Multifunctional fusion peptide modified 3D printing Ti implant constructed by fusion peptide platform and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides a multifunctional fusion peptide modified 3D printing Ti implant constructed by a fusion peptide platform and a preparation method and application thereof. The antibacterial peptide KR-12 and the adhesion peptide GFOGER are simultaneously loaded on the surface of the 3D printing titanium implant through the specific targeting peptide minTBP-1. The sequence of the fusion peptide platform is divided into four main components: bioactive polypeptide sequences KR-12 and GFOGER; a connection unit (Linker); a lysine residue; a specific anchoring group minTBP-1; the composite material has specific loading and slow release capabilities, and utilizes synergistic antibacterial and osteogenesis functions to cope with continuous challenges of implant infection and insufficient osseointegration in biomedical equipment. Meanwhile, sterilization and bone regeneration promotion are achieved, and the method is of great importance to bone healing and enhancement of bone combination around the implant. In addition to the antibacterial effect, the synergistic effect of the antibacterial peptide KR-12 and the adhesive peptide GFOGER can significantly promote osteogenesis of the BMSCs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multifunctional fusion peptide-modified 3D printed Ti implant constructed by a fusion peptide platform, and a preparation method and application thereof. Background Art

[0002] Bone defects caused by trauma, infection, or tumors can severely impair patients' health and quality of life. Conventional treatments, such as drug therapy or bone grafting, are often of limited effectiveness and are associated with serious complications. Titanium (Ti) and its alloys have become the standard material for orthopedic implants due to their excellent mechanical properties, corrosion resistance, and biocompatibility. However, challenges such as high elastic modulus, low surface bioactivity, and high risk of postoperative infection often hinder effective bone integration and increase the likelihood of implant failure. Studies have increasingly emphasized that aseptic loosening and infection are the main causes of implant failure. To improve the long-term stability and success of titanium-based implants, it is crucial to develop surfaces that can promote both bone integration and antimicrobial activity. In this context, surface modification has become a promising strategy to enhance the dual functions of titanium implants: promoting osteogenesis and preventing bacterial infection.

[0003] Functionalized antimicrobial strategies on implant surfaces can significantly reduce the risk of bacterial colonization and infection. In recent years, the main means of enhancing antimicrobial properties include: surface sharp morphology modification for physical sterilization, loading of antibiotics (such as vancomycin, metronidazole), application of bioactive molecules (such as antimicrobial peptides (AMPs), nano-precious metal coatings), and near-infrared (NIR)-assisted therapy. Among them, antimicrobial peptides, as natural antimicrobial agents, have attracted much attention due to their good biocompatibility, low drug resistance and broad-spectrum antimicrobial effects. The KR-12 peptide is a minimal antimicrobial motif (residues 18-29: KRIVQRIKDFLR) designed based on the only human kaesarin (LL-37). It has broad-spectrum antimicrobial activity, can avoid the potential cytotoxicity and hemolysis of the full-length LL-37 motif, and reduce production costs. The bactericidal mechanism of the KR-12 peptide may be due to its α -Helical conformation and positively charged structure, targeting negatively charged bacterial cell membranes through electrostatic interactions, using the "carpet model" to cause bacterial membrane destruction and dissolution, thereby exerting antibacterial effects. In addition to antibacterial activity, KR-12 peptide has been shown to have multiple functions such as immunomodulation, promoting epithelialization, and promoting new bone formation. Importantly, studies have shown that KR-12 can promote the osteogenic differentiation of human bone marrow stem cells (hBMSCs) by activating the BMP / SMAD signaling pathway, which highlights its application prospects in bone tissue regeneration.

[0004] While preventing bacterial colonization, antimicrobial peptides may also interfere with host cell adhesion, thereby hindering the rapid integration of implants into host tissues. How to effectively prevent bacterial infection without affecting the physiological function of osteoblasts is a key challenge in the biomedical field. Therefore, achieving a balance between bacterial inhibition and host cell compatibility is crucial for the success of bone integration. The ideal implant surface should promote the adhesion and proliferation of host cells while inhibiting bacterial adhesion.

[0005] Promoting interactions between host cells and the extracellular matrix (ECM) can direct specific cellular behaviors. α 2 β 1 Integrin is a protein receptor expressed in large quantities on the surface of osteoblasts, regulating cell adhesion, migration, proliferation and signal transduction by binding to ECM collagen. α 2 β 1 The binding of integrin to type I collagen activates signaling pathways that enhance osteoblast differentiation and promote matrix mineralization. GFOGER is derived from type I collagen. α 1-chain short peptide fragment. Its triple helical structure affects the cell membrane α 2 β 1 Integrins have high affinity and specificity. Compared with the commonly used adhesion peptide RGD, GFOGER has obvious advantages in osteogenic performance due to the lack of competitive binding sites and its inherent property of selectively targeting osteoblasts. As a multipotent cell, bone marrow mesenchymal stem cells can differentiate into osteoblasts and secrete growth factors that promote bone regeneration. This makes them an ideal model for studying the interaction between materials and cells. We expect that the application of GFOGER will significantly promote the osteogenic differentiation of bone marrow mesenchymal stem cells. However, precise control of the proportion and delivery of peptides on titanium surfaces remains a major challenge.

[0006] To date, many research strategies have attempted to graft bioactive peptides onto titanium surfaces. These strategies include physical and chemical methods. However, due to the weak nature of non-covalent bonds, physical binding often lacks stability or may lead to excessive release. In contrast, the chemical binding process is complex and often involves the use of toxic substances. Therefore, developing a simple, stable, and safe multifunctional high-density grafting modification method is an attractive research goal in the field of surface bioengineering.

[0007] Recent advances in phage and cell surface display technologies have enabled researchers to isolate a peptide called Mintitanium Binding Peptide -1 (minTBP-1, RKLPDA), which can specifically recognize Ti surfaces without the need for surface pretreatment or harsh reaction conditions. The peptide minTBP-1 specifically binds to the titanium oxide layer through electrostatic interactions. During this binding process, the -O - and -OH 2 + The groups interact with the R1 and D5 residues of minTBP-1, respectively. Our goal was to enhance the osteointegration of titanium implants by constructing a fusion peptide (FP) by combining minTBP-1 with peptides KR-12 and GFOGER. In addition, the introduction of the peptide platform can demonstrate two complementary or synergistic bioactive sequences through precise chemical control. The construction of a peptide platform with an orthogonally protected lysine (Lys) as a branch structure capable of connecting two functional sequences (such as RGD and PHSRN) has been shown to significantly promote the adhesion, spreading, proliferation and differentiation of osteoblasts. These studies provide a solid foundation for our subsequent experimental design and theoretical analysis. Therefore, can the combination of peptide platform technology and specific targeting peptides achieve one-step multifunctional modification of Ti implants and enhance their osteointegration ability? Three-dimensional (3D) printing technology offers a high degree of freedom and precise customizability, allowing the design of structures that closely mimic the characteristics of bone. This capability provides an optimal environment for cellular interaction and adaptation. Titanium implants (3DTi) produced via 3D printing have a nano-scale rough surface that enhances cell adhesion and effectively reduces the elastic modulus of the implant to a level comparable to that of human bone. This adjustment can minimize the stress shielding effect around the implant, thereby reducing the loss of supporting bone and implant failure rates. Therefore, the integration of 3D printing technology represents a major advancement in enhancing osseointegration. Summary of the invention

[0008] The present invention provides a multifunctional fusion peptide-modified 3D-printed Ti implant constructed on a fusion peptide platform, and a preparation method and application thereof.

