CN110433330B - Titanium-based active bone implant and preparation method thereof - Google Patents
Titanium-based active bone implant and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
本发明涉及一种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：在纯钛表面负载多巴胺后，依次旋涂明胶溶液、葡聚糖胺溶液、明胶溶液、α‑黑素细胞刺激素溶液，将依次旋涂明胶溶液、葡聚糖胺溶液、明胶溶液、α‑黑素细胞刺激素溶液称为一次完整旋涂，后续根据对α‑黑素细胞刺激素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，随后浸泡于八臂聚乙二醇氨基接枝硼酸酯键后生成的接枝聚合物的溶液中2h以上，制得钛基活性骨植入体。该骨植入体在短时间内能够大量释放α‑MSH，达到其效应浓度，且具有促进骨相关细胞成骨分化的能力。该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 and a preparation method thereof, belonging to the technical field of medical materials. The method is as follows: after dopamine is loaded on the surface of pure titanium, gelatin solution, dextranamine solution, gelatin solution, α-melanocyte-stimulating hormone solution, the sequential spin coating of gelatin solution, glucanamine solution, gelatin solution, and α-melanocyte-stimulating hormone solution is called a complete spin coating. The actual demand release determines the number of times of the complete spin coating. After the last complete spin coating is completed, a layer of gelatin solution is spin-coated, and then immersed in the eight-arm polyethylene glycol amino grafted boronate bond to generate In the solution of the grafted polymer for more than 2 hours, the titanium-based active bone implant was prepared. The bone implant can release a large amount of α-MSH in a short time to reach its effect concentration, and has the ability to promote the osteogenic differentiation of bone-related cells. The preparation method of the titanium-based active bone implant is simple and easy to operate, and is suitable for enlarged production.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a titanium-based active bone implant and a preparation method thereof.
Background
Titanium implants have been widely used in clinical orthopedic implant surgery due to their good mechanical properties and biocompatibility. However, the titanium implant itself has some defects, such as surface metal ion extravasation, elastic modulus mismatch, osteoinductive deficiency, etc., which make it difficult to repair osteoporotic fractures.
In order to enable the implant body and the surrounding bone tissues to be quickly integrated, and the technologies of acid-base corrosion, micro-arc oxidation, plasma spraying, layer-by-layer self-assembly and the like are introduced into the surface modification of the bone implant body, most bioactive molecules have positive charges or negative charges under physiological conditions and have good biological functions, so that the bioactive molecules assembled on the surface of a titanium material through charge interaction is an ideal method for modifying the surface of the titanium material, the method can stably release the bioactive molecules at the implanted part for a long time, improve the local concentration of effector molecules and reduce side effects.
Alpha-melanocyte stimulating hormone (alpha-MSH) is a bioactive molecule capable of promoting cAMP expression, and partial studies have shown that it can promote osteogenic differentiation of osteoblasts (MC3T3-E11), but its use in materials is very limited due to the high concentration required for its action.
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; the other purpose is to provide a titanium-based active bone implant.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a method of making a titanium-based active bone implant, the method comprising:
after dopamine is loaded on the surface of pure titanium, sequentially spin-coating a gelatin solution, a glucosamine solution, a gelatin solution and an alpha-melanocyte stimulating hormone solution, sequentially spin-coating the gelatin solution, the glucosamine solution, the gelatin solution and the alpha-melanocyte stimulating hormone solution to be called as one complete spin coating, subsequently determining the number of times of the complete spin coating according to the actual release amount of the alpha-melanocyte stimulating hormone, spin-coating a layer of gelatin solution after the last complete spin coating is finished, and then soaking the gelatin solution in a graft polymer solution generated after eight-arm polyethylene glycol amino grafting a boric acid ester bond for more than 2 hours to prepare the titanium-based active bone implant.
Preferably, the method for loading dopamine on the surface of the pure titanium comprises the following steps: soaking pure titanium in a Tris-HCl buffer solution containing dopamine for 10-16h to obtain the product; the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of dopamine in the Tris-HCl buffer solution is 2 mg/mL.
