阅读说明：本技术 一种具有复合涂层的钛基活性骨植入体及其制备方法 (Titanium-based active bone implant with composite coating and preparation method thereof ) 是由 胡燕 沐彩云 蔡开勇 罗忠 于 2020-05-08 设计创作，主要内容包括：本发明涉及一种具有复合涂层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。其方法如下：在纯钛表面喷涂MgSiO<Sub>3</Sub>微米颗粒,获得MgSiO<Sub>3</Sub>生物陶瓷层,然后在所述MgSiO<Sub>3</Sub>生物陶瓷层上依次旋涂壳聚糖溶液、明胶溶液、神经肽P物质溶液,随后再依次旋涂明胶溶液、壳聚糖溶液、明胶溶液、神经肽P物质溶液,将随后依次旋涂明胶溶液、壳聚糖溶液、明胶溶液、神经肽P物质溶液称为一次完整旋涂,后续根据对神经肽P物质的实际需求释放量确定所述完整旋涂的次数,待完成最后一次所述完整旋涂后再旋涂一层明胶溶液,即可。该植入体生物相容性好,能够有效促进新骨的形成,且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 a composite coating and a preparation method thereof, belonging to the technical field of medical materials. The method comprises the following steps: spraying MgSiO on the surface of pure titanium 3 Micron particles to obtain MgSiO 3 Biological organismsA ceramic layer, then on the MgSiO 3 Sequentially spin-coating a chitosan solution, a gelatin solution and a neuropeptide P substance solution on the biological ceramic layer, then sequentially spin-coating the gelatin solution, the chitosan solution, the gelatin solution and the neuropeptide P substance solution to be called as one complete spin-coating, then determining the number of times of the complete spin-coating according to the actual release amount required for the neuropeptide P substance, and then spin-coating a layer of gelatin solution after the last complete spin-coating is completed. The implant has good biocompatibility, can effectively promote the formation of new bones, has simple and easy operation preparation method, and is suitable for expanded production.)

1. A method of making a titanium-based active bone implant having a composite coating, the method comprising:

spraying MgSiO on the surface of pure titanium₃Micron particles to obtain MgSiO₃Bioceramic layer, then on said MgSiO₃Sequentially spin-coating a chitosan solution, a gelatin solution and a neuropeptide P substance solution on the biological ceramic layer, then sequentially spin-coating the gelatin solution, the chitosan solution, the gelatin solution and the neuropeptide P substance solution to be called as one complete spin-coating, then determining the number of times of the complete spin-coating according to the actual release amount required for the neuropeptide P substance, and then spin-coating a layer of gelatin solution after the last complete spin-coating is completed.

2. The method of claim 1, wherein the spray coating is plasma spray coating or supersonic spray coating.

3. The method of claim 1, wherein the MgSiO is₃The thickness of the bioceramic layer is 90-100 μm.

4. The method of claim 1, wherein the chitosan solution, the gelatin solution and the neuropeptide P substance solution are spin-coated by the following method: firstly spin-coating at the speed of 500-900r/min for 6-10s, and then spin-coating at the speed of 2000-2500r/min for 20-25s, wherein the spin-coating amount is 10-20 μ L/cm²。

5. The method of claim 1, wherein the concentration of chitosan in the chitosan solution is 2-10 mg/mL; the concentration of gelatin in the gelatin solution is 2-10 mg/mL; the concentration of the neuropeptide P substance in the neuropeptide P substance solution is 10-500 mu g/mL.

6. The method of any one of claims 1-5, wherein the MgSiO is₃The preparation method of the micron particles comprises the following steps: mixing Na₂SiO₃·9H₂O and Mg (NO)₃)₂·6H₂Dissolving O in water, respectively, and adding Mg (NO) under stirring₃)₂·6H₂Injecting Na into O solution₂SiO₃·9H₂And (3) generating a milky white substance in the O solution, continuously stirring for 8-12h, standing for 12-24h, performing suction filtration and washing, performing freeze drying, and finally calcining, grinding and sieving to obtain the product.

7. The method of claim 6, wherein said Na₂SiO₃·9H₂O and Mg (NO)₃)₂·6H₂The molar ratio of O is 1: 1.

8. The method according to claim 6, wherein the suction filtration washing is specifically: alternately filtering and washing with ethanol and water for 3-5 times.

