Orthopedic implant material with degradable time sequence as well as preparation method and application thereof

Abstract

The invention relates to a time sequence degradable orthopedic implant material which comprises a base material and a biodegradable coating formed on the surface of the base material, the surface of the base material is provided with a nanocone array microstructure, the biodegradable coating takes a biodegradable polymer as a matrix and loads a metal element for promoting bone repair, and the metal element is a metal element for promoting bone repair. And the surface of the biodegradable coating is also connected with an anti-inflammatory active factor. According to the invention, grafted anti-inflammatory active factors are matched with the biodegradable coating loaded with metal elements for promoting bone repair, and the sequential active regulation and control of the biological effect of the micro-morphology on the surface of the base material cope with effector cells in different repair stages; the time sequence regulation and control on the macrophage inflammation phenotype and bone marrow mesenchymal stem cell osteogenic differentiation in different stages of bone repair are realized, so that each stage of bone tissue repair is matched, and bone repair and osseointegration are promoted.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical materials, specifically relating to a time-degradable orthopedic implant material, its preparation method, and its application. Background Technology

[0002] Bone defects are a common phenomenon in orthopedics, usually caused by factors such as trauma or tumors. Human bone tissue possesses a certain self-repairing capacity, meaning that in cases of minor fractures or defects, bones can heal naturally without surgery. However, once a fracture or defect reaches 1.5 times the diameter of the bone shaft, exceeding the body's self-repairing ability, bone resorption and nonunion may occur. The bone defect will then be unable to repair itself, severely impacting the patient's quality of life. Therefore, effectively repairing bone defects remains a major challenge in clinical practice.

[0003] With advancements in materials science and engineering technology, treatment methods are constantly being updated. Current research both domestically and internationally indicates that clinical treatments for bone defects include autologous bone grafting and allogeneic bone grafting. While autologous bone grafting offers excellent integration, it suffers from limitations such as a limited donor site and the risk of secondary injury, failing to meet clinical needs. Allogeneic bone grafting, on the other hand, has the advantage of a wide range of sources and forms, allowing for the use of various biomaterials as bone substitutes. However, it also presents challenges such as low biocompatibility, pathogen infection, and difficulties in bone integration. Due to these drawbacks of both autologous and allogeneic bone grafting, tissue engineering has focused on researching bone graft substitutes over the past decade, designing biomaterials with different structures and properties.

[0004] Bone repair is a complex mechanism involving interactions among many different cell types, including endothelial cells, neurons, osteoblasts, osteoclasts, and osteoprogenitors generated through the secretion of soluble factors. It can be broadly divided into three phases: the initial inflammatory phase, the proliferative phase, and the remodeling phase. The inflammatory phase occurs immediately after fracture, with hematoma formation and the release of a series of mediators (damage-associated molecular patterns, DAMPs), pro-inflammatory cytokines, and chemokines by dying or dying cells to recruit and activate other inflammatory cells. Following the recruitment of immune cells, a series of mediators, including bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF), are released, which promote further recruitment and differentiation of reparative osteoprogenitors and mesenchymal cells at the fracture site. Studies on human fracture hematomas have shown elevated levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and IFN-γ, along with increased levels of chemokines, including monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES (which regulate T cell expression and secretion when activated normally), as well as vascular endothelial growth factor. Based on a deeper understanding of bone healing around orthopedic implants, the interaction between bone and implants is a dynamic process involving different stages. Current bone repair materials often focus only on certain stages of the bone repair process and cannot programmatically match different stages of bone repair, thus failing to achieve complete bone repair and excellent osseointegration.

[0005] In recent years, among these biomaterials, polyetheretherketone (PEEK), as a novel medical polymer, has been highly valued by researchers both domestically and internationally as a candidate material for bone implants. Its excellent biocompatibility, mechanical properties, stable chemical properties, and radiation permeability have shown great potential in the field of bone defect repair. However, the PEEK surface is bioinert; therefore, if pristine PEEK material is used solely as a bone implant, its in vivo cellular signaling response and integration with surrounding bone tissue are clinically unsatisfactory. Summary of the Invention

[0006] To address at least one of the shortcomings of the existing technology, the present invention aims to provide a time-degradable orthopedic implant material that, through a series of processes on the material surface, can achieve the time-sequential release of multiple active factors that promote bone repair. These active factors released at different stages can match the needs of different stages in the bone repair process, temporally matching the bone repair progress, thereby promoting the bone repair effect at the defect site.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a time-degradable orthopedic implant material comprising a substrate and a biodegradable coating formed on the surface of the substrate; the surface of the substrate has a nanocone array microstructure; the biodegradable coating is based on a biodegradable polymer and loaded with metal elements that promote bone repair; the surface of the biodegradable coating is also connected to anti-inflammatory active factors.

[0009] In some embodiments of the present invention, the substrate is selected from polymeric materials suitable for orthopedic implantation, including at least one of polyetheretherketone (PEEK), ultra-high molecular weight polyethylene (UHMWPE), polymethyl methacrylate (PMMA), and polytetrafluoroethylene (PTFE).

