3D printing PEEK-based implant CPat3DP and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and oral medicine, and provides a 3D printing PEEK-based implant CP and 3DP as well as a preparation method and application thereof. A 3D printing polyether-ether-ketone (PEEK) implant is used as a carrier, a 3, 4-dihydroxy-L-phenylalanine mediated osteogenic peptide sequence PFS and an antibacterial peptide sequence GL13K are loaded, namely a chimeric peptide sequence, and the CP and 3DP are synthesized. An in-vitro cell experiment verifies the cell compatibility, the adhesion capability and the osteogenic differentiation capability of BMSCs, and an in-vitro bacteria experiment evaluates the capability of inhibiting adsorption, growth, reproduction and biological membrane formation of staphylococcus aureus and escherichia coli. And then implanting the PEEK-based implant in a rat skull defect model infected by staphylococcus aureus to prove the osteogenic differentiation promoting capability of the material. The PEEK-based implant expected to be obtained has good BMSCs adhesion, osteogenic differentiation and microbial infection inhibition capabilities, and is applied to oral maxillofacial bone implantation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a 3D printed PEEK-based implant CP@3DP and its preparation method and application. Further, it relates to a 3D printed polyetheretherketone (PEEK)-based implant with antibacterial and osteogenic dual functions and its preparation method, which is applicable to the repair of oral and maxillofacial bone defects, orthopedic implants, and the treatment of infectious bone defects. Background Art

[0002] In the field of oral medicine, the repair of bone defects is a key link in treatments such as implantology, orthodontics, and maxillofacial surgery. With the aggravation of population aging and the improvement of oral health awareness, the demand for efficient and safe bone repair materials is increasing continuously. Oral and maxillofacial bone defects may be caused by various factors, including periodontal disease, trauma, tumor resection, etc. [1], and all these situations require effective bone repair materials to restore oral function and maxillofacial aesthetics. Traditional autologous bone and allogeneic bone transplantation have achieved certain effects clinically, but there are problems such as large trauma, limited donors, surgical risks, and immune rejection. Therefore, the development of new bone repair materials for oral and maxillofacial regions has important clinical significance.

[0003] Synthetic bone materials are considered ideal bone defect repair and bone implant materials due to their advantages such as less damage to patients, low risk of immune rejection, and low risk of disease transmission [2, 3]. The research and application of such materials can not only improve the treatment effect but also reduce the physical and psychological burden of patients, having important social and economic value.

[0004] As early as the beginning of the 19th century, there were reports of using laboratory-made materials to repair bone defects. So far, people have conducted in-depth research on the application of metals and their composites, bioactive glasses, and polymers in orthopedic implants. Metals and their composites such as stainless steel, cobalt-chromium alloy, and titanium alloy are widely used clinically in the field of bone defect repair due to their load-bearing performance and stability [4], and titanium alloy is widely used as an oral implant on the market. However, metal implants may cause allergies and the release of metal particles, and the elastic modulus of metal materials is much higher than that of bone tissue. Since metal implants bear much higher stress than the surrounding bone tissue, it is easy to cause the surrounding bone tissue to lack stress stimulation and undergo bone resorption, reducing the activity and strength of the surrounding bone tissue, and generating the stress shielding effect. This will reduce the stress borne by the bone tissue, leading to bone loss and poor bone bonding. Bioactive glass has good biocompatibility and long-term bone integration ability, but its brittleness limits its use as a bone implant scaffold.

[0005] Polyether-ether-ketone (PEEK) is a two-phase semi-crystalline polymer with high mechanical strength, stable chemical properties, wear resistance, high temperature resistance, fatigue resistance, and no interference with magnetic resonance imaging. More importantly, the elastic modulus of PEEK (about 3 GPa) is comparable to that of human bone tissue (3 - 20 GPa), so it can effectively alleviate the stress shielding problem at the surgical site and reduce the risk of bone loss [5, 6].

[0006] However, the surface of PEEK is hydrophobic and bio-inert, which easily leads to poor surface bone bonding

[11] . Current research shows that different pore size ranges of bone tissue engineering materials have different biological functions. Nanopores (<0.3 μm) promote cell attachment by inducing the formation of focal adhesions in cells; micropores (0.3 - 100 μm) improve the permeability of the scaffold and promote cell migration; macropores (>100 μm) provide space for nutrient supply, waste removal, and gas diffusion. A mutually connected macroporous structure of 300 - 500 μm is more suitable for capillary and bone ingrowth [12, 13]. Therefore, customized porous structures are of great significance for overcoming the inertness of PEEK materials and promoting in vivo bone integration. The construction of a three-dimensional porous structure in PEEK materials can provide a larger bone bonding area and bone tissue ingrowth space for bone implants, thereby improving the bonding strength between bone and implants.

[0007] Fused Deposition Modeling (FDM) is a common 3D printing method

[14] . Compared with traditional injection molding, it has the advantages of simplified process, improved timeliness, and reduced cost, and can fabricate customized implants that match the bone defect site

[15] . Nevertheless, 3D printing technology has not changed the bioactivity of PEEK materials, and further bioactivity modification is still required

[16] .

