CN117288793A - Titanium-containing test piece of cross-linked PLGA drug-loaded coating, and preparation method and application thereof - Google Patents
Titanium-containing test piece of cross-linked PLGA drug-loaded coating, and preparation method and application thereof Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
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- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to the technical field of biomedical materials, in particular to a titanium-containing test piece of a cross-linked PLGA drug-loaded coating, and a preparation method and application thereof. The preparation method comprises the following steps: preparation of a catalyst having MAO-TiO 2 The coating comprises a micro-arc oxidized pure titanium test piece, PLGA microspheres loaded with SDF-1 and PLGA microspheres loaded with Mino; dissolving PLGA microsphere loaded with SDF-1 and PLGA microsphere loaded with Mino in gelatin solution, ultrasonic resuspension treatment, and transferring to MAO-TiO 2 And (3) oxidizing the surface of the pure titanium test piece by micro-arc of the coating, vibrating and drying to obtain the titanium-containing test piece of the cross-linked PLGA drug-loaded coating. PLGA medicine carrying coating on titanium-containing test piece can effectively solve early occupation of periodontal pathogenic bacteria interfering host cells on implant surfaceThe problem is that interference of pathogenic bacteria to cell occupation is interfered in a golden period (within 6 h) after implantation, a functional cell layer is induced to cover the surface of the implant, and early bone union is accelerated.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a titanium-containing test piece of a cross-linked PLGA drug-loaded coating, and a preparation method and application thereof.
Background
Since the invention of the dental implant technology, dental implants have become the first choice treatment scheme for patients with dentition defects and dentition defects, almost completely solve the problems of unavoidable grinding of adjacent teeth or poor comfort of patients in traditional restoration modes, and greatly improve the chewing efficiency of patients, which is called as the third pair of teeth of human beings. Periodontal disease is the leading cause of adult tooth loss, accounting for about 80% of cases of loss of teeth. Studies have shown that peri-implant inflammation is significantly more prevalent in patients with a history of chronic periodontitis than in periodontal health, and that the risk of implant failure in bone bonding is 3.1-4.7 times higher in patients with periodontal health loss than in periodontal health even if the patient with a history of periodontal disease is implanted after treatment. Thus, the success of implant to bone integration in periodontal disease patients is a current difficult problem facing clinicians. The pathogenic bacteria in the oral cavity of periodontal disease patients are easy to be planted on the surface of the implant in an early stage, especially in the golden period of 6 hours after operation, the pathogenic bacteria interfere with the host cells to occupy the site on the surface of the implant in the early stage, thereby causing the delay in bone bonding time or the reduction in quality, and being the main reason of planting failure. Revealing the competition mechanism of the two and effectively intervening the competition occupation of pathogenic bacteria to improve the early bone integration rate of the implant is a difficult problem to be solved in the field.
The researches of the scholars are focused on improving the surface structure and chemical composition of pure titanium to improve the osteogenesis activity of the pure titanium, and the scholars adopt an infiltration antibacterial agent and silver-carrying or antibacterial short titanium modification means to realize the antibacterial performance. The current research on implant surface modification has the following problems: (1) The key period of preoccupation of peri-implant pathogenic bacteria and host cells on the surface of the implant in the early golden period after the implant operation is lack of attention; (2) Focusing on unilateral investigation of osteogenic activity and/or antibacterial performance, under the coexisting environment of host cells and periodontal pathogens, the situation of adhesion proliferation on the surface of an implant is not evaluated; (3) The surface competition of cells and bacteria is not interfered from the source, so that early and firm combination between the implant and the bone is realized.
The key to the success of dental implants is how to establish an early firm bond between the surface of the implant and the alveolar bone, with a delay in the bone bonding process or a decrease in quality, which can cause related infections around the implant and ultimately failure of the implant. Implant osseointegration is mainly affected by two factors: firstly, the microstructure and chemical components of the surface of the implant are not ideal enough, and a vascular-free fibrous tissue layer is easy to appear at the bonding interface of the implant and bone tissue, so that host cells are difficult to reach the surface of the implant; secondly, the interference of peri-implant pathogenic bacteria, the early colonization of bacteria on the surface of the implant can inhibit the crosslinking of host cells, and bacterial plaques are formed along with the increase of the number of the bacteria and the extension of time, and the bacteria in the bacterial plaque barriers have strong resistance to the immunity of the host and the antibacterial treatment and are difficult to remove. Furst et al scholars found that periodontal pathogenic bacteria colonized on the surface of the implant, which may naturally colonize or migrate from the natural teeth to the implant, appeared only after the implant was implanted in the body for 30 minutes, and that the presence of pathogenic bacteria severely affected early crosslinking of host cells on the implant surface and the adverse pathogenic microenvironment played an important role in the development and progression of peri-implant inflammation.
Therefore, an effective material modification intervention means is searched, so that the surface activity of the implant is easy to crosslink host cells, and the implant has effective antibacterial performance, thereby being a necessary means for ensuring successful implant bone integration of patients suffering from periodontal disease.
