Hydrogel for remodeling aging microenvironment to promote rotator cuff injury repair and preparation method thereof

Abstract

The invention discloses hydrogel for remodeling an aging microenvironment and promoting rotator cuff injury repair and a preparation method of the hydrogel. The hydrogel comprises ordered hydroxyapatite super-long nanowire / gelatin-hyaluronic acid composite hydrogel and quercetin loaded in the ordered hydroxyapatite super-long nanowire / gelatin-hyaluronic acid composite hydrogel. According to the ordered hydroxyapatite ultra-long nanowire (AHNW) / gelatin-hyaluronic acid (GH) hydrogel compound loaded with Quercetin, the repairing capacity of bone marrow-derived mesenchymal stem cells (BMSCs) and tendon-derived stem cells (TDSCs) is improved by remodeling an aging microenvironment, so that tendon-bone healing is promoted, the integration effect is improved, and the application prospect is wide. A new treatment strategy is provided for the patient with the rotator cuff injury, especially the patient with the osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, and particularly relates to a hydrogel for remodeling the aging microenvironment to promote rotator cuff injury repair and a preparation method thereof. Background Art

[0002] Rotator cuff injury is one of the most common musculoskeletal diseases, and its incidence rate has been rapidly increasing with the aggravation of global population aging. Rotator cuff injury often causes persistent pain, dysfunction and limited joint movement, seriously affecting the quality of life of patients. Although conservative treatment can be adopted in the initial stage, the effect is not ideal, and many patients ultimately still need surgical treatment. Despite the continuous progress of rotator cuff repair surgery techniques, the postoperative retear rate is still as high as 20 - 94%.

[0003] In order to promote tendon - bone healing after rotator cuff repair, researchers have adopted a variety of tissue engineering techniques, including stem cell therapy, tissue engineering scaffolds (such as hydrogels and rotator cuff patches), growth factors, and small - molecule drugs, etc. However, the treatment effects of these methods are still not ideal. Especially in the context of osteoporosis or aging, their treatment effects are very limited, and the main problems can be attributed to the following points: 1) Insufficient repair ability of stem cells: In the context of osteoporosis or aging, the repair potential of stem cells is limited; in addition, senescent stem cells often show functional decline and cannot effectively promote tendon - bone healing. 2) Limitations of tissue engineering scaffolds: Although tissue engineering scaffolds such as hydrogels and rotator cuff patches can provide support and guidance, their integration effect with native tissues is not ideal; the degradation rate, mechanical properties, and biocompatibility of scaffold materials in vivo still cannot perfectly simulate natural tissues. 3) Limitations of small - molecule drugs: Currently used small - molecule drugs mainly focus on aspects such as anti - inflammation, promoting osteogenesis or chondrogenesis, and ignore the regulation of the aging microenvironment. 4) Challenges of the aging microenvironment: Rotator cuff injury is common in middle - aged and elderly people, especially osteoporosis patients, and the aging microenvironment poses an important challenge to the repair process; factors such as cellular senescence, inflammatory response, and oxidative stress in the aging microenvironment will significantly reduce the repair potential of bone marrow mesenchymal stem cells and tendon - derived stem cells; in addition, senescent cells lack sufficient proliferation and differentiation ability, resulting in significant inhibition of the tendon - bone healing process.

[0004] Therefore, how to remodel the aging microenvironment and activate the repair ability of senescent cells is a major challenge in current tendon - bone injury treatment techniques. Currently, there is no ideal material that can remodel the aging microenvironment to promote the repair of osteoporotic rotator cuff injury. Summary of the Invention

[0005] The purpose of the present invention is to provide a hydrogel for remodeling the aging microenvironment to promote rotator cuff injury repair and a preparation method thereof in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, a hydrogel for remodeling the aging microenvironment to promote rotator cuff injury repair is provided, including an ordered hydroxyapatite ultra-long nanowire / gelatin-hyaluronic acid composite hydrogel, and quercetin loaded therein.

