阅读说明：本技术 锌修饰种植体及其制备方法 (Zinc-modified implant and preparation method thereof ) 是由 光梦凯 金洁琪 于 2020-07-28 设计创作，主要内容包括：本发明提供了一种锌修饰种植体及其制备方法。所述锌修饰种植体包括种植体基体和形成于所述种植体基体表面的氧化锌复合阵列结构,所述氧化锌复合阵列结构包括氧化锌阵列,和依次涂覆在所述氧化锌阵列表面的多巴胺层和接枝在所述多巴胺层上的质酸层。该锌修饰种植体在体内微环境下会释放锌离子,在不改变种植体原有表面形态的同时,可以控制阵列的形貌和长短,从而控制锌离子的缓慢长期释放行为,进而调控其生物学性能,提高其生物相容性,并减少种植体周围炎症的发生。(The invention provides a zinc modified implant and a preparation method thereof. The zinc modified implant comprises an implant substrate and a zinc oxide composite array structure formed on the surface of the implant substrate, wherein the zinc oxide composite array structure comprises a zinc oxide array, and a dopamine layer and an acid layer, wherein the dopamine layer is sequentially coated on the surface of the zinc oxide array, and the acid layer is grafted on the dopamine layer. The zinc modified implant can release zinc ions in a microenvironment in vivo, and the shape and length of an array can be controlled without changing the original surface form of the implant, so that the slow long-term release behavior of the zinc ions is controlled, the biological performance of the zinc modified implant is regulated, the biocompatibility of the zinc modified implant is improved, and the inflammation around the implant is reduced.)

1. The zinc-modified implant is characterized by comprising an implant substrate and a zinc oxide composite array structure formed on the surface of the implant substrate, wherein the zinc oxide composite array structure comprises a zinc oxide array, and a dopamine layer and an acid layer, wherein the dopamine layer and the acid layer are sequentially coated on the surface of the zinc oxide array, and the dopamine layer is grafted on the dopamine layer.

2. The zinc-modified implant of claim 1, the layer of gallic acid is grafted from a hydroformylated hyaluronic acid.

3. The zinc modified implant of claim 1 or 2, said implant matrix being titanium.

4. The zinc-modified implant of claim 3, having a titanium-zinc oxide-dopamine-hyaluronic acid structure.

5. The preparation method of the zinc modified implant is characterized by comprising the following steps:

(1) preparing seed crystals: dissolving a first zinc salt and alkanolamine in an organic solvent, uniformly stirring, heating to react until the solution is clear and completely reacted, cooling, coating the reacted solution on the surface of an implant matrix, and annealing to obtain an implant matrix-zinc oxide seed crystal;

(2) preparing a zinc oxide array: preparing a growth solution by using a second zinc salt as a zinc source and alkylamine as alkali, putting the growth solution into a synthesis reaction device, inversely suspending the seed crystal obtained in the step (1) in the growth solution, reacting at 85-95 ℃, cooling after the reaction is finished, taking out the implant, and annealing the implant to obtain an implant matrix-zinc oxide array;

(3) coating dopamine: placing the implant matrix-zinc oxide array prepared in the step (2) in a dopamine Tris-HCl buffer solution for reaction to obtain a dopamine-coated implant matrix-zinc oxide array;

(4) grafting of the acid: oxidizing hyaluronic acid by using an oxidant, dialyzing, freeze-drying to obtain hydroformylation hyaluronic acid, and grafting the hydroformylation hyaluronic acid on the dopamine-coated implant matrix-zinc oxide array through Schiff base reaction to obtain the hyaluronic acid modified implant matrix-zinc oxide-dopamine composite array.

6. The production method according to claim 5, wherein the first zinc salt is zinc acetate, the alkanolamine is monoethanolamine, and the organic solvent is ethylene glycol monomethyl ether;

the second zinc salt is zinc nitrate, and the alkylamine is hexamethylenetetramine.

