阅读说明：本技术 一种具有ros响应的钛基活性骨植入体及其制备方法 (Titanium-based active bone implant with ROS response and preparation method thereof ) 是由 胡燕 孙钰婷 陈茂华 罗忠 蔡开勇 于 2020-03-05 设计创作，主要内容包括：本发明涉及一种具有ROS响应的钛基活性骨植入体及其制备方法,属于医用材料技术领域。该植入体的制备方法包括制备表面沉积有羟基磷灰石的钛片、制备硼酸酯键、制备硼酸酯键-成骨生长肽复合物及制备植入体四个步骤。本发明中骨植入体具有针对性、智能性,以及更优的生物相容性和骨整合性,与传统的生物材料改性方法相比,该骨植入体中Apt 19S的释放量在24h就能达到MSCs最佳招募浓度,且体内实验和体外实验都进一步证明了修饰有Apt 19S的钛基材能够将MSCs迁移至骨缺损部位,具有更好的促进骨相关细胞成骨分化的能力,加速骨修复。该具有ROS响应的钛基活性骨植入体制备方法简单易操作,适合扩大化生产。(The invention relates to a titanium-based active bone implant with ROS response and a preparation method thereof, belonging to the technical field of medical materials. The preparation method of the implant comprises four steps of preparing a titanium sheet with hydroxyapatite deposited on the surface, preparing a borate bond-osteogenic growth peptide compound and preparing the implant. Compared with the traditional biomaterial modification method, the bone implant disclosed by the invention has pertinence, intelligence and better biocompatibility and osseointegration, the release amount of Apt19S in the bone implant can reach the optimal recruitment concentration of MSCs within 24 hours, and in-vivo experiments and in-vitro experiments further prove that the titanium substrate modified with Apt19S can migrate the MSCs to bone defect parts, so that the bone implant has better capacity of promoting osteogenic differentiation of bone-related cells, and bone repair is accelerated. The preparation method of the titanium-based active bone implant with ROS response is simple and easy to operate, and is suitable for expanded production.)

1. A method of making a titanium-based active bone implant having a ROS response, said method comprising the steps of:

(1) preparing a titanium sheet with hydroxyapatite deposited on the surface;

(2) preparing a borate bond;

(3) preparation of boronic ester bond-osteogenic growth peptide complexes: dissolving the boric acid ester bond prepared in the step (2) to form a solution I, dissolving the osteogenic growth peptide to form a solution II, mixing and stirring the solution I and the solution II for reaction for at least 10 hours, and dialyzing, freezing and drying to obtain the product; the 3 'end of the osteogenic growth peptide amino acid sequence is modified with an amino group, and the 5' end is modified with two phosphate radicals;

(4) dissolving the borate bond-osteogenic growth peptide compound prepared in the step (3) to form a solution III, dissolving the aptamer to form a solution IV, immersing the titanium sheet with the hydroxyapatite deposited on the surface prepared in the step (1) in the solution III for at least 10 hours, taking out and immersing in the solution IV for at least 10 hours; the 3' end of the aptamer gene sequence is modified with an RNA molecule with any base; the gene sequence of the aptamer is shown as SEO ID No: 1 is shown.

2. The method according to claim 1, wherein in the step (1), the method for preparing the titanium sheet with the hydroxyapatite deposited on the surface is an electrochemical deposition method.

3. The method according to claim 1, wherein in step (2), the method for preparing the borate bond is as follows:

A. dissolving p-nitrophenylchloroformate in tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution V;

B. dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, 4-dimethylaminopyridine and triethylamine in tetrahydrofuran to obtain a solution VI;

C. under the ice bath condition, the solution V is dropwise added into the solution VI under stirring for 10-20min, then the solution V is stirred and reacted for 3-4h at the speed of 300-800r/min at room temperature, the obtained product is dried in a spinning mode, the dried product is dissolved by dichloromethane to obtain a yellow solution, and the yellow solution is sequentially washed by 1M HCl and saturated NaCl solution and then NaHCO₃And washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, performing gradient elution by using a mixed solution of petroleum ether and dichloromethane as an eluent, and performing spin drying to obtain the borate bond.

