A method for preparing an antibacterial-cell adhesion promoting orthopedic implant

Abstract

The application discloses a preparation method of an antibacterial and cell adhesion promoting orthopedic implant, and steps are as follows: 1) configuring a water solution of polyphenol compounds, adding polyhexamethylene guanidine hydrochloride and arginine-glycine-aspartic acid into the water solution, wherein the mass concentration ratio of the polyhexamethylene guanidine hydrochloride and the arginine-glycine-aspartic acid is 50:1-200:1; 2) soaking the implant base material in the solution at room temperature for 12-24 hours; and 3) after cleaning, the antibacterial and cell adhesion promoting orthopedic implant is obtained. The application realizes competitive growth of bacteria and cells, i.e. cell> bacterial growth. The antibacterial and cell adhesion promoting orthopedic implant realizes a win-win of antibacterial and adhesion promoting and bone forming, and can reduce the problem of implant loosening caused by infection in orthopedic surgery to a certain extent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and relates to a preparation method of an antibacterial and cell adhesion promoting orthopedic implant. BACKGROUND

[0002] Titanium (Ti) implants have superior physical and chemical properties and are favored in orthopedic applications. However, these implants often face the challenges of bacterial infection and insufficient bone integration in clinical practice. Implant-related bone infection is a key and persistent clinical problem, mainly caused by bacterial colonization and contamination on the surface of the implant, which can lead to severe inflammation, tissue necrosis, implant loosening, and ultimately implant treatment failure. Infection is mainly attributed to the dominant position of bacteria in the adhesion competition with tissue cells on the surface of the implant. Therefore, it is crucial to construct an implant surface that can inhibit bacterial colonization and promote cell adhesion. However, obtaining superior bactericidal performance and satisfactory cell compatibility is sometimes contradictory, as antibacterial ability is inevitably associated with cytotoxicity. Balancing the antibacterial performance of the implant surface and the ability to promote osteogenesis is a major challenge. Therefore, developing a strategy that can eliminate bacteria while promoting cell attachment and proliferation on a single surface may provide an effective solution to implant-related problems.

[0003] Various surface functionalization manufacturing strategies have been widely developed, including anodization, copolymerization, co-immobilization / co-precipitation, layer-by-layer assembly, cross-linking, and loading techniques. Recently, in order to integrate antibacterial performance and enhance cell adhesion to promote the osteogenic performance of material surfaces, various surface bioactivation strategies have been developed. Notable advances include the creation of smart surfaces that evolve in three stages of the implant life cycle, thereby ensuring long-term antibacterial properties, stem cell adhesion, and osteogenesis promotion. Carbon dots derived from arginine show significant bactericidal and osteogenic effects. In addition, implant surfaces treated with copper ion charge conversion coatings show improved antibacterial and cell adhesion properties. Furthermore, biomimetic adaptive nanocomposite arrays provide antibacterial advantages and stimulate cell proliferation without relying on antibiotics. However, stable modification of bioactive materials often requires complex, multi-step processes and harsh conditions, such as the use of strong acids or strong bases and ultraviolet irradiation. Therefore, there is an urgent need for a universal and simple modification technique to transform material surfaces from bio-inert to bio-active.

[0004] To develop a simple bioactivation strategy to create multifunctional surfaces, polyphenol chemistry offers significant advantages by facilitating the attachment of functional components to the surface. In particular, the backbone structure of multiple phenolic hydroxyl groups enables covalent and non-covalent interactions with a variety of biomacromolecules and bioactive ingredients containing -NH2 groups, including the antibacterial agent polyhexamethylene guanidine hydrochloride (PHMG) and the osteogenesis-promoting compound arginine-glycine-aspartic acid (RGD). PHMG is an effective cationic antibacterial agent that acts on a wide range of microorganisms, including bacteria, viruses and fungi. In addition, PHMG is stable, cost-effective, and relatively safe to humans and the environment. In addition to its remarkable bactericidal properties, bio-inspired surfaces also need to promote cell adhesion and osteogenesis. The RGD sequence exists in many extracellular matrix (ECM) proteins, which can specifically bind to integrins on the cell membrane and thus promote cell adhesion to the implant. The integration of RGD peptides is particularly promising because it has been shown to accelerate bone osteogenesis and enhance cell adhesion. However, how to integrate the antibacterial agent PHMG and the osteogenesis-enhancing compound RGD into the implant surface to simultaneously achieve antibacterial and osteogenic properties remains a challenge. SUMMARY

[0005] Therefore, the present application provides a preparation method of an antibacterial-cell adhesion promoting orthopedic implant.

