阅读说明：本技术 在可降解金属表面制备促骨修复和再生功能涂层的方法 (Method for preparing bone repair and regeneration promoting functional coating on degradable metal surface ) 是由 万国江 钱军余 张文泰 曾培杰 王佳乐 毛金龙 黄楠 于 2021-01-04 设计创作，主要内容包括：本发明公开了一种在可降解金属表面制备促骨修复和再生功能涂层的方法,包括以下步骤：S1.将可降解金属打磨、抛光、清洗并干燥,得到金属基层；S2.在65-75℃将S1所得金属基层在硝酸锌和氢氧化钠混合溶液中浸泡2-4h,然后置于紫外下照射6-8h,得到氧化金属基底层；S3.在20-25℃避光条件下将S2所得氧化金属基底层于多巴胺溶液中浸泡6-8h,然后清洗并吹干；重复该浸泡-清洗-吹干过程3-5次；S4.在20-25℃将S3所得样品于磷酸缓冲液、Ⅰ型胶原蛋白和氯化钙的混合溶液中浸泡12-24h,清洗干燥,即得。本发明解决了现有技术中的涂层促骨修复和再生能力不足的问题,达到在降解过程中同时引导骨修复和再生的目的。(The invention discloses a method for preparing a bone repair and regeneration promoting functional coating on the surface of degradable metal, which comprises the following steps: s1, grinding, polishing, cleaning and drying degradable metal to obtain a metal base layer; s2, soaking the metal substrate obtained in the step S1 in a mixed solution of zinc nitrate and sodium hydroxide at 65-75 ℃ for 2-4h, and then placing the metal substrate under ultraviolet irradiation for 6-8h to obtain an oxidized metal substrate layer; s3, soaking the oxidized metal substrate layer obtained in the step S2 in a dopamine solution for 6-8 hours at the temperature of 20-25 ℃ in a dark place, and then cleaning and drying; repeating the soaking-cleaning-drying process for 3-5 times; s4, soaking the sample obtained in the step S3 in a mixed solution of phosphate buffer, type I collagen and calcium chloride at the temperature of 20-25 ℃ for 12-24h, and cleaning and drying to obtain the collagen. The invention solves the problem of insufficient capability of promoting bone repair and regeneration of the coating in the prior art, and achieves the purpose of simultaneously guiding bone repair and regeneration in the degradation process.)

1. A method for preparing a bone repair and regeneration promoting functional coating on a degradable metal surface is characterized by comprising the following steps:

s1, grinding, polishing, cleaning and drying degradable metal to obtain a metal base layer;

s2, soaking the metal substrate obtained in the step S1 in a mixed solution of zinc nitrate and sodium hydroxide at 65-75 ℃ for 2-4h, and then placing the metal substrate under ultraviolet irradiation for 6-8h to obtain an oxidized metal substrate layer;

s3, soaking the oxidized metal substrate layer obtained in the step S2 in a dopamine solution for 6-8 hours at the temperature of 20-25 ℃ in a dark place, and then cleaning and drying; repeating the soaking-cleaning-drying process for 3-5 times;

s4, soaking the sample obtained in the step S3 in a mixed solution of phosphate buffer, type I collagen and calcium chloride at the temperature of 20-25 ℃ for 12-24h, and cleaning and drying to obtain the collagen.

2. The method for preparing the bone repair and regeneration promoting functional coating on the surface of the degradable metal according to claim 1, wherein the degradable metal is zinc, magnesium or alloy thereof.

3. The method for preparing a bone repair and regeneration promoting functional coating on a degradable metal surface according to claim 1, wherein the degradable metal surface is polished to 2000# by using a water abrasive paper in S1.

4. The method for preparing the functional coating for promoting bone repair and regeneration on the surface of the degradable metal according to claim 1, wherein the step S1 is to sequentially wash the degradable metal with deionized water and absolute ethyl alcohol under ultrasonic conditions for 2-3 times, and to dry the degradable metal after 3-5min of washing each time.

5. The method for preparing the functional coating for promoting bone repair and regeneration on the surface of the degradable metal according to claim 1, wherein the concentration of zinc nitrate in the S2 mixed solution is 0.03-0.08M, and the concentration of sodium hydroxide is 0.2-0.7M.

6. The method for preparing the coating for promoting bone repair and regeneration on the surface of the degradable metal according to claim 1, wherein the concentration of the dopamine solution in the S3 is 1-3 mg/mL.

