Injectable bionic synovial fluid hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable bionic synovial fluid hydrogel as well as a preparation method and application thereof, and belongs to the technical field of bionic synovial fluid hydrogel. The injectable bionic synovial fluid hydrogel comprises a hyaluronic acid compound, thrombin and platelet-rich plasma (PRP), the hyaluronic acid compound is a brush polymer (HPA) which takes hyaluronic acid as a main chain and is rich in sulfonate radical polymer as a side chain. The hyaluronic acid compound and the PRP bionic synovial fluid hydrogel prepared by the invention can make up for the defects of individually using hyaluronic acid and individually using PRP to treat osteoarthritis clinically at present. On one hand, the hydrogel can recover the lubrication performance of articular cartilage, on the other hand, cartilage regeneration can be promoted, osteoarthritis is treated from the two aspects of physical and biological functions, and a potential application thought is provided for clinical treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of bionic synovial hydrogel, and particularly relates to a preparation method based on hyaluronic acid and its derivatives and PRP and an application in the biomedical field, and is particularly suitable for treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a common degenerative joint disease that is often associated with physical and biological dysfunction. It is currently estimated to affect more than 500 million people worldwide, seriously affecting people's normal life. Characteristics of OA include synovial inflammation, increased joint friction and reduced synovial fluid lubrication. Restoring the physical and biological functions of articular cartilage at the same time is the key to treating OA.

[0003] Natural synovial fluid has excellent lubricity, which is the result of the synergistic effect of the complex of hyaluronic acid, lubricin and lipids. It selectively adsorbs on the surface of cartilage to form a lubricating layer, providing long-lasting lubrication. More and more studies have confirmed that cartilage lubrication dysfunction is the main factor leading to OA. The bionic synovial fluids currently under research and development can effectively lubricate articular cartilage, restore articular cartilage lubrication to a certain extent, and delay the progression of osteoarthritis. However, the design of these bionic synovial fluids only considers restoring the physical function of articular cartilage, but does not restore the biological function of articular cartilage.

[0004] At present, the existing injectable bionic synovial hydrogels are usually prepared by physical or chemical cross-linking. Physical cross-linking, such as electrostatic interaction, ionic interaction, hydrogen bonding, etc., forms gels. This type of hydrogel is non-permanent and may have low stability. Chemical cross-linking is a polymer process in which macromolecular chains are linked by chemical bonds to form a network or body structure. However, due to the use of cross-linking agents, chemically cross-linked hydrogels may have certain toxicity. In order to restore the biological function of articular cartilage, we constructed a bioactive bionic synovial hydrogel based on the principle of biological cross-linking, with the aim of simultaneously restoring the physical and biological functions of articular cartilage. Summary of the invention

[0005] The present invention provides a bionic synovial hydrogel for restoring cartilage function from both physical and biological levels, and a preparation method and application thereof. Under the action of thrombin, an injectable bionic synovial hydrogel is prepared in a green and simple method. The bionic synovial hydrogel of the present invention can restore the lubrication properties of damaged cartilage, and on the other hand, promote cartilage regeneration through the sustained release of factors contained in PRP, which can well restore the physical and biological dysfunction of osteoarthritis cartilage, and provide a potential application idea for the clinical treatment of osteoarthritis.

[0006] The present invention provides the following technical solutions:

[0007] An injectable biomimetic synovial hydrogel comprises a hyaluronic acid compound, thrombin and platelet-rich plasma; the hyaluronic acid compound is a brush-loaded polymer (HPA) with hyaluronic acid as the main chain and a sulfonate-rich polymer as the side chain, and the structural formula is as follows:

[0008]

[0009] Here, n is an integer from 200 to 300, x is an integer from 280 to 427, and y is an integer from 90 to 150.

[0010] Preferably, the hyaluronic acid compound further includes hyaluronic acid (HA) or sulfonated hyaluronic acid (sHA).

[0011] Preferably, the volume fractions of the hyaluronic acid compound, thrombin and platelet-rich plasma in the injectable biomimetic synovial hydrogel are 10%-50%, 30%-60% and 5%-10%, respectively.

[0012] The above-mentioned method for preparing an injectable biomimetic synovial hydrogel comprises the following steps:

[0013] (1) dissolving a hyaluronic acid compound and thrombin in physiological saline or phosphate buffered saline (PBS) respectively to obtain a hyaluronic acid compound solution and a thrombin solution;

[0014] (2) mixing the hyaluronic acid compound solution and the thrombin solution to obtain solution A;

[0015] (3) Platelet-rich plasma (PRP) is defined as liquid B, and liquid A and liquid B are injected through double-chamber injection to obtain an injectable biomimetic synovial hydrogel.

[0016] Preferably, the concentration of the hyaluronic acid compound solution is 1-10 mg / ml.