[0009] The present invention is achieved by the following technical scheme: a multifunctional fusion peptide modified 3D printed Ti implant constructed by a fusion peptide platform, through which the antimicrobial peptide KR-12 and the adhesion peptide GFOGER are simultaneously loaded on the surface of the 3D printed titanium implant through the specific targeting peptide minTBP-1 through the fusion peptide platform technology.

[0010] The sequence of the fusion peptide platform is shown in Table 1, which is divided into four main components: (i) biologically active peptide sequences KR-12 and GFOGER; (ii) linker unit; (iii) lysine residues; (iv) specific anchor group minTBP-1; and a rigid linker EAAAK containing a large number of hydrogen bonds is used as a spacer unit; Table 1 .

[0011] The method for preparing the multifunctional fusion peptide-modified 3D printed Ti implant constructed by the fusion peptide platform is as follows: (1) Preparation of fusion peptides: Based on the listed sequences and synthesis methods, the antimicrobial peptide KR-12, adhesion peptide GFOGER and targeting peptide minTBP-1 were synthesized; the molecular structure of the fusion peptides was predicted and analyzed using protein analysis software PSIPRED and PEP-FOLD; (2) Preparation of Ti material: The titanium substrate was polished step by step with silicon carbide sandpaper No. 400, 800, 1000, 1200, 1500 and 2000, and then ultrasonically cleaned with acetone, 75% ethanol and deionized water for 20 minutes; finally, the substrate was sterilized with ultraviolet light for 3 hours after drying. (3) Peptide loading: FITC-labeled peptide powder was dissolved in sterile deionized water to prepare peptide solutions with concentrations of 1, 10, 100, and 1000 ug / ml. Subsequently, the peptide solutions were added dropwise to the surface of the 3DTi sheet placed in a 24-well plate, 300 ul per well, and incubated overnight at room temperature. The next day, unbound peptides were washed away with phosphate-buffered saline (PBS). 3DTi was incubated at room temperature for 5 min or 2 h to observe its binding ability; the three modified 3DTi were labeled as 3DTi-FP1, 3DTi-FP2 and 3DTi-NFP, respectively.

[0012] Furthermore, the polypeptide concentration is 100 ug / ml.

[0013] For the first time, the present invention uses peptide platform technology to simultaneously load the antimicrobial peptide KR-12 and the adhesion peptide GFOGER on the surface of 3D printed titanium implants through the specific targeting peptide minTBP-1, thereby achieving local and targeted effects and preventing them from being released into the blood. The introduction of the linker effectively separates the functional domains, maintains the structural stability of the fusion peptide, and promotes the positive function of the functional peptide. More importantly, the synergistic effect of KR-12 and GFOGER may promote an unreported bone integration mechanism. This study systematically explored the surface morphology, element distribution, roughness, wettability and biocompatibility of 3D printed titanium implants loaded with a novel multifunctional fusion peptide (NFP), as well as the release ability of the fusion peptide. In addition, the biocompatibility, cell adhesion, antibacterial activity and osteogenesis of the 3D Ti-loaded fusion peptide were evaluated by in vitro experiments and in vivo infected bone defect models. Finally, transcriptome sequencing was used to analyze the relevant cytokines and signaling pathways to explore the mechanism of bone integration. The construction of this new fusion peptide will achieve local delivery and targeting effects of active peptide molecules, while exerting antibacterial and osteogenic functions to promote effective bone integration around titanium implants.

