Antibacterial peptide functionalized bone cement, and preparation method and application thereof

Abstract

The application belongs to the technical field of bone repair implant materials, and particularly relates to an antibacterial peptide functionalized bone cement as well as a preparation method and application thereof. An amino acid sequence general formula of the functional antibacterial peptide is ZUX1X2UX3X4UZ0. The antibacterial peptide functionalized bone cement takes a protease stable cationic antibacterial peptide as a functional component, is prepared by in-situ blending polymerization with a commercial bone cement, can improve the problems of high drug resistance risk, no immune regulation ability and insufficient osteogenesis activity of a traditional antibiotic-loaded bone cement, simultaneously expands an orthopedic material application scene of an Aib modified antibacterial peptide, and realizes a synergistic treatment of infection clearance, immune remodeling and bone regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a bone repair implant material, specifically an antimicrobial peptide functionalized bone cement and its preparation method and application. Background Technology

[0002] Chronic osteomyelitis is a common and refractory complication of orthopedic trauma and internal fixation surgery, with methicillin-resistant Staphylococcus aureus (MRSA) as the main pathogen. A vicious cycle of bacterial biofilm formation, colonization of dormant bacteria, a persistently inflammatory microenvironment, and suppressed osteogenic function within the lesion forms, easily leading to recurrent infections and persistent bone defects. Currently, the mainstream clinical approach is to use vancomycin-loaded PMMA bone cement for local filling of the lesion. However, this approach has significant drawbacks: firstly, vancomycin has difficulty penetrating the extracellular matrix of the biofilm, making it difficult to eradicate deeply persistent bacteria, and long-term use can easily induce bacterial resistance; secondly, vancomycin-loaded PMMA bone cement lacks immunomodulatory functions and cannot improve the excessively inflammatory microenvironment dominated by M1 macrophages in the lesion area; and thirdly, the material lacks osteogenic induction activity, so even if the infection is controlled, the bone defect still cannot repair itself. Therefore, there is an urgent clinical need to develop bone repair implant materials that integrate multiple functions, including highly effective anti-infection, inflammatory immunomodulation, and bone repair promotion.

[0003] Antimicrobial peptides (AMPs) kill bacteria by physically disrupting bacterial cell membranes, are less likely to induce bacterial resistance, and are highly promising novel anti-infective active molecules to replace traditional antibiotics. The applicant and their collaborating team previously filed a Chinese patent with publication number CN115925802B, which discloses an antimicrobial peptide with enhanced antimicrobial activity and serum stability. This peptide is a cationic antimicrobial peptide modified with α-aminoisobutyric acid (Aib, represented by U in the sequence). By introducing Aib residues at sites i and i+3 of the peptide to constrain its spatial conformation, the peptide's serum stability and broad-spectrum antimicrobial activity can be significantly improved. Typical amino acid sequences of the antimicrobial peptide include WKKWUKKUKKUW-NH2 and WRRWURRURRUW-NH2. However, the prior study only optimized the antibacterial properties of the peptide molecules themselves, and did not explore material modification for orthopedic implantation scenarios, nor did it reveal that this type of antimicrobial peptide has multiple biological activities such as regulating macrophage polarization and inducing osteogenic differentiation of mesenchymal stem cells. Currently, there are no related studies or reports on the application of this conformation-constrained cationic antimicrobial peptide in the repair of defects in chronic osteomyelitis by combining it with commercial bone cement.

[0004] Based on this, the present invention continues to explore further, building upon the team's prior research on peptides. For the first time, the above-mentioned protease-stabilized cationic antimicrobial peptides are in situ blended with commercial bone cement to develop a multifunctional antimicrobial peptide-functionalized bone cement. This breakthrough overcomes the functional limitations of traditional drug-loaded bone cement, provides a novel bone repair implant material, and offers a new material solution for the clinical treatment of chronic osteomyelitis. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing bone cement materials and provide an antimicrobial peptide-functionalized bone cement, its preparation method and application, which improves the problems of high drug resistance risk, lack of immunomodulatory ability and insufficient osteogenic activity of traditional antibiotic-loaded bone cement. At the same time, it expands the application scenarios of orthopedic materials with prior Aib-modified antimicrobial peptides to achieve synergistic treatment of infection clearance, immune remodeling and bone regeneration.