9. The method according to claim 6, characterized in that the calcination is in particular: calcining at 800-1200 ℃ for 2-3 h; the sieving is 250 mesh sieving.

10. A titanium-based active bone implant with a composite coating prepared by the method of any one 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 a composite coating and a preparation method thereof.

Background

Often beyond the critical size of a bone defect, endogenous repair is limited, and bone grafting is inevitable, especially in the case of complex fractures and diseases. Titanium and titanium alloys are widely used in the orthopedic field due to their good mechanical properties, corrosion resistance and biocompatibility. However, the biological inertness of the pure titanium surface limits its osseointegration with the surrounding bone tissue and even impairs its long-term implantation performance. In addition, bone repair is a highly regulated and complex physiological process in which bone-derived Mesenchymal Stem Cells (MSCs) play an indispensable role. Therefore, the method has important practical significance for further improving the osseointegration performance of the titanium-based implant by accurately regulating and controlling the MSCs.

Recent studies have shown that elevated concentrations of various chemokines can induce migration of MSCs and other progenitor cells under damaging or pathological conditions, thereby enhancing the effect of in situ tissue regeneration. However, these endogenous repair processes are often insufficient to achieve complete tissue regeneration. To overcome these limitations, new strategies by combining bone substitute materials and exogenous chemokines have been used for bone tissue regeneration, and these strategies have been shown to have a good impact on in situ bone tissue repair. However, research on the recruitment strategy of MSCs by using titanium-based materials is very limited.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for preparing a titanium-based active bone implant with a composite coating; the other purpose is to provide a titanium-based active bone implant with a composite coating.

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 composite coating, the method comprising:

spraying MgSiO on the surface of pure titanium₃Micron particles to obtain MgSiO₃Bioceramic layer, then on said MgSiO₃Sequentially spin-coating a chitosan solution, a gelatin solution and a neuropeptide P substance solution on the biological ceramic layer, then sequentially spin-coating the gelatin solution, the chitosan solution, the gelatin solution and the neuropeptide P substance solution to be called as one complete spin-coating, then determining the number of times of the complete spin-coating according to the actual release amount required for the neuropeptide P substance, and then spin-coating a layer of gelatin solution after the last complete spin-coating is completed.

Preferably, the spraying mode is plasma spraying or supersonic spraying.

Preferably, the MgSiO₃The thickness of the bioceramic layer is 90-100 μm.

Preferably, when the chitosan solution, the gelatin solution and the neuropeptide P substance solution are spin-coated, the following methods are adopted: firstly spin-coating at the speed of 500-900r/min for 6-10s, and then spin-coating at the speed of 2000-2500r/min for 20-25s, wherein the spin-coating amount is 10-20 μ L/cm²。

Preferably, the concentration of the chitosan in the chitosan solution is 2-10 mg/mL; the concentration of gelatin in the gelatin solution is 2-10 mg/mL; the concentration of the neuropeptide P substance in the neuropeptide P substance solution is 10-500 mu g/mL.

Preferably, the MgSiO₃The preparation method of the micron particles comprises the following steps:

mixing Na₂SiO₃·9H₂O and Mg (NO)₃)₂·6H₂Dissolving O in water, respectively, and adding Mg (NO) under stirring₃)₂·6H₂Injecting Na into O solution₂SiO₃·9H₂In O solution, milky white substance appearedStirring for 8-12h, standing for 12-24h, vacuum filtering, washing, freeze drying, calcining, grinding, and sieving.

Preferably, the Na is₂SiO₃·9H₂O and Mg (NO)₃)₂·6H₂The molar ratio of O is 1: 1.

Preferably, the suction filtration washing specifically comprises: alternately filtering and washing with ethanol and water for 3-5 times.

Preferably, the calcination is specifically: calcining at 800-1200 ℃ for 2-3 h; the sieving is 250 mesh sieving.

