CN110974452A - Application of surface-modified titanium and titanium alloy in dental pulp coronal occlusion - Google Patents
Application of surface-modified titanium and titanium alloy in dental pulp coronal occlusion Download PDFInfo
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- CN110974452A CN110974452A CN201911325267.7A CN201911325267A CN110974452A CN 110974452 A CN110974452 A CN 110974452A CN 201911325267 A CN201911325267 A CN 201911325267A CN 110974452 A CN110974452 A CN 110974452A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
- A61C5/00—Filling or capping teeth
- A61C5/50—Implements for filling root canals; Methods or instruments for medication of tooth nerve channels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
- A61C19/00—Dental auxiliary appliances
- A61C19/06—Implements for therapeutic treatment
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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
- C23C26/00—Coating not provided for in groups C23C2/00 - C23C24/00
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D11/00—Electrolytic coating by surface reaction, i.e. forming conversion layers
- C25D11/02—Anodisation
- C25D11/026—Anodisation with spark discharge
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D11/00—Electrolytic coating by surface reaction, i.e. forming conversion layers
- C25D11/02—Anodisation
- C25D11/26—Anodisation of refractory metals or alloys based thereon
Abstract
The invention discloses an application of surface-modified titanium and titanium alloy in dental pulp coronal occlusion, wherein the surface modification is used for improving the adhesion and differentiation induction performance of the titanium and the titanium alloy on dental pulp cells; the surface-modified titanium and titanium alloy are used as materials for covering pulp tissues after live pulp dissection or pulp regeneration so as to enable pulp to form dentin integration. The surface-modified titanium and titanium alloy can be selected from micro-arc oxidation composite graphene oxide coating-modified titanium and titanium alloy. The invention provides a method for applying surface-modified titanium or titanium alloy as a dental pulp crown sealing material to promote adhesion, proliferation and dentinogenesis differentiation of dental pulp stem cells and form a physiological dentinal structure on the top of exposed dental pulp.
Description
Technical Field
The invention relates to the technical field of dental preparations, in particular to application of surface-modified titanium and titanium alloy in dental pulp coronal occlusion.
Background
At present, live pulp cutting or root canal therapy is mostly adopted to treat tooth body defects caused by decayed teeth, tooth trauma and the like. In the live-cutting operation, after a part of the contaminated pulp tissue is removed, a pulp capping material is used to cover the exposed pulp to prevent or eliminate contamination and control inflammation. The diagnosis and treatment process is complicated, the number of times of patient treatment is required to be large, the cost is high, and the technical sensitivity is high. Commonly used pulp capping materials are MTA and calcium hydroxide, among others. The current use of pulp capping material still has drawbacks: the pH value of MTA and calcium hydroxide rises in the process of solidification, and the MTA and the calcium hydroxide are strong in alkalinity and have cytotoxicity, so that contacted dental pulp tissues are subjected to cell necrosis to generate an inflammatory reaction; the dentin bridge generated by the pulp capping material to induce the dental pulp stem cells is bone-like dentin, lacks the structure of physiological dentinal tubules, has physical and mechanical properties which are difficult to match with normal dentin, is easy to generate cracks or tunnels when bearing occlusal force, and has dentin integration performance to be improved. Endodontics is the complete removal of contaminated pulp tissue. The scholars implant the deciduous tooth pulp stem cells into the root canal to regenerate the pulp with the vascular nerves at the root. Pulp regeneration is a future development trend of dentistry, and is expected to replace root canal therapy, but no special pulp sealing material is developed and applied at present, so that the problem of how to realize coronal sealing of pulp after cutting of new pulp and live pulp becomes an urgent need to be solved.
Titanium has abundant sources, low price and good biocompatibility, and is widely used for dental implantation, denture supports, crown bridges and the like. The hardness of pure titanium is between dentin and enamel, the heat conductivity coefficient is low, and the titanium inlay, the whole crown and the like have the functions of protecting dental pulp and avoiding cold and hot stimulation. Therefore, titanium is expected to be an ideal material for sealing dental pulp, but a surface modification method is required to improve dentin induction performance of a titanium substrate. At present, the surface-modified titanium alloy is not used for crown sealing of dental pulp.
Disclosure of Invention
The invention aims to provide surface-modified titanium or titanium alloy, a preparation method thereof and application thereof in dental pulp coronal occlusion, wherein the surface-modified titanium or titanium alloy can promote the adhesion, proliferation and dentinogenesis differentiation of dental pulp stem cells.