[0006] In view of this, the present invention provides an antimicrobial peptide-functionalized bone cement, comprising: a bone cement matrix and a functional antimicrobial peptide, wherein the functional antimicrobial peptide is a cationic antimicrobial peptide, and the general formula of the amino acid sequence of the functional antimicrobial peptide is: ZUX1X2UX3X4UZ0; Where Z is selected from X5Z'- and (Z''X6X7). n Z'-、X8X9Z'''Z''X 10 X 11 Z''X 12 X 13 Any one of Z'; U is a non-natural amino acid; Z0, Z', Z'', Z''' are independently selected from any hydrophobic amino acid; X1~X 13 It is independently selected from any one of arginine, lysine, and histidine; n is 1 or 2.

[0007] Furthermore, the cationic antimicrobial peptide carries 410 positive charges.

[0008] Furthermore, the length of the functional antimicrobial peptide is 10-15.

[0009] Furthermore, the length of the functional antimicrobial peptide is 12.

[0010] Furthermore, the functional antimicrobial peptide is selected from any one or more of the following sequences: WKKWUKKUKKUW-NH2, WRRWURRURRUW-NH2, WRKWUKRURKUW-NH2, where U is α-aminoisobutyric acid.

[0011] Furthermore, the functional antimicrobial peptide is WRRWURRURRUW-NH2.

[0012] Furthermore, the bone cement matrix is ​​an organic acrylic bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix through in-situ compounding and / or surface modification to form the antimicrobial peptide-functionalized bone cement.

[0013] Furthermore, the bone cement matrix is ​​PMMA bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix in situ to form the antimicrobial peptide-functionalized bone cement.

[0014] Furthermore, the amount of functional antimicrobial peptide added to the antimicrobial peptide-functionalized bone cement is 4 to 6 wt% of the PMMA powder phase.

[0015] Furthermore, the water contact angle of the cured antimicrobial peptide-functionalized bone cement is 35° to 40°, the surface zeta potential at pH 7.4 is +6 to +10 mV, and the compressive strength is ≥80 MPa.

[0016] This invention also provides a method for preparing antimicrobial peptide-functionalized bone cement, comprising the following steps: S1, Powder premixing: Mix the powder phase of the bone cement matrix with the functional antimicrobial peptide powder according to the formula to obtain composite powder; S2, In-situ blending: Add liquid phase to the composite powder and stir until a homogeneous elastic paste is formed; S3, Shaping and Curing: The paste is extruded or injected into a mold, polymerized and cured, and then trimmed into shape.

[0017] The present invention also provides the application of the above-mentioned antimicrobial peptide functionalized bone cement in the preparation of bone repair implant materials.

[0018] Furthermore, the bone repair implant material is used to treat chronic osteomyelitis, post-traumatic infected bone defects, or implantation infection after internal fixation surgery.

[0019] Compared with existing technologies, the antimicrobial peptide-functionalized bone cement, its preparation method, and its application described in this invention have the following advantages: First, this invention breaks through the limitations of prior research on single-molecule Aib-modified cationic antimicrobial peptides, expanding the application scenarios of peptides to the field of orthopedic implant repair materials. It combines this conformationally constrained cationic antimicrobial peptide with commercial bone cement in situ, uncovering novel activities of the antimicrobial peptide in regulating macrophage polarization and inducing osteogenic differentiation of mesenchymal stem cells. This upgrades peptide molecules with only antibacterial functions into multifunctional integrated bone repair composite materials that integrate anti-infection, immune regulation, and osteopromotion, significantly enhancing the clinical application boundaries and practical therapeutic value of peptides.

[0020] Secondly, this invention avoids the inherent defects of traditional antibiotic-loaded bone cement at the level of mechanism of action, and achieves synergistic effects of anti-infection and bone repair. The invention uses Aib-modified cationic antimicrobial peptides to replace traditional antibiotics. It exerts its bactericidal effect by relying on electrostatic adsorption and physical disturbance to destroy bacterial cell membranes. It is not easy to induce bacterial drug resistance and can effectively disintegrate mature bacterial biofilms and eliminate persistent bacteria. At the same time, it can regulate the polarization balance of macrophages in lesions, reverse the excessive inflammatory microenvironment dominated by M1 macrophages in chronic osteomyelitis lesions, and simultaneously activate the osteogenic differentiation pathway of stem cells and initiate endogenous bone regeneration, breaking through the technical bottleneck of traditional drug-loaded bone cement that "only inhibits bacteria, does not control inflammation, and is difficult to repair".

