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

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 scaffold 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 technique

As one of the most important organs of the human body, bone has a certain self-healing ability, but for bone defects exceeding the critical size, it must be treated with appropriate bone substitute materials. Under the background that traditional bone repair materials cannot meet the treatment needs, it is urgent to find an artificial bone repair material with excellent performance to solve the current predicament. At present, the mainstream artificial bone repair materials include metal materials, bioceramics, polymer materials, and composite materials. Among them, bioceramics have become a research hotspot due to their good biocompatibility, certain degradability, and potential osteoconduction and osteoinduction.

Generally, biomaterials used for bone repair require a three-dimensional porous structure. The through-hole porous structure not only provides space for new bone tissue and blood vessels to grow into, but also acts as a transport channel for nutrients and metabolites. At the same time, studies have shown that the pore size is greater than 300 μm The bone scaffold can also promote the growth of blood vessels. Additive manufacturing, also known as 3D printing technology, has developed rapidly in recent years due to its unique advantages in high-precision, personalized manufacturing and complex shape construction, and has gradually integrated into all walks of life. Among them, DLP-based light curing forming technology With the advantages of high precision and fast forming, it has become the first choice for the preparation of bioceramic bone scaffolds.

Due to its good biocompatibility and high affinity with human tissues, degradable biopolymer materials have been discovered and tried to be applied in the field of tissue engineering for a long time, but limited mechanical properties limit its development. It is used as a coating material for surface modification of bone repair materials. Coating a layer of polymer material on the surface of the ceramic stent can enhance the strength and toughness of the ceramic stent to a certain extent. After implanting in the human body, it will also swell to a certain extent, which can stabilize the stent and avoid aseptic infection. occurrence of inflammation. In addition, it can also be used as a carrier for drug delivery to endow the stent with certain functionality. However, as a gel-like material, how to effectively coat the surface of the ceramic stent without affecting the pore structure of the stent has become a key technical difficulty affecting the application of polymer coatings.

Postoperative infection is one of the main reasons for the failure of implantation surgery. In the past few decades, the abuse of antibiotics has led to strain mutations, and the number of surgical failures caused by antibiotic resistance has increased year by year. The researchers found that some metal cations, such as Ag ⁺ , Mg ²⁺ , Zn ²⁺ and Cu ²⁺ , have a killing effect on bacteria, and will not or minimally lead to strain evolution. At the same time, studies have shown that a small amount of trace element doping can improve the osteogenic and vascularization properties of scaffolds. However, low-concentration ion doping cannot have sufficient antibacterial effect, and high-concentration ion doping will produce certain toxicity to normal tissue cells. Therefore, how to balance the antibacterial and osteogenic properties of bone repair materials has become a difficult problem for researchers.

Studies have shown that infection mostly occurs in the early postoperative period, and the main reasons for bacterial infection are firstly the introduction of bacteria during the operation, and secondly, most bone repair scaffolds themselves are prone to bacterial colonization. Therefore, if the stent material itself has certain antibacterial properties, in the early stage of implantation, it can prevent bacterial colonization while killing bacteria in situ, which can undoubtedly improve the success rate of surgery to a large extent.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the inventors realized that two or more polymer materials with opposite charges can be alternately coated, and a good coating effect can be achieved by utilizing the adsorption between anions and cations. On the basis of this technology, multi-layer coating can be carried out, and the porous structure of the scaffold can still be retained, and at the same time, different layers of trace elements can be doped with gradient concentrations by means of ion cross-linking, so as to achieve different periods after stent implantation. Release different concentrations or different kinds of trace elements to meet the actual needs of the human body.

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

A preparation method of a bone repair scaffold with gradient coating, comprising the following steps:

Step 1: Mix the photosensitive resin, dispersant and bioceramic powder, the mass percentage of the photosensitive resin is 20% to 50%, the mass percentage of the dispersant is 2% to 4%, and the remaining components are bioceramic powder; Stir for 15 minutes to obtain a uniformly mixed slurry;

Step 2: The slurry obtained in the above step 1 is formed into a porous ceramic body by 3D printing, and after cleaning, the ceramic body is subjected to degreasing and high temperature sintering treatment to obtain a porous support matrix;

Step 3: preparing a coating solution, respectively preparing a cationic polymer solution with a mass concentration of 0.1% to 1% and an anionic polymer solution with a mass concentration of 0.1% to 1%;

Step 4: preparing a metal ion solution with a concentration range of 2 μmol/L to 40 μmol/L, and a series of gradient concentration metal ion solutions corresponding to the number of coating layers;

Step 5: Alternately soak the porous stent obtained in the above step 2 in the coating solution prepared in step 3 and step 4 in the order of cationic polymer solution, anionic polymer solution, and metal ion solution; in each round of coating, Replace the metal ion solution with the solution corresponding to the current number of layers; after each dip coating of a solution, the stent needs to be washed in deionized water to remove the excess solution that is not adsorbed, and dried in a drying oven at 50 °C for 5 to 10 minutes before proceeding. Dip coating of the next solution; after coating the set number of layers, place the scaffold in a drying oven at 50° C. to dry for 6-18 hours to obtain the bone repair scaffold.

