method of a bone joint injection solution, which is different from example 1 only in that the amount of dopamine hydrochloride in step (1) is adjusted to 3 g, and dopamine-modified hyaluronic acid with a dopamine grafting rate of 50% is obtained, and the other steps are the same as example 1.

[0053] Example 5

[0054] The present embodiment provides a preparation method of a bone joint injection solution, which is different from example 1 only in that the amount of dopamine hydrochloride in step (1) is adjusted to 1 g, and dopamine-modified hyaluronic acid with a dopamine grafting rate of 15% is obtained, and the other steps are the same as example 1.

[0055] Example 6

[0056] The embodiment provides a preparation method of bone joint injection solution, which is different from the embodiment 1 only in that the amount of amino-polyethylene glycol-amino is adjusted to 5 g in step (2), and other steps are the same as those in the embodiment 1.

[0057] Embodiment 7

[0058] The embodiment provides a preparation method of bone joint injection solution, which is different from the embodiment 1 only in that the amount of amino-polyethylene glycol-amino is adjusted to 0.01 g in step (2), and other steps are the same as those in the embodiment 1.

[0059] Embodiment 8

[0060] The embodiment provides a preparation method of bone joint injection solution, which is different from the embodiment 1 only in that the amino-polyethylene glycol-amino is replaced by O-(2-aminoethyl) polyethylene glycol in step (2), and other steps are the same as those in the embodiment 1.

[0061] Comparative example 1

[0062] The comparative example provides a preparation method of bone joint injection solution, which is different from the embodiment 1 only in that the amino-polyethylene glycol-amino is not added in step (2), and other steps are the same as those in the embodiment 1.

[0063] Test example 1

[0064] Rheological test

[0065] The mechanical properties of the bone joint injection solution are subjected to rheological test by using a MARS III (HAAKE MARS III) rheometer, and the test includes G' (storage modulus), G'' (loss modulus) and η (dynamic viscosity) viscosity test strain under different strains.

[0066] The test results are shown in Table 1, and the modulus and viscosity trend graph of the bone joint injection solution provided in the embodiment 1 with the change of strain is shown in Figure 2 .

[0067] Table 1

[0068]

[0069] Test results:

[0070] (1) As can be seen from the embodiment 1 to the embodiment 8, the bone joint injection solution provided by the application has certain mechanical properties under frequency scanning and strain scanning test, and the viscosity is appropriate, and can simulate the physiological condition of joint activity. In the preferred scheme, G'' / G' under each strain is in the range of 0.3-0.8, and the viscosity is in the range of 100-300 Pa·s, which particularly meets the product requirements.

[0071] (2) By comparing Example 1 with Examples 4-5, it can be seen that by controlling the grafting rate of dopamine in dopamine-modified hyaluronic acid, the viscosity and modulus of the modified hyaluronic acid can be adjusted. When the grafting rate is too low, the viscosity and modulus are too low, and the modified hyaluronic acid cannot effectively stay on the surface of the bone joint. When the grafting rate is too high, the viscosity is high, the injectability is poor, and the bone joint movement can be hindered. By comparing Example 1 with Examples 6-7, it can be seen that by controlling the ratio of dopamine-modified hyaluronic acid and amino-polyethylene glycol, the dynamic viscosity and modulus of the injection solution can be adjusted. When the ratio is too low, the viscosity and modulus are too low, and the injection solution cannot achieve good therapeutic effect. When the ratio is too high, the viscosity and modulus are too high, which hinders the movement of the bone joint and reduces the therapeutic effect. By comparing Example 1 with Example 8, it can be seen that by further optimizing the amino-polyethylene glycol-amino, the dynamic viscosity and shear modulus of the bone joint injection solution can be accurately adjusted to achieve better therapeutic effect.

[0072] (3) By comparing Example 1 with Comparative Example 1, it can be seen that by compounding dopamine-modified hyaluronic acid and amino-polyethylene glycol, the mechanical properties of the obtained bone joint injection solution can be improved, the loss modulus and storage modulus ratio is stable at about 0.5, which has good rigidity, and the viscosity stability is increased, the minimum viscosity can reach 115 Pas, which can effectively stay on the surface of the bone joint.

[0073] Test Example 2

[0074] Shear thinning test

[0075] A MARS III (HAAKE MARS III) rheometer was used to test the shear thinning of the bone joint injection solution, which included: taking 400 microliters of injection solution and placing it on the rheometer table, controlling the shear strain to be 1%, the shear frequency to be 1 Hz, and the shear rate to be 0-150 1 / s.

