WO2021143584A1 - 一种钛种植体表面的长效可再生抗菌涂层 - Google Patents

一种钛种植体表面的长效可再生抗菌涂层 Download PDF

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WO2021143584A1
WO2021143584A1 PCT/CN2021/070355 CN2021070355W WO2021143584A1 WO 2021143584 A1 WO2021143584 A1 WO 2021143584A1 CN 2021070355 W CN2021070355 W CN 2021070355W WO 2021143584 A1 WO2021143584 A1 WO 2021143584A1
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titanium
antibacterial
coating
renewable
implant
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PCT/CN2021/070355
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English (en)
French (fr)
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吴淑仪
李彦
吴丁财
邹蕾燕
许剑萌
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中山大学附属口腔医院
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    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C22/00—Chemical surface treatment of metallic material by reaction of the surface with a reactive liquid, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals
    • C23C22/02—Chemical surface treatment of metallic material by reaction of the surface with a reactive liquid, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals using non-aqueous solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A61L27/06—Titanium or titanium alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C22/00—Chemical surface treatment of metallic material by reaction of the surface with a reactive liquid, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals
    • C23C22/73—Chemical surface treatment of metallic material by reaction of the surface with a reactive liquid, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals characterised by the process
    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C22/00—Chemical surface treatment of metallic material by reaction of the surface with a reactive liquid, leaving reaction products of surface material in the coating, e.g. conversion coatings, passivation of metals
    • C23C22/78—Pretreatment of the material to be coated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/106—Halogens or compounds thereof, e.g. iodine, chlorite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/02—Methods for coating medical devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/06—Coatings containing a mixture of two or more compounds

Definitions

  • the invention relates to a long-acting reproducible antibacterial coating on the surface of a titanium implant, which belongs to the field of oral biomedical materials.
  • implant denture restoration has become the first choice for functional and aesthetic restoration of tooth loss. Because the tissue surrounding the implant is different from the natural periodontal tissue, its defense ability against bacterial invasion is relatively weak, especially when planting under bad micro-ecological conditions such as periodontal disease, maxillofacial defect and infected alveolar socket, it is more likely to produce implants. The tissue around the body is infected or peri-implantitis, and once it occurs, it is difficult to cure. The current incidence of peri-implant inflammation is as high as 18%-35%, which is an important reason for the long-term failure of implant restoration.
  • N-halamine compounds are organic non-antibiotic antibacterial agents, which have the characteristics of broad-spectrum antibacterial, strong activity, stable structure, good biological safety and low cost. Its antibacterial mechanism includes: halogen amine compound precursor can undergo chlorination reaction in sodium hypochlorite aqueous solution, turning NH bond into N-Cl bond, in which positively charged chlorine atoms can adsorb and act on negatively charged bacterial membranes through electrostatic action At the bacterial receptor, it destroys or inhibits the metabolic process of bacteria, and then achieves the effect of contact antibacterial.
  • the N-Cl structure can dissociate positively charged chlorine atoms in an aqueous solution, and release them to the surrounding environment of the material to play a release antibacterial effect.
  • N-halamine compounds are widely used in water and air purification, medical consumables modification, food packaging modification, textile modification, and various coating or dye modification due to their excellent biological safety and environmental safety. However, N-halamine compounds have not yet been used for antibacterial modification of titanium implant surfaces.
  • antibacterial modification of the surface of implants has been used to reduce bacterial adhesion and inhibit the formation of plaque biofilms, thereby preventing initial infections after implantation.
  • Good progress has been made.
  • the antibacterial coating formed by binding the antibacterial agent to the titanium surface through physical adsorption or hydrogen bonding has a low degree of firmness, and the antibacterial component can be analyzed quickly.
  • the traditional chemical coating method combines high molecular antibacterial agents and antibacterial peptides. Other substances are applied to the surface modification of titanium, and the slow-release rate and efficiency decrease rapidly over time. Therefore, the performance of antibacterial coatings on the surface of implants is still difficult to achieve the expected antibacterial aging, and it is difficult to effectively prevent infection after implantation.
  • the host or environmental factors cause peri-implant inflammation, bone resorption around the implant, exposure of the implant, and further adhesion of plaque will eventually cause the implant to loosen and fall off. This process is often irreversible, while the existing implants
  • the antibacterial coating on the surface has basically disappeared in the long-term implant restoration, and it is difficult to effectively treat peri-implantitis.
  • the antibacterial coating structure on the surface of implants has defects such as stability, controllability, and timeliness, and antibacterial properties cannot be regenerated.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a renewable antibacterial coating on the surface of a titanium implant.
  • Another object of the present invention is to provide a long-lasting reproducible antibacterial coating on the surface of the titanium implant prepared by the above method.
  • Another object of the present invention is to provide the application of the above-mentioned regenerable antibacterial coating on the surface of the titanium implant in the preparation of a medicine for preventing and/or treating peri-implant inflammation.
  • the invention constructs a reproducible antibacterial coating on the surface of the titanium implant with firm bonding and antibacterial time-lasting effect, so as to effectively prevent the occurrence of peri-implant inflammation during the early stage of implant implantation; and in the later use process of the implant
  • the peri-implantitis can be treated by reactivating the antibacterial activity on the surface of the implant.
  • the technical solution adopted by the present invention is: a method for preparing a renewable antibacterial coating on the surface of a titanium implant, including the following steps:
  • step (1) The titanium sheet treated in step (1) is subjected to alkali heat treatment, and then treated with a silane coupling agent KH570 solution;
  • step (4) The titanium sheet treated in step (4) is immersed in a sodium hypochlorite solution to prepare a renewable antibacterial coating on the surface of the titanium implant.