[0006] The present application specifically provides the following technical solutions:

[0007] A preparation method of an antibacterial-cell adhesion promoting orthopedic implant, comprising the following steps:

[0008] 1) configuring a water solution of polyphenols, adding polyhexamethylene guanidine hydrochloride and arginine-glycine-aspartic acid to the single water solution, wherein the mass concentration ratio of polyhexamethylene guanidine hydrochloride and arginine-glycine-aspartic acid is 50:1-200:1;

[0009] 2) soaking the implant substrate in the above solution at room temperature for 12-24 hours;

[0010] 3) after cleaning, obtaining the antibacterial-cell adhesion promoting orthopedic implant.

[0011] Further, the mass concentration ratio of polyhexamethylene guanidine hydrochloride and arginine-glycine-aspartic acid is 100:1.

[0012] The concentration of arginine-glycine-aspartic acid is 100 μg / mL.

[0013] Further, the polyphenolic compound is pyrogallol, tea polyphenol or tannic acid.

[0014] Further, the mass concentration ratio of polyhexamethylene guanidine hydrochloride and arginine-glycine-aspartic acid is 50:1, 100:1, 150:1, 200:1.

[0015] Further, the step 3) is washed in water and ethanol respectively and alternately.

[0016] Further, the implant substrate is a titanium sheet.

[0017] The beneficial effects of the present application are that the antibacterial agent kills bacteria while often producing similar toxicity to eukaryotic cells, affecting the normal physiological function of the cells. The antibacterial agent damages the bacterial cell membrane while the eukaryotic cell membrane is also damaged. Since there is a big difference in size between eukaryotic cells and prokaryotic bacteria, it is crucial to regulate the content of the antibacterial agent. Moreover, the antibacterial agent itself does not have cell proliferation adhesion-promoting properties, and for an orthopedic implant, bone binding performance is crucial. Therefore, how to deal with bacterial damage under infection conditions without affecting cell growth and promoting cell adhesion growth is crucial. The present application screens different ratios of antibacterial / osteogenic adhesion-promoting peptides in the coating to obtain the optimal ratio (100:1). Under this ratio, the coating can achieve good bactericidal effect (with a bactericidal rate of 90%) and significantly promote the adhesion, proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). The present application realizes the competitive growth of bacteria and cells: cell > bacterial growth. It realizes the win-win of antibacterial and adhesion-promoting osteogenic, which can to some extent reduce the problem of implant loosening caused by infection in orthopedic surgery. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to make the purpose, technical solutions and beneficial effects of the present application clearer, the present application provides the following drawings:

[0019] Figure 1 It is a cell-bacterial competitive culture diagram.

[0020] Figure 2 It is a cell survival rate and antibacterial rate statistical diagram.

[0021] Figure 3 It is a cell biocompatibility and cell proliferation-promoting diagram.

[0022] Figure 4 It is a cell adhesion-promoting spreading diagram.

[0023] Figure 5 It is an in vivo implant osteogenic-promoting test.

[0024] Figure 6 It is an in vivo implant and bone binding force pull-out test.

[0025] Figure 7 It is an antibacterial effect diagram. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] The antibacterial and cell adhesion promoting implant 1 was prepared according to the following steps:

[0029] 1) A polyphenol compound (pyrogallol or tea polyphenol or tannic acid) was dissolved in a buffer to form a 20 mg / mL tannic acid aqueous solution, and polyhexamethylene guanidine hydrochloride (PHMG) and arginine-glycine-aspartic acid (RGD) were added to the tannic acid aqueous solution, wherein the PHMG concentration was 5 mg / mL and the RGD concentration was 100 μg / mL.

