阅读说明：本技术 医用钛植入物及其制备方法、医用钛植入物的应用 (Medical titanium implant, preparation method thereof and application of medical titanium implant ) 是由 王子杰 王林 吴紫媚 蒋星竹 侯峤丹 王蕾 江恒锋 吴云潇潇 于 2021-07-27 设计创作，主要内容包括：本申请属于医学技术领域,尤其涉及一种医用钛植入物及其制备方法,以及一种医用钛植入物的应用。其中,医用钛植入物的制备方法,包括以下步骤：获取预处理后的钛基体；将所述钛基体分散在多巴胺溶液中进行聚合反应,得到表面结合有聚多巴胺层的复合钛基体；制备含有聚乳酸-羟基乙酸共聚物和褪黑素的前驱液,将所述前驱液在所述复合钛基体表面进行纺丝处理,得到医用钛植入物。本申请制备的医用钛植入物,具有抗氧化活性,能促进新骨形成,又具有合适的体内降解速率使褪黑素具有持续且长效的药物缓释速率,从而使医用钛植入物发挥长效的促进骨形成的作用。(The application belongs to the technical field of medicine, and particularly relates to a medical titanium implant, a preparation method thereof and application of the medical titanium implant. The preparation method of the medical titanium implant comprises the following steps: obtaining a pretreated titanium substrate; dispersing the titanium matrix in a dopamine solution for polymerization reaction to obtain a composite titanium matrix with a polydopamine layer combined on the surface; preparing a precursor solution containing polylactic acid-glycolic acid copolymer and melatonin, and spinning the precursor solution on the surface of the composite titanium matrix to obtain the medical titanium implant. The medical titanium implant prepared by the application has antioxidant activity, can promote the formation of new bones, has proper in vivo degradation rate, and ensures that the melatonin has sustained and long-acting drug slow release rate, thereby ensuring that the medical titanium implant plays a role in promoting the formation of bone for a long time.)

1. A preparation method of a medical titanium implant is characterized by comprising the following steps:

obtaining a pretreated titanium substrate;

dispersing the titanium matrix in a dopamine solution for polymerization reaction to obtain a composite titanium matrix with a polydopamine layer combined on the surface;

preparing a precursor solution containing polylactic acid-glycolic acid copolymer and melatonin, and spinning the precursor solution on the surface of the composite titanium matrix to obtain the medical titanium implant.

2. A method of making a medical titanium implant according to claim 1, wherein said spinning process conditions comprise: under the conditions that the positive voltage is 12-18 kV, the negative voltage is 1-3 kV, the material pushing rate is 0.001-0.002 mm/s, and the inner diameter of a pinhole is 0.5-0.8 mm, carrying out electrostatic spinning treatment on the surface of the composite titanium matrix for 15-30 minutes, carrying out vacuum drying, and forming a nanofiber layer on the composite titanium matrix to obtain the medical titanium implant.

3. The method for preparing a medical titanium implant according to claim 2, wherein the thickness of said nanofiber layer is 5 to 10 μm;

and/or the diameter of the fiber in the nanofiber layer is 400-850 nm.

4. A method of preparing a medical titanium implant according to any one of claims 1 to 3, wherein the step of preparing the precursor solution containing polylactic acid-glycolic acid copolymer and melatonin comprises: dissolving the polylactic acid-glycolic acid copolymer in an organic solvent, and adding the melatonin for mixing treatment to obtain the precursor solution;

and/or in the polylactic acid-glycolic acid copolymer, the mass ratio of lactic acid to glycolic acid is (1-3): 1.

5. The method for preparing a medical titanium implant according to claim 4, wherein the mass percentage concentration of the polylactic acid-glycolic acid copolymer in the precursor solution is 10-20%;

and/or in the precursor liquid, the mass of the melatonin is 5-20% of that of the polylactic acid-glycolic acid copolymer;

and/or in the precursor solution, the organic solvent comprises at least one of N, N-dimethylformamide, chloroform, dichloromethane and tetrahydrofuran.

6. The method for preparing a medical titanium implant according to claim 5, wherein the dopamine solution has a dopamine hydrochloride concentration of 1-3 mg/ml and is Tris-HCl buffer solution with pH value of 7-9;

and/or, the step of polymerizing comprises: and soaking the titanium substrate in the dopamine solution, and reacting for 12-48 hours.

7. The method for preparing a medical titanium implant according to claim 5 or 6, wherein the organic solvent in the precursor solution comprises (3-5) by volume: (5-7) N, N-dimethylformamide and chloroform;

and/or the step of obtaining the pretreated titanium substrate comprises the following steps: and polishing the pure titanium sheet, ultrasonically cleaning for 1-3 times, and drying to obtain the pretreated titanium substrate.

8. The medical titanium implant is characterized by comprising a titanium substrate, a polydopamine layer combined on the surface of the titanium substrate, and a nanofiber layer combined on the surface of the polydopamine layer away from the surface of the titanium substrate, wherein the nanofiber layer contains polylactic acid-glycolic acid copolymer and melatonin.