[0008] For PEEK-based biomaterials, various implant surface strategies have been adopted to enhance surface activity, including physical, chemical, and biological methods, such as surface topography, organic substances, and growth factors [17 - 19]. However, these methods also have their deficiencies. Acid etching and alkali heat treatment techniques are used to construct relatively complex topographies and may change the inherent properties of implants. Traditional chemical fixation processes are very complex, physical adsorption is non-specific and inefficient.

[0009] Inspired by the anchoring properties of marine mussels on various surfaces, 3,4-dihydroxy-L-phenylalanine (DOPA), which is rich in blue mussel foot proteins (Mefps), has attracted extensive attention

[20] . Current research shows that DOPA easily forms covalent and non-covalent bonds with substrates, and combined with the interaction between catechol groups, enables marine mussel organisms to adsorb onto the surfaces of almost all solid objects under wet conditions, such as rocks, ship bottoms, and cement. DOPA can provide strong adhesion on inorganic and organic surfaces and exhibits good long-term durability in aqueous environments. Therefore, compared with traditional chemical and physical bonding methods, using this for the modification of PEEK implants may provide a more convenient biological strategy for enhancing the substance loading efficiency.

[0010] As important mediators of biological processes, polypeptides have attracted extensive attention in the biomedical field due to their beneficial properties such as potency, receptor selectivity, and low toxicity of metabolites

[21] . The osteogenic peptide sequence (PFSSTKT, PFS) is an affinity peptide sequence of bone mesenchymal stem cells (BMSCs) extracted from platelet-rich plasma. This peptide sequence can significantly increase the adhesion rate of BMSCs on the implant surface by mimicking the growth factor environment released by platelets

[22] . PFS promotes cell spreading and proliferation by binding to specific receptors on the surface of BMSCs, and further enhances its differentiation into osteoblasts. Cao et al. constructed a quartz system loaded with PFS and verified that PFS can not only increase the number of adhered BMSCs but also has the potential to promote the osteogenic differentiation of BMSCs

[23] . This enhanced osteogenic and osteogenic differentiation ability helps to increase the density of mouse pre-osteoblast cells (MC3T3) around the implant, thereby promoting the formation and mineralization of bone tissue and enhancing the bone-bonding ability.

[0011] The antibacterial peptide sequence (GKIIKLKASLKLL, GL13K) is a polypeptide derived from the human salivary gland with antibacterial properties, and it shows bactericidal effects on oral-related pathogenic bacteria in both solid matrices and solution media [24 - 26]. GL13K kills bacteria by disrupting the integrity of the bacterial cell membrane, resulting in the leakage of cell contents. In addition, it can also inhibit the formation of bacterial biofilms, thereby reducing the risk of postoperative infection of implants. These properties of GL13K make it a valuable bioactive molecule for the development of oral implant materials with antibacterial functions.

[0012] Chimeric Peptide (CP) technology is a synthetic peptide molecule composed of two or more peptide segments derived from different sources with different biological activities. The design purpose of chimeric peptides is to combine the unique functions of each peptide segment to create new biological activities or enhance the original functions. Compared with polypeptide mixtures, chimeric peptides usually have better chemical and physical stability, easier dose control, allowing precise concentration of specific functional peptides on the material surface, effectively solving the limitations of grafting sites and challenges related to spatial or orientation control during the grafting process of polypeptide mixtures [27,28].

[0013] In summary, PEEK can be used as an ideal bone implant material for repairing oral and maxillofacial bone defects. The bone-bonding area can be increased through 3D printing technology, but bioactive modification is still required.

[0014] References: [1] Wang X. Fourth National Oral Health Epidemiological Survey Report [M]. People's Medical Publishing House,

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[28] Mu Y, Ma S, Wei P, et al. Multifunctional Modification of SIS Membrane with Chimeric Peptides to Promote Its Antibacterial, Osteogenic, and Healing-Promoting Abilities for Applying to GBR[J]. Advanced Functional Materials, 2021, 31(31): 2101452。 Summary of the Invention

[0015] The present invention provides a 3D printed PEEK-based implant CP@3DP, its preparation method and applications.

[0016] The present invention is realized by the following technical solutions: A 3D printed PEEK-based implant CP@3DP, wherein the 3D printed PEEK-based implant CP@3DP uses a polyether ether ketone (PEEK) scaffold with an interconnected pore structure of 300 - 500 μm prepared by fused deposition modeling (FDM) technology as a carrier. Mediated by mussel-inspired 3,4-dihydroxy-L-phenylalanine (DOPA), the surface is loaded with a bifunctional chimeric peptide (CP) formed by linking the antimicrobial peptide GL13K and the osteogenic peptide PFS through DOPA, forming a composite scaffold implant CP@3DP with antibacterial-osteogenic synergistic functionality; Among them: The amino acid sequence of the antimicrobial peptide GL13K is as shown in SEQ ID NO.9 in the sequence listing, which is GKIIKLKASLKLL; the amino acid sequence of the osteogenic peptide PFS is as shown in SEQ ID NO.10 in the sequence listing, which is PFSSTKT.

[0017] Furthermore, the loading amount of the chimeric peptide CP is 600 - 700 μg / cm². Surface stable modification is achieved by the solution impregnation method, and it exhibits kinetic characteristics of rapid release in the first 3 days and subsequent slow release in phosphate buffer solution.

