CN114366853A

CN114366853A - High bone-inducing active dental implant coating and preparation method thereof

Info

Publication number: CN114366853A
Application number: CN202210068086.6A
Authority: CN
Inventors: 刘昌胜; 何宏燕; 李春旺; 杨梓鹤
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-19
Anticipated expiration: 2042-01-20
Also published as: CN114366853B

Abstract

The invention provides a dental implant coating with high bone inducing activity and a preparation method thereof. Specifically, the invention provides a preparation method of a coating with high bone inducing activity, which comprises the following steps: firstly, functionalizing the surface of a substrate material by utilizing the self-polymerization reaction of dopamine hydrochloride; then coating an intermediate obtained by covalent bonding of a dihydrazide compound and chondroitin sulfate; and finally coating bone growth inducing factors to form the coating. The bone inducing growth factor of the present invention is fixed on the surface of the coating in a suspended manner in a specific direction to maintain the biological activity of the molecular structure thereof, so that the release rate is obviously reduced, and the dental implant coated with the coating has more excellent bone inducing activity compared with the traditional dental implant.

Description

High bone-inducing active dental implant coating and preparation method thereof

Technical Field

The invention relates to the field of biological manufacturing, in particular to a dental implant coating with high bone-inducing activity and a preparation method thereof.

Background

The dental implant usually adopts metallic titanium as a base material, which is the most widely applied and most approved dental implant in the market at present. However, the titanium implant is significantly different from natural dental tissue and its non-physiological surface is in direct contact with the surrounding alveolar bone at the initial stage of implantation. Due to the lack of connective fibrous connective tissue and vascular access for nutrient supply, a strong bond with the alveolar bone is not easily formed. Moreover, the insufficient surface bioactivity of the dental implant, together with the slow expression and insufficient expression of growth factors related to osteogenesis at the implanted site, often results in a slow healing rate of the titanium implant after implantation, and the integration effect and healing period in clinical application are not very ideal.

In order to improve the bioactivity of the surface of the titanium implant and further enhance the bonding with the surrounding bone tissue, researchers have conducted a great deal of research in recent years from the aspects of microstructure regulation of the surface of the material, loading of bioactive molecules, and the like. Common surface modification methods include: sand blasting, plasma spraying, mechanical processing, alkaline heat treatment, electrochemical deposition, sol-gel methods, introduction of bioactive factors or active DNA plasmids, and the like.

However, the conventional physical and chemical methods have insufficient surface biological activity, so that the introduction of bioactive factors or active DNA plasmids is generally required for improvement. The introduced method mainly comprises macromolecule adsorption, self-assembly technology and bionic deposition. The direct adsorption method has limited load capacity, short retention time and easy occurrence of burst release phenomenon; the layer-by-layer self-assembly technology is characterized in that polyelectrolyte ions with opposite charges are alternately adsorbed on the surface of a charged substrate through electrostatic action to form a multi-molecule bioactive coating, and the slow release, stability and bioactivity of a protein factor are controllable by regulating and controlling the number of assembly layers and the crosslinking degree. However, the preparation period of layer-by-layer self-assembly is long, the mechanical property is insufficient, and the composite material with other materials or the carrier immobilization is required.

Therefore, it is necessary to develop an active coating of dental implant, which has high bone inducing activity, can effectively avoid the loss of bone inducing activity of active factors, and has simple preparation process and low cost.

Disclosure of Invention

The invention aims to provide the dental implant active coating which has high bone inducing activity, can effectively avoid the bone inducing activity loss of active factors, has simple preparation process and low cost and the preparation method thereof.

In a first aspect of the invention, there is provided a method of preparing a coating, the method comprising the steps of:

(a) providing a base material;

(b) mixing a base material with a first solution containing a biological ligand to perform self-polymerization reaction, so as to obtain a first coating containing polydopamine on the surface of the base material, wherein the biological ligand contains dopamine hydrochloride;

(c) mixing the first coating layer containing polydopamine with a second solution containing a linking molecule to react, thereby coating the linking molecule on the first coating layer to form a second coating layer, wherein the linking molecule comprises a product of covalent bonding of a dihydrazide compound and chondroitin sulfate;

(d) and mixing the second coating with a third solution containing bone growth inducing factors, and reacting to coat the growth inducing factors on the second coating to form the coating, wherein the bone growth inducing factors are bone morphogenetic proteins, preferably BMP-2.

