Microenvironment responsive bionic coating modified implant as well as preparation method and application thereof

Abstract

The invention particularly discloses a microenvironment responsive bionic coating modified implant as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The invention provides an implant modified by a microenvironment responsive bionic coating. The implant comprises a dopa-PFS peptide and / or a dopa-MLT peptide, wherein the dopa-PFS peptide and / or the dopa-MLT peptide are / is doped with the dopa-PFS peptide; the structure of the dopa-MLT peptide is CH3CO-(DOPA) m-GIGAVLKVL TTGLPALISWEIKRKRQQ-NH2, and the structural formula of the dopa-MLT peptide is shown in the description; the structure of the dopa-PFS peptide is as follows: (DOPA) n-PFSSTKT, and the structural formula of the dopa-PFS peptide is shown in the specification, wherein m and n are respectively selected from natural numbers of 2-8, the implant provided by the invention can respond to the acid change of an infected microenvironment, realizes sequential antibacterial, immune regulation and bone regeneration functions, and further meets the clinical dual requirements of infection control and osseointegration promotion on the surface of the implant at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a microenvironment-responsive biomimetic coating modified implant, a preparation method and application thereof. BACKGROUND

[0002] Implant-related infection is a major challenge in orthopedic clinical treatment, seriously affecting the success rate of surgery and patient prognosis. With the intensification of population aging and the rising incidence of traumatic fractures, the clinical application of orthopedic implants is increasingly widespread. Titanium and its alloys have become the first choice for orthopedic implants due to their good mechanical strength, corrosion resistance and biocompatibility. However, the biological inertness of the surface of titanium-based materials makes them easy to become a carrier for bacterial colonization, and once infection occurs, bacteria will form a biofilm on the surface of the implant, producing strong resistance to antibiotic therapy.

[0003] The inflammatory response after infection not only directly damages the surrounding bone tissue, but also activates the body's immune system, triggering a sustained inflammatory cascade. In this process, macrophages play a key role, and their polarization state directly affects the development and healing process of infection. Pro-inflammatory M1 macrophages secrete large amounts of inflammatory factors, not only exacerbating local inflammatory response, but also inhibiting the recruitment and osteogenic differentiation of bone marrow mesenchymal stem cells, seriously hindering the bone integration process around the implant. More seriously, even if the infection is controlled, the continued existence of the inflammatory microenvironment will still have a long-term impact on the repair and reconstruction of bone tissue.

[0004] Existing implant infection prevention and treatment strategies mainly rely on antibiotic therapy and surface modification technology, but both have obvious shortcomings. Systemic antibiotic therapy can control infection, but is prone to produce drug-resistant strains, and has limited effect on established biofilms. Local antibiotic drug delivery systems can maintain a high drug concentration at the implant site, but the release kinetics is difficult to accurately control, and lack the ability to respond to the infection microenvironment. Surface modification techniques, including ion implantation, plasma treatment, coating technology, etc., can impart antibacterial properties to the material to some extent, but most have problems such as complex process, high cost, poor stability, etc.

[0005] More importantly, current technologies generally adopt a single functional design philosophy, focusing either on antibacterial function or on pro-osteogenic effect, making it difficult to meet the dual needs of infection control and bone integration. At different stages of infection, the body's demand for antibacterial, anti-inflammatory and pro-regenerative properties presents dynamic change characteristics, and existing technologies lack the ability to respond to such temporal and spatial changes in the pathological process. Therefore, there is an urgent need to develop an implant surface treatment technology that can sense changes in the infection microenvironment and achieve sequential therapy functions. SUMMARY

[0006] (I) Technical problems solved

[0007] In view of this, one of the main purposes of the present application is to provide a microenvironment-responsive biomimetic coating modified implant and a preparation method and application thereof. The implant provided by the present application can respond to the acid change of the infection microenvironment, realize sequential antibacterial, immunomodulatory and bone regeneration functions, and further meet the dual needs of infection control and bone integration promotion on the surface of the implant in clinic.

[0008] (II) Technical solutions

[0009] In order to achieve the above-mentioned purpose, the present application provides a microenvironment-responsive biomimetic coating modified implant, which comprises a dopa-PFS peptide and / or a dopa-MLT peptide.

[0010] The structure of the dopa-MLT peptide is CH3CO-(DOPA)m-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, and the structure of the dopa-PFS peptide is (DOPA)n-PFSSTKT.

[0011] Wherein, m and n are each a natural number selected from 2 to 8.

[0012] In one embodiment, m is selected from 4.

[0013] In one embodiment, n is selected from 4.

[0014] In one embodiment, the structure of the dopa-MLT peptide is CH3CO-(DOPA)4-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, and the structure of the dopa-PFS peptide is (DOPA)4-PFSSTKT.

[0015] In one embodiment, the implant further comprises metal ions and / or a metal substrate.

[0016] In one embodiment, the metal ions are magnesium ions.

[0017] In one embodiment, the metal substrate is a titanium substrate.

[0018] In one embodiment, the implant comprises metal ions, a metal substrate, a dopa-PFS peptide and / or a dopa-MLT peptide, the structure of the dopa-MLT peptide is CH3CO-(DOPA)m-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, and the structure of the dopa-PFS peptide is (DOPA)n-PFSSTKT.

[0019] In one embodiment, the implant comprises magnesium ions, a titanium substrate, a dopa-PFS peptide and / or a dopa-MLT peptide; the structure of the dopa-MLT peptide is CH3CO-(DOPA)m-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2; the structure of the dopa-PFS peptide is (DOPA)n-PFSSTKT;

[0020] The present application provides, in another aspect, a method for preparing the above-mentioned implant, the method comprising:

[0021] S1: preparing a dopa-PFS peptide modified metal substrate by placing a metal substrate in a dopa-PFS peptide solution;

[0022] S2: preparing a magnesium ion-coordinated metal substrate by placing the dopa-PFS peptide modified metal substrate in a magnesium ion solution;

[0023] S3: preparing the implant by placing the magnesium ion-coordinated metal substrate obtained in S2 in a dopa-MLT peptide solution.

[0024] In one embodiment, the pH of the dopa-PFS peptide solution is 8.0-9.5; preferably, the pH is 8.4-8.6.

[0025] In one embodiment, the concentration of the dopa-PFS peptide solution is 0.005-1.000 mg / mL.

[0026] In one embodiment, the concentration of the dopa-PFS peptide solution is 0.1 mg / mL.

[0027] In one embodiment, the soaking time in S1 is 12-36 h.

[0028] In one embodiment, the soaking time in S1 is 24 h.

[0029] In one embodiment, S1 is performed in the dark.

[0030] In one embodiment, the concentration of the magnesium ion solution in S2 is 0.01-1 mM.

[0031] In one embodiment, the concentration of the magnesium ion solution in S2 is 0.1 mM.

[0032] In one embodiment, the soaking time in S2 is 3-12 h.

[0033] In one embodiment, the soaking time in S2 is 6 h.

[0034] In one embodiment, the concentration of the DOPA-MLT peptide solution is 0.005-1.000 mg / mL.

[0035] In one embodiment, the concentration of the DOPA-MLT peptide solution is 0.1 mg / mL.

[0036] In one embodiment, the soaking time in S3 is 6-24 h.

[0037] In one embodiment, the soaking time in S3 is 12 h.

[0038] In one embodiment, the preparation method specifically comprises:

[0039] S1: under light-avoiding conditions, soaking a metal substrate in a dopa-PFS peptide solution with a pH of 8.0-9.5 and a concentration of 0.005-1.000 mg / mL for 12-36 h to prepare a dopa-PFS peptide-modified metal substrate;

[0040] S2: soaking the dopa-PFS peptide-modified metal substrate in a magnesium ion solution with a concentration of 0.01-1 mM for 3-12 h to prepare a magnesium ion-coordinated metal substrate;

[0041] S3: soaking the magnesium ion-coordinated metal substrate in a DOPA-MLT peptide solution with a concentration of 0.005-1.000 mg / mL for 6-24 h to prepare the implant.