[0010] In some embodiments of the present invention, the biodegradable polymer includes at least one of poly(trimethylene carbonate) (PTMC), poly(lactic acid) (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO), polyhydroxyalkanoates (PHA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyglycolic acid-dioxanone copolymers (Glycolide-Dioxanone Copolymers), poly(lactide-co-caprolactone) (PLCL), and polyglycolic acid-trimethylene carbonate copolymers (PGA-PTMC Copolymers).

[0011] In some embodiments of the present invention, the metal element that promotes bone repair includes at least one of magnesium (Mg), iron (Fe), cerium (Ce), lithium (Li), calcium (Ca), strontium (Sr), zinc (Zn), and copper (Cu).

[0012] In the early stages of implantation, magnesium participates in inflammatory activation processes at the implantation site, including protein adsorption, inflammatory cell recruitment, monocyte differentiation, macrophage M1 phenotype polarization, and phagocytosis. After degradation and stabilization, appropriate release of magnesium ions, hydroxide ions, and hydrogen tends to promote macrophage M2 phenotype polarization, thereby inducing the creation of an anti-inflammatory immune microenvironment conducive to tissue regeneration. Under this favorable immune microenvironment, M2 macrophages interact with bone healing-related cells such as mesenchymal stem cells, promoting bone regeneration and participating in angiogenesis at the defect site. Furthermore, magnesium participates in the entire angiogenesis process, including the production of angiogenesis-related factors such as hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF), degradation of the vascular basement membrane, endothelial cell proliferation, migration, tube formation, and vascular maturation and stabilization. Notably, sensory nerves and their secreted calcitonin gene-related peptide have been shown to play a crucial role in magnesium-promoted endothelial cell migration and tube formation, and their mediated activation of the CGRP-FAK-VEGF signaling axis participates in angiogenesis at the bone defect site. Furthermore, under the influence of appropriate concentrations of magnesium ions, the upregulation of platelet-derived growth factor-BB (PDGF-BB), a key factor in angiogenesis, can achieve angiogenesis-osteoogenesis coupling, thereby significantly promoting bone healing.

[0013] Lithium is a promising element for bone regeneration. It plays a role in bone defect repair through multiple pathways, including promoting osteoblast proliferation, osteogenic differentiation of bone marrow mesenchymal stem cells, angiogenesis, and upregulating the expression of osteogenic differentiation factors. In the biomedical field, lithium biomedical materials mainly include lithium-containing bone matrix materials, lithium-containing bioscaffold materials, and lithium-containing hydrogels. Therefore, utilizing lithium and related biomedical materials to achieve bone tissue regeneration and repair has become a new approach and method.

[0014] Calcium in hydroxyapatite (Ca 10 As a major component of bone minerals, the (PO4)6(OH)2 form can directly participate in the mineralization process of new bone matrix and act as an intracellular signaling molecule to regulate osteoblast differentiation and function.

[0015] Strontium has a bidirectional regulatory effect on osteoblasts and osteoclasts involved in bone repair, simultaneously promoting osteoblast activity and inhibiting osteoclast activity. It can enhance bone matrix protein expression by activating osteoblast calcium-sensitive receptors (CaSR) and inhibit osteoclast differentiation factor (RANKL) by upregulating osteoprotegerin (OPG). Commonly used strontium-containing drugs in clinical practice include strontium ranelate, used to treat osteoporosis.

[0016] Zinc can promote bone formation by enhancing the proliferation, differentiation and mineralization of osteoblasts; it has certain antibacterial properties, which can inhibit bacterial infection around implants, thereby reducing the interference of inflammatory response on bone healing; and it acts as a cofactor for many enzymes (such as alkaline phosphatase) to regulate the activity of bone metabolism enzymes.

[0017] Copper promotes angiogenesis by upregulating vascular endothelial growth factor (VEGF) to accelerate the formation of new blood vessels, thereby providing oxygen or nutrients for bone repair. In addition, copper has antibacterial and anti-inflammatory effects, regulating the inflammatory microenvironment by reducing the risk of infection. Copper can also promote osteoblast activity at low concentrations, thereby promoting osteoblast repair and stimulating osteoogenesis.

[0018] In some embodiments of the present invention, the bone-repair-promoting metal element is loaded in ionic form into a biodegradable coating.

[0019] In some embodiments of the present invention, the anti-inflammatory active factors have inflammatory regulatory functions, and the anti-inflammatory active factors include interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), TGF-β (transforming growth factor-β), lipoxin, resolvin, COX-2 (cyclooxygenase-2), HO-1 (heme oxygenase-1), and polypeptides with anti-inflammatory activity.

[0020] In some embodiments of the present invention, the peptides with anti-inflammatory activity include peptides with the sequences KVLDGQDP (SEQ ID NO:1), IVYPWTQR (SEQ ID NO:2), GEAGPAGPAGPAGPR (SEQ ID NO:3), GPAGPSGPAGK (SEQ ID NO:4), FDKPVSPLL (SEQ ID NO:5), GPETAFLR (SEQ ID NO:6), and IVPAS (SEQ ID NO:7).

[0021] In some embodiments of the present invention, the nanocone array microstructure on the substrate surface is constructed using a plasma etching method for nanospheres.

[0022] In some embodiments of the present invention, the nanospheres include polystyrene microspheres.

