CN115212354A

CN115212354A - Bone repair stent with gradient coating and preparation method thereof

Info

Publication number: CN115212354A
Application number: CN202210039664.3A
Authority: CN
Inventors: 沈理达; 何志静; 张寒旭; 焦晨; 吴俊男
Original assignee: Nanjing University of Aeronautics and Astronautics
Current assignee: Nanjing University of Aeronautics and Astronautics
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-10-21

Abstract

The invention relates to the technical field of medical materials, and particularly discloses a bone repair stent with a gradient coating and a preparation method thereof. The bone repair scaffold comprises a porous ceramic matrix and a polymer coating on the surface of the porous ceramic matrix, wherein the polymer coating loads trace elements in a gradient concentration mode, and the trace elements are selected from one or more of magnesium, zinc, copper, calcium, strontium and iron. According to the invention, the multi-layer polymer coating is coated on the basis of the porous ceramic support, so that the strength and toughness of the porous ceramic support are improved, and meanwhile, the polymer coating has certain swelling property after being implanted, and can play a role in stabilizing the support. Wherein, the microelements with special functions are doped in the polymer coating in the form of ionic crosslinking. Furthermore, the concentration of the trace elements is controlled to be changed from the outer layer to the inner layer of the coating in a gradient manner in a multi-layer coating manner, so that the trace elements with different concentrations or different types can be released at different periods after the stent is implanted, and the actual requirements of a human body can be met.

Description

Bone repair stent with gradient coating and preparation method thereof

Technical Field

The invention relates to the technical field of medical materials, in particular to a bone repair bracket with a gradient coating and a preparation method thereof.

Background

The skeleton as one of the most important organs of human body has certain self-repairing capability, but for bone defects exceeding critical dimension, proper bone substitute materials are required to be matched for auxiliary treatment. Under the background that the traditional bone repair material can not meet the treatment requirement, people urgently expect to find an artificial bone repair material with excellent performance to solve the current dilemma. Currently, the mainstream artificial bone repair materials include metal materials, biological ceramics, high polymer materials, composite materials and the like. Among them, bioceramics have become a hot point of research due to their good biocompatibility, certain degradability, and potential osteo-conduction and osteo-induction.

In general, biomaterials for bone repair are required to have a three-dimensional porous structure, the through porous structure not only provides a growing space for new bone tissues and blood vessels, but also can be used as a transport channel for nutrients and metabolites, and meanwhile, researches show that a bone scaffold with the pore size of more than 300 μm can also promote the growth of blood vessels. Due to the unique advantages of the additive manufacturing technology, the 3D printing technology in high precision, personalized manufacturing and complex shape construction, the development is rapid in recent years and gradually integrated into various industries, and the DLP-based photocuring forming technology becomes the first choice for preparing the biological ceramic bone scaffold with the advantages of high precision and rapid forming.

The degradable biopolymer material has been discovered and tried to be applied to the field of tissue engineering for a long time due to its good biocompatibility and extremely high affinity with human tissues, but its development is limited by limited mechanical properties, and at present, it is often applied to the surface modification of bone repair materials as a coating material. The surface of the ceramic bracket is coated with a layer of polymer material, so that the strength and toughness of the ceramic bracket can be enhanced to a certain degree, and the ceramic bracket can generate certain swelling after being implanted into a human body, thereby playing a role in stabilizing the bracket and avoiding the occurrence of aseptic infection and inflammation. In addition, the polymer can also be used as a carrier for drug delivery to endow the stent with certain functionality. However, how to effectively coat the polymer material on the surface of the ceramic stent as a gel-like material without influencing the pore structure of the stent becomes a key technical difficulty influencing the application of the polymer coating.

