A gradient biomimetic peek-ha-col i composite implant and its preparation method and application

Abstract

The application discloses a gradient biomimetic PEEK-HA-Col I composite implant and a preparation method and application thereof, relates to the technical field of medical biomaterials, and has the following scheme: a composite implant composed of a load-bearing inner layer, a bioactive intermediate layer and a biomimetic bone matrix layer is constructed; the load-bearing inner layer contains a PEEK matrix, the intermediate layer contains PDA modified interfaces and HA induced and deposited by the interfaces, and the outer layer contains cross-linked modified collagen type I, and a bioactive gradient distribution is formed along the thickness direction; the preparation method and the application of the composite implant in bone repair are simultaneously provided. The application improves the interface bioactivity and the bone integration capability on the basis of maintaining the PEEK load-bearing support, improves the interface structure stability and the bioactive spatial distribution, improves the surface hydrophilicity and the interface microenvironment, is favorable for promoting early osteogenesis and bone defect repair, improves bone fusion and application adaptability in a load-bearing bone implantation scene, and simultaneously considers the synergistic matching of the load-bearing demand and the biological demand of the bone tissue interface.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a gradient biomimetic PEEK-HA-COL I composite implant, its preparation method, and its application. Background Technology

[0002] Polyetheretherketone (PEEK), a high-performance engineering polymer, has been widely used in spinal fusion devices, orthopedic implants, and other load-bearing implant components in recent years due to its high mechanical strength, good chemical stability, corrosion resistance, and processing performance, as well as its elastic modulus being closer to that of human bone tissue to a certain extent. Meanwhile, in the development of bone repair and implant materials technology, improving the biological responsiveness of the implant-bone interface has always been a key issue affecting the efficiency and long-term stability of post-implantation osseointegration. Hydroxyapatite (HA), as the main inorganic component of natural bone minerals, has good biocompatibility and osteoconductive properties and is often used to enhance the bioactivity of the implant material interface. Bone collagen (especially type I collagen, Col I), as an important component of the organic matrix of bone tissue, plays a crucial role in the construction of the interfacial microenvironment related to cell adhesion, spreading, and proliferation. Therefore, improving the compatibility of the bone-implant interface by introducing bioactive components such as HA and bone collagen around load-bearing matrix materials such as PEEK has become an important direction for the continuous development of related technologies in this field.

[0003] However, existing technologies still have significant limitations in improving the osseointegration performance of load-bearing implants using the aforementioned material systems, and these limitations directly affect the synergistic balance between load-bearing performance and interfacial bioactivity. First, while PEEK materials have advantages in mechanical and chemical stability, their surface bioinertness is strong, easily forming a fibrous encapsulation layer after implantation. This makes it difficult for bone tissue to establish a sufficient and stable osseointegration interface with the implant surface, thus affecting osseointegration capacity and long-term stability. In other words, PEEK has a clear advantage in "load-bearing support," but has an inherent weakness in "interfacial biological response," a weakness that cannot be eliminated solely by the matrix material itself. Second, while HA has good biocompatibility and osteoconductivity, its inherent brittleness and insufficient mechanical properties make it difficult to use as a standalone load-bearing implant structure. ColI, while beneficial for constructing an organic interfacial environment closer to natural bone tissue and promoting cell adhesion and proliferation, has limited mechanical strength and long-term stability, and suffers from rapid degradation in the in vivo environment. Therefore, HA and ColI have advantages in inorganic osteoconduction and organic matrix simulation, respectively. However, using them alone or simply increasing their amount cannot naturally meet the comprehensive requirements of structural strength, service stability, and long-term interface integrity for load-bearing implant scenarios. Furthermore, common improvements in existing technologies involve coating the PEEK surface with HA or using simple filling with biomaterials to enhance surface bioactivity. However, these approaches often suffer from insufficient interfacial bonding stability, unreasonable spatial distribution of bioactivity, and limited early osteogenic promotion effects. This is because: firstly, if the surface active layer lacks a stable and reliable bonding relationship with the matrix, local delamination, structural loosening, or functional decline can easily occur under long-term stress, fluid infiltration, and micro-motion conditions; secondly, existing solutions often focus on enhancing the activity of the outermost layer, lacking functional allocation along the implant thickness direction that matches load-bearing and tissue interface requirements. This results in a lack of stable transition between the load-bearing area and the bone contact area, making it difficult to balance load-bearing reliability and rapid osseointegration in the same implant component. Furthermore, if only short-term surface activity improvements are pursued without simultaneously addressing interfacial structural stability, interfacial microenvironment optimization, and performance retention under long-term service, problems such as limited early improvements, insufficient later-stage integration stability, or unbalanced overall performance may still occur. Therefore, the main deficiency of existing technologies is not a lack of a single indicator, but rather the difficulty in simultaneously considering interfacial bioactivity, interfacial structural stability, rational spatial distribution of bioactivity, and comprehensive bone repair-related performance in load-bearing implant systems.