7. The method of claim 5, wherein the implant matrix is titanium.

8. The production method according to claim 7, step (1) being a method of producing a Ti — ZnO seed crystal, comprising: sequentially dissolving zinc acetate and monoethanolamine in ethylene glycol monomethyl ether, uniformly stirring, reacting the obtained solution for 30-50min under the condition of water bath at 55-65 ℃, cooling after the solution is clarified and completely reacted, refrigerating the solution at 1-4 ℃ to be used as a seed crystal coating solution, coating the seed crystal coating solution on the surface of a titanium implant matrix, and annealing at the high temperature of 380-420 ℃ to obtain the Ti-ZnO seed crystal.

9. The method of claim 8, wherein the step (2) of preparing the Ti-ZnO array comprises: preparing a growth solution by using zinc nitrate as a zinc source and hexamethylenetetramine as alkali, putting the growth solution into a hydrothermal synthesis reaction kettle, inversely suspending the Ti-ZnO seed crystal obtained in the step (1) in the growth solution, reacting for 5-7h at 85-95 ℃, cooling after the reaction is finished, taking out the titanium implant, and annealing at the temperature of 480-520 ℃ for 1-3h to obtain the Ti-ZnO array.

10. The preparation method according to claim 9, wherein the method of coating dopamine in step (3) comprises: and (3) placing the Ti-ZnO array prepared in the step (2) in a dopamine Tris-HCl buffer solution, and reacting at 35-40 ℃ to obtain the dopamine coated Ti-ZnO composite array.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a zinc modified implant and a preparation method thereof.

Background

In recent years, researches on a composite zinc coating of biological materials such as titanium implants and biological ceramics are increasing. The zinc-coated titanium sheet can enhance the adhesion, proliferation, differentiation and osteogenesis of primary rat osteoblasts and osteoblast strains MC3T3-E1 and RAW264.7

Expression of the gene of interest; in vivo experiments, the titanium Implant with the zinc coating can accelerate osseointegration and improve Bone-Implant Contact (BIC) and New Bone Formation (NBF). Zinc may also increase the torque and retention strength of the implant. Furthermore, in induced osteoporosis rats, the HA coating containing zinc was higher than the peri-implant NBF and BIC of the conventional HA coating. Yusa et al report that zinc-modified titanium sheets promote osteogenic differentiation of dental pulp stem cells. Yu et al report that the titanium sheet of the zinc-magnesium composite coating can improve the activity of vascular endothelial cells and promote the gene expression of VEGF and KDR (VEGF receptor); in addition, bone marrow stromal cells can differentiate into bone marrow stromal cells and have certain antibacterial effect. In conclusion, most of the current researches on zinc-coated implants pay attention to the influence on bone formation, the effect on blood vessel formation is less involved, and the action mechanism is rarely reported.

Zinc, known as "calcium in the 21 st century", directly regulates bone metabolism as a signaling mediator, and plays an important role in maintaining bone homeostasis and bone formation. Zinc can promote osteoblast proliferation and differentiation, improve alkaline phosphatase activity, increase osteoblast related gene and protein expression, increase bone formation, and promote osteoblast to secrete Osteoprotegerin (OPG) to block osteoclast differentiation. Zinc can promote proliferation, adhesion and osteogenic differentiation of bone marrow stromal cells. In addition, the regulation effect of zinc on angiogenesis-related cells has increased in recent two years. In 2017, Zhu et al reported that zinc can promote vascular endothelial cell proliferation and adhesion, PDGFRA and VEGFA gene expression, angiogenesis and the like. Shearier et al found that the half-lethal dose of zinc (LD50) to vascular endothelial cells was 265. mu.M, which is much higher than the physiological serum zinc concentration (10-30. mu.M). Ma et al report that the effect of zinc on vascular endothelial cells is bidirectional: promoting proliferation, adhesion, migration and differentiation at low concentration (60 μ M); inhibition was observed at high concentrations (140. mu.M). In general, zinc at physiological concentrations promotes angiogenesis-related cellular biological activity, but at concentrations in excess becomes inhibitory. Since zinc ions have a certain toxicity at high concentrations, the zinc-modified implant model should have the property of effectively, stably and slowly releasing zinc ions at low concentrations for a long time.