In order to achieve the above objects, the present invention provides the use of surface-modified titanium and titanium alloys for crown closure of tooth pulp, said surface modification for improving the induction of adhesion and differentiation of titanium and titanium alloys to dental pulp cells; the surface-modified titanium and titanium alloy are used as materials for covering pulp tissues after live pulp dissection or pulp regeneration so as to enable pulp to form dentin integration.
Preferably, the method for using the titanium and the titanium alloy comprises the following steps: a particulate cover, inlay or onlay.
Preferably, the method for surface modification comprises:
carrying out surface drug loading treatment by using a self-assembly technology;
a method of physical modification comprising: physical vapor deposition, ion implantation technology and hydrophilic treatment;
a method of chemical modification comprising: strong alkali treatment, hydrogen peroxide treatment, acid etching treatment, sol-gel treatment and chemical vapor deposition;
the laser processing method comprises the steps of etching a specific structure or cladding coating on the surface of titanium and titanium alloy;
a mechanical method, comprising: grinding, polishing and spraying to make the titanium and titanium alloy surface have specific roughness and remove the pollution layer;
an electrochemical process comprising: micro-arc oxidation and anodic oxidation, the method changes the surface appearance of titanium and titanium alloy, and introduces ions with biological activity function.
Preferably, the surface-modified titanium and titanium alloy is modified by a micro-arc oxidation composite graphene oxide coating.
The invention also provides a preparation method of the titanium and the titanium alloy modified by the micro-arc oxidation composite graphene oxide coating, which comprises the following steps:
step (1), carrying out micro-arc oxidation treatment on titanium or titanium alloy to enable the titanium or titanium alloy to have a porous surface;
step (2), soaking titanium or titanium alloy in NaOH solution;
step (3), drying the titanium or the titanium alloy, and soaking the titanium or the titanium alloy in a solution containing a coupling agent;
and (4) soaking the titanium or the titanium alloy in a solution containing graphene oxide to enable the graphene oxide to be loaded in the porous structure on the surface of the titanium or the titanium alloy.
Preferably, the electrolyte adopted in the micro-arc oxidation process is as follows: 0.01 mol/L-1 mol/L calcium acetate monohydrate, 0.01 mol/L-0.1 mol/L sodium dihydrogen phosphate, 200V-500V of electrical parameters and 50Hz of electrical parameters.
Preferably, the concentration of the NaOH solution is 5-10 mol/L.
Preferably, the coupling agent is 3-aminopropyltriethoxysilane.
Preferably, the concentration of the graphene oxide is more than 1 mg/mL.
Preferably, the time for soaking the titanium or the titanium alloy in the solution containing the graphene oxide is 1-24 hours.
Has the advantages that:
(1) based on the excellent physical and chemical properties of titanium or titanium alloy, the invention applies the titanium or titanium alloy with surface modification as dental pulp crown sealing material;
(2) the surface-modified titanium or titanium alloy is used for 'pulp closure', and has important significance for protecting the vitality of the new pulp and promoting the functions of nutrition, repair, feeling and the like;
(3) the titanium or titanium alloy of the micro-arc oxidation composite graphene oxide can be used as an excellent dental pulp sealing material, promotes the adhesion, proliferation and dentinogenesis differentiation of dental pulp stem cells, forms a physiological dentin structure at the top of the exposed dental pulp, and forms 'dentin integration', thereby playing a sealing role and avoiding generating cytotoxicity, dental pulp inflammation and intra-dental absorption.
Drawings
FIG. 1 is a graph of the results of cell adhesion experiment Integrin β 1 immunofluorescence.
FIG. 2 is a graph showing the results of staining extracellular matrix mineralized alizarin red.
FIG. 3 shows PCR markers associated with differentiation of cells into dentin.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The invention provides an application of surface-modified titanium and titanium alloy in dental pulp coronal occlusion, wherein the surface modification is used for improving the adhesion and differentiation induction performance of the titanium and the titanium alloy on dental pulp cells; the titanium and titanium alloy are used for covering pulp tissues after live pulp section surgery or pulp regeneration so as to enable the pulp to form dentin integration.