[0076] The bone joint injection solutions provided by Examples 1-8 and Comparative Example 1 were tested, and the test results of Example 1 are shown in Figure 3 The horizontal coordinate is the shear rate, and the vertical coordinate is the shear viscosity. The shear thinning experiment shows that the viscosity of the bone joint injection solution decreases rapidly at high shear rate, which proves that it has good injectability. The test results of Examples 2-8 and Comparative Example 1 are similar to those of Example 1.

[0077] Test Example 3

[0078] Shear thinning test

[0079] Biological safety CCK-8 experiment: after the bone joint injection liquid (HA-DA / PEG-NH2) and dopamine-hyaluronic acid (HA-DA) and the blank control group were co-cultured with 3T3 cells for 24 hours, the cell metabolic activity was detected by CCK-8 reagent.

[0080] The test results of Example 1 are shown in Figure 4 The results show that the micro-crosslinked bone joint injection liquid provided by the application has good biocompatibility.

[0081] Test Example 4

[0082] Micro-CT test of rat osteoarthritis model

[0083] The bone joint injection liquid (HA-DA / PEG-NH2) and dopamine-hyaluronic acid (HA-DA) of Example 1 were used to treat the osteoarthritis rat model, and CT scanner (AX-2000, China) was used to perform joint angiography on the left knee joint of the rat. The obtained micro-CT test of the rat osteoarthritis model is shown in Figure 5 and Figure 6

[0084] The results show that the dopamine-hyaluronic acid (HA-DA) treatment group shows many uneven pits on the bone surface, indicating severe bone and cartilage erosion caused by osteoarthritis. The bone joint injection liquid (HA-DA / PEG-NH2) treatment group shows significantly reduced bone surface pits, and a uniform smooth state is observed, indicating that the cartilage and bone are protected or repaired.

[0085] The applicant states that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the application.​

Claims

1. A bone and joint injection solution, characterized in that, The bone and joint injection solution includes dopamine-modified hyaluronic acid, amino-polyethylene glycol, and a dissolving agent.

2. The bone and joint injection solution according to claim 1, characterized in that, The grafting rate of dopamine in the dopamine-modified hyaluronic acid is 10-50%, preferably 20-40%; Preferably, the weight-average molecular weight of the hyaluronic acid in the dopamine-modified hyaluronic acid is 200,000 Da to 2,000,000 Da.

3. The bone and joint injection solution according to claim 1 or 2, characterized in that, The amino-polyethylene glycol is selected from amino-polyethylene glycol-amino and / or O-(2-aminoethyl)polyethylene glycol, preferably amino-polyethylene glycol-amino; Preferably, the amino-polyethylene glycol has a weight-average molecular weight of 2000-8000 Da.

4. The bone and joint injection solution according to any one of claims 1-3, characterized in that, The dissolving solution includes any one or a combination of at least two of the following: water, phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citrate-sodium hydroxide-hydrochloric acid buffer, citrate-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, or sodium carbonate-sodium bicarbonate buffer.

5. The bone and joint injection solution according to any one of claims 1-4, characterized in that, The ratio of the dopamine-modified hyaluronic acid, amino-polyethylene glycol, and solution is (0.001-5) g:(0.01-3) g:100 mL, preferably (2-5) g:(0.5-2) g:100 mL.

6. A method for preparing a bone and joint injection solution according to any one of claims 1-5, characterized in that, The preparation method includes: mixing dopamine-modified hyaluronic acid, amino-polyethylene glycol and a dissolving solution to obtain the bone and joint injection solution.

7. The preparation method according to claim 6, characterized in that, The preparation method of the dopamine-modified hyaluronic acid includes: mixing hyaluronic acid, activator, dopamine hydrochloride and solution, and reacting to obtain dopamine-modified hyaluronic acid.

8. The preparation method according to claim 7, characterized in that, The activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and / or N-hydroxysuccinimide; Preferably, the dissolving solution comprises any one or a combination of at least two of the following: water, phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citrate-sodium hydroxide-hydrochloric acid buffer, citrate-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, or sodium carbonate-sodium bicarbonate buffer. Preferably, the ratio of hyaluronic acid, activator, dopamine hydrochloride and solution is (0.001-1)g:(0.001-1)g:(0.001-1)g:100 mL, and more preferably (0.01-1)g:(0.01-0.8)g:(0.01-0.5)g:100 mL.

9. The preparation method according to claim 7 or 8, characterized in that, The reaction is carried out at a temperature of 20-40℃ for 6-15 hours. Preferably, the reaction further includes a post-processing step, which includes dialyzing the reaction system and then freeze-drying it to obtain dopamine-modified hyaluronic acid.

10. The use of the bone and joint injection solution according to any one of claims 1-5 in the preparation of bone repair materials.

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