Preferably, when the gelatin solution, the glucosamine solution and the alpha-melanocyte stimulating hormone 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 5-10 μ L/cm2。
Preferably, the mass ratio of the gelatin in the gelatin solution, the glucoseamine in the glucoseamine solution and the alpha-melanocyte stimulating hormone in the alpha-melanocyte stimulating hormone solution is 2-10:2-10: 1.
Preferably, the concentration of the graft polymer in the solution of the graft polymer is 5 to 10 mg/mL.
Preferably, the graft polymer is prepared as follows:
(1) dissolving p-nitrophenylchloroformate in tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution I;
(2) dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, 4-dimethylaminopyridine and triethylamine in tetrahydrofuran to obtain a solution II;
(3) under the ice bath condition, dropwise adding the solution I into the solution II under stirring for 10-20min, then stirring and reacting at the room temperature at the speed of 300-800r/min for 3-4h, dissolving the dried product with dichloromethane after spin-drying to obtain a yellow solution, washing with 1M HCl and saturated NaCl solution in sequence, and then using NaHCO solution to wash3Washing the solution until the color of the yellow solution becomes light yellow, and finally passing through a silica gel column to obtain a silica gelPerforming gradient elution by using a mixed solution of the oil ether and the dichloromethane as an eluent, and performing spin drying to obtain a borate bond;
(4) and (4) adding the borate bond prepared in the step (3) and the eight-arm polyethylene glycol amino into an organic solvent, stirring and reacting at the speed of 800-1200r/min for 10-16h, and removing the organic solvent.
Preferably, the mass-volume ratio of the p-nitrophenylchloroformate to the 4- (hydroxymethyl) phenylboronic acid pinacol ester to the 4-dimethylaminopyridine to the triethylamine is 0.47:0.5:0.04:0.6, and the unit of the mass-volume ratio is g: g: g: mL; the molar ratio of the borate bond to the eight-arm polyethylene glycol amino is 6-9: 1.
Preferably, in step (3), 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 the step (4), the organic solvent is one of dichloromethane, tetrahydrofuran or dimethyl sulfoxide.
2. A titanium-based active bone implant prepared by the method.
The invention has the beneficial effects that: the invention provides a titanium-based active bone implant and a preparation method thereof, wherein a graft polymer generated by grafting an octa-armed polyethylene glycol amino group with a boric acid ester bond is used as a connector to cross different layers to crosslink glucan glucosamine, and simultaneously, alpha-melanocyte stimulating hormone (alpha-MSH) with osteoporosis treatment effect is sandwiched in the middle. Compared with the bone implant prepared by the traditional layer-by-layer self-assembly technology, the titanium-based activated bone implant can fully release bioactive factors in a shorter time, the 7-day release amount of the titanium-based activated bone implant is 2 times of that of the traditional layer-by-layer self-assembly technology, and the optimal concentration of alpha-MSH for promoting osteogenic differentiation is achieved. In vivo experiments and in vitro experiments prove that the titanium-based active bone implant has good capacity of promoting osteogenic differentiation of bone-related cells. The preparation method of the titanium-based active bone implant 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 a graph showing the contact angle test results of each coating layer in the process of preparing a titanium-based active bone implant in example 1;
fig. 2 is an SEM image of titanium-based bone implants prepared in pure titanium rods, comparative example 1, comparative example 2, and example 1 without any modification; (a in FIG. 2 is an SEM photograph of a pure titanium rod without any modification, b in FIG. 2 is an SEM photograph of a titanium-based bone implant (Ti/LBL) prepared in comparative example 1, and c in FIG. 2 is an SEM photograph of a titanium-based bone implant (Ti/LBL) prepared in comparative example 2PEG-NBC) In FIG. 2, d is the titanium-based bone implant (Ti/LBL) prepared in example 1PEG-NBC-MSH) SEM picture
FIG. 3 is a graph of Ti/LBL in example 4PEG-NBC-release profiles of FITC-MSH and Ti/LBL-FITC-MSH for two titanium-based bone implants under different conditions;
FIG. 4 is a graph showing the results of the differentiation ability test of the stem cells regulated by the different titanium-based bone implants in example 5;
FIG. 5 is a graph showing the results of testing the bone formation around different titanium-based implants in the osteoporosis model implanted with different titanium-based bone implants of example 5;
FIG. 6 is a graph showing the results of the osteogenic differentiation ability test of bone-related cells around different titanium-based implants in the osteoporosis model implanted with different titanium-based bone implants in example 5.