[0017] Further preferably, the concentration of the thrombin solution is 50-500 U / ml.

[0018] Preferably, the concentration of the thrombin solution is 100-500 U / ml.

[0019] Preferably, the platelet concentration in the platelet-rich plasma is 300-1200×10^9 / L.

[0020] Preferably, the volume ratio of the hyaluronic acid compound solution to platelet-rich plasma is one of 1:2, 1:1, 2:1, 1:3, and 1:4.

[0021] Preferably, the platelet-rich plasma is prepared by three-step centrifugation, specifically comprising the following steps:

[0022] First, draw sodium citrate for injection with a syringe, and then collect rabbit whole blood from the ear vein of New Zealand rabbits, wherein the volume ratio of blood drawn: sodium citrate for injection is 9:1; then inject the blood in the syringe into a centrifuge tube, and centrifuge for the first time: speed 300g, centrifugation time 20min; after centrifugation, use a pipette to absorb all the supernatant and the middle layer (containing platelets, white blood cells and a small amount of red blood cells) and transfer them to another centrifuge tube; perform a second centrifugation at a speed of 1500g, centrifuge for 10min, take all the supernatant, and transfer it to another centrifuge tube; perform a third centrifugation at a speed of 3000g, centrifuge for 10min, at this time all the platelets are at the bottom of the centrifuge tube, and then resuspend the platelets with serum to obtain platelet-rich plasma.

[0023] The use of the above-mentioned injectable bionic synovial hydrogel in the preparation of drugs for treating osteoarthritis.

[0024] The hyaluronic acid compounds and PRP bionic synovial hydrogel prepared by the present invention can make up for the shortcomings of the existing clinical use of hyaluronic acid alone and PRP alone to treat osteoarthritis. The hydrogel can restore the function of articular cartilage from a physical and biological level. In terms of physical function, the bionic synovial hydrogel can reduce the friction coefficient of damaged cartilage and restore the lubrication performance of cartilage. At the same time, it plays a synergistic lubrication role when combined with PRP. In terms of biological function, the hydrogel can slowly release the cytokines of PRP, which can effectively control inflammation, promote cell proliferation, migration and differentiation, and the hydrogel loaded with bionic synovial fluid has a denser structure and can effectively control the burst release behavior of factors. Therefore, the combination of bionic synovial fluid and PRP shows great potential in the treatment of osteoarthritis.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The present invention constructs a bionic synovial hydrogel based on hyaluronic acid compounds and PRP, which has the advantages of a green and simple route and convenient operation.

[0027] (2) The bionic synovial hydrogel prepared by the present invention has dual functions, which can restore damaged articular cartilage from both physical and biological aspects, thus overcoming the shortcomings of the existing clinical treatment of osteoarthritis with the use of hyaluronic acid alone or PRP alone.

[0028] (3) The bionic synovial hydrogel obtained by the present invention provides a new idea for the treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a physical schematic diagram of the use of Solution A and Solution B obtained in Example 2.

[0030] Figure 2Schematic diagram of the gelation of bionic synovial hydrogel under different thrombin concentrations or different platelet concentrations.

[0031] Figure 3 This is the rheological diagram of the gelation of the bionic synovial fluid hydrogel obtained in Example 5.

[0032] Figure 4 This is a microscopic morphology of the gelation of the bionic synovial hydrogel obtained in Example 5.

[0033] Figure 5 This is a curve diagram of the release of insulin growth factor in the bionic synovial hydrogel obtained in Example 5 at different time points.

[0034] Figure 6 This is a bar graph showing the lubrication performance of the bionic synovial hydrogel obtained in Example 5 under different pressures.

[0035] Figure 7 This is a diagram of cell proliferation of the bionic synovial fluid hydrogel obtained in Example 2.

[0036] Figure 8 This is a live-dead staining experiment diagram of the bionic synovial hydrogel obtained in Example 2. DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with embodiments, but the present invention is not limited to these embodiments.

[0038] Example 1

[0039] Preparation of rabbit PRP: Use a 10ml syringe to extract 1ml of 2.5% sodium citrate for injection; Take 9ml of rabbit whole blood from the ear vein of New Zealand rabbit (blood volume: sodium citrate for injection is 9:1); Inject the blood in the syringe into a centrifuge tube, and centrifuge for the first time: speed 300g, centrifugation time 20min. After centrifugation, use a pipette to absorb all the supernatant and the middle layer (containing platelets, white blood cells and a small amount of red blood cells) and transfer them to another centrifuge tube; Perform a second centrifugation at a speed of 1500g, centrifuge for 10min, take all the supernatant, and transfer it to another centrifuge tube; Perform a third centrifugation at a speed of 3000g, centrifuge for 10min, at this time, all the platelets are at the bottom of the centrifuge tube, and then use 1ml of serum to resuspend the platelets, which is PRP. Table 1 is a blood routine table of PRP obtained in Example 1.