[0008] In the second aspect, a preparation method of the above hydrogel for remodeling the aging microenvironment to promote rotator cuff injury repair is provided, including the following steps:

[0009] Step 1: Completely dissolve gelatin and sodium hyaluronate in deionized water to obtain a uniform basic solution.

[0010] Step 2: Add quercetin to the basic solution, and use an alkali solution to adjust the pH to 8-11, and stir to completely dissolve quercetin to obtain a basic solution containing quercetin.

[0011] Step 3: Completely immerse the HAP nanowire tape in the basic solution containing quercetin.

[0012] Step 4: Place the solution obtained in Step 3 under the condition of 0-4°C to gelate it to form a preliminary composite hydrogel structure; then place the formed hydrogel in an aqueous solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and carry out chemical crosslinking at room temperature.

[0013] Step 5: Wash the crosslinked hydrogel repeatedly with deionized water, then peel it off, and appropriately remove the excess hydrogel matrix to expose the highly ordered HAP ultra-long nanowires on the surface to obtain the final Que-AHNW / GH hydrogel product.

[0014] Further, in Step 1, the concentration of gelatin in the basic solution is 50-100 g / L, and the concentration of sodium hyaluronate is 20-30 g / L.

[0015] Further, in Step 2, the mass percentage of quercetin relative to the total mass of gelatin and sodium hyaluronate is 0.5-10%; the alkali solution is a sodium hydroxide solution with a concentration of 1-10 mol / L.

[0016] Further, in Step 3, the mass percentage of the HAP nanowire tape relative to the total mass of gelatin and sodium hyaluronate is 10-50%.

[0017] Further, the preparation method of the HAP nanowire tape is specifically as follows: Inject the HAP ultra-long nanowire slurry into absolute ethanol through a syringe with a rectangular needle orifice, and let it stand for 6 hours. Replace the ethanol every hour during this period to form a continuous and macroscopically highly ordered HAP nanowire tape; then, soak it in deionized water for 1 hour to remove residual inorganic salt impurities for standby.

[0018] Further, in step four, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 3 - 8 mg / mL, and the concentration of N-hydroxysuccinimide is 0.5 - 2 mg / mL.

[0019] Further, in step four, the gelation time is 1 - 3 hours, and the chemical cross-linking time is 24 - 36 hours.

[0020] The present invention adopts the above technical solutions. Compared with the prior art, it has the following technical effects:

[0021] After implanting the quercetin (Quercetin)-loaded ordered hydroxyapatite ultra-long nanowire (HAP) / gelatin-hyaluronic acid (Que-AHNW / GH) hydrogel composite of the present invention into the rotator cuff injury repair model of osteoporotic rats for 8 weeks, the senescent cells positive for γ-H2AX were significantly cleared. The biomechanical strength of the supraspinatus tendon-humerus complex was significantly improved: the ultimate load of the direct suture control group was 11.06 ± 1.17 N, while the ultimate load of the Que-AHNW / GH implantation group was 28.04 ± 1.88 N, an increase of about 150%.

[0022] In summary, the present invention provides a quercetin (Quercetin)-loaded ordered hydroxyapatite ultra-long nanowire (AHNW) / gelatin-hyaluronic acid (GH) hydrogel composite. By remodeling the senescent microenvironment, it improves the repair ability of bone marrow-derived mesenchymal stem cells (BMSCs) and tendon-derived stem cells (TDSCs), thereby promoting tendon-bone healing and improving the integration effect, providing a new treatment strategy for patients with rotator cuff injury, especially in cases accompanied by osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the preparation of the quercetin-loaded ordered hydroxyapatite ultra-long nanowire / gelatin-hyaluronic acid hydrogel (Que-AHNW / GH) of the present invention.

[0024] Figure 2 It is the scanning electron microscope (SEM) photograph and energy dispersive spectrum (EDS) element distribution of Que-AHNW / GH.

[0025] Figure 3Results of the order analysis of Que-AHNW / GH.

