CN114558170B

CN114558170B - Growth factor-containing skull repair polyether-ether-ketone material and preparation method thereof

Info

Publication number: CN114558170B
Application number: CN202210078053.XA
Authority: CN
Inventors: 唐三; 王喆; 周雄
Original assignee: Asia Biomaterials Wuhan Co ltd
Current assignee: Asia Biomaterials Wuhan Co ltd
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2023-04-25
Anticipated expiration: 2042-01-24
Also published as: CN114558170A

Abstract

The invention particularly relates to a growth factor-containing skull repairing polyether-ether-ketone material and a preparation method thereof, belonging to the technical field of biomedical materials, and comprising the following steps: forming polyether-ether-ketone to obtain a matrix; modifying the matrix by using the polydopamine nanometer microsphere to obtain a modified matrix; mixing a mineralization auxiliary agent and a guided tissue regeneration layer solution to obtain a mineralization guided tissue regeneration layer solution; mixing microspheres loaded with growth factors with mineralized guided tissue regeneration layer solution to obtain mineralized guided tissue regeneration layer solution containing growth factors; mixing a cross-linking agent, a modified matrix and a mineralized guiding tissue regeneration layer solution containing a growth factor to obtain a mixture; reacting, drying, crosslinking, analyzing and sterilizing the mixture to obtain the skull repairing polyether-ether-ketone material; the poly-dopamine nanometer microsphere is used for crosslinking and modifying the polyether-ether-ketone, so that raw materials are easy to obtain, safe and environment-friendly, and hidden dangers brought to human bodies in the preparation process and the use of final products are avoided.

Description

Growth factor-containing skull repair polyether-ether-ketone material and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a growth factor-containing skull repairing polyether-ether-ketone material and a preparation method thereof.

Background

Skull defects are a common secondary disease in clinic. Mainly seen in various traumas and postoperations, such as electric injury, car accident injury, gun-bullet injury, cranium malignant tumor excision, congenital malformation, and after cranium flap decompression operation. In principle, a skull defect with a maximum diameter exceeding 3cm requires a skull reconstruction operation, and clinical symptoms can be generated when the skull defect exceeds 3 cm. Successful skull reconstruction required 3 requirements: (1) maintaining the integrity of the dura mater, i.e., the protection of the brain; (2) The barrier protection between the cranium and the outside, namely biomechanical stability; (3) Maintaining the normal dome-like shape of the head, an aesthetic requirement.

The ideal skull defect repair material meets the following characteristics: (1) convenient acquisition; (2) high biocompatibility; (3) can completely match the defect part and has good ductility; (4) The biological mechanical property is good, the brain barrier is protected, and the external force is resisted; (5) has osteogenic potential; (6) skull image inspection compatibility; (7) resistance to infection.

At present, the skull repairing materials applied to clinic mainly comprise autologous bone, allogeneic bone, xenogeneic allogeneic bone and artificial materials. Autologous bone repair is the gold standard for skull reconstruction. Autologous bone tissue has good bone conductivity and tissue compatibility, no immune rejection reaction and low leakage rate of the thigh after operation, but has the problems of limited supply area, difficult shaping, increased secondary trauma, high bone absorption rate of transplanted bone and the like, and has limited clinical application. Allogeneic bone is generally subjected to special sterilization treatment, and common infectious diseases and immunogenicity are avoided. The allograft can be biologically combined with autologous tissue after surgery, allowing tissue vascularization and ingrowth reconstruction of autologous tissue. However, the high infection rate, bone graft absorptivity, religious and ethical factors after surgery limit the clinical application of the allograft bone on the skull defect. The heterogeneous bone has rich sources, but strong immunogenicity, and the freeze-dried bone, calcined bone and deproteinized bone used clinically are prepared by respectively carrying out freeze-drying, high-temperature calcination, irradiation, decalcification and other treatments on animal bone tissues, so that organic components such as cells, collagen and the like are removed, natural pore structures are reserved, antigenicity is eliminated, but the tissues have small mechanical strength, are loose and fragile, have poor mechanical strength and reduce plasticity.

The artificial skull repairing material commonly used in clinic mainly comprises hydroxyapatite, polymethyl methacrylate, titanium mesh, polyether ether ketone and the like. Polyether-ether-ketone (PEEK) is a thermoplastic special engineering plastic with full aromatic semicrystalline, has excellent physical and chemical properties, mechanical properties, thermal properties and the like, and can keep higher wear resistance and lower friction coefficient at a high temperature of 250 ℃. Polyether-ether-ketone has the characteristics of high melting point (334 ℃), low creep amount, high elastic modulus, excellent friction performance, high temperature resistance, chemical corrosion resistance and the like, and is increasingly used as a biological material for orthopedic implants and prostheses. The polyether-ether-ketone has good biocompatibility, and compared with the traditional hard tissue implantation metal materials (stainless steel, titanium alloy and the like), the polyether-ether-ketone has the elastic modulus equivalent to that of human cortical bone, and can effectively reduce the stress shielding effect after implantation. In addition, polyether-ether-ketone as a biological material has the advantages of radiation permeability, no artifact generated in magnetic resonance scanning and the like, can better evaluate the postoperative recovery condition, and is currently used in the fields of skull, jawbone, vertebra lumbar vertebra, artificial joint, oral defect repair and the like.

The biggest limitation of Polyetheretherketone (PEEK) as a biomedical material is bio-inertness. PEEK material has poor hydrophilicity, and is unfavorable for adhesion and growth of osteoblasts. In addition, the PEEK material is not easy to induce the deposition of hydroxyapatite on bone tissues in a body fluid environment, so that the PEEK material cannot be well bonded with the bone tissues, has poor bonding property and poor implantation effect.

In order to achieve optimal osseointegration after implantation, the surface modification method is the preferred approach to enhance the bioactivity of the PEEK surface without affecting the advantages of the PEEK material. The most common approach is to functionalize PEEK by physically or chemically preparing a bioactive coating. Many of the coatings reported in the prior art (including hydroxyapatite, bioactive particles, etc.) enhance the osseointegration of PEEK implants to some extent. However, these techniques are carried out using 98% concentrated sulfuric acid or at elevated temperatures ((360 ℃ C. Or above)).

Disclosure of Invention

The invention aims to provide a growth factor-containing skull repairing polyether-ether-ketone material and a preparation method thereof, so as to solve the problem that potential safety hazards exist in the existing process of modifying the surface of a PEEK material.