7. The method for preparing a coating for promoting bone repair and regeneration on a degradable metal surface as claimed in claim 1, wherein the concentration of the phosphate buffer solution in the S4 mixed solution is 3-6mM, the concentration of the type I collagen is 0.002-0.5mg/mL, and the concentration of the calcium chloride is 150-250 mM.

8. A bone repair and regeneration promoting coating prepared by the method of any one of claims 1 to 7.

Technical Field

The invention belongs to the technical field of biomaterial surface modification, and particularly relates to a method for preparing a bone repair and regeneration promoting functional coating on a degradable metal surface.

Background

The degradable metal is expected to be applied to a new generation of degradable bone implant material due to the moderate corrosion rate and the potential biological functionality of zinc ions. However, the poor integration of degradable metal bone and the insufficient capability of promoting bone repair and regeneration are still the key to restrict the clinical application effect.

Currently, surface modification research aiming at degradable metals mainly focuses on regulating the corrosion behavior and improving the biocompatibility of the degradable metals, and research on promoting bone repair and regeneration is insufficient.

Disclosure of Invention

The invention aims to: aiming at the defects in the prior art, the method for preparing the coating with the functions of promoting bone repair and regeneration on the surface of the degradable metal is provided, the problem of insufficient capability of promoting bone repair and regeneration of the coating in the prior art is solved by constructing the hybrid functional coating with the micro-nano structure on the surface of the degradable metal substrate, and the purpose of simultaneously guiding bone repair and regeneration in the degradation process is achieved.

The technical scheme adopted by the invention is as follows:

a method for preparing a bone repair and regeneration promoting functional coating on a degradable metal surface comprises the following steps:

s1, grinding, polishing, cleaning and drying degradable metal to obtain a metal base layer;

s2, soaking the metal substrate obtained in the step S1 in a mixed solution of zinc nitrate and sodium hydroxide at 65-75 ℃ for 2-4h, and then placing the metal substrate under ultraviolet irradiation for 6-8h to obtain an oxidized metal substrate layer;

s3, soaking the oxidized metal substrate layer obtained in the step S2 in a dopamine solution for 6-8 hours at the temperature of 20-25 ℃ in a dark place, and then cleaning and drying; repeating the soaking-cleaning-drying process for 3-5 times;

s4, soaking the sample obtained in the step S3 in a mixed solution of phosphate buffer, type I collagen and calcium chloride at the temperature of 20-25 ℃ for 12-24h, and cleaning and drying to obtain the collagen.

The mechanism of the invention is as follows: taking degradable metal zinc as an example, a zinc-based layer is pretreated by a chemical conversion method to generate a zinc oxide-based bottom layer, and the surface active hydroxyl groups of the zinc oxide-based bottom layer are increased by ultraviolet irradiation, so that the subsequent deposition and assembly of dopamine are facilitated. And then soaking the coating in a mixed solution of PBS, type I collagen and calcium chloride to prepare the hybrid coating. Based on the principles of covalent grafting and chemical coordination, amino/hydroxyl active sites on the surfaces of collagen and dopamine are assembled in a covalent bonding or hydrogen bonding mode, and coordination chelation of zinc and calcium ions with dopamine and collagen constructs a metal organic compound, so that nucleation sites are provided for deposition and growth of calcium phosphate/zinc calcium phosphate, and a composite hybrid functional coating is formed.

Further, firstly, immersing the metal base layer in a mixed solution of a zinc nitrate solution and a sodium hydroxide solution, and performing liquid phase deposition at 70 ℃ for 2 hours to construct a zinc oxide base layer; increasing the surface active hydroxyl of the zinc oxide layer by ultraviolet irradiation for 6h so as to circularly deposit a dopamine layer; and mixing the PBS solution, the I-type collagen and the calcium chloride solution, and preparing the composite coating by adopting a liquid phase deposition method. Based on the coordination chemical principle, organic molecules and inorganic components are hybridized in a compounding way, a specific micro-nano structure is formed, and the growth of an inorganic phosphoric acid phase is induced on the premise of ensuring the activity and stability of the organic molecules. The biological activity, biocompatibility and biological functionality of the coating are improved under the synergistic effect of the two. Can induce the deposition of active calcium and phosphate salt and promote the growth, proliferation and adhesion of osteoblasts; promoting the proliferation and migration of endothelial cells, and having the potential of promoting angiogenesis; in addition, the coating can also effectively inhibit the corrosion degradation speed of the degradable metal matrix and regulate and control the release of zinc ions.