[0030] 2) The titanium sheet was immersed in the above solution at a temperature of 25 degrees for 12 hours;

[0031] 3) The modified titanium sheet was alternately washed in water and ethanol for 5 minutes respectively.

[0032] The antibacterial and cell adhesion promoting implant obtained in this example was denoted as sample 1.

[0033] Example 2

[0034] The antibacterial and cell adhesion promoting implant 2 was prepared according to the following steps:

[0035] 1) A polyphenol compound (pyrogallol or tea polyphenol or tannic acid) was dissolved in a buffer to form a 20 mg / mL tannic acid aqueous solution, and polyhexamethylene guanidine hydrochloride (PHMG) and arginine-glycine-aspartic acid (RGD) were added to the tannic acid aqueous solution, wherein the PHMG concentration was 10 mg / mL and the RGD concentration was 100 μg / mL.

[0036] 2) The titanium sheet of different specifications was immersed in the above solution at a temperature of 25 degrees for 12 hours;

[0037] 3) The modified titanium sheet was alternately washed in water and ethanol for 5 minutes respectively.

[0038] The antibacterial and cell adhesion promoting implant obtained in this example was denoted as sample 2, also referred to as Ti-PR.

[0039] Example 3

[0040] The antibacterial and cell adhesion promoting implant 3 was prepared according to the following steps:

[0041] 1) polyphenol compound (pyrogallol or tea polyphenol or tannic acid) is dissolved in buffer to prepare a tannic acid aqueous solution of 20 mg / mL, and polyhexamethylene guanidine hydrochloride (PHMG) and arginine-glycine-aspartic acid (RGD) are added to the tannic acid aqueous solution, wherein the concentration of PHMG is 15 mg / mL, and the concentration of RGD is 100 μg / mL.

[0042] 2) titanium sheets of different specifications are immersed in the above solution at a temperature of 25 degrees for 12 hours of reaction;

[0043] 3) the modified titanium sheets are alternately washed in water and ethanol for 5 minutes respectively.

[0044] The antibacterial and cell adhesion promoting implant obtained in this example is denoted as sample 3.

[0045] Example 4

[0046] An antibacterial and cell adhesion promoting implant 4 is prepared, and the steps are as follows:

[0047] 1) polyphenol compound (pyrogallol or tea polyphenol or tannic acid) is dissolved in buffer to prepare a tannic acid aqueous solution of 20 mg / mL, and polyhexamethylene guanidine hydrochloride (PHMG) and arginine-glycine-aspartic acid (RGD) are added to the tannic acid aqueous solution. The concentration of PHMG is 20 mg / mL, and the concentration of RGD is 100 μg / mL.

[0048] 2) titanium sheets of different specifications are immersed in the above solution at a temperature of 25 degrees for 12 hours of reaction;

[0049] 3) the modified titanium sheets are alternately washed in water and ethanol for 5 minutes respectively.

[0050] The antibacterial and cell adhesion promoting implant obtained in this example is denoted as sample 4.

[0051] Comparative Example 1

[0052] Pure titanium sheets (Ti) are used as a control group, denoted as sample Ti.

[0053] Comparative Example 2

[0054] Pure titanium is modified with antibacterial agent PHMG: polyphenol compound (pyrogallol or tea polyphenol or tannic acid) is dissolved in buffer (pH) to prepare a tannic acid aqueous solution of 20 mg / mL, and polyhexamethylene guanidine hydrochloride (PHMG) is added to the tannic acid solution at a proportion of 15 mg / mL, and titanium sheets are immersed in the above solution at a temperature of 25 degrees for 12 hours of reaction; the modified titanium sheets are alternately washed in water and ethanol for 5 minutes respectively. Denoted as sample Ti-P.

[0055] Comparative Example 3

[0056] Pure titanium was modified with the cell adhesion-promoting peptide RGD: Polyphenolic compounds (pyrogallol, tea polyphenols, or tannic acid) were dissolved in a buffer (pH 5.5) to prepare a 20 mg / mL tannic acid aqueous solution. Arginine-glycine-aspartic acid (RGD) was added to the tannic acid solution at a ratio of 100 μg / mL. A titanium sheet was immersed in the solution at 25°C for 12 hours. The modified titanium sheet was then rinsed alternately in water and ethanol for 5 minutes each. This was designated sample Ti-R.