[0040] Table 1

[0041] sample White blood cell concentration (10^9 / L) Red blood cell concentration (10^12 / L) Platelet concentration (×10^9 / L) Whole blood 9.43 5.44 354 PRP-1 0.07 0.01 1273 PRP-2 0.26 0.05 2396 PRP-3 0.35 0.04 1269 PRP-4 0.69 0.04 2473

[0042] The number of red blood cells, white blood cells and platelets was tested by routine blood tests. As shown in Table 1, the three-step centrifugation method can stably prepare PRP with low white blood cells, low red blood cells and high platelets. At the same time, the platelet content in the extracted PRP is 4-8 times that of whole blood, proving the successful extraction of PRP.

[0043] Example 2

[0044] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 1 mg / ml and 500 U / ml, respectively. Then, mix 50 μl of HPA solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP prepared in Example 1 with a platelet content of 1200×10^9 / L. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0045] Figure 1 This is a physical schematic diagram of the use of liquid A and liquid B. As can be seen from the figure, after liquid A and liquid B are mixed through a double-chamber syringe, a stable gel can be formed.

[0046] Example 3

[0047] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 5 mg / ml and 500 U / ml, respectively. Then, mix 50 μl of HPA solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 1200×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0048] Example 4

[0049] Preparation of HPA and PRP bionic synovial hydrogel: Configuration of liquid A: Use physiological saline to dissolve HPA and thrombin at concentrations of 10 mg / ml and 500 U / ml, respectively. Then, mix 50 μl of HPA solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 1200×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP bionic synovial hydrogel (HPA@PRP gel). The corresponding PRP gel only needs to replace 50 μl of HPA solution in liquid A with 50 μl of physiological saline (PRP gel).

[0050] Example 5

[0051] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 100 U / ml, respectively. Then, mix 50 μl of HPA solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 1200×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0052] Example 6

[0053] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 200 U / ml, respectively, then mix 50 μl of hyaluronic acid solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 1200×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0054] Example 7

[0055] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 300 U / ml, respectively, then mix 50 μl of hyaluronic acid solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 1200×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0056] Example 8

[0057] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 500 U / ml, respectively, then mix 50 μl of hyaluronic acid solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 300×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0058] Example 9

[0059] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 500 U / ml, respectively, then mix 50 μl of hyaluronic acid solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 600×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0060] Example 10

[0061] Preparation of HPA and PRP biomimetic synovial hydrogel: Preparation of liquid A: Dissolve HPA and thrombin in physiological saline to concentrations of 10 mg / ml and 500 U / ml, respectively, then mix 50 μl of hyaluronic acid solution and 10 μl of thrombin solution to obtain liquid A. Liquid B: Take 100 μl of PRP with a platelet content of 900×10^9 / L prepared in Example 1. Mix liquid A and liquid B by double-chamber injection to obtain HPA and PRP biomimetic synovial hydrogel.

[0062] Figure 2 Schematic diagram of the gelation of bionic synovial hydrogel under different thrombin concentrations or different platelet concentrations. It can be seen from the figure that in the system of the present invention, liquid A and liquid B can form a stable bionic synovial hydrogel after mixing under different thrombin concentrations or different platelet concentrations. Figure 3 It can be seen that the bionic synovial hydrogel has the property of rapid gelation, and the strength of HPA@PRP gel is better than that of PRP gel. Figure 4 These are the microscopic morphologies of PRP gel and HPA@PRP gel. It can be observed that platelets adhere to fibrin and the structure of HPA@PRP gel is denser than that of PRP gel. Figure 5 This is the sustained release behavior of insulin growth factor in PRP gel and HPA@PRP gel. As can be seen from the figure, both PRP gel and HPA@PRP gel can sustainably release insulin growth factor. Among them, HPA@PRP gel releases the factor more slowly, showing a relatively stable release behavior, which can effectively control the factor burst release phenomenon.

[0063] Embodiment 11

[0064] The lubrication performance of the bionic synovial hydrogel prepared in Example 5 was tested using a Bruker friction and wear tester. The specific method is as follows:

[0065] Take fresh pig joints, use a trephine to take several small cartilage discs with a diameter of 7mm, as the upper and lower friction pairs, and fix them on the friction tester. Then add equal volumes of saline, 1% HPA saline solution, PRP gel and HPA@PRP gel to the cartilage surface, measure the friction coefficient under different loads, and repeat the measurement 3 times. Divide into four groups, the control group is saline, and the experimental group is HPA saline solution, PRP gel and HPA@PRP gel.