Further, the degradable metal is zinc, magnesium or an alloy thereof.

Further, in S1, the degradable metal surface is polished to 2000# with water abrasive paper.

Further, in S1, deionized water and absolute ethyl alcohol are sequentially adopted to clean for 2-3 times under the ultrasonic condition, and the cleaning is carried out for 3-5min each time and then the drying is carried out.

Further, in S1, deionized water and absolute ethyl alcohol are sequentially adopted to clean for 3 times under the ultrasonic condition, and the cleaning is carried out for 3min each time and then the drying is carried out.

Furthermore, the concentration of zinc nitrate in the S2 mixed solution is 0.03-0.08M, and the concentration of sodium hydroxide is 0.2-0.7M.

Further, the concentration of zinc nitrate and the concentration of sodium hydroxide in the S2 mixed solution were 0.05M and 0.5M, respectively.

Further, the concentration of the dopamine solution in the S3 is 1-3 mg/mL; preferably 2 mg/mL.

Furthermore, the concentration of the phosphate buffer solution in the S4 mixed solution is 3-6mM, the concentration of the type I collagen is 0.002-0.5mg/mL, and the concentration of the calcium chloride is 150-250 mM.

Further, the concentration of the phosphate buffer solution in the S4 mixed solution was 5mM, the concentration of type I collagen was 0.5mg/mL, and the concentration of calcium chloride was 200 mM.

The coating for promoting bone repair and regeneration is prepared by the method.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the invention constructs a multi-component modified functional layer, which comprises dopamine, main components of natural bone tissues, namely calcium phosphate and type I collagen, and skillfully combines the multi-components together based on the material chemistry principle to form a multi-scale surface micro-nano topological structure;

2. in the invention, a metal organic compound is formed by coordination and chelation, the growth of an inorganic phosphate phase is induced, collagen and calcium phosphate are combined together, and a natural bone tissue composition and structure are simulated, so that the deposition of extracellular matrix (ECM) and the proliferation and adhesion of osteoblasts are facilitated, and a good bone integration promoting effect is achieved;

3. the dopamine, zinc ions and calcium ions adopted in the invention can promote the proliferation, adhesion and migration of endothelial cells, and have the potential effect of promoting angiogenesis;

4. the hybrid functional layer constructed by the invention can promote osseointegration and angiogenesis simultaneously, and achieve the capability of promoting bone repair and regeneration.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a scanning electron microscope image of the zinc-based surface of example 1 before and after modification;

FIG. 2 is an XRD pattern before and after modification of the zinc-based surface of example 1; the upper curve is PDA/Cap/Col, and the lower curve is Zn;

FIG. 3 is XPS high resolution spectra before and after modification of the zinc-based surface of example 1;

FIG. 4 is a plot of polarization before and after modification of the zinc-based surface of example 1;

FIG. 5 is a fluorescent staining pattern of osteoblasts (a) and a statistical result of cell activity before and after zinc-based surface modification in example 1;

FIG. 6 is the fluorescence staining pattern of endothelial cells and the statistics of cell activity (a) before and after zinc-based surface modification in example 1;

FIG. 7 is a photo-mirror image of (a) endothelial cell migration before and after zinc-based surface modification and (b) migration area statistics of example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The preferred embodiment of the invention provides a method for preparing a coating for promoting bone repair and regeneration on the surface of degradable metal zinc, which comprises the following specific steps:

a. grinding the metal zinc to 2000# by using water abrasive paper; sequentially cleaning the raw materials by deionized water and absolute ethyl alcohol under ultrasonic conditions for 3 times, 3 minutes each time, and drying for later use;

b. b, immersing the metal zinc base layer obtained in the step a in a mixed solution of 0.05M zinc nitrate solution and 0.5M sodium hydroxide solution at the temperature of 70 ℃ for 2 hours to obtain a zinc oxide layer;

c. b, placing the zinc oxide layer obtained in the step b in a dopamine solution with the concentration of 2mg/ml for soaking for 6 hours at room temperature in a dark condition, then cleaning and drying, and repeating the step for 3 times;

d. and c, placing the sample obtained in the step c in a mixed solution of 5mM phosphate PBS buffer solution, 0.2mg/mL I type collagen and 200mM calcium chloride at room temperature, standing for 12 hours, cleaning and drying to obtain the collagen.

