A piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles, and a preparation method and application thereof

Abstract

The application provides an injectable piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles and a preparation method and application thereof, and belongs to the technical field of repair materials.The application enhances the piezoelectric performance of ZnO by causing lattice distortion of ZnO through Mn metal doping and enhancing polarization, and improves the dispersibility of the Mn-ZnO in the hydrogel by performing surface amino-functionalization on the Mn-ZnO, promotes the combination of the Mn-ZnO nanoparticles and the hydrogel network, and improves the overall flexibility, thereby solving the problem of the mechanical flexibility reduction of the hydrogel caused by piezoelectric materials.The application starts from human bioelectricity, promotes cartilage repair through the effect of electric stimulation, and the prepared injectable piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles can be used for bone cartilage defect repair.

Description

Technical Field

[0001] This invention relates to the field of repair materials technology, and in particular to a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles, its preparation method, and its application. Background Technology

[0002] Osteochondrial injury is a prevalent but challenging condition to manage, with its incidence rising due to sports-related injuries and an aging population. Cartilage is avascular and nerveless, unable to obtain necessary nutrients and growth factors from the bloodstream, thus its intrinsic regenerative capacity is limited, and it typically cannot heal spontaneously. Furthermore, chondrocytes have poor regenerative capacity, making recovery difficult through normal tissue regeneration after injury, thus requiring external intervention. Current clinical strategies primarily focus on symptom relief with analgesics and anti-inflammatory drugs, without providing a cure. Although tissue engineering has been used to complement microfracture therapies for osteochondral repair, significant challenges remain in optimizing the delivery of bioactive factors and chemical modifications, and historically, insufficient attention has been paid to biomechanical factors.

[0003] Bioelectricity plays a fundamental role not only in neurology but also participates in various physiological processes. Collagen, the main protein in the extracellular matrix (ECM) of bone and cartilage cells, is inherently piezoelectric, with a piezoelectric coefficient ranging from 0.2 to 2 pC N. -1 During human activity, the internal structures of joints are compressed, leading to deformation caused by mechanical forces. Due to the piezoelectric properties of biological tissues, this deformation generates electrical charges. Cartilage responds to electrical stimulation, and various studies have reported the application of direct current stimulation to promote cartilage repair. Furthermore, electrically stimulated osteochondral implants have been shown to increase the content of cartilage-related proteins, and the piezoelectric properties of bone tissue have been well demonstrated.

[0004] Hydrogels are commonly used biomaterials in biomedical applications due to their softness, flexibility, and biocompatibility, as well as their ability to retain high water content, enabling them to achieve strong similarity to the extracellular matrix (ECM). Properties such as tunable biodegradability and bioadhesion make them excellent cell carriers / matrices and delivery vehicles for biomolecules and drugs, capable of retaining drugs for extended periods before delivery. Furthermore, through nanomaterial functionalization, hydrogels can respond to various stimuli, such as pH, temperature, magnetic fields, and chemical stimuli. However, due to this responsiveness, traditional and pristine hydrogels cannot provide stimulation on their own without additional functional components and cannot independently initiate processes such as cell regeneration, which require the action of endogenous bioelectricity. Moreover, most biomedical devices using pristine hydrogels as functional materials require a separate power source to operate, making the entire device rigid and bulky. Therefore, hydrogels lack self-powered capabilities and typically require external stimulation to initiate cell regeneration processes. In contrast, piezoelectric materials are another class of materials applicable to various engineering disciplines because of their unique ability to convert mechanical stress into electrical energy (self-powered potential). However, compared to hydrogels, which are more commonly used in biomedical applications, piezoelectric materials are generally more rigid and less flexible, and not all piezoelectric materials are biocompatible, making it difficult to use pristine piezoelectric materials in biomedical applications. This is especially true for piezoelectric hydrogels using synthetic polymers, where the introduction of additional piezoelectric materials inevitably affects their mechanical flexibility. Compared to flexible hydrogel matrices, most organic and inorganic piezoelectric materials are relatively rigid or brittle; the introduction of these materials affects the cross-linked network of the hydrogel, making it denser and less homogeneous due to the physical interactions between the hydrogel polymer chains and fillers. Increasing the content of piezoelectric elements correspondingly increases the density and inhomogeneity of the hydrogel network, leading to a decrease in mechanical flexibility. Summary of the Invention

[0005] The purpose of this invention is to provide an injectable piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles, its preparation method, and its application, thereby solving the problem of reduced mechanical flexibility of hydrogels caused by piezoelectric materials.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles, comprising the following steps:

[0008] Zinc salt alcohol solution was mixed with manganese salt aqueous solution for doping to obtain Mn-doped ZnO;

[0009] The Mn-doped ZnO was mixed with a silane modifier and an organic solvent for modification to obtain amino-functionalized Mn-ZnO.