Existing pulp capping materials are used to cover exposed pulp to prevent or eliminate infection, control inflammation, and are not used for pulp sealing. The invention provides a concept of 'pulp closure', aiming at protecting the vitality of new pulp and avoiding pulp necrosis caused by pulp reinfection. The material selected for sealing the dental pulp should have good biocompatibility, enable the dental pulp to form dentin integration, and have mechanical strength matched with the tooth tissue, so that after the dental pulp is sealed, the tooth can have a chewing function and bear occlusal force. The concept of "dentin integration" proposed in the present invention means that dental pulp stem cells directly contact with the surface of a material, adhere, proliferate, and differentiate into dentin, and new dentin is formed between the material and the new dental pulp, thereby achieving the effect of effectively sealing the dental pulp.
Because titanium has good biocompatibility, and the hardness of pure titanium is between dentin and enamel, the titanium and titanium alloy without surface modification have poor capacity of inducing the adhesion and differentiation of dental pulp stem cells. The surface modification method improves the dentin induction performance of the titanium substrate, and makes titanium and titanium alloy ideal materials for sealing dental pulp.
There are various methods for surface modification of titanium and titanium alloys. Preparing a functional coating on the surface of the titanium alloy by adopting methods such as physics, chemistry, machinery and the like. The method for surface modification comprises the following steps:
(1) carrying out surface drug loading treatment by using a self-assembly technology;
(2) by physical modification methods such as physical vapor deposition, ion implantation techniques, hydrophilic treatment;
(3) by chemical modification methods such as strong alkali treatment, hydrogen peroxide treatment, acid etching treatment, sol-gel treatment, chemical vapor deposition, and the like;
(4) etching a specific structure on the surface of titanium and titanium alloy or cladding other coatings on the surface of titanium and titanium alloy by a laser processing method;
(5) the surface of titanium and titanium alloy is provided with specific roughness by mechanical methods such as grinding, polishing, spraying and the like, and a pollution layer and the like are removed;
(6) the surface appearance of titanium and titanium alloy is changed by electrochemical methods, such as micro-arc oxidation, anodic oxidation and the like, and ions with biological activity function are introduced.
The method for surface modification of titanium and titanium alloys is not limited to the above method, and any method capable of inducing adhesion, proliferation and differentiation of dental pulp stem cells can be used. The endodontic sealing material can be used in the form of a granular covering, an inlay, an onlay, or the like.
In the following examples, the titanium sheet is a medical titanium alloy. The surface-modified titanium and titanium alloy is selected from titanium and titanium alloy modified by a micro-arc oxidation composite graphene oxide coating.
Micro-arc oxidation (MAO), also known as Plasma Electrolytic Oxidation (PEO), micro-plasma oxidation (MPO), and the like, in the method, a special micro-arc oxidation power supply is used for applying high voltage on a workpiece to enable metal on the surface of the workpiece to interact with an electrolyte solution, so that the purposes of strengthening the surface of the workpiece, greatly improving the hardness, and improving the wear resistance, corrosion resistance, pressure resistance, insulation and high-temperature impact resistance of the workpiece are achieved. The basic principle and the characteristics of the technology are as follows: based on the common anodic oxidation, the arc discharge is utilized to enhance and activate the reaction generated on the anode, thereby forming a high-quality strengthened ceramic film on the surface of a workpiece taking metal and alloy thereof as materials. The ceramic film formed is a porous structure.
Example 1
And (3) preparing the micro-arc oxidation composite graphene oxide coating on the surface of the titanium sheet.
Example 1 the procedure was as follows:
1. the titanium sheet with the specification of 20 × 2mm is sequentially polished by 320, 800 and 1200-mesh sand paper, and is placed in an ethanol solution and a propanol solution for ultrasonic cleaning for 1 hour respectively.
2. Preparing electrolyte containing 0.1mol/L calcium acetate monohydrate and 0.03mol/L sodium dihydrogen phosphate, immersing the titanium alloy in the electrolyte, and selecting proper electrical parameters, namely 400v, 50Hz and 10 min.
3. Washing the micro-arc oxidized titanium sheet with deionized water, drying with nitrogen, soaking in 10mol/L NaOH solution at 60 deg.C for 24 h. The effect of NaOH is to hydroxylate the metal surface for chemical reaction with the next solution, increasing hydrophilicity.
4. The titanium plate was washed until the pH was 7. And (3) drying by nitrogen, soaking the titanium sheet in a 5% 3-aminopropyltriethoxysilane solution for 2 hours, and washing by deionized water.