[0066] Figure 6 This is a bar chart comparing the lubrication properties of biomimetic synovial hydrogels. Figure 6 It can be seen that under a load of 1 N, the average friction coefficient of the control group was 0.094, the average friction coefficient of the HPA group was 0.052, the average friction coefficient of the PRP gel group was 0.043, and the average friction coefficient of the HPA@PRP gel group was 0.0300. At the same time, under loads of 1, 2, and 3 N, the lubrication of the HPA@PRP gel was still better than that of the control group, the HPA group, and the PRP gel. In addition, it can be observed that the lubrication performance of the HPA@PRP gel is significantly better than that of the HPA and PRP gels alone, indicating that the combination of bionic synovial fluid and PRP has a synergistic lubrication effect.

[0067] Example 12

[0068] Evaluation of cell proliferation of the bionic synovial hydrogel prepared in Example 2

[0069] The biomimetic synovial hydrogel prepared in Example 2 was sterilized and added to the Transwell chamber above the 24-well plate. Chondrocytes were seeded on the 24-well plate at a density of 5000 cells / well, and the biomimetic synovial hydrogel and chondrocytes were co-cultured at 37°C and 5% CO2. CCK8 assay and live-dead staining assay were performed at 24 hours and 72 hours of culture, respectively.

[0070] Figure 7 The results of CCK8 for the proliferation of chondrocytes by biomimetic synovial hydrogel. The results show that both PRP gel and HPA@PRP gel promote the proliferation of chondrocytes. At the same time, HPA@PRP gel has a better proliferation effect than PRP gel. The live-dead staining experiment is consistent with the experimental results of CCK8. It further illustrates that the biomimetic synovial hydrogel has good biocompatibility and can promote cell proliferation. It can meet the treatment requirements for osteoarthritis and can be predicted to have a good effect.

Claims

1. An injectable bionic synovial hydrogel, characterized in that: It comprises hyaluronic acid compounds, thrombin and platelet-rich plasma; the structural formula of the hyaluronic acid compounds is as follows: Here, n is an integer from 200 to 300, x is an integer from 280 to 427, and y is an integer from 90 to 150.

2. The injectable biomimetic synovial hydrogel according to claim 1, characterized in that: The volume fractions of the hyaluronic acid compound, thrombin and platelet-rich plasma in the injectable bionic synovial fluid hydrogel are 10%-50%, 30%-60% and 5%-10% respectively.

3. A method for preparing an injectable biomimetic synovial hydrogel according to claim 1 or 2, characterized in that: The following steps are involved: (1) dissolving a hyaluronic acid compound and thrombin in physiological saline or phosphate buffer respectively to obtain a hyaluronic acid compound solution and a thrombin solution; (2) mixing the hyaluronic acid compound solution and the thrombin solution to obtain solution A; (3) The platelet-rich plasma is defined as liquid B, and liquid A and liquid B are then injected through a double-chamber injection to obtain an injectable bionic synovial hydrogel.

4. The injectable biomimetic synovial hydrogel according to claim 3, characterized in that: The concentration of the hyaluronic acid compound solution is 1-10 mg / ml.

5. The injectable biomimetic synovial hydrogel according to claim 3, characterized in that: The concentration of the thrombin solution is 50-500 U / ml.

6. The injectable biomimetic synovial hydrogel according to claim 5, characterized in that: The concentration of the thrombin solution is 100-500 U / ml.

7. The injectable biomimetic synovial hydrogel according to claim 3, characterized in that: The platelet concentration in the platelet-rich plasma is 300-1200×10^9 / L.

8. The injectable biomimetic synovial hydrogel according to claim 3, characterized in that: The volume ratio of the hyaluronic acid compound solution to platelet-rich plasma is one of 1:2, 1:1, 2:1, 1:3, and 1:

4.

9. The injectable biomimetic synovial hydrogel according to claim 3, characterized in that: The platelet-rich plasma is prepared by three-step centrifugation, specifically comprising the following steps: First, draw sodium citrate for injection with a syringe, and then collect rabbit whole blood from the ear vein of New Zealand rabbits, wherein the volume ratio of blood drawn: sodium citrate for injection is 9:1; then inject the blood in the syringe into a centrifuge tube, and centrifuge for the first time: speed 300g, centrifugation time 20min; after centrifugation, use a pipette to absorb all the supernatant and the middle layer and transfer them to another centrifuge tube; perform a second centrifugation at a speed of 1500g, centrifuge for 10min, take all the supernatant, and transfer it to another centrifuge tube; perform a third centrifugation at a speed of 3000g, centrifuge for 10min, at this time all the platelets are at the bottom of the centrifuge tube, and then resuspend the platelets with serum to obtain platelet-rich plasma.

10. Use of the injectable bionic synovial hydrogel according to claim 1 or 2 in the preparation of a drug for treating osteoarthritis.

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