A biomimetic mineralized hydrogel coating on a poly (arylene ether sulfone ketone) surface, a preparation method thereof and application in bone repair

Abstract

The application provides a kind of polyarylether sulfone ketone surface biomimetic mineralization hydrogel coating, its preparation method and application in bone repair.The biomimetic mineralization hydrogel coating is obtained by simulating body fluid mineralization of hydrogel coating, and apatite layer is formed in the interior and surface of hydrogel coating, the hydrogel coating includes polymer network and calcium phosphate oligomer uniformly dispersed therein, which is anchored to the surface of polyarylether sulfone ketone base.The introduction of calcium phosphate oligomer effectively solves the problem of inorganic phase agglomeration and improves the uniformity of mineralization.The mineralized hydrogel coating can significantly promote the expression of osteoblast MC3T3-E1 osteogenic activity and inhibit the differentiation of RAW264.7 cells into osteoclasts.The biomimetic mineralization hydrogel coating described in the application has dual biological activity of promoting bone formation and inhibiting bone resorption, and is suitable for surface modification of polyarylether ketone bone implants, and has wide application prospect in the field of osteoporotic bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomimetic mineralized hydrogel coating on the surface of polyarylether sulfone ketone, its preparation method, and its application in bone repair. Background Technology

[0002] Trauma, surgical resection, osteoporosis, and congenital bone defects can all lead to bone tissue loss. When the extent of bone loss exceeds the body's self-repair limit, bone repair materials must be used for filling and inducing regeneration. While autologous bone grafting, commonly used in clinical practice, has good osteogenesis results, it suffers from limitations such as limited donor bone sources, secondary trauma, infection risks, and difficulties in morphological matching, thus restricting its widespread application. Artificial bone repair materials mainly include medical-grade metallic materials (such as titanium alloys), bioceramic materials (such as hydroxyapatite and tricalcium phosphate), and medical-grade polymeric materials (such as polylactic acid and polyetheretherketone). Among these, poly(aryl ether sulfone ketone) (PPESK) has become an ideal matrix material for orthopedic and dental implants due to its excellent mechanical properties, chemical stability, and elastic modulus similar to human bone. However, PPESK materials have strong surface bioinertness, making it difficult to form a strong osseointegration with the host bone tissue after implantation, easily leading to implant loosening or even failure. Therefore, how to bioactively modify the surface of PPESK implants is a current hot topic and challenge in bone repair material research.

[0003] Surface coating technology is an effective means to improve the bioactivity of implants. In recent years, hydrogel coatings have attracted widespread attention due to their high water content, three-dimensional porous network structure, and physicochemical properties similar to the extracellular matrix. Hydrogels can provide a suitable microenvironment for cell adhesion, proliferation, and differentiation, and can also serve as sustained-release carriers for bioactive factors or drugs. However, traditional hydrogel coatings have poor mechanical properties and lack osteoinductive properties, making them unsuitable for bone defect repair when used alone. To address this, researchers have attempted to introduce inorganic minerals such as hydroxyapatite (HAP) into hydrogels to prepare organic-inorganic composite coatings. HAP is the main inorganic component of natural bone tissue and has good biocompatibility and osteoconductivity. However, simply blending HAP nanoparticles with polymers easily leads to inorganic phase aggregation, resulting in poor coating uniformity. Furthermore, the lack of strong chemical bonds between HAP and polymers leads to insufficient long-term stability. In addition, the calcium phosphate crystals formed by traditional biomimetic mineralization materials are mainly deposited on the material surface, making it difficult to achieve uniform mineralization within the coating, resulting in easy peeling of the mineralized coating and low mineralization efficiency. Summary of the Invention

[0004] This invention aims to address the problems of uneven mineralization, poor adhesion to the substrate, and insufficient bioactivity in existing hydrogel coatings on bone implant surfaces. It provides a biomimetic mineralized hydrogel coating for the surface of polyarylether sulfone ketone (PAS-K), its preparation method, and its application in bone repair. The biomimetic mineralized hydrogel coating is obtained by mineralizing a hydrogel coating using simulated body fluids, forming a uniform, dense, and highly crystalline apatite layer within and on the surface of the hydrogel coating. The hydrogel coating comprises an organic polymer network and uniformly dispersed calcium phosphate oligomers, fixed to the surface of a PAS-K substrate modified by plasma treatment and double bond grafting. This coating exhibits excellent mechanical stability, cell compatibility, bone formation promotion, and bone resorption inhibition activity, providing a novel strategy for modifying bone implant surfaces.