[0018] The elastic modulus of the composite scaffold implant CP@3DP is 3 - 4 GPa, which matches the elastic modulus of human cancellous bone, and the surface contact angle ≤ 30°, significantly improving hydrophilicity.

[0019] The method for preparing the 3D printed PEEK-based implant CP@3DP includes the following steps: (1) Preparation of the carrier: Design a 3D model with a pore size of 300 - 500 μm and 100% pore connectivity through SolidWorks software, print a PEEK scaffold using FDM technology, and ultrasonically clean and vacuum dry the scaffold in acetone, ethanol, and ultrapure water in sequence, named 3DP; Specifically: Use a Polyga ENGINEER Q300 FDM printer and Polyga IG-1 PEEK wire. Set the nozzle temperature at 420 °C, the build plate temperature at 25 °C, the chamber temperature at 65 °C, the printing speed: 30 mm / min, the gap and fillet at 0.4 mm, and the layer thickness: 0.2 mm, with a porosity of 58.4%; after printing, place it in a vacuum oven at 120 °C for annealing for 2 h to eliminate internal stress. The 3D printed PEEK with a size of φ14×3 mm is polished successively with different grades of silicon carbide sandpaper of 800#, 1200#, and 2000#, and ultrasonically cleaned and dried in acetone, ethanol, and ultrapure water in sequence, named 3DP; (2)Preparation and characterization of the chimeric peptide: Using the Fmoc solid-phase synthesis strategy, the GL13K sequence (GKIIKLKASLKLL), DOPA small molecule unit, and PFS sequence (PFSSTKT) were sequentially linked. The results of peptide synthesis were confirmed by the Kaiser detection method. After cleavage with trifluoroacetic acid and an inducer, the crude peptide was obtained. The crude peptide was purified using a preparative high-performance liquid system (C18 column, acetonitrile / water gradient elution), and the main peak (retention time 12.3 min) was collected. The purity of the peptide was detected by an analytical high-performance liquid system. The purity of the obtained pure peptide after lyophilization was required to be ≥95%.

[0020] The molecular weight of the pure peptide was confirmed by MALDI-TOF-MS with an error ≤0.05%. The chemical formula of the chimeric peptide (CP) is as follows: (3)Preparation of CP@3DP: DOPA solution and chimeric peptide solution with a concentration of 1 mg / mL were respectively used to soak the surface of the 3DP obtained in step (1) for 6 hours. The peptide segment was covalently anchored through the catechol group of DOPA, and then dried with nitrogen to obtain CP@3DP.

[0021] The synthesis of the chimeric peptide adopted the solid-phase peptide synthesis method SPPS, with a purity >95%, and the molecular weight was verified by HPLC and TOF-MS, with a theoretical value of 2393.45 Da.

[0022] The present invention also provides the application of the 3D-printed PEEK-based implant CP@3DP in the preparation of infectious bone defect repair materials, oral implants or orthopedic implants.

[0023] The present invention uses a 3D-printed PEEK implant as a carrier to load the osteogenic peptide PFS and antibacterial peptide sequence GL13K linked by DOPA, that is, the chimeric peptide sequence, to synthesize CP@3DP. The adhesion ability and osteogenic differentiation ability of the PEEK-based implant to BMSCs were verified through in vitro cell and bacteria experiments, and the ability to inhibit the adsorption, growth, and reproduction of Staphylococcus aureus and Escherichia coli was evaluated through in vitro bacteria experiments. Subsequently, the PEEK-based implant was implanted in a rat cranial defect model to verify the osteogenic differentiation ability of the material. It is expected that the obtained PEEK-based implant has good adhesion to BMSCs, osteogenic differentiation, and the ability to inhibit microbial infection, and it will be applied to oral and maxillofacial bone implantation.

[0024] The present invention prepares a PEEK scaffold with an interconnected pore structure through fused deposition modeling technology. Using mussel-inspired 3,4-dihydroxy-L-phenylalanine (DOPA) molecules as a linking unit, the antimicrobial peptide GL13K and the osteogenic peptide PFS are integrated into a chimeric peptide (CP), and surface functionalization is achieved through the solution impregnation method. The CP@3DP scaffold exhibits both excellent antibacterial properties (bacteriostatic rates against Staphylococcus aureus and Escherichia coli ≥ 95%) and osteogenic activity (the expression of alkaline phosphatase gene is increased by 8 times, and the bone volume is increased by 18 times in animal experiments). Its elastic modulus matches that of bone tissue, and the surface hydrophilicity is significantly improved. In vitro experiments confirm its promotion of the adhesion, proliferation, and osteogenic differentiation of bone mesenchymal stem cells (BMSCs). The in vivo rat cranial infection model shows its significant inhibition of bacterial infection and promotion of new bone formation. The multifunctional scaffold of the present invention provides an innovative solution for the repair of infectious bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the preparation process of CP@3DP of the present invention; Figure 2 It is a real photo of Φ14*3mm and Φ8*1mm samples; In the figure: the upper figure is a real photo of Φ14*3mm sample; the lower figure is a real photo of Φ8*1mm sample; Figure 3 It is the analysis of the purity and molecular weight of the chimeric peptide (CP); Time-of-flight mass spectrometry (TOF-MS) and HPLC analysis; In the figure: the upper figure is the time-of-flight mass spectrometry (TOF-MS) of the chimeric peptide purity; the lower figure is the HPLC analysis result; Figure 4 It is a graph of the XPS analysis results; Figure 5 It is a graph of the water contact angle results of the material; Figure 6 It is a graph of the elastic modulus results of the material; Figure 7 It is a graph of the CCK-8 results; Figure 8 It is a graph of the in vitro bacterial plate coating results; Figure 9 It is a graph of the in vitro bacterial SEM results; Figure 10 It is a graph of the ALP, ARS staining results and semi-quantitative results; In the figure: the left figure is the ALP, ARS staining results; the right figure is the semi-quantitative results; Figure 11 It is a graph of the expression of osteogenesis-related genes; Figure 12Schematic diagram of in vivo experiments; Figure 13 Results of in vivo antibacterial smear; Figure 14 Results diagram of three-dimensional reconstruction of rat skull; Figure 15 CT quantitative analysis diagram; Figure 16 Results diagram of in vivo tissue staining. Specific implementation manners