Disclosure of Invention
The invention provides a titanium-containing test piece of a cross-linked PLGA drug-loaded coating, and a preparation method and application thereof, wherein the PLGA drug-loaded coating on the titanium-containing test piece can effectively solve the problem that periodontal pathogenic bacteria interfere with early occupation of host cells on the surface of an implant, interfere pathogenic bacteria in a golden period after implantation operation to interfere with the occupation of cells, induce a functional cell layer to cover the surface of the implant, and accelerate early bone union.
According to a first aspect of the present invention, the present invention provides a method for preparing a titanium-containing test piece of a crosslinked PLGA drug-loaded coating, comprising the steps of:
preparation of a catalyst having MAO-TiO 2 Micro-arc oxidation pure titanium test piece of the coating;
preparing PLGA microspheres loaded with SDF-1;
preparing PLGA microspheres loaded with Mino;
dissolving the PLGA microspheres loaded with SDF-1 and the PLGA microspheres loaded with Mino in a gelatin solution, and moving to the MAO-TiO after ultrasonic resuspension treatment 2 And (3) oxidizing the surface of the pure titanium test piece by micro-arc of the coating, vibrating and drying to obtain the titanium-containing test piece of the cross-linked PLGA drug-loaded coating.
In the scheme, the preparation method of the titanium-containing test piece with the cross-linked PLGA drug-loaded coating provided by the invention comprises the steps of respectively preparing MAO-TiO 2 Coating micro-arc oxidized pure titanium test piece, PLGA microsphere loaded with SDF-1 and PLGA microsphere loaded with Mino, and MAO-TiO 2 The PLGA microspheres loaded with the SDF-1 and the PLGA microspheres loaded with the Mino are loaded in the micro-arc oxidized pure titanium test piece of the coating, and the matrix derived factor SDF-1 definitely has a dose-dependent chemotactic effect on a large number of BMSCs bone marrow stromal cells in the alveolar bone of a host, and can be used as a first-choice induction factor to run up the BMSCs to the surface of an implant; minocycline (Mino) not only can effectively inhibit and kill the periodontal disease main pathogenic bacteria G-Porphyromonas gingivalis (Porphyromonas gingivalis, P.g) which are easy to colonize on the surface of the implant in early stage of planting, destroy the structure of important factor pilin related to P.g virulence and adhesion, but also has obvious killing effect on key bacteria G+ staphylococcus aureus detected in post-periendophyte; polylactic acid-glycolic acid copolymer (PLGA) drug-loaded microspheres are crosslinked in the micropore structure on the surface of the micro-arc titanium oxide through gelatin with excellent safety and operability, thereby creating a reliable condition for slow release of SDF-1 and Mino. The PLGA drug-loaded coating on the titanium-containing test piece of the cross-linked PLGA drug-loaded coating prepared by the preparation method of the invention has bidirectional regulation and control effect on the competition inhibition of cells and bacteria in the early gold period of implantation, thus realizing the aim of starting from the sourceThe surface competition occupying behavior is positively interfered, so that the problem that periodontal pathogenic bacteria interfere with early occupying of host cells on the surface of an implant is effectively solved, and a solid foundation is laid for success of early bone integration of periodontal disease patients.
Further, the preparation of the PLGA microspheres loaded with SDF-1 comprises the following steps:
dissolving PLGA in dichloromethane to obtain a first oil phase; dissolving SDF-1 and polyvinyl alcohol together in water to obtain a first aqueous phase; dropwise adding the first oil phase into the first water phase for ultrasonic resuspension to obtain a first oil-water emulsion, magnetically stirring the obtained first oil-water emulsion, centrifuging, and collecting a first precipitate; and washing and freeze-drying the collected first precipitate to obtain the PLGA microsphere loaded with the SDF-1.
In the scheme, the PLGA microsphere with the SDF-1 is prepared by a specific preparation method, wherein the PLGA microsphere is suitable for encapsulation rate and drug loading rate, and the SDF-1 is effectively deposited on the PLGA microsphere.
Further, in the first oil phase, the weight ratio of PLGA to dichloromethane is 1 (4-6);
and/or the first aqueous phase has a concentration of SDF-1 of 25ng/ml to 75ng/ml and a concentration of polyvinyl alcohol of 0.01g/ml;
and/or the weight ratio of the first oil phase to the first water phase is 1 (15-25);
and/or dropwise adding the first oil phase into the first water phase, wherein the ultrasonic resuspension power is 200w-300w, and the time is 5min-10min;
and/or the magnetic stirring speed of the obtained first oil-water emulsion is 900r/min-1100r/min, and the time is 5h-8h; performing magnetic stirring on the obtained first oil-water emulsion, and centrifuging at a rotating speed of 9000r/min-15000r/min for 10min-20min;
and/or, the particle size of the PLGA microsphere loaded with the SDF-1 is 350nm-650nm.
In the scheme, in the preparation process of the PLGA microsphere loaded with the SDF-1, the weight ratio of PLGA to dichloromethane in the first oil phase, the concentration of the SDF-1 and the concentration of the polyvinyl alcohol in the first water phase, the weight ratio of the first oil phase to the first water phase, the speed and time of magnetic stirring and the speed and time of centrifugation are limited within reasonable range values, so that the preparation of the PLGA microsphere loaded with the SDF-1 with proper encapsulation rate and drug loading rate is facilitated.