Example 2

The preferred embodiment of the invention provides a method for preparing a coating for promoting bone repair and regeneration on the surface of degradable metal zinc, which comprises the following specific steps:

a. grinding the metal zinc to 2000# by using water abrasive paper; sequentially cleaning the raw materials with deionized water and absolute ethyl alcohol under ultrasonic conditions for 4 times, 3 minutes each time, and drying for later use;

b. b, immersing the metal zinc base layer obtained in the step a in a mixed solution of 0.04M zinc nitrate solution and 0.6M sodium hydroxide solution at the temperature of 70 ℃ for 2 hours to obtain a zinc oxide layer;

c. b, placing the zinc oxide layer obtained in the step b in a dopamine solution with the concentration of 2.5mg/ml for soaking for 7 hours at room temperature in a dark condition, then cleaning and drying, and repeating the step for 3 times;

d. and c, placing the sample obtained in the step c in a mixed solution of 4mM phosphoric acid PBS buffer solution, 0.08mg/mL I type collagen and 220mM calcium chloride at room temperature, standing for 20 hours, cleaning and drying to obtain the collagen.

Example 3

The preferred embodiment of the invention provides a method for preparing a coating for promoting bone repair and regeneration on the surface of degradable magnesium metal, which comprises the following specific steps:

a. grinding the magnesium metal to 2000# by using water abrasive paper; sequentially cleaning the raw materials by deionized water and absolute ethyl alcohol under ultrasonic conditions for 3 times, 3 minutes each time, and drying for later use;

b. b, immersing the magnesium metal substrate obtained in the step a in a mixed solution of 0.06M zinc nitrate solution and 0.5M sodium hydroxide solution at the temperature of 70 ℃ for 3 hours to obtain a zinc oxide layer;

c. b, placing the zinc oxide layer obtained in the step b in a dopamine solution with the concentration of 1.5mg/ml for soaking for 7 hours at room temperature in a dark condition, then cleaning and drying, and repeating the step for 3 times;

d. and c, placing the sample obtained in the step c in a mixed solution of 6mM phosphate PBS buffer solution, 0.12mg/mL I type collagen and 200mM calcium chloride at room temperature, standing for 17h, cleaning and drying to obtain the collagen.

Experimental example 1

The microstructure of the product prepared in example 1 was observed using a scanning electron microscope. As can be seen from the scanning result (figure 1), the whole surface of the sample modified by the method is of a mutually staggered flaky micro-nano structure, and the whole coating is completely covered. The scanning structure indicates that the coating is successfully built on the surface of the zinc metal.

Experimental example 2

The inorganic phase composition and binding state of the product obtained in example 1 were observed using XRD and XPS survey spectra.

As can be seen from the XRD spectrum (fig. 2), the sample modified by the method of the present invention shows peaks of zinc oxide, calcium phosphate and zinc calcium phosphate compared to pure zinc metal, so that the main inorganic components of the hybrid coating are zinc oxide, calcium phosphate and zinc calcium phosphate.

As can be seen from the comparison of XPS high resolution (fig. 3), the sample modified by the method of the present invention has O C, O-C and N peaks, but the pure zinc surface does not appear; in addition, the appearance of Zn-O and N-Zn peaks is sufficient to illustrate the coordination chelation of zinc ions and O, N, which is sufficient to demonstrate the success of hybrid coatings on zinc surfaces.

Experimental example 3

The corrosion resistance of the product prepared in example 1 was tested using potentiodynamic polarization curves.

Compared with pure samples, the sample modified by the method has smaller self-corrosion current density and certain corrosion resistance, which shows that the coating can play a role in regulating and controlling the corrosion degradation of the zinc substrate (figure 4).

Experimental example 4

The zinc metal and the product obtained in example 1 were examined for their osseo-compatibility and their endothelialising capacity.

1. This example used MC3T3-E1(ATCC CRL-2594, US) to verify the osteogenic compatibility of the samples, and osteoblasts used α -MEM (Hyclone) medium supplemented with 10% FBS. HUVECs cells were used for the verification of the endothelial promoting ability, and F-12DMEM (Hyclone) medium supplemented with 10% FBS was used for the endothelial cells. After the cells were cultured until the monolayer was approximately 80% confluent, the cells were seeded.