9. The medical titanium implant according to claim 8, wherein the nanofiber layer has a thickness of 5 to 10 μm;

and/or the diameter of the fiber in the nanofiber layer is 400-850 nm;

and/or in the nanofiber layer, the mass ratio of the polylactic acid-glycolic acid to the melatonin is 1: (0.05-0.2).

10. Use of a medical titanium implant, characterized in that a medical titanium implant prepared by the method according to any one of claims 1 to 7 or a medical titanium implant according to any one of claims 8 to 9 is applied in a diabetic environment.

Technical Field

The application belongs to the technical field of medicine, and particularly relates to a medical titanium implant, a preparation method thereof and application of the medical titanium implant.

Background

The titanium alloy has good forming performance and mechanical property, and has wide application prospect in orthopedic implants. The stability of titanium implants depends on the ordered structural and functional connection of human bone tissue to the surface of the implant, i.e., Osteointegration at the "titanium-bone" interface, which is essentially the process of the neogenetic repair of the interfacial bone tissue and the direct apposition of the neogenetic bone to the surface of the implant. The modification of the surface of the titanium alloy implant increases the specific surface area, improves the biocompatibility, and even carries specific drugs to induce the mesenchymal stem cells to differentiate into osteoblasts, which is the important direction for the modification of the titanium alloy implant at present. Artificially building a transitional coating on the surface of the implant, which has a benign interaction with the bone tissue, is an effective means for more actively reducing the loosening of the bone implant. Currently, calcium phosphate (CaP) coatings are representative of inorganic coatings. Calcium phosphate is the main inorganic component of bone tissue and has very strong biocompatibility and osteointegrative properties. The nano-structured inorganic coating can be formed on the surface of a metal material such as a titanium alloy by various physical and chemical methods through calcium phosphates such as monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, octacalcium phosphate and hydroxyapatite. However, most of the preparation methods of inorganic coatings need to be carried out under extreme environments such as high temperature or high pressure, and some environment-sensitive biomolecules and drugs cannot be carried.

In the general population, titanium implants have a long term stability rate of over 5 years of over 90%. However, clinical studies have found that the Diabetic environment (diabetes mellitis) can inhibit angiogenesis around the Implant, resulting in reduction of osteoblasts on the Bone Implant Interface (Bone Implant Interface), significantly affecting Bone regeneration and osseointegration conditions, resulting in Bone Implant failure rates as high as 10% -20% for Diabetic hyperglycemic patients, and only 1% -3% for normal patients, compared with the normal environment.

Disclosure of Invention

The application aims to provide a medical titanium implant, a preparation method thereof and application of the medical titanium implant, and aims to solve the problems that the bone implant is high in loosening rate, the surface of the titanium implant is not good in bone forming condition and medicine is difficult to carry in a diabetes environment to a certain extent.

In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides a method for preparing a medical titanium implant, comprising the steps of:

obtaining a pretreated titanium substrate;

dispersing the titanium matrix in a dopamine solution for polymerization reaction to obtain a composite titanium matrix with a polydopamine layer combined on the surface;

preparing a precursor solution containing polylactic acid-glycolic acid copolymer and melatonin, and spinning the precursor solution on the surface of the composite titanium matrix to obtain the medical titanium implant.

Further, the spinning treatment conditions include: under the conditions that the positive voltage is 12-18 kV, the negative voltage is 1-3 kV, the material pushing rate is 0.001-0.002 mm/s, and the inner diameter of a pinhole is 0.5-0.8 mm, carrying out electrostatic spinning treatment on the surface of the composite titanium matrix for 15-30 minutes, carrying out vacuum drying, and forming a nanofiber layer on the composite titanium matrix to obtain the medical titanium implant.

Further, the thickness of the nanofiber layer is 5-10 mu m.

Furthermore, the diameter of the fibers in the nanofiber layer is 400-850 nm.

Further, the step of preparing the precursor liquid containing the polylactic acid-glycolic acid copolymer and the melatonin comprises the following steps: and dissolving the polylactic acid-glycolic acid copolymer in an organic solvent, and adding the melatonin for mixing treatment to obtain the precursor solution.

Further, in the polylactic acid-glycolic acid copolymer, the mass ratio of lactic acid to glycolic acid is (1-3): 1.

Further, in the precursor solution, the mass percentage concentration of the polylactic acid-glycolic acid copolymer is 10-20%.

Further, in the precursor liquid, the mass of the melatonin is 5-20% of the mass of the polylactic acid-glycolic acid copolymer.

Further, in the precursor liquid, the solvent includes at least one of N, N-dimethylformamide, chloroform, dichloromethane, and tetrahydrofuran.

Further, in the dopamine solution, the concentration of the dopamine hydrochloride is 1-3 mg/ml, and the solution is Tris-HCl buffer solution with the pH value of 7-9.

Further, the step of polymerizing comprises: and soaking the titanium substrate in the dopamine solution, and reacting for 12-48 hours.

Further, in the precursor liquid, the solvent comprises the following components in a volume ratio of (3-5): (5-7) N, N-dimethylformamide and chloroform.