  • step (1) the specific operation of step (1) is: take a round titanium sheet with a diameter of 9.5mm and a thickness of 0.3mm with #400, #800, #1000SiC sandpaper, and then use the solvent acetone and absolute ethanol to polish and polish it step by step. And double distilled water followed by ultrasonic cleaning for 20 minutes, and then dry.
  • the alkali heat treatment is to place the titanium sheet in a 5mol/L NaOH solution for 24 hours at 60°C; the volume fraction of the silane coupling agent KH570 is 40%.
  • the radical polymerization reaction conditions are at least one of 50°C for 12h, 50°C for 24h, 60°C for 12h, 60°C for 24h, 70°C for 12h, 70°C for 24h .
  • the free radical polymerization reaction conditions are 60°C for 24 hours.
  • step (4) the titanium flakes are reacted with the ethylenediamine at a temperature of 80° C. for 24 hours.
  • the titanium sheet reacts with ethylenediamine containing a compound with amino groups, and the carboxyl group of polyacrylic acid reacts with the amino group of ethylenediamine through an amidation reaction to form an amide bond.
  • the effective concentration of the sodium hypochlorite solution is 10%.
  • an N-halamine polymer antibacterial layer is constructed on the titanium surface.
  • the present invention provides a renewable antibacterial coating on the surface of a titanium implant prepared by the method, and the renewable antibacterial coating is an N-halamine polymer antibacterial layer.
  • the application of the regenerable antibacterial coating on the surface of the titanium implant in the preparation of a medicine for preventing and/or treating peri-implant inflammation in another aspect, the application of the regenerable antibacterial coating on the surface of the titanium implant in the preparation of a medicine for preventing and/or treating peri-implant inflammation.
  • a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention adopts a covalent bonding method and a polymer grafting method to construct a firm and stable polymer antibacterial layer on the surface of the titanium sheet to achieve Long-lasting antibacterial effect.
  • the present invention applies haloamine polymer to the surface modification of titanium implant for the first time to construct a reproducible antibacterial coating.
  • the antibacterial layer is firm and stable, the antibacterial age is long, and the antibacterial layer can be recycled.
  • the present invention constructs a reproducible antibacterial coating that is firmly bonded and has a long-lasting antibacterial effect on the surface of the titanium implant, so as to effectively prevent the occurrence of peri-implant inflammation during the initial stage of implant implantation;
  • peri-implant inflammation occurs during later use, the peri-implant inflammation can be treated by reactivating the antibacterial activity on the surface of the implant.
  • Figure 1 is a Fourier transform infrared spectrogram of the N-halamine coating prepared in Example 1 of the present invention
  • Example 2 is a scanning electron microscope observation of the surface morphology of the N-halamine coating prepared in Example 1 of the present invention
  • Example 3 is an element map observation of the element composition and distribution of the N-halamine coating prepared in Example 1 of the present invention on the titanium surface;
  • Example 4 is a diagram showing the results of atomic force microscope analysis of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 5 is a thermogravimetric (TG) curve diagram of the N-halamine coating prepared in Example 1 of the present invention.
  • Fig. 6 is a graph showing the results of testing the mechanical properties of the N-halamine coating prepared in Example 1 of the present invention.
  • Fig. 7 is a diagram showing the results of the N-halamine coating contact antibacterial test prepared in Example 1 of the present invention.
  • Fig. 8 is a diagram showing the results of the antibacterial release test of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 9 is a diagram of the antibacterial detection result of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 10 is a diagram of the antibacterial detection result of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 11 is a graph of the test results of the storage stability of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 12 is a diagram of the long-acting antibacterial test results of the N-halamine coating prepared in Example 1 of the present invention.
  • FIG. 13 is a diagram of repeated antibacterial detection results of the N-halamine coating prepared in Example 1 of the present invention.
  • Example 14 is a graph showing the effect of the N-halamine coating prepared in Example 1 of the present invention on osteoblasts;
  • Example 15 is a graph showing the effect of the N-halamine coating prepared in Example 1 of the present invention on osteoblasts;
  • FIG. 16 is a result diagram of the effect of the N-halamine coating prepared in Example 1 of the present invention on the cytoskeleton of osteoblasts;
  • Figure 17 is a graph showing the results of histocompatibility of the N-halamine coating prepared in Example 1 of the present invention.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) Perform radical polymerization reaction of the titanium flakes treated in step (2) with acrylic monomers at 60°C for 24 hours;
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through an amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) Perform radical polymerization of the titanium flakes treated in step (2) with acrylic monomers at 50°C for 12 hours;
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through an amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) Perform a radical polymerization reaction between the titanium sheet treated in step (2) and the acrylic monomer at 60°C for 12 hours;
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through an amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) Perform a radical polymerization reaction between the titanium sheet treated in step (2) and the acrylic monomer at 70°C for 12 hours;
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through an amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • This embodiment is a method for preparing a renewable antibacterial coating on the surface of a titanium implant provided by the present invention, which includes the following steps:
  • step (3) The titanium flakes treated in step (3) are reacted with ethylenediamine, a compound with amino groups, for 24h at 80°C, and the carboxyl group of polyacrylic acid and the amino group of ethylenediamine are reacted to form an amide bond through an amidation reaction;
  • step (4) The titanium sheet treated in step (4) is immersed in a 10% sodium hypochlorite solution with an effective concentration of 10%, and positively charged chlorine atoms are introduced to construct an N-halamine polymer antibacterial layer on the titanium surface, which can be prepared Renewable antibacterial coating on the surface of titanium implants.