[0023] The concept of this invention is as follows: In the early stage of bone repair, during the inflammatory regulation phase, by releasing anti-inflammatory active factors (such as peptides with anti-inflammatory activity) on the surface of the material, the inflammatory signaling pathway is actively regulated, the activity of related immune cells is modulated, the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages is promoted, the secretion of pro-inflammatory factors is reduced, and the expression of anti-inflammatory factors is increased, thereby effectively inducing the timely resolution of the inflammatory response and creating a microenvironment conducive to bone integration; in the middle and late stages of bone repair that promotes osteogenic differentiation and mineralization, by continuously releasing osteogenic metal elements loaded on a biodegradable coating based on a biodegradable polymer matrix (… For example, lithium, magnesium, calcium, strontium, zinc, copper, iron, and cerium are used to promote osteoblast differentiation, inhibit excessive osteoclast activity, improve bone matrix mineralization capacity, increase new bone mass, and ensure close integration between the implant and bone tissue. Finally, in the later stage of bone repair, in order to enhance bone integration, a nanocone array microstructure is constructed on the surface of the substrate. This structure significantly enhances the mechanical interlocking and biocompatibility between the material and the host bone tissue in the later stage. On the other hand, its physical morphology itself helps macrophages to polarize to the M2 type, while providing a tighter bonding interface for the above-mentioned biodegradable coating, ensuring the stability of the coating and the sequential release of its functions.

[0024] A second aspect of the present invention provides a method for preparing a time-degradable orthopedic implant material, the method comprising the following steps:

[0025] 1) Assemble nanospheres on a substrate and etch the nanospheres on the substrate by plasma etching to obtain a substrate with a nanocone array microstructure on the surface.

[0026] 2) The biodegradable polymer is uniformly dispersed in solvent A to obtain solution A, and the metal salt that promotes bone repair is uniformly dispersed in solvent B to obtain solution B. Solution A and solution B are uniformly mixed and then spin-coated onto the surface of the substrate obtained in step 1). After removing the solvent, a biodegradable coating loaded with metal elements is obtained.

[0027] 3) The surface of the biodegradable coating obtained in step 2) is activated by gas plasma immersion ion implantation;

[0028] 4) Load anti-inflammatory active factors onto the biodegradable coating surface activated in step 3).

[0029] In some embodiments of the present invention, step 1) of assembling nanospheres on a substrate includes:

[0030] 1a) Preparation of nanosphere dispersion: Disperse nanospheres in ethanol solution, ultrasonically disperse and vortex to obtain nano-suspension emulsion;

[0031] 1b) The nano-suspension emulsion obtained in step 1a) is slowly added to distilled water, where it self-assembles into a monolayer microsphere membrane on the water surface;

[0032] 1c) Transfer the monolayer microsphere film on the water surface to the substrate surface by dip-coating method, and dry the substrate surface.

[0033] In some embodiments of the present invention, step 1) of etching the nanospheres on the substrate by plasma etching includes:

[0034] 1d) Place the substrate obtained in step 1c) into the sample chamber of the plasma etching machine and evacuate it;

[0035] 1e) Set the RF power supply to 90-110W, introduce gas to make the pressure in the sample chamber reach 0.1-0.2mbar, activate plasma etching for 10-15min, and then cool.

[0036] In some embodiments of the present invention, the preparation method of the time-degradable orthopedic implant material further includes substrate pretreatment, the pretreatment steps of which include: sanding the substrate with sandpaper until it is semi-mirror-like, ultrasonically cleaning the substrate with acetone, anhydrous ethanol and deionized water in sequence, drying it in a drying oven, and finally performing hydrophilic treatment in plasma.

[0037] In some embodiments of the present invention, the power of the plasma hydrophilic treatment is 290-300W and the time is 170-180s.

[0038] In some embodiments of the present invention, the substrate is selected from polymeric materials suitable for orthopedic implantation, including at least one of polyetheretherketone (PEEK), ultra-high molecular weight polyethylene (UHMWPE), polymethyl methacrylate (PMMA), and polytetrafluoroethylene (PTFE).

[0039] In some embodiments of the present invention, the nanospheres in step 1) include polystyrene microspheres.

[0040] In some embodiments of the present invention, the size of the polystyrene microspheres ranges from 400 to 600 nm.

[0041] In some embodiments of the present invention, the volume concentration of the ethanol solution in step 1a) is 40-60%.

[0042] In some embodiments of the present invention, the gas introduced in step 1e) is selected from at least one of oxygen, nitrogen, and argon.

[0043] In some embodiments of the present invention, the biodegradable polymer in step 2) includes at least one of polytrimethylene carbonate (PTMC), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polydioxanone (PDO), polyhydroxyalkanoates (PHA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyglycolic acid-dioxanone copolymers (Glycolide-Dioxanone Copolymers), polylactic acid-co-caprolactone copolymers (PLCL), and polyglycolic acid-trimethylene carbonate copolymers (PGA-PTMC Copolymers).

[0044] In some embodiments of the present invention, the biodegradable polymer in step 2) includes PLGA, wherein the molecular weight of the PLGA is in the range of 20 to 100 kDa.

[0045] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) contains at least one metal element selected from magnesium, lithium, calcium, strontium, zinc, copper, iron, and cerium.