Further, the step of obtaining the pretreated titanium substrate comprises: and polishing the pure titanium sheet, ultrasonically cleaning for 1-3 times, and drying to obtain the pretreated titanium substrate.

In a second aspect, the application provides a medical titanium implant, which comprises a titanium substrate, a poly dopamine layer combined on the surface of the titanium substrate, and a nanofiber layer combined on the poly dopamine layer and away from the surface of the titanium substrate, wherein the nanofiber layer contains polylactic acid-glycolic acid copolymer and melatonin.

Further, the thickness of the nanofiber layer is 5-10 mu m.

Furthermore, the diameter of the fibers in the nanofiber layer is 400-850 nm.

Further, in the nanofiber layer, the mass ratio of the polylactic acid-glycolic acid to the melatonin is 1: (0.05-0.2).

In a third aspect, the application provides an application of the medical titanium implant, the medical titanium implant prepared by the method or the medical titanium implant is applied to a diabetic environment.

According to the preparation method of the medical titanium implant provided by the first aspect of the application, the precursor solution containing the polylactic acid-glycolic acid copolymer and the melatonin is prepared into the nanofiber layer on the surface of the polydopamine layer, and the nanofiber layer has extremely high porosity and specific surface area, so that the adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblastic cells and the growth of new blood vessels are facilitated, and the growth of bone tissues into pores is promoted. The nanofiber layer prepared by spinning contains melatonin which has antioxidant activity and contains gene expression of alkaline phosphatase (ALP), bone morphogenetic protein 2(BMP2), bone morphogenetic protein 6(BMP6), osteocalcin and osteoprotegerin, and can promote formation of new bones. And simultaneously inhibits Receptor Activator (RANKL) of NF-kB (nuclear factor activated K-light chain enhancement of B cells) ligand, thereby inhibiting osteolysis. In addition, the polylactic acid-glycolic acid copolymer not only has good biocompatibility, but also has excellent tensile elongation performance, and not only can form uniform small-diameter fibers through spinning, so that melatonin is stably and uniformly distributed in the nano fiber layer of the titanium matrix; the medical titanium implant has a proper in-vivo degradation rate, can approximately maintain the network structure of the nano fibers within 1-2 months, and enables the melatonin to have a sustained and long-acting drug release rate, so that the medical titanium implant can play a role in promoting bone formation for a long time.

The medical titanium implant comprises a titanium substrate, a polydopamine layer combined on the surface of the titanium substrate, and a nanofiber layer combined on the polydopamine layer away from the surface of the titanium substrate; the polydopamine layer can improve biocompatibility, the adhesion performance of the nanofiber layer and the titanium substrate and the combination stability. The nano-fiber layer contains polylactic acid-glycolic acid copolymer and melatonin, wherein the polylactic acid-glycolic acid copolymer has good biocompatibility, proper in-vivo degradation rate and excellent tensile elongation performance, so that the melatonin is stably and uniformly distributed in the nano-fiber layer; melatonin has antioxidant activity and can promote new bone formation. The medical titanium implant has extremely high porosity and specific surface area, is beneficial to the adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblast cells and the growth of new blood vessels, and promotes the growth of bone tissues to pores.

In the third aspect of the present application, the medical titanium implant provided by the above is applied to a diabetic environment, and research shows that the medical titanium implant provided by the present application can promote the release of antioxidant enzymes in cells in the diabetic environment, reduce the damage of oxidative stress to the cells, promote the differentiation, attachment and proliferation of osteoblasts, inhibit the differentiation of osteoclasts, and greatly improve the bone regeneration and osseointegration effects of a titanium-bone interface in the diabetic environment.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

Fig. 1 is a morphology diagram of a medical titanium implant provided in example 1 of the present application;

FIG. 2 is a test chart of the coating rate of melatonin in the medical titanium implant provided in embodiments 1 to 4 of the present application;

FIG. 3 is a test chart of the drug loading rate of melatonin in the medical titanium implant provided in embodiments 1-4 of the present application;

FIG. 4 is a cell proliferation test chart of the medical titanium implant provided in examples 1 to 4 of the present application and comparative examples 1 and 2;

FIGS. 5 and 6 are graphs for testing the expression level of the osteogenesis related cytokines by the medical titanium implant provided in examples 1 to 4 and comparative examples 1 and 2 of the present application.

FIGS. 7 and 8 are test charts of the medical titanium implant provided in examples 1 to 4 and comparative examples 1 and 2 of the present application for detecting the expression level of the antioxidase-related gene in the cell;

FIGS. 9 to 12 are graphs showing the diameter distribution of the nanofiber filaments spun in the medical titanium implants prepared in examples 1 to 4, respectively.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

In the present application, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (one) of a, b, or c," or "at least one (one) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, and c may be single or plural, respectively.

It should be understood that, in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the execution sequence, some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its function and inherent logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weight of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the mass in the description of the embodiments of the present application may be in units of mass known in the chemical industry, such as μ g, mg, g, and kg.