Further, preparing the Mino-loaded PLGA microspheres comprises the following steps:
dissolving PLGA in dichloromethane to obtain a second oil phase; dissolving Mino and polyvinyl alcohol together in water to obtain a second aqueous phase; dropwise adding the second oil phase into the second water phase for ultrasonic resuspension to obtain a second oil-water emulsion, magnetically stirring the obtained second oil-water emulsion, centrifuging, and collecting a second precipitate; and washing and freeze-drying the collected second precipitate to obtain the PLGA microspheres loaded with Mino.
In the above scheme, PLGA microspheres loaded with Mino with proper encapsulation rate and drug loading rate are prepared by a specific preparation method, and Mino is effectively deposited on the PLGA microspheres.
Further, in the second oil phase, the weight ratio of PLGA to dichloromethane is 1:4-6;
and/or, the concentration of Mino in the second aqueous phase is 5-50 mug/ml, and the concentration of polyvinyl alcohol is 0.01g/ml;
and/or the weight ratio of the second oil phase to the second water phase is 1 (15-25);
and/or dropwise adding the second oil phase into the second water phase, wherein the ultrasonic resuspension power is 200w-300w, and the time is 5min-10min;
and/or carrying out magnetic stirring on the obtained second oil-water emulsion at the speed of 900r/min-1100r/min for 5-8 h; performing magnetic stirring on the obtained second oil-water emulsion, and centrifuging at a rotating speed of 9000r/min-15000r/min for 10min-20min;
and/or the particle size of the PLGA microsphere loaded with Mino is 350nm-650nm.
In the scheme, in the preparation process of the PLGA microsphere loaded with the Mino, the weight ratio of PLGA to dichloromethane in the first oil phase, the concentration of Mino and the concentration of polyvinyl alcohol in the second water phase, the weight ratio of the second oil phase to the second water phase, the speed and time of magnetic stirring and the speed and time of centrifugation are limited within reasonable range values, so that the PLGA microsphere loaded with the Mino with proper encapsulation rate and drug loading rate is more favorably prepared.
Further, the weight percentage of the gelatin solution is 0.1%;
and/or the time of oscillation is 2 hours, and the frequency of oscillation is 50rpm-100rpm; and/or the temperature of drying is 4 ℃.
In the scheme, the technical parameters such as the weight percentage of the gelatin solution, the time and frequency of oscillation, the drying temperature and the like are limited within reasonable range values, so that the Mino-loaded PLGA microsphere with proper encapsulation efficiency and drug loading capacity can be prepared more conveniently.
Further, preparation of a catalyst having MAO-TiO 2 The micro-arc oxidation pure titanium test piece of the coating specifically comprises the following steps: the medical grade pure titanium wafer type test piece is adopted as an anode, a stainless steel sheet is adopted as a cathode, and the MAO-TiO is obtained through electrochemical reaction 2 And (3) coating the micro-arc oxidized pure titanium test piece.
In the above scheme, the invention processes the pure titanium sample, so as to prevent different metals in the electrolyte from reacting, the clamp used as the anode electrode is also made of pure titanium, and the part exposed in the electrolyte is protected by plastic spraying in advance.
Further, the electrolytic tank used for carrying out the electrochemical reaction mainly comprises a three-layer structure, wherein the outermost layer is a cooling tank and a cold water circulation system, the inner layer is an insulating porous plastic inner net and a stirring device, and the middle layer is a stainless steel tank; when in use, cooling water is added into the cooling pool, electrolyte is injected into the stainless steel tank, and meanwhile, the inner air pump and the outer water circulating power supply are started.
In the scheme, the purpose of the design is to keep the concentration of the electrolyte uniform and a certain oxygen content, and rapidly release the heat generated in the micro-arc oxidation process to ensure the basically constant temperature of the system, and a cooling system is adopted to always keep the temperature below 40 ℃.
According to a second aspect of the invention, the invention also provides a titanium-containing test piece of the cross-linked PLGA drug-loaded coating, which is prepared by the preparation method.
According to a third aspect of the present invention there is also provided the use of a titanium-containing test piece as described above for the preparation of a dental implant.