[0021] Third, the antimicrobial peptide functionalized bone cement of the present invention adopts an in-situ blending process, the preparation process of which is compatible with the existing clinical surgical system, while taking into account the mechanical properties of the material and the feasibility of clinical transformation. It does not require modification of the entire clinical operation process of existing commercial bone cement, including mixing, injection, and implantation. Moreover, after the composite modified bone cement is cured and molded, it still retains the core advantages of high mechanical load-bearing capacity and excellent plasticity of bone cement substrate. Furthermore, the compressive strength and curing characteristics of the material meet the mechanical standards of orthopedic implants. There is no need for additional modification of surgical instruments and clinical operation specifications. The process is simple, the production adaptability is strong, and it has the potential for rapid clinical transformation and implementation.

[0022] Fourth, a multi-target synchronous intervention and treatment system targeting the pathological closed loop of chronic osteomyelitis is specifically constructed, forming a complete chain-like repair paradigm. Chronic osteomyelitis is a vicious cycle formed by bacterial colonization and biofilm formation, persistent bacterial latency, persistent excessive inflammation, and osteogenic inhibition. Traditional materials only target a single antibacterial link and cannot break the complete pathological chain. The antimicrobial peptide functionalized bone cement provided by this invention can sequentially achieve simultaneous intervention throughout the entire process of bacterial electrostatic capture, complete biofilm lysis and removal, lesion immune microenvironment remodeling, and endogenous bone tissue regeneration and repair. It blocks the pathological closed loop of chronic osteomyelitis from multiple links, including the source of infection, the inflammatory microenvironment, and bone defect regeneration, and achieves integrated synergistic treatment of anti-infection, anti-inflammation, and bone repair. The comprehensive treatment effect on refractory chronic osteomyelitis is significantly better than that of existing vancomycin-loaded bone cement.

[0023] Fifth, the modified components exhibit excellent biocompatibility, ensuring long-term implantation safety. The Aib-modified cationic antimicrobial peptides used in this invention have been verified in vitro to possess low hemolysis and low cytotoxicity characteristics, and at clinically effective antibacterial concentrations, they have no significant negative impact on mammalian cell survival. When incorporated into bone cement, they do not exacerbate substrate defects such as exothermic curing of bone cement or free monomer toxicity, nor are they prone to inducing severe hemolysis, apoptosis, or other adverse implantation reactions, thus possessing both long-lasting antibacterial stability and in vivo implantation biosafety. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the preparation process and function of the antimicrobial peptide-functionalized PMMA bone cement described in this invention. Figure 2 The physicochemical properties characterization results of the various groups of bone cements prepared in this invention are as follows: A represents the SEM surface morphology of PMMA, VCM-PMMA, and AMP-PMMA; B represents the XPS full spectrum of PMMA and AMP-PMMA; C-E represent the high-resolution XPS peak spectra of O1s, C1s, and N1s, respectively; F and G represent the water contact angle representative diagrams and quantitative results, respectively; H represents the Zeta potential of PMMA, AMP-PMMA, and free AMP under pH 7.4 and 5.5 conditions; I represents the compressive stress-strain curves of each group; J represents the 14-day cumulative release curves of different AMP loadings; K represents the free AMP, trypsin, and the results of co-incubation of the two. 1 H NMR spectrum; Figure 3 The results of in vitro antibacterial and antibiofilm properties tests of the various groups of bone cement prepared by this invention are shown in the following figures: A is the MRSA colony map of each group; B is the CFU quantification; C is the MRSA live / dead fluorescence staining; D is the SEM image of MRSA on the bone cement surface; E is the MRSA intracellular ATP level; F is the 24-h MRSA growth curve; and G is the MIC change during 50 consecutive passages. Figure 4 These are the results of in vitro biofilm activity tests against methicillin-resistant Staphylococcus aureus (MRSA) in various groups of bone cement prepared according to the present invention. Among them, A is crystal violet staining of MRSA biofilm; B is quantitative biofilm biomass at 595 nm; and C is three-dimensional CLSM reconstruction map of live / dead MRSA biofilm in each group. Figure 5 The results of in vitro biocompatibility testing of the bone cements prepared in this invention are shown in Figure A, where A represents the live / dead fluorescence staining of BMSCs in each group; B represents the viability of BMSCs after treatment with different concentrations of AMP or VCM; and C represents the representative graphs of the hemolysis test and the quantitative results of the hemolysis rate in each group. Figure 6 These are the experimental results of bone cement prepared in this invention regulating osteogenic differentiation of BMSCs and macrophage polarization. Among them, A and B are ALP staining and Alizarin Red S staining of BMSCs, respectively; C and D are ALP activity and Alizarin Red S quantification, respectively; E and F are the relative expression levels of Runx2 and Col-1, respectively; G and H are the relative expression levels of iNOS and CD206, respectively; I and J are the Western blot results of osteogenic and immune markers, respectively. Figure 7These are the results of in vivo bone regeneration and immune remodeling experiments in rat models of chronic osteomyelitis using bone cement prepared in this invention. In this study, A represents Micro-CT reconstruction of the femoral defect area in each group; B-E represent the quantitative analysis of BV / TV, Tb.Th, Tb.N, and Tb.Sp, respectively; F represents immunohistochemical staining for OCN, RUNX2, iNOS, and CD206; and G-J represent the quantitative analysis of the positive area of ​​the above four markers. Figure 8 The results of transcriptomic pathway enrichment analysis of bone defect tissues of various groups of bone cement prepared in this invention are shown. Among them, A is the gene expression heatmap of femoral tissue of each group; B and C are volcano maps of differential genes of AMP-PMMA / PMMA and VCM-PMMA / PMMA, respectively; D and E are the KEGG pathway enrichment of the two groups compared; F and G are the GO functional enrichment of the two groups compared. Figure 9 These are the H&E and Masson staining results of local tissues after implantation of the bone cement prepared in this invention. Figure 10 These are the H&E staining results of major organs after implantation of bone cement prepared in this invention. Figure 11 These are the gross observation results of the femoral specimens after implantation of the bone cement prepared in this invention. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0026] It should be noted that all directional and positional terms used in this invention, such as "up," "down," "left," "right," "front," "back," "vertical," "horizontal," "inner," "outer," "top," "lower," "lateral," "longitudinal," and "center," are only used to explain the relative positional relationships and connections between components in a specific state (as shown in the accompanying drawings). They are merely for the convenience of describing the invention and do not require the invention to be constructed and operated in a specific orientation; therefore, they should not be construed as limitations on the invention. Furthermore, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0027] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0028] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0029] This invention provides a bone repair implant material, specifically a functionalized bone cement modified with functional antimicrobial peptides.