[0005] The technical solution of the present invention is as follows: A biomimetic mineralized hydrogel coating on the surface of polyarylether sulfone ketone (PPESK) is obtained by mineralizing the hydrogel coating through simulated body fluid, forming an apatite layer inside and on the surface of the hydrogel coating; the hydrogel coating includes an organic polymer network and calcium phosphate oligomers uniformly dispersed therein.

[0006] Furthermore, the calcium phosphate oligomer has a particle size of 1–2 nm and its molecular structure contains 2–3 Ca3(PO4)2 units.

[0007] Furthermore, the simulated body fluid is a simulated body fluid containing or not containing strontium.

[0008] Furthermore, the simulated body fluid is a strontium-containing simulated body fluid; even further, the strontium-containing simulated body fluid has the following formula: 213 mM Na + 7.5 mM K + 2.3 mM Mg 2+ 3.04 mM Ca 2+ 0.76 mM Sr 2+ 221.7 mM Cl - 6.3 mM HCO 3- 1.5 mM HPO4 2- and 0.8 mM SO4 2- The solution pH is 7.4.

[0009] Furthermore, the hydrogel coating is anchored to the surface of the polyarylethersulfone ketone substrate.

[0010] Furthermore, the substrate surface is treated with nitrogen plasma to graft amino groups, which are then reacted with acrylic acid to introduce carbon-carbon double bonds. The coating and the substrate are then covalently cross-linked through these double bonds with the acrylamide monomer to achieve a strong bond.

[0011] Furthermore, the organic polymer network and the calcium phosphate oligomer uniformly dispersed therein are formed by copolymerization of organic acrylamide monomer (AM) and calcium phosphate oligomer (CPO) through a crosslinking agent and photoinitiation.

[0012] Furthermore, the crosslinking agent is N,N'-bis(acryloyl)cysteamine, and the photoinitiator is Irgacure 2959.

[0013] Furthermore, the precursor solution used for polymerization has an acrylamide mass-volume concentration of 10-20% and a calcium phosphate oligomer mass-volume concentration of 1-4%, with both mass-volume concentrations being 0.1-0.4%.

[0014] The present invention also provides a method for preparing the biomimetic mineralized hydrogel coating, comprising the following steps: S1: The surface of the polyarylethersulfone ketone substrate is subjected to plasma treatment, followed by acrylic acid grafting to introduce carbon-carbon double bonds; S2: Dissolve acrylamide monomer, calcium phosphate oligomer, photoinitiator and crosslinking agent in water to prepare a precursor solution, spin-coat it onto the substrate surface treated in step S1, and cure it with ultraviolet light to form a hydrogel coating. S3: Immerse the coating substrate obtained in step S2 into simulated body fluid and mineralize it at 25-40 ℃ for 3-14 days. Take it out, wash and dry it to obtain a biomimetic mineralized hydrogel coating.

[0015] The method for preparing the calcium phosphate oligomer includes reacting calcium chloride, phosphoric acid, and triethylamine in a solvent, followed by centrifugation, washing, and drying.

[0016] Further, in step S1, the plasma treatment is nitrogen plasma with a treatment power of 40-120 W and a time of 3-10 min; the acrylic grafting involves immersing the plasma-treated substrate in a 50-80% acrylic acid aqueous solution and reacting at 20-40 °C for 30-60 min.

[0017] Furthermore, the UV curing conditions in step S2 are: wavelength 365 nm, irradiation time 90–150 s.

[0018] The present invention also provides an application of the above-mentioned biomimetic mineralized hydrogel coating in bone repair.

[0019] Used to prepare surface modification materials for bone implants, bone repair materials, or materials for treating osteoporotic bone defects.

[0020] The beneficial effects of this invention are as follows: 1. Uniform Organic-Inorganic Molecular-Level Composite: This invention uses calcium phosphate oligomers with a particle size of approximately 1-2 nm to copolymerize with acrylamide monomers, forming a uniformly dispersed organic-inorganic hybrid network at the molecular level, thus avoiding the problem of HAP particle agglomeration in traditional blending methods. This uniform dispersion provides a large number of uniformly distributed nucleation sites for subsequent biomimetic mineralization, thereby inducing the formation of a dense, continuous, and deep apatite layer within the coating.

[0021] 2. Strong adhesion due to covalent bonding between the coating and the substrate: This invention introduces amino groups into the PPESK surface through nitrogen plasma treatment, followed by grafting carbon-carbon double bonds with acrylic acid. This causes the acrylamide monomer in the hydrogel precursor to copolymerize with the double bonds on the substrate surface, forming a strong chemical bond. This solves the key problem of hydrogel coatings easily peeling off on hydrophobic polymer surfaces.