The first aspect of the embodiments of the present application provides a method for preparing a medical titanium implant, including the following steps:

s10, obtaining a pretreated titanium substrate;

s20, dispersing the titanium substrate in a dopamine solution for polymerization reaction to obtain a composite titanium substrate with a polydopamine layer combined on the surface;

s30, preparing a precursor solution containing polylactic acid-glycolic acid copolymer and melatonin, and spinning the precursor solution on the surface of the composite titanium matrix to obtain the medical titanium implant.

According to the preparation method of the medical titanium implant provided by the first aspect of the embodiment of the application, firstly, a titanium substrate is dispersed in a dopamine solution in a soaking mode and the like, and a polydopamine layer is modified on the surface of the titanium substrate, so that the biocompatibility of the titanium substrate is improved, a nanofiber layer is favorably constructed on the surface of the titanium substrate after dopamine pretreatment subsequently, the adhesion between the nanofiber layer and the titanium substrate is enhanced, and the combination of the nanofiber layer and the titanium substrate is firmer and more stable. Then, the precursor solution containing polylactic-co-glycolic acid (PLGA) and melatonin is spun on the surface of the polydopamine layer to prepare a nanofiber layer, and the nanofiber layer has extremely high porosity and specific surface area, is favorable for adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblasts and growth of new blood vessels, and promotes bone tissues to grow into pores. In the medical titanium implant prepared in the embodiment of the application, the nanofiber layer prepared by spinning contains melatonin which has antioxidant activity and contains gene expression of alkaline phosphatase (ALP), bone morphogenetic protein 2(BMP2), bone morphogenetic protein 6(BMP6), osteocalcin and osteoprotegerin, so that new bone formation can be promoted. And simultaneously inhibits Receptor Activator (RANKL) of NF-kB (nuclear factor activated K-light chain enhancement of B cells) ligand, thereby inhibiting osteolysis. In addition, the polylactic acid-glycolic acid copolymer not only has good biocompatibility, but also can form uniform small-diameter fibers through spinning, so that the melatonin is stably and uniformly distributed in the nano fiber layer of the titanium matrix; the medical titanium implant has a proper in-vivo degradation rate, can approximately maintain the network structure of the nano fibers within 1-2 months, and enables the melatonin to have a sustained and long-acting drug release rate, so that the medical titanium implant can play a role in promoting bone formation for a long time. In vitro experiments show that the medical titanium implant disclosed by the embodiment of the application can promote the release of antioxidant enzyme in cells in a diabetic environment, reduce the damage of oxidative stress to the cells, promote the differentiation, attachment and proliferation of osteoblasts, inhibit the differentiation of osteoclasts, and greatly improve the bone regeneration and osseointegration effects of a titanium-bone interface in the diabetic environment.

In some embodiments, in step S10, the step of obtaining the preprocessed titanium substrate includes: and polishing the pure titanium sheet, removing an oxide layer on the surface of the titanium substrate to expose the surface of the pure titanium, then removing surface impurities by ultrasonic cleaning for 1-3 times, and drying to obtain the pretreated titanium substrate. In some embodiments, after polishing the pure titanium sheet, the pure titanium sheet is ultrasonically cleaned for 2 times, 10-20 minutes each time.

In some embodiments, in step S20, the concentration of dopamine hydrochloride in the dopamine solution is 1-3 mg/ml, and the solution is Tris-HCl buffer solution with pH value of 7-9. According to the embodiment of the application, the dopamine hydrochloride is good in dissolving and dispersing effects in a Tris-HCl buffer solution with the pH value of 7-9, the formed dopamine solution is good in stability, the dopamine solution with the concentration of 1-3 mg/ml has the best polymeric film forming effect on the surface of a titanium substrate, and the adhesion of a spinning nanofiber layer and the titanium substrate is guaranteed through a polydopamine film layer. If the concentration is too low, a complete poly dopamine hydrochloride film layer is difficult to form on the surface of the titanium substrate; if the concentration is too high, the dopamine hydrochloride is easy to self-polymerize, and the formation of a compact poly-dopamine hydrochloride film layer with uniform thickness on the surface of the titanium substrate is also not facilitated. In some embodiments, the concentration of dopamine hydrochloride in the dopamine solution is 1-2 mg/ml or 2-3 mg/ml, and the solution is Tris-HCl buffer solution with pH value of 7, 8, 8.5 or 9.

In some embodiments, the step of polymerizing comprises: and soaking the titanium substrate in a dopamine solution, and reacting for 12-48 hours. According to the embodiment of the application, the titanium substrate is soaked in the dopamine solution to react for 12-48 hours, so that dopamine hydrochloride is fully polymerized on the surface of the titanium substrate to form a poly-dopamine hydrochloride film layer, and the composite titanium substrate is obtained. In some embodiments, the soaking time may be 12 to 24 hours, or 24 to 48 hours, etc.

In some embodiments, in step S30, the step of preparing a precursor solution containing a poly (lactic-co-glycolic acid) and melatonin includes: dissolving polylactic acid-glycolic acid copolymer in organic solvent, adding melatonin, and mixing to obtain precursor solution. In the embodiment of the application, the polylactic acid-glycolic acid copolymer is dissolved in the organic solvent to form the polymer slurry which is uniformly and stably dispersed, and then the melatonin is dispersed in the polymer slurry and is uniformly dispersed through mixing treatment, so that the precursor liquid which is uniformly mixed and dispersed is obtained.