  • the N-halamine polymer chains grow from the surface of the porous structure to form a polymer layer after alkaline heating, but still retain the original disordered grid structure; the surface of the alkali-heated titanium has a diameter of about 200nm-400nm
  • the disordered mesh structure, the pore structure obviously increases the specific area of the titanium surface; the modified titanium sheet still retains this grid structure, but the surface of the titanium mesh is covered with the N-halamine polymer layer, and the titanium mesh is obviously thickened.
  • the pore size is smaller than before, which proves that the N-halamine polymer chains start to grow from the surface of the titanium mesh to form a polymer layer after alkali heating.
  • Hydrophilicity test results show that pure titanium has the worst hydrophilicity, with a contact angle of 69.5°, alkaline-heated titanium has the best hydrophilicity, with an average contact angle of 18.7°, and the surface contact angle of the modified titanium sheet is 43.7° , Although higher than Ti-OH, it is still significantly lower than pure titanium (h) (p ⁇ 0.01).
  • the surface roughness of the modified titanium sheet is slightly higher than that of the alkali-heated titanium sheet, but the contact angle is 43.7°, which is higher than that of the alkali-heated titanium (18.7°C); the N-halamine polymer chains are evenly distributed on the titanium surface.
  • the results of gel permeation chromatography in Table 1 show that the number average molar mass (Mn) and weight average molar mass (MW) of the N-halamine polymer chains on the titanium surface are 10329 Daltons and 17932 Daltons, respectively.
  • the degree of dispersion is 1.736, which satisfies the characteristic that the degree of dispersion of the polymer formed by free radical polymerization is usually between 1.5-2.0.
  • thermogravimetric (TG) curve in Figure 5 it can be seen from the thermogravimetric (TG) curve in Figure 5 that when the temperature rises from room temperature to 121°C/min, and then keeps it at 121°C for 20 minutes, the weight percentage of the N-halamine coating sample always remains at About 100%, indicating that the N-halamine coating connected to the titanium surface through chemical bonds at high temperature has no obvious chemical change, which indicates that the N-halamine coating has no obvious quality change at 121°C for 30 minutes, indicating its thermal stability good.
  • TG thermogravimetric
  • Figure 6 shows that in the micro-scratch test combined with a typical acoustic emission (AE) curve, the average critical load of the N-halamine coating prepared by free radical polymerization is 34.8N, which is significantly higher than that constructed by the physical adsorption method.
  • N-halamine coating P ⁇ 0.01, namely 6.23N.
  • the results show that the bonding strength of the chemically grafted N-halamine coating is significantly higher than that of the N-halamine coating formed by the physical adsorption method (p ⁇ 0.01). It can resist the torsion force applied during conventional implant implantation.
  • This test example detects the antibacterial properties, antibacterial aging properties and antibacterial renewable properties of the renewable antibacterial coating (N-halamine coating) prepared in Example 1
  • N-halamine coating contact antibacterial test (bacterial coating counting method) are shown in Figure 7.
  • Staphylococcus aureus and Porphyromonas gingivalis were selected as the model bacteria for the coating count to verify the performance of the modified titanium sheet
  • Antibacterial performance After 12 hours of co-cultivation of the titanium sheet and bacteria, the counts of bacterial coatings on the titanium surface were collected for contact antibacterial testing; the contact antibacterial rate was shown on the modified titanium sheet, and the average antibacterial rate against Staphylococcus aureus was 96.22%. The rate of Porphyromonas gingivalis is 90.77%.
  • the results of the N-halamine coating release antibacterial test are shown in Figure 8.
  • the release antibacterial test results show that the average anti-Staphylococcus aureus rate on the modified titanium sheet is 67.32%, and the average anti-gingival rate is 67.32%.
  • the rate of Porphyromonas is 40.49%, and **** indicates p ⁇ 0.0001.
  • N-halamine coating antibacterial detection fluorescence staining of living and dead bacteria
  • results of living and dead bacteria fluorescence staining show that the surface of alkali-heated titanium is mostly live bacteria stained with green fluorescence, while the surface of modified titanium sheet Most of them are dead bacteria stained with red fluorescence.
  • the results of the N-halamine coating antibacterial detection are shown in Figure 10.
  • the number of Staphylococcus aureus and Porphyromonas gingivalis colonized on the alkali-heated surface is higher than that of the modified surface.
  • the bacteria on the alkaline-heated surface form a biofilm-like distribution and the morphology of the bacteria is relatively regular, while the bacteria on the modified titanium surface are scattered and some of the bacteria are very irregular, which shows that the N-halamine coating can be effective Inhibit the adhesion and colonization of bacteria.
  • the N-halamine coating has a contact antibacterial rate of more than 90% for Porphyromonas gingivalis and Staphylococcus aureus, and a release antibacterial rate of 50%-70%. Confocal laser microscope and scanning electron microscope were used to observe the survival and morphology of bacteria. It was found that most of the bacteria in contact with the coating died and their morphology was deformed.
  • Antibacterial aging store the modified titanium sheet in the dark for 1-8w, and detect the available chlorine content on the titanium surface by potassium iodide-sodium thiosulfate titration method.
  • the modified titanium sheet was soaked in artificial saliva at 37°C for 1-12 weeks, and 0.5% crystal violet was used for bacterial staining on the titanium surface. After dissolving the crystal violet, the OD value at 595nm was detected by a microplate reader to calculate the antibacterial rate. .