[0042] In one embodiment, the preparation method specifically comprises:

[0043] S1: under light-avoiding conditions, soaking a metal substrate in a dopa-PFS peptide solution with a pH of 8.5±0.1 and a concentration of 0.1 mg / mL for 24 h to prepare a dopa-PFS peptide-modified metal substrate;

[0044] S2: soaking the dopa-PFS peptide-modified metal substrate in a magnesium ion solution with a concentration of 0.1 mM for 6 h to prepare a magnesium ion-coordinated metal substrate;

[0045] S3: soaking the magnesium ion-coordinated metal substrate in a DOPA-MLT peptide solution with a concentration of 0.1 mg / mL for 12 h to prepare the implant.

[0046] In another aspect, the present application also provides an implant obtained by the above preparation method.

[0047] In another aspect, the present application also provides a grafting material comprising the above implant.

[0048] (III) Beneficial Effects

[0049] The present application provides a microenvironment-responsive biomimetic coating modified implant, and a preparation method and application thereof. Compared with the prior art, the following beneficial effects are achieved:

[0050] 1. The microenvironment-responsive biomimetic coating modified implant provided by the present application, by learning from the strong adhesion ability of marine mussels, a biomimetic coating with pH-responsive release ability is prepared, and by sensing the acid change of the infected microenvironment, the conversion from anti-infection to bone integration promotion is realized, thereby solving the major clinical challenge of implant-related infection.

[0051] 2. The MLT peptide and PFS peptide are combined to the titanium surface in a biomimetic modification manner through DOPA groups, and excellent surface adhesion performance is achieved by using the catechol group of the DOPA group. The introduced magnesium ions can be combined with the catechol group through metal phenolic coordination bond, thereby endowing the coating with immune regulation effect and pH-responsive release ability.

[0052] 3. The biomimetic coating modified implant exhibits excellent biological performance. Experimental results show that the biomimetic coating can quickly release the antibacterial components under acidic conditions, achieve a strong bactericidal effect, significantly promote the polarization conversion of M1 to M2 macrophages, greatly increase the recruitment of bone marrow mesenchymal stem cells, and significantly improve the bone volume fraction.

[0053] 4. The designed MLT peptide and PFS peptide have unique molecular structure and functional characteristics, the biomimetic coating has excellent biocompatibility and safety, provides a favorable microenvironment for stem cells, promotes cell survival and osteogenic differentiation, and exhibits excellent synergistic effect in infection control and bone integration promotion. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0055] Figure 1 is the HPLC analysis diagram of dopa-PFS peptide and dopa-MLT peptide.

[0056] Figure 2 is the mass spectrum analysis diagram of dopa-PFS peptide and dopa-MLT peptide.

[0057] Figure 3 is a schematic diagram of the implant preparation process.

[0058] Figure 4are surface topography variation graphs of different implants.

[0059] Figure 5 are atomic force microscope topography and roughness analysis graphs of different implants.

[0060] Figure 6 are water contact angle measurement graphs of implants.

[0061] Figure 7 are X-ray photoelectron spectroscopy full spectrum and high resolution spectrum graphs of implants.

[0062] Figure 8 are ultraviolet-visible absorption spectrum graphs of implants.

[0063] Figure 9 are magnesium ion cumulative release curve graphs under different pH conditions.

[0064] Figure 10 are crystal violet staining and biofilm 3D (biofilm observation) graphs.

[0065] Figure 11 are bacterial plating and bacterial transmission electron microscopy (plankton observation) graphs.

[0066] Figure 12 are flow cytometry analysis bacterial viability distribution graphs.

[0067] Figure 13 are macrophage iNOS and Arg-1 immunofluorescence staining graphs.

[0068] Figure 14 are macrophage M1 / M2 phenotype flow cytometry analysis graphs.

[0069] Figure 15 are macrophage polarization related gene expression detection graphs.

[0070] Figure 16 are macrophage secreted cytokine level detection graphs.

[0071] Figure 17 are BMSC migration experiments and SDF-1α chemotactic factor detection graphs.

[0072] Figure 18 are osteogenic differentiation ALP and ARS staining graphs.

[0073] Figure 19 are osteogenic related gene RT-qPCR heat maps.

[0074] Figure 20 are bacterial culture, tissue staining and electron microscopy observation graphs 7 days after surgery.

[0075] Figure 21is a graph of quantitative analysis of bacterial count and inflammation assessment.

[0076] Figure 22 is a graph of immunohistochemical staining and quantitative analysis at 14 days post-operation.

[0077] Figure 23 is a graph of Micro-CT three-dimensional reconstruction and bone parameter analysis at 8 weeks post-operation.

[0078] Figure 24 is a graph of histological staining and detection of osteogenesis markers. DETAILED DESCRIPTION

[0079] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0080] Terms and definitions

[0081] As used herein, "containing", "having" or "including" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of", "consisting essentially of", and "consisting of" are sub-concepts of "containing", "having" or "including".

[0082] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0083] Example 1 Preparation of dopa-PFS peptide and dopa-MLT peptide:

[0084] The amino acid sequence of PFS (SEQ ID No: 1): PFSSTKT;

[0085] The amino acid sequence of MLT (SEQ ID No: 2): GIGAVLKVLTTGLPALISWIKRKRQQ.

[0086] 1. Preparation of dopa-MLT peptide:

[0087] 1.1. Rink Amide-MBHA resin (degree of substitution 0.38 mmol / g, 100-200 mesh) was chosen as the solid support. 0.5 g of resin was weighed into a reaction column. The resin was pre-treated by soaking in DMF for 30 minutes to allow full swelling, then washed with DMF 3 times, 10 mL each, with shaking for 5 minutes.

[0088] 1.2. The amino acids were coupled in the reaction column in the order of the sequence from C-terminus to N-terminus (Gln-Gln-Arg-Lys-Arg-Trp-Ile-Lys-Ser-Trp-Ile-Leu-Ala-Pro-Leu-Gly-Thr-Thr-Leu-Val-Lys-Leu-Val-Ala-Val-Gly-Ile-Gly-DOPA-DOPA-DOPA-DOPA-CH3CO).

[0089] Coupling reaction conditions: 3 molar excess of Fmoc (1.5 mmol) protected amino acid (0.5 mmol), HBTU as coupling reagent (3 equivalents, 1.5 mmol), HOBt (3 equivalents, 1.5 mmol) and DIEA (10 equivalents, 5.0 mmol) in DMF, total reaction volume 15 mL, mechanical stirring at room temperature for 60 minutes. After completion of the reaction, the reaction solution was washed with 10 mL of DMF 5 times.

[0090] 1.3. Fmoc deprotection reaction: 15 mL of 20% piperidine / DMF solution was used in two steps: first step: 7.5 mL of 20% piperidine / DMF solution was added, stirred at room temperature for 5 minutes; second step: fresh 7.5 mL of 20% piperidine / DMF solution was added, stirred at room temperature for 15 minutes. After completion of the deprotection, the reaction solution was washed with DMF 5 times, 10 mL each, and DCM 3 times, 10 mL each.

[0091] 1.4. Cleavage reaction: 15 mL of cleavage solution (95% TFA, 2% water, 2% EDT, 1% TIS) was mixed with the deprotection reaction solution, stirred at room temperature for 180 minutes. The solution was shaken every 30 minutes to ensure complete cleavage. After completion of the cleavage, the filtrate was collected by filtration, and the crude peptide was precipitated with cold ether (-20°C), centrifuged (4000 rpm, 10 minutes), and washed with cold ether 3 times to obtain the dopa-MLT peptide.

[0092] 2. Preparation of dopa-PFS peptide:

[0093] 2.1. Select 2-chlorotrityl chloride resin (degree of substitution 0.84 mmol / g, 100-200 mesh) as solid support, weigh 0.3 g resin into the reaction column. Pretreatment of resin: first soak in DMF for 30 min to fully swell, then wash with DMF 3 times, 10 mL each time, shake for 5 min.