[0026] Figure 4 Fourier transform infrared spectrum of Que-AHNW / GH.

[0027] Figure 5 Shows the γ-H2AX immunofluorescence staining results 8 weeks after the repair of rotator cuff injury in osteoporotic rats implanted with Que-AHNW / GH.

[0028] Figure 6 Shows the biomechanical test results of the supraspinatus tendon-humerus complex 8 weeks after the repair of rotator cuff injury in osteoporotic rats implanted with Que-AHNW / GH; ***P<0.001. Specific implementation manner

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not intended to limit the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0030] Example 1

[0031] This example provides a preparation method of a hydrogel for remodeling the aging microenvironment to promote the repair of rotator cuff injury, specifically including:

[0032] 1. Synthesize hydroxyapatite (HAP) ultra-long nanowires by the calcium oleate precursor solvothermal method

[0033] Mix 135 mL of deionized water, 60 mL of methanol and 105 mL of oleic acid under mechanical stirring, then add 150 mL of sodium hydroxide solution with a concentration of 1.75 mol / L, and stir for 30 minutes to ensure that the sodium hydroxide is fully dissolved to form a strong alkaline solution; then slowly add 120 mL of calcium chloride solution with a concentration of 0.25 mol / L and continue to stir for 30 minutes; subsequently add 180 mL of sodium dihydrogen phosphate dihydrate solution with a concentration of 0.33 mol / L and continue to stir for 30 minutes; transfer the uniformly mixed solution to a 1 L polytetrafluoroethylene-lined stainless steel shell autoclave, ensure that the container is completely sealed to prevent material leakage during the reaction, keep it at 185 °C for 26 hours, during which a solvothermal reaction occurs to promote the growth of HAP ultra-long nanowires; after the reaction, turn off the heating source and cool the autoclave to room temperature, separate the yellow viscous supernatant and the bottom white precipitate, and the HAP ultra-long nanowires mainly exist in the white precipitate; obtain a uniform HAP ultra-long nanowire slurry by mixing the white precipitate and the supernatant in a volume ratio of 4:1 and stirring at 65 °C for 2 hours.

[0034] Inject 5 mL of HAP ultra-long nanowire slurry (concentration: 8 wt.%) into absolute ethanol through a syringe. During this process, the HAP ultra-long nanowires self-assemble into a strip-shaped ordered structure, namely a HAP nanowire band. The needle orifice of the syringe used is rectangular, with a length of 15 mm and a width of 1 mm.

[0035] Let the injected sample stand for 6 hours to ensure that the HAP ultra-long nanowires are completely assembled in ethanol to form a continuous and multi-scale (nano-scale - micro-scale - macro-scale) ordered ribbon, and to ensure that ethanol can penetrate into it, thereby removing impurities such as oleic acid (salt) and mediating the ordered assembly of HAP ultra-long nanowires. It should be noted that the ethanol is changed every hour during this process to ensure the high purity and highly ordered structure of the final product. Then, soak it in deionized water for 1 hour to remove residual inorganic salt impurities for standby.

[0036] 2. Preparation of Que-AHNW / GH

[0037] 2.1 Preparation of the basic solution:

[0038] Dissolve 1.000 g of gelatin and 0.300 g of sodium hyaluronate in 10 mL of deionized water, and stir magnetically at 70 °C until completely dissolved into a homogeneous solution.

[0039] 2.2 Addition of quercetin:

[0040] Accurately weigh the corresponding amount of quercetin according to different quercetin loading amounts required (0.5%, 1%, 2.5%, 5% and 10% respectively, relative to the total mass of gelatin and hyaluronic acid). Add the quercetin to the above-mentioned mixed solution of gelatin and sodium hyaluronate, and adjust the pH to 8 - 11 using an alkaline solution, and stir until the quercetin is completely dissolved.