  • the results of antibacterial aging are shown in Figure 11-12.
  • the storage stability test result of the N-halamine coating is shown in Figure 11.
  • the storage stability test result of the modified titanium shows that with the extension of the storage time, the effective chlorine content on the surface of the modified titanium remains basically unchanged. After weeks, the available chlorine content was 0.284%, which indicated that the N-Cl bond in the haloamine polymer layer on the titanium surface was very stable. After eight weeks of storage, the sample is re-chlorinated, and the available chlorine content can still be restored to 100%. This is because the halogen atoms in the haloamine polymer can be re-halogenated in a simple way after being consumed, thus illustrating the preparation of the present invention The available chlorine on the modified titanium surface can be regenerated in a simple way after being consumed.
  • the long-lasting antibacterial test results of N-halamine coating are shown in Figure 12.
  • the antibacterial rate is still above 60%; as the soaking time increases (1 week ⁇ 12 weeks), the antibacterial rate gradually decreased; the results showed that the available chlorine in the N-halamine coating prepared on the titanium surface stably exists in the liquid environment; therefore, the antibacterial adhesion ability can be maintained on the implant surface for a long time.
  • the antibacterial rate of the modified titanium can be restored to the original level (67.89%-91.45%), indicating that the antibacterial coating has a renewable ability.
  • the antibacterial rate of the N-halamine coating gradually decreases from the original 91.45% to 67.89%, indicating that the available chlorine in the N-halamine coating is stable and slow in the liquid environment. Lasting release.
  • the antibacterial rate of the coating can be restored to the initial level, indicating that the antibacterial coating has a renewable ability.
  • ALP test results showed that there was no statistical difference between the alkaline phosphatase activity of MC3T3-E1 cells on alkaline heated titanium and modified titanium surface 7 days and 14 days after osteogenic induction, which indicates that N-halamine coating It will not affect the early osteogenic differentiation activity of its surface cells.

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Abstract