In another preferred embodiment, the preparation process is carried out in a sterile clean bench.

In another preferred embodiment, in step (b), the biological ligand further optionally comprises a polyphenol compound.

In another preferred embodiment, the polyphenol compound is selected from the group consisting of: catechol, flavanone, anthocyanidin, catechol, pyrogallol, or a combination thereof.

In another preferred embodiment, in step (b), the self-polymerization reaction is carried out in the presence of a catalyst.

In another preferred example, the step (b) includes: the substrate material is first mixed with the catalyst solution and then self-polymerized in the first solution containing biological ligand.

In another preferred embodiment, in step (b), the catalyst is a basic catalyst selected from the group consisting of: sodium hydroxide, potassium carbonate, sodium carbonate and Tris-HCl.

In another preferred embodiment, in step (b), the pH of the catalyst solution is 8.5 to 9.0.

In another preferred example, in the step (b), the mass ratio of the substrate material to the biological ligand is 0.5-2:1, preferably 1-1.5: 1.

In another preferred example, in the step (b), the concentration of the biological ligand in the first solution is 1-10 mg/mL, preferably 3-5 mg/mL.

In another preferred example, in the step (b), the volume ratio of the first solution to the catalyst solution is 0.6-1.5: 0.6-1.5, preferably 0.8-1.2: 0.8-1.2, and more preferably 1: 1.

In another preferred embodiment, in step (b), the solvent of the catalyst solution and the first solution is water.

In another preferred embodiment, in step (b), the self-polymerization reaction is carried out at 20 to 45 ℃.

In another preferred embodiment, in step (b), the reaction time t1 of the self-polymerization reaction is 1-24h, preferably 3-10h, more preferably 6 h.

In another preferred embodiment, the step (b) further comprises a post-treatment process of washing with deionized water and drying under a nitrogen atmosphere.

In another preferred example, in step (c), the mass ratio of the material coating the first coating layer to the linker molecules is 0.5-2:1, preferably 1-2: 1.

In another preferred embodiment, in step (c), the dihydrazide compound is selected from the group consisting of: adipic acid dihydrazide, pimelic acid dihydrazide, suberic acid dihydrazide, sebacic acid dihydrazide, or a combination thereof.

In another preferred embodiment, in step (c), the linker molecule is the product of covalent bonding of adipic acid dihydrazide and chondroitin sulfate.

In another preferred embodiment, in step (c), the linker molecule is present in the second solution at a concentration of 0.5-24mg/mL, preferably 5-15mg/mL, more preferably 8-10 mg/mL.

In another preferred example, in step (c), the mass ratio of the material coating the first coating layer to the linker molecules is 1-3:1, preferably 1.4-2: 1.

In another preferred embodiment, in step (c), the reaction is carried out under mixing conditions, wherein the mixing is carried out at 50-100rpm, 20-45 ℃ (preferably 60rpm, 37 ℃).

In another preferred embodiment, in step (c), the mixing is selected from the group consisting of: stirring by hand, stirring mechanically, and oscillating in a constant temperature oscillating box.

In another preferred example, in step (c), the solvent of the second solution is water or aqueous PBS solution, preferably aqueous PBS solution.

In another preferred embodiment, in the step (c), the reaction time t2 is 8-15 h, preferably 12 h.

In another preferred example, the step (c) further comprises a post-treatment process of washing and vacuum drying.

In another preferred embodiment, during the post-treatment, the washing is performed by gently rinsing with PBS 2 times.

In another preferred embodiment, in the post-treatment process, the vacuum drying refers to drying in a vacuum drying oven at 37 ℃, preferably drying for 12-48h, for example 24 h.

