Patent Description:
With the development of human society and the increase of human activities, injuries to human bone and hard tissues are becoming more and more frequent, so there is an increasing need for corresponding bone tissue fixation, repair, and replacement biomaterials with higher and higher demand.

Conventional bone fixation and replacement materials (e.g., metal materials such as titanium alloy and stainless steel) would likely cause many problems once implanted into the body due to their great difference with human bone tissue in the mechanical property such as the elasticity modulus: stress shielding, centering infection or inflammatory reaction caused by local pH change resulted from the release of metal ions. As a result, the biocompatibility is poor and it is difficult to adapt to the bone healing process. Polymeric materials are unable to be widely used as bone replacement materials considering their poor mechanical properties, especially their poor plastic, flexible, and radial mechanical properties.

As a typical light alloy, magnesium alloy AZ31 has almost the same elasticity modulus as that of human bone, so its mechanical property is approximate to that of human bone and it is an ideal human bone replacement material. Besides, magnesium is a necessary component of human metabolism and biological reactions. Magnesium has a very good promotion effect on bone growth and strengthening when in combination with osteoblasts. Magnesium has a good biocompatibility as a bone replacement material.

Whereas, since magnesium is very chemically active and various ions are present in human body environment, the magnesium and magnesium alloy AZ31 graft materials have a high degradation rate, resulting in an significant increase in pH value in local body fluid environment, which may trigger alkalosis and then cause local inflammatory reaction, finally causing cell death. Thus, controlling the degradation rate of the magnesium alloy AZ31 in vivo becomes a key issue for the application of the magnesium alloy AZ31 as a bone graft material.

On the other hand, when the magnesium-based alloy is used as the replacement material in vivo and in vitro, since it has no significant antibacterial and anti-inflammatory effects, bacterial overgrowth tends to occur in vivo and in vitro, thereby causing additional inflammatory reaction. This further limits the wide application of the magnesium-based alloy.

In order to solve the problem of too fast degradation of the magnesium-based alloy in vivo, various methods have been used to improve the corrosion resistance of the magnesium. The commonly used methods involve strengthening the surface by various physical and chemical means. Currently, in order to make the materials more bioactive and biocompatible, various coatings with biofunctionality are created. However, the defects of the magnesium-based alloy material itself cause the decreases in the functionality of the surface coating and in the possessed activity, affecting the use of the material. Moreover, the current biofunctional coating can hardly take into account the antibacterial and anti-inflammatory effects while improving the corrosion resistance of the material. Therefore, there is an urgent need to prepare a biofunctional coating which not only can improve the corrosion resistance of the magnesium-based alloy, but also has the anti-inflammatory and antibacterial effects.

<CIT> provides a magnesium alloy pipe and a heat treatment method and an application thereof, wherein the heat treatment method for the magnesium alloy pipe comprises the following steps of: carrying out heat treatment to the draw forming magnesium alloy pipe to be treated in vacuum, and then cooling to obtain the magnesium alloy pipe; wherein the vacuum degree of the vacuum is -<NUM> MPa to <NUM> MPa; the heating rate of the heat treatment is <NUM> DEG C/min-20DEG C/min, the heat preservation temperature of the heat treatment is <NUM>-<NUM> DEG C, and the heat preservation time of the heat treatment is <NUM>-<NUM>.

<NPL>) discloses a heat treated magnesium alloy AZ31.

<CIT> discloses a method of forming an implantable article includes providing a biodegradable polymer including anti-thrombogenic groups along the length of the biodegradable polymer, biodegradable groups in the backbone of the biodegradable polymer and a plurality of functional groups adapted to react with reactive functional groups on a surface of the implantable article, and reacting at least a portion of the plurality of functional groups with the reactive functional groups on the surface of the implantable article.

The technical problem to be solved in the present disclosure is to provide a method for manufacturing a small-peptide-coated magnesium alloy biomaterial. The magnesium alloy biomaterial manufactured by this method has a biofunctional coating which is anti-inflammatory and antibacterial. The corrosion resistance of the magnesium alloy can also be improved. The magnesium alloy biomaterial has good biological activity and human body compatibility, and can be applied in the manufacture of a hard-tissue defect repair material.