[0094] 2.2. According to the sequence from C-terminal to N-terminal (Thr-Lys-Thr-Ser-Ser-Phe-Pro-DOPA-DOPA-DOPA-DOPA), the amino acids are sequentially coupled in the reaction column.

[0095] The coupling reaction conditions are the same as the above method.

[0096] 2.3. Fmoc deprotection reaction: the same as the above method.

[0097] 2.4. Cleavage reaction: mix 15 mL cleavage solution (94.5% TFA, 2.5% water, 2.5% EDT, 1% TIS) with the deprotection reaction solution, stir at room temperature for 120 min. Shake every 30 min during the cleavage process to ensure complete cleavage. After the cleavage is completed, filter the filtrate, precipitate the crude peptide with cold ether (-20°C), centrifuge to collect (4000 rpm, 10 min), and wash with cold ether 3 times to obtain the dopa-MLT peptide.

[0098] 3. Purification of dopa-PFS peptide and dopa-MLT peptide:

[0099] Purification was performed using preparative high performance liquid chromatography (Waters 2545, USA), and the chromatographic column was a C18 reversed-phase column (250 x 10 mm, 5 μm). The mobile phase A was 0.1% TFA aqueous solution, and the mobile phase B was 0.1% TFA acetonitrile solution. Gradient elution program: 0-5 min, 5% B; 5-25 min, 5%-40% B; 25-35 min, 40%-95% B; 35-40 min, 95% B; 40-45 min, 95%-5% B. Flow rate 15 mL / min, detection wavelength 220 nm and 280 nm, collect the target peak segment.

[0100] 4. Polypeptide characterization analysis:

[0101] Purity detection was performed using analytical HPLC (Agilent 1260, USA) under the same gradient conditions, and the flow rate was 1 mL / min. The retention times of DOPA-PFS peptide and DOPA-MLT peptide were 9.083 ± 0.05 and 11.625 ± 0.08 min, respectively, and the purity was calculated by peak area integration, both > 95% ( Figure 1 ).

[0102] The molecular weight was confirmed using an electrospray ionization mass spectrometer (API 4000, USA) in positive ion mode, with a spray voltage of 5500 V, a desolvation temperature of 450 °C, and a gas curtain gas of 35 psi. The actual molecular weights of DOPA-PFS and DOPA-MLT were m / z 1484.5 ± 0.3 and m / z 902.27 ± 0.2, respectively, which were consistent with the theoretical molecular weights. Figure 2 ).

[0103] It can be seen that the synthesized polypeptide has high purity, accurate molecular weight and correct structure. Figures 1-2

[0104] Example 2 Preparation of microenvironment-responsive biomimetic coating modified implants

[0105] 1. Pretreatment of metal substrate

[0106] Titanium sheets (TA2 pure titanium, diameter 15 mm, thickness 2 mm, purity 99.5%) were purchased from Baoji Titanium Meihua Metal Material Co., Ltd. The pretreatment steps were as follows: first, mechanical polishing was performed using 400 mesh, 800 mesh, and 1200 mesh silicon carbide sandpaper for wet grinding, with each grit polished for 10 minutes, and deionized water was used to rinse and cool during the process. After polishing, ultrasonic cleaning was performed: ultrasonic cleaning in acetone for 15 minutes (power 240 W, frequency 40 kHz) to remove surface oil; then ultrasonic cleaning in absolute ethanol for 15 minutes to remove organic residues; finally, ultrasonic cleaning in ultrapure water for 15 minutes to remove inorganic salts to obtain the titanium substrate. After each cleaning, nitrogen was used to dry and store in a desiccator for future use.

[0107] 2. Preparation of DOPA-PFS solution and surface modification

[0108] Accurately weigh 10.0 mg of DOPA-PFS peptide prepared in Example 1, and add it dropwise to 100 mL of ultrapure water (resistivity > 18.2 MΩ·cm) while stirring at low speed (200 rpm) with a magnetic stirrer until completely dissolved, to obtain a clear solution with a concentration of 0.1 mg / mL. Adjust the pH of the solution to 8.5 ± 0.1 using 1M NaOH or 1M HCl, which is beneficial for the adhesion of DOPA groups. Filter sterilization using a 0.22 μm sterile filter, and store at 4°C for future use.

[0109] Immerse the pretreated titanium sheets completely in the DOPA-PFS solution, ensuring that the titanium sheets do not touch each other. Gently shake at 50 rpm on an orbital shaker at room temperature (25 ± 2°C) for 24 hours. Perform the operation in the dark during the reaction process, and perform it under nitrogen protection to prevent oxidation of the DOPA groups. Seal the reaction container after replacing it with nitrogen three times to maintain an inert atmosphere. After the reaction is complete, remove the titanium sheets, rinse them thoroughly with ultrapure water for 5 minutes each time for a total of 5 times to remove unbound free peptides. Finally, dry them with a nitrogen stream to obtain DOPA-PFS modified titanium substrates.​

[0110] 3. Preparation of Mg2+ solution: 1.0 mg of magnesium chloride hexahydrate (MgCl2-6H2O, analytical pure) was accurately weighed and dissolved in 50 mL of phosphate buffered saline solution (PBS, pH 7.4, ionic strength 0.15 M) with sufficient stirring to completely dissolve, obtaining a MgCl2solution with a concentration of 0.1 mM. The solution was sterilized by filtration through a 0.22 μm filter and stored at 4 °C in the dark.

[0111] 4. Preparation of DOPA-MLT peptide solution: 5.0 mg of DOPA-MLT peptide prepared in Example 1 was accurately weighed and dissolved in 50 mL of PBS with sufficient stirring to completely dissolve, obtaining a DOPA-MLT peptide solution with a concentration of 0.1 mg / mL. The solution was sterilized by filtration through a 0.22 μm filter and stored at 4 °C in the dark.

[0112] 5. Preparation of different implants:

[0113] Preparation of the Control group: i.e. titanium sheet without pretreatment;

[0114] Preparation of the DP group: i.e. DOPA-PFS modified titanium substrate (DP) as described above;

[0115] Preparation of the DP@DM group: the DOPA-PFS modified titanium substrate was placed in a 0.1 mg / mL DOPA-MLT peptide solution and shaken at 30 rpm on an orbital shaker at room temperature for 12 hours to obtain DP@DM. The substrate was washed with PBS for 3 times and dried with nitrogen flow.

[0116] Preparation of the DPMg group: the DOPA-PFS modified titanium substrate was completely immersed in a 0.1 mM MgCl2solution and gently shaken at 30 rpm on an orbital shaker at room temperature for 6 hours to obtain a magnesium ion coordinated metal substrate (DPMg). A stable metal-phenol coordination bond was formed through the coordination of DOPA groups with magnesium ions. After the reaction was completed, the substrate was washed with PBS for 3 times and dried with nitrogen flow.

[0117] Preparation of the DPMg@DM group: the DPMg modified titanium substrate was completely immersed in a 0.1 mg / mL DOPA-MLT peptide solution and gently shaken at 30 rpm on an orbital shaker at room temperature for 12 hours to obtain a microenvironment-responsive biomimetic coating modified implant (DPMg@DM). The DOPA-MLT peptide was further coordinated with the surface magnesium ions through its DOPA groups, forming a multi-layer biomimetic coating. After the reaction was completed, the substrate was washed with PBS for 3 times and dried with nitrogen flow. The coating thickness was precisely adjusted by the immersion time and solution concentration, and the final coating thickness was about 50-100 nm, which ensured the effective loading of functional molecules and did not affect the mechanical properties of the implant. All preparation processes were carried out in a clean environment to ensure the sterility and stability of the coating.

[0118] Figure 3 is a schematic diagram of the coating preparation process, showing the step-by-step preparation process of titanium substrate (Ti) → DOPA-PFS peptide modification (Ti-DP) → magnesium ion coordination (Ti-DPMg) → DOPA-MLT peptide assembly (Ti-DPMg@DM); Figure 4 is a surface morphology change chart of different modification steps; from Figures 3-4 It can be seen that the multifunctional biomimetic coating is successfully constructed by the step-by-step assembly strategy, and the surface morphology presents a regular porous network structure.

