Hydrogel microspheres with high elasticity and superlubricity, preparation method and application thereof

Abstract

The application provides a hydrogel microsphere with high elasticity and super-lubricating performance, and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The application uniformly disperses the laponite nanosheet in the GelMA base section in an intercalation structure through electrostatic / hydrogen bond interaction, and enhances the elasticity of the microsphere.Meanwhile, the multilayer structure of the laponite creates 'living and movable' physical restrictions by filling the interlayer space, thereby limiting the free movement of water molecules.Subsequently, PMPC is grafted to the surface of the microsphere through surface graft polymerization technology, and through the 'active and dynamic' charge dipole interaction with water molecules, a hydrated lubricating layer is formed, and finally the construction of the degradable elastic and lubricating hydrogel microsphere is realized.The hydrogel microsphere has excellent performance in reducing cartilage friction and slowing the progression of osteoarthritis, and paves the way for the application of hydrogels in the fields of medicine, medical treatment and mechanical lubrication.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a hydrogel microsphere with both high elasticity and superlubricity, its preparation method, and its application. Background Technology

[0002] Traditional hydrogel materials have inherent mechanical limitations, such as easy deformation due to low strength and brittleness despite poor elasticity, which poses a significant obstacle to their widespread application. In the process of modifying hydrogels, researchers face a common challenge: how to strike a balance between rigidity and toughness, and between elasticity and lubrication.

[0003] Previous studies have shown that balancing the "toughness" and "stiffness" of hydrogels can be achieved through various methods, such as constructing multi-network hydrogel systems, crystal domain crosslinking, high-density dynamic bonding systems, or host-guest interactions. However, integrating "elasticity" and "lubrication" properties into hydrogel structures has rarely been successfully achieved. This gap mainly stems from the fact that enhancing "elasticity" largely depends on strengthening the entanglement of polymer chains and the intermolecular forces between chain segments, thereby reducing the likelihood of gel breakage during deformation through energy dissipation, resulting in high inter-chain friction and low water content. Conversely, "lubrication" properties depend on the polymer molecular chains adsorbing a large number of water molecules to promote hydration lubrication. Strong binding with water molecules inevitably weakens the intermolecular forces between polymer chains, leading to chain expansion, softening, increased deformation, and structural fragility. Therefore, from both a structural and theoretical perspective, the "dehydration" that enhances elasticity and the "hydration" that lubricates in hydrogels are essentially a contradictory relationship between "elasticity" and "lubrication."

[0004] The construction of elastic hydrogels mainly relies on methods such as increasing chain entanglement, inducing crystallization, and enhancing intermolecular forces. For example, Suo's team increased the entanglement density of long-chain polymers under low water content conditions. This dense entanglement network promotes tension transfer within the polymer chains, thereby preventing chain breakage caused by stress concentration and achieving significant gel elasticity. Qiu's team designed polyvinyl alcohol hydrogels, achieving high-density crosslinking within crystalline domains by reducing hydration and enhancing the salting-out effect. The dissipation of non-covalent energy in these regions significantly contributes to the observed elastic enhancement. Meanwhile, Yu's team created layered hydrogels through the careful assembly of nanostructure building blocks and a two-stage in-situ polymerization process. This unique structure, characterized by highly coiled interconnections between layers and an in-situ crosslinked polymer network, endows the hydrogel with significant elastic strain capacity. Therefore, the main strategies for enhancing elasticity generally include increasing interchain density and friction, enhancing hydrophobic elasticity, and reducing the thickness of the water layer and the total water content, usually at the expense of lubrication performance.

[0005] While existing methods have begun to attempt to improve the mechanical strength of hydrogel microspheres while maintaining a low coefficient of friction, the results achieved by current research are limited. They fail to achieve a good balance between high elasticity and low friction in hydrogel microspheres, thus affecting their effectiveness in bone repair.

[0006] For example, it has been reported that the use of nano-lithium saponite can enhance the mechanical properties of hydrogels; however, the improvement is very limited, and it is difficult to significantly increase the elastic modulus of composite hydrogels. At the same time, due to the trade-off of elasticity, the lubrication properties of hydrogels cannot achieve ultra-low friction.

[0007] Therefore, how to provide a method that has high elastic modulus and low coefficient of friction, and can simultaneously achieve high elasticity and super-lubricity of hydrogel microspheres, effectively resolve the contradiction between "elasticity" and "lubricity" of hydrogel microspheres, and better apply them to the field of bone repair, has become an urgent technical problem to be solved at this stage. Summary of the Invention

[0008] The present invention aims to solve the aforementioned technical problems, thereby providing a hydrogel microsphere with both high elasticity and superlubricity, its preparation method, and its application. The technical objective of this invention is to provide a hydrogel microsphere possessing a high elastic modulus and a low coefficient of friction, effectively integrating the contradictory properties of "elasticity" and "lubricity," thus achieving both high elasticity and superlubricity for better application in bone injury repair.

[0009] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention first provides a method for preparing hydrogel microspheres with both high elasticity and superlubricating properties, comprising the following steps:

[0011] (1) Synthesis of photocrosslinked GelMA monomer solution;

[0012] (2) Lithium saponite nanosheets were mixed with the GelMA monomer solution obtained in step (1) and lithium saponite crosslinked hybrid hydrogel microspheres were prepared by microfluidic technology.

[0013] (3) By using the surface grafting method, 2-methacryloyloxyethyl phosphocholine and acrylamide are used to modify the hydrogel microspheres obtained in step (2) to obtain hydrogel microspheres with both high elasticity and super lubricity.

[0014] The simultaneous existence of "high elasticity" and "high lubricity" is a unique characteristic of living organisms because they possess tunable biological activity. However, in inanimate hydrogels, the enhanced elasticity from "dehydration" and the lubricating properties from "hydration" inherently create a contradictory relationship between "elasticity" and "lubricity." This invention successfully develops a "living" biodegradable hydrogel microsphere using the aforementioned method to address this challenge. This microsphere is internally reinforced by disc-shaped crystalline lithium saponite nanosheets and externally lubricated by in-situ formed molecular brushes, thus resolving the paradox of mutually exclusive "elasticity" and "lubricity" in inanimate hydrogels.