To solve the above technical problem, the following technological means are adopted in the present disclosure. A method for manufacturing a polypeptide-coated magnesium-based alloy biomaterial includes: ultrasonically cleaning a magnesium-based alloy AZ31 to remove impurities on a surface of the magnesium-based alloy AZ31; dissolving polyurethane with chloroform, and then placing the treated magnesium-based alloy AZ31 into the chloroform solution dissolved with the polyurethane in a plasma reactor, so that the magnesium-based alloy AZ31 is fully enclosed by the solution; taking out the magnesium-based alloy AZ31, and letting the magnesium-based alloy AZ31 stand until the solution on the surface thereof is solidified; and then activating the polyurethane coated on the surface of the magnesium-based alloy AZ31 by using a click reaction in the plasma reactor; and finally placing the surface-activated polyurethane-coated magnesium-based alloy AZ31 into a sodium phosphate solution dissolved with polypeptide and applying vibration to allow them fully react to form the corresponding polypeptide coating. The magnesium alloy AZ31 is a cold-drawn magnesium alloy AZ31; or the magnesium alloy AZ31 is provided by the following steps before the ultrasonically cleaning: fully annealing an original cold-drawn magnesium alloy AZ31 in an interference-free atmosphere which eliminates the original processing stress and the texture structure specificity of the original cold-drawn magnesium alloy AZ31, the fully annealing includes:.

The stable biological coating is formed in the magnesium-based alloy biomaterial in the present disclosure, improving the bacterial inhibition property and the corrosion resistance of the magnesium alloy.

Further, the preferred technical solutions are as follows.

In the ultrasonic cleaning, the cleaning is performed for <NUM> to <NUM> minutes, and the cleaning liquid is pure water or <NUM>% ethyl alcohol, in order to remove the impurities on the surface of the magnesium-based alloy AZ31 and keep the surface of the magnesium-based alloy AZ31 clean.

The chloroform solution of polyurethane is a uniform, colourless, transparent, and viscous solution formed by mixing the white solid polyurethane with colourless and transparent chloroform solution having the purity not less than <NUM>% in a ratio of <NUM>: <NUM> and then being vibrated and stirred at normal temperature.

The magnesium-based alloy AZ31 is placed into the chloroform solution of polyurethane, fully immersed for <NUM> minutes, and intermittently vibrated and stirred.

The magnesium-based alloy AZ31 with the solution on its surface solidified is surface-treated with oxygen plasma at <NUM> for <NUM> minute in the plasma reactor and then stands in the atmospheric environment for <NUM> minutes to further promote the formation of peroxide groups and hydroxyl groups on the surface.

The magnesium-based alloy AZ31 is moved back into the plasma reactor after it is subjected to the further promoting the formation of peroxide groups and hydroxyl groups on the surface. The vacuum degree of the plasma reactor is adjusted to be <NUM>. Acrylic acid vapor is introduced until reaching <NUM> Pa. After reacting for <NUM> minute, the magnesium-based alloy AZ31 is taken out and cleaned for <NUM> minutes in an ultrasonic cleaner. Thereafter, the magnesium-based alloy AZ31 is transferred into a mixed aqueous solution, with pH of <NUM>, of <NUM>/ml N-hydroxysuccinimide and <NUM>/ml <NUM>-ethyl-<NUM>-(<NUM>-dimethylaminopropyl) carbodiimide, vibrated and stirred for <NUM> hours at <NUM>, and then taken out. Finally, the polyurethane coated on the surface of the magnesium-based alloy AZ31 is activated.

The sodium phosphate solution of polypeptide is a <NUM> uniform solution prepared by dissolving the polypeptide F3 with the purity greater than <NUM>% into <NUM> sodium phosphate solution.

The magnesium-based alloy AZ31 is enclosed by the sodium phosphate solution containing the polypeptide, taken out after <NUM> hours of vibration, ultrasonically cleaned in ultrapure water for <NUM> minutes for twice, and dried, so that the manufacture is completed.