[0119] Example 3 Characterization of microenvironment-responsive biomimetic coating modified implants:

[0120] 1. Atomic force microscope characterization:

[0121] The atomic force microscope (Dimension Icon, Bruker, Germany) was used to detect the surface morphology of each group in tapping mode. Experimental conditions: silicon cantilever probe (TESPA-V2, Bruker), elastic constant 42 N / m, resonance frequency 320 kHz, scanning area 5 x 5 μm, scanning rate 0.5 Hz, pixel resolution 512 x 512. Each sample was scanned at least 3 different areas to ensure the representativeness of the results. Data processing used NanoScope Analysis software to calculate the surface roughness parameters Ra (arithmetic mean roughness) and Rq (root mean square roughness) Figure 5 ).

[0122] 2. Water contact angle measurement:

[0123] The contact angle measuring instrument (DSA100, Germany) was used to measure the static water contact angle. Experimental conditions: super pure water droplet volume 2.0 ± 0.1 μL, room temperature (25 ± 1 ℃), relative humidity 45-55%. A microsyringe was used to gently drop water droplets onto the sample surface to avoid impact deformation. Immediately after dropping, a photograph was taken, and the contact angle change was recorded every 5 seconds for 2 minutes. Each sample was measured at least 6 different positions, and the average value and standard deviation were calculated. The Young-Laplace equation was used to fit the droplet profile to accurately calculate the contact angle Figure 6 ).

[0124] 3. X-ray photoelectron spectroscopy analysis:

[0125] The X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Fisher Scientific, USA) was used to analyze the surface chemical composition. Experimental conditions: Al Kα monochromatic X-ray source (1486.6 eV), power 150 W, vacuum degree <1 x 10 -9 mbar, analysis area diameter 500 pm. Survey scan: pass energy 200 eV, step 1 eV, number of scans 5. High resolution spectra: pass energy 20 eV, step 0.1 eV, scan C1s, N 1s, O 1s, Ti 2p and Mg 2p regions. All binding energies were corrected using the C1s peak (284.8 eV) as an internal standard. Data processing was performed using Avantage software, with Shirley background subtraction and Gaussian-Lorentzian mixed function fitting.

[0126] Elemental content quantitative analysis: Taking into account the relative sensitivity factors, the atomic percentage of each element was calculated. The successful grafting of the polypeptide was marked by the appearance and intensity increase of the N1s peak, as well as the increase of the C-N and C=O components in the C1s peak. The evidence of the magnesium ion coordination was the detection of the Mg 2p peak (about 1304 eV) Figure 7 ).

[0127] 4. UV-Vis absorption spectrum:

[0128] The formation of the metal-phenol coordination complex was confirmed using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan). The coating samples were cut into small pieces (5 x 5 mm) and immersed in PBS for 24 hours to extract, and the extract was collected for determination. The scanning wavelength range was 200-800 nm, the scanning rate was medium, the slit width was 2 nm, and PBS was used as a blank control. The characteristic absorption peak of the metal-phenol coordination complex usually appeared near 280 nm, and the absorption intensity was positively correlated with the coordination degree Figure 8 ).

[0129] From Figures 5-8 It can be seen that compared with ordinary titanium sheets, the surface roughness of the DPMg@DM group increased significantly, forming a nano-micro composite topological structure suitable for cell adhesion. The water contact angle changed from a hydrophobic titanium surface to a hydrophilic surface, indicating a significant increase in surface energy. XPS analysis showed that the nitrogen content of the DPMg@DM group increased significantly, and a clear peptide bond characteristic signal was detected at 400.1 eV, and a C=O bond was detected at 532.1 eV. The UV-Vis absorption spectrum detected a maximum absorption peak at a wavelength of 280 nm, and the DPMg@DM group had the highest absorption intensity, confirming the successful formation of the metal-phenol coordination complex.

[0130] Example 4 pH-responsive release performance evaluation:

[0131] 1. Construction of a simulated physiological environment:

[0132] Different pH buffer systems were prepared to simulate the microenvironment at different stages of infection: pH 7.4 buffer solution simulating normal physiological environment, using phosphate buffered saline (PBS); pH 6.5 buffer solution simulating early inflammatory environment, using MES buffer system (50 mM MES, 150 mM NaCl); pH 5.5 buffer solution simulating acute infection environment, using acetic acid buffer system (50 mM sodium acetate, 150 mM NaCl). All the buffers were sterilized by filtering with 0.22 μm filter membrane and used after equilibration at 37 °C.

[0133] 2. Release kinetics experiment design:

[0134] Control, DP, DPMg, DP@DM and DPMg@DM (n = 3) prepared in Example 2 were respectively immersed in 20 mL of different pH buffer solution and placed in a constant temperature shaker (37 °C, 100 rpm) to simulate the in vivo environment. At the predetermined time points (0.5, 1, 3, 5, 7, 14, 21, 28 days), samples were taken for analysis. At each sampling time, 500 μL of supernatant was carefully taken for detection, and an equal volume of fresh buffer was added to maintain the constant volume. During the sampling process, the sample was avoided to be disturbed to ensure the continuity of the release behavior.

[0135] 2.1 Quantitative detection of magnesium ions:

[0136] The inductively coupled plasma mass spectrometer (ICP-MS, NexION 300X, PerkinElmer, USA) was used to quantitatively detect the concentration of magnesium ions. Instrument parameters: radio frequency power 1600 W, plasma gas flow rate 15 L / min, auxiliary gas flow rate 1.2 L / min, atomizer gas flow rate 0.98 L / min, sampling depth 8 mm.

[0137] Standard curve preparation: A series of concentrations (0.1, 0.5, 1, 5, 10, 50, 100 μg / L) were prepared using a magnesium standard solution (1000 mg / L), and the correlation coefficient R 2 > 0.999.

[0138] Sample pretreatment: 200 μL of the above supernatant was taken and diluted with 1800 μL of 2% nitric acid solution. After mixing, the sample was directly injected for analysis. Each sample was measured in triplicate, and the average value was calculated. At the same time, blank control and standard addition recovery experiments were set up to ensure the accuracy of the detection results Figure 9 ).

[0139] 2.2 DOPA-MLT peptide release detection:

[0140] The release of DOPA-MLT peptide was detected by high performance liquid chromatography (HPLC, Agilent 1260, USA). The chromatographic conditions were as follows: C18 reversed-phase column (250 x 4.6 mm, 5 μm), mobile phase of 0.1% TFA aqueous solution (phase A) and 0.1% TFA acetonitrile solution (phase B), gradient elution (0-20 min, 10%-50% B), flow rate of 1 mL / min, detection wavelength of 280 nm, column temperature of 30 °C, and injection volume of 20 μL.

[0141] Preparation of standard curve: The DOPA-MLT peptide standard was accurately weighed and prepared into a series of concentrations (1, 5, 10, 25, 50, 100 μg / mL) to draw a standard curve of concentration-peak area.

[0142] Sample pretreatment: At each sampling time, 300 μL of supernatant was carefully taken out for ICP-MS detection (200 μL) and HPLC detection (100 μL), and at the same time, an equal volume of fresh buffer was supplemented, directly filtered through a 0.22 μm filter membrane, and then analyzed by injection.

[0143] 2.3 Release mechanism analysis:

[0144] A variety of mathematical models were used to fit the release data, including zero-order kinetics, first-order kinetics, Higuchi model, and Korsmeyer-Peppas model, to determine the best fitting model. The release mechanism was analyzed by fitting parameters: n value <0.43 represented Fick diffusion mechanism, n value >0.85 represented matrix erosion mechanism, and 0.43 < n < 0.85 represented anomalous diffusion mechanism.