[0010] Chondroitin sulfate, an oxidizing agent, and water are mixed to carry out an oxidation reaction, yielding oxidized chondroitin sulfate.

[0011] The oxychloride chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO, and PBS buffer were mixed and loaded to obtain a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles.

[0012] Preferably, the molar ratio of zinc salt in the zinc salt alcohol solution to manganese salt in the manganese salt aqueous solution is 8.74:0.07-0.7; the doping temperature is 160-220℃ and the time is 9-12h.

[0013] Preferably, the silane modifier includes (3-aminopropyl)triethoxysilane, 3-aminopropylmethyldiethoxysilane, aminopropyltrimethoxysilane, aminopropyltrifluoromethylsilane, 3-aminosilane, or TMS-AMP; the organic solvent includes n-hexane.

[0014] Preferably, the ratio of the amount of Mn-doped ZnO to the amount of silane modifier and organic solvent is 2.3g:30-45mL:75-85mL.

[0015] Preferably, the modification is carried out at a temperature of 60-70°C for 5-7 hours, and the modification is performed under light-protected conditions.

[0016] Preferably, the oxidant includes sodium periodate, hydrogen peroxide, or chlorine dioxide; the molar ratio of chondroitin sulfate to the oxidant is 1:1 to 1:5.

[0017] Preferably, the oxidation reaction is carried out at a temperature of 30–45°C for 8–12 hours.

[0018] Preferably, the mixing of chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO, and PBS buffer comprises: mixing chondroitin sulfate with PBS buffer to obtain a chondroitin sulfate solution; mixing carboxymethyl chitosan with PBS buffer to obtain a carboxymethyl chitosan solution; adding amino-functionalized Mn-ZnO to the chondroitin sulfate solution; and then adding the carboxymethyl chitosan solution for loading; the volume ratio of the chondroitin sulfate solution to the carboxymethyl chitosan solution is 1.1–1.5:1; the concentration of the carboxymethyl chitosan solution is 5–7 g / mL; the concentration of the chondroitin sulfate solution is 6–8 g / mL; the concentration of the amino-functionalized Mn-ZnO in the loading mixture is 0.1–0.6 wt%; and the loading temperature is room temperature for 2–3 hours.

[0019] This invention provides a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles prepared by the preparation method described above.

[0020] This invention provides the application of the piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles as described above in the preparation of osteochondral defect repair materials.

[0021] This invention provides a method for preparing piezoelectric hydrogels loaded with amino-functionalized Mn-ZnO nanoparticles. The method utilizes Mn metal doping to induce lattice distortion in ZnO, enhancing polarization and thus improving the piezoelectric properties of ZnO, resulting in biodegradable manganese-doped zinc oxide (Mn-ZnO) nanoparticles with enhanced piezoelectric effect. Furthermore, addressing the problem that Mn-ZnO nanoparticles are difficult to uniformly disperse in the hydrogel matrix due to their large weight and lack of interaction with polymer chains, this invention improves their dispersibility in the hydrogel by surface amino-functionalizing Mn-ZnO. Simultaneously, it enables the formation of chemical bonds with chondroitin sulfate (OCS) chains. In addition to the crosslinking between polymer chains, the nanoparticles also act as crosslinking sites during hydrogel formation. This interaction helps improve the uniform distribution of nanoparticles in the hydrogel matrix, thereby promoting the bonding of Mn-ZnO nanoparticles with the hydrogel network, improving overall flexibility, and solving the problem of reduced mechanical flexibility of hydrogels caused by piezoelectric materials. This invention starts from human bioelectricity and promotes cartilage repair through electrical stimulation. The prepared injectable piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles can be used for the repair of osteocartilage defects.