4. The method of claim 3, wherein the mass to volume ratio of p-nitrophenyl chloroformate, 4- (hydroxymethyl) benzeneboronic acid pinacol ester, 4-dimethylaminopyridine, and triethylamine is in units of g: g: g: m L, is 0.47:0.5:0.04: 0.6.

5. The method according to claim 3, wherein in step C, the gradient elution is specifically: eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 1:4, eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 3:7, and eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 2: 3.

6. The method according to claim 1, wherein in the step (3), the mass ratio of the borate bond to the osteogenic growth peptide is 1: 6-10.

7. The method according to claim 1, wherein in the step (3), the solvent for dissolving the borate bond is one of dimethylsulfoxide, dimethylformamide, dioxane or dichloromethane; the solvent used to dissolve the osteogenic growth peptide is saturated NaHCO₃Solution, phosphate buffer solution, triple distilled water or dimethylformamideAnd (4) seed preparation.

8. The method according to claim 1, wherein in the step (4), the mass ratio of the borate bond-osteogenic growth peptide complex to the aptamer is 2:1 to 4: 3.

9. The method according to claim 1, wherein in step (4), the solvent for dissolving the borate bond-osteogenic growth peptide complex or aptamer is saturated NaHCO₃One of a solution, a phosphate buffered solution or triple distilled water.

10. A titanium-based active bone implant with ROS response prepared by the method of any of claims 1-9.

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a titanium-based active bone implant with ROS response and a preparation method thereof.

Background

Medical titanium-based materials are widely used in clinical surgery based on their good mechanical properties, corrosion resistance and biocompatibility. However, the inert titanium dioxide surface makes it less conformable to the surrounding bone tissue, especially in pathological implant microenvironments, such as for osteoporotic patients, due to their low bone mass, high fragility of the bone, loosening and dislodging of the material at an early stage is more likely to occur, resulting in surgical failure.

To improve the integration of the implant with the surrounding bone tissue, a number of physical (e.g., ion implantation, physical vapor deposition), chemical (e.g., acid-base etching, anodization), and biochemical (e.g., extracellular matrix components, growth factors, polypeptides, etc.) methods have been introduced into the surface modification of bone implants. However, how to simultaneously impart good biocompatibility and osteoinductivity to titanium-based materials is a problem that researchers need to solve.

Mesenchymal Stem Cells (MSCs) have the property of differentiating into osteoblasts and provide a source of growth for bone growth and repair. It does not directly differentiate into osteogenesis related cells in a bone marrow environment due to the characteristics of bone marrow itself, but needs to be transferred from abundant capillaries of bone marrow to a bone injury site for differentiation under specific physiological conditions. Therefore, in bone tissue engineering, a limited number of MSCs need to be artificially promoted to be aggregated and directionally differentiated in a large amount at a bone injury part, so as to achieve the purpose of rapidly repairing the bone injury.

Apt19S, a DNA aptamer, can specifically recognize pluripotent stem cells and has the capacity of capturing stem cells. MSCs can be recruited to the tissue defect part in the bone tissue to promote bone repair. However, it is necessary to make advanced research on how to achieve recruitment of MSCs by Apt19S at a tissue defect site, and achieve reconstruction and healing of bone around an implant, thereby achieving better osseointegration.