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.
Example 1
Preparation of titanium-based active bone implant
(1) Dissolving 0.47g of p-nitrophenylchloroformate in 15mL of tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution I;
(2) dissolving 0.5g of 4- (hydroxymethyl) phenylboronic acid pinacol ester, 0.04g of 4-dimethylaminopyridine and 0.6mL of triethylamine in 5mL of tetrahydrofuran to obtain a solution II;
(3) dropwise adding the solution I in the step (1) into the solution II in the step (2) under stirring for 10min under the ice bath condition, then stirring and reacting at the room temperature at the speed of 600r/min for 3h, dissolving the dried product with dichloromethane after spin-drying to obtain a yellow solution, washing with 1M HCl and saturated NaCl solution in sequence, and then using NaHCO3Washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, sequentially carrying out gradient elution on a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 1:4, a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 3:7, and a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 2:3, and carrying out spin drying to obtain a borate bond;
(4) adding the borate bond prepared in the step (3) and the eight-arm polyethylene glycol amino into dichloromethane according to the molar ratio of 8:1, stirring and reacting at the speed of 1000r/min for 12h, and removing the dichloromethane to prepare a graft polymer (PEG-NBC);
(5) soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 12h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
(6) sequentially spin-coating a gelatin solution, a glucosamine solution, a gelatin solution and an alpha-melanocyte stimulating hormone solution on the surface of the dopamine-loaded pure titanium rod obtained in the step (5), wherein the sequential spin-coating of the gelatin solution (L), the glucosamine solution (B), the gelatin solution (L) and the alpha-melanocyte stimulating hormone solution (alpha-MSH) is called as one complete spin coating, then the two complete spin coatings are carried out, then a layer of the gelatin solution (L) is spin-coated, and then the solution is soaked in a solution of a graft polymer (PEG-NBC) generated after the eight-arm polyethylene glycol amino is grafted with the borate bond in the step (4) for 2 hours to prepare the titanium-based active bone implant (Ti/LBL)PEG-NBC-MSH); wherein the mass ratio of the gelatin in the gelatin solution to the glucoseamine in the glucoseamine solution to the alpha-melanocyte stimulating hormone in the alpha-melanocyte stimulating hormone solution is 10:10: 1; when the gelatin solution, the glucosamine solution and the alpha-melanocyte stimulating hormone solution are spin-coated, the following methods are adopted: firstly spin-coating at 500r/min for 6s, then spin-coating at 2000r/min for 20s, the spin-coating amount is 5 μ L/cm2(ii) a The concentration of the graft polymer in the solution of the graft polymer was 5 mg/mL.
In the process of preparing the titanium-based active bone implant, the construction of the multilayer film on the surface of the pure titanium rod is monitored by using contact angle detection, and as shown in fig. 1, as can be seen from fig. 1, from the coating on the layer 1, the contact angles of the material surface are respectively 61 degrees, 52 degrees, 62 degrees and 45 degrees, which correspond to the sizes of the contact angles of gelatin, glucoseamine, gelatin and alpha-MSH when the material surface is the outermost layer, the contact angles formed by the deposition of the subsequent multilayer film on the surface of the pure titanium also generate similar rules, and finally the contact angle is changed to 15 degrees after the cross-linking by PEG-NBC, which indicates that a ROS-responsive drug-loaded system is formed on the surface of the pure titanium rod.