In some embodiments, the mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is (1-3): 1. In the polylactic acid-glycolic acid copolymer in the embodiment of the application, the biocompatibility of polylactic acid (PLA) is good, the tensile strength of the material is high, the hydrophilicity of the polylactic acid-glycolic acid copolymer can be improved by copolymerizing the PLA with glycolic acid (glycolic acid), the degradation rate adjustability and the tensile elongation rate of the polylactic acid-glycolic acid copolymer are increased, the higher the glycolic acid ratio is, the better the tensile elongation performance of PLGA is, but the degradation rate is also accelerated, the network structure of the spinning nanofiber is easy to degrade and damage, and the drug slow release and bone-promoting action time are shortened. In the polylactic acid-glycolic acid copolymer in the embodiment of the application, the mass ratio of lactic acid to glycolic acid is (1-3): 1, so that the tensile property of PLGA in the spinning process is ensured, the PLGA can be stretched into fibers which are thin enough, a nanofiber layer is prepared favorably, the PLGA has moderate degradation rate, a fiber network structure can be maintained approximately in 1-2 months, melatonin can slowly and durably release medicines, and the formation of new bones is promoted. In some embodiments, the mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer can be 1:1, 2:1, or 3:1, etc.

In some embodiments, the mass percentage concentration of the polylactic acid-glycolic acid copolymer in the precursor solution is 10-20%, and the mass percentage concentration of the polylactic acid-glycolic acid copolymer ensures the spinning effect of the precursor solution, is beneficial to forming a nanofiber layer with small fiber diameter, has extremely high porosity and specific surface area, is beneficial to adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblastic cells and growth of new blood vessels, and promotes bone tissues to grow into pores. The concentration of the polylactic acid-glycolic acid copolymer can influence the viscosity of the precursor liquid, if the concentration of the polylactic acid-glycolic acid copolymer is too low, the viscosity of the precursor liquid is too low, and the precursor liquid can be sprayed in the form of mist liquid drops instead of continuous fibers in the spinning process; if the concentration of the poly (lactic-co-glycolic acid) is too high, it is difficult to form a taylor cone, and the precursor liquid flows down from the needle instead of being ejected in the form of an extremely fine jet, which is also disadvantageous for spinning. In some embodiments, the concentration of the poly (lactic-co-glycolic acid) in the precursor solution may be 10-13%, 13-16%, 16-20%, etc.

In some embodiments, the mass of melatonin in the precursor solution is 5-20% of the mass of the polylactic-co-glycolic acid copolymer, i.e., the mass ratio of melatonin to polylactic-co-glycolic acid (PLGA) in the precursor solution is (0.05-0.2): 1; the melatonin has optimal antioxidant activity and can promote formation of new bone. If the melatonin content is too low, the effect of promoting the formation of new bones is not good; if the content of the melatonin is too high, the spinning fiber outlines are unclear and are adhered and fused with each other, so that the porosity of a spinning film is reduced, the specific surface area is reduced, and the adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblasts are not facilitated. In some embodiments, the melatonin in the precursor solution may be 5-10%, 10-15%, 15-20%, etc. of the polylactic acid-glycolic acid copolymer.

In some embodiments, the solvent in the precursor solution comprises at least one of N, N-dimethylformamide, chloroform, dichloromethane, and tetrahydrofuran, and these polar organic solvents have good dissolving and dispersing effects on PLGA and melatonin.

In some embodiments, the solvent in the precursor solution comprises (3-5) by volume: (5-7) N, N-dimethylformamide and chloroform. PLGA can be well dissolved in N, N-Dimethylformamide (DMF) and chloroform (trichloromethane, TCM); the dissolving effect of melatonin in DMF is better, but the boiling point of DMF is 152.8 ℃, the volatilization is not easy, the volatilization rate of a solvent is required to be higher for subsequent spinning, otherwise, fibers cannot be cured in time and are easy to mutually fuse, the pore structure is reduced, and the specific surface area is obviously reduced, so that TCM with the boiling point of 61.2 ℃ is mixed with DMF for use, the dissolving effect of the solvent on PLGA and melatonin is ensured, the volatile removal of the solvent is also ensured, and the spinning effect of subsequent spinning is improved. In some embodiments, the solvent in the precursor solution comprises N, N-dimethylformamide and chloroform in a 3:7 volume ratio.