In another preferred embodiment, the linker molecule is prepared by the following steps:

(i) activated chondroitin sulfate: mixing chondroitin sulfate with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for reaction to obtain activated chondroitin sulfate;

(ii) covalent binding of dihydrazide compounds to chondroitin sulfate: mixing the activated chondroitin sulfate of the step (a) with a dihydrazide compound, and reacting to obtain the connecting molecule.

In another preferred embodiment, the molar ratio of the chondroitin sulfate, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the dihydrazide compound is 1: 2-3: 2-3: 4-7, preferably 1: 2.5: 2.5: 5.

in another preferred example, the step (i) includes: mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide with the chondroitin sulfate solution to obtain activated chondroitin sulfate.

In another preferred embodiment, the concentration of the chondroitin sulfate solution is 5-10 mg/mL, preferably 7.5 mg/mL.

In another preferred example, in step (i), the solvent of the chondroitin sulfate solution is water or aqueous PBS solution, preferably aqueous PBS solution.

In another preferred embodiment, in step (i), the pH of the aqueous PBS solution is 5.0-6.0.

In another preferred embodiment, in step (i), the reaction time t4 for the activation is 10 minutes to 2 hours, preferably 30 minutes to 1 hour.

In another preferred embodiment, in step (ii), a dihydrazide compound is added to the reaction mixture containing the activated chondroitin sulfate obtained in step (i) to perform covalent bonding.

In another preferred embodiment, in step (ii), the reaction time t5 of the reaction is 6 to 48h, preferably 12 to 24 h.

In another preferred embodiment, in step (ii), the reaction is carried out at 20-45 ℃.

In another preferred example, the method further comprises the step of post-treating (iii).

In another preferred embodiment, the post-treatment (iii) comprises subjecting the reaction mixture containing the linker molecule to dialysis, freezing and lyophilization to obtain the linker molecule.

In another preferred embodiment, in step (iii), the dialysis has a weight average molecular weight cut-off of 5-10kDa (preferably 7 kDa).

In another preferred embodiment, in step (iii), the dialysis time is 3 days.

In another preferred embodiment, in step (d), the bone morphogenic protein is selected from the group consisting of: BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, or a combination thereof, preferably BMP-2.

In another preferred embodiment, in step (d), the bone morphogenic protein is recombinant BMP-2.

In another preferred example, the step (d) includes: and dropwise adding a third solution containing bone inducing growth factors onto the second coating to obtain the coating.

In another preferred example, in step (d), the mass ratio of the material coating the second coating layer to the osteoinductive growth factor is 100-.

In another preferred example, in step (d), the concentration of said bone inducing growth factor in the third solution is 200. mu.g/ml.

In another preferred example, in the step (d), the dropping rate of the dropwise addition is 2. mu.L/s.

In another preferred embodiment, the step (d) comprises a post-treatment step of freezing and drying.

In another preferred embodiment, in step (d), the reaction is carried out at 20-45 ℃.

In another preferred example, the step (d) further includes: freezing the obtained material in a refrigerator at-20 deg.C overnight, and freeze-drying in a sterile freeze-dryer to obtain the dental implant coated with the coating.

In another preferred embodiment, the step (d) may be performed before the dental implant is used.

In a second aspect of the invention, there is provided a coating prepared using the method of the first aspect of the invention.

In another preferred embodiment, the coating is a sterile, active coating.

In another preferred embodiment, the bone inducing growth factor is suspended on the surface of the base material.

In another preferred example, the bone growth inducing factor is fixed on the surface of the base material in a specific direction.

In a third aspect of the present invention, there is provided a dental implant comprising: a dental implant base material and a coating as described in the second aspect of the invention.

In another preferred embodiment, the dental implant base material comprises modified titanium or an alloy thereof.

In another preferred embodiment, the dental implant base material is a dental implant subjected to sand blasting and acid etching (SLA).