本发明涉及一种钛种植体表面的长效可再生抗菌涂层，属于口腔生物医学材料领域。本发明提供一种在钛种植体表面制备可再生抗菌涂层的方法：取钛片用SiC砂纸打磨抛光，用溶剂清洗，晾干，然后进行碱热处理，用硅烷偶联剂KH570溶液处理，再与丙烯酸单体进行自由基聚合反应后，与乙二胺反应，浸泡于次氯酸钠溶液中，即可制备得到钛种植体表面的可再生抗菌涂层。本发明采用共价键结合法及聚合物接枝法在钛片表面构建牢固稳定的高分子抗菌层，达到了持久抗菌的作用，且本发明首次将卤胺高分子应用于钛种植体表面改性，构建可再生利用的抗菌涂层，抗菌层牢固稳定，抗菌时效长，抗菌层可再生利用。

Description

一种钛种植体表面的长效可再生抗菌涂层 技术领域
本发明涉及一种钛种植体表面的长效可再生抗菌涂层，属于口腔生物医学材料领域。
背景技术
目前种植义齿修复已成为牙缺失功能和美学修复的首选方式。由于种植体周围组织不同于天然牙周组织，其对细菌入侵的防御能力相对较弱，尤其在牙周病、颌面缺损及感染牙槽窝等不良微生态环境条件下种植，更容易产生种植体周围组织感染或种植体周围炎，且一旦发生难以治愈。目前种植体周围炎的发生率高达18％-35％，是种植修复远期失败的重要原因。
N-卤胺化合物属于有机非抗生素类抗菌剂，具有广谱抗菌、活性强、结构稳定、生物安全性好及成本低廉等特性。其抗菌机理包括：卤胺化合物前驱体在次氯酸钠水溶液中可发生氯化反应，使N-H键变成N-Cl键，其中带正电的氯原子可通过静电作用吸附带负电的细菌胞膜并作用于细菌受体，破坏或抑制细菌的代谢过程，进而达到接触性抗菌的效果。N-Cl结构在水溶液中可解离出带正电的氯原子，释放到材料周边环境发挥释放性抗菌作用。氯原子消耗完后，再次浸泡次氯酸钠溶液可实现再氯化，表现为抗菌性能的循环再生。N-卤胺化合物因具有优良的生物安全性和环境安全性而广泛应用于水及空气净化、医疗耗材改性、食品包装改良、纺织品改性和各种涂料或染料的改性等领域。但目前N-卤胺化合物尚未用于钛种植体表面抗菌改性。
目前通过种植体表面抗菌改性来减少细菌黏附、抑制菌斑生物膜形成，从而预防种植术后初期感染，已取得不错的进展。然而，现有的研究中通过物理吸附或氢键将抗菌剂结合到钛表面形成的抗菌涂层牢固程度较低，抗菌成分析出快；而传统化学涂层法将高分子抗菌剂及抗菌肽等物质应用于钛表面改性，缓释速率及效率随时间推移快速降低。因此目前种植体表面抗菌涂层性能尚难达到预期的抗菌时效，难以有效预防种植术后感染。另外，当宿主或环境因素导致种植体周围炎发生后，种植体周围骨吸收、种植体暴露、菌斑进一步黏附，最终导致种植体松动和脱落，这一过程往往不可逆转，而现有种植体表面抗菌涂层在种植修复远期，种植体表面的抗菌活性已基本消失，难以有效地治疗种植体周围炎。目前种植体表面抗菌涂层结构存在稳定性、可控性、时效性不理想，抗菌性不可再生等缺陷。
发明内容
本发明的目的在于克服现有技术的不足，提供一种在钛种植体表面制备可再生抗菌涂层的方法。
本发明的另一目的是提供上述方法制备得到的钛种植体表面的长效可再生抗菌涂层。
本发明的再一目的是提供上述钛种植体表面的可再生抗菌涂层在制备预防和/或治疗种植体周围炎的药物中的应用。
本发明通过在钛种植体表面构建结合牢固且抗菌时效持久的、可再生利用的抗菌涂层，以期在种植体植入初期能有效地预防种植体周围炎的发生；以期在种植体后期使用过程中出现种植体周围炎时，通过重新激活种植体表面的抗菌活性，治疗种植体周围炎。
为实现上述目的，本发明采取的技术方案为：一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取钛片用SiC砂纸打磨抛光，然后用溶剂清洗，晾干；
(2)将步骤(1)处理后的钛片进行碱热处理，然后用硅烷偶联剂KH570溶液处理；
(3)将步骤(2)处理后的钛片与丙烯酸单体进行自由基聚合反应；
(4)将步骤(3)处理后的钛片与乙二胺反应；
(5)将步骤(4)处理后的钛片浸泡于次氯酸钠溶液中，即可制备得到钛种植体表面的可再生抗菌涂层。
优选地，步骤(1)的具体操作为：取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干。
优选地，步骤(2)中，所述碱热处理为将钛片置于5mol/L NaOH溶液中在60℃下反应24h；所述硅烷偶联剂KH570的体积分数为40％。
碱热处理使钛片表面羟基化，硅烷偶联剂KH570溶液处理钛片，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
优选地，步骤(3)中，所述自由基聚合反应条件为50℃反应12h、50℃反应24h、60℃反应12h、60℃反应24h、70℃反应12h、70℃反应24h中至少一种。
通过设置50℃反应12h、50℃反应24h、60℃反应12h、60℃反应24h、70℃反应12h、70℃反应24h系列梯度的自由基聚合反应条件，使丙烯酸单体与钛表面KH570碳碳双键相连并聚合成聚丙烯酸从而引入羧基，筛选最佳反应条件；
优选地，所述自由基聚合反应条件为60℃反应24h。
优选地，步骤(4)中，所述钛片与所述乙二胺在80℃温度条件下反应24h。
所述钛片与含有带氨基的化合物乙二胺作用，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键。
优选地，步骤(5)中，所述次氯酸钠溶液的有效率浓度为10％。
通过浸泡次氯酸钠溶液引入氯原子，在钛表面构建出N-卤胺高分子抗菌层。
另一方面，本发明提供所述的方法制备得到的钛种植体表面的可再生抗菌涂层，所述可再生抗菌涂层为N-卤胺高分子抗菌层。
再一方面，所述的钛种植体表面的可再生抗菌涂层在制备预防和/或治疗种植体周围炎的药物中的应用。
与现有技术相比，本发明的有益效果为：
(1)本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，采用共价键结合法及聚合物接枝法在钛片表面构建牢固稳定的高分子抗菌层，达到了持久抗菌的作用。