Postoperative infection is one of the leading causes of implant surgery failure, and over the past few decades, antibiotic abuse has led to strain mutation, with annual growth in cases of surgery failure due to antibiotic resistance. The investigators have found some metal cations, e.g. Ag ⁺ 、Mg ²⁺ 、Zn ²⁺ And Cu ²⁺ The like has a killing effect on bacteria, and does not or extremely possibly cause the strain to evolve. Meanwhile, research shows that a small amount of trace elements can be doped to improve the osteogenic property and the vascularization promoting property of the scaffold material. However, the low concentration ion doping cannot achieve sufficient antibacterial effect, and the too high concentration ion doping can generate certain toxicity to normal tissue cells. Therefore, how to combine the antibacterial and osteogenic properties of the bone repair material becomes a difficult problem for researchers.

Researches show that the infection often occurs in the early postoperative period, and the main reasons for causing bacterial infection are bacterial introduction in the operation process and bacterial colonization easily caused by most of bone repair scaffolds. Therefore, if the scaffold material has a certain antibacterial property, bacteria can be killed in situ while the bacterial colonization is prevented in the early stage of implantation, so that the success rate of the operation can be improved to a great extent.

Disclosure of Invention

In order to solve the problems of the prior art, the present inventors have recognized that a good coating effect can be achieved by alternately coating two or more polymer materials having opposite charges using adsorption between cations and anions. On the basis of the technology, multilayer coating can be carried out, the porous structure of the stent is still kept, and meanwhile, the microelements with gradient concentration are doped in different layers in an ion crosslinking mode, so that the microelements with different concentrations or different types are released at different periods after the stent is implanted so as to meet the actual requirements of a human body.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

a preparation method of a bone repair scaffold with a gradient coating comprises the following steps:

step 1: mixing photosensitive resin, a dispersing agent and biological ceramic powder, wherein the mass percent of the photosensitive resin is 20-50%, the mass percent of the dispersing agent is 2-4%, and the rest components are the biological ceramic powder; stirring at high speed for 15min to obtain uniformly mixed slurry;

step 2: forming the slurry obtained in the step 1 into a porous ceramic blank by adopting a 3D printing mode, and degreasing and sintering the ceramic blank at high temperature after cleaning to obtain a porous support matrix;

and step 3: preparing coating solution, namely respectively preparing cationic polymer solution with the mass concentration of 0.1-1% and anionic polymer solution with the mass concentration of 0.1-1%;

and 4, step 4: preparing a metal ion solution, wherein the metal ion solution with a concentration range of 2-40 mu mol/L and a series of gradient concentrations corresponding to the number of coating layers is prepared;

and 5: alternately soaking the porous scaffold prepared in the step 2 in the coating solution prepared in the step 3 and the step 4 according to the sequence of the cationic polymer solution, the anionic polymer solution and the metal ion solution; in each round of coating, the metal ion solution is replaced by a solution corresponding to the current layer number; after each solution is dip-coated, the bracket needs to be cleaned in deionized water to remove the unadsorbed redundant solution, and is dried in a drying oven at 50 ℃ for 5-10 min before the next solution is dip-coated; and after the set number of layers is fully coated, placing the scaffold in a drying box at 50 ℃ for drying for 6-18 h to obtain the bone repair scaffold.

Further, in step 1, the bioceramic material includes one or more of zirconia, alumina, calcium phosphate, hydroxyapatite, calcium silicate, magnesium silicate, and calcium sulfate.

Furthermore, in the step 2, a porous support model with the pore size of 500-1500 μm is selected in consideration of the size shrinkage of the support after degreasing and sintering and the reserved space for coating the coating.

Further, in the step 3, the cationic polymer may be one or more selected from chitosan, poly (diallyldimethylammonium chloride) (PDDA), polylysine (PLL), and the anionic polymer may be one or more selected from sodium alginate, hyaluronic acid, and polyacrylic acid (PAA); the polymer material is selected with low or medium molecular weight to ensure proper viscosity, so that the thickness of the coating layer can be conveniently controlled in a thinner range; the polymer materials are all degradable type biocompatible materials.

Further, in the step 4, the metal ions include one or more of calcium, magnesium, zinc, copper, strontium and iron ions; as the coordinating anion of the metal cation, chloride ion or nitrate ion can be selected.