[0030] Furthermore, the functionalized bone cement comprises: a bone cement matrix and a functional antimicrobial peptide, wherein the general formula of the amino acid sequence of the functional antimicrobial peptide is: ZUX1X2UX3X4UZ0; Where Z is selected from X5Z'- and (Z''X6X7). n Z'-、X8X9Z'''Z''X 10 X 11 Z''X 12 X 13 Z' is any one of the following, U is a non-natural amino acid, Z0, Z', Z'', and Z''' are independently selected from any hydrophobic amino acid, and X1 to X 13 It is independently selected from any one of arginine, lysine, and histidine, and n is 1 or 2.

[0031] As a preferred example of the present invention, Z0 is selected from any one of leucine, isoleucine, and tryptophan; Z', Z'', and Z''' are independently selected from any one of leucine, isoleucine, and tryptophan; U is aminoisobutyric acid (Aib); and X1 to X13 are independently selected from arginine or lysine.

[0032] As a preferred example of the present invention, the functional antimicrobial peptide is a cationic antimicrobial peptide. More preferably, the cationic antimicrobial peptide carries 410 positive charges.

[0033] As a preferred example of the present invention, the sequence length of the functional antimicrobial peptide is 10 to 15, such as 10, 12, 15, etc., and more preferably 12.

[0034] As a preferred example of the present invention, the functional antimicrobial peptide is selected from any one or more of the following sequences: WKKWUKKUKKUW-NH2, WRRWURRURRUW-NH2, WRKWUKRURKUW-NH2, where U is α-aminoisobutyric acid.

[0035] Among the P1-P14 antimicrobial peptides listed in Table 1 of the prior patent (CN115925802B), the 12-length peptide exhibits a more balanced composition. It has three uniformly distributed Aib conformational constraint sites, a highly stable helical structure, and a significantly prolonged serum half-life, resisting long-term degradation by proteases within the lesion. Furthermore, its moderate net positive charge and hydrophobicity match the bacterial membrane lysis threshold, resulting in lower MICs against common osteomyelitis pathogens such as MRSA, Escherichia coli, and Pseudomonas aeruginosa. It also possesses excellent anti-biofilm capabilities, effectively clearing deep-seated bacteria from lesions and avoiding the problem of traditional antibiotic resistance. Moreover, its excellent salt stability prevents significant attenuation of antibacterial activity in the high-ionic environment of the lesion, ensuring stable in vivo anti-infection effects. In addition, the 12-length peptide demonstrates superior biocompatibility, lower hemolysis and cytotoxicity, and a more suitable cost, making it more suitable for the industrialization of orthopedic consumables. Therefore, it is the optimal choice for combining with commercial bone cement to prepare multifunctional osteomyelitis repair materials.