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

Further, in the step 2, considering the size shrinkage of the stent after degreasing and sintering of the stent and the space reserved for coating, a porous stent model with a pore size of 500 μm to 1500 μm is selected.

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

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, a series of gradient concentration solutions prepared in the step 4 are free to adjust the ion concentration and ion type of each layer according to the desired 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 immersed in the polymer solution for 1 to 12 hours, and then centrifuged in a centrifuge with a rotational speed of 500 to 1500 rmp for 5 to 12 hours. 10min, remove the excess solution to ensure the bonding strength of the coating and the substrate;

Further, in the step 5, the stent is soaked in each solution for 10-60s; the overall layer thickness of the polymer coating is controlled at 50μrn-200μm, and the number of coating layers depends on the thickness of the single-layer coating; each layer The coating comprises polymers of anionic and cationic nature adsorbed together by electrostatic interactions and metal cations doped in the polymers of anionic nature in cross-linked form.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The matrix material used in the present invention is a bioceramic, which has a wide research base and exhibits good biocompatibility, and some materials can be degraded, and the degradation products can promote bone repair and bone tissue growth. important role.

(2) The present invention adopts the 3D printing technology to prepare the porous scaffold, and the technology can produce the scaffold suitable for the pore size of the bone repair according to the actual needs. In addition, using different surface adhesion materials, due to their different rheological properties, 3D printing technology can prepare suitable pore sizes to meet the adhesion of materials.

(3) In the present invention, the strength and toughness of the porous ceramic bone repair stent are improved by coating the polymer coating doped with trace elements on the basis of the porous ceramic stent, and the selected polymer materials are all degradable Bioactive materials will not be toxic to cells after implantation in the human body, nor will it cause immune rejection in the human body. At the same time, after the stent with polymer coating is implanted into the human body, it can swell to a certain extent, which can stabilize the stent. effect.

(4) The present invention prepares the polymer coating of the multilayer structure through the electrostatic adsorption between the cationic and anionic polymers, which ensures the stability of the coating and the good bonding effect with the ceramic substrate, and at the same time, the single The layer coating thickness is controlled to a very small value to achieve multi-layer coating while preserving the pore structure of the porous ceramic scaffold.

(5) In the present invention, multi-functional (multi-purpose) trace elements are doped into polymer materials through the cross-linking effect of divalent metal cations and anionic groups in anionic polymers. The degradation of the polymer is gradually released, thereby achieving a stable and slow release of metal ions.

(6) In the present invention, the multi-layer polymer coating is applied on the surface of the porous ceramic stent, and the polymer coating is doped with multi-functional and multi-purpose trace elements. The best ion release effect can be achieved by changing the species; for example, by controlling the concentration of metal ions such as Mg ²⁺ , Zn ²⁺ , and Cu ²⁺ with antibacterial effect from the outer layer to the inner layer, the Ca ²⁺ ensures that the total metal ion concentration in each coating is constant, so as to reduce the influence of metal ion concentration changes on the properties of the polymer, and finally achieve high-concentration release of metal ions with antibacterial effect in the early stage of bone scaffold implantation to enhance the antibacterial effect. It is released at a low concentration in the later stage, reducing its toxic effect on cells and stimulating osteogenesis of cells at the same time.

Description of drawings

Fig. 1 is the preparation flow chart of the preparation method of the present invention;

Fig. 2 is the structural representation of the gradient coating of the present invention;

Fig. 3 is the partial enlarged schematic diagram of the gradient coating of the present invention;

4 is a cell growth state diagram of the porous scaffold with gradient antibacterial coating and MC3T3-E1 cells co-cultured for 7 days in Example 1 of the present invention;

FIG. 5 is a comparison diagram of the antibacterial effect of the porous stent with the gradient antibacterial coating and the porous stent without the gradient antibacterial coating according to Example 1 of the present invention.

Detailed ways

In order to more clearly understand the above objects, features and advantages of the present invention, the solution of the present invention will be further described below. It should be understood that the embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1:

A preparation method of a bone repair scaffold with a gradient antibacterial coating, comprising the following steps:

Step 1: Mix the photosensitive resin, dispersant and bioceramic powder, the mass percentage of the photosensitive resin is 50%, the mass percentage of the dispersant is 3%, and the rest components are β-tricalcium phosphate bioceramic powder; The mixer has been stirred at a high speed of 1100rmp for 15min to obtain a uniformly mixed slurry;