In another preferred example, the dental implant has one or more of the following features:

(1) the release rate of the osteoinductive growth factor is lower than 80%, preferably lower than 60%, more preferably lower than 50%;

(2) the bone density of the newly formed bone is more than 1400mg/cm³(week 8), preferably greater than 1600mg/cm³；

(3) The new bone formation has a bone area/bone volume of more than 60% (week 8), preferably more than 70%.

In a fourth aspect of the invention, there is provided a medical material comprising a coating according to the second aspect of the invention or a dental implant according to the third aspect of the invention.

In another preferred embodiment, the medical material is for use in mammals.

In another preferred embodiment, the mammal is a human.

In a fifth aspect of the present invention, there is provided a kit comprising:

(a) a first container, and a second coating of material located within the container;

(b) a second container, and an osteoinductive growth factor located within the container.

In another preferred embodiment, the kit comprises:

(b) a second container, and an osteoinductive growth factor in the container, wherein the osteoinductive growth factor can be in the form of powder or dispersed in the solution.

In a sixth aspect of the invention, there is provided the use of a coating according to the third aspect of the invention or a dental implant according to the second aspect of the invention for the preparation of a medical device for hard tissue repair.

In a seventh aspect of the present invention, there is provided a method of implanting a tooth, the method comprising implanting the dental implant of the second aspect of the present invention into a site of an edentulous part of a human body.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a flow chart for the preparation of a B/C-pSLA dental implant;

FIG. 2 shows scanning electron micrographs of SLA dental implants before (a, b) and after (c, d) coating;

FIG. 3 shows surface element spectroscopy of SLA, PDA-SLA, B/C-pSLA dental implants;

FIG. 4 shows the in vitro release rate of BMP-2 after SLA, PDA-CSA-SLA dental implants were surface loaded with BMP-2;

FIG. 5 shows bone density (BMD) of new bone formation of SLA, B/C-pSLA dental implants in beagle dogs;

FIG. 6 shows the bone area/bone volume (BV/TV) of the newly formed bone of SLA, B/C-pSLA dental implants in beagle dogs.

Detailed Description

The inventor of the invention has conducted extensive and intensive research and unexpectedly developed a dental implant coating which has high bone inducing activity, can effectively avoid the loss of the bone inducing activity of active factors, and has simple preparation process and low cost for the first time. Specifically, the surface functionalized polymer and the long-chain molecules are coated on the implant substrate in advance, and then a layer of bone inducing growth factor is coated, so that the growth factor is suspended and fixed on the substrate surface, the loading rate is high, and the activity of the growth factor is well maintained. Based on the method, the obtained dental implant coated by the coating has high osteoinductivity, high biological responsiveness, and proper biodegradability and mechanical property. On this basis, the inventors have completed the present invention.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the terms "EDC", "1- (3-dimethylaminopropyl) -3-ethylcarbodiimide" are used interchangeably.

As used herein, the terms "NHS", "N-hydroxysuccinimide" are used interchangeably.

As used herein, the terms "SLA", "sand blasting and acid etching treatment" are used interchangeably and refer to a common surface treatment method for implants, the treated modified titanium and titanium alloy surfaces have higher surface energy, and macromolecules are more easily adsorbed, bent and deformed, so that attachment points are increased.

As used herein, the terms "pSLA", "PDA-SLA" are used interchangeably and refer to a poly dopamine coated SLA dental implant that functionalizes the surface of the SLA dental implant.

As used herein, the terms "C-pSLA", "PDA-CSA-SLA" are used interchangeably and refer to SLA dental implants coated with chondroitin sulfate-adipic dihydrazide-polydopamine.

As used herein, the term "B/C-pSLA" refers to C-pSLA dental implants adsorbed with BMP-2, which have high osteoinductive activity.

As used herein, the terms "bone density", "BMD", used interchangeably, refer to bone mineral density, an important indicator of bone strength, expressed in grams per cubic centimeter.

As used herein, the terms "bone area/bone volume", "BV/TV" are used interchangeably to refer to the bone volume fraction, representing the ratio of bone tissue volume to tissue volume, which directly reflects changes in bone mass.