[0057] Test Example 1 Adhesion-Bactericidal Competition Test

[0058] The cell adhesion and antibacterial properties of the surface of the material were evaluated. BMSCs, S. aureus and E. coli were cultured at a ratio of 20:1 (1×10 cells per well, 5×10 cells per 48 wells). 8 CFUs) were inoculated into the control group (pure Ti) and sample 2 (Ti-PR) and incubated for 24 hours. The samples were rinsed three times with PBS and fixed with 4% PFA for 20 minutes. To observe the endocytosis of BMSCs and the survival of S. aureus and E. coli, BMSCs and S. aureus were labeled with actin 488 (green) and DAPI (blue), respectively. Finally, after rinsing with PBS, the number and morphology of BMSCs and bacteria on the samples were observed using CLSM.

[0059] Figure 1 This is a cell-bacteria competitive culture diagram. Figure 1 As can be seen in the figure, the number of bacteria on the surface of the pure titanium group (Ti) increased significantly, and the morphology of the cells shrank significantly, while the cells on the Ti-PR (sample 2) surface were in good condition and the number of bacteria decreased significantly, which shows that our surface can kill bacteria and promote cell adhesion, and the surface antibacterial and cell adhesion promoting surface was successfully constructed.

[0060] Figure 2 The cell survival rate and antibacterial rate statistics are shown in Figure 2. Figure 2 It can be seen that by regulating the ratio of different antimicrobial agents and cell adhesion promoting peptides, different materials are constructed. When the ratio of PHMG:RGD is 200:1, the bactericidal rate reaches more than 90%, but the survival rate is around 70%. By regulating the content of the antimicrobial agent PHMG, the antimicrobial activity is maintained at 90% without affecting the antimicrobial activity, and the cells have a good survival rate. Through ratio screening, the PHMG:RGD ratio of 100:1 has the best antimicrobial and cell adhesion promoting effect.

[0061] Test Example 2 Adhesion and Proliferation Test

[0062] 1. Cell Culture

[0063] Bone marrow mesenchymal stem cells (BMSCs) were isolated from the femurs and tibias of 120-g Sprague–Dawley (SD) rats. After excision of the metaphysis, the bone marrow cavity was flushed with MEM-α medium (Hyclone, USA) using a syringe. The cells were then collected by centrifugation and cultured in MEM-α medium supplemented with 20% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin-streptomycin (Hyclone, USA) at 37°C in a humidified atmosphere with 5% CO₂. After 72 hours of culture, the BMSCs were passaged, and the medium was replaced with MEM-α medium supplemented with 10% FBS and 1% penicillin-streptomycin. The following experiments were performed using passage 4 BMSCs.

[0064] 2. Cytocompatibility Test

[0065] To test biocompatibility, sterile Ti, Ti-P, Ti-R, and Ti-PR (sample 2) were placed in a 48-well plate, with three parallel samples per group. 25,000 BMSCs were inoculated in each sample and cultured for 24 hours. Then, 10 mg of MTT was weighed and dissolved in 1 mL of PBS, and then 9 mL of pure culture medium was added for dilution. 100 μL of MTT solution was added, and the well plate was placed in an incubator for 4 hours. The remaining culture medium in the well plate was absorbed, and 100 μL of DMSO was added to each well and shaken thoroughly. 100 μL of solution was taken from each well and measured at a wavelength of 490 nm using a microplate reader (Thermo Fisher, 51119200, USA). Afterwards, the survival rate of BMSCs was calculated, and the biocompatibility of each group was evaluated.

[0066] The cell counting kit-8 (CCK-8, Sigma-Aldrich) was used to evaluate the proliferative effect of titanium surfaces on BMSCs. The samples were placed in a 48-well plate and 200 μL of BMSC cell suspension was inoculated at a density of 1.0 × 10 cells per well. After culturing the titanium sheet with BMSCs for 1, 4, and 7 days, the samples were transferred to a new 48-well plate, 200 μL of CCK-8 solution was added, and the cells were incubated at 37°C for 3 hours. Finally, 100 μL of CCK-8 solution was removed and the absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher, 51119200, USA) to evaluate the proliferative effect of the titanium surface on cells.