The embodiment of the invention provides a preparation method of a growth factor-containing skull repair polyether-ether-ketone material, which comprises the following steps:

analyzing the head to be repaired to obtain skull defect position information;

forming polyether-ether-ketone according to the skull defect part information to obtain a matrix;

modifying the matrix by using polydopamine nano microspheres to obtain a modified matrix;

mixing a calcium nitrate tetrahydrate solution, a diammonium phosphate solution and a guided tissue regeneration layer solution to obtain a mineralized guided tissue regeneration layer solution;

Dissolving a polymer in methylene dichloride to obtain a polymer solution;

mixing the growth factor with the polymer solution to obtain a first emulsion;

mixing the first emulsion and the first polyvinyl alcohol to obtain a second emulsion;

mixing the second emulsion with second polyvinyl alcohol, and evaporating to obtain microspheres loaded with growth factors;

mixing the microspheres loaded with growth factors with the mineralized guided tissue regeneration layer solution to obtain the mineralized guided tissue regeneration layer solution containing growth factors;

mixing 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride, N-hydroxysuccinimide, the modified substrate and the mineralized guided tissue regeneration layer solution containing growth factors to obtain a mixture;

and (3) carrying out reaction, drying, crosslinking, analysis and sterilization on the mixture to obtain the skull repairing polyether-ether-ketone material containing the growth factors.

Alternatively, the polymer comprises at least one of polycaprolactone, polyhydroxyaliphatic carboxylic acid, polyhydroxybutyrate-polyhydroxyvalerate interpolymer, polylactic acid-polycaprolactone interpolymer, polyanhydride, polysaccharide, lectin, glycosaminoglycan, chitosan, cellulose, acrylate polymer, homopolymer of glycolic acid, homopolymer of lactic acid, and copolymers derived from poly (lactide-co-glycolide);

The growth factors include at least one of vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor, transforming growth factor-beta, and platelet-derived growth factor;

the solute of the guided tissue regeneration layer solution is a degradable membrane material, and the degradable membrane material comprises: hyaluronic acid, carboxymethyl chitosan, sodium carboxymethyl cellulose, chondroitin sulfate, modified cellulose, modified chitosan, alginate, type I collagen, silk fibroin, polylactide, polyglycolide, polycaprolactone, polyhydroxybutyrate, and copolymers thereof.

Optionally, each 100mL of the mineralized guided tissue regeneration layer solution containing the growth factors contains 0.1g to 20g of the microspheres, and the diameter of the microspheres is 1 μm to 100 μm;

in the mineralized guided tissue regeneration layer solution, the mass fraction of the degradable film material is 0.5% -20%; the ratio of the amount of calcium ion substance to the amount of phosphate ion substance is 1-2: 1.

optionally, in the mixture, the mass concentration of the 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride is 0.1 g/mL-0.3 g/mL, and the mass concentration of the N-hydroxysuccinimide is 0.01 g/mL-0.05 g/mL.

Optionally, the drying is freeze drying, wherein the freeze drying comprises first freeze drying and second freeze drying, the drying temperature of the first freeze drying is-80 ℃ to-20 ℃, and the drying time of the first freeze drying is 3h to 24h; the drying temperature of the second freeze drying is-50 ℃ to 37 ℃, the drying time of the second freeze drying is 24h to 72h, and the drying pressure of the second freeze drying is 0.1Pa to 50Pa.

Optionally, the crosslinking comprises glutaraldehyde steam crosslinking and thermal crosslinking, wherein the crosslinking temperature of glutaraldehyde steam crosslinking is 37-52 ℃, the glutaraldehyde steam concentration of glutaraldehyde steam crosslinking is 5-25%, and the glutaraldehyde steam crosslinking time is 2-12 h; the crosslinking temperature of the thermal crosslinking is 100-110 ℃, the crosslinking pressure of the thermal crosslinking is 10-150 Pa, and the crosslinking time of the thermal crosslinking is 12-48 h.

Optionally, the analysis temperature of the analysis is 37-52 ℃, and the analysis time of the analysis is 2-4 d.

Optionally, the reaction temperature of the reaction is 4-6 ℃, and the reaction time of the reaction is 40-60 h.

Optionally, the modification of the substrate by the polydopamine nano microsphere to obtain a modified substrate specifically includes:

Soaking the matrix in the solution of the polydopamine nanometer microsphere for stirring reaction to obtain a modified matrix;

the reaction time of the stirring reaction is 24-48 h, and the preparation method of the solution of the polydopamine nanometer microsphere comprises the following steps:

dissolving tris (hydroxymethyl) aminomethane powder in a solvent to obtain tris (hydroxymethyl) aminomethane solution;

and dissolving dopamine hydrochloride powder in the tris (hydroxymethyl) aminomethane solution to obtain the solution of the polydopamine nanometer microsphere.

Based on the same inventive concept, the embodiment of the invention also provides a skull repairing polyether-ether-ketone material, which is prepared by adopting the preparation method of the growth factor-containing skull repairing polyether-ether-ketone material.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

according to the preparation method of the growth factor-containing skull repairing polyether-ether-ketone material, provided by the embodiment of the invention, the poly-dopamine nano microsphere is used for crosslinking and modifying the polyether-ether-ketone, so that the raw materials are easy to obtain, safe and environment-friendly, and hidden danger to human bodies in the preparation process and in the use of a final product is avoided.

The foregoing description is only an overview of the present invention, and is intended to be implemented in accordance with the teachings of the present invention in order that the same may be more clearly understood and to make the same and other objects, features and advantages of the present invention more readily apparent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a flow chart of a method provided by an embodiment of the present invention.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

applicants found during the course of the invention that: the 3D printing titanium mesh technology solves the problems of shaping and fitting of the repairing material in skull repairing application, has high processing speed, can finish manufacturing the implant within 1-2 days, and reduces the waiting time of patients; the porous through structure similar to the skeleton of a human body can effectively overcome the problems of stress shielding and low biological activity commonly existing in the implant, and can simultaneously minimize heat dissipation in the cranial cavity and maintain the normal heat conduction level. However, a single 3D printing titanium mesh belongs to a biological inert material, has no biological activity, cannot be quickly fused with soft tissues, and cannot effectively promote bone tissue repair and regeneration.