[0036] More preferably, the functional antimicrobial peptide is WRRWURRURRUW-NH2.

[0037] As some examples of the present invention, the bone cement matrix is ​​an organic acrylic bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix through in-situ compounding and / or surface modification to form the antimicrobial peptide functionalized bone cement.

[0038] As a preferred example of the present invention, the bone cement matrix is ​​PMMA bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix in situ to form the antimicrobial peptide-functionalized bone cement.

[0039] It should be noted that the PMMA bone cement can be pure PMMA homopolymer bone cement, or it can be PMMA copolymer modified acrylic bone cement, such as methyl methacrylate-styrene copolymer bone cement, methyl methacrylate-polymethyl acrylate copolymer bone cement, methyl methacrylate-acrylate multi-element copolymer bone cement, etc.

[0040] As a preferred example of the present invention, the amount of functional antimicrobial peptide added to the antimicrobial peptide functionalized bone cement is 4 to 6 wt% of the mass of PMMA powder phase, more preferably 5 wt%.

[0041] Furthermore, the water contact angle of the antimicrobial peptide functionalized bone cement described in this invention after curing is 35°~40°, the surface Zeta potential at pH 7.4 is +6~+10mV, and the compressive strength is ≥80MPa.

[0042] Furthermore, the release of functional antimicrobial peptides in the antimicrobial peptide-functionalized bone cement of the present invention exhibits a biphasic characteristic: the cumulative release amount is approximately 80% within 4 days and approximately 90% within 14 days.

[0043] Furthermore, the present invention also provides a method for preparing antimicrobial peptide-functionalized bone cement, which includes the following steps: S1, Powder premixing: The powder phase (i.e. powder) that forms the bone cement matrix is ​​fully ground and mixed with the functional antimicrobial peptide powder according to the formula to obtain a composite powder; S2, In-situ blending: Add liquid phase (mainly MMA liquid monomer) to the composite powder and stir until a homogeneous elastic paste is formed; S3, Shaping and Curing: The paste is squeezed out or injected into a mold, fully polymerized and cured at room temperature, and then trimmed into the shape of a bone cement implant. Store at 4°C away from light for later use.

[0044] The preparation parameters, such as the selection of raw materials, proportions, and polymerization and curing processes of the bone cement matrix, can be found in relevant existing technologies, and will not be described in detail in this invention.

[0045] As some examples of the present invention, the antimicrobial peptide functionalized bone cement may also include additives such as coagulants, polymerization inhibitors, X-ray imaging fillers, and toners.

[0046] Furthermore, this invention also provides the application of the above-mentioned antimicrobial peptide-functionalized bone cement in the preparation of bone repair implant materials.

[0047] As a preferred example of the present invention, the antimicrobial peptide functionalized bone cement of the present invention is particularly suitable for preparing anti-infective bone repair implant materials.

[0048] As a preferred example of the present invention, the bone repair implant material can be used to treat chronic osteomyelitis, such as MRSA-induced chronic osteomyelitis, post-traumatic infected bone defects, or implantation infections after internal fixation surgery.

[0049] The antimicrobial peptide-functionalized bone cement of this invention achieves the repair of infected bone defects through the synergistic effects of electrostatic bacterial capture, removal of mature biofilms, regulation of macrophage polarization towards the M2 type, and promotion of osteogenic differentiation of bone marrow mesenchymal stem cells. For details of its mechanism of action, please refer to [link to relevant documentation]. Figure 1 .

[0050] The following specific examples illustrate the antimicrobial peptide-functionalized bone cement and its preparation method according to the present invention: Example 1: Preparation of antimicrobial peptide-functionalized PMMA bone cement Experimental materials: Commercial PMMA bone cement (powder phase containing PMMA, benzoyl peroxide, and zirconium dioxide; liquid phase containing methyl methacrylate (MMA), N,N-dimethyl-p-toluidine, and hydroquinone); functional antimicrobial peptide WRRWURRURRUW-NH2 (solid phase synthesis, purity >95%, C-terminal amidation).