The small-peptide coated magnesium-based alloy biomaterial has good biological activity and human body compatibility.

The polishing the surface of the magnesium alloy AZ31 by the waterproof abrasive paper includes initial polishing and finishing; the initial polishing is performed with a <NUM> (<NUM>-mesh) waterproof abrasive paper for <NUM> to <NUM> minutes to remove the original oxide layer, and then the finishing is immediately performed, wherein the finishing is performed with a <NUM> (<NUM>-mesh) to <NUM> (<NUM>-mesh) waterproof abrasive paper for <NUM> to <NUM> minutes, to keep the finish of the surface of the magnesium alloy AZ31.

The initial polishing includes grinding along one direction, with a strength effective to remove the oxide layer, the initial polishing is performed until the dark oxide layer on the surface of the magnesium alloy AZ31 is removed to expose the silver white magnesium metal itself, and a grinding direction in the finishing is perpendicular to the direction of the initial polishing, with a strength smaller than that in the initial polishing, until there is no scratch on the surface of the magnesium alloy AZ31.

Based on its good biological activity and human body compatibility, the small-peptide coated magnesium-based alloy biomaterial can be widely used in artificial prosthesis, implantable replacement material repair of open trauma of human tissue, intraoral dental implant, repair of damage of tissue in body, and manufacture of human biomaterial such as biological catheter, joint bowl, and tubular joint nail.

Referring to <FIG>, a heat treatment method for improving the mechanical property and the biofunctional stability of a magnesium alloy, including steps of:.

The texture structures of the AZ31 before and after the heat treatment are compared by observing metallographic structures via the field emission scanning microscopy. The annealed AZ31 magnesium alloy AZ31 has a grain size of <NUM>, while the original cold-drawn magnesium alloy AZ31 has a grain size of <NUM>. The hardness of the AZ31 is measured by Vickers micro hardness tester, and the change in Vickers hardness before and after the heat treatment is compared. The untreated AZ31 has a Vickers hardness of <NUM> HV, while the AZ31 fully annealed in the interference-free atmosphere has a Vickers hardness of <NUM> HV.

In the method in this Example <NUM>, the process is simple, the operation is convenient, the implementability is high, the new material formed is stable, and can be combined with other material to form a stable structure; the activity of the combined product can be maintained and enhanced; the energy consumption is small, it is easy to produce with short period and easy to be industrialized, and there is no pollution for environment.

The present disclosure will be further described in the below with reference to Example <NUM>.

This example provides a method for manufacturing a polypeptide bio-coating, which can improve the bacterial inhibition property and the corrosion resistance property of the magnesium alloy, via a chemical click reaction (see <FIG>), including the following steps.

The cold-drawn magnesium alloy AZ31 and the magnesium alloy AZ31 fully annealed in the interference-free atmosphere are processed into sheet pieces which have a diameter of <NUM> and a thickness of <NUM>.

The surfaces of the two magnesium alloy samples processed into sheet shapes are cleaned, wherein the cleaning is performed for <NUM> to <NUM> minutes by using ultrasonic wave, and the cleaning liquid is pure water (alternatively, <NUM>% ethyl alcohol), to remove the impurities and adhesions on the surfaces, so that the surfaces of the two samples are kept clean.

The polyurethane produced from Selectophore™ (class MQ100) is selected as a coating agent of the metal surface. First, the white solid polyurethane particles and the colourless and transparent chloroform solution (with the purity ≥ <NUM>%) are mixed in a ratio of <NUM>: <NUM> (W/V) and then vibrated and stirred at normal temperature until the polyurethane is fully dissolved and a uniform, colourless, transparent, and viscous solution is formed.

The two magnesium-based alloys AZ31 (in sheet shape, with diameter of <NUM> and thickness of <NUM>) which have been surface-treated by ultrasonic wave are placed into the chloroform solution of polyurethane, fully immersed for <NUM> minutes, and intermittently vibrated and stirred.

The two magnesium alloy materials with the polyurethane uniformed coated thereon are taken out from the solution, placed into watch-glass, and then put in the fume cupboard for <NUM> hours to allow chloroform to be quickly volatilized so that one uniform, dense, and stable polyurethane coating is formed on the metal surface.