(2)本发明首次将卤胺高分子应用于钛种植体表面改性，构建可再生利用的抗菌涂层，抗菌层牢固稳定，抗菌时效长，抗菌层可再生利用。
(3)本发明通过在钛种植体表面构建结合牢固且抗菌时效持久的、可再生利用的抗菌涂层，以期在种植体植入初期能有效地预防种植体周围炎的发生；以期在种植体后期使用过程中出现种植体周围炎时，通过重新激活种植体表面的抗菌活性，治疗种植体周围炎。
附图说明
图1为本发明实施例1制备得到的N-卤胺涂层的傅里叶变换红外光谱图；
图2为扫描电镜观察本发明实施例1制备得到的N-卤胺涂层的表面形貌结果图；
图3为元素地图观察本发明实施例1制备得到的N-卤胺涂层在钛表面的元素组成和分布结果图；
图4为原子力显微镜分析本发明实施例1制备得到的N-卤胺涂层的结果图；
图5为本发明实施例1制备得到的N-卤胺涂层的热重(TG)曲线图；
图6为本发明实施例1制备得到的N-卤胺涂层的机械性能检测结果图；
图7为本发明实施例1制备得到的N-卤胺涂层接触性抗菌检测结果图；
图8为本发明实施例1制备得到的N-卤胺涂层释放性抗菌检测结果图；
图9为本发明实施例1制备得到的N-卤胺涂层抗菌检测结果图；
图10为本发明实施例1制备得到的N-卤胺涂层抗菌检测结果图；
图11为本发明实施例1制备得到的N-卤胺涂层保存稳定性检测结果图；
图12为本发明实施例1制备得到的N-卤胺涂层长效抗菌性检测结果图；
图13为本发明实施例1制备得到的N-卤胺涂层反复抗菌检测结果图；
图14为本发明实施例1制备得到的N-卤胺涂层对成骨细胞的影响结果图；
图15为本发明实施例1制备得到的N-卤胺涂层对成骨细胞的影响结果图；
图16为本发明实施例1制备得到的N-卤胺涂层对成骨细胞细胞骨架的影响的结果图；
图17为本发明实施例1制备得到的N-卤胺涂层组织相容性结果图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在60℃进行自由基聚合反应24h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
实施例2
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在50℃进行自由基聚合反应12h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
实施例3
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在50℃进行自由基聚合反应24h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
实施例4
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在60℃进行自由基聚合反应12h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
实施例5
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在70℃进行自由基聚合反应12h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
实施例6
本实施例为本发明提供的一种在钛种植体表面制备可再生抗菌涂层的方法，包括如下步骤：
(1)取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000 SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干；
(2)将步骤(1)处理后的钛片用5mol/L NaOH溶液60℃反应24小时进行碱热处理，使钛片表面羟基化，然后用体积分数为40％的硅烷偶联剂KH570溶液处理，其能与钛表面羟基反应，从而将C＝C双键引入钛片表面；
(3)将步骤(2)处理后的钛片与丙烯酸单体在70℃进行自由基聚合反应24h；
(4)将步骤(3)处理后的钛片与含有带氨基的化合物乙二胺在80℃时反应24h，通过酰胺化反应使聚丙烯酸的羧基与乙二胺的氨基反应形成酰胺键；
(5)将步骤(4)处理后的钛片浸泡于有效率浓度为10％次氯酸钠溶液，引入带正电的氯原子，在钛表面构建出N-卤胺高分子抗菌层，即可制备得到钛种植体表面的可再生抗菌涂层。
试验例1
本试验例检测实施例1制备得到的可再生抗菌涂层(N-卤胺涂层)物理化学特性检测、机械性能检 测和热稳定性检测
N-卤胺涂层物理化学特性检测：
(1)傅里叶红外光谱分析抗菌涂层(N-卤胺涂层)的化学键及分子基团组成，结果如图1所示。
图1的傅里叶变换红外光谱结果表明，N-卤胺聚合物成功地接枝到钛表面；如图1所示，Ti-PAA在1704cm -1处的峰代表聚丙烯酸PAA羧基的C＝O吸收峰；Ti-PAA-NH在1540和1400cm -1处的两个峰可归结为C＝O的拉伸振动峰和N-H的弯曲振动峰，说明经过胺化反应，聚丙烯酸的-COOH已转化为-CONH。Ti-PAA-NCL的光谱表明，由于N-Cl的诱导作用，C＝O的振动峰向1552cm -1方向移动，由此表明N-卤胺聚合物成功地接枝到钛表面。
(2)用扫描电镜观察抗菌涂层(N-卤胺涂层)的表面形貌，通过扫描电镜(SEM)进一步观察了碱热钛(a，b)与接枝卤胺的改性钛片(c，d)的表面形态，结果如图2所示。
如图2所示，N卤胺聚合物链在碱性加热后从多孔结构表面开始生长形成聚合物层，但仍保留原有的无序栅格结构；碱热钛表面具有直径约200nm-400nm的无序网孔状结构，孔隙结构明显增加钛表面的比面积；改性钛片仍然保留着这种格栅结构，但钛网表面覆盖N-卤胺聚合物层，钛网明显增厚，孔径比以前小，证明N-卤胺聚合物链在碱热后从网状钛表面开始生长形成聚合物层。