[0015] This invention employs microfluidics and surface grafting polymerization techniques to develop biodegradable hydrogel microspheres with internal reinforcement and external lubrication. These microspheres aim to resolve the challenging paradox of the mutual exclusion of "elasticity" and "lubrication" by minimizing hydration permeation and preventing water loss. First, lithium saponite nanosheets are uniformly dispersed in an intercalated structure within GelMA segments through strong electrostatic / hydrogen bonding interactions. Polymer chains grow near the sheets via photo-initiated free radical polymerization, resulting in a uniform tension distribution between network segments and enhancing the elasticity of the microspheres. Simultaneously, the multilayer structure of lithium saponite creates "living and mobile" physical confinement by filling the interlayer spaces, thereby restricting the free movement of water molecules and preventing them from permeating into the polymer framework. Subsequently, poly(2-methacryloyloxyethylphosphocholine) molecules are chemically grafted onto the microsphere surface via surface grafting polymerization. Through "active and dynamic" charged dipole interactions with water molecules, a hydrated lubricating layer is formed, ultimately achieving the construction of biodegradable elastic / lubricating hydrogel microspheres.

[0016] The lithium saponite nanosheets in this invention serve as "living" and "mobile" crosslinking points, providing physical confinement by occupying interlayer space, thereby preventing active water from penetrating into the polymer framework and maintaining substantial cohesion. Simultaneously, the "active and dynamic" communication between the molecular brush and water molecules via charged dipole interactions significantly enhances sustainable lubrication. This "living" design allows for controllable elastic modulus of the microspheres between 14 Pa and 4000 Pa, and adjustable coefficient of friction between 0.12 and 0.04. Further in vitro and in vivo experiments have confirmed the superior performance of the hydrogel microspheres of this invention in reducing cartilage friction and slowing the progression of osteoarthritis. Therefore, this novel synergistic design strategy solves the challenge of balancing "elasticity" and "lubrication," paving the way for the application of hydrogels in pharmaceuticals, medical applications, and mechanical lubrication.

[0017] In addition, as shown in the comparative examples of the present invention, the inventors attempted to replace methacrylated gelatin with methacrylated hyaluronic acid during the research process. As a result, it was found that the elastic modulus of the microspheres prepared under the system of the present invention was significantly lower, and thus it was impossible to obtain hydrogel microspheres with high elasticity and high lubricity.

[0018] Meanwhile, the inventors also attempted to prepare hydrogel microspheres by directly mixing and crosslinking the raw materials in the system of the present invention using a one-pot method. The results showed that the elastic modulus of the hydrogel microspheres prepared by the one-pot method was also significantly lower, and it was still impossible to obtain hydrogel microspheres with high elasticity and high lubricity.

[0019] Furthermore, the preparation method of the photocrosslinked GelMA monomer in step (1) is to react the gelatin solution with methacrylic anhydride and mix it with a photoinitiator to obtain the photocrosslinked GelMA monomer solution.

[0020] Furthermore, in step (2), the weight ratio of lithium saponite nanosheets to GelMA monomer is 5:1 to 5. Experiments have shown that reducing this weight ratio prevents the microspheres from gaining more elasticity. Increasing the ratio fails to meet the viscosity requirements of the microfluidic liquid.

[0021] Furthermore, in step (2), the diameter of the lithium saponite nanosheets is 20-50 nm and the thickness is 1-2 nm.

[0022] Furthermore, the concentration of the GelMA monomer solution in step (2) is 4–7 wt%.

[0023] Furthermore, in step (3), the weight ratio of 2-methacryloyloxyethyl phosphocholine, acrylamide and lithium saponite nanosheets is 0.5-2:0.5:1-5.

[0024] A second objective of this invention is to provide hydrogel microspheres prepared by the method described above, wherein the hydrogel microspheres possess both high elasticity and superlubricating properties.

[0025] Furthermore, the elastic modulus of the hydrogel microspheres is between 14 Pa and 4000 Pa, and the coefficient of friction is between 0.04 and 0.12.

[0026] A third objective of this invention is to provide applications of the hydrogel microspheres described above in the pharmaceutical, medical, and mechanical lubrication fields.

[0027] Furthermore, the hydrogel microspheres described above can be used in the preparation of medical materials or drugs for the treatment of osteoarthritis.

[0028] The beneficial effects of this invention are as follows:

[0029] (1) This invention obtains a biodegradable hydrogel microsphere with both high elasticity and super lubricity, which solves the contradiction between "elasticity" and "lubricity" in hydrogel microspheres and successfully realizes the construction of biodegradable elastic and lubricating hydrogel microspheres.

[0030] (2) The hydrogel microspheres obtained by the present invention have the significant effect that the elastic modulus is controllable between 14 Pa and 4000 Pa and the friction coefficient is adjustable between 0.12 and 0.04.

[0031] (3) The hydrogel microspheres of the present invention have excellent performance in reducing cartilage friction and slowing the progression of osteoarthritis. Attached Figure Description

[0032] Figure 1 For: (A) GelMA monomers 1 (A) 1H NMR spectrum; (B) Hydrated particle size of GL1 microspheres; (C) Bright-field microscopy image of GL1 microspheres; (D) XRD spectrum; (E) SEM image of lithium saponite (left) and GL1 microspheres; (F) EDS spectrum of GL1; (G) Rheological curve of hydrogel, rheological experiments were conducted at 25℃ with a constant frequency of 10 rad / s and a strain of 1%.

[0033] Figure 2 The results are as follows: (A) Optical image of hydrogel under pressure and schematic diagram of viscoelastic properties of microspheres; (B) Compression curve of GL hydrogel; (C) Compressive strength and (D) Cyclic compression curve; The compression test was carried out at a speed of 2 mm / min; (E) Molecular dynamics simulation of GelMA-Laponite composite material and (FH) results of intermolecular forces and (IK) number of hydrogen bonds; (L) Schematic diagram of compression simulation of GeMA-Laponite composite material and (MN) compression curve.

[0034] Figure 3 The following are: (A) XPS spectrum of GL1M0.5; (B) hydrogel expansion curve; (C) rheological curve of GL4M2; (D) schematic diagram of hydrogel microsphere lubrication test; (EH) friction coefficient of hydrogel microsphere under different parameter conditions and (IJ) corresponding substrate wear volume.