The invention has the beneficial effects that:
the invention provides a titanium-containing test piece of a cross-linked PLGA drug-loaded coating and a preparation method thereof, wherein PLGA nano microspheres are used for wrapping SDF-1 capable of targeting chemotactic cell homing and Mino capable of resisting bacterial colonization, and loading the SDF-1 into micro-arc titanium oxide surface micropores, and a difunctional coating capable of inducing cell homing and targeting antibacterial colonization is constructed on the surface of a titanium implant, so that interference of pathogenic bacteria on cell occupation is interfered during a gold period (within 6 h) after implantation, a functional cell layer is induced to cover the surface of the implant, and early bone integration is accelerated. By establishing a periodontitis canine model and a bacterial/cell co-culture system, the bidirectional regulation and control effect and the molecular mechanism of the titanium implant PLGA drug-loaded coating on the competitive inhibition of cells and pathogenic bacteria in the early gold period of planting are researched, and a new thought and theoretical basis is provided for promoting early bone integration of periodontal disease patients.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows an embodiment 1 of the present invention with MAO-TiO 2 XRD patterns of the surface of the micro-arc oxidized pure titanium test piece of the coating;
FIG. 2 is a graph showing the expected PLGA 24h release profile achieved in example 1 of this invention;
FIG. 3 is a diagram of an experimental test piece for scanning electron microscope observation according to the present invention;
FIG. 4 shows the MAO-TiO composition of example 1 of the present invention 2 Relevant characterization and analysis graphs of the micro-arc oxidation pure titanium test piece of the coating;
FIG. 5 is a graph showing the comparison of migration conditions of SDF-1 at different time points of different target drug release concentrations in scratch experiments;
FIG. 6 is a graph showing comparison of proliferation of cells cultured in vitro with BMSCs and added with SDF-1 and 24h at different target drug release concentrations;
FIG. 7 is an SEM image of the growth of the mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of the control group after 2 hours under the scanning electron microscope;
FIG. 8 is an SEM image of growth of rat mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of experimental group 1 after 2 hours under a scanning electron microscope;
FIG. 9 is an SEM image of the growth of the mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of the control group after 6 hours under the scanning electron microscope;
FIG. 10 is an SEM image of growth of rat mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of experimental group 1 after 6 hours under a scanning electron microscope;
FIG. 11 is an SEM image of the growth of the mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of the control group after 24 hours under the scanning electron microscope;
FIG. 12 is an SEM image of growth of rat mesenchymal stem cells (arrow 1) and Porphyromonas gingivalis (arrow 2) on the surface of the test piece of experimental group 1 after 24 hours under a scanning electron microscope;
FIG. 13 is an SEM image of a medical grade pure titanium disc specimen co-cultured with bone marrow mesenchymal stem cells (BMSCs) and Porphyromonas gingivalis (P.g) in the golden period;
FIG. 14 is a graph showing the effect of CCK-8 on cell activity for a gold period and 24 hours in the early stage of a medical grade pure titanium wafer test piece;
FIG. 15 is an SEM image of a medical grade pure titanium disc type specimen co-cultured with bone marrow mesenchymal stem cells (BMSCs) and Porphyromonas gingivalis (P.g) after 24 hours;
FIG. 16 is a volcanic chart showing gene differential screening in the cell-bacteria co-culture group P.g of medical grade pure titanium disc type test pieces in which bone marrow mesenchymal stem cells (BMSCs) and Porphyromonas gingivalis (P.g) were co-cultured in the golden period.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a titanium-containing test piece of a cross-linked PLGA drug-loaded coating, which specifically comprises the following steps:
(1) Preparation of a catalyst having MAO-TiO 2 Micro-arc oxidation pure titanium test piece of coating: the medical grade pure titanium wafer type test piece is adopted as an anode, a stainless steel sheet is adopted as a cathode, and the MAO-TiO is obtained through electrochemical reaction 2 Coated micro-arc oxidized pure titanium test piece with MAO-TiO 2 XRD test was performed on the surface of the coated micro-arc oxidized pure titanium test piece, and the test results are shown in FIG. 1. The electrolytic tank used for carrying out the electrochemical reaction mainly comprises a three-layer structure, wherein the outermost layer is a cooling tank and a cold water circulation system, the inner layer is an insulating porous plastic inner net and a stirring device, and the middle layer is a stainless steel tank; when in use, cooling water is added into the cooling pool, electrolyte is injected into the stainless steel tank, and meanwhile, the inner air pump and the outer water circulating power supply are started.
(2) Preparation of PLGA microspheres loaded with SDF-1: under the action of ultrasound, 20mg of PLGA is dissolved in methylene dichloride to obtain a first oil phase, wherein the weight ratio of PLGA to methylene dichloride is 1:5; 1.0. Mu.g of SDF-1 and 0.2g of polyvinyl alcohol were dissolved together in 20ml of distilled water to obtain a first aqueous phase; dropwise adding the first oil phase into a first water phase (the weight ratio of the first oil phase to the first water phase is 1:20) at 25 ℃, carrying out ultrasonic resuspension for 350s-360s at 250w to obtain a first oil-water emulsion, magnetically stirring the obtained first oil-water emulsion at 1000r/min for 6h, removing dichloromethane, centrifuging at 10000r/min for 15min, and repeatedly centrifuging for three times to collect a first precipitate; and washing and freeze-drying the collected first precipitate to obtain PLGA microspheres loaded with SDF-1, wherein the particle size of the PLGA microspheres is 350-650 nm, and the target drug release concentration of the PLGA microspheres loaded with SDF-1 is 100ng/ml.