Example 2
Preparation of titanium-based active bone implant
(1) Dissolving 0.47g of p-nitrophenylchloroformate in 15mL of tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution I;
(2) dissolving 0.5g of 4- (hydroxymethyl) phenylboronic acid pinacol ester, 0.04g of 4-dimethylaminopyridine and 0.6mL of triethylamine in 5mL of tetrahydrofuran to obtain a solution II;
(3) dropwise adding the solution I in the step (1) into the solution II in the step (2) under stirring for 20min under the ice bath condition, then stirring and reacting at the room temperature for 4h at the speed of 300r/min, dissolving the dried product with dichloromethane after spin-drying to obtain a yellow solution, washing with 1M HCl and saturated NaCl solution in sequence, and then using NaHCO3Washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, sequentially carrying out gradient elution on a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 1:4, a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 3:7, and a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 2:3, and carrying out spin drying to obtain a borate bond;
(4) adding the borate bond prepared in the step (3) and the eight-arm polyethylene glycol amino into tetrahydrofuran according to the molar ratio of 9:1, stirring and reacting at the speed of 800r/min for 14 hours, and removing the tetrahydrofuran to prepare a graft polymer;
(5) soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 14h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
(6) sequentially spin-coating a gelatin solution, a glucosamine solution, a gelatin solution and an alpha-melanocyte stimulating hormone solution on the surface of the dopamine-loaded pure titanium rod obtained in the step (5), wherein the sequential spin-coating of the gelatin solution, the glucosamine solution, the gelatin solution and the alpha-melanocyte stimulating hormone solution is called as one complete spin coating, then the complete spin coating is carried out for three times, then a layer of gelatin solution is spin-coated, and then the solution is soaked in the solution of the graft polymer generated after the eight-arm polyethylene glycol amino is grafted with the boric acid ester bond in the step (4) for 4 hours to prepare the titanium-based active bone implant; wherein the mass ratio of gelatin in the gelatin solution, the glucoseamine in the glucoseamine solution and the alpha-melanocyte stimulating hormone in the alpha-melanocyte stimulating hormone solution is 5:5: 1; spin coating gelatin solution, glucosamine solution and alpha-melanocyte stimulating hormone solutionSpin coating was performed as follows: firstly spin-coating at 700r/min for 10s, and then spin-coating at 2200r/min for 25s, wherein the spin-coating amount is 7 μ L/cm2(ii) a The concentration of the graft polymer in the solution of the graft polymer was 8 mg/mL.
Example 3
Preparation of titanium-based active bone implant
(1) Dissolving 0.47g of p-nitrophenylchloroformate in 15mL of tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution I;
(2) dissolving 0.5g of 4- (hydroxymethyl) phenylboronic acid pinacol ester, 0.04g of 4-dimethylaminopyridine and 0.6mL of triethylamine in 5mL of tetrahydrofuran to obtain a solution II;
(3) dropwise adding the solution I in the step (1) into the solution II in the step (2) under stirring for 15min under an ice bath condition, then stirring at the room temperature at the speed of 800r/min for reacting for 3h, dissolving the dried product with dichloromethane after spin-drying to obtain a yellow solution, washing with 1M HCl and saturated NaCl solution in sequence, and then using NaHCO3Washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, sequentially carrying out gradient elution on a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 1:4, a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 3:7, and a mixed solution formed by petroleum ether and dichloromethane according to a volume ratio of 2:3, and carrying out spin drying to obtain a borate bond;
(4) adding the borate bond prepared in the step (3) and the eight-arm polyethylene glycol amino into dimethyl sulfoxide according to the molar ratio of 6:1, stirring at the speed of 1200r/min, reacting for 16 hours, and removing the dimethyl sulfoxide to prepare a graft polymer;
(5) soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 16h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
(6) sequentially spin-coating the surface of the dopamine-loaded pure titanium rod obtained in the step (5) with a gelatin solution, a glucosamine solution, a gelatin solution and an alpha-melanocyte stimulating hormone solution, and sequentially spin-coating the surface with the gelatin solution, the glucosamine solution, the gelatin solution and the alpha-melanocyte stimulating hormone solutionThe melanocyte stimulating hormone solution is called as one-time complete spin coating, a layer of gelatin solution is spin coated after four times of complete spin coating, and then the solution is soaked in the solution of the graft polymer generated after the eight-arm polyethylene glycol amino is grafted with the boric acid ester bond in the step (4) for 5 hours to prepare the titanium-based active bone implant; wherein the mass ratio of the gelatin in the gelatin solution to the glucoseamine in the glucoseamine solution to the alpha-melanocyte stimulating hormone in the alpha-melanocyte stimulating hormone solution is 2:2: 1; when the gelatin solution, the glucosamine solution and the alpha-melanocyte stimulating hormone solution are spin-coated, the following methods are adopted: firstly spin-coating at 900r/min for 8s, and then spin-coating at 2500r/min for 23s, wherein the spin-coating amount is 10 μ L/cm2(ii) a The concentration of the graft polymer in the solution of the graft polymer was 10 mg/mL.