[0004] Based on the aforementioned state of the prior art, the key technical problems that urgently need to be solved in this field are: in implant material systems based on load-bearing matrix materials such as PEEK, how to improve the biological response and osseointegration level of the implant-bone interface while ensuring necessary mechanical strength and long-term service stability; how to improve the bonding stability between the interfacial active structure and the load-bearing matrix to reduce the risks of post-implantation interface instability, functional decline, and insufficient long-term stability; how to make the implant material form a bioactive distribution relationship in the thickness direction that matches the load-bearing requirements and the requirements of the bone tissue interface to alleviate the problems of uneven bioactive distribution and insufficient structural transition in the prior art; and how to improve early osteogenic processes and subsequent bone repair performance by improving surface wettability and interfacial microenvironment, thereby better meeting the comprehensive performance requirements of bone defect repair, bone fusion, and load-bearing bone implantation scenarios. Summary of the Invention

[0005] The purpose of this invention is to provide a composite implant for load-bearing matrix implant materials such as PEEK and related technical solutions, so as to improve the biological response and osseointegration level of the implant-bone tissue interface, improve the bonding stability between the interfacial active structure and the load-bearing matrix, optimize the bioactive distribution relationship of the implant material along the thickness direction, and improve the interfacial microenvironment and early osteogenic processes on this basis, thereby enhancing its comprehensive application adaptability in bone defect repair, bone fusion and load-bearing bone implantation scenarios.

[0006] To achieve the aforementioned objectives and address the aforementioned technical problems, this invention provides a gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant. It includes a load-bearing inner layer, a bioactive intermediate layer, and a biomimetic bone matrix layer arranged sequentially from the inside out; in: The load-bearing inner layer contains a polyetheretherketone matrix; The bioactive intermediate layer is disposed on the surface of the load-bearing inner layer and includes a polydopamine (PDA) modified interface and hydroxyapatite formed by induced deposition of the polydopamine modified interface. The biomimetic bone matrix layer is disposed on the surface of the bioactive intermediate layer and contains cross-linked modified type I collagen; The composite implant forms a bioactive gradient distribution structure along its thickness direction.

[0007] Preferably, the composite implant has a gradient distribution structure along its thickness direction, with low bioactivity on the load-bearing inner layer side and high bioactivity on the biomimetic bone matrix layer side.

[0008] Preferably, the polyetheretherketone matrix has a microporous structure formed by sulfonation treatment on its surface.

[0009] Preferably, the polyetheretherketone matrix is ​​made of medical-grade PEEK material and processed into a preset shape. The processing method can be extrusion molding, injection molding or 3D printing.

[0010] Preferably, the polyetheretherketone matrix is ​​cylindrical.

[0011] Preferably, the hydroxyapatite in the bioactive intermediate layer is in the form of micron-sized or nano-sized particles.

[0012] More preferably, the hydroxyapatite particles are selected to be <100 nm.

[0013] The present invention also provides a method for preparing the above-mentioned composite implant, comprising the following steps: S1. Provide a polyetheretherketone matrix and perform surface treatment on the polyetheretherketone matrix to form a surface microporous structure through sulfonation treatment; S2. A polydopamine coating is constructed on the surface of the polyetheretherketone matrix after the treatment in step S1, and the polydopamine-modified polyetheretherketone matrix is ​​placed in simulated body fluid (SBF) for biomineralization treatment, so that calcium and phosphorus ions in the simulated body fluid form and deposit hydroxyapatite on the surface and pores of the polyetheretherketone matrix, thereby constructing a bioactive intermediate layer. S3. Introduce cross-linked modified type I collagen on the surface of the bioactive intermediate layer to form a biomimetic bone matrix layer, thereby obtaining a molded composite implant. S4. The resulting molded composite implant is cured and sterilized.

[0014] Preferably, step S1 includes roughening the surface of the polyetheretherketone matrix, wherein the roughening treatment is at least one of mechanical grinding, laser treatment, and sandblasting.

[0015] More preferably, the surface of the polyetheretherketone matrix is ​​mechanically polished.

[0016] More preferably, silicon carbide sandpaper is used for sanding.

[0017] More preferably, silicon carbide sandpaper is used for sanding in successive grades of 150, 240, 320, 400, 600, 800, 1000, 1200, 1500 and 2000 grit.

[0018] Preferably, the sulfonation treatment method described in step S1 involves soaking in a sulfuric acid solution with a mass fraction of 95 wt% for 5 minutes at 25°C.

[0019] Preferably, in step S1, the polyether ether ketone matrix is ​​rinsed and dried after sulfonation treatment.

[0020] More preferably, in step S1, the polyether ether ketone matrix is ​​repeatedly rinsed with deionized water after sulfonation treatment.

[0021] More preferably, the polyetheretherketone matrix is ​​ultrasonically cleaned to fully remove residual sulfuric acid, and then dried in an oven at 60 °C.

[0022] The method for preparing the simulated body fluid is as follows: Preferably, the method for preparing the simulated body fluid is as follows: weigh out 8.035 g / L NaCl, 0.355 g / L NaHCO3, 0.255 g / L KCl, 0.219 g / L K2HPO4·3H2O, 0.203 g / L MgCl2·6H2O, 0.147 g / L CaCl2·2H2O, and 0.072 g / L Na2SO4; Add each ingredient in sequence and dissolve it completely in deionized water, while maintaining the temperature at 36.5 ℃; The pH of the solution was adjusted to 7.35 using hydrochloric acid and tris(hydroxymethyl)aminomethane. After cooling, store in a 4°C refrigerator for later use.

[0023] Preferably, the pH value of the simulated body fluid in step S2 is 7.30 to 7.50, and the soaking and mineralization time is 24 to 96 hours.

[0024] More preferably, the pH value of the simulated body fluid is 7.35.

[0025] More preferably, the simulated body fluid is replaced every 24 hours to maintain the ion concentration.