2. The titanium-based active bone implant with the composite coating prepared by the method.

The invention has the beneficial effects that: the present invention provides a titanium-based active bone implant with a composite coating and a method for preparing the same, which overcomes the inherent defect of insufficient MSCs in the early tissue repair process by releasing a neuropeptide P substance to recruit a sufficient number of MSCs in an early stage, and employs MgSiO in the recruitment process of MSCs₃The bioceramic layer continuously releases magnesium ions and silicon ions, and the basic microenvironment caused by ion release promotes the proliferation and differentiation of the recruited MSCs, and the novel staged precise control design of the MSCs can maximally promote the formation of new bones around the implant. In addition, the titanium-based active bone implant is prepared by adding chitosan, gelatin and neuropeptide P material into MgSiO₃A multilayer film system is alternately formed on the biological ceramic layer, and the system ensures the slow release of the neuropeptide P substance on one hand and can also delay the MgSiO on the other hand₃The ions in the biological ceramic layer are released and the pH value is changed due to the released ions, so that the limitation effect on the activity of the MSCs due to too fast release of the ions and too high pH value in the initial stage is avoided, and the biological activity of the implant can be further improved. The preparation method of the titanium-based active bone implant with the composite coating is simple and easy to operate, and is suitable for expanded production.

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 MgSiO prepared before calcination after freeze-drying in step (1) of example 1₃Micron particle, MgSiO prepared after calcining₃Micron particles, pure titanium treated in the step (2) and MgSiO loaded in the step (3)₃XRD pattern of pure titanium of bioceramic layer (a in FIG. 1 is MgSiO prepared before calcination after freeze-drying in step (1)₃XRD pattern of the micrometer particles, and b in FIG. 1 is MgSiO prepared after calcination in step (1)₃XRD patterns of the micrometer particles, wherein c in FIG. 1 is the XRD pattern of the pure titanium after being treated in the step (2), and d in FIG. 1 is the XRD pattern of the MgSiO supported in the step (3)₃XRD pattern of pure titanium of bioceramic layer);

fig. 2 is an SEM image of pure titanium after treatment in step (2) of example 1, a bone implant (designated as MgSi) prepared in comparative example 1, a bone implant (designated as MgSi/LBL) prepared in comparative example 2, and a titanium-based active bone implant (designated as MgSi/LBL-SP) prepared in example 1 (a in fig. 2 is an SEM image of pure titanium after treatment in step (2) of example 1, b in fig. 2 is an SEM image of a bone implant prepared in comparative example 1, c in fig. 2 is an SEM image of a bone implant prepared in comparative example 2, and d in fig. 2 is an SEM image of an active titanium-based bone implant prepared in example 1);

fig. 3 is a white light interferometer test result graph of the pure titanium after the treatment in the step (2) of example 1, the bone implant (denoted as MgSi) prepared in comparative example 1, the bone implant (denoted as MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (denoted as MgSi/LBL-SP) prepared in example 1 (a in fig. 3 is a white light interferometer test result graph of the pure titanium after the treatment in the step (2) of example 1, b in fig. 3 is a white light interferometer test result graph of the bone implant prepared in comparative example 1, c in fig. 3 is a white light interferometer test result graph of the bone implant prepared in comparative example 2, and d in fig. 3 is a white light interferometer test result graph of the active titanium-based bone implant prepared in example 1);

FIG. 4 is a graph showing the results of a release behavior test of the neuropeptide P substance in the titanium-based active bone implant prepared in example 1;

FIG. 5 is a graph of results of tests on the release behavior of Mg ions from a bone implant prepared in comparative example 1 (designated as MgSi), a bone implant prepared in comparative example 2 (designated as MgSi/LBL), and a titanium-based activated bone implant prepared in example 1 (designated as MgSi/LBL-SP);

FIG. 6 is a graph of pH change in the incubation solutions of sets of pure titanium (designated Ti) after treatment in step (2) of example 1, the bone implant prepared in comparative example 1 (designated MgSi), the bone implant prepared in comparative example 2 (designated MgSi/LBL) and the titanium-based active bone implant prepared in example 1 (designated MgSi/LBL-SP);

fig. 7 is a graph of the test results of the secretion level of MMP2 in each of the groups of pure titanium (designated Ti) after treatment in step (2) of example 1, the bone implant (designated MgSi) prepared in comparative example 1, the bone implant (designated MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (designated MgSi/LBL-SP) prepared in example 1 (n ═ 5, × p <0.05, × p < 0.01);