[0041] As a preferred example, add 13 mg of quercetin and 50 μL of a sodium hydroxide solution with a concentration of 5 mol / L to the mixed solution of gelatin and sodium hyaluronate, and stir evenly. At this time, the color of the solution changes to light brown. The loading amount of quercetin is 1% (relative to the total mass of gelatin and hyaluronic acid).

[0042] 2.3 Infiltration treatment of nanowires

[0043] Completely immerse AHNW (mass percentage relative to the total mass of gelatin and sodium hyaluronate is 10 - 50%, preferably 20 - 30%) in the above-mentioned basic solution containing quercetin, and place it in an oven at 95 °C for 12 hours with sealing and heat preservation to ensure complete infiltration.

[0044] 2.4 Gelation and crosslinking

[0045] The heat-treated solution was placed in a 4 °C refrigerator for 1 hour to trigger in-situ gelation of the collagen inside it, forming a preliminary composite hydrogel structure. The formed hydrogel was placed in an aqueous solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 0.07668 g / 20 mL) and N-hydroxysuccinimide (NHS, 0.0184 g / 20 mL), and soaked at room temperature for 24 hours for chemical crosslinking to enhance the stability and durability of the hydrogel.

[0046] 2.5 Post-treatment

[0047] The crosslinked hydrogel was washed repeatedly with deionized water until the filtrate was neutral to remove unreacted EDC and NHS. The crosslinked hydrogel was peeled off, and the excess hydrogel matrix was appropriately removed to expose the highly ordered HAP ultra-long nanowires on the surface, obtaining the final Que-AHNW / GH hydrogel product.

[0048] Example 2

[0049] The obtained Que-AHNW / GH hydrogel (with a quercetin loading of 1% and the mass percentage of AHNW relative to the total mass of gelatin and sodium hyaluronate being 21%) was characterized and applied to verify its biosafety, structural stability, and effectiveness in the repair of osteoporotic rotator cuff injuries.

[0050] 2.1 Energy Dispersive X-ray Spectroscopy (EDS) Analysis

[0051] As Figure 2 shown in B-F: The results of the elemental surface distribution of the energy dispersive spectrum of the quercetin-loaded ultra-long hydroxyapatite nanowire / gelatin-hyaluronic acid (Que-AHNW / GH) composite hydrogel showed that the calcium (Ca) and phosphorus (P) of the HAP ultra-long nanowires, the nitrogen (N) of gelatin and sodium hyaluronate, and the oxygen (O) element commonly contained in all components were evenly distributed, verifying the successful composite and uniform distribution of these components.

[0052] 2.2 Order Degree Analysis of Que-AHNW / GH

[0053] The results were as Figure 2 shown in A and Figure 3 as follows: Scanning electron microscope (SEM) photos combined with their two-dimensional fast Fourier transform (FFT) and orientation analysis confirmed the highly oriented arrangement of HAP ultra-long nanowires in the hydrogel.

[0054] 2.3 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0055] The results were as Figure 4It is described that Fourier transform infrared spectroscopy (FTIR) shows the characteristic peaks of HAP ultra-long nanowires (1027 cm -1 : PO 4 3 stretching vibration; 603 cm -1 and 561 cm -1 : bending vibration of O-P-O), as well as the characteristic peaks of the gelatin / hyaluronic acid matrix (2922 cm -1 : -CH 3 and -CH 2 groups; 1556 cm -1 : amino group), confirming the successful compounding of these components.

[0056] 2.4 Animal experiments

[0057] SD rats were induced to anesthesia, and bilateral ovariectomy was performed on female SD rats (body weight 250 ± 30 g). After the operation, pain relief and anti-infection treatments were routinely carried out. After 13 weeks, a rotator cuff injury repair model was further constructed. The method for constructing the rotator cuff injury repair model is as follows: SD rats were induced to anesthesia, and the hair on both shoulders was shaved after anesthesia. The skin and deltoid muscle were incised successively to expose the insertion point of the supraspinatus tendon. After cutting the supraspinatus tendon, the footprint area of the humerus was polished. At this time, the study was divided into two groups. In the control group, the cut supraspinatus tendon was directly sutured to the footprint area of the humerus. Que-AHNW / GH was placed between the supraspinatus tendon and the humerus before suture. Holding an electric drill with a drill bit diameter of 0.5 mm, a bone tunnel was drilled from the proximal end to the distal end in the footprint area of the humerus. Then, the cut supraspinatus tendon was sutured to the footprint area of the humerus using a 4-0 suture. After the operation, histopathological detection and biomechanical detection were routinely carried out.