[0026] Preferably, in step S3, the type I collagen solution used to construct the biomimetic bone matrix layer is cross-linked with a polylysine solution, the concentration of which is 0.05–0.2 mg / mL, and the concentration of the type I collagen solution is 0.1–1 mg / mL.

[0027] More preferably, the concentration of the polylysine solution is 0.1 mg / mL.

[0028] More preferably, the concentration of type I collagen solution is 1 mg / mL.

[0029] Preferably, in step S4, the formed composite implant is sterilized using ethylene oxide or low-temperature plasma. The present invention also provides the use of the above-mentioned gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant in bone defect repair, bone fusion or weight-bearing bone implantation.

[0030] Preferably, the above-mentioned gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant is used in craniofacial bone reconstruction and spinal fusion.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects: (1) While maintaining the load-bearing support of the PEEK matrix, improve the interfacial bioactivity of the implant surface, thereby improving the problem of insufficient osseointegration caused by the bioinertness of the traditional PEEK implant surface.

[0032] This invention forms a microporous structure by sulfonating the surface of a PEEK matrix and then performing biomineralization based on a polydopamine-modified interface, which simulates body fluids to form and deposit hydroxyapatite on the matrix surface and within the pores, thus constructing a bioactive intermediate layer and providing a basis for osteoconduction-related activities at the bone tissue interface.

[0033] (2) By layered construction and interface co-design, the stability of the interface structure is improved and the spatial distribution of bioactivity is optimized, thereby alleviating the problems of insufficient interface bonding stability and uneven distribution of bioactivity in the existing simple surface coating or simple filling methods.

[0034] This invention employs a gradient composite structure consisting of a load-bearing inner layer, a bioactive intermediate layer, and a biomimetic bone matrix layer. The microporous structure facilitates the bonding and intercalation of the interface layers, the polydopamine-modified interface promotes stable mineralization deposition, and PLL-mediated type I collagen fixation enhances the adhesion stability of the biomimetic bone matrix layer, thereby achieving improved interface structure stability and the construction of a bioactive gradient distribution.

[0035] (3) Improve the wettability of the material surface and the cell-material interface microenvironment, thereby promoting early osteogenic processes and improving early bone integration.

[0036] In this invention, after sequentially constructing a HA bioactive intermediate layer and a ColⅠ biomimetic bone matrix layer on the surface of a PEEK matrix, the hydrophilicity of the material surface is enhanced, which is beneficial to the adhesion, proliferation and differentiation of osteoblast-related cells, and also facilitates the subsequent mineralization process.

[0037] (4) Improve the bone repair-related performance of implants in bone defect repair and load-bearing bone implantation scenarios, so as to better meet the comprehensive requirements of load-bearing performance, interfacial bioactivity and bone integration ability.

[0038] The results of in vitro osteogenic evaluation and in vivo bone defect model show that the three-layer gradient composite implant of the present invention is superior to the control group in terms of osteogenic induction and bone regeneration, indicating that the technical solution of the present invention can further improve the adaptability of bone repair applications on the basis of load-bearing matrix. Attached Figure Description

[0039] Figure 1This is a schematic diagram of the structure of an embodiment of the present invention; Figure 2 This is a SEM test image from Embodiment 1 of the present invention; Figure 3 This is an XRD test image of Embodiment 1 of the present invention; Figure 4 The figures show the CCK-8 experimental test results of Embodiment 1 and Comparative Examples 1 and 2 of the present invention; Figure 5 The graph shows the results of the alkaline phosphatase quantitative experiment in Comparative Examples 1 and 2 of Example 1 of the present invention. Figure 6 The figures show the results of the alizarin red staining quantitative experiment of Comparative Examples 1 and 2 of Example 1 of the present invention; Figure 7 The image shows the RT-qPCR test results of Comparative Examples 1 and 2 of Example 1 of this invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Of course, the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Example 1 Preparation of a gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant 1. PEEK matrix molding and dimensional preparation Medical-grade pure polyether ether ketone (PEEK) material was selected and processed into a cylindrical structure with a diameter of 10 mm and a height of 10 mm.

[0042] The polyetheretherketone matrix is ​​made of medical-grade PEEK material, processed into a preset shape, and then formed by 3D printing.

[0043] 2. Mechanical grinding treatment of PEEK substrate surface The PEEK substrate is successively polished using silicon carbide sandpaper with grit sizes of 150 mesh, 240 mesh, 320 mesh, 400 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh and 2000 mesh to obtain a substrate surface with a smooth surface and controllable roughness.

[0044] 3. PEEK substrate cleaning treatment The polished PEEK substrate was ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence. The ultrasonic cleaning time for each solvent was 15 minutes to remove processing residues and organic contaminants from the substrate surface.

[0045] 4. Sulfuric acid sulfonation treatment to construct a porous structure The cleaned PEEK matrix was immersed in a 95 wt% sulfuric acid solution at 25°C for 5 min to induce sulfonation on the surface of the PEEK matrix and form a porous structure.

[0046] 5. Cleaning and drying after sulfonation The sulfonated PEEK matrix was removed and repeatedly rinsed with deionized water to thoroughly remove residual sulfuric acid. The sample was then placed in a 60 °C oven for drying to obtain a sulfonated porous PEEK matrix.

[0047] 6. Preparation of Simulated Body Fluid (SBF) Solution SBF solution was prepared according to the simulated body fluid formula, and the pH value of the SBF solution was adjusted to 7.35, and then stored at 4°C for later use.