[0035] Figure 4 The following images are included: (A) Representative images of live / dead cell staining in microspheres co-cultured with bone marrow mesenchymal stem cells (BMSCs); and (BC) corresponding live cell counts; (D) Representative images of live / dead cell staining in microspheres co-cultured with chondrocytes; and (EF) corresponding live cell counts; (n=3, * and # indicate comparison with the control group and sham-operated group, respectively, p<0.05).

[0036] Figure 5The data are as follows: (A) Representative images of microspheres co-cultured with bone marrow mesenchymal stem cells stained with Alcian Blue and Safranin O, with (BC) related statistical data; (D) Expression level of type II collagen in bone marrow mesenchymal stem cells and representative immunofluorescence images; and (EF) corresponding fluorescence intensity; (G) qRT-PCR detection results of cartilage-specific genes Col II, Aggrecan and Sox9; (n=3, * indicates p<0.05 compared with the control group).

[0037] Figure 6 The results are as follows: (A) Application of microspheres in OA cartilage regeneration; the viscoelasticity and lubrication properties of microspheres effectively reduce cartilage friction and wear, while the dissociation of lithium saponite releases KGN, which has a significant effect on promoting cartilage regeneration; (B) Macroscopic assessment of knee joint specimens, showing representative views of the femoral condyle and tibial plateau of each experimental group, with cartilage defects or damage areas indicated by blue arrows; (CD) Outerbridge scores and relative cartilage defect areas of each experimental group; (n=3, * and # indicate comparison with the control group and sham surgery group, respectively, p<0.05).

[0038] Figure 7 The following are included: (A) X-ray images; AP: frontal view, LAT: lateral view; (B) KL score results for each group; (C) Micro-CT images; showing representative three-dimensional reconstructions and horizontal sections of the tibial side for each group; (D) Bone mineral density results for each group; (E) BV / TV results for each group; (n=3, * and # indicate comparison with the control group and sham surgery group, respectively, p<0.05).

[0039] Figure 8 The following are representative images of H&E staining, TB staining, Safranin O staining, and type II collagen immunohistochemistry in each group: (A) Quantitative analysis of MMP13, Collagen II, and gag positive cells; (B) n=3, * and # indicate comparison with the control group and sham-operated group, respectively, p<0.05).

[0040] Figure 9 Rheological curves of HAMA-Laponite hydrogel microspheres prepared after replacing the system in Comparative Example 1.

[0041] Figure 10 The rheological curves of the hydrogel microspheres prepared by the one-pot method in Comparative Example 2 are shown.

[0042] Figure 11This is a schematic diagram illustrating the design and application of the hydrogel microspheres of the present invention; (A) Degradable elastic and lubricating hydrogel microspheres are prepared using microfluidic technology. Lithium saponite nanodiscs, as multifunctional physical crosslinking agents and nano-reinforcing agents, are entangled with GelMA-based polymer chains through electrostatic and hydrogen bonding interactions, serving as physical crosslinking agents for the hydrogel microspheres; subsequently, PMPC molecules are chemically grafted onto the surface of the microspheres using surface grafting polymerization technology; (B) The polymerization process of monomers in an aqueous solution containing lithium saponite. During free radical polymerization, polymer chains initially grow from the surface of lithium saponite disks and gradually extend to the surfaces of other lithium saponite disks, forming an organic / inorganic polymer network bridged by nanodiscs; (C) A schematic diagram illustrating the lubrication and viscoelasticity of the microspheres. The heterogeneous charge distribution of lithium saponite is entangled with polymer chains through electrostatic and hydrogen bonding interactions, significantly improving the mechanical strength of the microspheres; in addition, the PMPC molecular brushes on the surface of the microspheres form a short-range ordered hydration layer through interaction with the charged dipoles of water, thereby achieving shear lubrication. Furthermore, the layered structure of lithium saponite acts as a physical barrier for molecular transport, restricting the free diffusion of water molecules. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0044] Example 1

[0045] I. Experimental Methods and Characterization

[0046] (I) Experimental Materials and Sources

[0047] The small molecule compound Kartogenin (KGN) was purchased from Med Chem Express (USA); reagents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), ammonium persulfate, 2-methacryloyloxyethylphosphocholine (MPC), methacrylic anhydride (MA), and acrylamide were prepared using raw materials provided by Aladdin; gelatin was purchased from Maclean's; and lithium saponite nanosheets were purchased from Nanocor.

[0048] (II) Preparation method of microspheres

[0049] (1) Synthesis of GelMA monomer

[0050] The synthesis method of GelMA monomer is as follows: 20 g of gelatin was dissolved in 200 mL of PBS. Then, 6 mL of MA was gradually introduced into the gelatin solution using a syringe pump (Lead Fluid, China). After reacting for 2 hours, the resulting GelMA solution was dialyzed with deionized water before lyophilization.

[0051] (2) Preparation of GL and GLK microspheres

[0052] Microspheres were prepared using microfluidic technology, and the detailed composition of each group of microspheres is shown in Table 1. In short, an aqueous hydrogel precursor solution (5 wt% GelMA, 0.5 wt% acrylamide, 0.5 wt% photoinitiator, a certain amount of lithium saponite and KGN) and a microfluidic oil phase (paraffin oil mixed with 5 wt% Span 80) were introduced into the inlet of a flow-focusing microfluidic device. At the intersection, pre-gel droplets formed through shear forces and hydrophobic interactions. Subsequently, the generated droplets were exposed to ultraviolet radiation to induce cross-linking of the microgel. The collected microspheres were then washed three times with acetone and deionized water to remove excess oil phase and other additives.

[0053] (3) Grafting MPC onto microspheres

[0054] 100 mg of microspheres were dissolved in 10 ml of distilled water. Then, 30 mg of ammonium persulfate and 100-300 mg of MPC were added sequentially to the solution at 60 °C. The resulting mixture was stirred for 8 hours under a nitrogen atmosphere. The solution was then centrifuged to remove impurities.

[0055] Table 1 Composition of hydrogel microspheres

[0056]

[0057] The raw material codes in the table above are as follows: GelMA is methacrylated gelatin, Acrylamide is acrylamide, Laponite is lithium soapstone, and MPC is 2-methacryloyloxyethyl phosphorylcholine.