[0145] From Figure 9 It can be seen that under the condition of pH 7.4, the coating maintains a stable low level of magnesium ion release, indicating the stability of the coordination bond. Under the condition of pH 6.5, it shows a moderate degree of release kinetics, reflecting the gradual influence of the slightly acidic environment on the coordination bond. Under the condition of pH 5.5, it triggers rapid magnesium ion release, which is rapid in the early stage and then stabilizes at a higher release level. The release behavior of DOPA-MLT peptide is basically consistent with that of magnesium ion, confirming the pH sensitivity of the metal-phenol coordination bond. Kinetic analysis shows that the release behavior under acidic conditions conforms to the anomalous diffusion mechanism. This pH-dependent release mechanism verifies the response ability of the coating, which can achieve rapid release of the antibacterial component in the infected microenvironment.

[0146] Example 5: Evaluation of in vitro antibacterial performance:

[0147] 1. Bacterial culture and preparation:

[0148] E. coli (ATCC 25922) and methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) were purchased from the China Center for Type Culture Collection.

[0149] Bacterial activation: Strains were taken from -80 °C cryotubes, inoculated on Luria-Bertani (LB) agar plates, and incubated at 37 °C for 18-24 hours until single colony formation. Single colonies were picked and inoculated in LB broth, and incubated at 37 °C, 180 rpm for 12-16 hours until log phase (OD 600 = 0.6-0.8).

[0150] Bacterial suspension standardization: Bacteria incubated to log phase were collected by centrifugation (3000 rpm, 10 minutes), washed with sterile PBS for 3 times, and resuspended in PBS. Spectrophotometer was used to adjust OD 600 to 0.1, corresponding to a bacterial concentration of about 1 x 10 8 CFU / mL. Further dilution was made with PBS to a working concentration of 1 x 10 6 CFU / mL, prepared extemporaneously. Actual bacterial concentration was verified by plate counting method to ensure experimental accuracy.

[0151] 2. Suspended bacteria killing experiment:

[0152] Control, DP, DPMg, DP@DM, and DPMg@DM (15 mm in diameter) prepared in Example 2 were placed in 24-well plates, and 1 mL of the above prepared bacterial suspension (1 x 10 6 CFU / mL) was added to each well, ensuring complete immersion of the implants. Six replicates were set for each group, and incubated at 37 °C in a 5% CO2 incubator for 24 hours, avoiding shaking to prevent disruption of bacteria-material interface interactions.

[0153] Bacterial viability detection: Bacterial plating and transmission electron microscopy were used. After 24 hours of incubation, the supernatant was mixed gently by blowing, and 100 μL was taken for serial 10-fold dilutions (10 -1 to 10 -5 ). 100 μL of each dilution was plated on LB agar plates, and colonies were counted after 24 hours of incubation at 37 °C. Bacterial survival rate (%) = (CFU of experimental group / CFU of control group) x 100%. Bactericidal efficiency was expressed as log reduction value: log 10 (control group CFU / experimental group CFU) Figure 11 .

[0154] 3. Biofilm formation inhibition experiment:

[0155] Modified microplate method was used to evaluate the biofilm formation inhibition ability. Control, DP, DPMg, DP@DM and DPMg@DM prepared in Example 2 were placed in a 24-well plate, 1 mL of fresh LB medium and 100 μL of the above prepared working concentration bacterial suspension (final concentration 1 x 10 5 CFU / mL) were added. Incubate at 37°C for 48 hours, replace half of the medium every 24 hours to maintain nutrition supply and promote mature biofilm formation.

[0156] Crystal violet staining quantification: remove the medium, gently rinse with PBS for 3 times to remove planktonic bacteria. Add 500 μL of 0.1% crystal violet solution, stain at room temperature for 15 minutes. Rinse thoroughly with PBS until the rinse solution is no longer purple, and dry. Add 500 μL of 33% acetic acid solution, dissolve at room temperature for 30 minutes. Transfer 200 μL of the dissolved solution to a 96-well plate, and measure the absorbance at 590 nm wavelength using a microplate reader. Biofilm inhibition rate (%) = (1- experimental group OD / control group OD) x 100% Figure 10

[0157] Biofilm three-dimensional structure analysis: obtain biofilm three-dimensional structure information by laser confocal microscope Z-axis scanning. Scan parameters: Z-axis step 0.5 μm, scan range 0-50 μm, pixel resolution 1024 x 1024. Use the 3D plug-in of ImageJ to reconstruct the biofilm three-dimensional structure, calculate the biofilm thickness, volume and coverage rate, etc. Figure 10

[0158] Flow cytometry quantitative analysis: after the incubation, gently scrape the biofilm and disperse into a single cell suspension with PBS. Remove cell clumps through a 40 μm cell screen, and adjust the cell concentration to 1 x 10 6 cells / mL. Add SYTO 9 and PI mixed staining solution, incubate at 4°C for 30 minutes in the dark. Use a flow cytometer (CytoFLEX, Beckman Coulter, USA) to analyze, detect 10,000 events for each sample. Laser settings: 488 nm laser excitation, FITC channel detects SYTO 9, PE channel detects PI Figure 12

[0159] The DPMg@DM group showed excellent antibacterial performance. The planktonic bacteria killing experiment showed that it had strong killing effect on E. coli and MRSA, meeting the clinical antibacterial requirements. The biofilm formation inhibition experiment showed that the DPMg@DM group had significant inhibition effect on E. coli and MRSA biofilm, and the biofilm thickness was greatly reduced. Flow cytometry analysis confirmed that the dead cell population increased significantly.

[0160] Example 6 verification of immunomodulatory function: ​​​

[0161] 1. Macrophage culture and polarization induction:

[0162] RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in high-glucose DMEM medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell passage: When cell confluence reached 80-90%, cells were gently scraped off with a cell scraper, centrifuged at 1000 rpm for 5 minutes, resuspended in fresh medium, and passaged at a 1:4 ratio. The day before the experiment, cells were cultured at 4 × 10⁻⁶ cells / mL. 4 The cells / well were seeded at a density of cells / well in 24-well plates containing Control, DP, DPMg, DP@DM and DPMg@DM prepared in Example 2.

[0163] M1 polarization induction: To simulate the pro-inflammatory environment under infection, a classic M1-activated macrophage model was established by stimulating macrophages with lipopolysaccharide (LPS, 100 ng / mL, derived from E. coli O111:B4, Sigma) and interferon-γ (IFN-γ, 20 ng / mL, PeproTech) for 24 hours. LPS activates the NF-κB signaling pathway through Toll-like receptor 4 (TLR4), and IFN-γ activates the STAT1 signaling pathway; both synergistically promote M1 polarization.

[0164] 2. Immunofluorescence staining detection:

[0165] Cell fixation: Remove the culture medium and gently wash three times with pre-cooled PBS (4°C), 5 minutes each time. Add 500 μL of 4% paraformaldehyde fixative and fix at 4°C for 15 minutes. Wash three times with PBS to remove the fixative.

[0166] Cell membrane permeability: Add 200 μL of 0.1% Triton X-100 solution and incubate at room temperature for 10 minutes to increase cell membrane permeability and allow antibody entry. Wash three times with PBS.

[0167] Blocking nonspecific binding: Add 300 μL of 5% normal goat serum blocking solution and block at room temperature for 1 hour to prevent antibody nonspecific binding.

[0168] Primary antibody incubation: Prepare the primary antibody working solution. Dilute the iNOS antibody (1:200 dilution, Abcam) and Arg-1 antibody (1:300 dilution, Abcam) separately with PBS containing 1% BSA. Add 200 μL of the primary antibody working solution and incubate overnight at 4°C. Recover the primary antibody and wash three times with PBS for 10 minutes each time.

[0169] Secondary antibody incubation: Prepare working solution of fluorescent secondary antibody, Alexa Fluor 488 labeled anti-rabbit IgG (1:1000 dilution, for iNOS) and Alexa Fluor 594 labeled anti-mouse IgG (1:1000 dilution, for Arg-1). Incubate at room temperature for 1 hour in the dark. Wash with PBS for 3 times.