[0048] The SBF solution has the following composition: NaCl (8.035 g / L), NaHCO3 (0.355 g / L), KCl (0.255 g / L), K2HPO4·3H2O (0.219 g / L), MgCl2·6H2O (0.203 g / L), CaCl2·2H2O (0.147 g / L), and Na2SO4 (0.072 g / L).

[0049] The specific preparation process is as follows: Weigh each component using an electronic balance; add an appropriate amount of deionized water to a plastic measuring cup, add each component in sequence and fully dissolve it in the deionized water, and maintain the temperature at 36.5 ℃ using a water bath; adjust the pH of the solution to 7.35 using 1 mol / L hydrochloric acid and tris(hydroxymethyl)aminomethane (Tris); make up to volume using a 1 L volumetric flask, cool the solution after bottling, and store it in a 4 ℃ refrigerator for later use.

[0050] 7. PEEK matrix pretreatment The PEEK substrate was sequentially immersed in acetone and pure water, and ultrasonically cleaned for 20 minutes each time. After repeating the above steps twice, the sample was dried in an environment of 60 °C for 24 h to remove surface contaminants and improve the bonding stability of subsequent coatings.

[0051] 8. Preparation of polydopamine solution 20 mg of polydopamine was dissolved in 10 mL of Tris buffer solution with a concentration of 10 mM; the pH of the solution was adjusted to 8.50 by adding hydrochloric acid dropwise to obtain a polydopamine solution for surface modification.

[0052] 9. Construction of polydopamine coating on PEEK substrate surface The pretreated PEEK matrix was placed in 1 mL of the polydopamine solution and continuously shaken in a shaker at 37 °C for 24 h to allow the polydopamine to undergo a self-polymerization reaction on the PEEK matrix surface and form a continuous polydopamine coating. After polymerization, the sample was ultrasonically cleaned with deionized water to remove the polydopamine particles physically adsorbed on the matrix surface.

[0053] 10. Biomineralized sedimentary HA The polydopamine-modified PEEK matrix sample was placed in 1 mL of the SBF solution and subjected to biomineralization treatment by continuous shaking under shaking conditions. The SBF solution was replaced every 24 h to maintain the effective concentration of calcium and phosphorus ions in the solution and promote the continuous deposition of HA on the matrix surface. After 72 h of biomineralization treatment, the sample was taken out and ultrasonically cleaned with deionized water to remove unbound salts or loose deposits.

[0054] Through the above steps, a bioactive intermediate layer containing HA is formed on the surface of the PEEK matrix.

[0055] 11. Poly-L-L (PLL) treatment: The above-mentioned PEEK-loaded HA bilayer composite implant was immersed in a polylysine (PLL) solution with a concentration of 0.1 mg / mL and left to stand at room temperature for 24 hours to allow the polylysine to adhere to the surface of the implant and the inner wall of the pores through electrostatic adsorption. After the treatment, the implant was ultrasonically cleaned with deionized water to remove unbound polylysine molecules.

[0056] 12. Collagen cross-linking fixation treatment: The PLL-treated composite implant was transferred to a 1 mg / mL type I collagen (Col I) solution, completely immersed, and left to stand at room temperature for 24 h. Through PLL-mediated action, collagen molecules were attached and cross-linked to the implant surface and pore structure. After treatment, the implant was ultrasonically cleaned again with deionized water to remove unfixed collagen components.

[0057] After the above treatment, a three-layer gradient composite implant of PEEK-HA-ColⅠ was obtained.

[0058] Example 2 Preparation of a gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant 1. PEEK matrix molding and dimensional preparation Medical-grade pure polyether ether ketone (PEEK) material was selected and processed into a cylindrical structure with a diameter of 10 mm and a height of 10 mm.

[0059] 2. Mechanical grinding treatment of PEEK substrate surface The PEEK substrate is successively polished using silicon carbide sandpaper with grit sizes of 150 mesh, 240 mesh, 320 mesh, 400 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh and 2000 mesh to obtain a substrate surface with a smooth surface and controllable roughness.

[0060] 3. PEEK substrate cleaning treatment The polished PEEK substrate was ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence. The ultrasonic cleaning time for each solvent was 15 minutes to remove processing residues and organic contaminants from the substrate surface.

[0061] 4. Sulfuric acid sulfonation treatment to construct a porous structure The cleaned PEEK matrix was immersed in a 95 wt% sulfuric acid solution at 25°C for 5 min to induce sulfonation on the surface of the PEEK matrix and form a porous structure.

[0062] 5. Cleaning and drying after sulfonation The sulfonated PEEK matrix was removed and repeatedly rinsed with deionized water to thoroughly remove residual sulfuric acid. The sample was then placed in a 60 °C oven for drying to obtain a sulfonated porous PEEK matrix.

[0063] 6. Preparation of Simulated Body Fluid (SBF) Solution SBF solution was prepared according to the simulated body fluid formula, and the pH value of the SBF solution was adjusted to 7.35, and then stored at 4°C for later use.

[0064] The SBF solution has the following composition: NaCl (8.035 g / L), NaHCO3 (0.355 g / L), KCl (0.255 g / L), K2HPO4·3H2O (0.219 g / L), MgCl2·6H2O (0.203 g / L), CaCl2·2H2O (0.147 g / L), and Na2SO4 (0.072 g / L).