First coating

In the invention, the substrate material is coated with functional polymer which can be effectively adsorbed with the substrate material in advance, so that the surface of the substrate material is functionalized to obtain the first coating.

The functional polymer has one or more of the following characteristics:

(i) has a polyphenol structure;

(ii) the molecular weight is 500Da-10 kDa;

(iii) the coating rate is in the range of 30% to 100%, preferably 50% to 100%, more preferably 70% to 100%.

In another preferred embodiment, the functional polymer comprises polydopamine.

In another preferred embodiment, the structural formula of the polydopamine is shown as the following formula I:

wherein n is 2 to 10000, preferably 2 to 1000.

In another preferred embodiment, the polydopamine is a three-dimensional cross-linked high molecular polymer.

In another preferred embodiment, the substrate material is connected to the first coating layer by electrostatic interaction.

As used herein, the terms "polydopamine", "PDA" are used interchangeably.

Second coating layer

In the present invention, the linker molecules are coated on the first coating layer to form a second coating layer.

The connecting molecule is a product formed by covalently bonding a dihydrazide compound and chondroitin sulfate, and is preferably a product formed by covalently bonding adipic acid dihydrazide and chondroitin sulfate (CS-ADH).

In another preferred embodiment, the dihydrazide compound is selected from the group consisting of: adipic acid dihydrazide, pimelic acid dihydrazide, suberic acid dihydrazide, sebacic acid dihydrazide, or a combination thereof.

As used herein, the terms "ADH", "adipic dihydrazide" are used interchangeably and are of the structure shown in formula II. ADH is often used as a cross-linking agent. It has a long-chain structure.

In the invention, chondroitin sulfate contains sulfonate and has strong electrostatic interaction with bone-inducing growth factors, so that the bone-inducing growth factors can be fixed on the surface of the material in a suspended manner like ship anchors.

In another preferred embodiment, the chondroitin sulfate has one or more of the following characteristics:

(i)-SO₃the grafting rate of H is 5-50%;

(ii) the grafting rate of-COOH is between 5% and 50%;

(iii) the molecular weight is in the range of 7kDa to 100 kDa.

In another preferred embodiment, the chondroitin sulfate further comprises a pharmaceutically acceptable salt of chondroitin sulfate.

As used herein, the terms "chondroitin sulfate", "chondroitin", "CS" are used interchangeably and are structurally represented by formula III. The chondroitin sulfate contains sulfonic group, has stronger electrostatic acting force with the bone-inducing growth factor, and can be used for fixing the bone-inducing growth factor molecules. Chondroitin sulfate has effects of relieving pain and promoting cartilage regeneration.

As used herein, the terms "chondroitin sulfate-adipic acid dihydrazide", "CS-ADH", used interchangeably, refer to the linking macromolecule used to prepare the coating of the present invention resulting from the covalent attachment of CS to ADH.

Coatings of the invention

In the present invention, the bone inducing growth factor can be suspended and fixed on the dental implant, thereby achieving excellent bone inducing effect and lower release rate of the active ingredient.

The bone induction growth factor is preferably an osteoblast growth factor.

The bone induction growth factor is bone morphogenetic protein, especially bone morphogenetic protein 2.

The bone-inducing growth factor is connected with the chondroitin sulfate through electrostatic interaction.

As used herein, the terms "BMP-2", "bone morphogenetic protein 2" and "BMP-2" are used interchangeably. Bone morphogenetic proteins stimulate DNA synthesis and cellular replication, thereby promoting the directed differentiation of mesenchymal cells into osteoblasts. It is also a major factor inducing bone and cartilage formation in vivo, and is expressed in limb growth, endochondral ossification, early stage of fracture, cartilage repair, and plays an important role in embryonic development and regenerative repair of bones. It has been shown that BMP can exert its osteogenesis inducing activity as much as possible only when it is loaded on a carrier with a certain structure and good affinity.

As used herein, the terms "rhBMP-2", "recombinant BMP-2", "recombinant bone morphogenetic protein 2" are used interchangeably. The bone morphogenetic protein used in the present invention is rhBMP-2.