[0014] The present invention successfully loaded the novel fusion peptide onto 3D-printed titanium-based implants, endowed them with specific loading and sustained release capabilities, and utilized their synergistic antibacterial and osteogenic functions to address the persistent challenges of implant infection and insufficient bone integration in biomedical devices. The 3DTi-NFP implant surface was able to simultaneously achieve bactericidal and promote bone regeneration, which is essential for bone healing and enhancing bone integration around implants. In particular, in addition to the antibacterial effect, the synergistic effect of the antimicrobial peptide KR-12 and the adhesion peptide GFOGER significantly promoted the osteogenesis of BMSCs, one of the molecular mechanisms of which may involve focal adhesion and activation of the PI3K-Akt signaling pathway. This mechanism accelerated the regeneration of new bone around the implant. Therefore, the present invention established a promising method to successfully endow 3DTi-based implants with antibacterial and osteogenic properties, providing significant clinical potential for alleviating surgical complications and implant failure caused by bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the design and manufacture of the 3DTi implant functionalized with the novel multifunctional fusion peptide according to the present invention, which is used to repair the bacterially infected rat skull defect model; Figure 2Schematic diagram of the structure, loading and release of the fusion peptides of the present invention; as shown in the figure: (A) The secondary and tertiary structures of the fusion peptides FP1 and FP2 were predicted using PSIPRED and PEP-FOLD software; (B) The optimal concentrations of FP1, FP2 and NFP fusion peptides were screened for quantification of pixel density; (C) Images of FP1, FP2 and NFP fusion peptides loaded on the 3DTi surface after 5 minutes and 2 hours; (D) Images of FP1, FP2 and NFP fusion peptides released from the 3DTi surface after 3d and 7d; Data are expressed as mean ± SD. * p<0.05, ** p<0.01, ** p<0.001, ** p<0.0001, ns: not meaningful; (*Due to the need for structure prediction, the linker of FP2 was extended to (EAAAK) 4 . P = Proline, O = Hydroxyproline); Figure 3 Characterization of the modified titanium substrate of the present invention; in the figure: (A) is the SEM image of the Ti substrate treated by different modification methods (top: scale bar = 100 μm; bottom: scale bar = 10 μm); (B) is the XPS spectrum of the Ti substrate after different modifications; (C) is the AFM image showing the surface morphology of different samples; (D) is the surface roughness measurement of the titanium substrate; (E) is the EDS spectrum of the surface of the 3DTi sample loaded with NFP; (F) is the water contact angle image and quantitative results after modification; (G) is the elastic modulus of the Ti substrate after different modifications; data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * ** p<0.0001, ns: not significant; Figure 4 The biocompatibility and adhesion activity of the modified titanium substrate of the present invention; (AB) are CCK-8 test results showing the proliferation of bone marrow mesenchymal stem cells on different sample surfaces; (C) is a fluorescence image of live / dead staining of bone marrow mesenchymal stem cells cultured on different surfaces for 1 day and 3 days (scale bar, 100 μm); (D) is a fluorescence microscope image of bone marrow mesenchymal stem cell cytoskeleton staining (scale bar, 20 μm); (E) is immunofluorescence staining showing the focal adhesion of bone marrow mesenchymal stem cells on different Ti matrices (scale bar, 100 μm); (F) is the quantitative immunofluorescence protein staining of different samples; data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * * * p<0.0001, ns: not significant; Figure 5In vitro antibacterial analysis of the modified titanium substrate of the present invention; (A) is a representative image of Staphylococcus aureus and Escherichia coli colonies after 24 hours of culture of different samples; (B) is a quantitative analysis of the antibacterial effect (%) of different groups on Escherichia coli and Staphylococcus aureus; (C) is a representative fluorescent image of live / dead stained bacteria, green is live bacteria, and red is dead bacteria (scale bar, 100 μm); (D) is a representative image of the inhibition zone of Staphylococcus aureus and Escherichia coli after 24 hours of culture of different groups; (E) is a quantitative analysis of the diameter of the inhibition zone; (F) is a representative SEM image of bacterial morphology in different groups (scale bar, 100 nm); data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * * * p<0.0001, ns: not significant; Figure 6 Figure 2 shows the in vitro osteogenesis of the modified titanium substrate of the present invention; (A) is a microscopic (scale bar = 100 μm) and optical image of alkaline phosphatase-stained cells after 7 days of culture in osteogenic induction medium; (B) is a quantitative analysis of cell alkaline phosphatase activity; (C) is a microscopic (scale bar = 100 μm) and optical image of alizarin red-stained cells after 14 days of osteogenic differentiation; (D) is a quantitative analysis of calcium mineralization; (E) is an immunofluorescence staining of bone marrow mesenchymal stem cells cultured on different Ti matrices, showing the cytoskeleton (red), ALP and OCN proteins (green) and cell nuclei (blue) (scale bar = 20 μm); (F) is a semi-quantitative analysis of immunofluorescence staining of ALP and (G) OCN; (HK) is a qRT-PCR analysis of the expression levels of ALP, BMP2, OPN and OCN genes in bone marrow mesenchymal stem cells cultured on different titanium matrices in osteogenic induction medium (n = 4); data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * * * p<0.0001, ns: not significant; Figure 7 The in vitro osteogenesis of the modified titanium substrate of the present invention; in the figure: (A) is the immunofluorescence staining of bone marrow mesenchymal stem cells cultured on different Ti matrices, showing the cytoskeleton (red), BMP-2 and OPN proteins (green) and cell nuclei (blue) (scale = 20 μm); (B) is the semi-quantitative analysis of immunofluorescence staining of BMP-2 and (C) OPN. Data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * * * p<0.0001, ns: not significant; Figure 8Informatics analysis of the gene expression profile of the present invention; in the figure: (A) is the principal component analysis (PCA) of different samples in the 3DTi-NFP group and the Ti control group; (B) is a volcano map comparing the differentially expressed genes (DEGs) of 3DTi-NFP and Ti; (C) is a heat map showing all up-regulated and down-regulated DEGs in the 3DTi-NFP group and the Ti group; (D) is a bar chart showing the GO enrichment analysis of up-regulated DEGs; (E) is a bubble map of up-regulated KEGG pathway enrichment terms in 3DTi-NFP and Ti; the cluster heat map shows the DEGs involved in focal adhesion (F) and PI3K-AKT signaling pathway (G); Fig. 9 The antibacterial and osteogenesis evaluation of the 3DTi implant of the present invention in the SD rat skull defect model infected with Staphylococcus aureus; in the figure: (A) is an image of colonies collected from the wound exudate around the 3DTi implant site 3 days after surgery; (B) is a quantitative analysis of the antibacterial effect of different 3DTi substrates after implantation; (C) is a Micro-CT three-dimensional reconstruction image showing the newly formed bone (pink) around the implant (gray) (lower left: coronal CT view; lower right: top view); (D) is a fluorescence quantitative analysis; (E) is a histological image of the newly formed bone around the implant by H&E and Masson-Goldner trichrome staining (scale bar = 200 μm); (F-I) are Micro-CT quantitative parameters: BMD, BV / TV, Tb.N, and Tb.Th. (n = 5) Data are expressed as mean ± SD. * p<0.05, * * p<0.01, * * * p<0.001, * * * * p<0.0001, ns: not significant; Fig.10 The performance of the 3D titanium implants loaded with different fusion peptides of the present invention in the hemolysis test; Fig.11 H&E staining of the heart, liver, spleen, lung, and kidney described in the present invention. Scale bar = 100 μm. DETAILED DESCRIPTION

[0016] In order to make the purpose, 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. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0018] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0019] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0020] 1. Experimental Methods 1. Preparation of fusion peptides: FP1, FP2 and NFP were synthesized by Qiangyao Biotechnology Co., Ltd. (Shanghai, China) using the solid phase method and Fmoc (9-fluorenylmethyloxycarbonyl) chemical method. Their sequences are shown in Table 1. The purity of non-fluorescent peptides exceeded 99%, while the purity of fluorescent labeled peptides (fluorescein isothiocyanate, FITC, green) was above 95%. The molecular structure of the fusion peptides was predicted and analyzed using protein analysis software PSIPRED and PEP-FOLD.

[0021] Table 1: 2. Preparation of Ti materials: Ti substrates (purity 99.89%) were provided by Jiangsu Jinwu New Materials Co., Ltd. Two sizes were prepared: 14mm × 14mm × 1mm discs (labeled as Ti / 3DTi) for in vitro studies and 7mm × 7mm × 0.5mm discs for in vivo studies. The Ti substrates were polished with silicon carbide sandpaper with grits of 400, 800, 1000, 1200, 1500 and 2000 in sequence. Subsequently, the substrates were ultrasonically cleaned with acetone, 75% ethanol and ddH2O for 20 minutes. After drying, they were sterilized under UV light for 3 hours before use.

[0022] 3. Loading and release of peptides: Fluorescein isothiocyanate-labeled peptide powder was dissolved in ddH2O to prepare peptide solutions with concentrations of 1, 10, 100, and 1000 μg / mL. These solutions (300 μL per well) were then applied to 3DTi substrates placed on 24-well plates and incubated overnight at room temperature. The next day, unbound peptides were washed away with phosphate-buffered saline (PBS) to determine the optimal concentration. The selected optimal concentration was then used to incubate the 3DTi substrate at room temperature for 5 minutes or 2 hours to evaluate the binding efficiency. The three modified 3DTi samples were named 3DTi-FP1, 3DTi-FP2, and 3DTi-NFP.

[0023] BMSCs were seeded on 3DTi-FP1, 3DTi-FP2, and 3DTi-NFP samples to observe peptide release behavior. The samples were washed with PBS after culture and fixed with 4% paraformaldehyde for 30 min at room temperature after 3 or 7 days. The cells were then permeabilized with 0.1% TritonX-100 (Beyotime, China) for 5 min and washed three times with PBS. Actin filaments were stained with rhodamine-phalloidin for 30 min in the dark. Finally, 4′, 6-diamidino-2-phenylindole (DAPI, 5 mg / mL; Invitrogen, USA) was used for 10 min. All loading and release experiments were performed using confocal laser scanning microscopy (CLSM; FV1000, Olympus, Japan), and the most representative images were selected for each sample.

[0024] The 3DTi substrate was immersed in 100 μg / mL NFP peptide solution (2 mL) at room temperature for 2 h. After incubation, the treated samples were washed with PBS and ddH 2 O was thoroughly rinsed and then dried in an argon atmosphere. The surface morphology was observed by field emission scanning electron microscopy (FE-SEM; JSM-7001F, JEOL, Japan) with an accelerating voltage of 10 kV. Energy dispersive X-ray spectroscopy; QX200, Bruker Optics, Germany) was used to analyze the element distribution and concentration on the sample surface. The surface roughness (Ra) was measured in the range of 10 μm × 10 μm by atomic force microscopy (AFM; SPA-300HV, NSK, Japan). The surface wettability was evaluated by the hanging drop method using a contact angle analyzer (JC2000DF, Powereach, China). In addition, X-ray photoelectron spectroscopy (XPS; K-α, Thermo Fisher, USA) was used to evaluate the elemental composition and chemical state of the sample surface.