In some embodiments, in the step S30, the spinning process conditions include: under the conditions that the positive voltage is 12-18 kV, the negative voltage is 1-3 kV, the material pushing rate is 0.001-0.002 mm/s and the inner diameter of a pinhole is 0.5-0.8 mm, electrostatic spinning treatment is carried out on the surface of the composite titanium matrix for 15-30 minutes, nanofibers are formed on the surface of the composite titanium matrix, solvent components are removed through vacuum drying, and a nanofiber layer is formed on the composite titanium matrix, so that the medical titanium implant is obtained. In the embodiment of the application, electrostatic spinning treatment is adopted, so that a nanofiber layer with a uniform small diameter can be formed more conveniently, the diameter of the nanofiber can be flexibly controlled, and the stability of the fiber is good. In the electrostatic spinning process, the faster the material pushing speed of electrostatic spinning is, the smaller the electric field between the needle head and the receiving plate is, the larger the fiber diameter is, and the specific surface area is reduced therewith. The excessively small specific surface area is not favorable for the timely volatilization of the solvent, so that the fiber structures of the film are easy to mutually fuse, and the specific surface area and the porosity in the formed nanofiber layer are reduced, thereby reducing the adhesion, proliferation and differentiation of osteoprogenitor cells and the growth efficiency of new blood vessels.

In some embodiments, the diameter of the fibers in the nanofiber layer is 400-850 nm, and the fibers with the diameter ensure that the medical titanium implant has high porosity and specific surface area, and simultaneously ensure that the fibers have a long degradation rate and a sustained melatonin release rate. If the diameter of the fiber is too small, the fiber degradation in the nanofiber layer and the drug release rate are accelerated too fast, and the effective action time of the coating of the titanium implant is shortened. If the fiber diameter is too large, the porosity and specific surface area of the nanofiber layer are reduced, which is not favorable for adhesion, proliferation and differentiation of osteoprogenitors and osteoblasts and growth of new blood vessels, and is not favorable for bone tissue to grow into pores. In some embodiments, the diameter of the fibers in the nanofiber layer may be 400-450 nm, 450-500 nm, 500-550 nm, 550-600 nm, 600-700 nm, 700-800 nm, 800-850 nm, etc.

In some embodiments, the thickness of the nanofiber layer is 5-10 μm, and if the nanofiber layer is too thin, the drug loading is insufficient, the action time is shortened, and the space for bone tissue adhesion to grow into is insufficient; if the nanofiber layer is too thick, the nanofiber layer is prone to fall off to form fragments, and inflammation hidden danger is caused. In some embodiments, the nanofiber layer may have a thickness of 5 to 6 μm, 6 to 7 μm, 7 to 8 μm, 8 to 10 μm, or the like.

The second aspect of the embodiment of the application provides a medical titanium implant, which comprises a titanium substrate, a poly-dopamine layer combined on the surface of the titanium substrate, and a nanofiber layer combined on the surface of the poly-dopamine layer away from the surface of the titanium substrate, wherein the nanofiber layer contains polylactic acid-glycolic acid copolymer and melatonin.

The medical titanium implant comprises a titanium substrate, a polydopamine layer combined on the surface of the titanium substrate, and a nanofiber layer combined on the polydopamine layer away from the surface of the titanium substrate; the polydopamine layer can improve biocompatibility, the adhesion performance of the nanofiber layer and the titanium substrate and the combination stability. The nano-fiber layer contains polylactic acid-glycolic acid copolymer and melatonin, wherein the polylactic acid-glycolic acid copolymer has good biocompatibility, proper in-vivo degradation rate and excellent tensile elongation performance, so that the melatonin is stably and uniformly distributed in the nano-fiber layer; melatonin has antioxidant activity and can promote new bone formation. The medical titanium implant provided by the embodiment of the application has extremely high porosity and specific surface area, is beneficial to adhesion, proliferation and differentiation of osteoprogenitor cells and osteoblasts and growth of new blood vessels, and promotes bone tissues to grow into pores.

In some embodiments, the thickness of the nanofiber layer is 5-10 μm, and if the nanofiber layer is too thin, the drug loading is insufficient, the action time is shortened, and the space for bone tissue adhesion to grow into is insufficient; if the nanofiber layer is too thick, the nanofiber layer is prone to fall off to form fragments, and inflammation hidden danger is caused.

In some embodiments, the diameter of the fibers in the nanofiber layer is 400-600 nm, and the fibers with the diameter ensure that the medical titanium implant has high porosity and specific surface area, and simultaneously ensure that the fibers have a long degradation rate and a sustained melatonin release rate.

In some embodiments, the mass ratio of polylactic-glycolic acid to melatonin in the nanofiber layer is 1: (0.05-0.2), the mass ratio of polylactic acid-glycolic acid to melatonin in the nanofiber layer is ensured, and the pores, the specific surface area and the new bone promoting effect of the nanofiber layer are ensured.

The medical titanium implant of the embodiment can be prepared by the method of the embodiment.

In a third aspect of the embodiments of the present application, a medical titanium implant prepared by the above method or the above medical titanium implant is applied to a diabetic environment.

In the third aspect of the present application, the medical titanium implant provided in the above embodiment is applied to a diabetic environment, and research shows that the medical titanium implant provided in the above embodiment of the present application can promote release of antioxidant enzymes in cells in the diabetic environment, reduce damage of oxidative stress to the cells, promote differentiation, attachment and proliferation of osteoblasts, inhibit differentiation of osteoclasts, and greatly improve bone regeneration and osseointegration effects of a titanium-bone interface in the diabetic environment.