Method for producing coatings

The invention provides a method for preparing the coating, which comprises the following steps:

the preparation method comprises the following steps:

(a) placing the SLA dental implant into a 100mL beaker, adding 25mL of Tris-HCl solution into the beaker, and stirring by using a small rotor to fully mix the SLA dental implant and the solution; then adding 25mL of dopamine hydrochloride solution into a beaker, and reacting for 1-24 h; then taking out the dental implant after dopamine polymerization reaction and washing off redundant polydopamine on the surface by deionized water; drying in nitrogen atmosphere to obtain SLA dental implant (pSLA) modified by PDA;

(b) putting the PDA modified SLA dental implant obtained in the step (a) into a 50mL centrifuge tube, and then adding 10mL of CS-ADH solution with the concentration of 0.5-24 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain a CS-PDA modified SLA dental implant (C-pSLA);

(c) slowly and uniformly dripping 1-20 mu g of rhBMP-2 solution on the surface of the SLA dental implant modified by the CS-PDA obtained in the step (b); the resulting mixture was frozen in a refrigerator at-20 ℃ overnight and freeze-dried in a sterile freeze-dryer to obtain SLA dental implants (B/C-pSLA) comprising the coating of the present invention.

The preparation process of the coating is carried out on a sterile super clean bench, and the obtained coating is a sterile active coating.

The pH of the Tris-HCl solution in the step (a) is 8.5-9.0.

The concentration of the dopamine hydrochloride solution in the step (a) is 4-5 mg/mL.

Wherein the preparation method of the CS-ADH solution in the step (b) comprises the following steps:

(b1) adding 300mg of CS into 40mL of PBS solution, then adding 383.4mg of EDC and 230mg of NHS, mixing and stirring for 30min to activate carboxyl on CS chain;

(b2) adding 700mg of ADH and continuously stirring at normal temperature for overnight;

(b3) dialyzing the mixture obtained in the step (2) by using a dialysis bag with the weight-average molecular weight cutoff of 7kDa for 3 days, and changing water in a beaker every 8 hours;

(b4) the dialyzed residue was frozen overnight, vacuum freeze-dried for 3 days, and redissolved in PBS to give a CS-ADH solution.

The pH of the PBS solution in the step (b1) is 5.0-6.0.

The molar ratio of CS, EDC, NHS and ADH is 1: 2.5: 2.5: 5 or 1:2:2.5: 4.

In the step (c), the freeze-drying time is 24 hours.

The freeze-dried dental implant containing the coating is sealed and then stored in a refrigerator at the temperature of-20 ℃.

The preparation method is shown in figure 1.

The preparation method comprises the steps of firstly adopting a self-polymerization reaction of a polydopamine precursor to be adsorbed on the surface of the dental implant, then taking a connecting molecule ADH as a cross-linking agent, connecting chondroitin sulfate to the surface of the dental implant through the reaction of polydopamine and CS-ADH, and then utilizing the special non-covalent interaction existing between chondroitin sulfate molecules and BMP-2 to ensure that the BMP-2 molecules are fixed on the surface of a coating in a suspended manner in a specific direction, and simultaneously obtaining a lower BMP-2 release rate and maintaining the bioactivity of the molecular structure of the BMP-2.

The main advantages of the invention include:

1) the invention designs and constructs a coating with osteoinductivity, high biological responsiveness, suitable biodegradability and mechanical property, effectively combines each key factor influencing rapid repair, and explores the topological structure, the coating property and the active factor on the surface of the dental implant to cooperatively promote the growth of osteoblasts from the micro-nano scale/material property/protein factor level;

2) the coating integrates the three components, not only endows the substrate material with proper immune regulation function, promotes the integration of a graft-bone interface through immune regulation and improves the early osseointegration capability, but also fixes the growth factors on the surface of the coating in a specific direction so as to more efficiently play a role

3) The invention utilizes the characteristic that other components except the cell growth factor can be hermetically stored at normal temperature, so that the cell growth factor can be loaded on the surface of the dental implant just before implantation, the activity loss of the growth factor in the storage process is avoided, and the cost of the implant is reduced.