[0022] 3. Uniform and Deep Biomimetic Mineralization: The nanoscale nucleation sites provided by CPO, combined with simulated calcium and phosphorus ions in body fluids, enable uniform mineralization from the surface to the interior of the coating under mild conditions of 37℃. This allows for greater mineral deposition and a significant increase in the inorganic phase content. Sr in the strontium-containing mineralized coating... 2+ Successful doping can endow the coating with additional biological functions.

[0023] 4. Dual bioactivity of promoting bone formation and inhibiting bone resorption: In vitro cell experiments showed that the mineralized coating significantly promoted the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts (increased alkaline phosphatase (ALP) activity and increased calcium nodule formation). Simultaneously, the strontium-containing coating effectively inhibited RANKL-induced differentiation of RAW264.7 cells into osteoclasts (reduction of TRAP-positive cells). This invention is the first to achieve a mineralized hydrogel coating with both osteogenic and anti-osteoclast functions on the PPESK surface, making it particularly suitable for bone defect repair in patients with osteoporosis.

[0024] 5. Mild and controllable preparation process: This invention uses ultraviolet light curing, which is under mild conditions (365 nm, 2 minutes) and does not damage the substrate material; biomimetic mineralization is carried out at 37°C in simulated body fluid, without the need for high temperature and high pressure, making it easy to scale up production. The coating thickness can be adjusted by spin coating parameters, making it suitable for implants with complex shapes. Attached Figure Description

[0025] Figure 1 Structural characterization of calcium phosphate oligomers (CPO): (a) FT-IR spectrum; (b) DLS particle size distribution; (c) TEM image.

[0026] Figure 2 Performance comparison of three hydrogels (PAM, PAMH, PAMC): (a) SEM surface morphology; (b) FT-IR spectrum.

[0027] Figure 3 Characterization of PPESK substrate surface modification: (a) N1s XPS spectrum of PPESK; (b) N1s XPS spectrum of PPESK-NH2; (c) C1s XPS spectrum of PPESK; (d) C1s XPS spectrum of PPESK-DB; (e) Photographs of cross-cut adhesion test.

[0028] Figure 4 Structure and composition of the mineralized hydrogel coating: (a) SEM images of the surface and cross-section (mineralization depth marked); (b) XRD pattern; (c) FT-IR pattern; (d) TGA curve.

[0029] Figure 5 This study aims to test the MTT cytotoxicity of the material relative to MC3T3-E1 cells.

[0030] Figure 6 To evaluate the osteogenic activity of the material relative to MC3T3-E1 cells: ALP staining (3, 7, 14 days) and Alizarin Red ARS staining (14 days).

[0031] Figure 7 Evaluation of the relative osteoclast inhibitory activity of the material against RAW264.7 cells: TRAP staining of RAW264.7 cells (10 days). Detailed Implementation

[0032] The following specific embodiments further illustrate the essential features and significant advancements of the present invention. However, the content of the present invention is not limited to the following embodiments and can be adjusted according to actual circumstances.

[0033] Example 1 Step 1: Synthesis of calcium phosphate oligomer (CPO). 4.704 g of CaCl₂·2H₂O was dissolved in 1.6 L of ethanol to form a clear alcoholic solution. 88.716 mL of TEA was added to the above solution, and the mixture was stirred at room temperature for 30 minutes. An alcoholic solution of H₃PO₄ (1.672 mL of H₃PO₄ dissolved in 80 mL of ethanol) was added dropwise to the above solution, and the mixture was stirred at room temperature. The CPO gel was obtained by centrifugation at 8000 rpm for 5 min and washed several times with ethanol to remove residual TEA. The CPO was redispersed in ethanol to form a homogeneous emulsion with a concentration of approximately 10 mg / mL for future use.

[0034] The second step involved surface modification of the PPESK substrate. PPESK sheets (15 mm × 10 mm × 1 mm) were sequentially ultrasonically cleaned with acetone, ethanol, and deionized water for 15 min each, and then dried at 60°C. They were then placed in a plasma treatment chamber, purged with nitrogen (50 mL / min), and treated at 60 W for 5 min to obtain PPESK-NH2. Immediately after removal, the sheets were immersed in a 70% acrylic acid aqueous solution and reacted at 25°C for 40 min. After removal, they were rinsed three times with deionized water and vacuum dried at 30°C to obtain PPESK-DB with surface-grafted double bonds.