[0051] Preparation process: (1) Weigh 500mg of powder phase and 25mg of functional antimicrobial peptide powder, place them in an agate mortar and grind them thoroughly for 10min until the powder color is uniform and there are no particles agglomerating, to obtain composite powder; (2) Add 250 μL of liquid phase to the composite powder and stir rapidly for 30 s to form a homogeneous elastic paste without particles; (3) The paste is drawn into a sterile syringe and extruded into a fine filament with a diameter of 1 mm through a custom needle. Before complete polymerization, it is cut into a standard rod with a length of 12 ± 1 mm. (4) Let stand at room temperature for 2 hours until completely cured, then seal and store in a refrigerator at 4°C away from light for later use.

[0052] In subsequent experiments, the antimicrobial peptide-functionalized PMMA bone cement sample prepared in Example 1 was named AMP-PMMA.

[0053] Comparative Example 1: Sample preparation for the control group: The blank PMMA group (without added antimicrobial peptide) and the VCM-PMMA group (using 25 mg vancomycin hydrochloride to replace the functional antimicrobial peptide) were prepared using the exact same process.

[0054] Experimental Example 1: Characterization of the physical and chemical properties of materials The physicochemical properties of the samples prepared in Example 1 and Comparative Example 1 were tested, and the results are shown in the figure. Figure 2 : (I) Surface morphology and elemental characterization: Scanning electron microscopy (SEM) showed that the surface of pure PMMA was dense and smooth; VCM-PMMA had irregular large pores and phase separation; AMP-PMMA had a dense and homogeneous microstructure with uniformly dispersed micropores and no obvious agglomeration.

[0055] (ii) X-ray photoelectron spectroscopy (XPS) showed that AMP-PMMA had a distinct N1s characteristic peak, confirming that the antimicrobial peptide was successfully incorporated into the material matrix.

[0056] (III) Hydrophilicity and Surface Potential: Static water contact angle test results: pure PMMA 65°±3°, VCM-PMMA 112°±4°, AMP-PMMA 38°±3°, showing a significant improvement in hydrophilicity. Zeta potential at pH 7.4: pure PMMA -15mV, AMP-PMMA +8mV, achieving a change from negative to positive surface charge.

[0057] (iv) Mechanical properties: The results of the compression test by the universal testing machine are as follows: the compressive strength of pure PMMA is 93.2 MPa, VCM-PMMA is 86.5 MPa, and AMP-PMMA is 80.3 MPa, all of which meet the clinical threshold of >70 MPa for orthopedic implant materials.

[0058] (v) In vitro release behavior: PBS immersion release experiment showed that 5% load of AMP-PMMA exhibited biphasic release characteristics: approximately 80% was released cumulatively in the first 4 days, achieving rapid anti-infection; sustained release continued from 4 to 14 days, with approximately 90% released cumulatively on the 14th day, maintaining long-term biological activity.

[0059] Experimental Example 2: Validation of in vitro antibacterial and anti-biofilm properties Using MRSAUSA300 strain as the experimental subject, the antibacterial properties of the material were tested, and the results are as follows: Figures 3-4 As shown: (a) Plate colony counting: After co-culturing the samples with bacterial suspension for 24 hours, the surface bacteria were washed off and plated for counting. The blank group and the pure PMMA group had dense colonies, while the VCM-PMMA and AMP-PMMA groups had significantly reduced colony counts, and the bactericidal efficiency of the two groups was comparable.

[0060] (ii) Resistance induction experiment: MRSA was passaged 50 times at sub-MIC concentration, and the MIC value was measured every 5 generations. The results showed that the MIC / MIC0 ratio of the VCM group exceeded 8 times, indicating significant drug resistance; the MIC / MIC0 ratio of the AMP-PMMA group was only about 2 times, and the risk of drug resistance induction was significantly lower.

[0061] (III) Mature biofilm removal experiment: Mature MRSA biofilms were formed by pre-culturing for 48 hours, and then each group of samples was added for 24 hours of treatment. The results of quantitative crystal violet and laser confocal three-dimensional reconstruction showed that VCM could only kill bacteria on the surface of the biofilm and could not destroy the biofilm structure; AMP-PMMA could completely disintegrate the biofilm skeleton and kill most of the bacteria inside the membrane, with a significantly better removal effect.

[0062] Experimental Example 3: Validation of In Vitro Immune Regulation and Osteogenic Activity Results of in vitro immune regulation and osteogenic activity validation experiments are shown in Figures 5-6 Specifically: (I) Regulation of macrophage polarization: An inflammation model was constructed using RAW264.7 macrophages induced by LPS, and each group of materials was treated with extracts for 24 h. Western blot analysis showed that the pure PMMA group and the VCM-PMMA group had high expression of the M1 marker iNOS and low expression of the M2 marker CD206; the AMP-PMMA group showed significant downregulation of iNOS and significant upregulation of CD206, which can effectively drive macrophage polarization from pro-inflammatory M1 to repair M2.