Further, in the step 4, the ion concentration and the ion type of each layer of the solution with the gradient concentration prepared in the step 4 are freely adjusted according to the expected ion release effect;

further, in the step 5, when the stent is coated with the first layer of the first polymer solution, the stent is soaked in the polymer solution for 1 to 12 hours, and then is centrifuged in a centrifuge with the rotating speed of 500 to 1500rmp for 5 to 10 minutes, so that the redundant solution is removed, and the bonding strength of the coating and the matrix is ensured;

further, in the step 5, the stent is soaked in each solution for 10 to 60 seconds; the integral layer thickness of the polymer coating is controlled to be 50-200 mu m, and the number of the coating layers depends on the thickness of the single-layer coating; each coating comprises a polymer of anionic and cationic nature adsorbed together by electrostatic interaction and metal cations in cross-linked form doped in the polymer of anionic nature.

Compared with the prior art, the invention has the beneficial effects that:

(1) The matrix material adopted by the invention is biological ceramic, the material has wide research foundation and shows good biocompatibility, and some materials can be degraded, and the degradation product plays an important role in promoting bone repair and bone tissue growth.

(2) According to the invention, the porous scaffold is prepared by adopting a 3D printing technology, and the technology can produce the scaffold with a pore size suitable for bone repair according to actual requirements. In addition, different surface adhesion materials are used, and due to different rheological properties, the 3D printing technology can prepare a proper pore size to meet the adhesion of the materials.

(3) The polymer coating doped with trace elements is coated on the basis of the porous ceramic scaffold, so that the strength and the toughness of the porous ceramic bone repair scaffold are improved, and the polymer material is degradable bioactive material, so that the scaffold can not generate toxicity to cells and cause immunological rejection reaction of a human body after being implanted into the human body.

(4) The invention prepares the polymer coating with a multilayer structure through the electrostatic adsorption between the polymers with cationic properties and anionic properties, ensures the stability of the coating and the good fitting effect with a ceramic matrix, simultaneously can control the thickness of a single-layer coating to be a very small value, and retains the pore structure of the porous ceramic support while realizing multilayer coating.

(5) The invention dopes multifunctional (multipurpose) microelements in a polymer material through the crosslinking action of divalent metal cations and anion groups in an anionic polymer, and the multifunctional (multipurpose) microelements are gradually released along with the degradation of the polymer after being implanted into a body, thereby realizing the stable and slow release of metal ions.

(6) The invention realizes the optimal ion release effect by coating a plurality of polymer coatings on the surface of the porous ceramic bracket, doping multifunctional and multipurpose trace elements in the polymer coatings and regulating the concentration and variety change of the trace elements in each coating; for example, by controlling Mg having an antibacterial effect ²⁺ 、Zn ²⁺ 、Cu ²⁺ The concentration of the plasma metal ions is reduced from the outer layer to the inner layer in a gradient way, and then Ca without antibacterial effect is matched ²⁺ The total metal ion concentration in each layer of coating is ensured to be constant, so that the influence of the concentration change of the metal ions on the performance of the polymer is reduced, the metal ions with the antibacterial effect are released at a high concentration in the initial stage of the bone scaffold implantation to strengthen the antibacterial effect, and released at a low concentration in the later stage, the toxic effect of the metal ions on cells is reduced, and the cells are stimulated to form bone.

Drawings

FIG. 1 is a production flow chart of the production method of the present invention;

FIG. 2 is a schematic structural view of a gradient coating of the present invention;

FIG. 3 is an enlarged partial schematic view of the gradient coating of the present invention;