[0022] In the piezoelectric hydrogel of amino-functionalized Mn-ZnO nanoparticles prepared in this invention, chondroitin sulfate oxidized and carboxymethyl chitosan crosslink to form a Schiff base reaction, constituting a hydrogel network. Nanoparticles with amino-grafted surfaces can also react with chondroitin sulfate oxidized to form Schiff base crosslinks, participating in the hydrogel network and forming a unified whole. This invention uses chondroitin sulfate as one of the components of the hydrogel. Chondroitin sulfate (CS), as one of the three major components of cartilage, is beneficial for cartilage repair and possesses good biocompatibility and biodegradability. By oxidizing chondroitin sulfate to form aldehyde groups, a Schiff base reaction is formed between CS and the amino groups of carboxymethyl chitosan, resulting in dynamic chemical crosslinking and injectability. Chitosan, a natural polysaccharide, has excellent biocompatibility and biodegradability. ZnO was chosen as the piezoelectric material because its hexagonal wurtzite structure makes it a well-known piezoelectric semiconductor material with good biocompatibility and biodegradability. It is also low in cost and can be prepared through a simple hydrothermal synthesis reaction. Its piezoelectric properties are improved by doping with Mn, and its surface amino functionalization is combined with the hydrogel network to form crosslinking sites, which improves dispersibility and enhances overall flexibility. At the same time, ZnO has good antibacterial properties.

[0023] The piezoelectric hydrogel prepared in this invention combines the advantageous properties of both piezoelectric materials and hydrogels. Through its inherent piezoelectric properties, it can release a weak electric field, stimulating cell growth and promoting cartilage tissue repair without requiring an external power source, thus reducing side effects. Simultaneously, it possesses good biocompatibility and similar biomechanical properties, effectively replicating the mechanical properties of cartilage and integrating better with surrounding tissues. This piezoelectric hydrogel can promote cell proliferation and differentiation, as well as in vivo cartilage repair. Attached Figure Description

[0024] Figure 1 The FTIR spectra of CS and OCS in Example 1, and OCS / N, O-CMC in Comparative Example 2 and OCS / N, O-CMC in Comparative Example 3 are shown.

[0025] Figure 2 The FTIR spectra of APTES, Mn-ZnO and A(Mn-ZnO) in Example 1 are shown below.

[0026] Figure 3 The XRD patterns are of ZnO in Comparative Example 1, Mn-ZnO and A(Mn-ZnO) in Example 1;

[0027] Figure 4 TEM images of ZnO(a) in Comparative Example 1 and Mn-ZnO(b) in Example 1;

[0028] Figure 5The images are SEM images of Mn-ZnO in Example 1 at different magnifications, where (a) is 1 μm and (b) is 100 nm.

[0029] Figure 6 The image shows the EDS diagram of Mn-ZnO in Example 1;

[0030] Figure 7 SEM images of the OCS / N,O-CMC hydrogel without amino-functionalized Mn-ZnO in Comparative Example 2 at different magnifications;

[0031] Figure 8 SEM images of the CMCS / OCS@A(Mn-ZnO) (0.3%) hydrogel in Example 1 at different magnifications;

[0032] Figure 9 EDS image of CMCS / OCS@A(Mn-ZnO) (0.3%) hydrogel in Example 1;

[0033] Figure 10 The swelling ratios of different hydrogels in Examples 1-3 and Comparative Example 2 are shown in the graph.

[0034] Figure 11 The graph shows the degradation loss of different hydrogels in Examples 1-3 and Comparative Example 2;

[0035] Figure 12 The results show the live and dead staining of the hydrogels in Comparative Example 2 and Example 1. Detailed Implementation

[0036] In this invention, unless otherwise specified, the raw materials or reagents required for preparation are all commercially available products well known to those skilled in the art.

[0037] This invention provides a method for preparing a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles, comprising the following steps:

[0038] Zinc salt alcohol solution was mixed with manganese salt aqueous solution for doping to obtain Mn-doped ZnO;

[0039] The Mn-doped ZnO was mixed with a silane modifier and an organic solvent for modification to obtain amino-functionalized Mn-ZnO.

[0040] Chondroitin sulfate, an oxidizing agent, and water are mixed to carry out an oxidation reaction, yielding oxidized chondroitin sulfate.