[0063] (II) Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells: Osteogenic induction was performed using rat BMSCs, with extracts from each group added. ALP activity was detected after 14 days of induction, and Alizarin Red S mineralization staining was performed after 21 days. Results showed that the ALP activity and mineralized nodule quantity in the AMP-PMMA group were significantly higher than the other three groups. Western blot analysis confirmed a significant upregulation of the osteogenic core proteins Runx2 and Col-1, indicating that the material possesses direct osteogenic differentiation-promoting activity.

[0064] (III) Biocompatibility test: The hemolysis test showed that the hemolysis rate of AMP-PMMA was 1.8%, which was lower than that of VCM-PMMA (2.8%) and far below the clinical safety limit of 5%; CCK-8 and live / dead staining showed that the material extract could promote the proliferation of BMSCs without significant cytotoxicity.

[0065] Experiment 4: In vivo treatment of chronic osteomyelitis in rats Animal model and grouping: 8-week-old female SD rats, bone defects were established by drilling holes in the distal femur, and 1×10⁻⁶ oz. medullary cavity was injected. 8 A chronic osteomyelitis model was established by sealing the defect with CFU / mL MRSA bacterial solution and bone wax for 2 weeks. Rats with successful modeling were randomly divided into 4 groups: infection control group, pure PMMA group, VCM-PMMA group, and AMP-PMMA group, with 6 rats in each group. After debridement, corresponding bone cement rods were implanted into the defect, and the wound was sutured layer by layer.

[0066] Results of in vivo treatment experiments for chronic osteomyelitis in rats are shown below. Figure 7 , Figure 9 , Figure 10 and Figure 11 As shown, specifically: (I) Imaging evaluation of bone regeneration: Micro-CT scans and three-dimensional reconstructions were performed on samples taken 2 months postoperatively. The results showed that the infected control group and the pure PMMA group had significant bone defects and sparse and disordered trabeculae; the VCM-PMMA group had a small amount of new bone formation; the AMP-PMMA group had almost closed bone defects, abundant new bone formation, and dense trabecular structure. Quantitative parameters showed that the AMP-PMMA group had significantly higher bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) than the other three groups, and significantly lower trabecular separation (Tb.Sp).

[0067] (II) Histological and Immunohistochemical Evaluation: H&E staining showed that the AMP-PMMA group had the least inflammatory cell infiltration and well-preserved tissue morphology; Masson's trichrome staining showed that the AMP-PMMA group had abundant collagen deposition and formation of mature new bone matrix. Immunohistochemical results showed that the AMP-PMMA group had significantly increased positive expression of bone markers OCN and RUNX2, upregulated M2 macrophage marker CD206, and downregulated pro-inflammatory M1 marker iNOS.

[0068] (III) System safety evaluation: Two months after the operation, the heart, liver, spleen, lungs and kidneys were taken for H&E staining. No obvious inflammatory infiltration, tissue necrosis or structural damage was observed in any group, which confirmed that the material had good systemic biosafety.

[0069] Experimental Example 5: Verification of Transcriptomics Mechanism Transcriptome sequencing was performed on femoral bone defect tissue from rats two months post-surgery. Bioinformatics analysis results are shown below. Figure 8 The results showed: Compared to the pure PMMA group, the differentially expressed genes in the AMP-PMMA group were significantly enriched in the PI3K-Akt signaling pathway, focal adhesion pathway, and autophagy pathway. These pathways are directly related to osteogenic differentiation and macrophage polarization regulation. In contrast, the differentially expressed genes in the VCM-PMMA group were mainly enriched in basic metabolic pathways such as ribosome synthesis and RNA splicing, without activation of specific bone repair and immune regulation pathways. These results confirm at the molecular level that the material of this invention achieves dual regulation of immunity and osteogenicity through multi-pathway synergy, and its functional mechanism far exceeds the single antibacterial activity disclosed in prior patents.