Comparative example 1
(1) Soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 12h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
(2) sequentially spin-coating a gelatin solution and a glucosamine solution on the surface of the dopamine-loaded pure titanium rod obtained in the step (1), wherein the sequential spin-coating of the gelatin solution (L) and the sequential spin-coating of the glucosamine solution (B) are called as one complete spin coating, and then a layer of the gelatin solution (L) is spin-coated after five complete spin coatings are carried out, so as to obtain a titanium-based bone implant (Ti/LBL); wherein the mass ratio of gelatin in the gelatin solution to the glucosamine in the glucosamine solution is 1: 1; when the gelatin solution and the glucosamine solution are spin-coated, the following methods are adopted: firstly spin-coating at 500r/min for 6s, then spin-coating at 2000r/min for 20s, the spin-coating amount is 5 μ L/cm2。
Comparative example 2
(1) Soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 12h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
(2) negatives obtained in step (1)The surface of the pure titanium rod loaded with dopamine is sequentially coated with gelatin solution and glucosamine solution in a spin mode, the gelatin solution (L) and the glucosamine solution (B) are sequentially coated in a spin mode to form a complete spin mode, a layer of gelatin solution (L) is further coated in a spin mode after five complete spin modes are carried out, and then the pure titanium rod is soaked in a solution of a graft polymer (PEG-NBC) generated after eight-arm polyethylene glycol amino is grafted with a boric acid ester bond in the step (4) in the example 1 for 2 hours to obtain the titanium-based bone implant (Ti/LBL)PEG-NBC) (ii) a Wherein the mass ratio of gelatin in the gelatin solution to the glucosamine in the glucosamine solution is 1: 1; when the gelatin solution and the glucosamine solution are spin-coated, the following methods are adopted: firstly spin-coating at 500r/min for 6s, then spin-coating at 2000r/min for 20s, the spin-coating amount is 5 μ L/cm2(ii) a The concentration of the graft polymer in the solution of the graft polymer was 5 mg/mL.
The titanium-based bone implants prepared in the pure titanium rod without any modification, comparative example 1, comparative example 2 and example 1 were respectively tested by using a scanning electron microscope, and the test results are shown in fig. 2, wherein a in fig. 2 is an SEM picture of the pure titanium rod without any modification, b in fig. 2 is an SEM picture of the titanium-based bone implant (Ti/LBL) prepared in comparative example 1, and c in fig. 2 is a titanium-based bone implant (Ti/LBL) prepared in comparative example 2PEG-NBC) In FIG. 2, d is the titanium-based bone implant (Ti/LBL) prepared in example 1PEG-NBCMSH), as can be seen from FIG. 1, the surface of the pure titanium rod without any modification was rough and slightly scratched, the Ti/LBL surface prepared in comparative example 1 was smooth, and the Ti/LBL prepared in comparative example 2 was smoothPEG-NBCAnd Ti/LBL prepared in example 1PEG-NBCMSH surface smooth with no significant difference, but comparable to Ti/LBL, Ti/LBLPEG-NBCMSH appears more compact due to the surface covered multilayer film.