[0065] The specific preparation process is as follows: Weigh each component using an electronic balance; add an appropriate amount of deionized water to a plastic measuring cup, add each component in sequence and fully dissolve it in the deionized water, and maintain the temperature at 36.5 ℃ using a water bath; adjust the pH of the solution to 7.3 using 1 mol / L hydrochloric acid and tris(hydroxymethyl)aminomethane (Tris); make up to volume using a 1 L volumetric flask, cool the solution after bottling, and store it in a 4 ℃ refrigerator for later use.

[0066] 7. PEEK matrix pretreatment The PEEK substrate was sequentially immersed in acetone and pure water, and ultrasonically cleaned for 20 minutes each time. After repeating the above steps twice, the sample was dried in an environment of 60 °C for 24 h to remove surface contaminants and improve the bonding stability of subsequent coatings.

[0067] 8. Preparation of polydopamine solution 20 mg of polydopamine was dissolved in 10 mL of Tris buffer with a concentration of 10 mM; the pH of the solution was adjusted to 8.50 by adding hydrochloric acid dropwise to obtain a polydopamine solution for surface modification.

[0068] 9. Construction of polydopamine coating on PEEK substrate surface The pretreated PEEK matrix was placed in 1 mL of the polydopamine solution and continuously shaken in a shaker at 37 °C for 24 h to allow the polydopamine to undergo a self-polymerization reaction on the PEEK matrix surface and form a continuous polydopamine coating. After polymerization, the sample was ultrasonically cleaned with deionized water to remove the polydopamine particles physically adsorbed on the matrix surface.

[0069] 10. Biomineralized sedimentary HA The polydopamine-modified PEEK matrix sample was placed in 1 mL of the SBF solution and subjected to biomineralization treatment by continuous shaking under shaking conditions. The SBF solution was replaced every 24 h to maintain the effective concentration of calcium and phosphorus ions in the solution and promote the continuous deposition of HA on the matrix surface. After 24 h of biomineralization treatment, the sample was taken out and ultrasonically cleaned with deionized water to remove unbound salts or loose deposits.

[0070] Through the above steps, a bioactive intermediate layer containing HA is formed on the surface of the PEEK matrix.

[0071] 11. Poly-L-L (PLL) treatment: The above-mentioned PEEK-loaded HA bilayer composite implant was immersed in a polylysine (PLL) solution with a concentration of 0.05 mg / mL and left to stand at room temperature for 24 hours to allow the polylysine to adhere to the surface of the implant and the inner wall of the pores through electrostatic adsorption. After the treatment, the implant was ultrasonically cleaned with deionized water to remove unbound polylysine molecules.

[0072] 12. Collagen cross-linking fixation treatment: The PLL-treated composite implant was transferred to a 0.1 mg / mL type I collagen (Col I) solution, completely immersed, and left to stand at room temperature for 24 hours. The collagen molecules were then attached and cross-linked to the implant surface and pore structure through the PLL-mediated process. After treatment, the implant was ultrasonically cleaned again with deionized water to remove any unfixed collagen components.

[0073] After the above treatment, a three-layer gradient composite implant of PEEK-HA-ColⅠ was obtained.

[0074] Example 3 Preparation of a gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant 1. PEEK matrix molding and dimensional preparation Medical-grade pure polyether ether ketone (PEEK) material was selected and processed into a cylindrical structure with a diameter of 10 mm and a height of 10 mm.

[0075] 2. Mechanical grinding treatment of PEEK substrate surface The PEEK substrate is successively polished using silicon carbide sandpaper with grit sizes of 150 mesh, 240 mesh, 320 mesh, 400 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh and 2000 mesh to obtain a substrate surface with a smooth surface and controllable roughness.

[0076] 3. PEEK substrate cleaning treatment The polished PEEK substrate was ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence. The ultrasonic cleaning time for each solvent was 15 minutes to remove processing residues and organic contaminants from the substrate surface.

[0077] 4. Sulfuric acid sulfonation treatment to construct a porous structure The cleaned PEEK matrix was immersed in a 95 wt% sulfuric acid solution at 25°C for 5 min to induce sulfonation on the surface of the PEEK matrix and form a porous structure.

[0078] 5. Cleaning and drying after sulfonation The sulfonated PEEK matrix was removed and repeatedly rinsed with deionized water to thoroughly remove residual sulfuric acid. The sample was then placed in a 60 °C oven for drying to obtain a sulfonated porous PEEK matrix.

[0079] 6. Preparation of Simulated Body Fluid (SBF) Solution SBF solution was prepared according to the simulated body fluid formula, and the pH value of the SBF solution was adjusted to 7.35, and then stored at 4°C for later use.

[0080] The SBF solution has the following composition: NaCl (8.035 g / L), NaHCO3 (0.355 g / L), KCl (0.255 g / L), K2HPO4·3H2O (0.219 g / L), MgCl2·6H2O (0.203 g / L), CaCl2·2H2O (0.147 g / L), and Na2SO4 (0.072 g / L).

[0081] The specific preparation process is as follows: Weigh each component using an electronic balance; add an appropriate amount of deionized water to a plastic measuring cup, add each component in sequence and fully dissolve it in the deionized water, and maintain the temperature at 36.5 ℃ using a water bath; adjust the pH of the solution to 7.35 using 1 mol / L hydrochloric acid and tris(hydroxymethyl)aminomethane (Tris); make up to volume using a 1 L volumetric flask, cool the solution after bottling, and store it in a 4 ℃ refrigerator for later use.

[0082] 7. PEEK matrix pretreatment The PEEK substrate was sequentially immersed in acetone and pure water, and ultrasonically cleaned for 20 minutes each time. After repeating the above steps twice, the sample was dried in an environment of 60 °C for 24 h to remove surface contaminants and improve the bonding stability of subsequent coatings.