[0058] The results are as Figure 5 shown: Compared with the control group with direct suture, the implantation of Que-AHNW / GH significantly reduced the number of senescent cells positive for γ-H2AX.

[0059] The results are as Figure 6 shown: The biomechanical strength of the supraspinatus tendon-humerus complex was significantly improved 8 weeks after the implantation of Que-AHNW / GH. The ultimate load of the supraspinatus tendon-humerus complex in the direct suture control group was only 11.06 ± 1.17 N, while that in the Que-AHNW / GH implantation group was 28.04 ± 1.88 N, an increase of about 150%.

[0060] As mentioned above, the above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be able to realize that all the equivalent replacements and obvious changes made by using the content of the specification and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. A hydrogel for reshaping the aging microenvironment and promoting the repair of rotator cuff injury, characterized in that: It includes ordered hydroxyapatite ultra-long nanowire / gelatin-hyaluronic acid composite hydrogel and quercetin loaded therein.

2. A method for preparing a hydrogel for reshaping the aging microenvironment and promoting the repair of rotator cuff injuries as claimed in claim 1, characterized in that: The steps include: Step 1, completely dissolving gelatin and sodium hyaluronate in deionized water to obtain a uniform basic solution; Step 2, adding quercetin to the basic solution, adjusting the pH to 8-11 with an alkali solution, and stirring to completely dissolve the quercetin to obtain a basic solution containing quercetin; Step 3, completely immersing the HAP nanowire tape in the basic solution containing quercetin; Step 4, placing the solution obtained in step 3 at 0-4°C to gelate it and form a preliminary composite hydrogel structure; then placing the formed hydrogel in an aqueous solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to perform chemical crosslinking at room temperature; Step 5: The cross-linked hydrogel is repeatedly washed with deionized water, then peeled off and the excess hydrogel matrix is ​​appropriately removed to expose the highly ordered HAP ultra-long nanowires on the surface to obtain the final Que-AHNW / GH hydrogel product.

3. The preparation method according to claim 2, characterized in that: In step 1, the concentration of gelatin in the base solution is 50-100 g / L, and the concentration of sodium hyaluronate is 20-30 g / L.

4. The preparation method according to claim 2, characterized in that: In step 2, the mass percentage of quercetin relative to the total mass of gelatin and sodium hyaluronate is 0.5-10%; the alkali solution is a sodium hydroxide solution with a concentration of 1-10 mol / L.

5. The preparation method according to claim 2, characterized in that: In step 3, the mass percentage of the HAP nanowire ribbon relative to the total mass of gelatin and sodium hyaluronate is 10-50%.

6. The preparation method according to claim 2, characterized in that: The preparation method of the HAP nanowire tape is specifically as follows: injecting the HAP ultra-long nanowire slurry into anhydrous ethanol through a syringe with a rectangular needle port, leaving it to stand for 6 hours, during which the ethanol is replaced every hour, to form a continuous and macroscopically highly ordered HAP nanowire tape; then, soaking it in deionized water for 1 hour to remove residual inorganic salt impurities, and setting it aside.

7. The preparation method according to claim 2, characterized in that: In step 4, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 3-8 mg / mL, and the concentration of the N-hydroxysuccinimide is 0.5-2 mg / mL.

8. The preparation method according to claim 2, characterized in that: In step 4, the gelation time is 1-3 hours, and the chemical cross-linking time is 24-36 hours.

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