[0067] Figure 3 Figure 2 shows cell biocompatibility and cell proliferation promotion.

[0068] from Figure 3 As can be seen in (left), the cell survival rate of the Ti-PR group (sample 2) is above 80%, indicating good biocompatibility.

[0069] from Figure 3 As can be seen in (right), after co-culturing the cells on the titanium surface for 1 day, 4 days and 7 days, the Ti-PR group (sample 2) surface promoted cell proliferation. This is mainly because the RGD structure and phenolic hydroxyl structure on the coating surface of sample 2 promoted cell proliferation.

[0070] 3. Cell Adhesion

[0071] A 1 × 1 cm titanium plate was used to observe cell adhesion in each group. BMSCs were plated at 5 × 10 cells per cm². 3 Cells were seeded at a density of 100 cells per sample in a 48-well plate. After 48 hours of cell culture, the cells were fixed with 4% formaldehyde (PFA, Biosharp, BL539A, China) for 20 minutes and permeabilized with 0.1% (v / v) Triton-X 100 (Sigma, 9002-93-1, USA) for 3 minutes. After rinsing twice with PBS, the cells were blocked with 1% (w / v) bovine serum albumin (BSA, Sigma, B2064, USA) in PBS for 30 minutes. The cells were then treated with 0.165 μmol L⁻¹ of rhodamine phalloidin (Invitrogen, R415, USA) and 4,6-diamino-2-phenylindole (DAPI, Invitrogen, D1306, USA) in the dark for 20 minutes to visualize filamentous actin (F-actin) and nuclei, respectively. After rinsing with PBS, BMSCs on the samples were observed using CLSM. BMSCs were seeded onto each sample in a 48-well plate, cultured for 48 hours, and then fixed with 4% PFA. After rinsing twice with PBS, the cells were dehydrated using a gradient of ethanol (10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and 100%), followed by critical point drying and platinum plating. The morphology of the cells on the samples was observed using SEM.

[0072] Figure 4 This is a diagram to promote cell adhesion. Figure 4 It can be seen from the figure that the spreading and adhesion effect of BMSC cells on the surfaces of different materials is that compared with pure titanium (Ti), the cell surface spreading area of ​​Ti-PR (sample 2) is larger, and the SEM test surface has similar results. The good cell spreading and adhesion is mainly because PHMG does not affect cell activity, while RGD promotes cell adhesion and spreading.

[0073] Test Example 3 In vivo implantation osteogenic test

[0074] 1. In vivo micro-computed tomography analysis of peri-implant bone tissue morphology

[0075] Male Sprague-Dawley rats weighing approximately 280 g were selected for femoral implant placement experiments to evaluate the material's in vivo anti-infection and osteogenic properties. The modeling method was as follows: After effective intraperitoneal anesthesia with 10% chloral hydrate, the right femur of each rat was removed, the skin prepared, and a titanium post (1 mm in diameter and 2 mm in length) was implanted proximal to the metaphysis near the knee joint. The rats were divided into two groups based on the surface modification of the titanium post: a control group (unmodified pure titanium) and an experimental group (Ti-PR (sample 2)). After implantation, the skin was sutured and sutured. At 4, 8, and 12 weeks after surgery, the rats were euthanized, and the femurs containing the implants were collected and fixed in 10% neutral buffered formaldehyde for 48 hours. To evaluate the new bone tissue surrounding the implants, the femurs containing the implants were scanned using a micro-computed tomography system (SkyScan 1276 scanner, USA). The scanning protocol settings were 85 kV, 130 μA, and a resolution of 9.00 μm. New bone formation around the implants was assessed in a volume of interest (VOI) based on a hollow cylindrical area with a radius of 200 μm from the implant surface. Furthermore, quantitative micro-computed tomography analyses were performed, including bone volume fraction (bone volume / total volume, BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Furthermore, three-dimensional (3D) reconstructions of each sample were performed using Mimics Medical 21.0 software (Materialise, Belgium) to visualize new bone formation around the implants.