[0046] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) contains two or more metal elements selected from magnesium, lithium, calcium, strontium, zinc, copper, iron, and cerium.

[0047] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) includes at least one of magnesium salt, lithium salt, calcium salt, strontium salt, zinc salt, copper salt, iron salt, and cerium salt.

[0048] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) includes silicates and phosphates containing at least one metal element selected from magnesium, lithium, calcium, strontium, zinc, copper, iron, and cerium.

[0049] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) includes silicates and phosphates containing two or more of the following metal elements: magnesium, lithium, calcium, strontium, zinc, copper, iron, and cerium.

[0050] In some embodiments of the present invention, the metal salt that promotes bone repair in step 2) includes at least one of lithium magnesium silicate, potassium bicarbonate, potassium citrate, polyphosphate, ranelate, calcium phosphate, and sodium alendronate.

[0051] In some embodiments of the present invention, solvent A in step 2) includes at least one of dichloromethane, trichloromethane, acetone, and tetrahydrofuran.

[0052] In some embodiments of the present invention, solvent B in step 2) includes at least one of aqueous ethanol, aqueous methanol, and dimethyl sulfoxide.

[0053] In some embodiments of the present invention, solvent B in step 2) comprises a 40-60% aqueous ethanol solution.

[0054] In some embodiments of the present invention, the mass concentration of the biodegradable polymer in solution A in step 2) ranges from 0.05 to 0.1 g / mL.

[0055] In some embodiments of the present invention, the mass concentration of the bone-repairing metal salt in solution B in step 2) ranges from 0.05 to 0.1 g / mL.

[0056] In some embodiments of the present invention, the mixing ratio of solution A and solution B in step 2) is 3:1 to 1:1.

[0057] In some embodiments of the present invention, the gas in the gas plasma immersion ion implantation in step 3) includes at least one of oxygen, nitrogen, and argon.

[0058] In some embodiments of the present invention, the activation process in step 3) includes: adjusting the gas inlet flow rate to make the pressure range of the sample chamber between 0.15 and 0.25 mbar, setting the radio frequency power supply to 90 to 110 W, and activating the plasma for 5 to 10 minutes.

[0059] In some embodiments of the present invention, the anti-inflammatory active factors in step 4) include interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1Ra), TGF-β (transforming growth factor-β), lipoxin, resolvin, COX-2 (cyclooxygenase-2), HO-1 (heme oxygenase-1), and polypeptides with anti-inflammatory activity.

[0060] In some embodiments of the present invention, the peptides with anti-inflammatory activity include peptides with the sequences KVLDGQDP (SEQ ID NO:1), IVYPWTQR (SEQ ID NO:2), GEAGPAGPAGPAGPR (SEQ ID NO:3), GPAGPSGPAGK (SEQ ID NO:4), FDKPVSPLL (SEQ ID NO:5), GPETAFLR (SEQ ID NO:6), and IVPAS (SEQ ID NO:7).

[0061] In some embodiments of the present invention, step 4) specifically includes: incubating the biodegradable coating activated in step 3) with an anti-inflammatory active factor solution to obtain the time-degradable orthopedic implant material.

[0062] In some embodiments of the present invention, the concentration of the anti-inflammatory active factor solution is 0.4 to 0.6 mg / mL.

[0063] In some embodiments of the present invention, the incubation time is 12 to 24 hours.

[0064] A third aspect of the present invention provides the use of the time-degradable orthopedic implant material described in the first aspect or the time-degradable orthopedic implant material prepared by the preparation method described in the second aspect in the preparation of bioengineering scaffolds.

[0065] In some embodiments of the present invention, the bioengineered scaffold includes bone injury repair materials.

[0066] A fourth aspect of the present invention provides a medical product comprising the time-degradable orthopedic implant material described in the first aspect above, or the time-degradable orthopedic implant material prepared by the preparation method described in the second aspect.

[0067] The beneficial effects of this invention are as follows:

[0068] This invention utilizes grafted anti-inflammatory active factors, combined with a biodegradable coating loaded with metal elements that promote bone repair, and the temporal active regulation of the biological effects of the substrate surface micromorphology to address effector cells at different repair stages. This enables the temporal regulation of macrophage inflammatory phenotypes and osteogenic differentiation of bone marrow mesenchymal stem cells at different stages of bone repair, thereby matching each stage of bone tissue repair and promoting bone repair and bone integration.

[0069] The temporal regulatory effects of this application specifically include: in the early stage of bone repair, during the inflammation regulation phase, by releasing anti-inflammatory active factors (such as peptides with anti-inflammatory activity) on the surface of the material, actively regulating inflammatory signaling pathways, modulating the activity of related immune cells, promoting the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, reducing the secretion of pro-inflammatory factors, and increasing the expression of anti-inflammatory factors, thereby effectively inducing the timely resolution of the inflammatory response and creating a microenvironment conducive to bone integration; in the middle and late stages of bone repair that promotes osteogenic differentiation and mineralization, by continuously releasing pro-inflammatory factors loaded on a biodegradable coating based on a biodegradable polymer, the effects of this method are further enhanced. Osteogenic metal elements (such as lithium and magnesium) promote osteoblast differentiation, inhibit excessive osteoclast activity, enhance bone matrix mineralization, increase new bone mass, and ensure close integration of the implant with bone tissue. Finally, in the later stages of bone repair, to enhance osseointegration, a nanocone array microstructure is constructed on the substrate surface. This structure significantly enhances the mechanical interlocking and biocompatibility between the material and the host bone tissue in the later stages. On the other hand, its physical morphology itself helps macrophages polarize to the M2 type, while providing a tighter bonding interface for the aforementioned biodegradable coating, ensuring coating stability and the sequential release of functions.