The coated metal sheets are placed into a plasma reactor (manufactured by MiniFlecto®, Plasma Technology GmbH) and surface-treated with oxygen plasma at <NUM> for <NUM> minute, and then stand in the atmospheric environment for <NUM> minutes to further promote the formation of peroxide groups and hydroxyl groups on the surface.

The metals are moved back into the plasma reactor. The vacuum degree is adjusted to <NUM>. Acrylic acid vapor is slowly introduced until reaching <NUM> Pa. After reacting for <NUM> minute, the metals are taken out and cleaned for <NUM> minutes in an ultrasonic cleaner. Thereafter, the metals are transferred into a mixed aqueous solution (with pH of <NUM>) of <NUM>/ml N-hydroxysuccinimide and <NUM>/ml <NUM>-ethyl-<NUM>-(<NUM>-dimethylaminopropyl) carbodiimide, vibrated and stirred for <NUM> hours at <NUM>, and then taken out. Finally, the polyurethanes coated on the surfaces of the magnesium-based alloys AZ31 are activated.

The synthetic high purity (><NUM>%) polypolypeptides F1 and F3 (which were found in the gland at the back of the Australian tree frog) are separately dissolved into <NUM> sodium phosphate solution to prepare an <NUM> uniform solution. The surface-activated polyurethane-coated metal sheets are immersed into the sodium phosphate solution of polypeptide at <NUM>, taken out after <NUM> hours of vibration, ultrasonically cleaned for <NUM> minutes in ultrapure water for twice, and dried in the fume cupboard, so that the maufacture is completed.

The experiment results are shown in Table <NUM>.

With reference to Table <NUM> showing the experiment results, the observation results for specific pH values are as follows.

As shown in <FIG>, in the process of <NUM> hours of in-vitro corrosion, taken a set of samples of annealed AZ31 as example, in the process of <NUM> hours of in-vitro corrosion of pure metal AZ31, the pH value of the DMEM solution corresponding thereto is surged from an initial value of <NUM> to <NUM>. Moreover, in the whole corrosion process, its pH value is highest in this set of samples, suggesting its corrosion speed is most fast. The pH value of the sample of the annealed AZ31 coated with polypeptide F1 coating is significantly higher than the sample of the AZ31 coated with polypeptide F3 coating within the first <NUM> hours. It is suggested that its corrosion speed in this process is always faster than the sample of the AZ31 coated with the F3 coating. At the <NUM> hours, its pH value may be in turn lower than the sample of the AZ31 coated with the F3 coating, which may be resulted from the slight bacterial pollution, and this does not suggest that in the later period its corrosion speed is slower than the latter. Therefore, it is obvious that the sample of the annealed AZ31 coated with the F3 coating has the slowest corrosion speed in the whole process, and the sample of the annealed AZ31 coated with the F1 coating also has a corrosion speed significantly smaller than that of the sample of bare metal annealed AZ31. Therefore, both of the two polypeptide coatings significantly inhibits the release of the metal ions into the DMEM solution, thereby inhibiting the corrosion and significantly increase the corrosion resistance of the material.

With reference to Table <NUM>, the observation results for sample weights are as follows.

As shown in <FIG>, the analysis for the weights of samples in the corrosion process shows obvious weight-decreasing process of three samples in the first <NUM> hours and the significant decrease in the weight of the sample of the bare metal annealed AZ31 after <NUM> hours, which is in consistent with the change of pH value. As the corrosion speed is accelerated, the weight loss of sample is aggravated. While the changes of the weights of the samples of the annealed AZ31 coated with the two polypeptides are not significant within <NUM> hours, especially, the change of the weight of the sample of the annealed AZ31 coated with the polypeptide F3 is still not significant at <NUM> hours. Thus, the observation result for changes of sample weights is consistent with the change of pH values: they suggests the materials coated with the two polypeptides can effectively inhibit the corrosion of the material.

The SEM surface morphology analysis suggests the following.