[0170] Nucleus and cytoskeleton staining: Label F-actin cytoskeleton with Phalloidin-iFluor 647 (1:200 dilution), and counterstain nucleus with DAPI (1 μg / mL), incubate at room temperature for 30 minutes in the dark. Finally, mount with anti-fluorescence quenching mounting medium (Prolong Gold with DAPI, Invitrogen, USA) Figure 13

[0171] 3. Laser confocal microscope observation:

[0172] Observe using laser confocal microscope (LSM 880, Zeiss, Germany), laser settings: 405 nm excite DAPI (blue), 488 nm excite Alexa Fluor 488 (green), 561 nm excite Alexa Fluor 594 (red), 633 nm excite Alexa Fluor 647 (far-red). Objective lens: 63x oil lens, numerical aperture 1.4. Image acquisition parameters: pixel resolution 1024x1024, pixel dwell time 4 μs, average 4 times. Randomly select 8-10 fields of view for each sample to take pictures, to ensure the representativeness of the results.

[0173] 4. Flow cytometry phenotype analysis:

[0174] Cell collection: After 48 hours of culture, gently scrape the cells with a cell scraper, and collect the cells by centrifugation at 1000 rpm for 5 minutes. Wash with PBS containing 2% FBS for 2 times.

[0175] Surface antigen staining: Adjust the cell concentration to 1x10 6 cells / mL, take 100 μL of cell suspension per tube. Add Fc receptor blocker (1:100 dilution), incubate at 4°C for 10 minutes to block non-specific binding. Add fluorescently labeled antibodies: FITC-CD11b (1:100 dilution, macrophage marker), PE-CD86 (1:100 dilution, M1 marker), APC-CD206 (1:100 dilution, M2 marker), respectively, incubate at 4°C in the dark for 30 minutes. Wash with PBS for 2 times.

[0176] ​Flow cytometry: analyzed using flow cytometer (CytoFLEX, Beckman Coulter, USA), lasers: 488nm and 633nm. Detection parameters: FSC / SSC gating to remove dead cells and debris, 20,000 events were detected for each sample. Data analysis was performed using FlowJo software, set CD11b positive gate, further analyze CD11b+CD86 + (M1 phenotype) and CD11b+CD206 + (M2 phenotype) cell proportion Figure 14 ).

[0177] 5. Quantitative analysis of gene expression:

[0178] RNA extraction: total RNA was extracted using TRIzol reagent, strictly following the instructions. Add 1 mL of TRIzol, lyse at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 seconds, stand for 3 minutes, centrifuge at 4°C for 15 minutes (12,000g). Take the upper aqueous phase, add isopropanol to precipitate RNA, centrifuge at 4°C for 10 minutes. Wash with 75% ethanol, dry and dissolve with DEPC water.

[0179] RNA quality detection: use NanoDrop 2000 spectrophotometer to detect RNA concentration and purity, A260 / A280 ratio in the range of 1.8-2.0 is qualified. Use agarose gel electrophoresis to detect RNA integrity, 28S and 18S bands are clear and the ratio is about 2:1.

[0180] Reverse transcription reaction: use PrimeScript RT Master Mix kit, reaction system: total RNA 1 μg, 5×PrimeScript Buffer 4 μL, PrimeScript RT Enzyme Mix I 1 μL, Random 6mers 1 μL, Oligo dT Primer 1 μL, RNase Free dH2O to 20 μL. Reaction conditions: 37°C, 15 minutes, 85°C, 5 seconds, 4°C storage.

[0181] Real-time quantitative PCR: SYBR Green Master Mix was used. The reaction system consisted of 2 μL cDNA, 10 μL 2×SYBR Green Mix, 1 μL each of forward and reverse primers (final concentration 0.5 μM), and dH2O to a final volume of 20 μL. Reaction conditions: 95℃ pre-denaturation for 30 seconds, 40 cycles (95℃, 5 seconds, 60℃, 30 seconds), followed by melting curve analysis. Target genes included M1 marker genes (TNF-α, iNOS, CD86) and M2 marker genes (IL-10, CD206, ARG-1), with GAPDH as an internal control gene. Figure 15 ).

[0182] 6. Cytokine secretion detection:

[0183] ELISA Assay: Collect cell culture supernatant, centrifuge to remove cell debris, and store at -80℃. Detect IL-4 and IL-13 concentrations using a commercial ELISA kit. Standard Curve Preparation: Prepare a series of concentration standards (0, 15.6, 31.25, 62.5, 125, 250, 500, 1000 pg / mL), with double replicates for each concentration. Sample Dilution: Based on preliminary experimental results, dilute the samples appropriately to the detection range. Strictly follow the kit instructions, including coating, blocking, primary antibody incubation, secondary antibody incubation, and substrate development. Read the absorbance at 450 nm using a microplate reader, and calculate the sample concentration using the standard curve. Figure 16 ).

[0184] Depend on Figures 13-16 It is evident that the magnesium-containing coating significantly promotes the polarization transition from M1 to M2 macrophages. Immunofluorescence results showed that macrophages on the surface of the Control, DP, and DP@DM groups mainly expressed iNOS (M1 marker, green fluorescence), while macrophages on the surface of the DPMg and DPMg@DM groups mainly expressed Arg-1 (M2 marker, red fluorescence), indicating successful polarization transition. Flow cytometry quantitative analysis confirmed that the M1 cell population was significantly reduced and the M2 cell population was significantly increased in the DPMg@DM group. RT-qPCR analysis showed that the expression of M1-related genes TNF-α, iNOS, and CD86 was significantly downregulated, while the expression of M2-related genes IL-10, CD206, and ARG-1 was significantly upregulated. ELISA detection showed that the secretion levels of IL-4 and IL-13 were significantly increased in the DPMg@DM group, further confirming the functionality of M2 polarization.

[0185] Example 7: Stem cell recruitment and osteogenic differentiation function:

[0186] 1. Isolation and culture of bone marrow mesenchymal stem cells:

[0187] Purchase 6-8 weeks old SD rats, anesthetized to death to extract primary bone marrow mesenchymal stem cells (BMSCs) in long bone. The specific extraction method is as follows: under sterile conditions, separate the femur and tibia of the rat, use ophthalmic scissors to cut off both ends of the long bone, and expose the bone marrow cavity. Use a 5 mL sterile syringe to extract α-MEM culture medium containing 10% FBS to repeatedly flush the bone marrow cavity until the bone is white, collect the bone marrow fluid and filter it using a 40 μm cell screen to remove bone fragments and cell clumps. Centrifuge the filtered bone marrow fluid (1500 rpm, 10 minutes), discard the supernatant, and resuspend the cell pellet with fresh culture medium. Culture to the third generation. Cell identification: use flow cytometry to detect mesenchymal stem cell surface markers, CD29, CD90, CD105 positive rate > 95%, hematopoietic stem cell markers CD34, CD45 negative rate > 95%, and confirm as mesenchymal stem cells. Multidirectional differentiation ability verification: alizarin red staining is positive after 14 days of osteogenic induction, and oil red O staining is positive after 21 days of adipogenic induction, confirming pluripotency.

[0188] 2. Cell recruitment ability evaluation:

[0189] Transwell migration experiment: use a 24-well Transwell insert (pore size 8 μm, Corning), place Control, DP, DPMg, DP@DM and DPMg@DM prepared in Example 2 in the lower chamber respectively, and add 600 μL of α-MEM medium containing 10% FBS. Seed BMSCs (5 x 10 4 cells / 200 μL serum-free medium) in the upper chamber, and incubate at 37°C for 48 hours. After the experiment, gently wipe off the cells on the surface of the upper chamber with a cotton swab, fix the lower chamber surface with 4% paraformaldehyde for 15 minutes, and 0.1% crystal violet staining for 10 minutes. Count the number of cells that have migrated under a microscope, and randomly count 5 fields of view (400x) for each insert. Figure 17

[0190] SDF-1α chemokine detection: seed BMSCs (1 x 10 4 cells / well) in a 24-well plate containing each group of coating samples, add 500 μL of α-MEM medium, and incubate at 37°C for 24 hours. The coating material stimulates BMSCs to secrete SDF-1α chemokine. After the culture is completed, centrifuge the supernatant at 4°C (3000 rpm, 10 minutes) to remove cell debris, and filter it through a 0.22 μm filter membrane. Use an SDF-1α ELISA kit to detect the concentration of SDF-1α in the supernatant, with a standard curve range of 15.6-1000 pg / mL. After appropriate dilution of the sample, detect each sample in triplicate.