[0083] 8. Preparation of polydopamine solution 20 mg of polydopamine was dissolved in 10 mL of Tris buffer with a concentration of 10 mM; the pH of the solution was adjusted to 8.50 by adding hydrochloric acid dropwise to obtain a polydopamine solution for surface modification.

[0084] 9. Construction of polydopamine coating on PEEK substrate surface The pretreated PEEK matrix was placed in 1 mL of the polydopamine solution and continuously shaken in a shaker at 37 °C for 24 h to allow the polydopamine to undergo a self-polymerization reaction on the PEEK matrix surface and form a continuous polydopamine coating. After polymerization, the sample was ultrasonically cleaned with deionized water to remove the polydopamine particles physically adsorbed on the matrix surface.

[0085] 10. Biomineralized sedimentary HA The polydopamine-modified PEEK matrix sample was placed in 1 mL of the SBF solution and subjected to biomineralization treatment by continuous shaking under shaking conditions. The SBF solution was replaced every 24 h to maintain the effective concentration of calcium and phosphorus ions in the solution and promote the continuous deposition of HA on the matrix surface. After 96 h of biomineralization treatment, the sample was taken out and ultrasonically cleaned with deionized water to remove unbound salts or loose deposits.

[0086] Through the above steps, a bioactive intermediate layer containing HA is formed on the surface of the PEEK matrix.

[0087] 11. Poly-L-L (PLL) treatment: The above-mentioned PEEK-loaded HA bilayer composite implant was immersed in a polylysine (PLL) solution with a concentration of 0.2 mg / mL and left to stand at room temperature for 24 hours to allow the polylysine to adhere to the surface of the implant and the inner wall of the pores through electrostatic adsorption. After the treatment, the implant was ultrasonically cleaned with deionized water to remove unbound polylysine molecules.

[0088] 12. Collagen cross-linking fixation treatment: The PLL-treated composite implant was transferred to a 1 mg / mL type I collagen (Col I) solution, completely immersed, and left to stand at room temperature for 24 h. Through PLL-mediated action, collagen molecules were attached and cross-linked to the implant surface and pore structure. After treatment, the implant was ultrasonically cleaned again with deionized water to remove unfixed collagen components.

[0089] After the above treatment, a three-layer gradient composite implant of PEEK-HA-ColⅠ was obtained.

[0090] Comparative Example 1 1. PEEK matrix molding and dimensional preparation: Medical-grade pure polyether ether ketone (PEEK) material was selected and processed into a cylindrical structure with a diameter of 10 mm and a height of 10 mm.

[0091] 2. Mechanical grinding treatment of PEEK substrate surface: The PEEK substrate is successively polished using silicon carbide sandpaper with grit sizes of 150 mesh, 240 mesh, 320 mesh, 400 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh and 2000 mesh to obtain a substrate surface with a smooth surface and controllable roughness.

[0092] 3. PEEK substrate cleaning treatment: The polished PEEK substrate was ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence. The ultrasonic cleaning time for each solvent was 15 minutes to remove processing residues and organic contaminants from the substrate surface.

[0093] After the above processing, a PEEK implant was obtained.

[0094] Comparative Example 2 1. PEEK matrix molding and dimensional preparation: Medical-grade pure polyether ether ketone (PEEK) material was selected and processed into a cylindrical structure with a diameter of 10 mm and a height of 10 mm.

[0095] 2. Mechanical grinding treatment of PEEK substrate surface: The PEEK substrate is successively polished using silicon carbide sandpaper with grit sizes of 150 mesh, 240 mesh, 320 mesh, 400 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh and 2000 mesh to obtain a substrate surface with a smooth surface and controllable roughness.

[0096] 3. PEEK substrate cleaning treatment: The polished PEEK substrate was ultrasonically cleaned in acetone, anhydrous ethanol and ultrapure water in sequence. The ultrasonic cleaning time for each solvent was 15 minutes to remove processing residues and organic contaminants from the substrate surface.

[0097] 4. Sulfonation treatment to construct a porous structure: The cleaned PEEK matrix was immersed in a 95 wt% sulfuric acid solution at 25 °C for 5 min to induce sulfonation on the surface of the PEEK matrix and form a porous structure.

[0098] 5. Cleaning and drying after sulfonation: The sulfonated PEEK matrix was removed and repeatedly rinsed with deionized water to thoroughly remove residual sulfuric acid. The sample was then placed in a 60 °C oven for drying to obtain a sulfonated porous PEEK matrix.

[0099] 6. Preparation of simulated body fluid (SBF) solution: Prepare an SBF solution according to the method described in step 6 of Example 1, adjust the pH of the SBF solution to 7.35, and store it at 4 ℃ for later use.

[0100] 7. PEEK matrix pretreatment: Process according to the method described in step 7 of Example 1.

[0101] 8. Preparation of polydopamine solution: The polydopamine solution was prepared according to the method described in step 8 of Example 1.

[0102] 9. Construction of polydopamine coating on PEEK substrate surface: A polydopamine coating was constructed on the surface of the PEEK substrate according to the method described in step 9 of Example 1.

[0103] 10. Biomineralized sedimentary deposits (HA): Biomineralization treatment is performed according to the method described in step 10 of Example 1, so that HA is continuously deposited on the substrate surface and a bioactive intermediate layer containing HA is formed.