According to an exemplary embodiment of the present invention, there is provided a method for preparing a growth factor-containing skull repair polyether-ether-ketone material, the method comprising:

s1, analyzing a head to be repaired to obtain skull defect position information;

s2, forming polyether-ether-ketone according to the skull defect part information to obtain a matrix;

in some embodiments, the matrix may be a two-dimensional braid of polyetheretherketone material or a polyetheretherketone material made by 3D printing, the matrix being prepared by 3D printing only as follows:

Firstly, performing CT (computed tomography) flat scanning and enhanced scanning on a skull defect part, and then performing three-dimensional reconstruction to determine the position, size, shape and the like of the skull defect; and then pouring the scanning data into software to design a bone defect model, storing the model in an STL format, importing the model into a 3D printer, correcting and calibrating the skull defect model by using the software, and molding the model by using digital equipment according to the data of the model to prepare the polyether-ether-ketone matrix matched with the defect part. The polyether-ether-ketone raw material meets the standard of YY/T0660-2008 'Standard Specification of polyether-ether-ketone (PEEK) polymers for surgical implants'.

S3, modifying the matrix by using the polydopamine nanometer microspheres to obtain a modified matrix;

the polydopamine is mainly secreted by mussel foot glands, contains a large amount of adhesive proteins, is secreted into seawater, gradually solidifies, forms foot filaments and is firmly adhered to the surface of a base material. Mussel-inspired polydopamine coating technology is able to provide strong adhesive interactions with a variety of materials and biomolecules containing amine and thiol functionalities. The polydopamine can promote the adhesion of cells, has good biocompatibility and biodegradability, and can be rapidly developed and widely applied as a simple and general functional surface modification method. The polydopamine not only can be used for modifying regular surfaces, but also can be used for modifying three-dimensional surfaces with higher complexity, such as metal, cardiovascular stent surfaces, carbon nanotubes and the like. After the three-dimensional surfaces are modified by polydopamine, the polydopamine has secondary reactivity, and can be directly modified by connecting biomolecules and medicines or combined with other coating technologies to prepare multifunctional composite coatings. The polydopamine can realize thin thickness when coating the surface of the substrate material, is firmly combined, and can obtain good hydrophilicity and adhesiveness on the surface of the substrate material. The literature reports that the polydopamine coating can promote the in vitro osteogenesis differentiation and calcium mineralization, and the in vivo experiment can promote the osteogenesis and increase the osseointegration. The poly dopamine nanometer microsphere modification is carried out on the polyether-ether-ketone, so that the surface hydrophilicity and the biological activity of the porous polyether-ether-ketone material can be improved, and the surface of the porous polyether-ether-ketone material is modified by a secondary coating, so that the adhesion, proliferation and secretion of extracellular matrixes of vascular endothelial cells on the surface of the material are facilitated, the rapid fusion of the repair material and soft tissues is accelerated, and the surface morphology and the biological performance of the material are more in line with the requirements of the skull repair clinical application.

In some embodiments, the polydopamine nanospheres modify the matrix to obtain a modified matrix, specifically comprising:

s3.1, soaking the substrate in the solution of the polydopamine nanometer microsphere for stirring reaction to obtain a modified substrate;

s3.1.1, dissolving the tris (hydroxymethyl) aminomethane powder in a solvent to obtain a tris (hydroxymethyl) aminomethane solution;

s3.1.2, dissolving dopamine hydrochloride powder in the tris (hydroxymethyl) aminomethane solution to obtain the solution of the polydopamine nanometer microsphere.

Specifically, the preparation method of the dopamine solution comprises the following steps: dissolving tris (hydroxymethyl) aminomethane powder in deionized water, titrating with dilute hydrochloric acid to adjust the pH value to 7.5-10, dissolving dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring for 30-120 min to form dopamine solution. The mass concentration of the dopamine in the dopamine solution is 0.1-20 mg/mL. The dopamine can be subjected to oxidative polymerization reaction in an alkaline (pH is more than 7.5) aerobic environment to form the polydopamine nanometer microsphere. As the pH increases, dopamine gradually undergoes self-polymerization to form polydopamine, which gradually changes color from light brown to dark brown. Adding the prepared polyether-ether-ketone into a dopamine solution, magnetically stirring at room temperature for reaction for 24-48 hours, repeatedly washing the poly-dopamine microsphere modified polyether-ether-ketone with pure water for 2-3 times, and drying in a blast drying oven at 37-52 ℃ for 12-24 hours to obtain a modified matrix.

S4, mixing a calcium nitrate tetrahydrate solution, a diammonium hydrogen phosphate solution and a guided tissue regeneration layer solution to obtain a mineralized guided tissue regeneration layer solution;

specifically, the method for preparing the mineralized guided tissue regeneration layer solution comprises the steps of mixing a calcium nitrate tetrahydrate solution, a diammonium hydrogen phosphate solution and a guided tissue regeneration layer solution to obtain the mineralized guided tissue regeneration layer solution, wherein the mineralized guided tissue regeneration layer solution comprises the following components:

s4.1, preparing a tissue regeneration layer solution;

s4.2, preparing mineralized guided tissue regeneration layer solution; and (3) dropwise adding a tetrahydrate calcium nitrate solution and a diammonium phosphate solution into the guided tissue regeneration layer solution, regulating the pH value to 7-9 by using ammonia water, uniformly mixing, standing the solution, separating out precipitate and washing impurity ions, and thus obtaining the mineralized guided tissue regeneration layer solution.

In some embodiments, the solute of the guided tissue regeneration layer solution is a degradable membrane material comprising: at least one of hyaluronic acid, carboxymethyl chitosan, sodium carboxymethyl cellulose, chondroitin sulfate, modified cellulose, modified chitosan, alginate, type I collagen, silk fibroin, polylactide, polyglycolide, polycaprolactone, polyhydroxybutyrate, and copolymers thereof.

In some embodiments, the mass fraction of the degradable film material in the mineralized guided tissue regeneration layer solution is 0.5% -20%; the ratio of the amount of calcium ion substance to the amount of phosphate ion substance is 1-2: 1.

Preferably, the degradable membrane material is selected from the group consisting of type I collagen and silk fibroin. Specifically, the preparation method of the guided tissue regeneration layer solution comprises the following steps: obtaining a silk fibroin solution and a type I collagen solution; uniformly mixing the silk fibroin solution and the type I collagen solution to obtain a guided tissue regeneration layer solution; wherein the type I collagen in the type I collagen solution is obtained by dissolving type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 0.5% -5%; the silk fibroin in the silk fibroin solution is decomposed in a lithium bromide solution or a calcium chloride ternary system solution, and the mass fraction is 0.5% -20%; the mass ratio of silk fibroin and type I collagen in the guided tissue regeneration layer solution is (0.3-3): 1, a step of; the mass ratio of the added substance of the calcium ions in the mineralized guiding tissue regeneration layer to the protein in the protein solution is 0.002 mol/g-0.02 mol/g, and the molar ratio of the added substance of the calcium ions to the added substance of the phosphate ions is 1-2: 1.