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. The materials cited herein and their cited materials will be incorporated by reference.

[0028] Equivalent technologies of the specific embodiments described that can be understood by those skilled in the art through conventional experiments will be included in this application.

[0029] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0030] I. Experimental animals: SD rats at 6 - 8 weeks old, weighing 250 - 300 g, purchased from the Animal Experiment Center of Shanxi Medical University, and raised for one week before the operation. The feeding process and operation process strictly comply with ethical requirements.

[0031] II. Experimental methods 1. Sample preparation: The Polyga ENGINEER Q300 FDM printer and Polyga IG-1 PEEK wire were used. The nozzle temperature was set at 420 °C, the build plate temperature at 25 °C, and the chamber temperature at 65 °C. The printing speed was 30 mm / min, the gap and shoulder were 0.4 mm, the layer thickness was 0.2 mm, and the porosity was 58.4%. After printing, the samples were annealed in a vacuum oven at 120 °C for 2 h to eliminate internal stress. The disc-shaped PEEK and 3D printed PEEK (φ8×1 mm, φ14×3 mm) were polished with different grades of silicon carbide sandpaper (800#, 1200#, 2000#), and then ultrasonically cleaned and dried in acetone, ethanol, and ultrapure water in sequence, named PEEK and 3DP respectively.

[0032] The Fmoc solid-phase synthesis strategy was adopted to sequentially connect the GL13K sequence (GKIIKLKASLKLL), DOPA small molecule unit, and PFS sequence (PFSSTKT). The results of peptide synthesis were confirmed by the Kaiser detection method. After cleavage with trifluoroacetic acid and an inducer, the crude peptide was obtained. The crude peptide was purified by a preparative high-performance liquid system (C18 column, gradient elution with acetonitrile / water), and the main peak (retention time 12.3 min) was collected. The purity of the peptide was detected by an analytical high-performance liquid system. The purity of the obtained pure peptide after lyophilization was required to be ≥95%. The molecular weight of the pure peptide was confirmed by MALDI-TOF-MS with an error ≤0.05%. Subsequently, the scaffolds were immersed in a solution of 1 mg / mL DOPA and CP at 25 °C and shaken for 6 h, and then dried with nitrogen. Covalent anchoring of the peptide segments was achieved through the catechol groups of DOPA to obtain D@3DP and CP@3DP.

[0033] The actual pictures of the samples were taken using a digital camera to record the sample surface.

[0034] 2. Cell and bacteria preparation: Rat bone marrow mesenchymal stem cells (BMSCs) were used as the cell model. BMSCs were cultured in high-glucose Dulbecco’s Modified Eagle medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37 °C in a 5% carbon dioxide environment. During the culture process, the medium was changed every 48 hours to ensure the stability of cell growth. In in vitro experiments, material samples with a diameter of 14 mm were placed in 24-well plates, and BMSCs of passages 4-7 were inoculated on the sample surface at a density of 2×10 4 cells / well.

[0035] Staphylococcus aureus and Escherichia coli were used as the bacteria models. The single colonies were isolated using the streak plate technique. The amplification and subculture of bacteria were completed using tryptic soy agar medium. The colonies were dissolved in TSA medium to prepare a bacterial suspension, which was diluted to 1×10 7 -108 CFU / mL.

[0036] 3. Sample characterization: The surface elemental composition of PEEK, 3DP, D@3DP, and CP@3DP was obtained using an X-ray photoelectron spectrometer (XPS).

[0037] 4. Water contact angle: The surface hydrophilicity or hydrophobicity of the samples was evaluated using a contact angle measuring instrument. Ultra-pure water (5 μL) was dropped onto the surface of the samples, and photos were taken using a digital camera to record the contact angle (WCA).

[0038] 5. Elastic modulus: The elastic modulus of the samples was determined using a nanoindenter. Five nanoindentation measurements with a depth of 1000 nm were performed at a speed of 10 nm / s, and the test positions were at least 50 μm apart.