[0104] After the above treatment, a PEEK-HA dual-functional layer composite implant was obtained.

[0105] Experimental test: 1. Morphological characterization test 1.2 The surface morphology of the samples was observed using a scanning electron microscope (SEM) under both low and high magnification conditions; 1.2 Energy dispersive spectroscopy (EDS) was used to perform qualitative analysis of the elemental composition of the sample surface; 1.3 X-ray diffraction (XRD) was used to analyze the phase composition of the sample surface to verify the loading of HA on the PEEK surface.

[0106] 2. Hydrophilicity test The samples were tested using a water contact angle tester. 3. In vitro osteogenic performance test Bone marrow mesenchymal stem cells (BMSCs) from SD rats were selected, and different groups of implants were co-cultured with BMSCs. Osteogenic differentiation of cells was induced during the culture process using osteogenic induction medium.

[0107] Osteogenic induction medium: Osteogenesis induction medium was based on DMEM containing 10% fetal bovine serum, with the following added: Dexamethasone (used to activate osteogenic gene expression); Sodium β-glycerophosphate (used to provide a phosphoric acid source and promote mineralization); Ascorbic acid (used to induce collagen synthesis).

[0108] 3.1 Quantitative test of alkaline phosphatase (ALP) BMSCs were co-cultured with each group of scaffolds until day 7 and day 14. ALP assays were performed according to the instructions of the assay kit, and total protein was standardized using the BCA method to evaluate the level of early osteogenic differentiation.

[0109] 3.2 Alizarin Red Staining (ARS) Quantitative Test BMSCs were co-cultured with each group of scaffolds until day 14 and day 21. Staining and elution quantification were performed according to the instructions of the alizarin red staining kit. The absorbance value (OD) was measured at a wavelength of 550 nm to evaluate the formation of mineralized nodules.

[0110] 3.3 RT-qPCR gene expression analysis The expression levels of osteogenic-related genes in BMSCs co-cultured with each group of scaffolds for 7 days were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The genes detected included ColⅠ, Runx-2, OPN and OCN, in order to evaluate the induction effect of the materials on osteogenic differentiation.

[0111] 4. In vivo bone regeneration performance test Healthy male SD rats (12 weeks old, weighing 250±20 g) were selected to create a circular full-thickness bone defect with a diameter of 5 mm in the parietal region of the skull, thus constructing a critical-size skull defect model. Different groups of implants were implanted into the defect site, with 8 animals in each group. Postoperatively, the animals were fed and observed according to standard animal experimental procedures.

[0112] Micro-CT detection methods Twelve weeks after the operation, the experimental animals were sacrificed and skull specimens were obtained. Micro-CT was used to scan and reconstruct the defect area in three dimensions, and the newly formed bone tissue was quantitatively analyzed using analysis software.

[0113] Table 1. Water contact angle test results

[0114] Table 2 Comparison of skull defect repair effects in rats (12 weeks)

[0115] (1) The technical solution of the present invention can stably construct a bioactive intermediate layer containing HA on the surface of PEEK matrix, and the formation of mineralized layer has clear structural and phase evidence to support it.

[0116] SEM observation revealed dense spherical deposits on the sample surface. EDS analysis showed that the deposited area was rich in Ca and P elements, with a Ca / P ratio of approximately 1.60, close to the theoretical Ca / P ratio of hydroxyapatite (HA) (approximately 1.67). XRD analysis showed a characteristic peak at 2θ = 32.4°, consistent with the (211) crystal plane of HA. These results corroborate each other, indicating that after polydopamine modification and simulated body fluid biomineralization treatment, a bioactive intermediate layer containing HA was successfully formed on the PEEK surface, providing a structural basis for the subsequent construction of a biomimetic bone matrix layer and the improvement of interfacial biological properties.

[0117] (2) The three-layer gradient composite implant constructed in this invention is superior to ordinary PEEK implants and PEEK-HA dual-functional composite implants in terms of surface wettability, and can improve the hydrophilicity of the material surface.

[0118] Water contact angle tests showed that the water contact angles of the ordinary PEEK implant, the PEEK-HA dual-layer composite implant, and the PEEK-HA-ColⅠ three-layer gradient composite implant were 79.10±2.13°, 68.33±2.33°, and 52.11±2.09°, respectively. The results indicate that after sequentially constructing the HA bioactive intermediate layer and the ColⅠ biomimetic bone matrix layer on the PEEK matrix surface, the surface contact angle of the material continuously decreased, and the surface hydrophilicity gradually increased. These results demonstrate that the composite structure design of this invention can effectively improve the surface wettability of PEEK-based materials, providing more favorable surface conditions for cell adhesion and interfacial biological responses.

[0119] (3) The three-layer gradient composite implant of the present invention showed superior comprehensive performance to the control group in terms of in vitro osteogenic induction, indicating that it can effectively promote cell proliferation, osteogenic differentiation and mineralization process.

[0120] In in vitro osteogenic validation experiments, compared with the ordinary PEEK implant group and the PEEK-HA dual-layer composite implant group, the PEEK-HA-ColⅠ three-layer gradient composite implant group showed better BMSC adhesion and proliferation trends in CCK-8 assays; ALP quantitative analysis results showed improved early osteogenic differentiation capacity; ARS quantitative analysis results showed enhanced mineralized nodule formation capacity; RT-qPCR results showed that it could upregulate the expression of osteogenic-related genes such as ColⅠ, Runx-2, OPN, and OCN in BMSCs. These results indicate that the three-layer gradient composite interface constructed in this invention not only improves the initial interaction of the cell-material interface but also further promotes the expression of osteogenic-related signals and the mineralization process, demonstrating good in vitro osteogenic induction ability.