After <NUM> hours of in-vitro corrosion, large crack phenomenon occurs on the surface of the annealed AZ31 sample (<FIG>). Whereas no such phenomenon occurs on the AZ31 coated with the two polypeptides, with the surface morphologies not too different from the surfaces before the corrosion (<FIG>). This suggests that the corrosion processes of those two materials coated with the coatings is not significant.

The EDS analysis result suggests the following.

Two components, carbon and magnesium oxide, are selected to be analyzed. This is because that the polypeptide contains the carbon element and the situation of the polypeptide coating can be estimated by detecting the change of the carbon element. The product of the corrosion of the magnesium alloy is the magnesium oxide, so the corrosion situation of the material can be estimated from the another aspect by analyzing the change in the magnesium oxide component. From <FIG>, it can be seen that after <NUM> hours of in-vitro corrosion, the sample of the annealed AZ31 coated with the F3 coating produces fewest magnesium oxide, followed by the sample of the annealed AZ31 coated with the F1 polypeptide coating, and the bare metal anneal AZ31 produces most magnesium oxide. Therefore, it is obvious that the corrosion of the bare metal anneal AZ31 is fastest, followed by the annealed AZ31 coated with the F1 coating, the corrosion of the sample of the annealed AZ31 coated with the F3 polypeptide coating is slowest. This suggests that the two coatings can effectively decrease the corrosion speed of the magnesium alloy.

Moreover, it can be found that the carbon content of the AZ31 coated with the F1 polypeptide coating is significantly higher than that coated with the F3 coating, suggesting that the amount of the F1 polypeptide coating is larger than the F3 polypeptide coating. This suggests from the another aspect that the F3 polypeptide coating coated on the metal surface can inhibit the corrosion of the metal material more effectively than the F1 polypeptide coating.

For the method in the Example <NUM>, the process is simple, the operation is convenient, the implementability is high, the new material formed is stable and can be combined with other material to form a stable structure; the activity of the combined product can be maintained and enhanced; the energy consumption is small, it is easy to produce with short period and easy to be industrialized, and there is no pollution for environment.

Claim 1:
A method for manufacturing a polypeptide-coated biomaterial based on a magnesium alloy AZ31, comprising steps of: ultrasonically cleaning the magnesium alloy AZ31 to remove impurities on the surface of the magnesium alloy AZ31; dissolving polyurethane with chloroform, and then placing the treated magnesium alloy AZ31 into the chloroform solution dissolved with the polyurethane, so that the magnesium alloy AZ31 is fully enclosed in the solution; taking out the magnesium alloy AZ31, and letting the magnesium alloy AZ31 stand until the solution on the surface thereof is solidified; and then activating the polyurethane coated on the surface of the magnesium alloy AZ31 by using a click reaction in a plasma reactor; and finally placing the surface-activated polyurethane-coated magnesium alloy AZ31 into a sodium phosphate solution dissolved with polypeptide and applying vibration to allow them fully react to form the corresponding polypeptide coating; wherein:
the magnesium alloy AZ31 is a cold-drawn magnesium alloy AZ31; or
the magnesium alloy is provided by the following steps before the ultrasonically cleaning:
fully annealing an original cold-drawn magnesium alloy AZ31 in an interference-free atmosphere which eliminates the original processing stress and the texture structure specificity of the original cold-drawn magnesium alloy AZ31, the fully annealing comprises:
(<NUM>) polishing a surface of the magnesium alloy AZ31 by a waterproof abrasive paper to remove an original oxide layer on the surface while maintaining or improving the finish of the surface;
(<NUM>) heating the magnesium alloy AZ31 obtained from the step (<NUM>) in an interference-free atmosphere created by an inert gas or a vacuum, wherein the magnesium alloy AZ31 is heated to a temperature of <NUM> to <NUM> in the interference-free atmosphere and then the temperature is kept for <NUM> to <NUM> hours;
(<NUM>) letting the magnesium alloy AZ31 obtained from the step (<NUM>) complete the fully annealing in the interference-free atmosphere, by cooling the magnesium alloy AZ31 obtained from the step (<NUM>) along with a furnace to room temperature, to obtain an equiaxed crystal structure which is texture-even and isotropic.