FIG. 4 is a diagram showing the cell growth state of the porous scaffold with a gradient antibacterial coating of example 1 of the present invention and MC3T3-E1 cells cultured together for 7 days;

fig. 5 is a graph comparing the antibacterial effects of the porous scaffold having the gradient antibacterial coating according to example 1 of the present invention and the porous scaffold without the gradient antibacterial coating.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, the scheme of the present invention will be further described below, with the understanding that the examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1:

a preparation method of a bone repair scaffold with a gradient antibacterial coating comprises the following steps:

step 1: mixing photosensitive resin, a dispersing agent and biological ceramic powder, wherein the mass percent of the photosensitive resin is 50%, the mass percent of the dispersing agent is 3%, and the rest is beta-tricalcium phosphate biological ceramic powder; stirring at high speed of 1100rmp for 15min in a vacuum stirrer to obtain uniformly mixed slurry;

step 2: preparing a porous ceramic blank from the slurry obtained in the step 1 by adopting a DLP photocuring forming mode; the porous support model adopts a diagonal porous model, the aperture of the porous support model is 1500 mu m, and a cylindrical model with the size of 9mm in diameter and 4.5mm in height is selected; putting the ceramic blank obtained by printing into a beaker filled with absolute ethyl alcohol, and putting the beaker into an ultrasonic cleaning machine to clean for 5min so as to remove redundant slurry; then, blowing off residual alcohol and slurry on the surface of the ceramic by using an air gun, carrying out secondary curing under ultraviolet light with the wavelength of 405nm, and drying; and finally, carrying out degreasing and high-temperature sintering treatment on the ceramic blank, wherein the degreasing curve is as follows: at the stage of 25-120 ℃, the heating rate is 0.5 ℃/min, and the temperature is kept at 120 ℃ for 2h; in the stage of 120-360 ℃, the heating rate is 0.5 ℃/min, and the temperature is kept for 5h at 360 ℃; at the stage of 360-420 ℃, the heating rate is 0.5 ℃/min, and the temperature is kept at 420 ℃ for 5h; at the stage of 420-510 ℃, the heating rate is 0.5 ℃/min, and the temperature is preserved for 5h at 510 ℃; at the stage of 510-900 ℃, the heating rate is 0.5 ℃/min, and the temperature is preserved for 5h at 900 ℃; and cooling the furnace to room temperature. The sintering curve is: at the stage of 25-1300 ℃, the heating rate is 1 ℃/min, and the temperature is kept at 1300 ℃ for 5h; at the stage of 1300-900 ℃, the cooling rate is 2 ℃/min; and cooling the furnace to room temperature.

And 3, step 3: preparing a coating solution, namely preparing a chitosan solution with the mass concentration of 0.2%, wherein the solvent is an acetic acid solution with the mass fraction of 1wt%, the deacetylation degree of the chitosan is more than or equal to 95%, and the viscosity is between 100 and 200mpa.s; preparing a sodium alginate solution with the mass concentration of 0.2%, wherein the solvent is deionized water, and the sodium alginate is selected to be analytically pure;

and 4, step 4: preparation of ZnCl ₂ And CaCl ₂ Mixed solution, solution a: znCl of 5 mu mol/L ₂ And 20. Mu. Mol/L of CaCl ₂ Mixing the solution; solution B: znCl of 15 mu mol/L ₂ And 10. Mu. Mol/L of CaCl ₂ Mixing the solution; solution C:25 mu mol/L ZnCl ₂ A solution; the solvent is deionized water.

And 5: soaking the porous support prepared in the step 2 in a chitosan solution for 12 hours, then centrifuging for 5min in a centrifuge with the rotating speed of 1500rmp, and drying for 5min in a drying box at the temperature of 50 ℃; then placing the stent into a sodium alginate solution for soaking for 20s, and placing the stent into a drying box at 50 ℃ for drying for 5min; then placing the stent into the solution A to be soaked for 20s, and placing the stent into a drying oven at 50 ℃ to be dried for 5min, thus finishing the coating operation of the first coating; then, the coating mode of each layer of coating is similar, only 20s of soaking is needed when the chitosan solution is soaked, and the centrifugal operation is not needed; the total number of the coating layers is 24, the inner 8 layers are soaked in the solution A, the middle 8 layers are soaked in the solution B, and the outer 8 layers are soaked in the solution C; and after the coating is finished, drying the scaffold in a drying box at the temperature of only 50 ℃ for 12 hours to obtain the bone repair scaffold.