[0058] (III) Material Characterization Methods

[0059] 1. Characterization of the appearance and morphology of microspheres

[0060] The surface morphology of the microspheres was characterized using scanning electron microscopy (SEM, Hitachi S-4800), and the same samples were analyzed by EDS (Thermo Scientific, USA) using SEM, with elemental surface chemistry evaluated. The chemical structure of the GelMA was determined using nuclear magnetic resonance (NMR, 400 MHz, Avance III, Germany). Furthermore, the morphology and particle size of the microspheres in aqueous solution were observed using phase-contrast optical microscopy (PCOM, Nikon, Japan). The elemental composition of the GLKM microspheres was analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Nexsa, US).

[0061] 2. Rheological property analysis

[0062] All rheological analyses were performed using an AR2000 rheometer (TA Instruments, New Castle, DE). Hydrogel tests employed a 40 mm steel cone and plate geometry with a 200 μm gap. Rheological experiments were conducted at a constant frequency of 10 rad / s and a strain of 1% at 25 °C. All rheological experiments were performed in triplicate.

[0063] 3. Compression test

[0064] Compression tests were conducted on hydrogel cylinders with a diameter of 10 mm and a height of 6 mm using an Instron 5542 mechanical testing machine equipped with a 100 N load cell, at a compression rate of 2 mm / min. At least three samples were tested under each condition, and the compressive stress-strain curves were recorded.

[0065] 4. X-ray diffraction (XRD)

[0066] Using wavelength The crystal structure of the sample was analyzed by X-ray diffraction (XRD, Rigaku MiniFlex 600, Japan) under Cu Kα radiation at 40 kV and 200 mA. The scanning range was 5°–60° (2θ), with a step size of 1° / min. The interplanar spacing of each diffraction plane was determined using Bragg's law.

[0067] 2dsinθ=nλ (1)

[0068] In the formula, λ is the wavelength of the copper anode source. θ is the diffraction angle of each refractive index diffraction surface.

[0069] 5. Tribological testing

[0070] Lubrication performance was measured using a universal material testing instrument (UMT-2, Bruker, Germany) in reciprocating mode (amplitude: 3 mm), as described in previous studies. All tribological experiments employed a ball-and-disc friction test mode, using Si3N4 balls and polytetrafluoroethylene (PTFE) as the upper and lower friction pairs. PBS was used as a buffer solution to ensure good stretching of the microspheres. Friction tests were conducted for 10 minutes at specific lubricant concentrations (5.0 mg mL⁻¹), different loads (1–3 N), and frequencies (0.5–3 Hz). The time versus coefficient of friction (COF) curves were recorded during the tests. After the friction tests, the surface roughness of the samples was observed using confocal phase shift-microxam-3d to evaluate wear performance.

[0071] 6. Expansion test

[0072] A 1 cm diameter spherical hydrogel, freeze-dried, was immersed in 3 ml of PBS solution. After a specified time, it was removed and weighed. The mass difference represents the amount of water absorbed by the dried hydrogel. At least three samples were tested.

[0073] 7. Molecular Dynamics (MD) Simulation

[0074] MD simulations were performed using GROMACS (version 2020.6), and structures of 100 repeating unit polymers and laponite were constructed in Materials Studio. The polymer and laponite structures were generated in Materials Studio software. The GelMA polymer was modeled using the Amber99sb force field, while the laponite structure was described using a generalized force field. First, the polymer repeating units were geometrically optimized using density functional theory calculations via the b3lyp / 6-31g(d) method, followed by RESP charge calculations using the Multiwfn program. Subsequently, the polymer structure was relaxed at 500 K using stochastic mechanics calculations. Eight randomly coiled chains were then inserted into a 10 × 10 nm cubic box centered on laponite. After energy minimization through thousands of steps, there was a 10 ns equilibrium period before water was added to dissolve the entire system. Under the NPT ensemble, the production run time was 100 ns, and non-equilibrium box deformation was performed at a rate of 1 nm / ns within the 10 ns period to measure mechanical properties. The temperature was controlled at 300 K using the Nose-Hoover method. Non-bonded interactions were handled using a 1.2 nm cutoff scheme, while long-range electrostatic interactions were treated using the Ewald method with a particle mesh and a 0.1 nm Fourier spacing. The LINCS algorithm was used to constrain covalent bonds involving hydrogen atoms.

[0075] 8. Cell experiments

[0076] 1) Cell Culture: Cell culture was performed using SD rat third and fourth generation bone marrow mesenchymal stem cells (BMSCs) and first generation chondrocytes. 10% bovine serum and 100 U / mL were added to DMEM / F-12 (Gibico, USA) medium. -1 Penicillin and 100g·mL -1 Streptomycin was used to prepare complete cell culture media. A Transwell co-culture system was used to introduce the hydrogel microspheres into the system. In this experiment, cells were seeded in the lower chamber, and hydrogel microspheres with different drug loadings were added to the upper chamber. Because the pore diameter on the Transwell membrane is 0.4 μm, the therapeutic drugs and degradation products of the hydrogel microspheres can pass through the pores into the lower chamber, thereby exerting their therapeutic effects.

[0077] 2) Biocompatibility assessment: Live / dead cell staining was performed (Beyotime, China). Bone marrow mesenchymal stem cells or chondrocytes were seeded into Transwell 12-well plates at a density of 1*10⁴ cells / well, with GM, GLM, and GLKM supplemented in the upper cavity. Live / dead cell staining was performed on days 1, 3, and 5. CCK-8 assay was performed (Beyotime, China): Cells were seeded into the lower cavity of Transwell 96-well plates at a density of 2×10⁴ cells / well. 3 Cells / well. Add drug-loaded hydrogel microspheres to the upper cavity following the steps described above. Perform CCK8 staining on days 1, 3, and 5 to analyze OD values.

[0078] 9. Evaluation of chondrocyte differentiation

[0079] To induce chondrocyte differentiation, 10 ng / mL TGF-β3 and 1% ITS were added to DMEM / F-12 to reduce FBS from 10% to 5%. Bone marrow mesenchymal stem cells were seeded at a density of 1 × 10⁴ cells per well in Transwell 12-well plates, and drug-loaded hydrogel microspheres were added to the upper cavity of the culture system. A control group was established by adding PBS, and a positive control group consisted of chondrocytes. The culture medium was changed every two to three days. The Transwell co-culture system allows for the sustained release of encapsulated drugs in the hydrogel microspheres. Bone marrow mesenchymal stem cells were induced for two weeks, and then evaluated using the following methods:

[0080] a) Alcian Blue Staining (Solarbio). Immerse in acidifying solution for 3 minutes, then add Alcian Blue staining solution and react for 30 minutes. Rinse the sample with running distilled water for 5 minutes and observe under a bright-field microscope. The intensity of the blue indicates the degree of cartilage differentiation. The deeper the blue, the more pronounced the cartilage differentiation.