The invention will be further illustrated with reference to the following specific examples. 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 experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 preparation of B/C-pSLA

EXAMPLE 1.1 preparation of CS-ADH solution

Then 300mg of CS is added into 40mL of PBS solution, 383.4mg of EDC and 230mg of NHS are added, and the mixture is mixed and stirred for 30min to activate the carboxyl on the CS chain; then 700mg of ADH is added and the mixture is stirred at normal temperature for a night; dialyzing the reaction mixed solution by using a dialysis bag with the weight-average molecular weight cutoff of 7kDa for 3 days, and changing water in a beaker every 8 hours; finally freezing the dialysis raffinate overnight, and carrying out vacuum freeze drying for 3 days to obtain CS-ADH.

EXAMPLE 1.2 preparation of B/C-pSLA

(1) A commercially available dental implant of vicarikang, which has been subjected to sand blasting and acid etching, was used. Putting 130mg of SLA dental implant into a 100mL beaker, adding 25mL of prepared Tris-HCl solution (with the pH of 8.5-9.0 and the preferred pH of 8.5) into the beaker, and stirring by using a small rotor to fully mix the SLA dental implant and the solution; then 25mL of dopamine hydrochloride solution (4-5mg/mL) is added into the beaker, and the reaction time is 1-24h (6 h is preferred); then taking out the SLA dental implant after dopamine polymerization reaction, and washing off redundant polydopamine on the surface by using deionized water; drying in nitrogen atmosphere to obtain SLA dental implant (pSLA) modified by PDA;

(2) putting the PDA modified SLA dental implant obtained in the step (1) into a 50mL centrifuge tube, and then adding 10mL CS-ADH solution with the concentration of 9 mg/mL; incubating the centrifuge tube in a constant temperature shaking box at 60rpm and 37 ℃ overnight; gently washing the incubated product with PBS for 2 times, and drying in a vacuum drying oven at 37 ℃ for 24h to obtain a CS-PDA modified SLA dental implant (C-pSLA);

(3) slowly and uniformly dripping 18 mu L of rhBMP-2 solution (the concentration of the solution is 200 mu g/mL) on the surface of the SLA dental implant modified by the CS-PDA; freezing the obtained mixture in a refrigerator at-20 deg.C overnight, and lyophilizing in a sterile lyophilizer to obtain B/C-pSLA.

A schematic flow chart of the preparation of the B/C-pSLA dental implant is shown in FIG. 1.

Scanning electron micrographs of SLA dental implants before (a, b) and after (c, d) coating are shown in FIG. 2. The results show that the coating has been successfully applied to the surface of the SLA dental implant and that the porous structure of the coated surface is not destroyed.

Surface elemental energy profiling of SLA, pSLA and B/C-pSLA dental implants is shown in FIG. 3. The results showed that the Ti element peak on the surface of pSLA and B/C-pSLA disappeared, indicating that the coating had been successfully applied to the surface of the SLA implant.

Example 2.

18 mu L of rhBMP-2 protein solution (200 mu g/mL) is slowly and uniformly dripped on the SLA surface, the pSLA surface and the C-pSLA surface respectively, the obtained mixture is put into a refrigerator at the temperature of-20 ℃ for freezing overnight, and freeze-dried in a sterile freeze dryer to obtain three samples of surface immobilized protein, which are respectively named as B-SLA, B-pSLA and B/C-pSLA, and three parallel samples are prepared for each group.

The implant loaded with the rhBMP-2 is placed into a centrifuge tube containing 1mL of PBS solution, the centrifuge tube is placed into a constant temperature oscillation box at 37 ℃ after being sealed by a sealing film, and the implant is taken out according to time points of 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h and 144h and is placed into new 1mL of PBS solution.