At present, most of implants are mainly made of pure titanium. Researches show that the titanium metal has good biocompatibility, high mechanical strength and stable chemical property, and can form good osseointegration with normal bone tissues. However, pure titanium, as an inert metal, lacks the ability to stimulate osteoblast and osteocyte proliferation and relies primarily on mechanical locking with the alveolar bone to provide retention. In order to enhance the bonding between the implant and the bone, prevent the bone tissue absorption around the implant, and ensure the long-term stability, researchers have tried many implant surface modification techniques, such as using large-particle sand blasting and acid etching (SLA), Plasma Spraying (PSP), Micro Arc Oxidation (MAO), alkali treatment after MAO, etc. to form a surface physicochemical coating capable of changing the surface composition, morphology, energy, roughness, and corrosion resistance of the implant, or forming rivet points and a three-dimensional growth space capable of providing osteoblast adhesion and growth by electrochemical deposition, dip coating-sintering, integration-sintering, sol-gel, etc., to perfect the osteoinductive bioactive coating of the implant.

At present, the implant surface coating has the problems of insufficient bonding strength, easy peeling, insufficient comprehensive biological performance, unknown long-term stability and the like, forms a perfect surface coating which has firm mechanical performance, good biocompatibility, firm osseointegration and antibacterial property, and becomes a problem to be solved by the researchers.

Disclosure of Invention

The invention aims to provide a zinc modified implant with simple process and stable controlled release effect and a preparation method thereof, and particularly, a zinc oxide array structure with a dopamine and hyaluronic acid coating layer is formed on an implant substrate, so that the bonding strength, biocompatibility and antibacterial performance of the implant are improved.

According to one aspect of the invention, the zinc modified implant comprises an implant substrate and a zinc oxide composite array structure formed on the surface of the implant substrate, wherein the zinc oxide composite array structure comprises a zinc oxide array, a dopamine layer coated on the surface of the zinc oxide array in sequence and a humic acid layer grafted on the dopamine layer.

Further, the acid layer is formed by grafting of a hydroformylated hyaluronic acid.

Further, the implant matrix is titanium.

Further, the zinc-modified implant has a titanium-zinc oxide-dopamine-hyaluronic acid structure.

According to another aspect of the present invention, there is provided a method for preparing a zinc-modified implant, comprising the steps of:

(1) preparing seed crystals: dissolving a first zinc salt and alkanolamine in an organic solvent, uniformly stirring, heating to react until the solution is clear and completely reacted, cooling, coating the reacted solution on the surface of an implant matrix, and annealing to obtain an implant matrix-zinc oxide seed crystal;

(2) preparing a zinc oxide array: preparing a growth solution by using a second zinc salt as a zinc source and alkylamine as alkali, putting the growth solution into a synthesis reaction device, inversely suspending the seed crystal obtained in the step (1) in the growth solution, reacting at 85-95 ℃ (preferably 90 ℃), cooling after the reaction is finished, taking out the implant, and annealing the implant to obtain an implant matrix-zinc oxide array;

(3) coating dopamine: placing the implant matrix-zinc oxide array prepared in the step (2) in a dopamine Tris-HCl buffer solution for reaction to obtain a dopamine-coated implant matrix-zinc oxide array;

(4) grafting of the acid: oxidizing hyaluronic acid by using an oxidant, dialyzing, freeze-drying to obtain hydroformylation hyaluronic acid, and grafting the hydroformylation hyaluronic acid on the dopamine-coated implant matrix-zinc oxide array through Schiff base reaction to obtain the hyaluronic acid modified implant matrix-zinc oxide-dopamine composite array.

Further, the first zinc salt is zinc acetate, the alkanolamine is monoethanolamine, and the organic solvent is ethylene glycol monomethyl ether;

the second zinc salt is zinc nitrate, and the alkylamine is hexamethylenetetramine.

Further, the implant matrix is titanium.