[0081] b) Saffron O staining (Solarbio). Incubate with 4% paraformaldehyde for 10 minutes, then treat with Safranine O staining solution for 2 minutes. Rinse with distilled water for 1 minute and observe under a bright-field microscope. The intensity of the red color indicates the degree of cartilage differentiation. The deeper the red, the more pronounced the cartilage differentiation.

[0082] c) Immunofluorescence staining. Cells were fixed with 4% paraformaldehyde for 20 minutes, then treated with 0.2% Triton X-100 for 10 minutes, and then with 5% skim milk for 2 hours. They were then incubated with rat type II collagen antibody (1:200) and secondary antibody (1:500) at 4°C for 1 hour. The cytoskeleton and nuclei were stained, and the cells were observed under a laser scanning confocal microscope.

[0083] d) Quantitative reverse transcription polymerase chain reaction (qRT-PCR). RNA was extracted using the chloroform method and isopropanol, and reverse transcribed into cDNA using a reverse transcription kit (Takara, Japan). The cDNA was then amplified using a qRT-PCR kit (Takara, Japan). Finally, using GAPDH as a reference gene, the expression of type II collagen (Col II), Aggrecan, and SOX9 was calculated using the CT method. The primer sequences for these four genes are shown in Table 2 below.

[0084] Table 2

[0085]

[0086]

[0087] 10. Animal experiments

[0088] All animal experiments were approved by the Animal Research Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, approval number: SYXK 018-0027.

[0089] a) The establishment of a medial meniscus instability (DMM) osteoarthritis model in SD rats was conducted according to the guidelines formulated by the Animal Research Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Anesthesia was performed via intraperitoneal injection of 3% sodium pentobarbital (40 mg kg⁻¹). After successful anesthesia, the DMM model was surgically constructed, with the right knee joint selected as the experimental side. At weeks 2, 4, and 6 post-modeling, drug-loaded hydrogel microspheres were injected into the knee joint. Animals were divided into 6 groups: sham-operated group, control group, GelMA group, GM group, GLM group, and GLKM group. The injection volume was 200 μL; the sham-operated group did not receive further intra-articular injection. The control group received PBS at pH 7.4.

[0090] b) Radiographic evaluation: The knee joint was removed 8 weeks after modeling. The severity of knee osteoarthritis was assessed by radiographs using the Kellgren-Lawrence (KL) score. In addition, micro-CT was used to measure tibial bone mineral density (BMD) and subchondral bone volume fraction (BV / TV).

[0091] c) Gross Appearance Evaluation of the Knee Joint: To more intuitively investigate the therapeutic effect of GLKM on knee osteoarthritis, we evaluated the gross appearance of the knee joints of SD rats. After carefully removing surrounding muscles, ligaments, and meniscus tissue, the femoral condyle and tibial plateau were separated, and the articular cartilage was then assessed under direct visual inspection. The Outerbridge score was used to quantify the condition of the tibial plateau cartilage. Subsequently, the specimens were photographed, and the relative lesion area of ​​the tibial plateau was quantified using ImageJ software. This software measures the percentage of damaged cartilage area on the tibial plateau relative to the total area of ​​the tibial plateau.

[0092] d) Histological and immunohistochemical evaluation: Decalcified knee joint specimens were paraffin-embedded, sectioned, and stained sequentially with H&E, saffron O-fast green, and toluidine blue, followed by type II collagen immunohistochemical staining. The articular cartilage degeneration was then observed and evaluated under a bright-field microscope, and quantitative analysis was performed.

[0093] 11. Data Statistics and Analysis

[0094] All experimental data are expressed as mean ± standard deviation, and independent experiments were repeated at least three times. Statistical analysis was performed on the experimental data, and corresponding graphs were generated. Tukey's multiple comparison test was used to perform pairwise comparisons between multiple groups. p < 0.05 was considered statistically significant. For quantitative images, at least three images were randomly observed, and one representative image was selected for display.

[0095] II. Experimental Results and Discussion

[0096] 1. Preparation and characterization results of GL elastic microspheres

[0097] First, a photocrosslinked GelMA monomer was successfully synthesized. Its structure was determined through... 1 H NMR spectrum ( Figure 1 The presence of characteristic peaks in (A) was confirmed, particularly between 5.34 ppm and 5.65 ppm, corresponding to vinyl methacrylate of methacrylic anhydride. Subsequently, customizable inorganic lithium saponite crosslinked hybrid hydrogel microspheres were prepared using microfluidic technology. The microspheres in Table 1 are named according to their respective compositions, and the obtained microsphere size is approximately 230 μm. Figure 1 (Chinese BC).

[0098] like Figure 1As shown, lithium saponite nanosheets are artificially synthesized layered polyanionic nanosheets composed of silicate layers. They are disk-shaped with a diameter of approximately 20-50 nm and a thickness of 1-2 nm. These disks have a negatively charged surface and a positively charged edge, exhibiting a unique heterogeneous charge distribution and interlayer interactions. Due to their hydrophilicity and electrostatic interactions, lithium saponite can be dispersed in aqueous solutions as a monolayer sheet structure or form an intercalation structure similar to a "house of cards," thereby forming a tight bond with polymer chains.

[0099] To determine the interaction mechanism between lithium saponite and the polymer chains, XRD analysis was performed on microspheres GL1 and GL4 containing 1 wt% and 4 wt% lithium saponite, respectively. Figure 1 As shown in Figure D, pure lithium saponite exhibits characteristic diffraction peaks at 2θ = 6.05, 19.99, 26.30, 34.89, and 53.17, corresponding to the five crystal planes (001), (100), (005), (110), and (511), respectively. For GL1, no obvious lithium saponite peaks were observed, indicating that at low concentrations, lithium saponite detaches from its layered structure and is uniformly dispersed in the polymer network. In contrast, GL4, except for the absence of the (005) crystal plane, shows obvious lithium saponite peaks at 2θ = 19.51, 34.63, and 53.10.