[0039] 6. CCK-8 assay: The proliferation effects of PEEK, 3DP, D@3DP, and CP@3DP samples on BMSC cells on days 1, 3, and 5 were evaluated using CCK-8. BMSC cells were seeded on the surface of each sample and cultured at 37 °C until days 1, 3, and 5. The culture medium was replaced with a pre-mixed solution of DMEM / CCK8 at a volume ratio of 10:1. After incubation at 37 °C for 1 hour, 100 μL of the solution from each group was transferred to a 96-well plate. The absorbance value at 450 nm was measured using a multi-functional microplate reader.

[0040] 7. In vitro counting analysis: Colony counting Each sample was co-cultured with a bacterial suspension of 1×10 7 CFU / mL for 12 hours, diluted by a certain multiple until the number of colonies was between 30 and 300. The bacterial suspension was spread on an LB plate, and after culturing for 12 hours, photos were taken and recorded, and the colony-forming units (CFU) were counted. The bacterial survival rate (P) was calculated using the following formula: P (%) = (B / A) × 100%. Where P represents the bacterial survival rate, A is the average colony-forming unit (CFU) count of the control group (PEEK), and B is the colony-forming unit (CFU) count of the experimental groups (3DP, D@3DP, and CP@3DP).

[0041] 8. In vitro bacterial SEM analysis: After co-incubating the bacteria with PEEK, 3DP, D@3DP, and CP@3DP samples for 12 hours, the samples were washed, fixed, dehydrated in gradient, and sputter-coated with gold for imaging.

[0042] 9. ALP staining, ARS staining, and semi-quantitative analysis: Osteogenic induction medium was prepared and used. BMSCs were cultured on the surface of the samples (2×10 4After 7, 14, and 21 days (cells / well), the cells were fixed with 4% paraformaldehyde. For ALP staining, BCIP / NBT working solution was used for 30 minutes, and the stained images were taken and collected under an upright optical microscope. At the same time, cell lysates were collected, and ALP activity was measured at a wavelength of 405 nm using an ALP detection kit. For alizarin red staining, the cells were fixed with 4% paraformaldehyde. After staining with 2% ARS solution for 10 minutes, the images of mineralized nodules were washed and taken. After the stained samples were further soaked in 1% cetylpyridinium chloride solution, the absorbance was measured at a wavelength of 562 nm.

[0043] 10. Detection of osteogenesis-related gene expression of rBMSCs on scaffolds in each group by RT-PCR: Total RNA was isolated using a total RNA extraction kit, and the RNA density was quantified using a NanoDrop 2000 spectrophotometer. cDNA was synthesized using a complementary DNA synthesis kit. RT-PCR was performed using primers and SYBR green M5 HiPer real-time PCR mixture in an ABI Prism 7300 thermal cycler. The 2-ΔΔCt method was used to calculate the mRNA level. The primer sequences are shown in Table 1.

[0044] Table 1: Primer sequences used in RT-PCR experiments 11. In vivo osteogenesis experiment A. Establishment of rat cranial defect model and specimen acquisition: The animal experiment of this invention was approved by the Animal Ethics Committee of the Medical Center of Shanxi Medical University. All procedures were carried out in accordance with the standards described in the Guide for the Care and Use of Laboratory Animals. Ethical number: KQDW-2024-001. Twenty healthy male SD rats weighing about 300 g were used to construct an 8-mm cranial defect model, with 5 rats in each group.

[0045] All rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (2 ml / kg body weight). An 8-mm drill bit was used to create a defect at the cranial suture, removing the entire bone thickness. Then, the samples of each group were randomly implanted into the defect site, with a pure PEEK implant as the control group. After injecting a suspension of Staphylococcus aureus, the muscle and skin were sequentially sutured with absorbable 4-0 surgical sutures.

[0046] B. In vivo antibacterial analysis: Three days after surgery, secretions from the implantation sites were collected from 3 randomly selected SD rats in each group using sterile swabs to evaluate peri-implant infection. The bacterial suspensions from different groups were transferred to 1 mL of sterile PBS containing 0.1% Tween 80 and sonicated. Serial 10-fold dilutions (100 μL) were plated on agar plates and observed and recorded after 24 hours of incubation.

[0047] C. Micro-CT Detection: After 8 weeks, all rats were euthanized by overdose anesthesia. The skull samples were harvested, all soft tissues were removed, and fixed with 4% paraformaldehyde. To evaluate bone formation, 20 circular regions of interest were selected around each implant, and the scanning resolution was set to 10 μm. The bone tissue was three-dimensionally reconstructed using CTvox software. Meanwhile, bone mineral density (BMD), bone volume (BV), bone volume fraction (BV / TV), bone surface (BS), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were measured.

[0048] D. Histological Analysis: After the Micro-CT test, the non-calcified specimens were embedded in polymethyl methacrylate (PMMA) to make sections. Then the tissue blocks were cut into 200-μm sections using an EXAKT300CP hard tissue slicer (EXAKT, Germany), and then ground to a thickness of 20 μm using an EXAKT400S grinding system (EXAKT, Germany). The sections were stained with H&E and Goldner's trichrome, and images were captured with a microscope.

[0049] 12. Statistical Analysis: All experiments were repeated three times, and all data were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 10 software. *p < 0.05 and **p < 0.01 indicate statistically significant results.