Performing high-temperature sterilization treatment on the bone repair scaffold with the gradient antibacterial coating at the temperature of 120 ℃, then putting the scaffold into a 24-hole cell culture plate, adding 1mL of cell culture medium with the MC3T3-E1 cell concentration of 104 cells/mL into each hole, putting the 24-hole plate into a cell culture plate at the temperature of 37 ℃, and adding CO ₂ The 5% cell culture box was cultured for 7 days, and the culture medium was replaced every two days after washing with PBS, as shown in fig. 4, in which the scaffold showed good growth state and adhesion effect under an optical microscope after 7 days of cell culture.

The concentration of 100. Mu.L of the bacteria was 10 ⁶ And (3) inoculating the Escherichia coli bacterial liquid of CFU/mL to the surface of the bone repair scaffold with the gradient antibacterial coating obtained in the step (the bacterial liquid is diluted by PBS), culturing for 2 hours at 37 ℃, then putting the scaffold into 30mL of PBS solution, fully shaking, uniformly coating 100 mu L of the scaffold on the surface of a solid agar culture medium, culturing for 24 hours in a bacterial incubator at 37 ℃, observing the growth condition of bacterial colonies, selecting an uncoated scaffold as a control group, and performing the same operation, wherein the antibacterial effect comparison graph of the porous scaffold with the gradient antibacterial coating and the porous scaffold without the gradient antibacterial coating is shown in figure 5.

The bone repair scaffold with the gradient antibacterial coating obtained in example 1 was subjected to cell compatibility and antibacterial performance tests, and the test results are shown in fig. 3 and 4, and the test results show that the scaffold has antibacterial effect and hardly generates toxic effect on cells by coating a small amount of zinc ion-doped polymer coating on the porous ceramic scaffold in multiple layers.

The above is only a preferred embodiment of the present invention, but the technical features of the present invention are not limited thereto. It should be noted that any simple changes, equivalent substitutions and the like based on the present invention to achieve substantially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A preparation method of a bone repair scaffold with a gradient coating is characterized by comprising the following steps:

step 2: forming the slurry obtained in the step 1 into a porous ceramic blank by adopting a 3D printing mode, and degreasing and sintering the ceramic blank at a high temperature after cleaning;

and 3, step 3: preparing a coating solution, namely respectively preparing a cationic polymer solution with the mass concentration of 0.1-1% and an anionic polymer solution with the mass concentration of 0.1-1%;

2. The method according to claim 1, wherein the bioceramic material in step 1 comprises one or more of zirconia, alumina, calcium phosphate, hydroxyapatite, calcium silicate, magnesium silicate and calcium sulfate.

3. The method according to claim 1, wherein the porous scaffold model used in step 2 has a pore size of 500 to 1500 μm.

4. The preparation method of claim 1, wherein the cationic polymer in step 2 is one or more selected from chitosan, poly (diallyldimethylammonium chloride) (PDDA) and Polylysine (PLL), and the anionic polymer is one or more selected from sodium alginate, hyaluronic Acid (HA) and polyacrylic acid (PAA); the polymer materials are all degradable type biocompatible materials.

5. The preparation method according to claim 1, wherein the metal ions in step 4 comprise one or more of calcium, magnesium, zinc, copper, strontium and iron ions; as coordinating anion of the metal cation, chloride or nitrate may be used.

6. The method of claim 1, wherein the ion concentration and ion type of each layer are freely adjusted according to the desired ion release effect in the series of gradient concentration solutions prepared in step 4.

7. The method according to claim 1, wherein the coating of the first polymer solution on the stent in step 5 is performed by immersing the stent in the polymer solution for 1 to 12 hours and then centrifuging the stent in a centrifuge at a rotation speed of 500 to 1500rmp for 5 to 10 minutes to remove the excess solution.

8. The method according to claim 1, wherein the soaking time of the stent in each solution in the step 5 is 10 to 60s; the overall layer thickness of the polymer coating is controlled to be 50-200 mu m, and the number of the coating layers depends on the thickness of a single-layer coating.