[0025] For biomechanical testing, the compressive strength of smooth Ti and 3DTi samples was evaluated on a universal testing machine (Model 880, MTS, USA) according to ISO 13314. The maximum load capacity of the machine was 500 kN and the test speed was set to 0.5 mm / min. Five independent samples were tested per experiment and three discs were analyzed for each surface treatment.

[0026] 2. Verification Experiment 1. Cell culture: Bone marrow mesenchymal stem cells were obtained from the femoral bone marrow of Sprague-Dawley (SD) rats. The third-generation cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) under standard incubation conditions (37°C, 5% CO 2 For the experiment, 1 × 10 4-7 passage bone marrow mesenchymal stem cells were used for each sample. 4 All in vitro assays were performed in three independent replicates to ensure statistical reliability.

[0027] 2. Sample biocompatibility assessment: The proliferation of bone marrow mesenchymal stem cells was assessed using the CCK-8 method (Solaibao, China). On days 1, 4, and 7, 500 μL of 10% CCK-8 solution was added to each well and incubated at 37°C for 2 hours. After incubation, 100 μL of supernatant was transferred from each well to a 96-well plate, and the optical density (OD) was recorded at 450 nm using a spectrophotometer.

[0028] The viability of BMSCs was assessed using a live / dead cell staining kit (CA1630, Solebo, China). After 1 and 3 days of culture, cells were stained according to the manufacturer's protocol. In live cells, esterase cleaves calcein-AM into calcein, producing green fluorescence, while ethidin-1 (EthD-1) only penetrates the cell membrane of dead cells and binds to nucleic acids to produce red fluorescence. Finally, a fluorescence microscope was used to observe and capture cell images.

[0029] 3. Cell adhesion activity: To study the adhesion and morphology of BMSCs, cells were initially cultured in serum-free medium for 4 h. After culture, the samples were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 (Beyotime, China) for 5 min and then washed three times with PBS. Actin filaments were stained with rhodamine-phalloidin for 30 min in the dark. Finally, 4′, 6-diamino-2-phenylindole (DAPI, 5 mg / mL; Invitrogen, USA) was used for 10 min. All loading and release experiments were performed using confocal laser scanning microscopy (CLSM; FV1000, Olympus, Japan), and the most representative images were selected for each sample.

[0030] 4. Bacterial culture: To evaluate the antibacterial effect, Staphylococcus aureus (S. aureus, ATCC 29213) and Escherichia coli (E. coli, ATCC 43300) were selected for the test and initially cultured in tryptic soy broth (TSB). A single colony of each bacterium was transferred to a TSB agar plate and incubated overnight. After incubation, one colony was transferred to TSB and shaken at 220 rpm at 37°C for 6 hours. The bacterial culture was then diluted with sterile PBS to a concentration of 1 × 10 8 CFU / mL. All experiments were performed in triplicate, and each group of samples had three independent replicates to ensure statistical validity.

[0031] 5. In vitro antibacterial test: The plate count method was used to determine its antibacterial activity. Initially, 1 µL of bacterial suspension was placed on different substrates in a 24-well plate and incubated at 37°C for 12 hours. After incubation, the bacteria in each group were removed by ultrasound and collected in 1 mL of sterile PBS containing 0.1% Tween-80. The suspension was diluted 10 times in series, and 100 µL of each dilution was applied to the agar plate. Then it was incubated at 37°C for another 12 hours. The CFU was then counted and recorded by photography. The antibacterial effect (R) was calculated as follows: R = (A - B) / A × 100%, where R is the antibacterial effect, A is the average CFU count of the control group (Ti), and B is the average CFU count of the experimental groups (3DTi, 3DTi- FP1, 3DTi- FP2, 3DTi- NFP).

[0032] In addition, 1 × 10 8 CFU / mL bacterial suspension was evenly inoculated on a 100 mm agar plate. Each Ti substrate was placed in the center of the plate, and untreated Ti was used as a control. After incubation at 37°C for 24 hours, the inhibition zone was observed and photographed.

[0033] 6. Live / dead bacterial staining test: Bacterial viability was assessed using the Live / Dead BacLight Bacterial Viability Kit (Thermo Fisher, Shanghai, China). After incubation at 37°C for 12 h, the bacterial suspension was rinsed with PBS. Next, a staining solution containing 10 µM propidium iodide (PI) and 10 µg / mL SYTO9 was used, followed by another 30 min incubation. Finally, bacterial viability on the substrate surface was observed and recorded by CLSM.

[0034] 7. Observation of bacterial adhesion morphology: Field emission scanning electron microscopy was used to detect the bacterial morphology on different titanium surfaces. 1 mL of bacterial suspension was added to various substrates arranged in a 24-well plate and incubated at 37°C for 12 hours. Then it was gently rinsed three times with PBS, fixed in 2.5% glutaraldehyde at 4°C for 4 hours, and then washed with PBS. Next, the samples were dehydrated in a graded ethanol series ranging from 30% to 100% and then air-dried. After gold plating, the bacterial morphology was observed under a field emission scanning electron microscope.

[0035] 8. Osteogenesis: Alkaline phosphatase (ALP) activity: ALP activity of BMSCs was analyzed using a 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium (BCIP / NBT) staining kit and an ALP assay kit (Beyotime, China). BMSCs were cultured on titanium surfaces with various modifications in osteogenic (OB) medium by adding 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate to the basal medium. After 7 and 14 days of culture, BMSCs were fixed in 4% paraformaldehyde for 30 min. BCIP / NBT staining solution was then added and incubated for 1 h to visualize ALP. After staining, the BCIP / NBT solution was discarded and the samples were rinsed with PBS to stop the reaction. ALP staining images were obtained using an optical microscope.

[0036] For quantitative analysis of ALP activity, bone marrow mesenchymal stem cells were lysed with 1% Triton X-100 on ice for 30 minutes. The lysate was centrifuged at 12,000 rpm for 4 minutes at 30°C, and the resulting supernatant was collected. 50 μL of the supernatant was mixed with 50 μL of ALP working solution and incubated for 30 minutes. ALP activity was recorded at 405 nm absorbance using a microplate reader.

[0037] 9. Alizarin red (ARS) staining: On days 14 and 21 of culture, cells were fixed in 4% paraformaldehyde for 15 min and then stained with 0.1% ARS (Solarbio, China) at 37°C for 30 min. After staining, cells were rinsed with PBS and observed under an optical microscope. For quantification, mineralized deposits were dissolved in 10% hexadecylpyridinium chloride (Sigma-Aldrich, South Korea), and the absorbance at 570 nm was recorded using a microplate reader.