fig. 8 is a graph of the results of MSCs migration capability tests on pure titanium (denoted as Ti) after treatment in step (2) of example 1, the bone implant prepared in comparative example 1 (denoted as MgSi), the bone implant prepared in comparative example 2 (denoted as MgSi/LBL), and the titanium-based active bone implant prepared in example 1 (denoted as MgSi/LBL-SP) (n-5, p <0.05, p < 0.01);

fig. 9 is a graph showing the results of cell activity measurements in each of the groups of pure titanium (denoted as Ti) after treatment in step (2) of example 1, the bone implant (denoted as MgSi) prepared in comparative example 1, the bone implant (denoted as MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (denoted as MgSi/LBL-SP) prepared in example 1 (n-5, p <0.05, p < 0.01);

fig. 10 is a graph of the results of testing the alkaline phosphatase activity of each of the groups of pure titanium (denoted as Ti) after treatment in step (2) of example 1, the bone implant (denoted as MgSi) prepared in comparative example 1, the bone implant (denoted as MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (denoted as MgSi/LBL-SP) prepared in example 1 (n-5, p <0.05, p < 0.01);

fig. 11 is a graph of the results of testing the levels of collagen secretion for each of the groups of pure titanium (designated Ti) after treatment in step (2) of example 1, the bone implant (designated MgSi) prepared in comparative example 1, the bone implant (designated MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (designated MgSi/LBL-SP) prepared in example 1 (n-5, p <0.05, p < 0.01);

fig. 12 is a graph of the results of matrix mineralization level tests (n ═ 5, p <0.05, p <0.01) for each of the groups of pure titanium (denoted Ti) after treatment in step (2) of example 1, the bone implant prepared in comparative example 1 (denoted MgSi), the bone implant prepared in comparative example 2 (denoted MgSi/LBL), and the titanium-based activated bone implant prepared in example 1 (denoted MgSi/LBL-SP);

fig. 13 is a graph showing the results of the test of the expression level of proteins (OCN, OPN, Col-1, BMP2) related to MSCs grown on each group of the pure titanium (denoted as Ti) after the treatment in step (2) of example 1, the bone implant (denoted as MgSi) prepared in comparative example 1, the bone implant (denoted as MgSi/LBL) prepared in comparative example 2, and the titanium-based active bone implant (denoted as MgSi/LBL-SP) prepared in example 1 (n-3, p <0.05, p < 0.01);

fig. 14 is a representation of TUNEL staining and a quantitative result of the staining after implantation surgery 7d of pure titanium (denoted as Ti) treated in step (2) of example 1, the bone implant prepared in comparative example 1 (denoted as MgSi/LBL-SP) and the titanium-based active bone implant prepared in example 1 (denoted as MgSi/LBL-SP) (n-5, p <0.05, p <0.01, fig. 14 a is a representation of TUNEL staining, and fig. 14B is a quantitative statistical representation of TUNEL staining);

FIG. 15 is CD29 surrounding a Ti-based implant after implantation surgery 7d and 14d for pure titanium (designated Ti) treated in step (2) of example 1, a bone implant prepared in comparative example 1 (designated as MgSi), and a titanium-based active bone implant prepared in example 1 (designated as MgSi/LBL-SP)⁺/CD90⁺Immunofluorescence mapping of double positive cellsAnd positive cell quantitation results (n-5, p)<0.05，**p<0.01, Panel A in FIG. 15 is the immunofluorescence profile after implantation 7d, Panel B in FIG. 15 is the immunofluorescence profile after implantation 14d, and Panel C in FIG. 15 is the CD29 for each implant⁺/CD90⁺Double positive cells were quantitated);

fig. 16 is a graph of results of micro-CT analysis evaluating new bone formation 1 month after implantation of pure titanium (denoted as Ti) treated in step (2) of example 1, the bone implant (denoted as MgSi) prepared in comparative example 1, and the titanium-based active bone implant (denoted as MgSi/LBL-SP) prepared in example 1 (a graph in fig. 16 is a 3D graph of bone formation conditions at interfaces of each group of new bones and the implant, and a B graph in fig. 16 is a quantitative analysis graph of BV/TV (new bone mass/total bone mass) of each group of new bones).

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.