Disclosure of Invention

In view of the above, the present invention is directed to a method for preparing a titanium-based active bone implant having ROS response; the other purpose is to provide a titanium-based active bone implant with ROS response.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a method of making a titanium-based active bone implant having a ROS response, said method comprising the steps of:

(1) preparing a titanium sheet with hydroxyapatite deposited on the surface;

(2) preparing a borate bond;

(3) preparation of boronic ester bond-osteogenic growth peptide complexes: dissolving the boric acid ester bond prepared in the step (2) to form a solution I, dissolving the osteogenic growth peptide to form a solution II, mixing and stirring the solution I and the solution II for reaction for at least 10 hours, and dialyzing, freezing and drying to obtain the product; the 3 'end of the osteogenic growth peptide amino acid sequence is modified with an amino group, and the 5' end is modified with two phosphate radicals;

(4) dissolving the borate bond-osteogenic growth peptide compound prepared in the step (3) to form a solution III, dissolving the aptamer to form a solution IV, immersing the titanium sheet with the hydroxyapatite deposited on the surface prepared in the step (1) in the solution III for at least 10 hours, taking out and immersing in the solution IV for at least 10 hours; the 3' end of the aptamer gene sequence is modified with an RNA molecule with any base; the gene sequence of the aptamer is shown as SEO ID No: 1 is shown.

Preferably, in the step (1), the method for preparing the titanium sheet with hydroxyapatite deposited on the surface is an electrochemical deposition method.

Preferably, the method for preparing the titanium sheet with the hydroxyapatite deposited on the surface comprises the following steps: the method comprises the following steps of polishing a pure titanium sheet smoothly by using abrasive paper of No. 400, No. 1000 and No. 2000 in sequence, then respectively ultrasonically cleaning the pure titanium sheet for 20-40min by using alkali liquor, absolute ethyl alcohol and deionized water in sequence, etching the pure titanium sheet by using a fluoric acid/nitric acid solution until a topological structure with a rough surface appears on the pure titanium sheet after ultrasonic cleaning, finally, using the etched pure titanium sheet as a cathode of an electrochemical reaction, using a platinum sheet as an anode, and performing cathodic oxidation by using direct current in an electrolyte solution containing calcium chloride, ammonium dihydrogen phosphate, sodium chloride and sodium citrate.

Preferably, in the step (2), the method for preparing the borate bond is as follows:

A. dissolving p-nitrophenylchloroformate in tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution V;

B. dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, 4-dimethylaminopyridine and triethylamine in tetrahydrofuran to obtain a solution VI;

C. under the ice bath condition, the solution V is dropwise added into the solution VI under stirring for 10-20min, then the solution V is stirred and reacted for 3-4h at the speed of 300-800r/min at room temperature, the obtained product is dried in a spinning mode, the dried product is dissolved by dichloromethane to obtain a yellow solution, and the yellow solution is sequentially washed by 1M HCl and saturated NaCl solution and then NaHCO₃And washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, performing gradient elution by using a mixed solution of petroleum ether and dichloromethane as an eluent, and performing spin drying to obtain the borate bond.

Preferably, the mass-to-volume ratio of the p-nitrophenylchloroformate, the 4- (hydroxymethyl) phenylboronic acid pinacol ester, the 4-dimethylaminopyridine and the triethylamine is 0.47:0.5:0.04:0.6, and the unit of the mass-to-volume ratio is g: g: m L.

Preferably, in step C, the gradient elution is specifically: eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 1:4, eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 3:7, and eluting with a mixed solution of petroleum ether and dichloromethane in a volume ratio of 2: 3.

Preferably, in step (3), the osteogenic growth peptide amino acid sequence is as defined in SEO ID No: 2, respectively.

Preferably, in the step (3), the mass ratio of the borate bond to the osteogenic growth peptide is 1: 6-10.

Preferably, in the step (3), the solvent for dissolving the borate bond is one of dimethyl sulfoxide, dimethylformamide, dioxane or dichloromethane; the solvent used to dissolve the osteogenic growth peptide is saturated NaHCO₃One of a solution, a phosphate buffer solution, triple distilled water or dimethylformamide.

Preferably, in the step (3), the cut-off molecular weight of the dialysis bag during dialysis is 500-1000D.