5. The sample was immersed in 1mg/mL GO solution (graphene oxide solution) for 24h and dried in vacuo.
Example 2
Influence of micro-arc oxidation composite graphene oxide on in-vitro bioactivity of human dental pulp stem cells (hDPSCs)
Step one, isolated culture of human dental pulp stem cells
(1) Collecting the teeth which need to be pulled out for clinical reasons of impacted and orthodontic treatment, and flushing the teeth for 3 times by using sterile PBS solution; cutting the neck of the tooth with a dental split drill to expose the pulp chamber;
(2) picking out the dental pulp tissue by using a sterile broach, and placing the dental pulp tissue in a culture dish;
(3) cutting the pulp tissue to about 1 x 1mm size with sterile ophthalmic scissors; dipping vaseline at four corners of a cover glass, and covering the cover glass on the cut pulp tissues to prevent the tissue blocks from floating;
(4) finally, MEM culture medium containing 20% fetal bovine serum and 1% double antibody was added to the culture dish, and 5% CO was added at 37 ℃2After the cells are cultured in the incubator for 7 days and observed to creep out of the tissue block to 80% and be fused, trypsinized and subcultured. The dental pulp cells crawl out of the tissue mass in the form of long spindle, resembling fibroblasts.
Step two, the effect of the micro-arc oxidation composite graphene oxide titanium alloy surface coating on human dental pulp stem cells in vitro
(1) The cell adhesion capability detection method comprises the steps of placing a sample in a 6-well plate, inoculating 2mL of human dental pulp stem cell suspension, after 4 hours, sucking and discarding culture solution, washing 3 times with PBS, sucking and discarding, fixing for 30 minutes by using 4 wt% paraformaldehyde solution, allowing for 20 minutes by using 0.1% TritonX-100 solution, washing 3 times with PBS, treating with BSA blocking solution for 1 hour, discarding the solution, adding integrin β 1 antibody, standing overnight at 4 ℃, washing 3 times with PBS, adding green fluorescent secondary antibody, treating for 1 hour at room temperature, dyeing cell skeletons for 2 hours at 37 ℃ by using TRITC-Phalloidin (rhodamine-labeled Phalloidin), dyeing cell nucleuses for 5 minutes by using DAPI solution, observing under a fluorescence microscope, wherein TRITC-Phalloidin is a reagent for dyeing the cell skeletons and can present the cell morphology, and determining that the dyeing content is cells, wherein integrin β 1 is an important index for mediating early cell adhesion.
The experimental result is shown in figure 1, Ti represents a titanium sheet, MAO represents a micro-arc oxidized titanium sheet, 1.0GO represents a titanium sheet with a micro-arc oxidized composite graphene oxide coating on the surface, the concentration of GO solution is 1.0mg/mL when the titanium sheet is prepared, the representation method of other figures is the same as that in figure 1, Actin refers to a cell skeleton structure, Merge represents a fused picture, the fused picture can show the intracellular expression level of integrin, and as can be seen from figure 1, the micro-arc oxidized composite graphene oxide titanium alloy surface coating promotes the high expression of integrin β 1 and promotes the early adhesion of cells.
(2) Cytotoxicity assays
Placing the sample in a 6-well plate, inoculating 2ml of human dental pulp stem cell suspension in the well, culturing for 24h, staining with a cell live-dead kit, staining dead cells in red, staining live cells in green, observing with a fluorescence microscope, and calculating the ratio of viable cells by using ImageJ software.
The experimental result shows that the micro-arc oxidation composite graphene oxide titanium alloy surface coating has no cytotoxicity, the living cell ratio (Viable cells) is increased compared with the control group (the Ti group and the MAO group), and the cell proliferation is promoted.
(3) Extracellular matrix mineralization detection
Placing the sample in a 6-well plate, inoculating 2ml of human dental pulp stem cell suspension in the well, culturing in MEM culture solution for 14 days, changing the culture solution every 5 days, staining with alizarin red kit, and taking pictures under a fluorescent microscope. In alizarin red staining, red represents mineralized nodules, and the mineralized nodules can be judged according to the area size and the fluorescence intensity of a red area.
The experimental result is shown in figure 2, and the micro-arc oxidation composite graphene oxide coating promotes the formation of extracellular matrix mineralized nodules.