It should be noted that, the PLGA microsphere carrying SDF-1 is prepared by adopting an emulsification-solvent volatilization method: under the ultrasonic action, dissolving a certain amount of PLGA in dichloromethane to obtain a first oil phase; SDF-1 and 0.2g of polyvinyl alcohol were dissolved in 20ml of distilled water as a first aqueous phase. Dropwise adding the first oil phase into the first water phase at 25 ℃ in proportion, carrying out ultrasonic resuspension for a certain time to obtain a first oil-water emulsion, magnetically stirring for 6h at 1000r/min, and removing CH 2 Cl 2 . Centrifuging at 10000r/min for 15min, repeatedly centrifuging for three times, and collecting first precipitate; and washing and freeze-drying the collected first precipitate to obtain PLGA microspheres loaded with SDF-1 and having the particle size of 350-650 nm, and measuring the drug loading and encapsulation efficiency. By combining PLGA molecular weight, monomer proportion, encapsulation rate (79.84+/-1.23)%, drug loading rate (26.7+/-0.19) ng/mg and 24h drug release rate of 16% -18%, and repeatedly searching and adjusting parameters, as shown in figure 2, an expected PLGA 24h drug release curve (average value is obtained through 3 detection) is realized, and it is clear that if the target drug release concentration of the PLGA microsphere loaded with SDF-1 is 100ng/ml, in the process of preparing the PLGA microsphere loaded with SDF-1, the dosage of SDF-1/PLGA is 1 mug/20 mg, and the ultrasonic resuspension time is 350s-360s.
(3) Preparing PLGA microspheres loaded with Mino: dissolving 20mg of PLGA in dichloromethane to obtain a second oil phase, wherein the weight ratio of PLGA to dichloromethane is 1:5; 1mg of Mino and 0.2g of polyvinyl alcohol were dissolved together in 20ml of distilled water to obtain a second aqueous phase; dropwise adding the second oil phase into the second water phase (the weight ratio of the second oil phase to the second water phase is 1:20) and carrying out ultrasonic resuspension for 6min to obtain a second oil-water emulsion, magnetically stirring the obtained second oil-water emulsion at 1000r/min for 6h to remove dichloromethane, centrifuging at 10000r/min for 15min, and repeatedly centrifuging for three times to collect a second precipitate; and washing and freeze-drying the collected second precipitate to obtain Mino-loaded PLGA microspheres with the particle size of 350-650 nm, wherein the target drug release concentration of the Mino-loaded PLGA microspheres is 1000ng/ml.
(4) Dissolving 0.3ml PLGA microsphere loaded with SDF-1 and 0.7ml PLGA microsphere loaded with MinoDissolving in 1ml of 0.1% gelatin solution, and ultrasonic resuspension treating to obtain MAO-TiO 2 And (3) micro-arc oxidizing the surface of the pure titanium test piece of the coating, oscillating for 2 hours at the frequency of 70rpm, and drying at the temperature of 4 ℃ to obtain the titanium-containing test piece of the cross-linked PLGA drug-loaded coating.
Example 2
The difference from example 1 is that SDF-1 is used in the amount of 1.5. Mu.g in step (2), and the target drug release concentration of the PLGA microspheres loaded with SDF-1 is 150ng/ml.
Example 3
The difference from example 1 is that SDF-1 is used in the amount of 0.5. Mu.g in step (2), and the target drug release concentration of the PLGA microspheres loaded with SDF-1 is 50ng/ml.
Experimental example
Characterization analysis was performed on the titanium-containing test piece prepared in example 1 of the present invention. The titanium-containing test piece is characterized, analyzed and observed, and the surface morphology, microstructure and X-ray diffraction (XRD) of the test piece are observed by adopting a high-resolution scanning electron microscope (Scanning Electronic Microscopy, SEM), so that the components and phase components of the coating and the changes of chemical components are clearly detected by a scattering energy spectrometer (Energy Dispersive Spectroscope, EDS); the surface roughness of the titanium-containing test piece is analyzed by a surface roughness tester (TR 240), and the surface contact angle is measured by a surface contact angle tester (SL 200B); the relative proliferation rate (RGR) is calculated according to the OD value measured by the MTT method, the relative proliferation rate of cells RGR% = OD test group/OD negative control group X100%, cytotoxicity Classification (CTS) is carried out according to the RGR value, and the experimental result must meet the requirement on cytotoxicity of materials in the national medical instrument biological evaluation Standard GB/T16886.1-2001. And conventionally cleaning and sterilizing the titanium-containing test piece, and then air-drying for later use.
1. As shown in fig. 3, wherein a diagram in fig. 3 shows a titanium sheet and a titanium implant diagram, and B diagram shows an experimental test piece diagram for scanning electron microscope observation.
MAO-TiO with high resolution scanning electron microscope observation 2 The surface morphology and microstructure of the coated micro-arc oxidized pure titanium test piece, the PLGA microsphere loaded with SDF-1 and the PLGA microsphere loaded with Mino are shown in FIG. 4, wherein the C diagram in FIG. 4 is MAO-TiO 2 Micro-arc of coatingSEM image of pure titanium oxide test piece, which has micropores with diameter of 2-7 μm on its surface. Panel D in FIG. 4 is an SEM image of PLGA microspheres loaded with SDF-1.