[0041] The oxychloride chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO, and PBS buffer were mixed and loaded to obtain a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles.

[0042] This invention involves mixing a zinc salt alcohol solution with a manganese salt aqueous solution for doping to obtain Mn-doped ZnO.

[0043] In this invention, the zinc salt in the zinc salt alcohol solution is preferably Zn(NO3)2·6H2O, the solvent is preferably glycol, and the concentration of the zinc salt alcohol solution is preferably 0.017 g / mL; the manganese salt in the manganese salt aqueous solution is preferably Mn(NO3)2, and the mass concentration of the manganese salt aqueous solution is preferably 500 mg / mL.

[0044] In this invention, the molar ratio of zinc salt in the zinc salt alcohol solution to manganese salt in the manganese salt aqueous solution is preferably 8.74:0.07 to 0.7, more preferably 8.74:0.07 to 0.5, and even more preferably 8.74:0.07.

[0045] In this invention, a manganese salt aqueous solution is added to a zinc salt alcohol solution, and the mixture is stirred at 70°C for 20 minutes to obtain a homogeneous mixture. The stirring is carried out at 2°C / min. - Doping is performed by heating at a rate of 1.

[0046] In this invention, the doping temperature is preferably 160–220°C, more preferably 160–200°C, and the doping time is preferably 9–12 h, more preferably 10–11 h.

[0047] After the doping is completed, the present invention preferably centrifuges the obtained product at 7000 rpm·min. -1 After 8 min, the collected solid was washed with distilled water and ethanol in sequence, and dried in a vacuum oven at 60 °C for 5 h to obtain Mn-doped ZnO, denoted as Mn-ZnO.

[0048] After obtaining Mn-doped ZnO, the present invention mixes the Mn-doped ZnO with a silane modifier and an organic solvent for modification to obtain amino-functionalized Mn-ZnO.

[0049] In this invention, the silane modifier preferably includes (3-aminopropyl)triethoxysilane (APTES), GPTMS (3-aminopropylmethyldiethoxysilane), AMPTES (aminopropyltrimethoxysilane), APS (aminopropyltrifluoromethylsilane), 3-aminosilane, or TMS-AMP; the organic solvent preferably includes n-hexane.

[0050] In this invention, the preferred ratio of the amount of Mn-doped ZnO to the silane modifier and the organic solvent is 2.3g:30-45mL:75-85mL, and more preferably 2.3g:30-40mL:75-80mL.

[0051] In this invention, the silane modifier is preferably dissolved in an organic solvent, and Mn-ZnO is added for modification under stirring conditions; the modification temperature is preferably 60-70℃, more preferably 65℃; the modification time is preferably 5-7h, more preferably 6h, and the modification is preferably carried out under light-protected conditions.

[0052] After the modification is completed, the product is preferably washed sequentially with ethanol and water, then centrifuged and vacuum dried at 65°C to obtain amino-functionalized Mn-ZnO. This invention achieves subsequent binding with hydrogels through amino functionalization of the Mn-ZnO particle surface.

[0053] This invention involves mixing chondroitin sulfate, an oxidant, and water to carry out an oxidation reaction, thereby obtaining oxidized chondroitin sulfate.

[0054] In this invention, the oxidant preferably includes sodium periodate (NaIO4), hydrogen peroxide, or chlorine dioxide; the molar ratio of chondroitin sulfate to the oxidant is preferably 1:1 to 1:5, more preferably 1:2 to 4, and even more preferably 1:4.

[0055] In this invention, chondroitin sulfate (CS) is preferably dissolved in water and stirred with a magnetic stirrer to obtain a CS solution. Sodium periodate is then added to the CS solution and stirred in the dark to oxidize it.

[0056] In this invention, the temperature of the oxidation reaction is preferably 30-45°C, more preferably 36-40°C, and the time is preferably 8-12 hours, more preferably 8 hours.

[0057] After the oxidation reaction is completed, the present invention preferably adds ethylene glycol to the obtained product and continues stirring in the dark for 1 hour to terminate the reaction. The collected product mixture is placed in a dialysis bag (molecular weight cutoff MWCO: 3500 Da) that has been boiled thoroughly. Dialysis solution (deionized water) of 100 times the volume of the product mixture is poured into the dialysis device and dialyzed for 3 days, with the dialysis solution being replaced every 8 hours. After dialysis, the sample is freeze-dried in a freeze dryer for 36 hours. The powder sample (oxidized chondroitin sulfate) is collected, sealed and stored at 4°C.