the type I collagen is main structural protein of a spinal animal, is extracellular matrix secreted by osteoblasts in the process of bone formation, and is a scaffold deposited by calcium salt, a bone matrix double-layer promoter and a double-layer template; can promote cell migration, adsorption and differentiation, and can regulate cell growth, and has been approved by the FDA as a biomaterial in the united states, and has a series of collagen bone implant products. The type I collagen has the advantages of low immunogenicity, no toxic or side effect of in vivo degradation products, and the like, but has poor mechanical properties and high degradation rate. Silk fibroin has excellent biocompatibility, biodegradability and better mechanical properties, is easy to sterilize and shape, and is widely applied to ligament tissue repair, vascular tissue transplantation, cartilage tissue repair, skin tissue regeneration, nerve tissue engineering and other aspects, but the mechanical strength of the silk fibroin is far less than that of bone tissue, and the degradation speed of the pure silk fibroin is too slow. The nano-scale hydroxyapatite has good bone conductivity and biocompatibility, but the single hydroxyapatite has larger brittleness and low toughness. Therefore, the composite use of the hydroxyapatite, the type I collagen and the silk fibroin can solve the problem of insufficient performance of a single material, realize the advantage complementation of various materials, ensure that the obtained bone repair material has good mechanical property and controllable biodegradation time, ensure that the bone repair material can maintain the morphological structure for a certain time or a long time and is matched with the biomechanical property of the original bone tissue of the skull at the implantation position; the type I collagen and the silk fibroin are natural fiber proteins, have good biocompatibility and bone induction performance, are favorable for adhesion, proliferation and secretion of extracellular matrixes of seed cells on the surface of the material, accelerate rapid fusion of the repair material and soft tissues, and can stimulate the differentiation of chondrocytes and osteoblasts around an implantation position to form new bone tissues; has good osteoinductive property, and can stimulate the differentiation of chondrocytes and osteoblasts around the implantation site to form new bone tissues.

S5, obtaining microspheres loaded with growth factors;

s5.1, dissolving a polymer in methylene dichloride to obtain a polymer solution;

in some embodiments, the polymer is a biocompatible and biodegradable polymer selected from at least one of polycaprolactone, polyhydroxyaliphatic carboxylic acid, polyhydroxybutyrate-polyhydroxyvalerate interpolymer, polylactic acid-polycaprolactone interpolymer, polyanhydride, polysaccharide, lectin, glycosaminoglycan, chitosan, cellulose, acrylate polymer, homopolymers of glycolic acid or lactic acid, and copolymers derived from poly (lactide-co-glycolide) (abbreviated PLGA). Preferably, the polymer is poly (lactide-co-glycolide) (abbreviated PLGA).

S5.2, mixing the growth factors with the polymer solution to obtain a first emulsion;

in some embodiments, the growth factor may be selected from at least one of Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), transforming growth factor-beta (TGF-beta) (including transforming growth factor-beta 1 (TGF-beta 1), bone morphogenic protein-2 (BMP-2), and the like) or platelet-derived growth factor (PDGF). Preferably, the growth factor is bone morphogenic protein-2 (BMP-2).

Bone morphogenic protein-2 (BMP-2) is considered to be a growth factor having the strongest osteoinductive capacity and promoting bone regeneration, and is capable of inducing proliferation and differentiation of undifferentiated mesenchymal stem cells toward osteoblasts across species, thereby promoting bone repair. The bone morphogenetic protein-2 (BMP-2) is contained in poly (lactide-co-glycolide) (PLGA) microspheres, and the growth factors can be released for a long time through slow diffusion and slow degradation of microsphere carriers, so that the bone morphogenetic protein-2 maintains the bone formation activity for a long time and accelerates the repair and healing of skull defects.

S5.3, mixing the first emulsion with first polyvinyl alcohol to obtain a second emulsion;

s5.4, mixing the second emulsion with second polyvinyl alcohol, and evaporating to obtain microspheres loaded with growth factors;

in some embodiments, the microspheres have a diameter of 1 μm to 100 μm and the growth factor loaded microspheres comprise 0.1% to 10% growth factor and 90% to 99.9% biopolymer by mass fraction.

Specifically, the preparation method of the microsphere comprises the following steps: an amount of the polymer from which the microspheres were prepared was dissolved in Dichloromethane (DCM) to prepare a polymer solution. 1mL of the polymer solution was added to a glass vessel, and a certain amount of growth factor was added. The above mixture was sonicated with an ultrasonic probe for 30 seconds. The first emulsion was added to a volume of 1% polyvinyl alcohol and the phases were vigorously stirred at 14000rpm to give a second emulsion. The emulsion was added to a volume of 0.1% polyvinyl alcohol 30000-70000 (Sigma) and stirred with a homogenizer at 300rpm for 1 hour to evaporate the methylene chloride. Finally, the microspheres were collected by filtration, washed several times with distilled water and lyophilized in a freeze dryer. The dried microspheres were stored at 4 ℃ until use.

S6, mixing the microspheres loaded with the growth factors with the mineralized guide tissue regeneration layer solution to obtain the mineralized guide tissue regeneration layer solution containing the growth factors;

in some embodiments, the mineralized guided tissue regeneration layer solution containing growth factors contains 0.1g-20g of the microspheres per 100 mL.

The bone morphogenetic protein-2 (BMP-2) of the growth factor is contained in poly (lactide-co-glycolide) (PLGA) microspheres, the growth factor can be released for a long time through slow diffusion and slow degradation of microsphere carriers, the number of new capillaries and fibroblasts of a wound surface are promoted, the repair and healing of skull tissues are accelerated, the microsphere concentration is controlled to be 0.1-20 g/100mL, the microsphere can release the growth factor for a long time through degradation, the repair and healing of skull tissues are accelerated, the adverse effect of the excessive concentration value is that the degradation speed of the microsphere is slow, the release of the growth factor is influenced, the repair and healing of skull tissues are influenced, the adverse effect of the excessive concentration is that the degradation speed of the microsphere is fast, the content of the growth factor is low, the release speed is fast, and the repair and the healing of skull tissues are influenced.

S7, mixing 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride, N-hydroxysuccinimide, the modified matrix and the mineralized guided tissue regeneration layer solution containing growth factors to obtain a mixture;

The mineralized guiding tissue regeneration layer containing the growth factors is fixed on the polyether-ether-ketone material in a chemical bonding mode, so that the problems that biomolecules cannot act on the surface of the polyether-ether-ketone material for a long time and are easy to fall off in a physical fixing method are solved, the mineralized guiding tissue regeneration layer can be slowly degraded, the bone induction function is fully exerted in the formation of new bones, and the bone formation capacity of the defect part of the skull is improved.