The surface morphology and microstructure of the titanium-containing test piece of the crosslinked PLGA drug-loaded coating are observed by adopting a high resolution scanning electron microscope, and as shown in an E diagram in fig. 4, the PLGA microsphere loaded with SDF-1 and the PLGA microsphere loaded with Mino are crosslinked with MAO-TiO 2 In the micropores of the micro-arc oxidized pure titanium test piece of the coating, the invention shows that the nanometer microspheres are successfully anchored in the micro-arc oxidized surface micropore structure by adopting an oscillation infiltration coating crosslinking method. The micro-arc oxidation electrochemical surface is firmly combined with the titanium substrate, and the high-strength micro-arc oxidation structure on the surface of the titanium implant can protect the nano-microspheres in the micropores from being damaged and bring the safety belt into target tissues for slow release and play a role in the process of implanting the implant into the body. In addition, the physical and chemical properties of the surface of the material on the titanium-containing test piece of the cross-linked PLGA drug-loaded coating of the present example were primarily examined, and the surface of the pure titanium wafer-type test piece, MAO-TiO, is shown in Table 1 below 2 The results of comparison of porosity, roughness and contact angle of the coating surface and the PLGA drug-loaded coating are shown. FIG. 4, panel F, shows a Pictol software analysis of PLGA drug-loaded coating surface pore patterns.
TABLE 1
2. Biological property evaluation of PLGA drug-loaded coating of titanium-containing test piece of crosslinked PLGA drug-loaded coating
2.1, research on the effects of PLGA drug-loaded coatings on inducing host cell migration homing and proliferation regulation
Isolation and culture of bone marrow mesenchymal stem cells: obtained from femur and tibia marrow of 4 week old SD rats. The multiple differentiation capacity of cells was demonstrated using osteogenic and lipogenic induction experiments. 2-4 surrogate was used for cell experiments.
Setup ofUncrosslinked PLGA drug-loaded coating with MAO-TiO 2 The micro-arc oxidized pure titanium test pieces of the coating are used as a control group, and the titanium-containing test pieces of the cross-linked PLGA drug-loaded coatings of the embodiment 1, the embodiment 2 and the embodiment 3 of the invention are respectively used as an experimental group 1, an experimental group 2 and an experimental group 3.
(1) Cell adhesion experiments: DAPI-labeled cell nucleus fluorescent microscope photographing and counting cells
(2) Cell migration experiments: tanswell cell method and scratch method
Tanswell cell method: a filter membrane with the diameter of 8 μm is selected, 200 mu L of serum-free cell suspension is added into an upper chamber, and each group of test pieces and 800 mu L of DMEM cell culture solution containing 10% FBS are respectively added into a lower chamber. 37 ℃ and 5% CO 2 Culturing in incubator for 24 hr, taking out, fixing 4% paraformaldehyde, and dyeing with crystal violet. Cells on the filter (toward the lower chamber side) were counted under an inverted microscope and randomly selected 5 fields.
Scoring method: the culture solution containing cells is respectively planted on the surface of a titanium test piece in a porous plate, the center of the surface of the test piece is scraped by using a gun head to form a cell-free area, and after the culture is carried out for 15 hours, the cell migration analysis is carried out by using an ImageXpress system.
(3) Cell proliferation assay: MTT method and CCK-8 method detection
(4) Statistical analysis: statistical descriptions of quantitative data were used. In the comparison of the differences between the groups, a two-way ANOVA analysis was used if the normalization and variance alignment were satisfied, otherwise a non-parametric Scheirer-Ray-Hare test was selected. Statistical analysis using SAS 9.4 and STATA 15.0 software, a two-sided assay was used to verify levels.
FIG. 5 shows the results of scratch experiments to observe the migration of PLGA microspheres loaded with SDF-1 at different time points at different target drug release concentrations. The target drug release concentrations of example 1 and example 2 showed significantly increased BMSCs migration capacity over the control, but no significant difference between the two groups. FIG. 6 shows that BMSCs were cultured in vitro, SDF-1 was added at different target drug release concentrations, proliferation of 24h cells was observed, BMSCs was proliferated optimally under the conditions of example 1, and proliferation capacity was slightly decreased when the target drug release concentration was increased to 150ng/ml. The invention mainly examines the occupation and proliferation of the cells planted in the golden period on the surface of the implant by combining the proliferation and migration of BMSCs, and therefore, the effect of the embodiment 1 under the condition of the target drug release concentration is relatively suitable.
2.2 study of the antibacterial Properties of the surface of the PLGA drug-loaded coating
Setting up uncrosslinked PLGA drug-loaded coating with MAO-TiO 2 The micro-arc oxidized pure titanium test pieces of the coating are used as a control group, the titanium-containing test pieces of the cross-linked PLGA drug-loaded coating of the embodiment 1, the embodiment 2 and the embodiment 3 are respectively used as an experiment group 1, an experiment group 2 and an experiment group 3, a bacteria adhesion experiment and an antibacterial experiment are carried out, and an antibacterial ring experiment is carried out to evaluate the antibacterial performance of the surface of the PLGA drug-loaded coating.