Further, the step (1) is a method for preparing the Ti-ZnO seed crystal, which comprises the following steps: sequentially dissolving zinc acetate and monoethanolamine in ethylene glycol monomethyl ether, uniformly stirring, reacting the obtained solution for 30-50min under the water bath condition of 55-65 ℃ (preferably 60 ℃), cooling after the solution is clarified and completely reacted, refrigerating the solution at 1-4 ℃ to be used as a seed crystal coating solution, coating the seed crystal coating solution on the surface of a titanium implant matrix, and annealing at the high temperature of 380-420 ℃ to obtain the Ti-ZnO seed crystal.

Further, the method for preparing the Ti-ZnO array in the step (2) comprises the following steps: preparing a growth solution by using zinc nitrate as a zinc source and hexamethylenetetramine as alkali, putting the growth solution into a hydrothermal synthesis reaction kettle, inversely suspending the Ti-ZnO seed crystal obtained in the step (1) in the growth solution, reacting for 5-7h at 85-95 ℃, cooling after the reaction is finished, taking out the titanium implant, and annealing at the temperature of 480-520 ℃ for 1-3h to obtain the Ti-ZnO array.

Further, the method for coating dopamine in the step (3) comprises the following steps: and (3) placing the Ti-ZnO array prepared in the step (2) in a dopamine Tris-HCl buffer solution, and reacting at 35-40 ℃ to obtain the dopamine coated Ti-ZnO composite array.

According to the zinc modified implant, the dental implant is functionalized by a simple array synthesis method, the introduced zinc oxide array has the advantages of antibiosis and osteogenesis promotion, meanwhile, the coating of dopamine can remove high-toxicity free radicals generated by zinc oxide, the biocompatibility of the zinc modified implant is greatly improved by combining with natural polysaccharide hyaluronic acid, and the zinc ions are slowly released for a long time due to rich carboxyl groups. Therefore, the zinc modified implant has the advantages of improving mechanical performance and biocompatibility, enhancing antibacterial property and improving osseointegration fastness.

Drawings

Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic process flow diagram of the present invention;

FIG. 2 shows an SEM image of a coating prepared according to example 1, A being titanium based array zinc oxide (Ti-ZnO) and B being titanium based array zinc oxide coated hyaluronic acid (Ti-ZnO-HA);

FIG. 3 shows confocal laser images of E.coli dead and live staining of coatings prepared according to example 1;

FIG. 4 shows a confocal laser image of dead and live staining of Staphylococcus aureus with a coating prepared according to example 1;

FIG. 5 is the inhibition rate of E.coli bacteria by the coating prepared according to example 1;

FIG. 6 is a graph of the bacteriostatic rate of Staphylococcus aureus for coatings prepared according to example 1;

FIG. 7 shows different Zn²⁺The effect on osteoblastic differentiation of Abmsc;

FIG. 8 shows a comparison of different concentrations of zinc ion coating to promote OPN protein secretion by osteoblasts;

in fig. 9, a is the inhibition rate of zinc sulfate with different concentrations, B is the release of zinc ions with different concentrations of growth solution, C is the inhibition rate of supernatant released by Ti-ZnO coating for 12 hours, and D is the inhibition effect and inhibition zone experiment of coating after introducing dopamine on bacteria in the solution.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

The zinc-modified implant comprises an implant substrate and a zinc oxide composite array structure formed on the surface of the implant substrate, wherein the zinc oxide composite array structure comprises a zinc oxide array, a dopamine layer and an acid layer, the dopamine layer is sequentially coated on the surface of the zinc oxide array, and the acid layer is grafted on the dopamine layer.

Preferably, the hyaluronic acid layer is formed by grafting of a hydroformylated hyaluronic acid, and the implant matrix is titanium. A zinc-modified implant having a titanium-zinc oxide-dopamine-hyaluronic acid structure.