[0070] The technologies involved in this invention have significant advantages such as simplicity, efficiency, and high repeatability, which ensure their subsequent batch and industrial production. Attached Figure Description

[0071] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0072] Figure 1 This is a flowchart illustrating the preparation process of the time-degradable orthopedic implant material in Example 1.

[0073] Figure 2 The images are planar scanning electron microscope images of samples PEEK, ETCH, PLGA, LAP, and PEP from Example 1.

[0074] Figure 3 The hydrophilicity and hydrophobicity of the sample surfaces in Example 1 are represented by "*" for p < 0.05, "**" for p < 0.01, "***" for p < 0.001, and "****" for p < 0.0001.

[0075] Figure 4 The figures show the full X-ray photoelectron spectroscopy (XPS) spectra of each group of samples in Example 1 and the atomic percentage of the surface elements of each group of materials. Figure 4 (a) shows the XPS full spectrum of each group of samples. Figure 4 (b) represents the atomic percentage of each element on the surface of each group of materials.

[0076] Figure 5The release curves of anti-inflammatory peptides and magnesium and lithium ions from sample PEP in Example 1 at different time points are shown.

[0077] Figure 6 The cell viability of RAW264.7 cells cultured on the surfaces of PEEK, PLGA, LAP, and PEP samples in Example 1 for 1, 3, and 5 days was measured.

[0078] Figure 7 The expression of M1 marker genes (TNF-α, iNOS) and M2 marker genes (TGF-β, VEGF) in RAW264.7 cells cultured on the surfaces of PEEK, PLGA, LAP, and PEP samples in Example 1 is shown.

[0079] Figure 8 The results show the fluorescence staining of RAW264.7 cells cultured on the surfaces of PEEK, PLGA, LAP, and PEP samples in Example 1.

[0080] Figure 9 Alkaline phosphatase staining of rat bone marrow mesenchymal stem cells after 7 days of culture on PEEK, ETCH, PLGA and LAP samples in Example 1 (group (a)) and alizarin red staining after 14 days of culture (group (b)). Detailed Implementation

[0081] To better illustrate the purpose, technical solution, and advantages of the present invention, the technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0082] Unless otherwise specified, the raw materials, reagents or apparatus used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0083] Example 1

[0084] This embodiment aims to prepare a time-degradable orthopedic implant material from polyetheretherketone (PEEK). The preparation process is as follows: Figure 1 As shown, the specific preparation steps are as follows:

[0085] 1.1 PEEK Material Pretreatment: Polyetheretherketone (PEEK) was prepared into circular discs with dimensions of Ф15mm×3mm. The disc surfaces were then polished sequentially with 1000, 1200, 2000, 3000, 4000, and 5000 grit sandpaper until they reached a semi-mirror finish. Next, the PEEK was ultrasonically cleaned for 30 minutes each with acetone, anhydrous ethanol, and deionized water, respectively. It was then dried in a forced-air drying oven at 60℃. Finally, it underwent hydrophilic treatment in plasma at a power of 290–300W for 170–180s to facilitate subsequent microsphere loading, thus obtaining the original substrate material. The sample was labeled as PEEK.

[0086] 1.2 Preparation of Nanocone Morphology on PEEK Material Surface: Polystyrene (PS) microspheres with a particle size of 400–600 nm were ultrasonically dispersed and vortexed in a 50% ethanol solution to prepare a nano-suspension emulsion. A clean glass slide was taken, and one side was immersed in a petri dish containing distilled water, while the other side was supported at an angle of approximately 50°. The PS nanosphere suspension emulsion was then slowly dripped onto the tilted glass slide. After the microsphere mixture came into contact with the distilled water surface, it uniformly self-assembled into a monolayer microsphere film on the water surface under the influence of Brownian motion and surface tension. Finally, the PEEK sheet obtained in step 1.1 was immersed below the water surface along the side of the petri dish using tweezers. After transferring the PEEK sheet to the area below the monolayer microsphere film, the PEEK sheet was vertically pulled upwards. This immersion-pulling method transferred the microsphere film from the liquid surface to the PEEK sheet surface. The PEEK sheet was then laid flat and dried at room temperature. Next, the PEEK sheet carrying nanospheres was placed into the plasma device. The RF power supply was set to 90-110W. The RF power supply was turned on and the frequency was maintained at about 40kHz. After oxygen was introduced, the pressure in the sample chamber reached 0.1-0.2mbar. The plasma etching process was activated for 10-15 minutes to obtain a modified PEEK sheet with micro-nano structures on the surface. The sample was denoted as ETCH.

[0087] 1.3 Preparation of PLGA coating: PLGA with a molecular weight of 100 kDa was dissolved in dichloromethane to prepare a PLGA solution with a mass concentration of 0.1 g / mL. Then, the PLGA dichloromethane solution was uniformly spin-coated onto the surface of the ETCH sample obtained in step 1.2 using a spin coater. The sample was then dried at room temperature overnight to obtain a sample with a PLGA coating, which was denoted as the PLGA sample.