In some embodiments, the mass concentration of 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride in the mixture is from 0.1g/mL to 0.3g/mL and the mass concentration of N-hydroxysuccinimide is from 0.01g/mL to 0.05g/mL.

S8, carrying out reaction, drying, crosslinking, analysis and sterilization on the mixture to obtain the skull repairing polyether-ether-ketone material.

In some embodiments, the drying is freeze-drying, including a first freeze-drying and a second freeze-drying, the first freeze-drying having a drying temperature of-80 ℃ to-20 ℃ and a drying time of 3h to 24h; the drying temperature of the second freeze drying is-50 ℃ to 37 ℃, the drying time of the second freeze drying is 24h to 72h, and the drying pressure of the second freeze drying is 0.1Pa to 50Pa.

The reason for controlling the drying temperature of the first freeze drying to be minus 20 to minus 80 ℃ and the time to be 3 to 24 hours is to meet the requirements of the actual freeze dryer equipment parameters and the product freeze drying process, the time can be longer due to the fact that the temperature is higher, the temperature is lower, and the product structure of the subsequent freeze drying process can be influenced due to the fact that the equipment temperature parameters are not reached.

The reason that the temperature of the second freeze drying is controlled to be-50-37 ℃, the pressure is controlled to be 0.1-50 Pa, and the time is controlled to be 24-72 h is that the requirements of the actual freeze dryer equipment parameters and the product freeze drying process are met, the freezing temperature is higher, the freezing time is longer, the freezing temperature is lower, the freezing speed is too high, and the product structure of the subsequent freeze drying process is influenced.

In some embodiments, crosslinking includes glutaraldehyde steam crosslinking and thermal crosslinking, the glutaraldehyde steam crosslinking having a crosslinking temperature of 37 ℃ to 52 ℃, a glutaraldehyde steam concentration of 5% to 25%, and a glutaraldehyde steam crosslinking time of 2 hours to 12 hours; the crosslinking temperature of the thermal crosslinking is 100-110 ℃, the crosslinking pressure of the thermal crosslinking is 10-150 Pa, and the crosslinking time of the thermal crosslinking is 12-48 h.

The temperature of glutaraldehyde steam crosslinking is controlled to be 37-52 ℃, the volume concentration of glutaraldehyde steam is 5-25%, and the time is 2-12 h, because the volume concentration of glutaraldehyde steam is too large to be achieved, the volume concentration of glutaraldehyde steam is too low, the crosslinking temperature is low, and the crosslinking time is too long.

The thermal crosslinking temperature is controlled to be 100-110 ℃, the pressure is controlled to be 10-150 Pa, and the crosslinking time is controlled to be 12-48 h, so that the thermal crosslinking temperature is low, the crosslinking time is long, and the thermal crosslinking temperature is high, thereby influencing the structural performance of the product.

In some embodiments, the resolution temperature of the resolution is from 37 ℃ to 52 ℃ and the resolution time of the resolution is from 2d to 4d.

The analysis temperature is controlled to be 37-52 ℃ and the analysis time is controlled to be 2-4 d, so that the analysis temperature is low, the analysis time is long, and the analysis temperature is high, thereby influencing the structural performance of the product.

In some embodiments, the reaction temperature of the reaction is 4 ℃ to 6 ℃ and the reaction time of the reaction is 40 hours to 60 hours.

The reason for controlling the reaction temperature to be 4-6 ℃ and the reaction time to be 40-60 h is to keep the long-time activity of the cross-linking agent and promote the sufficient cross-linking reaction between the polydopamine microsphere modified matrix and the mineralized guided tissue regeneration layer.

The following describes the preparation method of the growth factor-containing skull repairing polyether-ether-ketone material according to the present application in detail by referring to examples, comparative examples and experimental data.

Example 1

A preparation method of a growth factor-containing skull repair polyether-ether-ketone material comprises the following steps:

the polyether-ether-ketone material is obtained or prepared. And obtaining the polyether-ether-ketone two-dimensional braided fabric material or preparing the polyether-ether-ketone material through 3D printing. The method for preparing the polyether-ether-ketone material by 3D printing comprises the following steps: firstly, performing CT (computed tomography) flat scanning and enhanced scanning on a skull defect part, and then performing three-dimensional reconstruction to determine the position, size, shape and the like of the skull defect; and then pouring the scanning data into software to design a bone defect model, storing the model in an STL format, importing the model into a 3D printer, correcting and calibrating the skull defect model by using the software, and molding the model by using digital equipment according to the data of the model to prepare the polyether-ether-ketone matched with the defect part. The polyether-ether-ketone raw material meets the standard of YY/T0660-2008 'Standard Specification of polyether-ether-ketone (PEEK) polymers for surgical implants';

Polydopamine microsphere modified polyether-ether-ketone. The dopamine can be subjected to oxidative polymerization reaction in an alkaline (pH is more than 7.5) aerobic environment to form the polydopamine nanometer microsphere. Preparing a dopamine solution: 0.121g of tris (hydroxymethyl) aminomethane powder is taken and dissolved in 100mL of deionized water, the pH is adjusted to 8.5 by titration with dilute hydrochloric acid, 200mg of dopamine hydrochloride powder is taken and dissolved in tris (hydroxymethyl) aminomethane solution, and the mixture is stirred for 60min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, magnetically stirring at room temperature for reaction for 36 hours, repeatedly washing the poly-dopamine microsphere modified polyether-ether-ketone with pure water for 2-3 times, and drying in a forced air drying oven at 40 ℃ for 24 hours;

the poly dopamine microsphere is modified on polyether ether ketone to chemically crosslink mineralized guiding tissue regeneration layer containing growth factors. The preparation method comprises the following steps:

leading the preparation of a tissue regeneration layer solution; the preparation method comprises the following steps: obtaining a silk fibroin solution and a type I collagen solution; uniformly mixing the silk fibroin solution and the type I collagen solution to obtain a guided tissue regeneration layer solution; wherein the type I collagen in the type I collagen solution is obtained by dissolving type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1%; the silk fibroin in the silk fibroin solution is decomposed in a lithium bromide solution or a calcium chloride ternary system solution, and the mass fraction is 5%; the mass ratio of silk fibroin to type I collagen in the guided tissue regeneration layer solution is 7:3, a step of;