The preparation method of the zinc modified implant comprises the following steps:

(1) preparing seed crystals: dissolving a first zinc salt and alkanolamine in an organic solvent, uniformly stirring, heating to react until the solution is clear and completely reacted, cooling, coating the reacted solution on the surface of an implant matrix, and annealing to obtain an implant matrix-zinc oxide seed crystal;

(2) preparing a zinc oxide array: preparing a growth solution by using a second zinc salt as a zinc source and alkylamine as alkali, putting the growth solution into a synthesis reaction device, inversely suspending the seed crystal obtained in the step (1) in the growth solution, reacting at 85-95 ℃, cooling after the reaction is finished, taking out the implant, and annealing the implant to obtain an implant matrix-zinc oxide array;

(3) coating dopamine: placing the implant matrix-zinc oxide array prepared in the step (2) in a dopamine Tris-HCl buffer solution for reaction to obtain a dopamine-coated implant matrix-zinc oxide array;

(4) grafting of the acid: oxidizing hyaluronic acid by using an oxidant, dialyzing, freeze-drying to obtain hydroformylation hyaluronic acid, and grafting the hydroformylation hyaluronic acid on the dopamine-coated implant matrix-zinc oxide array through Schiff base reaction to obtain the hyaluronic acid modified implant matrix-zinc oxide-dopamine composite array.

Preferably, the first zinc salt is zinc acetate, the alkanolamine is monoethanolamine, and the organic solvent is ethylene glycol monomethyl ether; the second zinc salt is zinc nitrate, and the alkylamine is hexamethylenetetramine.

More specifically, step (1) is a method for preparing a Ti — ZnO seed crystal, comprising: sequentially dissolving zinc acetate and monoethanolamine in ethylene glycol monomethyl ether, uniformly stirring, reacting the obtained solution for 30-50min under the condition of water bath at 55-65 ℃, cooling after the solution is clarified and completely reacted, refrigerating the solution at 1-4 ℃ to be used as a seed crystal coating solution, coating the seed crystal coating solution on the surface of a titanium implant matrix, and annealing at the high temperature of 380-420 ℃ to obtain the Ti-ZnO seed crystal.

The method for preparing the Ti-ZnO array in the step (2) comprises the following steps: preparing a growth solution by using zinc nitrate as a zinc source and hexamethylenetetramine as alkali, putting the growth solution into a hydrothermal synthesis reaction kettle, inversely suspending the Ti-ZnO seed crystal obtained in the step (1) in the growth solution, reacting for 5-7h at 85-95 ℃, cooling after the reaction is finished, taking out the titanium implant, and annealing at the temperature of 480-520 ℃ for 1-3h to obtain the Ti-ZnO array.

The method for coating dopamine in the step (3) comprises the following steps: and (3) placing the Ti-ZnO array prepared in the step (2) in a dopamine Tris-HCl buffer solution, and reacting at 35-40 ℃ to obtain the dopamine coated Ti-ZnO composite array.

FIG. 1 is a schematic process flow diagram of the process of the present invention.

The method mainly comprises the following steps:

(1) preparing Ti-ZnO seed crystal: dissolving zinc acetate and monoethanolamine in ethylene glycol monomethyl ether, stirring uniformly, heating for reaction, cooling after the solution is clarified and reacts completely, coating the solution as a seed crystal on the surface of the titanium implant, and annealing to obtain the Ti-ZnO seed crystal.

(2) Preparing Ti-ZnO: preparing a growth solution by using zinc nitrate as a zinc source and hexamethylenetetramine as alkali, putting the growth solution into a hydrothermal synthesis reaction kettle, inverting and suspending the Ti-ZnO seed crystal obtained in the step (1) in the growth solution, placing the reaction kettle in an oven for reaction, cooling after the reaction is finished, taking out the titanium implant, washing, and finally placing the titanium implant in a muffle furnace for annealing to remove residual organic matters to obtain the Ti-ZnO.

(3) Coating dopamine: and (3) placing the Ti-ZnO prepared in the step (2) in a dopamine Tris-HCL buffer solution, and reacting overnight to obtain the dopamine coated Ti-ZnO.

(4) Grafting hyaluronic acid: oxidizing hyaluronic acid by using sodium periodate, dialyzing, freeze-drying to obtain hydroformylation hyaluronic acid, and grafting the hydroformylation hyaluronic acid on the Ti-ZnO coated with dopamine through Schiff base reaction to obtain the Ti-ZnO-PDA-HA modified by hyaluronic acid.