In order to make the above implementation details and operations of the present application clearly understood by those skilled in the art and to make the progress of the medical titanium implant and the manufacturing method and application thereof obvious, the above technical solution is illustrated by the following examples.

Example 1

A medical titanium implant, the preparation of which comprises the steps of:

firstly, surface pretreatment of a pure titanium matrix: the polished titanium sheet was ultrasonically cleaned twice for 20 minutes each.

Surface modification of polydopamine: the titanium sheet after polishing and cleaning was immersed in a dopamine solution (2mg/ml) prepared with Tris-HCl buffer solution (10mmol, pH 8.5), reacted for 24 hours, taken out, ultrasonically cleaned 3 times with deionized water for 10 minutes each, and then vacuum-dried at 27 ℃ for 24 hours to prepare a titanium sheet (PDA-Ti) having a polydopamine film on the surface.

③ modifying the electrostatic spinning PLGA nano fiber film: dissolving PLGA with a TCM/DMF (7/3, v/v) mixed solvent at a concentration of 16% (m/v), adding a certain amount of melatonin to prepare melatonin with a concentration of 5% to obtain a melatonin-PLGA solution, and stirring for two hours to obtain a precursor solution. The precursor solution was filled in a 5ml syringe and electrospun using a 20G needle under conditions of a positive pressure of 16kV, a negative pressure of 2kV and a stock pushing speed of 0.0016 mm/s. And (3) taking PDA-Ti as a receiving substrate, electrospinning for 20 minutes, and then putting the substrate into a vacuum drying oven to dry for 24 hours to obtain the medical titanium implant 5% PLGA @ MT sample.

Example 2

A medical titanium implant, which differs from example 1 in that: and step three, the concentration of the melatonin is 10%, and 10% PLGA @ MT is obtained.

Example 3

A medical titanium implant, which differs from example 1 in that: and step three, the concentration of the melatonin is 15%, and 15% PLGA @ MT is obtained.

Example 4

A medical titanium implant, which differs from example 1 in that: and step three, the concentration of the melatonin is 20%, and 20% PLGA @ MT is obtained.

Comparative example 1

A medical titanium implant, which differs from example 1 in that: the pure Ti is obtained without the treatment of the step two and the step three.

Comparative example 2

A medical titanium implant, which differs from example 1 in that: and step three, the concentration of the melatonin is 0%, and 0% PLGA @ MT is obtained.

Further, in order to verify the advancement of the medical titanium implant in the embodiment of the present application, the medical titanium implant prepared in the embodiments 1 to 4 and the comparative examples 1 to 2 was subjected to the following performance test.

1. Surface topography detection: the morphology of the medical titanium implant prepared in example 1 after the gold spraying was observed by a scanning electron microscope (FEI, NanoSEM 450).

And (3) testing results: as shown in A and B in the attached drawing 1, the fiber wires are distributed on the titanium sheet in a staggered manner to form a dense high-porosity nanofiber mesh structure, and the extremely large specific surface area is favorable for the adhesion and growth of bone cells.

2. In vitro experiments: a high-sugar culture medium is used for simulating a type I diabetes cell culture environment, an MC3T3 cell line (a mouse osteoblast cell line) is respectively inoculated on the surfaces of the treated medical titanium implants of the examples 1-4 and the comparative examples 1-2, three groups are respectively made, and a titanium-bone interface in-vitro model of the diabetes environment is constructed.

3. Testing of melatonin loading rate of medical titanium implants:

the experimental method comprises the following steps: dissolving melatonin in DMF to prepare a melatonin standard solution, measuring the absorbance (A) of the standard solution at the maximum absorption wavelength of the melatonin by using an ultraviolet spectrophotometer, and drawing a standard curve of the melatonin content by linear regression analysis by using the melatonin concentration as a horizontal coordinate and the absorbance as a vertical coordinate. Taking 100mg of each of 4 drug-loaded nanofiber membranes with different melatonin concentrations (5%, 10%, 15% and 20%), respectively putting the drug-loaded nanofiber membranes into 500mL of DMF, stirring until the drug-loaded nanofiber membranes are completely dissolved, measuring the absorbance of the solutions of the four groups at the maximum absorption wavelength of the melatonin, and calculating the concentration of the melatonin according to a standard curve. The melatonin loading rates of the nanofiber coating samples at the four concentrations can be calculated by the following formula:

melatonin loading rate (%) (actual melatonin content (m) in coating)₁) /(fiber coating quality (m)₂))

Each of 40mg of the 4 samples was soaked in 40mL of PBS buffer, sealed and placed in a constant temperature water bath shaker (T ═ 37.0 ± 0.5 ℃, shaking speed 120 r/min). Then 3ml of each solution was taken periodically at different time points and poured back after measuring the absorbance. The PBS buffer was replaced every 5 days. And calculating the content of the melatonin in the PBS buffer solution by using the measured absorbance through a standard curve, and finally calculating accumulated data to draw an in vitro melatonin slow release curve.