Preferably, in the step (4), the mass ratio of the borate bond-osteogenic growth peptide complex to the aptamer is 2:1 to 4: 3.

Preferably, in step (4), the solvent used for dissolving the borate bond-osteogenic growth peptide complex or aptamer is saturated NaHCO₃One of a solution, a phosphate buffered solution or triple distilled water.

2. A titanium-based active bone implant having ROS response prepared by the method.

The invention has the beneficial effects that: the invention provides a titanium-based active bone implant with ROS response and a preparation method thereof, the bone implant has pertinence, intelligence, better biocompatibility and osseointegration, compared with the traditional biomaterial modification method, the release amount of Apt19S in the bone implant can reach the optimal recruitment concentration of MSCs within 24h, and in vivo experiments and in vitro experiments further prove that the titanium substrate modified with Apt19S can migrate the MSCs to bone defect parts, so that the titanium-based active bone implant has better capability of promoting osteogenic differentiation of bone-related cells, and bone repair is accelerated. The preparation method of the titanium-based active bone implant with ROS response is simple and easy to operate, and is suitable for expanded production.

The construction schematic diagram of the titanium-based active bone implant with ROS response in the method is shown in figure 1, wherein a boric acid ester bond is taken as a bridge, through the coordination and combination of a boric acid group at one end of the boric acid ester bond and an ortho hydroxyl group of an aptamer (Apt19S) modified with RNA molecules with any basic group at the 3 ' end, the reaction and combination of an osteogenic growth peptide aminoamide modified with two phosphate groups at the 5 ' end, the osteogenic growth peptide is connected with Apt19S, then through the reaction of two phosphate groups modified at the 5 ' end of the osteogenic growth peptide and hydroxyapatite deposited on the surface of a titanium sheet, Apt 19S-boric acid ester bond-osteogenic growth peptide is successfully introduced to the surface of the titanium sheet deposited with hydroxyapatite on the surface, and the ROS-responsive stem cell recruitment system is constructed. Because an amido bond generated by the reaction of an ester bond in a boric acid ester bond and the amino amide of the osteogenic growth peptide has ROS responsiveness, when the ROS level in the environment of the implant is higher, the amido bond is broken under the continuous action of the ROS, so that Apt19S is released, the local concentration of the Apt19S is increased, the recruitment concentration of MSCs is reached, the MSCs are recruited to the bone defect part, osteogenic differentiation is promoted under the synergistic action of the osteogenic growth peptide and hydroxyapatite, and the reconstruction and healing of the bone around the implant are finally realized.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic representation of the ROS-responsive titanium-based active bone implant construction of the present invention;

FIG. 2 is a graph of the results of testing the cumulative release of Apt19S in a microenvironment simulating the pathology of an osteoporotic patient for titanium-based active bone implants made in accordance with the present invention having ROS response;

FIG. 3 is an SEM photograph of Ti sheets, Ti/HA/OGP and Ti/HA/OGP/Ap prepared in example 1; (in FIG. 3, a is an SEM picture of a Ti plate, b is an SEM picture of Ti/HA in FIG. 3, c is an SEM picture of Ti/HA/OGP in FIG. 3, and d is an SEM picture of Ti/HA/OGP/Apt in FIG. 3.)

FIG. 4 is a graph showing the results of testing the ability of the titanium-based active bone implant with ROS response to modulate the migration of MSCs;

FIG. 5 is a graph showing the results of the ability of the titanium-based active bone implant with ROS response prepared in the present invention to regulate the osteogenic differentiation of MSCs; (in FIG. 5, A is a WB statistical chart of osteogenesis-related proteins, and in FIG. 5, B and C are both gray-scale-value statistical quantitative analysis charts)

Fig. 6 is a graph of the results of an in vivo osteogenesis test in an animal model of ROS-responsive titanium-based activated bone implant induced osteoporosis prepared in accordance with the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.