[0088] 1.4 Preparation of PLGA coating loaded with magnesium and lithium elements: Synthetic lithium magnesium silicate powder (Laponite) was dissolved in 50% ethanol solution to prepare a lithium magnesium silicate ethanol solution with a mass concentration of 0.1 g / mL. The PLGA dichloromethane solution from step 1.3 was taken, and the two solutions were mixed at a ratio of 1:1 (v / v) to prepare a PLGA solution containing synthetic lithium magnesium silicate with a mass fraction of 0.05 g / mL. The mixed solution was then uniformly coated onto the surface of the PLGA sample obtained in step 1.3 using a spin coater and dried at room temperature overnight to obtain a composite material with a PLGA coating loaded with magnesium and lithium element powder. The sample was denoted as LAP.

[0089] 1.5 Grafting of Anti-inflammatory Peptide Segments: The surface of the composite material obtained in step 1.4 was activated using gas plasma immersion ion implantation technology. Oxygen was used as the gas source, and the pressure in the sample chamber was adjusted to be within the range of 0.15–0.25 mbar. The radio frequency power supply was turned on, the frequency was maintained at approximately 40 kHz, and the power was set to 90–110 W for 5–10 min. The peptide powder was dissolved in ultrapure water to prepare an anti-inflammatory peptide (KVLDGQDP, SEQ ID NO:1) solution with a concentration of 0.5 mg / mL and a volume of 1 mL. The activated composite material was then immersed in the anti-inflammatory peptide solution and reacted at 4 °C for 12 hours to graft the anti-inflammatory peptide segments. The resulting sample was labeled as PEP.

[0090] Figure 2 Scanning electron microscope (SEM) images of samples PEEK, ETCH, PLGA, LAP, and PEP are shown. As shown in the figure, the PEEK group exhibits a completely smooth initial surface compared to the other three groups. The ETCH group shows a nano-array topology etched into the material surface. The PLGA group has a biodegradable film coated on the substrate; the small black pores on the surface are left from the condensation and air-drying process and do not affect subsequent steps. The LAP group shows increased metal salt particles compared to the ETCH group, indicating the successful preparation of a bone repair material with a micro-nano morphology and a biodegradable coating. The PEP group, based on the LAP group, has short polypeptide chains grafted onto it; no significant morphological difference is shown between the two groups in the SEM images.

[0091] Example 2

[0092] The static contact angles of the samples (PEEK, ETCH, PLGA, LAP, PEP) in Example 1 were measured using a contact angle meter to characterize the hydrophilicity and hydrophobicity of the sample surfaces. A clean sample was fixed on the sample stage of the contact angle meter, and a droplet method was used to add 20 μL of ultrapure water to the sample surface via a syringe. The contact angle between the droplet and the material interface was then measured.

[0093] The results are as follows Figure 3 As shown, the average contact angle of the original PEEK sample was 88.4°; the average contact angle of the ETCH sample with nano-morphology on the substrate surface was 82.43°; the average contact angle of the PLGA sample coated with a PLGA coating was 70.42°; the average contact angle of the LAP sample coated with a PLGA coating containing magnesium and lithium was 65.58°; and the average contact angle of the PEP sample grafted with anti-inflammatory peptide segments was 59.1°.

[0094] Overall, the contact angles of the sample surfaces in each group showed a trend of PEEK > ETCH > PLGA > LAP > PEP, indicating that changing the surface roughness, coating with PLGA, loading magnesium and lithium elements, using gas plasma immersion ion implantation technology, and grafting anti-inflammatory peptide segments can significantly improve the hydrophilicity of the bone repair material surface, thereby effectively improving the adhesion of cells to the implanted material. Combined with the analysis of Example 5, it can be seen that the compatibility between cells and materials can be improved.

[0095] Example 3

[0096] X-ray photoelectron spectroscopy (XPS) wide-field scanning was performed on the surface of the samples (PEEK, ETCH, PLGA, LAP, PEP) in Example 1 to obtain... Figure 4 (a) shows the XPS full spectrum, where the intensity of the characteristic peaks represents the abundance of the element on the surface. Figure 4 (b) shows the atomic percentage of each element on the material surface obtained from XPS analysis. The results indicate that the main elements on the surface of each sample group are C and O, while other elements are below the detection limit and can be ignored. Figure 4 (a) and Figure 4 (b) Combined, it can be seen that compared with the PEEK group, the carbon content of the EHCH group and PLGA group samples decreased, while the oxygen content increased significantly. This change is attributed to the chemical reaction between active oxygen ions and the sample surface during the oxygen plasma etching and hydrophilic treatment of PEEK, which introduced a large amount of oxygen elements onto the surface. Compared with the previous three groups, the LAP group showed peaks of lithium, magnesium, and silicon, and the atomic content of the corresponding elements also increased accordingly, proving that the PLGA coating loaded with magnesium and lithium elements was successfully covered on the PEEK surface. The XPS full spectrum of the PEP group showed an N1s peak, and the N atom content also increased significantly, indicating that the anti-inflammatory polypeptide chain was successfully grafted onto the material surface through covalent grafting.