Preparing mineralized guiding tissue regeneration layer solution; dropwise adding a tetrahydrate calcium nitrate solution and a diammonium phosphate solution into the guided tissue regeneration layer solution, regulating the pH value to 7 by using ammonia water, uniformly mixing, standing the solution, separating out precipitate and washing impurity ions, and obtaining a mineralized guided tissue regeneration layer solution; the mass ratio of the added substance of calcium ions to the protein in the protein solution in the mineralized guiding tissue regeneration layer is 0.01mol/g, and the molar ratio of the added substance of calcium ions to the added substance of phosphate ions is 1.67;

preparing microspheres loaded with growth factors;

adding the microspheres loaded with the growth factors into the mineralized guide tissue regeneration layer solution, and uniformly mixing to obtain a mineralized guide tissue regeneration layer solution containing the growth factors; the diameter of the microsphere loaded with the growth factor is 1-100 mu m, the concentration of the microsphere in the mineralized guiding tissue regeneration layer solution is 10g/100mL, and the microsphere loaded with the growth factor comprises 0.5% of growth factor and 99.5% of biopolymer by mass fraction;

adding 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC.HCl), N-hydroxysuccinimide (NHS) and polydopamine microsphere modified polyether ether ketone into the mineralized guiding tissue regeneration layer solution containing the growth factors, and mixing and reacting for 48 hours at the temperature of 5 ℃; the concentration of the cross-linking agent 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC & HCl) in the mineralized guiding tissue regeneration layer solution is 0.2g/ml, and the concentration of the N-hydroxysuccinimide (NHS) is 0.03g/ml;

Freeze drying, glutaraldehyde steam crosslinking, heat crosslinking, analysis and cobalt 60 irradiation sterilization are carried out to obtain the skull repairing polyether-ether-ketone material. Specifically, the process conditions of freeze-drying are: pre-freezing at-60deg.C for 12 hr, and drying at 10deg.C under 10Pa for 48 hr; the process conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 12h at 40 ℃ under the condition that glutaraldehyde steam concentration is 10%; the process conditions of thermal crosslinking are: crosslinking for 48h at 100 ℃ under 100Pa in a vacuum drying oven; the analysis process conditions are as follows: in the forced air drying oven, the analysis temperature was 37℃and the analysis time was 2d.

Example 2

Polydopamine microsphere modified polyether-ether-ketone. The dopamine can be subjected to oxidative polymerization reaction in an alkaline (pH is more than 7.5) aerobic environment to form the polydopamine nanometer microsphere. Preparing a dopamine solution: 0.121g of tris (hydroxymethyl) aminomethane powder is taken and dissolved in 100mL of deionized water, the pH is adjusted to 8.5 by titration with dilute hydrochloric acid, 250mg of dopamine hydrochloride powder is taken and dissolved in tris (hydroxymethyl) aminomethane solution, and the mixture is stirred for 80min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, magnetically stirring at room temperature for reaction for 24 hours, repeatedly washing the poly-dopamine microsphere modified polyether-ether-ketone with pure water for 2-3 times, and drying in a blast drying oven at 50 ℃ for 12 hours;

leading the preparation of a tissue regeneration layer solution; the preparation method comprises the following steps: obtaining a silk fibroin solution and a type I collagen solution; uniformly mixing the silk fibroin solution and the type I collagen solution to obtain a guided tissue regeneration layer solution; wherein the type I collagen in the type I collagen solution is obtained by dissolving type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1%; the silk fibroin in the silk fibroin solution is decomposed in a lithium bromide solution or a calcium chloride ternary system solution, and the mass fraction is 10%; the mass ratio of silk fibroin to type I collagen in the guided tissue regeneration layer solution is 3:2;

Preparing mineralized guiding tissue regeneration layer solution; dropwise adding a tetrahydrate calcium nitrate solution and a diammonium phosphate solution into the guided tissue regeneration layer solution, regulating the pH value to 7.5 by using ammonia water, uniformly mixing, standing the solution, separating out precipitate and washing out impurity ions to obtain a mineralized guided tissue regeneration layer solution; the mass ratio of the added substance of calcium ions to the protein in the protein solution in the mineralized guiding tissue regeneration layer is 0.015mol/g, and the molar ratio of the added substance of calcium ions to the added substance of phosphate ions is 1.67;

preparing microspheres loaded with growth factors;

adding the microspheres loaded with the growth factors into the mineralized guide tissue regeneration layer solution, and uniformly mixing to obtain a mineralized guide tissue regeneration layer solution containing the growth factors; the diameter of the microsphere loaded with the growth factor is 1-100 mu m, the concentration of the microsphere in the mineralized guiding tissue regeneration layer solution is 6g/100mL, and the microsphere loaded with the growth factor comprises 0.5% of growth factor and 99.5% of biopolymer by mass fraction;

adding 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC.HCl), N-hydroxysuccinimide (NHS) and polydopamine microsphere modified polyether ether ketone into the mineralized guide tissue regeneration layer solution, and mixing and reacting for 48 hours at the temperature of 5 ℃; the concentration of the cross-linking agent 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC & HCl) in the mineralized guiding tissue regeneration layer solution is 0.2g/ml, and the concentration of the N-hydroxysuccinimide (NHS) is 0.03g/ml;

Freeze drying, glutaraldehyde steam crosslinking, heat crosslinking, analysis and cobalt 60 irradiation sterilization are carried out to obtain the skull repairing polyether-ether-ketone material. Specifically, the process conditions of freeze-drying are: pre-freezing at-50deg.C for 12 hr, and drying at 20deg.C under pressure of 20Pa for 48 hr; the process conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 6h at 40 ℃ under the condition that glutaraldehyde steam concentration is 20%; the process conditions of thermal crosslinking are: crosslinking for 24h at 105 ℃ under 50Pa in a vacuum drying oven; the analysis process conditions are as follows: in the forced air drying oven, the analysis temperature was 50℃and the analysis time was 3d.