[0070] In summary, this invention discloses an antimicrobial peptide-functionalized bone cement, its preparation method, and its applications. The antimicrobial peptide-functionalized bone cement is prepared by in-situ co-polymerization of protease-stabilized cationic antimicrobial peptides with commercial bone cement. This antimicrobial peptide-functionalized bone cement material exhibits a positively charged surface, excellent hydrophilicity, and compressive strength meeting clinical implantation standards. The peptides are released in a biphasic manner. It can efficiently kill MRSA and disintegrate mature biomembranes through physical membrane disruption mechanisms with low drug resistance risk. Simultaneously, it can regulate macrophage polarization towards M2 repair and promote osteogenic differentiation of bone marrow mesenchymal stem cells. In vivo experiments have confirmed that this material can significantly accelerate bone regeneration in chronic osteomyelitis lesions, with good systemic biocompatibility. This invention overcomes the limitations of traditional antibiotic-loaded bone cements that "only fight infection and do not promote repair," constructing an integrated treatment system of "capture-bactericidal-immune remodeling-bone regeneration." It is a bone repair implant material with excellent comprehensive performance, particularly suitable for the clinical treatment of refractory MRSA chronic osteomyelitis, and has promising prospects for translational application.

[0071] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. An antimicrobial peptide-functionalized bone cement, characterized in that, include: Bone cement matrix and functional antimicrobial peptide, wherein the functional antimicrobial peptide is a cationic antimicrobial peptide, and the general formula of the amino acid sequence of the functional antimicrobial peptide is: ZUX1X2UX3X4UZ0; Where Z is selected from X5Z'- and (Z''X6X7). n Z'-、X8X9Z'''Z''X 10 X 11 Z''X 12 X 13 Any one of Z'; U is a non-natural amino acid; Z0, Z', Z'', Z''' are independently selected from any hydrophobic amino acid; X1~X 13 It is independently selected from any one of arginine, lysine, and histidine; n is 1 or 2.

2. The antimicrobial peptide-functionalized bone cement according to claim 1, characterized in that, The cationic antimicrobial peptide carries 410 positive charges.

3. The antimicrobial peptide-functionalized bone cement according to claim 1, characterized in that, The length of the functional antimicrobial peptide is 10-15.

4. The antimicrobial peptide-functionalized bone cement according to claim 3, characterized in that, The length of the functional antimicrobial peptide is 12.

5. The antimicrobial peptide-functionalized bone cement according to claim 4, characterized in that, The functional antimicrobial peptide is selected from any one or more of the following sequences: WKKWUKKUKKUW-NH2, WRRWURRURRUW-NH2, WRKWUKRURKUW-NH2, where U is α-aminoisobutyric acid.

6. The antimicrobial peptide-functionalized bone cement according to claim 5, characterized in that, The functional antimicrobial peptide is WRRWURRURRUW-NH2.

7. The antimicrobial peptide-functionalized bone cement according to claim 1, characterized in that, The bone cement matrix is ​​an organic acrylic bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix through in-situ compounding and / or surface modification to form the antimicrobial peptide-functionalized bone cement.

8. The antimicrobial peptide-functionalized bone cement according to claim 7, characterized in that, The bone cement matrix is ​​PMMA bone cement, and the functional antimicrobial peptide is compounded with the bone cement matrix in situ to form the antimicrobial peptide-functionalized bone cement.

9. The antimicrobial peptide-functionalized bone cement according to claim 8, characterized in that, The amount of functional antimicrobial peptide added to the antimicrobial peptide-functionalized bone cement is 4 to 6 wt% of the PMMA powder phase.

10. The antimicrobial peptide-functionalized bone cement according to claim 1, characterized in that, The water contact angle of the antimicrobial peptide-functionalized bone cement after curing is 35°~40°, the surface zeta potential at pH 7.4 is +6~+10mV, and the compressive strength is ≥80MPa.

11. A method for preparing antimicrobial peptide-functionalized bone cement, characterized in that, The preparation of the antimicrobial peptide-functionalized bone cement according to any one of claims 1 to 10 includes the following steps: S1, Powder premixing: Mix the powder phase of the bone cement matrix with the functional antimicrobial peptide powder according to the formula to obtain composite powder; S2, In-situ blending: Add liquid phase to the composite powder and stir until a homogeneous elastic paste is formed; S3, Shaping and Curing: The paste is extruded or injected into a mold, polymerized and cured, and then trimmed into shape.

12. The use of the antimicrobial peptide functionalized bone cement according to any one of claims 1 to 10 in the preparation of bone repair implant materials.

13. The application according to claim 12, characterized in that, The bone repair implant material is used to treat chronic osteomyelitis, post-traumatic infected bone defects, or implantation infections after internal fixation surgery.

Images