[0038] 10. Immunofluorescence staining: BMSCs cultured on different sample surfaces for 7 days were first rinsed with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Next, the cells were permeabilized with 0.1% Triton X-100 for 5 minutes, then rinsed three times with PBS and blocked with 5 mg / mL bovine serum albumin (BSA, Beyotime, China) for 30 minutes at room temperature to reduce nonspecific binding. Samples were then incubated overnight at 4°C with primary antibodies targeting vinculin (VIN, 1:150; ab129002, Abcam, Cambridge, UK), ALP (1:50; ab7817, Abcam, Cambridge, UK), bone morphogenetic protein 2 (BMP-2, 1:100; ab284387, Abcam, Cambridge, UK), osteopontin (OPN, 1:250; Affinity Biosciences, AF0227, Cincinnati, USA) and osteocalcin (OCN, 1:250; Affinity Biosciences, DF12303, Cincinnati, USA). After primary antibody incubation, fluorescent secondary antibodies (Alexa Fluor 488, ab150077, Abcam, UK) were used for 1 hour at room temperature in the dark. Actin filaments were stained with rhodamine-phalloidin for 30 min at room temperature, and nuclei were labeled with DAPI for 10 min, with PBS washes between each staining. Finally, fluorescence images were captured using CLSM.

[0039] 11. qRT-PCR assay: To evaluate the osteogenic differentiation of BMSCs, we analyzed the gene expression levels of ALP, BMP-2, OPN, and OCN by qRT-PCR. BMSCs were seeded on each sample and cultured for 14 days under different conditions. Total RNA was isolated using TRIzol reagent (Beyotime, China), and complementary DNA (cDNA) was synthesized using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR was performed on an ABI Prism 7300 thermal cycler (AppliedBiosystems, Australia), and the detection dye was SYBR Green. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene. Each sample was run in triplicate to ensure accuracy. The relative expression level was determined using the 2^(-ΔCt) method, where ΔCt is the average Ct value adjusted to the reference gene. The primer sequences are shown in Table 2.

[0040] Table 2: Primer sequences used for qRT-PCR 12. RNA-seq and data analysis: To investigate the potential mechanisms of the observed effects, transcriptome sequencing and bioinformatics analysis were performed. BMSCs were cultured on Ti and 3DTi-NFP surfaces, and total RNA was extracted using TRIzol reagent after 14 days of osteogenic induction. RNA sequencing libraries were constructed and sequenced on the BGISEQ-500 platform at BGI (Shenzhen, China). Genes with p-values ​​less than 0.05 above 1.5 or below -1.5 were identified as differentially expressed genes (DEGs). Functional annotations of genes and gene products were obtained using Gene Ontology (GO) (http: / / www.geneontology.org). Bioinformatics analysis was performed using the Bio-Cloud platform (https: / / bio-cloud.aptbiotech.com / tool).

[0041] 3. In vivo study of multifunctional fusion peptide-modified 3D printed Ti implants constructed using the prepared fusion peptide platform 1. Implantation surgery: The animal experiments in this study were approved by the Animal Ethics Committee of Shanxi Medical University Medical Center. Twenty healthy male SD rats, each weighing about 300 g, were selected to construct an 8 mm skull defect model, with 5 rats in each group. To establish an implant-related infection model, Staphylococcus aureus (1 × 108 CFU, 20 μL PBS) was injected into the defect site, and all surgeries were performed under sterile conditions.

[0042] All rats were anesthetized with 2% sodium pentobarbital (45 mg / kg body weight) injected intraperitoneally. Defects were created at the suture line of the skull using an 8 mm drill, and the full thickness of the bone was removed. Then the 3DTi substrates loaded with fusion peptides were randomly implanted into the defect site, and the unmodified 3DTi implants were used as the control group. After the injection of Staphylococcus aureus suspension, the muscle and skin were closed sequentially with absorbable 4-0 surgical sutures. After surgery, each rat was housed individually and penicillin (150,000 units) was injected intramuscularly once a day for 3 consecutive days. After 8 weeks, all rats were euthanized by overdose of anesthesia, and the skull specimens were removed, all soft tissues were removed, and fixed with 4% paraformaldehyde.

[0043] 2. In vivo antibacterial analysis: Three days after surgery, three SD rats were randomly selected from each group, and peri-implant secretions were collected with sterile swabs to evaluate peri-implant infection. The bacterial suspensions of different groups were transferred to 1 mL of sterile PBS containing 0.1% Tween 80 and ultrasonically treated. The bacterial suspensions (100 μL) diluted 10 times were evenly spread on agar plates and incubated at 37°C for 12 hours. CFUs were counted and recorded by photography.

[0044] 3. Evaluation of general condition and hematological indicators of animals: After surgery, the rats were returned to their cages, provided with a standard diet, and allowed to move freely. Wound healing, body weight, and body temperature were monitored regularly. Eight weeks after implantation, cardiac blood samples were taken for routine hematological analysis. In addition, the heart, liver, spleen, lungs, and kidneys were taken for hematoxylin and eosin (H&E) staining to assess systemic infection.

[0045] 4. Micro-computed tomography (Micro-CT) detection: To evaluate bone formation, 20 50 μm wide circular regions of interest (ROI) were selected around each implant. High-resolution micro-CT scanning was performed using a Bruker Skyscan 1172 system (Kontich, Belgium), with a voxel size of 13.73 μm, a voltage of 100 kV, a current of 100 μA, a full 360° rotation, a 0.5 mm aluminum filter, a rotation step of 0.7°, and an exposure time of 250 ms. After scanning, the data were three-dimensionally reconstructed, and the micro-CT images were quantitatively analyzed to determine the fluorescence density, bone mineral density (BMD), bone volume to total volume ratio (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th).

[0046] 5. Histological analysis: After fixation in 4% paraformaldehyde for 48 h, the specimens were embedded in resin by a light-curing process. The tissue blocks were then 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 Masson-Goldner trichrome, and images were captured using a microscope.

[0047] 6. Statistical analysis: All experiments were performed three times unless otherwise stated. Data are presented as mean ± standard deviation (SD) (in vitro: n = 3; in vivo: n = 5). Statistical analysis and quantitative imaging were performed using GraphPad Prism 9.5 (GraphPad, USA). Statistical significance was assessed using t-test and one-way ANOVA, followed by Tukey post hoc test. p Value < 0.05 ( * ) considered significant.

[0048] IV. Experimental Results 1. Design and synthesis of novel multifunctional fusion peptides: The sequence and main structural features of the fusion peptide platform proposed in the present invention are as follows: Figure 1 As shown in the figure, it is divided into four main components: (i) biologically active peptide sequences KR-12 and GFOGER; (ii) linker unit; (iii) lysine residues; and (iv) specific anchoring group minTBP-1. This is the first time that the antimicrobial peptide KR-12 is combined with the adhesion peptide GFOGER to study the effects of these two active motifs on cell behavior after being grafted onto the surface of titanium-based materials.

[0049] The sequences of the proposed fusion peptide platform (see Table 1 for details) and their key structural features are as follows Figure 1As shown in Figure 2. The platform consists of four main components: (i) bioactive peptide sequences KR-12 and GFOGER; (ii) linker unit; lysine residues; (iv) specific anchoring group minTBP-1. This study combined the antimicrobial peptide KR-12 with the adhesion peptide GFOGER for the first time, aiming to study the effects of these two active motifs on cell behavior after grafting on titanium-based materials.

[0050] In order to maintain the optimal spatial distance between the functional peptide and the Ti substrate while ensuring that the peptide has sufficient distance to exert its stable biological activity, a rigid spacer unit EAAAK composed of a large number of hydrogen bonds was selected. The introduction of lysine residues is essential for the synthesis of branched conformations, which improves the accessibility and orientation of the two functional peptides compared to linear structures. The inorganic binding peptide minTBP-1 shows a specific affinity for Ti and can quickly and accurately recognize and anchor to the Ti substrate, thereby facilitating the efficient loading of the novel fusion peptide onto the Ti surface in a one-step process. Figure 2 As shown in A, the secondary structures of FP1 and FP2 consist of random coils and α - Helical composition. The random coil structure allows the chimeric peptides to exhibit a greater range of motion at the Ti interface, while the shared helical structure enhances their functional activity.