[0076] Figure 5 Micro-CT cross-sectional images and 3D reconstruction images of peri-implant bone tissue as well as bone tissue morphometric analysis results of the region of interest, including bone volume / total volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). Figure 5It can be seen that in the early stage of healing at 4 weeks, the Ti-PR group (sample 2) showed better peri-implant osteogenesis, which continued to rise at 8 weeks and reached its highest point after 12 weeks. The implant surface was covered with a large amount of new bone tissue. In contrast, there was only a small amount of new bone on the Ti surface of the control group. Compared with the Ti group, the BV / TV values ​​of the Ti-PR group (sample 2) were significantly higher at 4 weeks, 8 weeks and 12 weeks. Similarly, the Ti-PR (sample 2) group also showed better Tb.Th, Tb.N and Tb.Sp values ​​than the Ti group. These results show that the implant of the present invention can promote higher quality and larger amounts of new bone formation compared to the Ti implant. Taking into account the time dimension, the implant of the present invention showed enhanced bone integration ability over time compared to the Ti implant, indicating that the modified surface of the present invention not only has superior early osteogenesis ability, but also has long-term bone regeneration ability. It shows that the good bone integration performance of the implant of the present invention is largely due to its excellent anti-infection properties. The results show that the material of the present invention can significantly promote peri-implant osteogenesis.

[0077] 2. In vivo biomechanical testing

[0078] The animal modeling method was the same as above, and the biomechanical pull-out test of the implant bone integration strength was performed to quantify the bone integration strength between bone tissue and implant. At 4 weeks, 8 weeks and 12 weeks after surgery, the rats were euthanized, and the right femur containing the implant was collected and fixed with 10% neutral buffered formaldehyde for 48 hours. A custom three-valve collar was connected that could clamp the implant and connected to a universal testing machine (EZ-LX 50N, Shimadzu Instrument Co., LTD). An axial tensile force was applied to the implant at a rate of 5.0 mm min-1, and the pull-out force was recorded by the universal testing machine (the pull-out force was defined as the maximum value of the load-displacement curve).

[0079] To evaluate the bonding strength of the implants, biomechanical testing was performed by recording the maximum pull-out force of the implants. Figure 6 The above is the pull-out experiment. The three-piece chuck is used to clamp the titanium column and pull it out from the femoral tissue. Figure 6 As can be seen from the figure below, compared with the Ti group, the pull-out force of the Ti-PR (sample 2) group after 4 weeks, 8 weeks and 12 weeks of implantation was 50.9%, 34% and 24% higher than that of the control group, respectively, indicating that the implant of the present invention has better bone integration ability, thereby increasing the long-term stability of the bone implant.

[0080] Test Example 4 Sterilization Test

[0081] The antibacterial properties of Ti, Ti-P, Ti-R, and Ti-PR (sample 2) were tested using a film-sticking method, surface dead-alive staining, and bacterial scanning electron microscopy.

[0082] (1) Adhesive film method: The adhesive film method (QB / T 2591-2003) was used to detect the antibacterial performance of the samples. The samples were cut into 2 cm x 2 cm squares and sterilized under UV light for 2 hours, then placed in a 6-well plate. 32 μL of S. aureus or E. coli suspension (2 x 10 5 CFU mL-1) was added to each sample in the well, and incubated at 37°C for 24 hours. Then, the samples were transferred to a 15 mL centrifuge tube containing 4 mL of sterile PBS, and vortexed for 30 seconds to separate the bacteria from the surface of the samples. Next, the obtained bacterial suspension was diluted at a ratio of 1:200, and 50 μL of the diluted bacterial suspension was spread on a standard agar plate, which was incubated at 37°C for 24 hours. The number of bacteria on the surface was evaluated by counting the colonies on the agar plate. The inhibition efficiency (η) was determined by equation (1):

[0083] η = [(A0-A1) / A0] x 100% (1)

[0084] wherein A1 is the colony count value of the culture plate containing the titanium surface, and A0 is the blank positive control.