[0058] The oxychloride chondroitin prepared in this invention has multiple aldehyde functional groups.

[0059] In this invention, chondroitin sulfate, carboxymethyl chitosan (CMCS), and PBS buffer are mixed with amino-functionalized Mn-ZnO and loaded to obtain a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles.

[0060] In this invention, the mixing of chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO, and PBS buffer preferably comprises: mixing chondroitin sulfate with PBS buffer to obtain a chondroitin sulfate solution; mixing carboxymethyl chitosan with PBS buffer to obtain a carboxymethyl chitosan solution; adding amino-functionalized Mn-ZnO to the chondroitin sulfate solution; and then adding the carboxymethyl chitosan solution for loading; the volume ratio of the chondroitin sulfate solution to the carboxymethyl chitosan solution is 1.1–1.5:1, more preferably 1.3–1.4:1; the concentration of the carboxymethyl chitosan solution is 5–7 g / mL, more preferably 6 g / mL; and the concentration of the chondroitin sulfate solution is 6–8 g / mL, more preferably 7 g / mL.

[0061] In this invention, the concentration of the amino-functionalized Mn-ZnO in the loaded mixture is preferably 0.1 to 0.6 wt%, more preferably 0.3 to 0.5 wt%.

[0062] In this invention, the temperature of the load is preferably room temperature, and the time is preferably 2 to 3 hours, more preferably 2 hours.

[0063] During the loading process, amino-functionalized Mn-ZnO particles first undergo a Schiff base reaction with chondroitin sulfate oxidized (aldehyde group), and then carboxymethyl chitosan solution is added to react with chondroitin sulfate oxidized (amino and aldehyde groups undergo a Schiff base reaction).

[0064] This invention provides a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles prepared by the preparation method described above.

[0065] This invention provides the application of the piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles described above in the preparation of bone and cartilage defect repair materials. This invention does not impose any particular limitation on the method of application; any method well-known in the art can be used.

[0066] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0067] Unless otherwise specified, the experimental and testing methods described below are conventional methods; unless otherwise specified, the reagents and raw materials described below are commercially available.

[0068] Example 1

[0069] 1) Weigh 2.5g (5mmol) of chondroitin sulfate CS powder and dissolve it in 80mL of deionized water. Stir with a magnetic stirrer to obtain a CS solution. Add 4.5g (20mmol) of NaIO4 to the CS solution and oxidize it by stirring in the dark at 36℃ for 8h. After the reaction is completed, add 5mL of ethylene glycol and continue stirring in the dark for 1h to terminate the reaction. Place the collected OCS solution in a boiled dialysis bag (MWCO: 3500Da). Pour 100 times the volume of the dialysis solution (deionized water) into the dialysis apparatus and dialyze for 3 days. Change the dialysis solution every 8h. After the dialysis is completed, freeze dry the sample for 36h. Collect the powdered sample, seal it and store it at 4℃ for later use. Oxidized chondroitin sulfate is obtained and denoted as OCS.

[0070] 2) Disperse 2.6 g Zn(NO3)2·6H2O in 150 mL diethylene glycol (DEG), add 25 μL of a 500 mg / mL Mn(NO3)2 (12.5 mg) aqueous solution to the above solution, stir at 70 °C for 20 min, and then add the resulting mixture at 2 °C·min under stirring. - 1. Heat the mixture to 160℃ and react for 10 hours. Centrifuge the resulting product at 7000 rpm. -1 After 8 min, the collected solid was washed with distilled water and ethanol in sequence, and dried in a vacuum oven at 60 °C for 5 h to obtain Mn-ZnO;

[0071] 3) Dissolve 30 mL of APTES in 75 mL of n-hexane, add 2.3 g of the Mn-ZnO prepared above, stir at 65 °C in the dark for 6 h, wash the obtained product with ethanol and water in sequence, then centrifuge and dry under vacuum at 65 °C to obtain amino-functionalized Mn-ZnO, denoted as A(Mn-ZnO).