[0121] (4) The three-layer gradient composite implant of the present invention showed good bone regeneration ability in the in vivo bone defect repair model, and could promote the formation of new bone and the reconstruction of bone trabecular structure.

[0122] In a rat model of critical-sized skull defects, micro-CT quantitative analysis at 12 weeks post-surgery showed that the bone volume fraction (BV / TV) and trabecular bone number (Tb.N) of the PEEK-HA-ColⅠ three-layer gradient composite implant group were significantly higher than those of the conventional PEEK implant group and the PEEK-HA two-layer composite implant group. Specifically, the BV / TV of the PEEK-HA-ColⅠ three-layer gradient composite implant group was 26.22±2.12%, and the Tb.N was 3.75±0.41 1 / mm, demonstrating superior new bone formation and trabecular bone structure construction. These results indicate that the present invention can effectively improve bone tissue regeneration in an in vivo bone defect repair environment and has good potential for bone repair applications.

[0123] (5) Based on the comprehensive structural characterization, surface properties, in vitro cell experiments and in vivo animal experiments, the effectiveness of the technical solution of the present invention has been verified.

[0124] This invention constructs a gradient composite interface structure by sequentially building a HA bioactive intermediate layer and a ColⅠ biomimetic bone matrix layer on the surface of a PEEK load-bearing matrix. While maintaining the fundamental load-bearing properties of the PEEK matrix, it achieves a comprehensive effect of surface mineralization layer construction, improved hydrophilicity, enhanced in vitro osteogenic induction, and promoted in vivo bone regeneration. The above test results verify the feasibility and effectiveness of the composite implant of this invention in bone repair and load-bearing implant applications from different perspectives.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A gradient biomimetic polyetheretherketone-hydroxyapatite-type I collagen composite implant, characterized in that, It includes a load-bearing inner layer, a bioactive intermediate layer, and a biomimetic bone matrix layer arranged sequentially from the inside out; in: The load-bearing inner layer contains a polyetheretherketone matrix; The bioactive intermediate layer is disposed on the surface of the load-bearing inner layer and includes a polydopamine-modified interface and hydroxyapatite formed by deposition induced by the polydopamine-modified interface. The biomimetic bone matrix layer is disposed on the surface of the bioactive intermediate layer and contains cross-linked modified type I collagen; The composite implant forms a bioactive gradient distribution structure along its thickness direction.

2. The composite implant according to claim 1, characterized in that, The polyetheretherketone matrix has a microporous structure formed by sulfonation treatment on its surface.

3. The composite implant according to claim 2, characterized in that, The composite implant forms a gradient distribution structure along its thickness, gradually changing from low bioactivity on the load-bearing inner layer side to high bioactivity on the biomimetic bone matrix layer side.

4. A method for preparing the composite implant according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Provide a polyetheretherketone matrix and perform surface treatment on the polyetheretherketone matrix to form a surface microporous structure through sulfonation treatment; S2. Construct a polydopamine coating on the surface of the polyetheretherketone matrix after step S1, and place the polydopamine-modified polyetheretherketone matrix in a simulated body fluid for biomineralization treatment, so that calcium and phosphorus ions in the simulated body fluid form and deposit hydroxyapatite on the surface and pores of the polyetheretherketone matrix, thereby constructing a bioactive intermediate layer. S3. Introduce cross-linked modified type I collagen on the surface of the bioactive intermediate layer to form a biomimetic bone matrix layer, thereby obtaining a molded composite implant; S4. The resulting molded composite implant is cured and sterilized.

5. The preparation method according to claim 4, characterized in that, Step S1 includes roughening the surface of the polyetheretherketone matrix, wherein the roughening treatment is at least one of mechanical grinding, laser treatment, and sandblasting.

6. The preparation method according to claim 4, characterized in that, In step S1, the polyether ether ketone matrix is ​​rinsed and dried after sulfonation treatment.

7. The preparation method according to claim 4, characterized in that, In step S2, the bioactive intermediate layer is constructed using a simulated body fluid immersion method. The pH value of the simulated body fluid is 7.30 to 7.50, and the immersion mineralization time is 24 to 96 hours.

8. The preparation method according to claim 4, characterized in that, The method for preparing the simulated body fluid is as follows: Weigh NaCl 8.035 g / L, NaHCO3 0.355 g / L, KCl 0.255 g / L, K2HPO4·3H2O 0.219 g / L, MgCl2·6H2O 0.203 g / L, CaCl2·2H2O 0.147 g / L, Na2SO4 0.072 g / L; Add each ingredient in sequence and dissolve it completely in deionized water, while maintaining the temperature at 36.5 ℃; The pH of the solution was adjusted to 7.35 using hydrochloric acid and tris(hydroxymethyl)aminomethane. After cooling, store in a 4°C refrigerator for later use.

9. The preparation method according to claim 4, characterized in that, In step S3, the type I collagen solution used to construct the biomimetic bone matrix layer is cross-linked with polylysine solution. The concentration of polylysine solution is 0.05-0.2 mg / mL, and the concentration of type I collagen solution is 0.1-1 mg / mL.

10. The use of the gradient biomimetic polyetheretherketone-hydroxyapatite-collagen composite implant according to any one of claims 1-3 in bone defect repair, bone fusion or weight-bearing bone implantation.

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