[0085] (2) Surface live / dead staining: The samples were sterilized under UV light at room temperature for 2 hours, and then placed in a 48-well plate. In each well of the 48-well plate, 200 μL of bacterial suspension (S. aureus or E. coli, 10 8 CFU mL-1) was added, and incubated at 37°C for 12 hours. After that, the samples were removed from the 48-well plate, rinsed with sterile PBS for 3 times, and then placed on a glass slide. 1 mL of DMAO and 2 mL of EthD-III in the live / dead bacterial staining kit were mixed in a microcentrifuge tube, and after thoroughly mixed, 8 mL of 0.85% NaCl solution was added to obtain a 100 x dye solution, which was then diluted to 1 x with LB liquid medium. 200 μL of the dye solution was added to the surface of the samples and incubated at room temperature in the dark for 15 minutes, and then rinsed with sterile PBS for 3 times. Observation was performed by CLSM using a 63 x oil objective. Live (green fluorescence) and dead (red fluorescence) bacteria can be observed in the FITC and Cy3 (or Texas Red) channels, respectively.

[0086] (3) Bacterial scanning electron microscopy observation: To evaluate the antibacterial effect, the morphology of the bacteria was studied. A 1 x 1 cm titanium sheet was used. 200 μL of bacterial suspension (S. aureus or E. coli, concentration of 10 8CFU / mL) and incubated at 37°C for 12 hours, followed by fixation with 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) at 4°C for 12 hours. After two PBS rinses, the samples were dehydrated with graded ethanol concentrations (10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, and 100%), followed by critical point drying and platinum sputtering (Leica, EM CPD300, Germany). The bacterial morphology of the samples was observed using scanning electron microscopy (SEM).

[0087] Figure 7 The results of colony culture, bacterial scanning electron microscopy and live-death staining are shown. Figure 7 As can be seen in the figure, the number of bacteria on the surfaces of Ti, Ti-P, Ti-R, and Ti-PR (sample 2) is significantly reduced, and the antibacterial rate can reach over 99%. This is also confirmed by bacterial live / dead staining, which shows a significant increase in the number of dead bacteria on the surface. The antibacterial properties of Ti, Ti-P, Ti-R, and Ti-PR (sample 2) are not affected, and the antibacterial rate exceeds 99%. They have excellent killing effects on different positive and negative bacteria. As can be seen from the SEM images, the bacteria on the Ti-P and Ti-PR surfaces show obvious shrinkage and damage to the bacterial cell membrane. This is mainly due to the presence of PHMG on the surface. Its guanidine group destroys the bacterial cell membrane, further demonstrating the excellent antibacterial properties of the coating.

[0088] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing an antibacterial and cell adhesion-promoting orthopedic implant, characterized in that: Here are the steps: 1) preparing an aqueous solution of a polyphenol compound, and adding polyhexamethyleneguanidine hydrochloride and arginine-glycine-aspartic acid to the aqueous solution of the polyphenol compound, wherein the mass concentration ratio of polyhexamethyleneguanidine hydrochloride to arginine-glycine-aspartic acid is 100:1; 2) Soak the implant substrate in the above solution at room temperature for 12 to 24 hours; 3) After cleaning, an antibacterial and cell-adhesion-promoting orthopedic implant is obtained; The antibacterial and cell-adhesion-promoting orthopedic implant is used for bone implant implantation to achieve competitive growth of bacteria and cells: cells > bacteria growth, with a sterilization rate of 90%, promoting the adhesion, proliferation and differentiation of bone marrow mesenchymal stem cells, and promoting osteogenesis around the implant. The implant substrate is a titanium sheet.

2. The method for preparing an antibacterial and cell adhesion-promoting orthopedic implant according to claim 1, characterized in that: The concentration of the arginine-glycine-aspartic acid is 100 μg / mL.

3. The method for preparing an antibacterial and cell adhesion-promoting orthopedic implant according to claim 1, characterized in that: The polyphenol compound is pyrogallol, tea polyphenol or tannic acid.

4. The method for preparing an antibacterial and cell adhesion-promoting orthopedic implant according to claim 1, characterized in that: Step 3) is to wash alternately in water and ethanol.

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