[0035] The third step is the preparation of the hydrogel coating. 1.5 g of acrylamide, 0.2 g of CPO / HAP, 0.02 g of Irgacure 2959, and 0.02 g of N,N'-bis(acryloyl)cysteine ​​were dissolved in 10 mL of deionized water and stirred in the dark for 30 min to obtain a precursor solution. This solution was cured under a 365 nm UV lamp for 120 s to obtain PAM. The solution with added HAP was called PAMH, and the solution with added CPO was called PAMC. This solution was spin-coated onto the surface of PPESK-DB (500 rpm, 30 s), and then cured under a 365 nm UV lamp for 120 s to obtain PAM@PPESK, PAMH@PPESK, and PAMC@PPESK.

[0036] Step 4: Biomimetic mineralization. PPESK, PAM@PPESK, PAMH@PPESK, and PAMC@PPESK were immersed in 40 mL of SBF or Sr-SBF, respectively, and left to stand at 37°C for 7 days, with the mineralization solution replaced every 2 days. After removal, they were rinsed three times with deionized water and freeze-dried. These were designated as AP / PPESK, AP / PAM@PPESK, AP / PAMH@PPESK, AP / PAMC@PPESK, and SrAP / PAMC@PPESK, respectively.

[0037] The fifth step involves in vitro cell compatibility experiments. The material undergoes an MTT toxicity test to demonstrate its cell compatibility. Alkaline phosphatase staining and Alizarin Red staining are performed on MC3T3-E1 cells co-cultured with the material to verify its in vitro osteogenic properties. Tartrate-resistant acid phosphatase (TRAP) staining is performed on RAW264.7 cells co-cultured with the material to demonstrate its ability to inhibit osteoclast differentiation.

[0038] like Figure 1 As shown, TEM observation revealed a particle size of approximately 1.5 nm, while DLS measured an average particle size of approximately 1.3 nm. The FT-IR spectrum at 1194 cm⁻¹... -1 The presence of a CN stretching vibration peak indicates that triethylamine was successfully capped, proving that CPO was successfully synthesized.

[0039] like Figure 2 As shown, Figure 2 It can be observed that all three hydrogels exhibit a typical porous structure. The magnified image shows HAP aggregation in PAMH, with the red arrow pointing to the HAP particles in PAMH. PAMC, on the other hand, exhibits a similar morphology to PAM hydrogel, and no inorganic material is observed. Figure 2 In sample b, characteristic peaks of amide I and amide II, belonging to polyacrylamide hydrogels, can be observed at 562 cm⁻¹. -1 The signal at that location can be attributed to the bending vibration of the PO, but it shifts to 544 cm in the PAMC. -1 The slight blue shift of the PO bond can be attributed to the interaction between CPO and AM. This demonstrates the successful synthesis of the PAMC hydrogel.

[0040] like Figure 3 As shown, a represents PPESK and b represents PPESK-NH2. The increased content of amide bonds (N*─C═O) indicates the introduction of hydrophilic amino groups on its surface. Figure 3 c is the C1s spectrum of PPESK. Figure 3 d is the C1s spectrum of PPESK-DB. Figure 3 The π-π* satellite peak appearing at approximately 291.5 eV is a typical characteristic of conjugated double bond systems, proving that carbon-carbon double bonds were successfully introduced through grafting with acrylic acid. Cross-cut adhesion testing showed that the modified PPESK-DB surface coating exhibited almost no peeling, while the unmodified PPESK surface coating peeled off over a large area.

[0041] like Figure 4 As shown in Figure a, the AP / PPESK surface has almost no mineral deposition; the AP / PAM@PPESK surface has a small amount of scattered particles; the AP / PAMH@PPESK surface is covered with a relatively dense particle layer, but the cross-section shows that the minerals are only distributed in about half the thickness of the coating surface; the AP / PAMC@PPESK and SrAP / PAMC@PPESK surfaces form continuous and dense flaky apatite crystals, and the cross-section shows that the minerals penetrate the entire coating thickness (more than 3 / 4) and are tightly bonded to the coating. Figure 4 XRD tests of b showed that AP / PAMC@PPESK and SrAP / PAMC@PPESK exhibited typical HAP characteristic peaks at 2θ = 25.9°, 31.8°, 32.9°, and 46.8°, consistent with the standard card PDF#84-1998, while the HAP peaks of AP / PAM@PPESK and AP / PAMH@PPESK were significantly weaker. Figure 4 The infrared spectrum of c indicates that the mineralized coating is at 1108, 1025, and 957 cm⁻¹. -1 PO stretching vibration peaks were observed at 601 and 561 cm⁻¹.-1 The presence of a PO bending vibration peak confirms the formation of apatite crystals. Figure 4 Thermogravimetric analysis showed that the residual mass of the AP / PAMC group at 800℃ reached 43%, which was significantly higher than that of the control group, confirming that it had a higher degree of mineralization and a richer content of inorganic phase.