Example 3

Polydopamine microsphere modified polyether-ether-ketone. The dopamine can be subjected to oxidative polymerization reaction in an alkaline (pH is more than 7.5) aerobic environment to form the polydopamine nanometer microsphere. Preparing a dopamine solution: 0.121g of tris (hydroxymethyl) aminomethane powder is taken and dissolved in 100mL of deionized water, the pH is adjusted to 9.0 by titration with dilute hydrochloric acid, 300mg of dopamine hydrochloride powder is taken and dissolved in tris (hydroxymethyl) aminomethane solution, and the mixture is stirred for 90min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, magnetically stirring at room temperature for reaction for 48 hours, repeatedly washing the poly-dopamine microsphere modified polyether-ether-ketone with pure water for 2-3 times, and drying in a forced air drying oven at 40 ℃ for 24 hours;

leading the preparation of a tissue regeneration layer solution; the preparation method comprises the following steps: obtaining a silk fibroin solution and a type I collagen solution; uniformly mixing the silk fibroin solution and the type I collagen solution to obtain a guided tissue regeneration layer solution; wherein the type I collagen in the type I collagen solution is obtained by dissolving type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1.5%; the silk fibroin in the silk fibroin solution is decomposed in a lithium bromide solution or a calcium chloride ternary system solution, and the mass fraction is 7.5%; the mass ratio of silk fibroin to type I collagen in the guided tissue regeneration layer solution is 1:1, a step of;

Preparing mineralized guiding tissue regeneration layer solution; dropwise adding a tetrahydrate calcium nitrate solution and a diammonium phosphate solution into the guided tissue regeneration layer solution, regulating the pH value to 8.0 by using ammonia water, uniformly mixing, standing the solution, separating out precipitate and washing out impurity ions to obtain a mineralized guided tissue regeneration layer solution; the mass ratio of the added substance of calcium ions to the protein in the protein solution in the mineralized guiding tissue regeneration layer is 0.02mol/g, and the molar ratio of the added substance of calcium ions to the added substance of phosphate ions is 1.67;

preparing microspheres loaded with growth factors;

adding the microspheres loaded with the growth factors into the mineralized guide tissue regeneration layer solution, and uniformly mixing to obtain a mineralized guide tissue regeneration layer solution containing the growth factors; the diameter of the microsphere loaded with the growth factor is 1-100 mu m, the concentration of the microsphere in the mineralized guiding tissue regeneration layer solution is 15g/100mL, and the microsphere loaded with the growth factor comprises 0.6% of growth factor and 99.4% of biopolymer by mass fraction;

Freeze drying, glutaraldehyde steam crosslinking, heat crosslinking, analysis and cobalt 60 irradiation sterilization are carried out to obtain the skull repairing polyether-ether-ketone material. Specifically, the process conditions of freeze-drying are: pre-freezing at-60deg.C for 24 hr, and drying at 5deg.C under 30Pa for 48 hr; the process conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 3h at 40 ℃ under the condition that glutaraldehyde steam concentration is 25%; the process conditions of thermal crosslinking are: crosslinking for 24h at 110 ℃ under 30Pa in a vacuum drying oven; the analysis process conditions are as follows: in the forced air drying oven, the analysis temperature was 45℃and the analysis time was 3d.

Example 4

the polyether-ether-ketone material is obtained or prepared. And obtaining the polyether-ether-ketone two-dimensional braided fabric material or preparing the polyether-ether-ketone material through 3D printing. The method for preparing the polyether-ether-ketone material by 3D printing comprises the following steps: firstly, performing CT (computed tomography) flat scanning and enhanced scanning on a damaged part of the skull defect, and then performing three-dimensional reconstruction to determine the position, size, shape and the like of the skull defect; and then pouring the scanning data into software to design a bone defect model, storing the model in an STL format, importing the model into a 3D printer, correcting and calibrating the skull defect model by using the software, and molding the model by using digital equipment according to the data of the model to prepare the polyether-ether-ketone matched with the defect part. The polyether-ether-ketone raw material meets the standard of YY/T0660-2008 'Standard Specification of polyether-ether-ketone (PEEK) polymers for surgical implants';

Polydopamine microsphere modified polyether-ether-ketone. The dopamine can be subjected to oxidative polymerization reaction in an alkaline (pH is more than 7.5) aerobic environment to form the polydopamine nanometer microsphere. Preparing a dopamine solution: 0.121g of tris (hydroxymethyl) aminomethane powder is taken and dissolved in 100mL of deionized water, the pH is adjusted to 9.5 by titration with dilute hydrochloric acid, 200mg of dopamine hydrochloride powder is taken and dissolved in tris (hydroxymethyl) aminomethane solution, and the mixture is stirred for 60min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, magnetically stirring at room temperature for reaction for 36 hours, repeatedly washing the poly-dopamine microsphere modified polyether-ether-ketone with pure water for 2-3 times, and drying in a forced air drying oven at 45 ℃ for 12 hours;

leading the preparation of a tissue regeneration layer solution; the preparation method comprises the following steps: obtaining a silk fibroin solution and a type I collagen solution; uniformly mixing the silk fibroin solution and the type I collagen solution to obtain a guided tissue regeneration layer solution; wherein the type I collagen in the type I collagen solution is obtained by dissolving type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1%; the silk fibroin in the silk fibroin solution is decomposed in a lithium bromide solution or a calcium chloride ternary system solution, and the mass fraction is 5%; the mass ratio of silk fibroin to type I collagen in the guided tissue regeneration layer solution is 3:7, preparing a base material;

Preparing mineralized guiding tissue regeneration layer solution; dropwise adding a tetrahydrate calcium nitrate solution and a diammonium phosphate solution into the guided tissue regeneration layer solution, regulating the pH value to 7.5 by using ammonia water, uniformly mixing, standing the solution, separating out precipitate and washing out impurity ions to obtain a mineralized guided tissue regeneration layer solution; the mass ratio of the added substance of calcium ions to the protein in the protein solution in the mineralized guiding tissue regeneration layer is 0.01mol/g, and the molar ratio of the added substance of calcium ions to the added substance of phosphate ions is 1.67;

preparing microspheres loaded with growth factors;

adding the microspheres loaded with the growth factors into the mineralized guide tissue regeneration layer solution, and uniformly mixing to obtain a mineralized guide tissue regeneration layer solution containing the growth factors; the diameter of the microsphere loaded with the growth factor is 1-100 mu m, the concentration of the microsphere in the mineralized guiding tissue regeneration layer solution is 20g/100mL, and the microsphere loaded with the growth factor comprises 1% of growth factor and 99% of biopolymer by mass fraction;

Freeze drying, glutaraldehyde steam crosslinking, heat crosslinking, analysis and cobalt 60 irradiation sterilization are carried out to obtain the skull repairing polyether-ether-ketone material. Specifically, the process conditions of freeze-drying are: pre-freezing at-50deg.C for 24h, and drying at 25deg.C under 15Pa for 72h; the process conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 6h at 40 ℃ under the condition that glutaraldehyde steam concentration is 20%; the process conditions of thermal crosslinking are: crosslinking for 48h at 110 ℃ under 100Pa in a vacuum drying oven; the analysis process conditions are as follows: in the forced air drying oven, the analysis temperature was 45℃and the analysis time was 4d.

Comparative example 1

In the comparative example, the polyetheretherketone two-dimensional braid material was obtained or the polyetheretherketone material was prepared by 3D printing without performing surface modification.