[0051] 2. Binding and release ability of fusion peptides on 3DTi surface: Three new fusion peptides were successfully synthesized in this paper. In the process of optimizing the binding concentration, 3DTi samples were incubated overnight in peptide solutions of different concentrations, and the surface loading was evaluated using confocal laser scanning microscopy. The fluorescence intensity of FITC-labeled peptides on the 3DTi surface gradually increased with the increase of peptide concentration ( Figure 2 B). However, when the concentration was further increased to 1000 μg / mL, the fluorescence signal was not significantly different from that at 100 μg / mL, indicating that FP1, FP2, and NFP reached saturation at 100 μg / mL. Therefore, 100 μg / mL was selected as the optimal concentration for subsequent experiments.

[0052] like Figure 2As shown in Figure C, approximately 5% of the peptides were rapidly bound to the 3DTi surface after incubation for 5 minutes at a concentration of 100 μg / mL. After 2 hours of incubation, approximately 15% of the peptides were successfully loaded, indicating that a large portion of the peptides achieved specific binding in a short period of time. It is worth noting that 3DTi presents a three-dimensional printed structure with a high surface area, and because the CLSM focuses on a specific plane, the actual number of bound peptides may be higher than observed. In addition, the loading of the newly constructed multifunctional fusion peptide was not adversely affected at different time points, and the binding amount gradually increased over time. Studies on the release behavior showed that the fusion peptide was still present and functional after 7 days, promoting the complete extension of the cytoskeleton in bone marrow mesenchymal stem cells ( Figure 2 D). This confirms the sustained release and functional efficacy of the fusion peptide.

[0053] 3. Characterization of the binding and release ability of the fusion peptide on the 3DTi surface: The surface morphology of each sample was examined using SEM ( Figure 3 A). The smooth Ti surface exhibits a relatively flat structure with slight scratches, while the 3DTi surface exhibits a microporous network and Ti powder particles can be seen at higher magnification. After incubation of 3DTi with NFP peptide solution, SEM images show that the fusion peptide is evenly distributed on the 3DTi surface, forming a small particle structure without changing the original surface morphology. Due to the small molecular size of the fusion peptide, SEM mainly captures peptide aggregates, which may explain the slight difference in binding rate observed with CLSM.

[0054] XPS analysis shows the characteristic peaks of elements on the surface of each sample ( Figure 3 B), including Ti 2p (456.0 eV), O 1s (530.8 eV), C 1s (284.8 eV), and N 1s (399.9 eV). After the fusion peptide bound to the 3DTi surface, the signal intensities of Ti and O decreased, while those of C and N increased. The N / Ti atomic ratio increased from 0.217 in bare Ti to approximately 15.304 in 3DTi-NFP (Table 3), confirming the successful attachment of the peptide coating. The C content observed in the Ti and 3DTi control samples was attributed to organic pollutants in the atmosphere, as well as trace amounts of nitrogen contamination. It is noteworthy that the N peak at 399.9 eV belongs to the amide group and other nitrogen-containing functional groups characteristic of the peptide sequence. The deposition of the peptide layer also reduced the TiO detected by XPS. 2 signal. This effect is particularly pronounced on the NFP platform, consistent with its larger molecular weight. However, due to the presence of C and N impurities in all control samples, it is challenging to determine the exact ratios by XPS to quantitatively compare the attachment efficiency of different biomolecules. In addition, EDS of 3DTi-NFP samples showed that Ti, O, C, and N were evenly distributed on the surface ( Figure 3 E).

[0055] Table 3: XPS method to determine the percentage of surface elements in different samples The morphology and roughness (Ra) of different Ti surfaces were evaluated using atomic force microscopy (AFM). Figure 3 C and D), the results showed that the roughness of the Ti surface was 21.6 ± 8.9 nm, while the roughness of the 3DTi surface was significantly higher, 65.3 ± 9.6 nm. After loading the fusion peptide, the roughness of 3DTi-FP1, 3DTi-FP2, and 3DTi-NFP reached 107.0 ± 7.8 nm, 123.0 ± 3.0 nm, and 76.3 ± 7.8 nm, respectively. The increase in roughness enhanced the hydrophilicity of the Ti surface, as confirmed by contact angle measurements ( Figure 3 F). The water contact angles of Ti and 3DTi were 81±0.53° and 56.8±0.78°, respectively, while the contact angle of the 3DTi surface loaded with fusion peptides was significantly reduced, indicating that the hydrophilicity was improved. Among them, the contact angles of 3DTi-FP1, 3DTi-FP2, and 3DTi-NFP were 45.1±0.26°, 41.53±0.71°, and 30.27±0.80°, respectively. This enhancement in hydrophilicity facilitated the interaction between the implant and the surrounding cells, promoting cell adhesion and proliferation.

[0056] The high elastic modulus of titanium implants can have an adverse effect on bone integration with surrounding bone tissue. Compression tests were performed on different samples using a universal testing machine, and the results showed that the elastic modulus of 3DTi was 11.63±3.29 GPa, which was significantly lower than that of Ti (94.03±6.14 GPa). It is worth noting that the transplantation of fusion peptides had no significant effect on the elastic modulus of 3DTi (15.20±2.38 GPa), which is very close to the elastic modulus of human cortical bone (14.4±0.85 GPa) ( Figure 3 G).

[0057] In summary, through various surface characterization techniques, we demonstrated that a thin adhesive peptide layer rapidly and spontaneously formed on the 3DTi surface after simple immersion in the fusogenic peptide solution. After peptide conjugation, the surface properties of 3DTi remained largely unchanged, indicating the importance of the specific Ti-binding peptide minTBP-1 in surface adhesion and functionalization. Thus, we successfully developed a simple and effective strategy for multifunctional surface engineering of titanium implants.

[0058] 4. Evaluation of biocompatibility and adhesion and proliferation activity: The interaction between cells and materials is crucial to the ability of materials to enhance bone regeneration. To evaluate the biocompatibility of various Ti and 3DTi substrates, CCK-8 assay, live / dead staining, and immunofluorescence assay were used to evaluate the cytotoxicity, adhesion, and proliferation of BMSCs on Ti substrates. BMSCs were inoculated with Ti matrices and cultured for 1, 4, and 7 days, followed by CCK-8 evaluation. Figure 4 A and B show the time-dependent increase in cell proliferation in all groups. A significant increase in cell proliferation was observed with the extension of culture time, especially on days 4 and 7, which highlights the positive effects of the peptides on cell viability and proliferation. Notably, the cell counts in the 3DTi-NFP group on days 4 and 7 were slightly higher than those in the other groups, indicating that the combined effects of GFOGER and KR-12 enhanced cell proliferation. In addition, the live / dead staining results after 1 and 3 days of culture ( Figure 4 C) showed that there was no obvious cytotoxicity on the Ti substrate and cell proliferation was significantly enhanced, which was consistent with the findings of CCK-8. These results confirmed that the prepared Ti substrate had good cytocompatibility and could significantly promote cell adhesion and proliferation.