2. The specific preparation method of the sample leaching solution used in the experiment comprises the following steps of carrying out ultraviolet sterilization on a sample for 30 minutes according to the length of 1.25cm²The serum-containing medium was added dropwise to the medium in a standard/mL format and allowed to stand in an incubator (37 ℃ C., 5% CO2) for 24h before being removed for use.

3. The cell inoculation step is as follows:

(a) preparation before experiment: carrying out high-temperature sterilization treatment on the experimental equipment;

(b) placing the sterilized experimental equipment (a centrifuge tube, tweezers, an injector, a suction tube, a gun head and the like) into a super clean bench for ultraviolet irradiation for more than 30 minutes;

(c) washing osteoblast/endothelial cell with good growth condition and normal shape with normal saline solution three times, adding pancreatin to ensure complete contact of pancreatin and cell, and digesting for 1-2min (observing under optical microscope, adding culture medium containing serum to stop digestion if cell shape is shrunk, rounded and shiny). Blowing the cells uniformly by using a suction tube, adding the cell suspension into a centrifuge tube, centrifuging for 5min at 1200rpm/min, and carrying out cell resuspension;

(d) dropwise adding 1mL of cell resuspension liquid to a cell counting plate for counting; the planting density of osteoblasts and endothelial cells adopted in the experiment is 2 multiplied by 10⁴cells/mL；

(e) The diluted cell suspension was inoculated to the surface of a 96-well plate at 200. mu.L per well using a pipette gun, and the plate was placed in an incubator (37 ℃ C., 5% CO)₂) After one day of culture, the medium was replaced with the aforementioned cell extract and cultured for 1, 3,5 days.

4. The experiment detects the cell activity through CCK-8, and comprises the following specific steps: cells were incubated for 3h at each time point with media containing 10% CCK-8, removed and tested for absorbance using a microplate reader and converted to percent cell activity.

5. Cytoskeleton was stained with Rhodamine 123 (Rhodamine-phaloidin) to analyze the growth morphology and the adhesion amount of osteoblasts on the sample surface, and observation and analysis were performed under a fluorescent microscope. The dyeing steps are as follows:

(a) taking out the culture plate, sucking out the culture medium, washing with normal saline for three times, and adding 2.5% glutaraldehyde solution for fixation for 4 h;

(b) sucking out glutaraldehyde, washing with normal saline for three times, blow-drying, dripping 60 mu L rhodamine under the condition of keeping out of the sun, dyeing for 15 minutes, washing with normal saline for three times, blow-drying, and observing through a fluorescence microscope.

Osteoblast culture results as shown in FIG. 5, the unmodified pure zinc surface cells were shriveled at three time points and the number of cells was very small. The modified sample surface cells showed better cell morphology, spread well, and the number of cell adhesion increased with increasing culture time. The results of the cell activity test also show a similar trend, and the cell activity is higher than 75% by 5 days of culture (dotted line in the figure), which indicates that the modified sample has good biocompatibility and can promote the proliferation, adhesion and growth of osteoblasts.

The results of endothelial cell culture are shown in FIG. 6, in which the number of unmodified pure zinc surface cells was very small and the cells were wrinkled. The cell adhesion quantity of the modified sample is close to that of the control group, and the cell spreading form is good. The same trend was shown for the detection of cell viability, all above 75%, approaching and even exceeding the control group. Indicating that the modified sample can promote the proliferation, adhesion and growth of endothelial cells.

Experimental example 5

The ability of zinc metal and the product prepared in example 1 to promote endothelial cell migration was examined.

The cell inoculation steps are consistent with the above steps, the endothelial cells are inoculated in a 24-well plate in the experiment, and the planting density is 5 multiplied by 10⁴cells/ml, after culturing to a fully plated well plate, scarification with a 1ml tip and washing three times with sodium chloride, adding the above leaching solution to replace the medium, in an incubator (37 ℃, 5% CO)₂) The culture was carried out for 8h and 24h, and photographed by observing with an optical microscope. The area of endothelial cell migration was calculated using Image J.

As shown in FIG. 7, it can be seen from the optical microscope that the number and area of the endothelial cells migrated were higher in the modified sample after 8h of culture than in the control sample, and the modified sample spread uniformly after 24h of culture. Migration area statistics showed the same trend. The sample after surface modification has good capacity of promoting endothelial cell migration. Indicating the potential angiogenic potential of the coating.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.