(1) Bacterial culture: porphyromonas gingivalis ATCC33277 was inoculated on the surface of the sample for bacterial culture study. Rat mesenchymal stem cells (5000 pieces/hole, 24 hole plates) and porphyromonas gingivalis (0.5 CFU/hole, 24 hole plates) were used for co-culturing on round titanium plates (diameter 11.5mm, thickness 0.2 mm) of a control group and an experimental group for 2 hours, 6 hours and 24 hours, and then fixed for 4 hours, 30%, 60%, 70%, 80%, 90%, 95% and 100% gradient concentration ethanol is used for dehydration, ventilation drying and scanning electron microscope observation after metal spraying on the surfaces of test pieces.
Fig. 7 to 12 show growth of rat mesenchymal stem cells (arrow 1) and porphyromonas gingivalis (arrow 2) on the surfaces of the test pieces of the control group and the test piece of the experimental group 1 after 2h, 6h and 24h under the scanning electron microscope.
As can be seen from the results of fig. 7 to 12, the PLGA drug-loaded coating on the test piece surface of experimental group 1 can reduce the adhesion and colonization of porphyromonas gingivalis ATCC33277 at an early stage, and guide the adhesion of the mesenchymal stem cells of rats to the test piece surface.
(2) Early bacterial adhesion and proliferation observations: and designing adhesion and proliferation conditions of bacteria on the surface of a test piece at different time points (2 h,4h,6h and 24 h), and observing the changes of the morphology and the quantity of the bacteria by a scanning electron microscope.
As can be seen from the results of fig. 7-12, at 2h, both test pieces had bacterial adhesion on the surface, but the control group had significantly more bacterial adhesion than the experimental group 1; only a small amount of bacteria is adhered to the surface of the PLGA drug-loaded coating of the experimental group 1 in 6 hours, and the bacteria are more on the surface of the pure MAO; at 24h, the control group had significantly more bacterial adhesion proliferation than experimental group 1. The finding shows that the early adhesion and proliferation of bacteria on the surface of the uncrosslinked PLGA drug-loaded coating are obviously higher than those of the PLGA drug-loaded coating, and the PLGA nano microsphere wrapping SDF-1/Mino has the effect of inhibiting the adhesion and colonization of bacteria on the surface of a test piece.
3. Pre-experiment: construction of a bacterial/cell Co-culture System
The co-culture system condition is basically groped and matured, a separate incubator is needed to be selected, the cell culture condition is taken as the main condition, and BMSCs cells in logarithmic growth phase are taken; the anaerobic condition of the incubation process of the co-cultured porphyromonas gingivalis P.g is very high, in order to ensure the activity of bacteria, the anaerobic bag is adopted to carry out sealed culture for 48-72 hours in advance in a laboratory, and the bacteria are required to be suspended and centrifuged, the supernatant is discarded and the cell culture medium is adopted to carry out resuspension during the co-culture.
The project mainly aims to observe the 'cell and bacteria competition inhibition behavior' on the surface of a medical grade pure titanium wafer type test piece, observe the co-culture condition of BMSCs and P.g in a gold planting period and 24h in the early period under a pre-experimental light microscope, and detect the toxic effect of bacteria on cells.
FIG. 13 shows the microscopic co-culture of bone marrow mesenchymal stem cells (BMSCs) with Porphyromonas gingivalis (P.g) at various time points during the golden period after implantation: p.g bacteria are arranged in the gray frame, BMSCs bacteria are arranged in the black frame, the low quantity P.g is 10000/test piece, and the high quantity P.g is 100000/test piece. As can be seen in FIG. 13, the presence of pathogenic bacteria directly interferes with the host cell's adhesive spreading and proliferative activity.
FIG. 14 shows the effect of CCK-8 on cell activity in the golden period and early 24h, and FIG. 14 shows that the early cell activity of high-number bacterial groups is reduced, which is statistically significant.
FIG. 15 shows SEM images after 24 hours of co-culture, and it can be seen from FIG. 15 that BMSCs cells of a large-area low-number bacterial group are spread in adhesion, and cells of a high-number bacterial group P.g are in a fusiform shape, and the cells have good activity (P.g bacteria are indicated by arrows in FIG. 15).
The experimental results that can be obtained from fig. 13-15: a P.g and BMSCs co-culture system was successfully constructed on a medical grade pure titanium wafer type test piece. The results in FIG. 13 indicate that the presence of pathogenic bacteria directly interfere with the adhesive spreading and proliferative activity of the host cell. The results from FIG. 14 show that the early cell activity of the high number bacterial groups was reduced, statistically significant with the low number groups. The results in FIG. 15 show that BMSCs cells of the large-area low-number bacterial group are adhered and spread, and cells of the high-number bacterial group P.g bacterial group are in a fusiform shape, and the cells have good activity.