[0059] Cell morphology plays a crucial role in regulating cell phenotype. Figure 4 As shown in Figure 4, after 3 days of co-culture, BMSCs adhered to all Ti matrices; however, clear differences were observed in cell morphology. Compared with the elongated cells and fewer numbers on the control Ti surface, the cells on the 3DTi surface were polygonal, with enhanced diffusion and extensive pseudopodia. This spindle-shaped morphology is closely associated with the adhesion, proliferation, and differentiation of osteoblasts, indicating that the increased roughness of the 3DTi surface supports cell growth. With the loading of the peptide, the number of cells increased further, the cell morphology became more stretched, the cytoskeletal structure became more developed, the pseudopodia were bifurcated, and the cell-cell connections were tight, confirming the powerful regulatory effects of 3DTi and its fusion peptide on cell adhesion and morphology.

[0060] To further verify the affinity of 3DTi and fusion peptides for cells, we performed immunofluorescence staining of vinculin, as vinculin is commonly used as a marker for focal adhesions. Focal adhesion formation was observed on all 3DTi substrates, indicating that the fusion peptides have good cell affinity due to their hydrophilicity ( Figure 4 E). Furthermore, the presence of the adhesion peptide GFOGER promoted greater cell spreading and the formation of more focal adhesions on both 3DTi-FP2 and 3DTi-NFP groups, indicating that these surfaces actively support cell growth ( Figure 4 F). Taken together, these findings indicate that 3DTi loaded with fusion peptides significantly enhanced the adhesion, proliferation, and spreading of early stage cells, confirming good cytocompatibility.

[0061] 5. Antibacterial effect: Preventing bacterial colonization and proliferation is a prerequisite for promoting bone formation. The antibacterial effect of the samples on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli was evaluated by colony counting, inhibition zone determination, live / dead staining and scanning electron microscopy imaging. Figure 5 As shown in A, almost no colony formation was observed in the 3DTi-FP1 and 3DTi-NFP groups, and the inhibition rates against Staphylococcus aureus and Escherichia coli exceeded 98% and 90%, respectively ( Figure 5 B). This finding suggests that samples lacking KR-12 are more susceptible to bacterial proliferation. Figure 5 In C, abundant green fluorescence was observed in the Ti, 3DTi, and 3DTi-FP2 groups, indicating that Staphylococcus aureus and Escherichia coli had good survival ability on these surfaces, while almost all bacteria in the 3DTi-FP1 and 3DTi-NFP groups showed red fluorescence, indicating that most bacteria were dead. These findings were further confirmed by inhibition zone assays ( Figure 5 D). After 24 h of culture, obvious inhibition zones were formed around the 3DTi-FP1 and 3DTi-NFP groups, while no inhibition zones were found around the Ti, 3DTi, and 3DTi-FP2 groups, indicating that the release of KR-12 had a strong bactericidal effect. The diameters of the inhibition zones of the 3DTi-FP1 and 3DTi-NFP groups against Staphylococcus aureus and Escherichia coli were 31.35±0.23 mm, 31.76±0.21 mm, 24.52±0.17 mm, and 25.02±0.34 mm, respectively ( Figure 5 E). Unexpectedly, there was no significant difference in the inhibition zone size between the 3DTi-FP1 group and the 3DTi-NFP group, indicating that the presence of GFOGER did not affect the antibacterial activity of KR-12.

[0062] SEM observations of bacterial morphology and membrane integrity further confirmed these results ( Figure 5 F). On Ti, 3DTi, and 3DTi-FP2 samples, S. aureus and E. coli showed typical spherical and rod-like shapes with smooth and intact membranes, indicating no adverse effects on bacterial viability. In contrast, bacteria attached to the 3DTi-FP1 and 3DTi-NFP surfaces showed irregular shapes with signs of shrinkage, deformation, and even membrane rupture. These findings clearly demonstrate the potential of KR-12-loaded 3DTi implants to significantly reduce the risk of bacterial infection in vitro.

[0063] 6. Osteogenesis: Effective bone integration is a key strategy to solve peri-implant bone defects and implant loosening. In order to evaluate the osteogenic potential of different samples on BMSCs, we performed a comprehensive analysis, including early osteogenic marker ALP activity, late osteogenic marker ARS staining, qRT-PCR, and immunofluorescence staining. Figure 6 A and Figure 6 C shows the ALP staining results after 7 days and ARS staining results after 14 days, respectively. The 3DTi-NFP group had the highest staining intensity and density, followed by the 3DTi-FP2 and 3DTi-FP1 groups. On day 14, the expression of ALP in the 3DTi-NFP group was 3.6 times that of the Ti control group. ARS staining results also showed a similar trend, with the 3DTi-NFP group having the darkest staining and the most abundant mineralized nodules. Semi-quantitative analysis of ALP and ARS staining further confirmed this observation ( Figure 6 B and 6D).

[0064] Immunofluorescence staining was used to detect the expression of bone-specific ECM proteins (ALP, BMP-2, OPN, and OCN) on each Ti substrate. After 14 days, the cell density on the 3DTi surface was high and the cells were polygonal in morphology, which showed the characteristics of osteoblasts. Figure 6 EG and Figure 7 As shown, the protein expression of the 3DTi-NFP group was significantly enhanced, and the fluorescence was bright green, which was particularly obvious compared with the other groups, and 3DTi-FP2 ranked second. This suggests that the GFOGER peptide has a powerful effect in promoting osteogenic differentiation, and the synergistic effect of KR-12 and GFOGER may promote osteogenesis by upregulating key osteogenic genes.

[0065] To further elucidate the molecular mechanism of osteogenic differentiation, we analyzed the expression levels of key osteogenic-related genes ALP, BMP-2, OPN, and OCN. Overall, the expression of these osteogenic genes was significantly increased on day 14 compared with the control group ( Figure 6 HK), among which the gene expression level in the 3DTi-NFP group was the highest.

[0066] Finally, comprehensive analysis of qRT-PCR and staining results showed that 3DTi-NFP exhibited strong osteogenic activity at both molecular and cellular levels, enhancing the expression of ALP, BMP-2, OPN, and OCN. The combination of KR-12 and GFOGER peptide showed a synergistic effect and significantly enhanced osteogenic activity. This peptide-functionalized 3D printed Ti substrate significantly promoted osteoblast activity and increased bone matrix protein expression. Therefore, 3DTi-NFP is expected to become a functional implant for bone regenerative medicine and effectively promote bone defect repair.

[0067] 7. RNA-seq analysis of osteogenic mechanism: To clarify the mechanism by which 3DTi-NFP promotes osteogenic differentiation of bone marrow mesenchymal stem cells, we performed RNA sequencing. PCA of the samples showed a clear separation between the 3DTi-NFP and Ti groups, confirming that both groups met the quality control standards ( Figure 8 A). Volcano plot and heat map of DEGs identified 606 up-regulated genes and 475 down-regulated genes in the 3DTi-NFP group compared with the Ti control group ( Figure 8 B and C). GO enrichment analysis of DEGs was divided into three main areas: biological process (BP), molecular function (MF), and cellular component (CC) ( Figure 8 D). Notably, relative to the Ti group, the upregulated genes in the 3DTi-NFP group were significantly enriched in the categories of developmental process, extracellular matrix organization, integrin binding, Wnt protein binding, and extracellular matrix.