[0097] Example 4

[0098] The PEP sample prepared in Example 1 was immersed in 10 mL of ultrapure water and placed in a constant temperature incubator at 37°C. Then, 5 mL of the extract was collected at time points of 1, 4, 7, 14, 21, and 28 days, and 5 mL of ultrapure water was added each time. The concentrations of anti-inflammatory peptides, magnesium, and lithium ions in the extract were then measured, and release curves were plotted. The results are as follows: Figure 5 As shown in the release curve, the anti-inflammatory peptides are released rapidly in the first four days, while magnesium and lithium ions are released at approximately 20% on the first day of immersion incubation and continue to be released until about 28 days. This result demonstrates that PEP material can resolve the inflammatory response through the rapid release of anti-inflammatory peptides in the early stages, and promote macrophage anti-inflammatory polarization through the continuous release of magnesium and lithium elements in the middle and later stages, creating a suitable environment for the differentiation of mesenchymal stem cells into osteoblasts in the later stages.

[0099] Example 5

[0100] Mouse macrophage-like cell line RAW264.7 was seeded onto the surfaces of the samples (PEEK, PLGA, LAP, PEP) in Example 1 and cultured for 1, 3, and 5 days. The original culture medium was then replaced with basal medium containing 10% CCK-8 reagent, and the cells were incubated for another hour in a cell culture incubator under dark conditions. Finally, the absorbance of each sample in the corresponding well was measured at 450 nm using a microplate reader to assess the effect of the material on cell viability.

[0101] The results are as follows Figure 6 As shown, on day 1, there was no significant difference in macrophage proliferation among the different groups. This is because RAW264.7 cells are adherent cells, requiring time to adhere to the material surface and adapt to the changed culture environment after seeding. Therefore, their initial growth rate is relatively slow, and the surface morphology of the material does not show any influence on macrophage proliferation. By day 3, macrophage proliferation in the LAP and PEP groups was higher than that in the PEEK and PLGA groups. This phenomenon may be related to the higher surface roughness of the LAP and PEP groups, as this surface feature facilitates effective cell adhesion. Therefore, the LAP and PEP groups showed better cell proliferation than the PEEK group. The CCK-8 assay results on day 5 were consistent with the trend on day 3, further validating the above conclusions regarding the influence of material surface morphology and composition on macrophage adhesion and proliferation.

[0102] Example 6

[0103] Mouse macrophage-like cell line RAW264.7 was seeded onto the surfaces of the samples (PEEK, PLGA, LAP, PEP) in Example 1 and cultured for 3 days. Cells were lysed using Trizol lysis buffer and total RNA was extracted. The RNA was then reverse-programmed to obtain cDNA. Finally, the obtained cDNA was used for real-time quantitative PCR (qPCR) analysis to determine the expression of macrophage M1 marker genes: inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α), and M2 marker genes: transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF).

[0104] The results are as follows Figure 7 As shown, compared with the PEEK and PLGA materials, the PEP and LAP materials showed higher expression of M2 marker genes (TGF-β, VEGF) and lower expression of M1 marker genes (TNF-α, iNOS). This indicates that the grafted anti-inflammatory peptides and the biodegradable coating containing magnesium and lithium can inhibit macrophage polarization to M1 type while promoting macrophage polarization to M2 type, thereby inhibiting the inflammatory response.

[0105] Example 7

[0106] RAW264.7 cells were seeded onto sterilized PEEK, PLGA, LAP, and PEP substrates. After one day of cell culture, the culture medium was removed, and the cells were washed three times with 1×PBS to remove any residue. Subsequently, the cells were fixed in 4% paraformaldehyde solution at room temperature and incubated for 30 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. After permeabilization, blocking buffer (Beyotime Biotechnology QuickBlock blocking buffer, catalog number: P0231) was added and the cells were incubated for 10 min to reduce non-specific antibody binding.

[0107] Cells from different groups were incubated overnight at 4°C with iNOS primary antibody (M1) (Abogen (Shanghai) Trading Co., Ltd., catalog number: ab115819) and CD163 (M2) primary antibody (Abogen (Shanghai) Trading Co., Ltd., catalog number: ab182422), respectively. Then, Cy3-labeled secondary antibody (Abogen (Shanghai) Trading Co., Ltd., catalog number: ab150081) was added, and the cells were incubated for 1 hour in the dark. After staining, DAPI working solution was added and the cells were incubated for 5 minutes to label the cell nuclei. Finally, the cells were washed three times with 1×PBS to remove unbound dye, mounted with anti-fluorescence quenching mounting medium, and observed and imaged using a confocal fluorescence microscope. Results are as follows: Figure 8 As shown, the fluorescence intensity of CD163 (M2) in the LAP and PEP groups was higher than that in the PEEK and PLGA groups, while the fluorescence intensity of iNOS (M1) was lower than that in the PEEK and PLGA groups. These results indicate that the array micromorphology of nanocones can not only inhibit the polarization of macrophages towards the pro-inflammatory M1 phenotype, but also promote the transformation of macrophages towards the anti-inflammatory M2 phenotype.