Fig. 16 shows that FimA is a gene with significant fold of differential expression in P.g bacteria of the cell-bacteria co-culture group found by a differential screening method, suggesting that FimA is highly correlated with cell-bacteria competitive adhesion and is an adhesion key factor.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the titanium-containing test piece of the cross-linked PLGA drug-loaded coating is characterized by comprising the following steps of:
preparation of a catalyst having MAO-TiO 2 Micro-arc oxidation pure titanium test piece of the coating;
preparing PLGA microspheres loaded with SDF-1;
preparing PLGA microspheres loaded with Mino;
dissolving the PLGA microspheres loaded with SDF-1 and the PLGA microspheres loaded with Mino in a gelatin solution, and moving to the MAO-TiO after ultrasonic resuspension treatment 2 And (3) oxidizing the surface of the pure titanium test piece by micro-arc of the coating, vibrating and drying to obtain the titanium-containing test piece of the cross-linked PLGA drug-loaded coating.
2. The method of claim 1, wherein preparing the SDF-1 loaded PLGA microspheres comprises the steps of:
dissolving PLGA in dichloromethane to obtain a first oil phase; dissolving SDF-1 and polyvinyl alcohol together in water to obtain a first aqueous phase; dropwise adding the first oil phase into the first water phase for ultrasonic resuspension to obtain a first oil-water emulsion, magnetically stirring the obtained first oil-water emulsion, centrifuging, and collecting a first precipitate; and washing and freeze-drying the collected first precipitate to obtain the PLGA microsphere loaded with the SDF-1.
3. The method of claim 2, wherein the first oil phase comprises PLGA to methylene chloride in a weight ratio of 1 (4-6);
and/or the first aqueous phase has a concentration of SDF-1 of 25ng/ml to 75ng/ml and a concentration of polyvinyl alcohol of 0.01g/ml;
and/or the weight ratio of the first oil phase to the first water phase is 1 (15-25);
and/or dropwise adding the first oil phase into the first water phase, wherein the ultrasonic resuspension power is 200w-300w, and the time is 5min-10min;
and/or the magnetic stirring speed of the obtained first oil-water emulsion is 900r/min-1100r/min, and the time is 5h-8h; performing magnetic stirring on the obtained first oil-water emulsion, and centrifuging at a rotating speed of 9000r/min-15000r/min for 10min-20min;
and/or, the particle size of the PLGA microsphere loaded with the SDF-1 is 350nm-650nm.
4. The method of claim 1, wherein preparing the minio-loaded PLGA microspheres comprises the steps of:
dissolving PLGA in dichloromethane to obtain a second oil phase; dissolving Mino and polyvinyl alcohol together in water to obtain a second aqueous phase; dropwise adding the second oil phase into the second water phase for ultrasonic resuspension to obtain a second oil-water emulsion, magnetically stirring the obtained second oil-water emulsion, centrifuging, and collecting a second precipitate; and washing and freeze-drying the collected second precipitate to obtain the PLGA microspheres loaded with Mino.
5. The method of claim 4, wherein the weight ratio of PLGA to methylene chloride in the second oil phase is 1 (4-6);
and/or, the concentration of Mino in the second aqueous phase is 5-50 mug/ml, and the concentration of polyvinyl alcohol is 0.01g/ml;
and/or the weight ratio of the second oil phase to the second water phase is 1 (15-25);
and/or dropwise adding the second oil phase into the second water phase, wherein the ultrasonic resuspension power is 200w-300w, and the time is 5min-10min;
and/or carrying out magnetic stirring on the obtained second oil-water emulsion at the speed of 900r/min-1100r/min for 5-8 h; performing magnetic stirring on the obtained second oil-water emulsion, and centrifuging at a rotating speed of 9000r/min-15000r/min for 10min-20min;
and/or the particle size of the PLGA microsphere loaded with Mino is 350nm-650nm.
6. The method of claim 1, wherein the volume ratio of the PLGA microsphere loaded with SDF-1 to the PLGA microsphere loaded with Mino is (20-30): (70-80); the volume ratio of the sum of the volumes of the PLGA microspheres loaded with SDF-1 and the PLGA microspheres loaded with Mino to the gelatin solution is as follows: 1:1; the weight percentage of the gelatin solution is 0.1%;
and/or the time of oscillation is 2 hours, and the frequency of oscillation is 50rpm-100rpm; and/or the temperature of drying is 4 ℃.
7. The method according to claim 1, wherein MAO-TiO is prepared 2 The micro-arc oxidation pure titanium test piece of the coating specifically comprises the following steps: the medical grade pure titanium wafer type test piece is adopted as an anode, a stainless steel sheet is adopted as a cathode, and the MAO-TiO is obtained through electrochemical reaction 2 And (3) coating the micro-arc oxidized pure titanium test piece.
8. The preparation method according to claim 7, wherein the electrolytic tank used for carrying out the electrochemical reaction mainly comprises a three-layer structure, wherein the outermost layer is a cooling tank and a cold water circulation system, the inner layer is an insulating porous plastic inner net and a stirring device, and the middle layer is a stainless steel tank; when in use, cooling water is added into the cooling pool, electrolyte is injected into the stainless steel tank, and meanwhile, the inner air pump and the outer water circulating power supply are started.
9. A titanium-containing test piece of a cross-linked PLGA drug-loaded coating, characterized in that it is prepared by the preparation method of any one of claims 1-8.
10. Use of a titanium-containing test piece according to claim 9 for the preparation of a dental implant.
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