(3)通过元素地图进一步观察了N-卤胺涂层在钛表面的元素组成和分布。
图3结果表明，碳(a)，氧(b)，氯(c)元素在改性钛片表面分布均匀，表明N-卤胺聚合物链在钛表面分布均匀；粗糙度测试结果表明，纯钛(d)的表面最光滑，PSA约为0.4896，碱热钛(e)和改性钛(f)的表面粗糙度高于纯钛(p<0.01).，其PSA值分别为0.6936和0.741，但两者之间无显着性差异(g)。亲水性试验结果表明，纯钛的亲水性最差，接触角为69.5°，碱热钛的亲水性最好，平均接触角为18.7°，改性钛片的表面接触角为43.7°，虽然高于Ti-OH，但仍明显低于纯钛(h)(p<0.01)。综上表明，改性钛片表面粗糙度略高于碱热钛片，但接触角为43.7°，高于碱热钛(18.7℃)；N-卤胺聚合物链在钛表面分布均匀。
(4)用凝胶渗透色谱分析抗菌涂层(N-卤胺涂层)高分子链的长度及分散度，结果如表1所示。
表1.聚合物的分子质量和分散性
反应条件 Mn(Daltons) Mw(Daltons) 分散度
60℃24h 10329 17932 1.736
表1的凝胶渗透色谱结果表明，钛表面N-卤胺聚合物链的数均摩尔质量(Mn)和重均摩尔质量(MW)分别为10329道尔顿和17932道尔顿，分子链的分散度为1.736，满足了自由基聚合反应形成的聚合物的分散度通常在1.5-2.0之间的特点。
(5)原子力显微镜分析N-卤胺涂层，结果如图4所示。
由图4的原子力显微镜结果表明，N-卤胺聚合物层在钛表面的杨氏模量为261MPa。
(6)N-卤胺涂层热稳定性检测：模拟口腔器械效度进行热重分析检测抗菌涂层的热稳定性，结果如图5所示。
由图5的热重(TG)曲线可以看出，当温度从室温升高到121℃/min，然后在121℃保持20min时，)N-卤胺涂层样品的重量百分比总是保持在约100％，表明在高温下通过化学键与钛表面连接的N-卤胺涂层没有明显的化学变化，由此说明N-卤胺涂层在121℃30min无明显质量变化，说明其热稳定性良好。
(7)N-卤胺涂层机械性能检测：微划痕实验结合典型声发射曲线分析N-卤胺涂层的抗剥脱性能，结果如6所示。
图6显示，在微划痕试验中结合典型声发射(AE)曲线，通过自由基聚合反应制备的N-卤胺涂层的平均临界载荷为34.8N，明显高于通过物理吸附法所构建的N-卤胺涂层(P<0.01)，即6.23N。结果表明，化学接枝N-卤胺涂层的结合强度明显高于物理吸附法形成的N-卤胺涂层(p<0.01).，其可抵抗常规种植体植入时施加的扭力。
试验例2
本试验例检测实施例1制备得到的可再生抗菌涂层(N-卤胺涂层)的抗菌性、抗菌时效性及抗菌可再生性能检测
(1)抗菌性能：以牙龈卟啉单胞菌和金黄色葡萄球菌作为模型菌，调整菌液浓度至10 6，钛片与菌液共培养12h后用涂板计数法检测涂层的抗菌性能，同时行钛片表面细菌活死荧光染色及扫描电镜观察，结果如图7-10所示。
N-卤胺涂层接触性抗菌检测(细菌涂板计数法)结果如图7所示，选用金黄色葡萄球菌及牙龈卟啉 单胞菌作为涂板计数的模型菌来验证改性钛片的抗菌性能；钛片与细菌共培养12h后，收集钛表面的细菌涂板计数进行接触性抗菌检测；接触性抗菌率显示在改性钛片上，平均抗金黄色葡萄球菌率为96.22％，平均抗牙龈卟啉单胞菌率为90.77％，对照组碱热钛(Ti-OH)与实验组改性钛片(Ti-CONCl))间存在显著的统计学差异(p<0.0001)，改性钛片对金黄色葡萄球菌以及牙龈卟啉单胞菌均表现出了优异的抗菌活性，说明本发明所制备的N-卤胺涂层能有效的抑制细菌在其表面的粘附与定植。
N-卤胺涂层释放性抗菌检测(细菌涂板计数法)结果如图8所示，释放性抗菌检测的结果显示在改性钛片上平均抗金黄色葡萄球菌率为67.32％，平均抗牙龈卟啉单胞菌率为40.49％，****表明p<0.0001。
N-卤胺涂层抗菌检测(活死细菌荧光染色)结果如图9所示，细菌活死荧光染色结果显示，碱热钛表面多数为染成绿色荧光的活菌，而改性钛片表面大多为染成红色荧光的死菌。
N-卤胺涂层抗菌检测(扫描电镜观察)结果如图10所示，细菌的电镜结果中可以看到金黄色葡萄球菌以及牙龈卟啉单胞菌定植于碱热表面的数量均高于改性钛表面；碱热表面的细菌形成生物膜状分布且细菌形态较为规则，而改性钛表面细菌均为散在分布且有些细菌形态非常不规则，由此可说明N-卤胺涂层可以有效的抑制细菌的粘附与定植。
综上结果表明，N-卤胺涂层对牙龈卟啉单胞菌和金黄色葡萄球菌这两种菌的接触性抗菌率均达90％以上，释放性抗菌率在50％-70％。激光共聚焦显微镜和扫描电镜观察细菌的存活情况及形态，发现与涂层接触的细菌大部分死亡，且形态变形。
(2)抗菌时效性：将改性钛片避光保存1-8w，通过碘化钾-硫代硫酸钠滴定法检测钛表面的有效氯含量。另外将改性钛片在人工唾液中37℃浸泡1-12周，采用0.5％的结晶紫进行钛表面细菌染色，将结晶紫溶解后通过酶标仪检测595nm处的OD值进行抗菌率的计算。抗菌时效性的结果如图11-12所示。
N-卤胺涂层保存稳定性检测结果如图11所示，改性钛的储存稳定性测试结果显示，随着存放时间的延长，改性钛表面的有效氯含量基本保持不变，存放八周后，有效氯含量为0.284％，这说明钛表面卤胺高分子层中的N-Cl键很稳定。存放八周之后将样品重新氯化，有效氯含量仍能恢复100％，这是因为卤胺高分子中的卤素原子在被消耗后能通过简单的方式再卤素化，由此说明本发明所制备的改性钛表面的有效氯在被消耗后是能够通过简单的方式来进行再生的。
N-卤胺涂层长效抗菌性检测结果如图12所示，将样品在人工唾液中37℃浸泡12周后，抑菌率仍在60％以上；随着浸泡时间的延长(1周～12周)，抗菌率逐渐下降；结果表明，在钛表面制备的N-卤胺涂层中的有效氯在液体环境中稳定存在；因此，在种植体表面可以长期保持抗菌粘附能力。在次氯酸钠浸泡 12周后，改性钛的抗菌率可恢复到原来的水平(67.89％～91.45％)，说明该抗菌涂层具有可再生能力。