Example 4
Titanium-based bone implant (Ti/LBL) according to the inventionPEG-NBC-MSH) alpha-MSH bioactive factor Release Capacity test
(1) Preparation of titanium-based active bone implant (Ti/LBL) containing FITC labeled alpha-MSHPEG-NBC-FITC-MSH)
Differences from example 1Replacing the alpha-melanocyte stimulating hormone solution in the step (6) with a FITC-labeled alpha-melanocyte stimulating hormone solution (FITC-MSH) to prepare a titanium-based active bone implant (Ti/LBL) containing the FITC-labeled alpha-MSHPEG-NBC-FITC-MSH)；
(2) Preparation of titanium-based bone implant containing FITC-MSH (Ti/LBL-FITC-MSH)
1) Soaking a pure titanium rod with the diameter of 1mM and the height of 10mM in a Tris-HCl buffer solution containing dopamine for 12h to load the dopamine on the surface of the pure titanium rod, wherein the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of the dopamine in the Tris-HCl buffer solution is 2 mg/mL;
2) sequentially spin-coating a gelatin solution, a glucosamine solution, a gelatin solution and a FITC-labeled alpha-melanocyte stimulating hormone solution (FITC-MSH) on the surface of the dopamine-loaded pure titanium rod obtained in the step 1), wherein the sequential spin-coating of the gelatin solution (L), the glucosamine solution (B), the gelatin solution (L) and the FITC-labeled alpha-melanocyte stimulating hormone solution (FITC-MSH) is called as one complete spin coating, and then the complete spin coating is carried out twice and then a layer of the gelatin solution (L) is spin-coated to prepare a titanium-based bone implant (Ti/LBL-FITC-MSH) containing the FITC-MSH; wherein the mass ratio of gelatin in the gelatin solution, the glucoseamine of the glucoseamine solution and the FITC-labeled alpha-melanocyte stimulating hormone in the FITC-labeled alpha-melanocyte stimulating hormone solution is 10:10: 1; when gelatin solution, glucosamine solution and FITC marked alpha-melanocyte stimulating hormone solution are spin-coated, the following methods are adopted: firstly spin-coating at 500r/min for 6s, then spin-coating at 2000r/min for 20s, the spin-coating amount is 5 μ L/cm2(ii) a The concentration of the graft polymer in the solution of the graft polymer was 5 mg/mL.
(3) Mixing the Ti/LBL prepared in the step (1)PEG-NBCFITC-MSH and Ti/LBL-FITC-MSH prepared in step (2) were each placed in PBS buffer at pH 7.4 and 500. mu. M H2O2The two titanium-based bone implants were incubated at 37 ℃ in PBS buffer solution, all the solutions were taken out at corresponding time points for fluorescence intensity detection, and the release curves of FITC-MSH of the two titanium-based bone implants under different conditions were made according to the released fluorescence intensity and the previous deposition amount, and the results are shown in FIG. 3, from which FIG. 3 shows that H exists2O2In the presence of Ti/LBL within 36h and 72hPEG-NBCFITC-MSH release amounts of 60% and 80% in FITC-MSH, respectively, and FITC-MSH release amounts of 25% and 40% in Ti/LBL-FITC-MSH, respectively; in the absence of H2O2Under the conditions of (1), Ti/LBL within 36h and 72hPEG-NBCFITC-MSH release amounts of 15% and 23% in FITC-MSH, respectively, and approximately 25% and 40% in Ti/LBL-FITC-MSH, respectively, followed by sustained FITC-MSH release; in the presence of H2O2After incubation for 7 days under the conditions of (1), Ti/LBLPEG-NBCThe FITC-MSH surface had almost no residual FITC-MSH, while the Ti/LBL-FITC-MSH surface still had about 50% FITC-MSH residual; in the absence of H2O2After 7 days of incubation under conditions of (3) Ti/LBLPEG-NBCThe surface of FITC-MSH had about 70% FITC-MSH residue, while the surface of Ti/LBL-FITC-MSH had about 50% FITC-MSH residue. The results show that there is H2O2Under the condition of (2) Ti/LBLPEG-NBCFITC-MSH surface FITC-MSH showed a faster release rate, but in the absence of H2O2When Ti/LBL is caused by the cross-linking of PEG-NBCPEG-NBCFITC-MSH among FITC-MSH is difficult to release from the surface of titanium implant.