[0068] We performed enrichment analysis of DEGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and identified the top 20 pathways with the highest enrichment scores ( Figure 8 E). These pathways include those related to positive regulation of cell adhesion, proliferation, and matrix formation, such as focal adhesion and ECM receptor interaction pathways, and pathways closely related to osteogenesis, such as Wnt signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway. These genes were further clustered to explore interactions at the protein level. Figure 8 As shown in F and G, these genes are involved in cell proliferation and differentiation (such as Igf1, Fgfr2), extracellular matrix components (such as Lama1, Col1a1), and osteogenesis-related processes (such as Col1a1, Itga1, Itga11). Figure 6 The in vitro experimental results shown are consistent, indicating that 3DTi-NFP implants may promote new bone formation by enhancing the adhesion, proliferation, differentiation and ECM organization of BMSCs.

[0069] 8. Evaluation of in vivo antibacterial and osteogenic abilities: To evaluate the in vivo antibacterial and osteogenic abilities of implants, we used a skull defect model of 3DTi-based implants. After creating a critical-size defect with a diameter of 8 mm in the skull of SD rats, the implant was placed and Staphylococcus aureus was injected into the defect site to simulate post-implantation infection. Exudate from the implant site was collected 3 days after implantation to evaluate the antibacterial effect. The colony count results (98A and B) showed that more bacteria were attached in the 3DTi and 3DTi-FP2 groups. In contrast, the 3DTi-FP1 and 3DTi-NFP groups had the least bacterial growth, with inhibition rates of 98.49% and 95.48%, respectively, which were consistent with the results of in vitro studies.

[0070] Eight weeks after surgery, the antibacterial and osteogenic properties of rat skull specimens were evaluated using micro-CT, and the defect area was 3D reconstructed using IMARIS software ( Fig. 9 C and D). New bone formation was limited in the 3DTi group over time, indicating that residual S. aureus hindered bone regeneration. In contrast, bone formation was enhanced in the 3DTi-FP1, 3DTi-FP2, and 3DTi-NFP groups loaded with fusion peptides, indicating that KR-12 effectively inhibited S. aureus infection. Notably, bone formation in the 3DTi-NFP group was not affected by the adhesion peptide and showed greater bone regeneration than the 3DTi-FP1 group. This result suggests that the complex immune environment in vivo promotes osteoblast adhesion rather than bacterial adhesion. Analysis of key parameters based on quantitative micro-CT data, including bone mineral density (BMD), bone volume / total volume ratio (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), demonstrated the superior performance of 3DTi-NFP. This success may be attributed to the favorable microenvironment created by 3DTi-NFP, which promotes the anti-infection properties and bone synergistic induction of the implant ( Fig. 9 F-I).

[0071] H&E staining and Masson-Goldner trichrome staining were then performed to observe the bone formation process within 8 weeks ( Fig. 9 E). In the figure, green is trabeculae and cortical bone, orange is collagen fibers, blue-purple is osteocyte nuclei, and black is 3DTi implants. The results showed that in the 3DTi-FP1 and 3DTi-FP2 groups, there were obvious collagen fibers between the newly formed bone and the implant, indicating mixed repair of fibrous tissue and bone-like tissue. In contrast, the 3DTi-NFP group showed more newly mineralized bone, achieving true "osseointegration".

[0072] Importantly, at the end of the 8-week experimental period, pathological examination with H&E staining revealed no abnormalities in the major metabolic organs (heart, liver, spleen, lungs, and kidneys) associated with the presence of S. aureus and peptide-loaded Ti implants. This finding further confirmed the good biocompatibility of all implant materials ( Fig.10 and 11 ).

[0073] In summary, these results indicate that 3DTi-NFP implants can effectively inhibit S. aureus infection while promoting bone healing, showing the best skull defect repair effect. The results include: 1) eradication of S. aureus infection; 2) the composition and structure of the new bone are similar to natural bone tissue, and new blood vessels can be observed; 3) increased coverage of the defect, transition from a predominantly fibrous structure to a bone-like structure, and bone integration.

[0074] The present invention successfully integrated a novel fusion peptide into 3D-printed titanium-based implants, endowing them with specific loading and sustained release capabilities. This approach exploits the synergistic antimicrobial and osteogenic properties of peptides to address the persistent challenges of infection and insufficient bone integration in biomedical devices. The surface of the 3DTi-NFP implant effectively achieved bactericidal and bone regeneration promotion, which is essential for bone healing and enhanced bone integration around implants. Notably, in addition to the antimicrobial properties, the synergistic effect of the antimicrobial peptide KR-12 and the adhesive peptide GFOGER also significantly enhanced the osteogenic potential of bone marrow mesenchymal stem cells, which may involve focal adhesion and activation of the PI3K-Akt signaling pathway. This mechanism accelerated the regeneration of newly formed bone around the implant. Therefore, this study established a promising strategy to successfully endow 3DTi-based implants with antimicrobial and osteogenic properties, providing important clinical potential for alleviating surgical complications and implant failure caused by bacterial infection.

[0075] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multifunctional fusion peptide modified 3D printed Ti implant constructed by a fusion peptide platform, characterized in that: Through the fusion peptide platform technology, the antimicrobial peptide KR-12 and the adhesion peptide GFOGER were simultaneously loaded on the surface of the 3D printed titanium implant through the specific targeting peptide minTBP-1.

2. The multifunctional fusion peptide modified 3D printed Ti implant constructed by a fusion peptide platform according to claim 1, characterized in that: The sequence of the fusion peptide platform is shown in Table 1, which is divided into four main components: (i) biologically active peptide sequences KR-12 and GFOGER; (ii) linker unit; (iii) lysine residues; (iv) specific anchor group minTBP-1; and a rigid linker EAAAK containing a large number of hydrogen bonds is used as a spacer unit; Table 1 。 3. A method for preparing a multifunctional fusion peptide modified 3D printed Ti implant constructed by the fusion peptide platform of claim 1 or claim 2, characterized in that: The specific method is as follows: (1) Preparation of fusion peptides: Based on the listed sequences and synthesis methods, the antimicrobial peptide KR-12, adhesion peptide GFOGER and targeting peptide minTBP-1 were synthesized; the molecular structure of the fusion peptides was predicted and analyzed using protein analysis software PSIPRED and PEP-FOLD; (2) Preparation of Ti material: The titanium substrate was polished step by step with silicon carbide SiC sandpaper No. 400, 800, 1000, 1200, 1500 and 2000, and then ultrasonically cleaned with acetone, 75% ethanol and deionized water for 20 minutes; finally, the substrate was dried and sterilized with ultraviolet light for 3 hours before use; (3) Peptide loading: FITC-labeled peptide powder was dissolved in sterile deionized water to prepare peptide solutions with concentrations of 1, 10, 100, and 1000 ug / ml. Subsequently, the peptide solutions were added dropwise to the surface of the 3DTi sheet placed in a 24-well plate, 300 ul per well, and incubated overnight at room temperature. The next day, unbound peptides were washed away with phosphate-buffered saline (PBS). 3DTi was incubated at room temperature for 5 min or 2 h to observe its binding ability; the three modified 3DTi were labeled as 3DTi-FP1, 3DTi-FP2 and 3DTi-NFP, respectively.

4. The preparation method according to claim 3, characterized in that: The modified 3DTi is labeled as 3DTi-NFP, and the concentration of the polypeptide solution is 100 ug / ml.

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