[0100] Furthermore, calculations based on the Bragg equation revealed varying degrees of change in the interplanar distance. For example, the interplanar spacing d(100) increased from 0.085 nm to 0.126 nm. This is likely due to some lithium saponite particles infiltrating the polymer chains in a "house of cards" embedding manner, acting as physical crosslinking agents and providing physical crosslinking sites for the growth and connection of molecular chains. That is, the polymer chains grow on the disk surface, forming a chain network bridged by disk particles until they terminate by combining with free radicals of other chain segments. The extension and expansion of the interlayer polymer chains increase the interplanar distance. In addition, SEM / EDS analysis showed that the lithium saponite disks and GL1 microspheres exhibited a multilayered sheet-like structure, while the GL1 microspheres exhibited a porous honeycomb structure with no obvious sheet-like particle accumulation on the surface, indicating that lithium saponite is uniformly embedded in the GelMA-based polymer chains as crosslinking points.

[0101] To investigate the effect of lithium saponite concentration on the viscoelastic properties of GelMA-based networks, dynamic frequency sweep tests were conducted on cylindrical samples with a diameter of 40 mm and a thickness of 2 mm. Figure 1Figure G shows the changes in the elastic modulus (G′) and loss modulus (G″) of the hydrogels over time. It can be seen that the G′ values ​​for all hydrogels are greater than the G″ values, confirming the elastic behavior of the hydrogels. With increasing lithium saponite concentration, the G′ value significantly increases from 14 Pa to 4000 Pa, while the loss factor tanδ (tanδ = G′ / G″) decreases from 0.22 to 0.052, indicating a strong interaction between lithium saponite and GelMA, thereby enhancing the viscoelasticity of the gel network. Furthermore, the increase in G″ indicates higher viscous dissipation in the hydrogels and reversible chemical bonds.

[0102] To observe the viscoelasticity of microspheres from a macroscopic perspective, such as Figure 2 As shown in Figure A, spherical hydrogels with a diameter of 10 mm were fabricated, containing 2 wt% (for staining) and 4 wt% lithium saponite, namely GL2 and GL4 microspheres, and a certain pressure was applied in the vertical direction. It can be clearly seen from the figure that under almost the same pressure, GL2 is softer and deforms more than GL4, indicating that increasing the lithium saponite content effectively enhances the viscoelasticity of the hydrogel microspheres. Furthermore, after the external force is removed, the spherical structure of GL4 can rapidly self-recover.

[0103] In addition, the elastic properties of the microspheres were verified using a general-purpose testing machine. Figure 2 (BD). From the compressive stress-strain curve ( Figure 2 As can be seen from Figure B, the compressive strength of the hydrogel gradually increases with the increase of lithium saponite content. Among them, the GL4 hydrogel exhibits a compressive strength as high as 55 kPa at 40% strain. Furthermore, in the stress-strain cycle curve (… Figure 2 The near-overlapping of different cycle curves (CD) indicates low hysteresis. This demonstrates that GL4 effectively avoids stress concentration and energy dissipation under external pressure, exhibiting excellent fatigue resistance. This can be attributed to lithium saponite acting as a reversible non-covalent active center, providing abundant hydrogen bonding sites and electrostatic interactions with the polymer chains, increasing the number of crosslinking points and forming layered chain entanglements. Under the synergistic effect of hydrogen bonding / electrostatic interactions, the polymer segments are effectively fixed, preventing excessive slippage and breakage under pressure, thereby minimizing energy dissipation under load and effectively improving the mechanical properties of the hydrogel.

[0104] To further elucidate the interaction between lithium saponite and the GelMA polymer chains, molecular dynamics simulations were performed. For example... Figure 2As shown in Figure E, a monolayer consisting of 100 lithium saponite and GelMA molecules was constructed through calculation. An Amber99sb force field was used to simulate the GelMA polymer, while a universal force field was used to characterize the lithium saponite structure. It can be seen that after relaxation treatment, the GelMA polymer chains are uniformly dispersed in the aqueous environment, surrounded by abundant water molecules. However, the addition of lithium saponite significantly enhances the cohesive force within the GelMA polymer chains, leading to aggregation. Furthermore, with the extension of the contact time between GelMA and water molecules, the Coulomb force, van der Waals force, and number of hydrogen bonds between GelMA and water molecules begin to decrease and gradually stabilize, indicating that the GelMA-water configuration gradually transitions to an equilibrium state. Figure 2 (F).

[0105] When lithium saponite is introduced into the system, GelMA rapidly binds to it, exhibiting a rapid increase in Coulomb forces, van der Waals forces, and the number of hydrogen bonds. Figure 2 (middle GH). In addition, from Figure 2 As can be seen from the IK, the total interaction energy between the GelMA-lithium saponite mixture and water is lower than that of the pure dispersion, and the number of hydrogen bonds between them decreases from approximately 1000 initially to around 665, indicating that GelMA binds more readily to lithium saponite. These results show that in aqueous solution, GelMA polymer chains tend to aggregate on and around the surface of lithium saponite disks through strong electrostatic, Coulombic, and hydrogen bonding interactions, thus forming a denser polymer layer. This also indicates that during free radical polymerization, GelMA polymer chains initially grow from the surface of lithium saponite disks and gradually extend to the surface of other lithium saponite disks, forming a nanodisk-bridged organic / inorganic polymer network.

[0106] In addition, computer compression simulations were performed on GelMA-lithium saponite and the GelMA system. Figure 2 (LN). Specifically, eight chains were initially placed in a 10×10 nm cubic box centered on a gluconite with a random coil structure. After numerous energy minimization steps, there was a 10 ns equilibrium period before more than 24,000 water molecules were introduced to solvate the entire system. In the NPT system, the production time was extended by 100 ns. After this, the box was subjected to non-equilibrium stretching and compression at a rate of 1 nm / ns for 10 ns and 5 ns, respectively. Strain-stress simulation curves corresponding to these processes show that the mechanical properties of the GelMA network are significantly enhanced due to the presence of lithium saponite.

[0107] 2. Preparation and characterization of GLM elastic and lubricating microspheres

[0108] Hydrogels are polymeric materials composed of hydrophilic polymer networks. However, the hydration capacity and load-bearing capacity of traditional hydrogels are inversely proportional. Highly hydrated hydrogel materials are too soft and easily deformed during high-load shearing processes. Conversely, hydrogels with excellent mechanical properties have poor surface hydration properties, which hinders their lubrication capabilities.