综上说明，随着浸泡时间的延长，N-卤胺涂层的抗菌率逐渐由原来的91.45％下降到67.89％，表明N-卤胺涂层中的有效氯在液体环境中稳定存在并缓慢持久释放。
(3)抗菌可再生性：将改性钛片与牙龈卟啉单胞菌菌液反复共培养后结晶紫染色计算抗菌率，待抗菌率降低至一定程度后将消耗后的涂层再次浸泡次氯酸钠，检测N-卤胺涂层的抗菌可再生性，结果如图13所示。
N-卤胺涂层反复抗菌检测结果如图13所示，在重复接触细菌1～14次时，抑菌率可达80％以上，经反复接种、染色、乙醇浸泡、超声波清洗29次后，改性仍具有抗菌性能。
当将消耗后的涂层再次浸泡次氯酸钠后，涂层的抗菌率可恢复到初始水平，说明该抗菌涂层具有可再生能力。
试验例3
本试验例检测实施例1制备得到的可再生抗菌涂层(N-卤胺涂层)的生物相容性评估
(1)细胞相容性实验：取第三代骨髓间充质干细胞接种于改性钛片上，发现涂层对细胞的增殖能力、成骨能力及细胞骨架均无明显影响，证明该涂层的细胞相容性良好，且对细胞的成骨功能没有明显影响。
N-卤胺涂层对成骨细胞的影响，结果如图14-15所示。
由图14可知，碱热钛(Ti-OH)及改性钛(Ti-PAA-NCl)表面MC3T3-E1细胞增殖检测结果显示1天，3天，7天时改性钛与碱热钛表面细胞的CCK8检测结果相近，相互之间均无统计学差异。说明接枝了卤胺高分子的钛表面并不会产生细胞毒性，细胞能在其表面正常定植及生长增殖。ALP检测结果显示在MC3T3-E1细胞在碱热钛及改性钛表面分别成骨诱导7天及14天后两组的碱性磷酸酶活性间无统计学差异，由此说明N-卤胺涂层不会影响其表面细胞的早期成骨分化活性。
由图15可知，碱热钛(Ti-OH)及改性钛(Ti-PAA-NCl)表面MC3T3-E1细胞成骨诱导后茜素红染色结果显示碱热钛及改性钛表面均被染成均匀的红色区域，表面均出现较多的大小不一的红色颗粒状钙结节。钙含量的半定量分析结果显示碱热钛组与改性钛组间表面钙含量无明显差异，证明接枝卤胺高分子后并不会降低碱热钛表面的成骨性能。
N-卤胺涂层对成骨细胞细胞骨架的影响，如图16所示。
由图16可知，碱热钛(Ti-OH)及改性钛(Ti-PAA-NCl)表面粘附的MC3T3-E1细胞的荧光染色图像结果显示碱热钛及改性钛表面细胞均呈方圆形，细胞核呈现蓝色荧光，细胞骨架呈现绿色荧光，细胞骨架呈丝状清晰地沿同一方向排列，细胞核较居中。证明MC3T3-E1能在接有卤胺高分子的钛表面正常的粘附伸展。
(2)组织相容性实验：将改性钛片植入裸鼠背部皮下，四周后处死动物取材，行苏木素伊红染色及巨噬细胞的标记物CD68荧光染色，结果如图17所示。
由图17可知，将碱热钛(Ti-OH)及改性钛(Ti-PAA-NCl)分别植入裸鼠背部皮下一月，取材时可见所有植入体表面均覆盖有一层纤维结缔组织膜，未见炎性组织及脓性分泌物，创口愈合良好，取材后行HE染色观察胞膜及CD68免疫荧光染色观察巨噬细胞数量及分布。CD68荧光染色结果中巨噬细胞特异性CD68染色表现为绿色，蓝色为所有细胞核染色。可见碱热钛组与改性钛组表面包膜内均没有出现巨噬细胞聚集或成团的现象。HE染色结果显示碱热钛组与改性钛组表面所覆盖的纤维包膜均无明显增厚，其内有血管穿过，无明显的炎性细胞浸润。这说明接枝N-卤胺高分子的钛片在生物体内具有较好的相容性，不会引起明显的炎症等排异反应，结果证明该涂层的组织相容性良好。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种在钛种植体表面制备可再生抗菌涂层的方法，其特征在于，包括如下步骤：
    (1)取钛片用SiC砂纸打磨抛光，然后用溶剂清洗，晾干；
    (2)将步骤(1)处理后的钛片进行碱热处理，然后用硅烷偶联剂KH570溶液处理；
    (3)将步骤(2)处理后的钛片与丙烯酸单体进行自由基聚合反应；
    (4)将步骤(3)处理后的钛片与乙二胺反应；
    (5)将步骤(4)处理后的钛片浸泡于次氯酸钠溶液中，即可制备得到钛种植体表面的可再生抗菌涂层。
  2. 如权利要求1所述的方法，其特征在于，步骤(1)的具体操作为：取直径为9.5mm、厚为0.3mm的圆形钛片用#400、#800、#1000SiC砂纸逐级打磨抛光，然后用溶剂丙酮、无水乙醇和双蒸水依次超声清洗20min，晾干。
  3. 如权利要求1所述的方法，其特征在于，步骤(2)中，所述碱热处理为将钛片置于5mol/LNaOH溶液中在60℃下反应24h；所述硅烷偶联剂KH570的体积分数为40％。
  4. 如权利要求1所述的方法，其特征在于，步骤(3)中，所述自由基聚合反应条件为50℃反应12h、50℃反应24h、60℃反应12h、60℃反应24h、70℃反应12h、70℃反应24h中至少一种。
  5. 如权利要求4所述的方法，其特征在于，所述自由基聚合反应条件为60℃反应24h。
  6. 如权利要求1所述的方法，其特征在于，步骤(4)中，所述钛片与所述乙二胺在80℃温度条件下反应24h。
  7. 如权利要求1所述的方法，其特征在于，步骤(5)中，所述次氯酸钠溶液的有效率浓度为10％。
  8. 采用如权利要求1-7任一项所述的方法制备得到的钛种植体表面的可再生抗菌涂层。
  9. 如权利要求8所述的钛种植体表面的可再生抗菌涂层，其特征在于，所述可再生抗菌涂层为N-卤胺高分子抗菌层。
  10. 如权利要求8-9任一所述的钛种植体表面的可再生抗菌涂层在制备预防和/或治疗种植体周围炎的药物中的应用。
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