[0072] 4) A 6 g / mL carboxymethyl chitosan solution was prepared using 0.41 mL PBS buffer (pH=7.4), and a 7 g / mL oxychondroitin sulfate solution was prepared using 0.59 mL PBS buffer (pH=7.4). 3 mg of amino-functionalized Mn-ZnO particles were added to the prepared oxychondroitin sulfate solution, followed by the prepared carboxymethyl chitosan solution. The concentration of amino-functionalized Mn-ZnO in the mixture was 0.3 wt%. The reaction was carried out at room temperature for 2 h to obtain a piezoelectric hydrogel loaded with A(Mn-ZnO) nanoparticles, denoted as CMCS / OCS@A(Mn-ZnO) (0.3%).

[0073] Example 2

[0074] The only difference from Example 1 is that in step 4), 1 mg of amino-functionalized Mn-ZnO particles were added to obtain CMCS / OCS@A(Mn-ZnO) (0.1%).

[0075] Example 3

[0076] The only difference from Example 1 is that in step 4), 5 mg of amino-functionalized Mn-ZnO particles were added to obtain CMCS / OCS@A(Mn-ZnO) (0.5%).

[0077] Comparative Example 1

[0078] The only difference from Example 1 is that, following the method of Example 1, bare ZnO is synthesized without adding Mn(NO3)2.

[0079] Comparative Example 2

[0080] The only difference from Example 1 is that A(Mn-ZnO) is not added, resulting in OCS / N,O-CMC hydrogel.

[0081] Comparative Example 3

[0082] Commercially available carboxymethyl chitosan (Macklin / Macklin carboxymethyl chitosan C902396-100g) was used as Comparative Example 3, denoted as N,O-CMC.

[0083] Characterization and performance testing

[0084] 1) Figure 1 The FTIR spectra of CS and OCS in Example 1, OCS / N,O-CMC in Comparative Example 2, and N,O-CMC in Comparative Example 3 are shown below. Figure 1 As shown, at 1732cm -1 The formation of aldehyde groups on both sides proves oxidation. (From 1630cm) -1 The disappearance of the imine structure (-CN-) peak and aldehyde signal detected at the site can reasonably be inferred as a Schiff base reaction between OCS and CMC.

[0085] Figure 2 The FTIR spectra of APTES, Mn-ZnO, and A(Mn-ZnO) in Example 1 are shown below; Figure 2 As shown, at 3424cm -1 The NH stretching vibration at 2917 and 2850 cm -1 The CH stretching vibration at the point indicates that amino groups were successfully grafted onto the surface of Mn-ZnO nanoparticles.

[0086] Figure 3 The XRD patterns are shown for ZnO in Comparative Example 1, Mn-ZnO and A(Mn-ZnO) in Example 1; from Figure 3As can be seen, the synthesized sample has a typical hexagonal wurtzite structure, confirming its composition as ZnO. Furthermore, no metallic Mn or oxide phases were detected, indicating that Mn doping into the ZnO lattice did not alter the ZnO phase structure. The amino-functionalized particles obtained through APTES also did not change the ZnO phase structure.

[0087] Figure 4 TEM images of ZnO(a) in Comparative Example 1 and Mn-ZnO(b) in Example 1; as shown Figure 4 As shown, undoped ZnO exhibits irregular rod-shaped crystal aggregates. TEM images of Mn-ZnO nanoclusters reveal a uniform particle distribution (approximately 140 nm) and spherical shapes with mesoporous structures. The nanoclusters are composed of smaller nanocrystals (7-9 nm). The porous structure of the nanoclusters can be understood through the spacing between primary nanocrystals and adjacent nanocrystals.

[0088] Figure 5 The images show SEM images of Mn-ZnO at different magnifications in Example 1, where (a) is 1 μm and (b) is 100 nm; Figure 5 As shown, ZnO doped with Mn exhibits a uniform particle distribution (approximately 140 nm) and a spherical shape with a mesoporous structure.

[0089] Figure 6 The image shows the EDS diagram of Mn-ZnO in Example 1, indicating that Mn, Zn, and O elements are evenly distributed.

[0090] Figure 7 SEM images of the OCS / N,O-CMC hydrogel without amino-functionalized Mn-ZnO in Comparative Example 2 at different magnifications; Figure 8 The images show SEM images of the CMCS / OCS@A(Mn-ZnO) (0.3%) hydrogel from Example 1 at different magnifications; Figures 7-8 It is known that the average pore size of the undoped hydrogel is about 160 μm, while the doping makes the hydrogel network denser, with an average pore size of about 10 μm. The overall structure of the hydrogel is more stable, which improves its mechanical properties.