[0109] To obtain GLM microspheres with elasticity and lubricity, GL microspheres were modified with 2-methacryloyloxyethyl phosphocholine (MPC). As an amphoteric polymer, poly(2-methacryloyloxyethyl phosphocholine) (PMPC) achieves lubrication by forming a short-range ordered hydration layer on its surface through strong interactions between its anionic and cationic groups and water. XPS spectroscopy (…) Figure 3 A) confirms the successful introduction of PMPC, with its characteristic peak located at 133.1 eV.

[0110] Typically, increased gel hydration leads to polymer chain segment swelling, thereby reducing its load-bearing capacity. We analyzed, through swelling tests, whether the introduction of PMPC resulted in extensive water permeation into the microspheres and subsequently into the polymer network. Figure 3 As shown in Figure B, the addition of PMPC enhances the water absorption capacity of the gel, while the increase in lithium saponite content reduces the swelling degree of the gel. This may be due to the following reasons: (1) Molecular simulation results show that the polymer chains tend to bind tightly with lithium saponite, thereby inhibiting water molecules from entering the polymer framework; (2) The layered structure of lithium saponite acts as a physical barrier for molecular transport, restricting the free migration of water molecules. Therefore, due to the inhibition of swelling, the elastic modulus of GL4M1 is similar to that of GL4, indicating that the addition of PMPC does not significantly reduce the mechanical strength of the gel. Figure 3 (C)

[0111] Friction experiments were conducted using a UMT-3 reciprocating friction testing apparatus to analyze the effect of PMPC on the lubrication performance of microspheres. Figure 3 As shown in Figure DH, the coefficient of friction (COF) of the microspheres was tested under different frequencies, loads (simulating the frequency and load of joints during human walking / running), and substrates. We found that under these test conditions, the COF of the GLM microspheres was lower than that of the GL. This is because the zwitterionic ions of PMPC lock in a large number of water molecules through charged dipole interactions, forming a stable hydrated lubricating layer. Furthermore, with increasing reciprocating friction frequency and load, the COF value of GLM was significantly lower than that of GL, which may be due to the combined effects of its lubrication mechanism, hydrodynamic lubrication, and boundary lubrication.

[0112] In addition, the surface morphology of the steel substrate after the friction test was observed using a profilometer. Figure 3Among the three phases (IL), GelMA microsphere solution exhibited the highest wear as a lubricant, while GLM showed the lowest wear. The results indicate that GLM possesses good elastic and lubricating properties.

[0113] 3. Assessment of in vitro cartilage regeneration capacity

[0114] Osteoarthritis (OA) is a common joint disease characterized by cartilage wear and tear. Its pathogenesis may be related to excessive mechanical load and insufficient joint lubrication. Mechanical overload leads to cartilage damage, induces the overexpression of catabolic enzymes, causes chondrocyte apoptosis and extracellular matrix degeneration, thereby increasing articular cartilage friction, further aggravating joint inflammation, and forming a vicious cycle.

[0115] To evaluate the feasibility of using the aforementioned GLM elastic and lubricating microspheres for OA treatment, the biocompatibility of the microspheres was assessed by co-culturing them with bone marrow mesenchymal stem cells (BMSCs) and chondrocytes using CCK-8 and live / dead staining methods. Notably, to synergistically promote cartilage repair and regeneration, we utilized the high specific surface area and double charge properties of lithium saponite to efficiently load Kartogenin (KGN) (in the form of GL4M2 microspheres) to prepare GLKM. KGN is a small molecule drug with pharmacological effects promoting MSC chondrocyte differentiation. Figure 4 As shown, all experimental groups exhibited positive effects on cell growth and proliferation, indicating that the above-mentioned biomaterials have good biocompatibility. Preliminary results from Alcian Blue and Safranin O staining showed ( Figure 5 GLKM (from AC) has the ability to promote chondrogenesis of bone marrow mesenchymal stem cells. Simultaneously, immunocytochemical analysis showed that the GLKM group had the highest Collagen II expression level. Figure 5 qRT-PCR results showed that the expression of Collagen II, Aggrecan, and SOX9 was also increased in the Laponite / KGN(LK) and GLKM groups. Figure 5 (G). These results can be explained by the sustained release of KGN, the synergistic interaction between cells and lithium saponite, and the dissociation of lithium saponite ions. Therefore, GLKM exhibits the best chondrogenic capacity because its 3D porous network enables sustained drug release.

[0116] 4. Evaluation of cartilage protection and regeneration in a rat model of osteoarthritis

[0117] Elastic and lubricating properties are widely present in nature and are crucial for the physiological function of biological interfaces, especially in load-bearing components such as joints, which have complex biomechanical environments. The stress buffering and friction reduction provided by the high elasticity and lubrication properties of articular cartilage are indispensable. An imbalance between the elasticity and lubrication of cartilage leads to stress concentration on the cartilage surface during mechanical loading, resulting in increased friction and wear, irreversible cartilage degeneration, and ultimately, osteoarthritis.

[0118] To verify the protective and repairing effects of the aforementioned elastic lubricating microspheres on articular cartilage in an osteoarthritis (OA) environment, a rat OA model based on medial meniscus instability (DMM) was established. Intra-articular injections were administered to the knee joints of rats at weeks 2 and 4 post-modeling, and all rats were euthanized at week 8 to further evaluate the treatment efficacy. Figure 6 (A) First, the gross appearance of the rat knee joint was evaluated to determine the protective effect of each group on the articular cartilage. For example... Figure 6 As shown in the BD results, the tibial plateau cartilage damage was significant in the control group, while the damage area and cartilage surface damage depth were reduced in the GLKM group. The Outerbridge score was 1.2±0.20 points, while that in the control group was 3.60±0.25 points, indicating that the cartilage damage was significantly improved in the GLKM group.

[0119] Radiological assessment ( Figure 7 The results (A / B) showed that the knee joint in the control group exhibited extensive osteophyte formation, subchondral bone destruction, significant narrowing of the joint space, and enhanced epiphyseal radioactivity, indicating successful OA modeling. However, after GLKM treatment, the knee joint showed mild joint space narrowing, minimal osteophyte formation, and significantly increased subchondral and epiphyseal bone density, indicating that GLKM has a good cartilage-protective effect.