Step 2: Use the slurry obtained in the above step 1 to prepare a porous ceramic body by DLP photocuring; the porous support model is a body diagonal porous model, the aperture is 1500 μm, and a cylindrical model is used, and the size is 9 mm in diameter. The height is 4.5mm; put the printed ceramic body into a beaker filled with absolute ethanol, and put the beaker into an ultrasonic cleaner for 5 minutes to remove excess slurry; after that, use an air gun to blow off the residual alcohol on the ceramic surface And slurry, secondary curing and drying under ultraviolet light of 405nm wavelength; finally, the ceramic body is degreasing and high-temperature sintering. In the stage of 120～360℃, the heating rate is 0.5℃/min, and the temperature is kept at 360℃ for 5h; in the stage of 360～420℃, the heating rate is 0.5℃/min, and the temperature is kept at 420℃ for 5h; in the stage of 420～510℃, the heating rate is 0.5°C/min, hold at 510°C for 5h; in the stage of 510-900°C, the heating rate is 0.5°C/min, hold at 900°C for 5h; furnace cool to room temperature. The sintering curve is as follows: 25～1300℃ stage, heating rate is 1℃/min, 1300℃ hold for 5h; 1300～900℃ stage, cooling rate is 2℃/min; furnace cooled to room temperature.

Step 3: Prepare a coating solution, prepare a chitosan solution with a mass concentration of 0.2%, the solvent is an acetic acid solution with a mass fraction of 1 wt%, the chitosan is selected with a degree of deacetylation ≥ 95%, and the viscosity is between 100-200mpa.s ; The preparation mass concentration is 0.2% sodium alginate solution, the solvent is deionized water, and the sodium alginate is analytically pure;

Step 4: prepare a mixed solution of ZnCl ₂ and CaCl ₂ , solution A: mixed solution of 5 μmol/L ZnCl ₂ and 20 μmol/L CaCl ₂ ; solution B: mixed solution of 15 μmol/L ZnCl ₂ and 10 μmol/L CaCl ₂ ; Solution C: 25 μmol/L ZnCl ₂ solution; the solvent is deionized water.

Step 5: Soak the porous scaffold prepared in the above step 2 in the chitosan solution for 12 hours, then centrifuge it in a centrifuge with a rotation speed of 1500 rpm for 5 minutes, and put it into a drying box at 50 °C for 5 minutes; then put the scaffold into Soak in sodium alginate solution for 20s, put it in a drying oven at 50°C for 5min; then put the stent in solution A, soak it for 20s, put it in a drying oven at 50°C for 5min, and complete the coating of the first layer. After that, the coating method of each layer is similar, except that the chitosan solution only needs to be soaked for 20s, and no centrifugal operation is required; the total number of layers of the coating is 24 layers, and the inner 8 layers are soaked in solution A. , the middle 8 layers were immersed in solution B, and the outer 8 layers were immersed in solution C; after the coating was completed, the scaffold was dried in a drying oven at 50° C. for 12 hours to obtain the bone repair scaffold.

The bone repair scaffold with gradient antibacterial coating obtained in the above steps was subjected to high temperature sterilization at a temperature of 120 ° C, and then the scaffold was placed in a 24-well cell culture plate, and MC3T3-E1 cells were added to each well at a concentration of 104 cells/well. 1mL of cell culture medium, put the 24-well plate into a cell incubator with a temperature of 37 °C and a _CO content of 5% for 7 days, and the culture medium was replaced every two days after washing with PBS. , as shown in Figure 4, after 7 days of cell culture, under the light microscope, the scaffolds showed a good growth state and adhesion effect.

Inoculate 100 μL of Escherichia coli bacterial solution with a bacterial concentration of 10 ⁶ CFU/mL on the surface of the bone repair scaffold with a gradient antibacterial coating obtained in the above step (the bacterial solution is diluted with PBS), cultured at 37 ° C for 2 hours, and then inoculated. After the scaffold was fully shaken in 30 mL of PBS solution, 100 μL was taken from it and spread evenly on the surface of the solid agar medium, placed in a bacterial incubator at 37 °C for 24 hours, and the colony growth was observed, and an uncoated scaffold was selected as the The control group, the same operation, as shown in Figure 5, is a comparison diagram of the antibacterial effect of the porous scaffold with the gradient antibacterial coating and the porous scaffold without the gradient antibacterial coating.

The cytocompatibility and antibacterial properties of the bone repair scaffold with gradient antibacterial coating obtained in Example 1 were tested. The test results are shown in Figure 3 and Figure 4. A polymer coating doped with a small amount of zinc ions can achieve an antibacterial effect on the stent with little toxicity to cells.

The above are only preferred embodiments of the present invention, but the technical features of the present invention are not limited thereto. It should be pointed out that any simple changes, equivalent replacements, etc. made based on the present invention in order to achieve basically the same technical effect are all covered within the protection scope of the present invention.

Claims (8)

1. A preparation method of a bone repair scaffold with a gradient coating is characterized by comprising 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 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%;

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.

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.

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