[0108] Example 8

[0109] Rat bone marrow-derived mesenchymal stem cells (BMSCs) were seeded onto the surface of various sample groups (PEEK, ETCH, PLGA, and LAP). The BMSCs were cultured in osteoinduction medium to induce osteoblast differentiation. After 7 days of culture, the BMSCs were fixed with 4% paraformaldehyde, and alkaline phosphatase (ALP) expression was semi-quantitatively studied using an alkaline phosphatase (ALP) qualitative assay kit. Results are as follows: Figure 9 As shown in group (a) above. After 14 days of culture, the osteogenic mineralization capacity of the samples was tested using alizarin red staining solution, and the results are as follows. Figure 9 As shown in group (b) above, it can be seen that among the four groups of samples, the LAP group showed the most blue-purple staining spots on the surface of cultured BMSCs, indicating that the ALP expression level of the LAP group was the highest. In addition, the LAP group also showed the most red-purple staining spots, indicating that the osteogenic mineralization ability of the LAP group was the strongest. This shows that the osteogenic differentiation of BMSCs was effectively promoted by the dual effects of nanoarray morphology and osteodegradable coating.

[0110] The applicant declares that the above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A time-degradable orthopedic implant material, characterized in that, The time-degradable orthopedic implant material includes a substrate and a biodegradable coating formed on the surface of the substrate; the surface of the substrate has a nanocone array microstructure; the biodegradable coating is based on a biodegradable polymer and loaded with metal elements that promote bone repair; the surface of the biodegradable coating is also linked to anti-inflammatory active factors.

2. The time-degradable orthopedic implant material according to claim 1, characterized in that, The substrate is selected from polymeric materials suitable for orthopedic implantation, including at least one of polyetheretherketone (PEEK), ultra-high molecular weight polyethylene (UHMWPE), polymethyl methacrylate (PMMA), and polytetrafluoroethylene (PTFE).

3. The time-degradable orthopedic implant material according to claim 1, characterized in that, The biodegradable polymers include at least one of the following: poly(trimethylene carbonate) (PTMC), poly(lactic acid) (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO), polyhydroxyalkanoates (PHA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyglycolic acid-dioxanone copolymers (Glycolide-Dioxanone Copolymers), poly(lactide-co-caprolactone) (PLCL), and polyglycolic acid-trimethylene carbonate copolymers (PGA-PTMC Copolymers).

4. The time-degradable orthopedic implant material according to claim 1, characterized in that, The metal elements that promote bone repair include at least one of magnesium, iron, lithium, cerium, calcium, strontium, zinc, and copper.

5. The method for preparing the time-degradable orthopedic implant material according to any one of claims 1-4, characterized in that, Includes the following steps: 1) Assemble nanospheres on a substrate and etch the nanospheres on the substrate by plasma etching to obtain a substrate with a nanocone array microstructure on the surface. 2) The biodegradable polymer is uniformly dispersed in solvent A to obtain solution A, and the metal salt that promotes bone repair is uniformly dispersed in solvent B to obtain solution B. Solution A and solution B are uniformly mixed and then spin-coated onto the surface of the substrate obtained in step 1). After removing the solvent, a biodegradable coating loaded with metal elements is obtained. 3) The surface of the biodegradable coating obtained in step 2) is activated by gas plasma immersion ion implantation; 4) Load anti-inflammatory active factors onto the biodegradable coating surface activated in step 3).

6. The preparation method according to claim 5, characterized in that, The step of assembling nanospheres on the substrate in step 1) includes: 1a) Preparation of nanosphere dispersion: Disperse nanospheres in ethanol solution, ultrasonically disperse and vortex to obtain nano-suspension emulsion; 1b) The nano-suspension emulsion obtained in step 1a) is slowly added to distilled water, where it self-assembles into a monolayer microsphere membrane on the water surface; 1c) Transfer the monolayer microsphere film on the water surface to the substrate surface by dip-coating method, and dry the substrate surface.

7. The preparation method according to claim 5, characterized in that, The step 1) of etching the nanospheres on the substrate by plasma etching includes: 1d) Place the substrate obtained in step 1c) into the sample chamber of the plasma etching machine and evacuate it; 1e) Set the RF power supply to 90-110W, introduce gas to make the pressure in the sample chamber reach 0.1-0.2mbar, activate plasma etching for 10-15min, and then cool.

8. The preparation method according to claim 5, characterized in that, The activation process in step 3) includes: adjusting the gas inlet flow rate to make the pressure range of the sample chamber 0.15 to 0.25 mbar, setting the radio frequency power supply to 90 to 110 W, and activating the plasma for 5 to 10 minutes.

9. The preparation method according to claim 5, characterized in that, Step 4) includes: incubating the biodegradable coating activated in step 3) with an anti-inflammatory active factor solution to obtain the time-degradable orthopedic implant material.

10. The use of the time-degradable orthopedic implant material as described in any one of claims 1-4, or the time-degradable orthopedic implant material prepared by the preparation method as described in any one of claims 5-10, in the preparation of bioengineering scaffolds; preferably, the bioengineering scaffold includes bone injury repair materials.

11. A medical product, characterized in that, Includes the time-degradable orthopedic implant material according to any one of claims 1-4, or the time-degradable orthopedic implant material prepared by the preparation method according to any one of claims 5-9.

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