And (3) selecting an ELISA kit of human BMP-2 of Xinbo Sheng company for the sample solution to carry out protein content determination. The protein concentration detection steps are as follows:

(1) first, a standard curve is drawn, and rhBMP-2 standard solutions (2000pg/mL, 100pg/mL, 500pg/mL, 250pg/mL, 125pg/mL) with different concentrations are prepared. Drawing a linear equation of two-dimensional curve and fitting R²(0.995-1) to calculate the concentration of rhBMP-2 in the sample.

(2) Samples containing rhBMP-2 taken at each time point were measured and converted to rhBMP-2 concentration on a fitted curve based on the measured OD values.

(3) The rhBMP-2 concentration of each extraction time point is added with the previous concentration in sequence to become the real accumulated concentration of the extraction point, and a release curve is drawn to observe the protein release condition.

The in vitro protein release of rhBMP-2 after the surface immobilization of rhBMP-2 by SLA, pSLA and C-pSLA dental implants is shown in FIG. 4.

The results showed that the cumulative release of SLA and pSLA-loaded rhBMP-2 was over 50% over 24 hours, whereas the release of C-pSLA was only 30%. It can be seen that the C-pSLA material used for loading protein factors helps to release protein factors continuously, ensures that rhBMP-2 is used as much as possible during in vivo implantation, and in addition, the SLA loading protein burst is about 22%, the pSLA material loading protein burst is 11%, and the C-pSLA material loading protein burst is only 7%, indicating that the effect on protein adsorption is limited when protein is directly loaded on the material, and there is no strong binding property between the loaded protein and the matrix. Compared with direct immobilization, the protein immobilization amount of the SLA surface PDA coating is improved, which shows that PDA has a certain slow release effect on protein release, but the action time is limited. The C-pSLA group immobilized proteins successfully slowed the rate of protein release.

Example 3.

SLA and B/C-pSLA dental implants prepared in example 1.2 were implanted in adult male beagle dogs, and material was drawn at 8, 12 weeks post implantation and the area of implant surface within the bone marrow cavity was analyzed for Bone Mineral Density (BMD), bone body score (BV/TV).

Bone mineral density analysis at 8, 12 weeks of implantation of SLA and B/C-pSLA dental implants in beagle dogs is shown in FIG. 5. The results show that the bone density of B/C-pSLA is higher than that of SLA, indicating that the osteogenic activity of the implant in animals is significantly increased after loading with the active coating.

The analysis of the bone volume fraction at 8 and 12 weeks of implantation of SLA and B/C-pSLA dental implants in beagle dogs is shown in FIG. 6. The results show that bone volume of B/C-pSLA is higher than that of SLA, indicating a significant increase in osteogenic activity of implants in animals after loading with the active coating.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method for producing a coating, comprising the steps of:

(a) providing a base material;

(d) and mixing the second coating with a third solution containing bone inducing growth factors, and reacting to coat the growth factors on the second coating to form the coating, wherein the bone inducing growth factors are bone morphogenetic proteins.

2. The method of claim 1, wherein in step (b), the mass ratio of the substrate material to the bioligand is from 0.5 to 2:1, preferably 1-1.5: 1.

3. The method of claim 1, wherein in step (c), the dihydrazide compound in the linker molecule is selected from the group consisting of: adipic acid dihydrazide, pimelic acid dihydrazide, suberic acid dihydrazide, sebacic acid dihydrazide, or a combination thereof.

4. The method of claim 1, wherein the linker molecule is prepared by:

5. The method of claim 4, wherein the molar ratio of chondroitin sulfate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dihydrazide compound is 1: 2-3: 2-3: 4-7, preferably 1: 2.5: 2.5: 5.

6. the method of claim 1, wherein step (d) comprises: and dropwise adding a third solution containing bone inducing growth factors onto the second coating to obtain the coating.

7. The method as claimed in claim 1, wherein in step (d), the mass ratio of the material for coating the second coating layer to the bone inducing growth factor is 100: 200.001-0.02.

8. A coating produced using the method of claim 1.

9. A dental implant, comprising: a dental implant base material and a coating according to claim 8.

10. A medical material comprising the coating of claim 8 or the dental implant of claim 9.