[0120] Furthermore, Micro-CT showed similar results ( Figure 7 (CE). Compared with the control group (bone mineral density = 0.44 ± 0.05), the bone mineral density of the GLKM group was significantly increased to 0.68 ± 0.04. A similar trend was observed in BV / TV. The observed phenomena may be attributed to the elastic properties of GLKM, which contribute to stress buffering of articular cartilage. Furthermore, its lubricating properties help prevent frictional wear caused by an increased coefficient of friction on the cartilage surface. In addition, the synergistic effect of KGN promotes cartilage repair, ultimately leading to successful attenuation of cartilage degeneration.

[0121] Simultaneously, histological evaluation was performed to further assess the treatment efficacy of GLKM. H&E and toluidine blue staining (TB) were also conducted. Figure 8The results showed that the degree of cartilage damage in the GelMA group was comparable to that in the control group, with disruption of the entire cartilage fibrous structure and a decrease in chondrocyte count. However, in the GM, GLM, and GLKM groups, we observed reduced cartilage damage depth and an increased number of chondrocytes forming clumps. Safranin O / fast green and Col II immunofluorescence staining showed that the GLKM-treated group had the highest expression of glycosaminoglycans (GAG) and type II collagen. These results indicate that the elastic and lubricating properties of therapeutic microspheres play an important role in protecting cartilage.

[0122] In summary, GLKM demonstrates remarkable efficacy in promoting cartilage tissue repair, combining its elastic properties to buffer stress, its lubricating properties to reduce cartilage friction, and the synergistic effect of KGN in promoting cartilage formation.

[0123] III. Conclusion

[0124] This invention combines microfluidics with surface grafting polymerization to construct elastic lubricating hydrogel microspheres. The microspheres are internally cross-linked with inorganic lithium saponite nanosheets and externally grafted with PMPC. The heterogeneous charge distribution of lithium saponite enhances the elastic modulus of the microspheres by two orders of magnitude through strong electrostatic and hydrogen bonding interactions. The hydration effect of PMPC reduces the coefficient of friction (COF) of the elastic microspheres from 0.12 to 0.04. By utilizing the water-locking capacity of PMPC and the physical barrier effect of lithium saponite, the permeation and softening of the polymer chains by water molecules are effectively inhibited, resulting in a hydrogel microsphere that combines elasticity and lubricity.

[0125] In vitro experiments showed that the dissociation of lithium saponite and the sustained release of KGN synergistically promoted chondrogenesis in bone marrow mesenchymal stem cells. In a rat OA model, these elastic and lubricating hydrogel microspheres effectively slowed the progression of OA by providing hydration lubrication and mechanical support under shear forces, reducing frictional wear in the knee joint, and synergistically promoting cartilage regeneration. In conclusion, this type of elastic and lubricating microsphere holds promise for treating medical conditions related to mechanical and lubrication imbalances.

[0126] Comparative Example 1

[0127] Referring to the method in Example 1, the 5wt% GelMA in the GL4 hydrogel precursor solution formulation (5wt% GelMA, 0.5wt% Acrylamide, 0.5wt% photo-initiator, 4wt% Laponite, and KGN) was replaced with 5wt% HAMA, while keeping other conditions unchanged. Rheological curves of the resulting HAMA-Laponite microspheres were plotted, and the results are as follows: Figure 9 The results show that its elastic modulus is significantly smaller than that of GL4.

[0128] Comparative Example 2

[0129] Referring to the method in Example 1, GL4M2 hydrogel was prepared using a one-pot method. The specific method is as follows: 5 wt% GelMA, 0.5 wt% Acrylamide, 0.5 wt% photo-initiator, 4 wt% Laponite, and 2 wt% MPC were directly mixed uniformly and then subjected to photocrosslinking to obtain GL4M2 microspheres. The rheological curves were obtained as shown below. Figure 10 The results showed that its elastic modulus was significantly smaller than that of the GL4M2 hydrogel prepared in steps in Example 1.

Claims

1. A method for preparing hydrogel microspheres with both high elasticity and superlubricating properties, characterized in that, Includes the following steps: (1) Dissolve 20 g of gelatin in 200 mL of PBS. Then, use a syringe pump to gradually introduce 6 mL of methacrylic anhydride into the gelatin solution. After reacting for 2 hours, dialyze the resulting GelMA solution with deionized water before lyophilizing. (2) The aqueous hydrogel precursor solution and the microfluidic oil phase are introduced into the inlet of the flow focusing microfluidic device, respectively; at the intersection, pregel droplets are formed through shear force and hydrophobic interaction; the precursor solution consists of 5 wt% GelMA, 0.5 wt% acrylamide, 0.5 wt% photoinitiator LAP, 1, 2, 3, 4 or 5 wt% lithium saponite nanosheets and small molecule compound Kartogenin; the oil phase is obtained by mixing paraffin oil with 5 wt% Span 80; subsequently, the generated droplets are exposed to ultraviolet radiation to induce cross-linking of the microgel. The collected microspheres were then rinsed three times with acetone and deionized water to remove excess oil phase and other additives. (3) Dissolve 100 mg of microspheres in 10 ml of distilled water; Subsequently, at 60°C, 30 mg of ammonium persulfate and 100-300 mg of 2-methacryloyloxyethyl phosphocholine were added sequentially to the solution. The resulting mixture was stirred for 8 hours under a nitrogen atmosphere, and then the solution was centrifuged to remove impurities.

2. The method according to claim 1, characterized in that, In step (2), the diameter of the lithium saponite nanosheets is 20-50 nm and the thickness is 1-2 nm.

3. The hydrogel microspheres prepared by the method of claim 1 or 2, characterized in that, The hydrogel microspheres possess both high elasticity and super lubrication properties.

4. The hydrogel microspheres according to claim 3, characterized in that, The elastic modulus of the hydrogel microspheres is between 14 Pa and 4000 Pa, and the coefficient of friction is between 0.04 and 0.

12.

5. The application of the hydrogel microspheres prepared by the method of claim 1 or 2, or the hydrogel microspheres of claim 3 or 4, in the field of mechanical lubrication.

6. The application of the hydrogel microspheres prepared by the method of claim 1 or 2, or the hydrogel microspheres of claim 3 or 4, in the preparation of medical materials or drugs for the treatment of osteoarthritis.

Images