[0050] III. Results and Analysis 1. Construction of the 3D Printing Mechanism: 3D printing technology can prepare PEEK samples with specific shapes, pore sizes, and porosities. Studies have shown that an interconnected macroporous structure of 300 - 500 μm is more conducive to the ingrowth of capillaries and bone tissue. As can be seen from the actual pictures of the samples ( Figure 2 ), the pore diameter of the 3D-printed PEEK samples is about 300 - 500 μm and shows an interconnected structure. This design provides a suitable space for angiogenesis and the ingrowth of bone tissue.

[0051] 2. Design and Integration of CP: The scheme illustrates the sequence of the proposed chimeric peptide (Table 2) and its key structural features.

[0052] Table 2: Sequences Used for CP The chimeric peptide consists of three main components: (i) the antimicrobial peptide sequence GL13K; (ii) the osteogenic peptide PFS; (iii) the linking and anchoring unit DOPA.

[0053] This study represents the first chemical bond integration of the antimicrobial peptide GL13K and the osteogenic peptide PFS, aiming to investigate the effects of grafting these two active motifs onto 3D PEEK materials on bacterial and cellular behaviors. To maintain the grafting between the two functional peptides and the 3D PEEK samples while ensuring sufficient spatial separation for the functional peptides to exert their stable bioactivities, the mussel-derived bioactive molecule DOPA was selected as the linker.

[0054] Time-of-flight mass spectrometry (TOF-MS) and HPLC analysis ( Figure 3 confirmed the correct synthesis and purity of the chimeric peptide. XPS analysis (Figure 4) showed a significant increase in the nitrogen (N 1s) content on the surface of the modified scaffold, further confirming the successful introduction of CP. In summary, the introduction of DOPA not only enhanced the loading efficiency of the chimeric peptide on the PEEK surface but also provided more active sites through its chemical bonding and structural characteristics, significantly improving the bioactivity and osteointegration potential of the PEEK scaffold.

[0055] The surface hydrophilicity test (Figure 5) showed that the PEEK and 3DP scaffolds had large contact angles, indicating hydrophobicity. For the scaffolds modified with DOPA and CP (D@3DP and CP@3DP), the contact angles were significantly reduced, especially for CP@3DP, showing a significant enhancement in hydrophilicity. This change laid the foundation for subsequent cell adhesion and biocompatibility improvement.

[0056] The elastic modulus ( Figure 6 ) further verified the changes in the mechanical properties of the scaffold during the modification process. The results showed that there was no significant difference in the elastic modulus between the modified scaffold and the original scaffold, indicating that the modification process did not significantly change the mechanical properties of the scaffold, thus ensuring its structural stability and functional durability. In summary, the above experiments fully demonstrated that the introduction of the chimeric peptide effectively improved the surface properties of the PEEK scaffold without affecting its mechanical properties. This provided a solid experimental basis for the potential of the scaffold in antimicrobial and osteogenic applications.

[0057] 3. Biocompatibility evaluation of the samples: CCK-8 experiment ( Figure 7) showed significant differences in cell proliferation ability among different scaffolds. On the 1st day, the cell proliferation abilities of all groups were similar, and the D@3DP and CP@3DP groups were slightly higher than the PEEK and 3DP groups. However, on the 3rd and 5th days, the cell proliferation rate of the CP@3DP scaffold group was significantly higher than that of other groups. This trend benefited from the promoting effect of the chimeric peptide modification layer, which enhanced cell adhesion and nutrient supply, creating a more favorable microenvironment for cell proliferation. In contrast, the unmodified PEEK and 3DP scaffolds showed lower cell proliferation levels.

[0058] 4. In vitro antibacterial evaluation: The results of in vitro bacterial plating ( Figure 8 ) indicated that the chimeric peptide-modified CP@3DP scaffold exhibited significant antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The CP@3DP scaffold group showed a significant reduction in the colony numbers of both bacteria, while the colony numbers of the D@3DP and 3DP groups were close to those of the control PEEK group and showed almost no antibacterial effect. This indicated that the CP modification layer effectively reduced the growth and reproduction of bacteria through its GL13K antibacterial component.

[0059] The SEM results ( Figure 9 ) further revealed the morphological characteristics of bacteria on the surfaces of different scaffolds. On the surface of the CP@3DP scaffold, both bacteria showed a shrunken or even ruptured state (indicated by the red arrows), suggesting that the bacteriostatic effect of CP might be attributed to the interaction between the chemical functional groups provided by the chimeric peptide modification layer and the bacterial membrane, thus destroying the integrity of the bacterial membrane and leading to bacterial death. The bacteria on the surfaces of the D@3DP and 3DP scaffolds maintained normal morphology, and the bacteria on the surface of the PEEK scaffold were densely distributed and all showed normal morphology without any damage. Considering the above results, CP modification significantly enhanced the antibacterial performance of the 3DP scaffold and could effectively inhibit the growth and reproduction of Staphylococcus aureus and Escherichia coli. This enhanced antibacterial performance might be attributed to the interaction between the chemical functional groups provided by the chimeric peptide modification layer and the bacterial membrane, thus destroying the integrity of the bacterial membrane and leading to bacterial death. These results indicated that the CP@3DP scaffold had excellent antibacterial potential in orthopedic implants, laying a solid foundation for future clinical applications.