Example 5
Titanium-based bone implant (Ti/LBL) according to the inventionPEG-NBCMSH) differentiation Capacity test of regulatory Stem cells
Primary culture of bone marrow mesenchymal stem cells was first performed, and fourth-generation bone marrow mesenchymal stem cells were seeded on a polystyrene plate (TCPS), a pure titanium rod (Ti) without any modification, a titanium-based bone implant (Ti/LBL) prepared in comparative example 1, and a titanium-based bone implant (Ti/LBL) prepared in comparative example 2, respectivelyPEG-NBC) And the titanium-based bone implant prepared in example 1 (Ti/LBL)PEG-NBCMSH) surface, cell seeding density 2X 104Per cm2After 7 days of culture, cells were collected and total RNA was extracted using Trizol kit, the total RNA was reverse transcribed into cDNA for subsequent quantitative PCR (qPCR) detection, and the expression of the target gene was normalized using GAPDH gene and passed through 2(-DD CT)The calculation result of the method is shown in FIG. 4, and it can be seen from FIG. 4，Ti/LBLPEG-NBCMSH surface MSCs showed high levels of osteogenesis related gene expression including nuclear transcription related factor 2, alkaline phosphatase, osteoprotegerin, osteopontin, type I collagen, osteocalcin. Wherein, Ti/LBLPEG-NBCGenes such as nuclear transcription associated factor 2, alkaline phosphatase, osteoprotegerin, osteopontin, type I collagen, osteocalcin and the like in MSCs of the MSH group are respectively up-regulated by 1.3 times, 1.5 times, 1.6 times, 1.8 times and 1.8 times compared with the Ti group. The ROS-responsive alpha-MSH release functional interface constructed on the surface of pure titanium is shown to be beneficial to inducing the differentiation of stem cells into osteoblasts and promoting osteogenesis.
Example 6
Titanium-based bone implant (Ti/LBL) according to the inventionPEG-NBCMSH) induced osteoporosis animal model in vivo bone formation ability test an osteoporosis model of SD rat was constructed using the ovariectomy method by separately preparing a titanium-based bone implant (Ti/LBL) prepared in comparative example 1, a titanium-based bone implant (Ti/LBL) prepared in comparative example 2 and a pure titanium rod (Ti) without any modificationPEG-NBC) And the titanium-based bone implant prepared in example 1 (Ti/LBL)PEG-NBCMSH) is implanted into the femoral head epiphysis of the established osteoporosis model, in particular: anesthetizing a rat by intraperitoneal injection of chloral hydrate, shaving and disinfecting a rat operation site, rotating a hole at the femoral head epiphysis of the rat by using an operation electric switch (the diameter is 1.2cm) along the direction parallel to the femoral head, inserting the different titanium-based bone implants into the hole, and observing the generation condition of bone around the different titanium-based bone implants at the femoral head epiphysis of an osteoporosis model by using micro-CT after implanting for 4 weeks, wherein as shown in figure 5, the middle red area in figure 5 is the implantation position of the titanium-based bone implant, and as can be known from figure 5, Ti/LBL (titanium/boron) is adoptedPEG-NBCMore new bone formation in the 1mm region around MSH, Ti/LBL as seen in the longitudinal cut of femoral headPEG-NBCBest bone formation around MSH.
Meanwhile, massson and H are carried out on bone tissues around different titanium-based bone implants&E staining to observe osteogenic differentiation ability of bone-related cells around the Ti-based bone implant as shown in FIG. 6, Ti/LBLPEG-NBCMore new osteogenesis around the MSH implant, indicating titaniumFunctional interface (LBL)PEG-NBCMSH) promotes bone remodeling at the implant interface more than untreated pure titanium rods, probably because of LBLPEG-NBCThe MSH surface contains alpha-MSH bioactive molecules, and the molecules can regulate osteogenic differentiation of mesenchymal stem cells in a positive feedback manner and finally promote osteogenesis.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. A method of making a titanium-based active bone implant, the method comprising:
after dopamine is loaded on the surface of pure titanium, sequentially spin-coating a gelatin solution, a glucosamine solution, a gelatin solution and an alpha-melanocyte stimulating hormone solution, sequentially spin-coating the gelatin solution, the glucosamine solution, the gelatin solution and the alpha-melanocyte stimulating hormone solution to be called as one complete spin coating, subsequently determining the number of times of the complete spin coating according to the actual release amount of the alpha-melanocyte stimulating hormone, spin-coating a layer of gelatin solution after the last complete spin coating is finished, and then soaking the gelatin solution in a graft polymer solution generated after eight-arm polyethylene glycol amino grafting a boric acid ester bond for more than 2 hours to prepare the titanium-based active bone implant.