And (3) testing results: as shown in fig. 2, the melatonin coating rate of the medical titanium implants loaded with melatonin of different concentrations in examples 1 to 4 is above 70%, which indicates that the melatonin is effectively loaded in the nanofiber coating of the medical titanium implant. As shown in fig. 3, in the medical titanium implants of examples 1 to 4 carrying melatonin of different concentrations, the melatonin is released rapidly (about 80%) in 1 to 15 days, and the release rate is slowed down in the following 15 to 35 days, but the melatonin is still released continuously, which indicates that the PLGA nanofiber layer is a good drug sustained release carrier.

4. Cell survival and growth were measured by the CCK8 method: the cell proliferation status was measured on days 1, 4 and 7 by the CCK8 method.

Medical titanium implants covered with MC3T3 were trypsinized, cell suspensions (100. mu.l/well) were seeded in 96-well plates, 10. mu.l of CCK solution was added to each well, the plates were incubated in an incubator for 2-3 hours, and the absorbance at 450nm was measured with a microplate reader.

And (3) testing results: as shown in fig. 4, the MC3T3 cell number attached to the nanofiber coating of the medical titanium implant with 15% melatonin concentration was significantly increased at the fourth day in examples 1-3 compared to comparative example 2 (0% @ PLGA) and comparative example 1 (pure Ti, control). In addition, on day seven, MC3T3 cell proliferation activity was enhanced in the nanofiber coating attached at a melatonin concentration of 10% (example 2), 15% (example 3), and particularly MC3T3 cell proliferation activity was significantly enhanced in the nanofiber coating at a melatonin concentration of 15% (example 3).

And (4) analyzing results: compared with 0% @ PLGA of comparative example 2 and the pure titanium sheet of comparative example 1, the titanium surface composite melatonin PLGA electrostatic spinning nanofiber layer in the medical titanium implant of the embodiment 1-3 of the application has better effect of promoting proliferation to MC3T3 cells.

5. Expression of key factors associated with osteogenic differentiation: real-time quantitative PCR detection of the expression level of mRNA of the osteogenesis related cytokine: runx2, ALP, OPN, GAPDH were used as housekeeping genes for the control.

And (3) testing results: as shown in fig. 5 (compare comparative example 1) and fig. 6 (compare comparative example 2), compared with comparative example 2 (0% @ PLGA) and comparative example 1 (pure Ti, control group), the expression of the bone formation related factor of MC3T3 cells attached to the medical titanium implant with melatonin concentrations of 5% (example 1), 10% (example 2), 15% (example 3) and 20% (example 4) respectively is significantly increased in the diabetic environment, which indicates that the titanium surface composite melatonin PLGA electrospun nanofiber layer in the medical titanium implant of examples 1 to 4 of the present application has a significantly improved effect on the inhibition of osteogenic differentiation of "titanium-bone" interface osteoblasts in the diabetic environment.

6. Detecting the mRNA expression level of the related gene of the antioxidant enzyme in the cells by real-time quantitative PCR: SOD (superoxide dismutase), SIRT1 (deacetylase), CAT (catalase), GAPDH were used as housekeeping genes for control.

And (3) testing results: as shown in fig. 7 (comparative example 1), fig. 8 (comparative example 2), MC3T3 intracellular CAT, SOD, SIRT1 gene expression was significantly enhanced in the diabetic environment attached to the medical titanium implant with melatonin concentrations of 5% (example 1), 10% (example 2), and 15% (example 3), respectively, as compared to comparative example 2 (0% @ PLGA) and comparative example 1 (pure Ti, control). Experimental results show that the melatonin PLGA electrostatic spinning nanofiber layer compounded on the surface of the titanium in the medical titanium implant in the embodiment 1-4 promotes the release of osteoblast antioxidase at a titanium-bone interface in a diabetes environment, and weakens the oxidative stress in cells.

7. The diameter distribution of nanofiber spinning in the medical titanium implants prepared in the embodiments 1 to 4 is measured, wherein the diameter of the fiber in the embodiment 1 is shown in figure 9, and the diameter distribution is 400-700 nm; the diameter of the fiber in the embodiment 2 is shown in figure 10, and the diameter distribution is 550-750 nm; the diameter of the fiber in the embodiment 3 is shown in figure 11, and the diameter distribution is 550-800 nm; the diameter of the fiber in example 4 is shown in figure 12, and the diameter distribution is 550-850 nm. As can be seen from the attached drawings, the diameter of the nano-fibers in the medical titanium implant prepared in the embodiments 1-4 of the application is small and the distribution is uniform.

8. The thickness of the nanofiber-spun film in the medical titanium implant prepared in examples 1 to 4 and comparative example 1 (0% @ PLGA) was measured using a KLAP-7 probe type surface profiler using a step profiler, and the results are shown in table 1 below. The film thickness of example 1 was 6.98. mu.m, the film thickness of example 2 was 7.55. mu.m, the film thickness of example 3 was 6.15. mu.m, the film thickness of example 4 was 6.39. mu.m, and the film thickness of comparative example 1 was 6.12. mu.m. As can be seen from the attached drawings, the thickness of the nanofiber layer in the medical titanium implant prepared in the examples 1-4 of the application is uniform and ranges from 6 μm to 7 μm.

TABLE 1

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.