[0060] 5. In vitro osteogenesis experiment: ALP staining ( Figure 10), at 7 days, the staining intensity and density of the CP@3DP scaffold group were significantly higher than those of other groups, indicating that it could effectively promote early osteogenic differentiation. Semi - quantitative analysis further verified this. The ALP expression level in the CP@3DP group was significantly higher than that in the D@3DP, 3DP, and PEEK groups, and there was no statistical difference among the three at 7 days. At 14 days, the ALP expression in the D@3DP group was slightly higher than that in the 3DP and PEEK groups. Alizarin red staining showed that the formation of mineralized nodules was consistent with the ALP staining trend. Semi - quantitative analysis showed that the mineralization ability of the CP@3DP group was significantly higher than that of other groups, while the D@3DP was slightly higher than the 3DP and PEEK groups, and there was no statistical difference between the two. These results indicate that chimeric peptide modification significantly improves the osteogenic potential of the material by promoting the formation of the mineralized microenvironment.

[0061] RT - PCR quantitative analysis ( Figure 11 ) showed that the CP@3DP scaffold significantly up - regulated the expression levels of osteogenesis - related genes such as ALP, Runx2, OCN, and OPN, further verifying its promoting effect on osteogenic differentiation. Although there was no statistical difference in the expression of these genes between the D@3DP and 3DP groups, the expression of Runx2 and OCN was higher than that in the PEEK group and was statistically significant. This may be because the D@3DP and 3DP groups, due to insufficient surface modification, could only weakly promote early osteogenic differentiation (such as the expression of ALP), but had limited influence on late differentiation (such as the expression of Runx2, OCN, and OPN).

[0062] Based on the above results, the CP@3DP scaffold significantly enhanced its in vitro osteogenic performance by promoting cell adhesion, osteogenic differentiation, and mineralization. These results indicate that CP modification provides a more suitable osteogenic microenvironment for the 3D PEEK scaffold, demonstrating its great application potential in bone repair and regeneration.

[0063] 6. Bone defect repair under in - vivo microbial infection conditions: In a skull defect model of SD rats under an infection model, we systematically evaluated the in - vivo antibacterial and osteogenic abilities of the implanted scaffolds. By creating a critical - sized skull defect with a diameter of 8 mm and injecting Staphylococcus aureus to simulate the infected environment ( Figure 12 ), the exudates at the scaffold site were collected 3 days after implanting the material for colony counting. The results showed ( Figure 13 ) that except for the CP@3DP scaffold, the colony counts of the other three groups (PEEK, 3DP, and D@3DP) were all in the range of 600 - 700, while the colony count of the CP@3DP group was significantly reduced, only 22, indicating that the CP@3DP scaffold has extremely high antibacterial ability and can effectively inhibit Staphylococcus aureus infection.

[0064] Micro-CT imaging results (Figure 14) showed that the most new bone was formed in the defect area in the CP@3DP group. These new bones not only had significantly higher quantity and volume than other groups, but also could extensively grow into the interconnected macroporous structures of the 3D scaffold, achieving good bone tissue scaffold ingrowth and spatial integration. In contrast, less new bone was formed in the remaining groups and failed to grow into the interconnected pores of the scaffold. Quantitative analysis of Micro-CT data (Figure 15) further supported this conclusion. The CP@3DP group was significantly superior to other groups in key parameters such as bone mineral density (BMD), bone volume (BV), bone volume to tissue volume ratio (BV / TV), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp), demonstrating excellent osteogenic performance.

[0065] HE and Goldner trichrome histological analysis (Figure 16) showed that only the CP@3DP group formed true bone tissue in the defect area, filling the interconnected pore area of the scaffold and achieving true bone bonding with the scaffold material (red arrow), while the other groups mainly repaired with fibrous tissue and less mature bone tissue was formed.

[0066] In summary, the CP@3DP scaffold demonstrated excellent antibacterial and osteogenic performance in the infectious cranial defect model. It could not only significantly inhibit bacterial infection, but also achieve sufficient ingrowth into the macroporous structure of the scaffold material by promoting new bone formation, and finally achieve true bone bonding between bone tissue and the scaffold. These results provided strong support for the clinical application of the CP@3DP scaffold in the repair of infectious bone defects.

[0067] With the antibacterial and osteogenic performance verified by the present invention, the CP@3DP material demonstrated its great potential in the field of bone implants. Its significant antibacterial ability effectively inhibited the infection risk of pathogenic bacteria such as Staphylococcus aureus and Escherichia coli, providing an important guarantee for postoperative infection management. At the same time, its excellent osteogenic performance achieved sufficient filling of the scaffold material by new bone tissue and true bone integration by promoting the adhesion, proliferation, and differentiation of bone cells. The synergistic effect of this antibacterial and osteogenic dual function made CP@3DP an ideal candidate material for the treatment of infectious bone defects.