[0191] 3. Osteogenic differentiation induction culture:

[0192] ​Preparation of conditioned medium: RAW 264.7 cells (2 × 10⁻⁶) were cultured in the medium. 4 Implants (number per well) were seeded into 24-well plates containing the implants prepared in Example 2, and 1 mL of complete culture medium was added. The plates were incubated at 37°C for 48 hours. During this period, factors secreted by cells on the implant surface diffused into the culture medium. After incubation, the implants were removed, and the supernatant was collected. The supernatant was centrifuged at 4°C (3000 rpm, 10 minutes) to remove cell debris. The supernatant was then filtered through a 0.22 μm filter to obtain conditioned medium, which was stored at -20°C for later use.

[0193] Preparation of induction medium: Conditioned medium and fresh α-MEM were mixed in a 1:1 ratio and osteogenic induction factors were added: 10 mM β-glycerophosphate (as a phosphate source), 0.1 μM dexamethasone (to promote osteogenic differentiation), and 0.25 mM L-ascorbic acid (to promote collagen synthesis) to obtain the induction medium.

[0194] Osteogenesis induction: BMSCs at 2×10 4 Inoculate one well per cell into a 24-well plate containing induction medium, and change the induction medium every 3 days.

[0195] 4. Osteogenesis marker detection:

[0196] Alkaline phosphatase (ALP) staining: After 7 days of induction, remove the culture medium, wash with PBS, and fix with 4% paraformaldehyde for 10 minutes. Use the BCIP / NBT alkaline phosphatase staining kit according to the instructions, and react at room temperature in the dark for 15-30 minutes until a blue-purple precipitate appears. Observe under a microscope and take pictures for recording. Figure 18 ).

[0197] ALP activity quantification: Cells were lysed with 0.1% Triton X-100 for 1 hour, and the supernatant was collected by centrifugation. p-nitrophenyl phosphate (pNPP) was used as a substrate, and the reaction was carried out at 37°C for 30 minutes. The reaction was terminated with 1M NaOH, and the absorbance was measured at 405 nm. Protein concentration was determined using the BCA method, and ALP activity was expressed as U / mg protein.

[0198] Alizarin Red staining: After 21 days of induction, fix with 4% paraformaldehyde and wash with deionized water. Stain with 1% alizarin red staining solution (pH 4.2) for 30 minutes, and wash thoroughly until the rinsing solution is clear. Observe the formation of calcium nodules under a microscope and take photographs for recording. Figure 18 ).

[0199] Osteogenesis-related gene expression:

[0200] RNA extraction, reverse transcription, RT-qPCR detection were performed according to the method of Example 6. Target genes include early osteogenic marker ALP, transcription factor RUNX2, mid-stage marker osteocalcin (OCN), late-stage marker bone collagen COL1A1, with GAPDH as the internal reference gene Figure 19 ).

[0201] From Figures 17-19 It can be seen that the DP and DPMg groups showed excellent BMSC recruitment ability. The Transwell experiment showed that the number of cells penetrating the membrane in the PFS peptide-containing group was significantly increased. In the scratch healing experiment, the healing rate was significantly improved. SDF-1a concentration detection showed that the concentration of the DP group and the DPMg group was significantly higher than that of the control group. Osteogenic differentiation experiment showed that the conditioned medium containing magnesium significantly promoted BMS C osteogenic differentiation, and the ALP staining area and activity were significantly improved. Alizarin red staining showed extensive formation of mineralized matrix, and the degree of mineralization was significantly increased. RT-qPCR analysis showed that the expression levels of osteogenic-related genes ALP, RUNX2, OCN, COL1A1 were significantly up-regulated.

[0202] Example 8: Establishment of animal model and in vivo antibacterial evaluation:

[0203] 1. Animal feeding and grouping:

[0204] Male SD rats (8-10 weeks old, 250-300g) were purchased from Vantianlihua Experimental Animal Technology Co., Ltd. with license number SCXK (Jing) 2016-0006. The animal feeding conditions were temperature 22±2℃, humidity 55±10%, 12-hour light-dark cycle, free feeding and free water. After 1 week of adaptive feeding, the experiment began. All animal experiments strictly followed the 3R principle and were approved by the Animal Ethics Committee of China University of Science and Technology (Approval No.: USTC AUC1901013).

[0205] 2. Establishment of infection model:

[0206] Implant pretreatment: Control, DP, DPMg, DP@DM and DPMg@DM (diameter 1.5mm, length 8mm) prepared in Example 2 were soaked in 75% ethanol for 30 minutes for disinfection, irradiated with ultraviolet lamp for 30 minutes, and washed with sterile PBS for 3 times.

[0207] Bacterial inoculation: MRSA was cultured to the logarithmic phase, washed with PBS to adjust to 1×10 8 CFU / mL. The implants were soaked in the bacterial solution for 30 minutes to ensure a bacterial load of about 10 6 CFU / implant on the surface.

[0208] 3. Surgical operation:

[0209] Anesthesia: Anesthesia was induced by intraperitoneal injection of sodium pentobarbital (35 mg / kg), and the eyes were kept moist with saline to prevent drying. The surgical area was shaved and disinfected with iodophor three times.

[0210] Implantation: A 15 mm long incision was made on the lateral side of femoral condyle, and the muscle was bluntly separated to expose the femoral condyle. A 1.5 mm diameter drill was used to drill a vertical hole 5 mm from the articular surface of the femoral condyle, with a depth of 8 mm, and the hole was flushed with saline to cool it down. The pre-infected implant was slowly implanted, ensuring that it was completely embedded in the bone. The muscle and skin were sutured in layers, and penicillin was injected intramuscularly for 3 days after the operation to prevent soft tissue infection.

[0211] 4. Animal grouping and sampling:

[0212] Randomly divided into 5 groups (n=8 / group): Control group, DP group, DPMg group, DP@DM group, DPMg@DM group. Part of the animals were sacrificed at 7 days, 14 days, and 56 days after the operation for analysis.

[0213] 5. Evaluation of antibacterial effect in vivo:

[0214] Implant retrieval: The animals were sacrificed under anesthesia at 7 days after the operation, and the implants were removed under sterile conditions and immediately placed in 2 mL of sterile PBS. The surface bacteria were dispersed by ultrasonic treatment (40 kHz, 5 minutes) and vortexed for 1 minute.

[0215] Bacterial quantification: The implant eluate was serially diluted by 10 times, and 100 μL of each dilution was spread on blood agar plates. After 24 hours of incubation at 37°C, the colonies were counted. The results were expressed as log 10 CFU / implant. Figure 21 ).

[0216] Histological evaluation: The bone tissue around the implant was fixed with 4% paraformaldehyde for 48 hours, decalcified with 10% EDTA for 4 weeks, embedded in paraffin, and 6 μm thick sections were prepared. Inflammation cell infiltration was observed by H&E staining ( Figure 21 ), and bacterial distribution was observed by Giemsa staining ( Figure 20 ). Inflammation scoring criteria: 0 points-no inflammation; 1 point-mild inflammation (a small amount of inflammatory cells); 2 points-moderate inflammation (obvious inflammatory cell infiltration); 3 points-severe inflammation (a large number of inflammatory cells, tissue necrosis) Figure 20 ).

[0217] SEM observation: After fixation with 2.5% glutaraldehyde, dehydration with ethanol, critical point drying, and gold ion sputtering coating, the surface bacterial colonization of the implant was observed by SEM Figure 20 ).