(4) Real-time PCR detection of expression of related genes
Placing the sample in a 6-well plate, inoculating 2mL of human dental pulp stem cell suspension in the well, culturing for 7 days, collecting total RNA of each group of samples by using Trizol reagent respectively, and detecting the expression of related genes. And measuring the concentration and purity of the extracted RNA under 260nm and 280nm of an ultraviolet spectrophotometer, and synthesizing cDNA according to the specification of the reverse transcription kit. And then detecting the expression of mineralization related genes ALP, OCN, DSPP and DMP-1. The housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control.
The experimental result is shown in figure 3, and the micro-arc oxidation composite graphene oxide coating up-regulates the expression of related genes such as ALP, OCN, DSPP, DMP-1 and the like. In FIG. 3, Relative mRNAexpression indicates the Relative expression level of mRNA.
In conclusion, the titanium alloy with the modified surface is beneficial to improving the dentin induction performance of the titanium alloy; the surface-modified titanium or titanium alloy can be used as an excellent dental pulp sealing material to promote the adhesion, proliferation and dentinogenesis of dental pulp stem cells.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (10)
1. The application of the surface-modified titanium and titanium alloy for sealing the pulp crown is characterized in that the surface modification is used for improving the adhesion and differentiation induction performance of the titanium and titanium alloy to pulp cells; the surface-modified titanium and titanium alloy are used as materials for covering pulp tissues after live pulp dissection or pulp regeneration so as to enable pulp to form dentin integration.
2. The use of the surface-modified titanium and titanium alloy of claim 1 for coronal obturation of a tooth marrow comprising the steps of: a particulate cover, inlay or onlay.
3. The use of the surface-modified titanium and titanium alloys of claim 1 for coronal obturation of a tooth marrow comprising the steps of:
carrying out surface drug loading treatment by using a self-assembly technology;
a method of physical modification comprising: physical vapor deposition, ion implantation technology and hydrophilic treatment;
a method of chemical modification comprising: strong alkali treatment, hydrogen peroxide treatment, acid etching treatment, sol-gel treatment and chemical vapor deposition;
the laser processing method comprises the steps of etching a specific structure or cladding coating on the surface of titanium and titanium alloy;
a mechanical method, comprising: grinding, polishing and spraying to make the titanium and titanium alloy surface have specific roughness and remove the pollution layer;
an electrochemical process comprising: micro-arc oxidation and anodic oxidation, the method changes the surface appearance of titanium and titanium alloy, and introduces ions with biological activity function.
4. The use of the surface-modified titanium and titanium alloy of claim 1 for coronal obturation of a dental pulp, wherein the surface-modified titanium and titanium alloy is micro-arc oxidation composite graphene oxide coating-modified titanium and titanium alloy.
5. A preparation method of micro-arc oxidation composite graphene oxide coating modified titanium and titanium alloy is characterized by comprising the following steps:
step (1), carrying out micro-arc oxidation treatment on titanium or titanium alloy to enable the titanium or titanium alloy to have a porous surface;
step (2), soaking titanium or titanium alloy in NaOH solution;
step (3), drying the titanium or the titanium alloy, and soaking the titanium or the titanium alloy in a solution containing a coupling agent;
and (4) soaking the titanium or the titanium alloy in a solution containing graphene oxide to enable the graphene oxide to be loaded in the porous structure on the surface of the titanium or the titanium alloy.
6. The method for preparing the micro-arc oxidation composite graphene oxide coating modified titanium and the titanium alloy according to claim 5, wherein the electrolyte adopted in the micro-arc oxidation process is as follows: 0.01 mol/L-1 mol/L calcium acetate monohydrate, 0.01 mol/L-0.1 mol/L sodium dihydrogen phosphate, 200V-500V of electrical parameters and 50Hz of electrical parameters.
7. The method for preparing the titanium and the titanium alloy modified by the micro-arc oxidation composite graphene oxide coating according to claim 5, wherein the concentration of the NaOH solution is 5-10 mol/L.
8. Use of the surface-modified titanium and titanium alloys for obturation of the coronal plane of a tooth marrow according to claim 5, characterized in that said coupling agent is 3-aminopropyltriethoxysilane.
9. The method for preparing titanium and titanium alloy modified by micro-arc oxidation composite graphene oxide coating according to claim 5, wherein the concentration of the graphene oxide is more than 1 mg/mL.
10. The method for preparing titanium and titanium alloy modified by micro-arc oxidation composite graphene oxide coating according to claim 5, wherein the time for soaking titanium or titanium alloy in the solution containing graphene oxide is 1-24 hours.
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