[0042] like Figure 5 As shown, after co-culturing with MC3T3-E1 cells for one day and three days, the cell survival rate was above 80%, proving that the prepared mineralized hydrogel coating had no obvious cytotoxicity.

[0043] like Figure 6 As shown, ALP activity was significantly increased in AP / PAMC@PPESK and SrAP / PAMC@PPESK, and the number of red calcium nodules stained with Alizarin Red was significantly increased compared with the control group, indicating that the materials can promote osteoblast osteogenic differentiation in vitro.

[0044] like Figure 7 As shown, RAW264.7 cells were co-cultured with the samples for 10 days under RANKL (100 ng / mL) induction, and the osteoclast-inhibiting activity of the materials was evaluated. The results showed that the positive control group formed abundant purple-red staining, corresponding to inhibition of osteoclast formation; the AP / PPESK group was similar to the positive control; while the SrAP / PAMC@PPESK group showed a significant reduction in TRAP purple-red staining, approaching that of the negative control group (without RANKL). This indicates that SrAP / PAMC@PPESK can effectively inhibit the activity of RAW264.7 cells.

Claims

1. A biomimetic mineralized hydrogel coating on the surface of polyarylether sulfone ketone, characterized in that: The biomimetic mineralized hydrogel coating is obtained by mineralizing a hydrogel coating through simulated body fluid, forming an apatite layer inside and on the surface of the hydrogel coating; the hydrogel coating includes an organic polymer network and calcium phosphate oligomers uniformly dispersed therein.

2. The biomimetic mineralized hydrogel coating on the surface of polyarylether sulfone ketone as described in claim 1, characterized in that: The calcium phosphate oligomer has a particle size of 1–2 nm and its molecular structure contains 2–3 Ca3(PO4)2 units; and / or, The simulated body fluid is a strontium-containing simulated body fluid.

3. The biomimetic mineralized hydrogel coating as described in claim 1, characterized in that: The hydrogel coating is anchored to the surface of the polyarylether sulfone ketone substrate.

4. The biomimetic mineralized hydrogel coating on the surface of polyarylether sulfone ketone as described in claim 1, characterized in that: The hydrogel coating is formed by copolymerization of organic acrylamide monomers and calcium phosphate oligomers through a crosslinking agent and photoinitiation.

5. The biomimetic mineralized hydrogel coating as described in claim 4, characterized in that: The precursor solution used for copolymerization has an acrylamide concentration of 10-20% by mass and volume, a calcium phosphate oligomer concentration of 1-4% by mass and volume, and a photoinitiator and crosslinking agent concentration of 0.1-0.4% by mass and volume.

6. A method for preparing the biomimetic mineralized hydrogel coating according to claim 1, characterized in that: Includes the following steps: S1: The surface of the polyarylethersulfone ketone substrate is subjected to plasma treatment, followed by acrylic acid grafting to introduce carbon-carbon double bonds; S2: Dissolve acrylamide monomer, calcium phosphate oligomer, photoinitiator and crosslinking agent in water to prepare a precursor solution, spin-coat it onto the substrate surface treated in step S1, and cure it with ultraviolet light to form a hydrogel coating. S3: Immerse the coating substrate obtained in step S2 into simulated body fluid and mineralize it at 25-40 ℃ for 3-14 days. Take it out, wash it, and dry it to obtain a biomimetic mineralized hydrogel coating.

7. The method for preparing the biomimetic mineralized hydrogel coating as described in claim 6, characterized in that: In step S1, the plasma treatment is nitrogen plasma with a power of 40-120 W and a time of 3-10 min; the acrylic grafting involves immersing the plasma-treated substrate in a 50-80% acrylic acid aqueous solution and reacting at 20-40 °C for 30-60 min.

8. The method for preparing the biomimetic mineralized hydrogel coating as described in claim 6, characterized in that: The UV curing conditions in step S1 are: wavelength 365 nm, irradiation time 90–150 s.

9. The application of the biomimetic mineralized hydrogel coating of claim 1 in bone repair.

10. The application of the biomimetic mineralized hydrogel coating as described in claim 9 in bone repair, characterized in that: It is used to prepare surface modification materials for bone implants, bone repair materials, or materials for the treatment of osteoporotic bone defects.

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