[0218] From the above results, it can be seen that the implantation of the pre-infected implant in the DP@DM group and the DPMg@DM group can effectively inhibit the bacterial colonization on the implant surface and the surrounding bone tissue, and the inflammation is significantly reduced. The implantation of the pre-infected implant in the DP@DM group and the DPMg@DM group can effectively inhibit the bacterial colonization on the implant surface and the surrounding bone tissue, and the inflammation is significantly reduced. Figures 20-21It can be seen that the DPMg@DM group achieved significant antibacterial effect at day 7. The bacterial quantitative results showed that the control group implant surface had high bacterial load, while the DPMg@DM group had a significant reduction in bacterial number, achieving strong bactericidal effect. Histological evaluation showed that the control group presented severe inflammatory response, a large number of neutrophil infiltration and tissue necrosis; the DPMg@DM group had mild inflammation, normal tissue structure, and only slight lymphocyte infiltration. Giemsa staining showed that the control group had a large number of bacteria accumulated in the tissue, while the DPMg@DM group had almost no bacteria detected. Scanning electron microscope observation confirmed that the control group implant surface formed a dense bacterial biofilm with dense distribution of bacteria; while the DPMg@DM group surface was smooth and clean, with only sporadic bacteria remaining and abnormal morphology, indicating the effective killing effect of the antibacterial peptide.

[0219] Example 9 In vivo immune regulation and bone integration evaluation:

[0220] 1. Immune regulation evaluation (14 days after operation):

[0221] Immunohistochemical staining: tissues were fixed with 4% paraformaldehyde, decalcified with EDTA, and embedded in paraffin to make continuous sections. Antigen retrieval: microwave retrieval in citric acid buffer (pH 6.0) for 15 minutes. Endogenous peroxidase blocking: 3% H2O2 treatment for 10 minutes. Serum blocking: 5% normal goat serum blocking at room temperature for 30 minutes.

[0222] Primary antibody incubation: TNF-a antibody (1:200), IL-10 antibody (1:150), CD86 antibody (1:300, M1 marker), CD163 antibody (1:250, M2 marker), incubated overnight at 4°C. Secondary antibody incubation: HRP-labeled universal secondary antibody, incubated at room temperature for 1 hour. DAB color development: color development time controlled at 3-5 minutes, hematoxylin counterstaining, dehydration and mounting.

[0223] Image analysis: 5 fields of each section were randomly taken using a microscope (x200) and the percentage of positive staining area was analyzed using ImageJ software. Immunocyte counting: CD86 + and CD163 + cells were counted respectively, and the M1 / M2 ratio was calculated. Figure 22 ).

[0224] 2. Bone integration evaluation (8 weeks after operation):

[0225] Micro-CT scanning: High-resolution micro-CT (SkyScan 1176, Bruker, Belgium) was used to scan the entire femur specimen. Scanning parameters: X-ray source voltage 60 kV, current 500 μΑ, pixel size 18 μm, rotation step 0.4°, aluminum filter 0.5 mm thickness, exposure time 240 ms. The scanning time for each sample was about 45 minutes.

[0226] Image reconstruction: 3D reconstruction was performed using NRecon software, reconstruction parameters: ring artifact correction 20, beam hardening correction 35%, smoothing factor 2. High-quality 3D image data was obtained after reconstruction.

[0227] Region of interest (ROI) definition: centered on the implant, extending 500 pm outward as the analysis region, excluding cortical bone, only analyzing cancellous bone area. 3D morphometric analysis was performed using CTAn software.

[0228] Bone parameter calculation: bone volume fraction (BV / TV, %)=bone volume / tissue volume x 100%; bone surface / bone volume (BS / BV, mm -1 ); trabecular thickness (Tb.Th, pm); trabecular number (Tb.N, mm -1 ); trabecular separation (Tb.Sp, pm); bone mineral density (BMD, g / cm 3 )( Figure 23 )。

[0229] 3. Histological evaluation:

[0230] Specimen preparation: femoral specimens after Micro-CT scanning were continued to be decalcified in 10% EDTA for 2 weeks until completely softening, paraffin-embedded, and made into 6 pm thick continuous sections.

[0231] Routine staining: Masson trichrome staining distinguishes collagen fibers (blue) and muscle fibers (red); Toluidine blue staining specifically shows cartilage matrix and newly formed bone tissue.

[0232] Immunohistochemical detection of osteogenic markers: bone sialoprotein (BSP, osteoblast marker), type I collagen (COL-1, main component of bone matrix) were detected according to standard immunohistochemical procedures Figure 24 )。

[0233] 4. Histomorphometric analysis:

[0234] Image analysis method was used for quantitative evaluation of tissue sections. Microscope was used to take tissue section images at standard magnification, and quantitative analysis was performed by image analysis software such as ImageJ Figure 24 )。

[0235] Bone-implant contact rate: the proportion of the area of the implant surface directly contacted with bone tissue to the total surface of the implant was measured.

[0236] Bone tissue area percentage: the proportion of the area of newly formed bone tissue in the region of interest to the total tissue area.

[0237] Osteoblast distribution density: osteogenic activity was evaluated by counting the number of osteoblasts per unit area.

[0238] Fibrous tissue thickness: the average thickness of the fibrous capsule around the implant was measured to evaluate the degree of foreign body reaction.

[0239] By Figures 22-24 It can be seen that the coating can effectively regulate the immune microenvironment around the implant and promote the transition of inflammation to repair. The inflammatory infiltration area of the DPMg@DM group was significantly reduced, and the expression of pro-inflammatory factor TNF-α was down-regulated, while the expression of anti-inflammatory factor IL-10 was up-regulated. Macrophage phenotype analysis showed that the density of M1 type macrophages was significantly reduced, the density of M2 type macrophages was significantly increased, and the M1 / M2 ratio was greatly reduced, confirming the effective immunomodulatory effect. The results of bone integration evaluation showed that the DPMg@DM group performed excellently in all bone morphometric parameters, and the bone volume fraction, trabecular thickness, trabecular number and bone density were significantly better than those of the control group. Histological analysis showed that the bone-implant contact in the DPMg@DM group was tight, and the expression of osteogenic markers BSP and COL-1 was significantly increased, indicating an active bone formation process. Masson trichrome staining showed that the coating group had abundant collagen fiber deposition and good bone matrix tissue around the implant, while the Control group mainly had loose fibrous tissue. These results confirmed that the coating can effectively promote bone integration in an infected environment.

[0240] It should be noted that the relational terms herein such as first and second and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises... a" does not, without more limitations, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the stated elements.

[0241] The above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the foregoing embodiments of the present application have been described in detail, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A microenvironment-responsive, biomimetic coating-modified implant, characterized in that, The implant comprises a dopa-PFS peptide and / or a dopa-MLT peptide. The structure of the dopa-MLT peptide is CH3CO-(DOPA) m -GIGAVLKVLTTGLPALISWIKRKRQQ-NH2; the structure of the dopa-PFS peptide is: (DOPA) n -PFSSTKT; m and n are each a natural number from 2 to 8.

2. The implant of claim 1, wherein, The implant further comprises metal ions and a metal substrate.

3. The implant of claim 2, wherein, The metal ions are magnesium ions.

4. The implant of claim 2, wherein, The metal substrate is a titanium substrate.

5. A method of producing the implant according to any one of claims 1 to 4, characterized in that, The preparation method comprises: S1: placing a metal substrate in a dopa-PFS peptide solution to prepare a dopa-PFS peptide modified metal substrate; S2: placing the dopa-PFS peptide modified metal substrate in a magnesium ion solution to prepare a magnesium ion coordinated metal substrate; S3: placing the magnesium ion coordinated metal substrate obtained in S2 in a dopa-MLT peptide solution to prepare the implant.

6. The production method according to claim 5, wherein The pH of the dopa-PFS peptide solution is 8.0-9.5; preferably, the pH is 8.4-8.

6.

7. The preparation method according to claim 5, characterized in that, The S1 operation is performed in the dark.

8. The preparation method according to claim 5, characterized in that, The thickness of the dopa-PFS peptide in S3 is 50-100 nm.

9. An implant, characterized in that, The implant is prepared by the preparation method of any one of claims 5-8.

10. A graft material, characterized in that, The implant material comprises the implant of any one of claims 1-4 or claim 9.

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