2. The method of claim 1, wherein the dopamine is loaded on the surface of the pure titanium by the following method: soaking pure titanium in a Tris-HCl buffer solution containing dopamine for 10-16h to obtain the product; the concentration of the Tris-HCl buffer solution is 10mM, the pH value is 8.5, and the concentration of dopamine in the Tris-HCl buffer solution is 2 mg/mL.
3. The method of claim 1, wherein the gelatin solution and the glycosaminoglycan solution are spin coatedBoth the solution and the alpha-melanocyte stimulating hormone solution were spin coated as follows: 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 5-10 μ L/cm2。
4. The method according to claim 1, wherein the mass ratio of gelatin in the gelatin solution, the glycosaminoglycan in the glycosaminoglycan solution, and the α -melanocyte-stimulating hormone in the α -melanocyte-stimulating hormone solution is 2-10:2-10: 1.
5. The method of claim 1, wherein the concentration of graft polymer in the solution of graft polymer is from 5 to 10 mg/mL.
6. The method of claim 1, wherein the graft polymer is prepared by:
(1) dissolving p-nitrophenylchloroformate in tetrahydrofuran, and filling nitrogen at 0 ℃ to obtain a solution I;
(2) dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, 4-dimethylaminopyridine and triethylamine in tetrahydrofuran to obtain a solution II;
(3) under the ice bath condition, dropwise adding the solution I into the solution II under stirring for 10-20min, then stirring and reacting at the room temperature at the speed of 300-800r/min for 3-4h, dissolving the dried product with dichloromethane after spin-drying to obtain a yellow solution, washing with 1M HCl and saturated NaCl solution in sequence, and then using NaHCO solution to wash3Washing the solution until the color of the yellow solution becomes light yellow, finally passing through a silica gel column, performing gradient elution by taking a mixed solution of petroleum ether and dichloromethane as an eluent, and performing spin drying to obtain a borate bond;
(4) and (4) adding the borate bond prepared in the step (3) and the eight-arm polyethylene glycol amino into an organic solvent, stirring and reacting at the speed of 800-1200r/min for 10-16h, and removing the organic solvent.
7. The method of claim 6, 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: mL, is 0.47:0.5:0.04: 0.6; the molar ratio of the borate bond to the eight-arm polyethylene glycol amino is 6-9: 1.
8. The method according to claim 6, wherein in step (3), 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.
9. The method of claim 6, wherein in step (4), the organic solvent is one of dichloromethane, tetrahydrofuran, or dimethylsulfoxide.
10. A titanium-based active bone implant prepared by the method of any one of claims 1-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910888826.9A CN110433330B (en) | 2019-09-19 | 2019-09-19 | Titanium-based active bone implant and preparation method thereof |
Applications Claiming Priority (1)
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| CN111420118B (en) * | 2020-03-05 | 2022-05-17 | 重庆大学 | Titanium-based active bone implant with ROS response and preparation method thereof |
| CN111407930B (en) * | 2020-03-19 | 2021-01-08 | 中国科学院长春应用化学研究所 | Polymer bionic coating and preparation method thereof |
| CN111529754B (en) * | 2020-05-08 | 2021-10-26 | 重庆大学 | Titanium-based active bone implant with composite coating and preparation method thereof |
| IT202000016576A1 (en) * | 2020-07-08 | 2022-01-08 | Univ Degli Studi Di Brescia | THREE-DIMENSIONAL BIOACTIVE POROUS BODY FOR THE REGENERATION OF BONE TISSUE AND PROCESS FOR ITS PRODUCTION |
| CN114504677B (en) * | 2022-01-11 | 2023-03-10 | 武汉亚洲生物材料有限公司 | 3D printing skull repairing titanium mesh and preparation method thereof |
| CN116407677B (en) * | 2023-05-30 | 2023-11-03 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | A magnesium alloy layered coating with wear-resistant self-healing function and preparation method thereof |
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