[0068] In future clinical applications, the CP@3DP material is expected to be extended to the repair of complex bone defects, bone grafting surgeries, and other orthopedic surgeries and oral implant fields related to antibacterial requirements. Its good biocompatibility and excellent bone repair performance provide the possibility of reducing secondary surgeries and improving the postoperative rehabilitation quality of patients. In addition, the design concept of this material can be further applied to personalized medicine to achieve precise repair of complex bone structures through customized scaffold design.

[0069] The present invention successfully prepared a 3D printed polyetheretherketone (PEEK) scaffold functionalized with a chimeric peptide (CP) - CP@3DP. This scaffold demonstrated dual functions in antibacterial and osteogenic properties, significantly enhancing its application potential in the repair of infectious bone defects. The design of the chimeric peptide enables it to effectively inhibit the attachment and growth of bacteria (such as Staphylococcus aureus and Escherichia coli), and at the same time promote the adhesion, proliferation and osteogenic differentiation of osteoblasts. The connection of DOPA ensures its loading amount and loading rate on the PEEK surface, and also ensures the spatial stability of GL13K and PFS respectively. The scaffold constructed by 3D printing technology has an ideal pore structure and mechanical properties, providing a good microenvironment for cell infiltration and new bone formation. At the same time, the sustained - release mechanism of the chimeric peptide further enhances its long - term biological activity.

[0070] The in - vivo experimental results further verified the superior performance of CP@3DP. In the infectious cranial defect model, this material not only significantly cleared the local bacterial infection, but also significantly promoted the formation of new bone and the effective ingrowth of bone tissue into the scaffold, ultimately achieving true bone integration. These results fully demonstrate that the CP@3DP scaffold has significant advantages in dealing with the repair of infectious bone defects. Its excellent antibacterial and bone tissue regeneration capabilities provide new possibilities for the development of clinical bone implants.

[0071] The present invention provides an important reference for the design of future bone repair materials, indicating that antibacterial and osteogenic synergy can be achieved through functionalized 3D printed scaffolds. This strategy not only provides the possibility for the clinical translation of personalized bone regeneration implants, but also opens up a new way for the development of bone tissue engineering materials, promoting the development of the bone repair field.

[0072] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A 3D printed PEEK-based implant CP@3DP, characterized in that: The 3D-printed PEEK-based implant CP@3DP is a polyetheretherketone (PEEK) scaffold with an interconnected pore structure of 300-500 μm prepared by fused deposition modeling (FDM) technology. Mediated by mussel-inspired 3,4-dihydroxy-L-phenylalanine (DOPA), the surface is loaded with a bifunctional chimeric peptide CP formed by linking the antimicrobial peptide GL13K and the osteogenic peptide PFS through DOPA, forming a composite scaffold implant CP@3DP with antibacterial-osteogenic synergistic functionalization; Among them: the sequence of the antimicrobial peptide GL13K is as shown in Sequence Listing SEQ ID NO.9, which is GKIIKLKASLKLL; the sequence of the osteogenic peptide PFS is as shown in Sequence Listing SEQ ID NO.10, which is PFSSTKT.

2. The 3D printed PEEK-based implant CP@3DP according to claim 1, wherein: The loading amount of the chimeric peptide CP is 600-700 μg / cm².

3. The 3D printed PEEK-based implant CP@3DP according to claim 1, wherein: The elastic modulus of the composite scaffold implant CP@3DP is 3-4 GPa, and the surface water contact angle ≤ 30°.

4. Method for preparing the 3D printed PEEK-based implant CP@3DP according to any one of claims 1-3, characterized in that: It includes the following steps: (1) Preparation of the carrier: Design a 3D model with a pore size of 300-500 μm and 100% pore connectivity by SolidWorks software, print a PEEK scaffold using FDM technology, ultrasonically clean the scaffold in acetone, ethanol, and ultrapure water in sequence, and vacuum dry it, named 3DP; (2) Preparation and characterization of the chimeric peptide: Adopt the Fmoc solid-phase synthesis strategy, sequentially connect the GL13K sequence, DOPA small molecule unit, and PFS sequence, and the reaction is confirmed by the Kaiser detection method for the polypeptide synthesis result. After cleavage with trifluoroacetic acid and an inducer, a crude peptide is obtained; the crude peptide is purified by a preparative high-performance liquid system, a C18 column, and gradient elution with acetonitrile / water, and the main peak is collected, with a retention time of 12.3 min. The purity of the polypeptide is detected by an analytical high-performance liquid system, and the purity of the obtained pure peptide after freeze-drying is ≥ 95%; (3) Preparation of CP@3DP: Immerse the 3DP obtained in step (1) in a DOPA solution with a concentration of 1 mg / mL and a chimeric peptide solution on the surface for 6 hours respectively, and realize covalent anchoring of the peptide segment through the catechol group of DOPA, and then dry it with nitrogen to obtain CP@3DP.

5. The preparation method according to claim 4, characterized in that: The synthesis of the chimeric peptide adopts solid-phase peptide synthesis (SPPS), with a purity > 95%, and the molecular weight is verified by HPLC and TOF-MS, with a theoretical value of 2393.45 Da.

6. Use of the 3D-printed PEEK-based implant CP@3DP according to any one of claims 1-3 in the preparation of materials for repairing infectious bone defects, oral implants, or orthopedic implants.

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