[0091] Figure 9 The image shows the EDS spectrum of the CMCS / OCS@A(Mn-ZnO) (0.3%) hydrogel in Example 1. According to the EDS spectrum, the A(Mn-ZnO) nanoparticles are uniformly distributed in the hydrogel.

[0092] Figure 10 The swelling ratios of different hydrogels in Examples 1-3 and Comparative Example 2 are shown in the graph. Figure 11 The graph shows the degradation loss of different hydrogels in Examples 1-3 and Comparative Example 2;

[0093] Depend on Figures 10-11 It can be seen that as the proportion of doped nanoparticles increases, the swelling rate of the hydrogel decreases, the degradation loss of the hydrogel is reduced, and it becomes more stable.

[0094] Figure 12 To compare the live and dead cell staining results of the hydrogels in Example 2 and Example 1, the Beyotime calcein / PI cell viability and cytotoxicity assay kit was used. PI represents dead cells, and merge represents a fluorescent staining pattern of combined live and dead cells. Figure 12 The hydrogel doped with A(Mn-ZnO) nanoparticles exhibits good biocompatibility.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles for use as a material for repairing osteochondral defects, characterized in that, Includes the following steps: Zinc salt alcohol solution was mixed with manganese salt aqueous solution for doping to obtain Mn-doped ZnO; The Mn-doped ZnO was mixed with a silane modifier and an organic solvent for modification to obtain amino-functionalized Mn-ZnO. Chondroitin sulfate, an oxidizing agent, and water are mixed to carry out an oxidation reaction, yielding oxidized chondroitin sulfate. The oxidized chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO and PBS buffer were mixed and loaded to obtain a piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles. The molar ratio of zinc salt in the zinc salt alcohol solution to manganese salt in the manganese salt aqueous solution is 8.74:0.07~0.7; The doping temperature is 160~220 ℃, and the time is 9~12 h; The modification is performed at a temperature of 60-70 °C for 5-7 h. The mixing of chondroitin sulfate, carboxymethyl chitosan, amino-functionalized Mn-ZnO, and PBS buffer comprises: mixing chondroitin sulfate with PBS buffer to obtain a chondroitin sulfate solution; mixing carboxymethyl chitosan with PBS buffer to obtain a carboxymethyl chitosan solution; adding amino-functionalized Mn-ZnO to the chondroitin sulfate solution; and then adding the carboxymethyl chitosan solution for loading. The volume ratio of the chondroitin sulfate solution to the carboxymethyl chitosan solution is 1.1~1.5:1; the concentration of the carboxymethyl chitosan solution is 5~7 g / mL; the concentration of the chondroitin sulfate solution is 6~8 g / mL; the concentration of the aminofunctionalized Mn-ZnO in the loading mixture is 0.1~0.6 wt%; the loading temperature is room temperature, and the loading time is 2~3 h.

2. The preparation method according to claim 1, characterized in that, The silane modifier includes (3-aminopropyl)triethoxysilane, 3-aminopropylmethyldiethoxysilane, aminopropyltrimethoxysilane, aminopropyltrifluoromethylsilane, 3-aminosilane, or TMS-AMP; the organic solvent includes n-hexane.

3. The preparation method according to claim 1, characterized in that, The ratio of Mn-doped ZnO to silane modifier and organic solvent is 2.3 g: 30~45 mL: 75~85 mL.

4. The preparation method according to claim 2 or 3, characterized in that, The modification was carried out under light-protected conditions.

5. The preparation method according to claim 1, characterized in that, The oxidant includes sodium periodate, hydrogen peroxide, or chlorine dioxide; the molar ratio of chondroitin sulfate to the oxidant is 1:1 to 1:

5.

6. The preparation method according to claim 1 or 5, characterized in that, The oxidation reaction is carried out at a temperature of 30-45°C for 8-12 hours.

7. The piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the piezoelectric hydrogel loaded with amino-functionalized Mn-ZnO nanoparticles as described in claim 7 in the preparation of osteochondral defect repair materials.

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