Comparative example 2

In the comparative example, the cross-linking agent 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) are not added in the process of compounding the mineralized guiding tissue regeneration layer containing the growth factors on the polydopamine microsphere modified polyether ether ketone; the rest of the procedure is the same as in example 1.

Comparative example 3

In the comparative example, the cross-linking agent 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) are not added in the process of compounding the mineralized guiding tissue regeneration layer containing the growth factors on the polydopamine microsphere modified polyether ether ketone; the rest of the procedure is the same as in example 4.

Experimental example

A skull repair polyether ether ketone material obtained in example 1 and comparative example 1 was subjected to surface contact angle detection, cell adhesion and proliferation capacity detection, and alkaline phosphatase (ALP) activity detection.

Surface contact angle detection: the contact angle of each set of surfaces was measured using a contact angle tester at room temperature, 3 samples were tested for each set of samples, 2 positions were tested for each sample, and the average was calculated.

Cell adhesion and proliferation potency assay: MG-63 osteoblasts were inoculated on the surface of the samples in 24-well plates for culture, and after the 1 st day and 7 th day of culture, a human cholecystokinin/cholecystokinin octapeptide (CCK-8) reagent was added, and absorbance (OD) was measured at a wavelength of 450nm using an enzyme-labeled instrument.

Alkaline phosphatase (ALP) Activity assay: samples in 24 well plates were surface seeded with MG-63 osteoblasts for culture and ALP activity assays were performed on day 7 and day 14 of culture, respectively: the washing was repeated 3 times with PBS, 0.1% Triton-X was added, and the mixture was left in a refrigerator to cleave for 40min at 4℃after which the ALP activity was examined by performing an operation according to the instructions of the biquinine acid (BCA) kit.

The test results are shown in the following table:

note that: in comparison with comparative example 1, (1)p < 0.05; in comparison with comparative example 1, (2)p < 0.05)

As can be seen from the data in the table, compared with the simple polyetheretherketone material of comparative example 1, the poly dopamine microsphere of example 1 is used for chemically crosslinking the mineralized guiding tissue regeneration layer containing growth factors, and the contact angle of the polyetheretherketone material is obviously reduced, which indicates that the hydrophilicity is improved and the adhesion of cells is facilitated.

Compared with the simple polyether-ether-ketone material of comparative example 1, after the poly dopamine microsphere is used for modifying the mineralized guiding tissue regeneration layer containing the growth factors on the polyether-ether-ketone, CCK test results show that the chemically modified polyether-ether-ketone material has good biocompatibility, the adhesion and proliferation of cells after modification are obviously improved, and the surface activity is effectively improved.

Compared with the simple polyether-ether-ketone material of comparative example 1, after the poly dopamine microsphere is used for modifying the mineralized guiding tissue regeneration layer containing the growth factors, ALP activity test results prove that ALP activity of the chemically modified polyether-ether-ketone material is effectively improved, surface osteogenesis activity is improved, and surface biological activity of the chemically modified polyether-ether-ketone material is effectively improved.

(1) The raw materials adopted by the method provided by the embodiment of the invention are easy to obtain, safe and environment-friendly, and hidden danger to human bodies in the preparation process and the use of the final product is avoided;

(2) According to the method provided by the embodiment of the invention, the mineralized guiding tissue regeneration layer containing the growth factors is fixed on the polyether-ether-ketone material in a chemical bonding mode, so that the problems that biomolecules cannot act on the surface of the polyether-ether-ketone material for a long time and are easy to fall off in a physical fixing method are overcome, the mineralized guiding tissue regeneration layer can be slowly degraded, the osteoinduction effect is fully exerted in the formation of new bones, and the bone formation capacity of the defect part of the skull is improved.

Finally, it is also noted that 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.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for preparing a growth factor-containing skull repair polyether-ether-ketone material, which is characterized by comprising the following steps:

analyzing the head to be repaired to obtain skull defect position information;

dissolving a polymer in methylene dichloride to obtain a polymer solution;

mixing the growth factor with the polymer solution to obtain a first emulsion;

reacting, drying, crosslinking, analyzing and sterilizing the mixture to obtain a skull repairing polyether-ether-ketone material containing the growth factors;

the solute of the guided tissue regeneration layer solution is a degradable membrane material, each 100mL of the mineralized guided tissue regeneration layer solution containing growth factors contains 0.1g-20g of microspheres, and the mass fraction of the degradable membrane material in the mineralized guided tissue regeneration layer solution is 0.5% -20%; the ratio of the amount of calcium ion substance to the amount of phosphate ion substance is 1-2: 1, in the mixture, the mass concentration of the 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride is 0.1 g/mL-0.3 g/mL, the mass concentration of the N-hydroxysuccinimide is 0.01 g/mL-0.05 g/mL, the crosslinking comprises glutaraldehyde steam crosslinking and thermal crosslinking, the crosslinking temperature of the glutaraldehyde steam crosslinking is 37-52 ℃, the glutaraldehyde steam concentration of the glutaraldehyde steam crosslinking is 5-25%, and the crosslinking time of the glutaraldehyde steam crosslinking is 2-12 h; the crosslinking temperature of the thermal crosslinking is 100-110 ℃, the crosslinking pressure of the thermal crosslinking is 10-150 Pa, the crosslinking time of the thermal crosslinking is 12-48 h, the resolving temperature of resolving is 37-52 ℃, the resolving time of resolving is 2-4 d, the reaction temperature of the reaction is 4-6 ℃, and the reaction time of the reaction is 40-60 h.

2. A method of preparing a growth factor-containing skull repair polyetheretherketone material according to claim 1, wherein the polymer comprises at least one of polycaprolactone, polyhydroxyaliphatic carboxylic acid, polyhydroxybutyrate-polyhydroxyvalerate interpolymer, polylactic acid-polycaprolactone interpolymer, polyanhydride, lectin, glycosaminoglycan, chitosan, cellulose, acrylate polymer, homopolymers of glycolic acid, homopolymers of lactic acid, and copolymers derived from poly (lactide-co-glycolide);

the degradable film material comprises: at least one of hyaluronic acid, chondroitin sulfate, modified cellulose, modified chitosan, alginate, type I collagen, silk fibroin, polylactide, polyglycolide, polycaprolactone, polyhydroxybutyrate, and copolymers thereof.

3. A method of preparing a growth factor-containing skull repair polyetheretherketone material according to claim 2, wherein the microsphere has a diameter of 1 μm to 100 μm.

4. The method for preparing the growth factor-containing skull repairing polyether-ether-ketone material according to claim 1, wherein the drying is freeze drying, the freeze drying comprises first freeze drying and second freeze drying, the drying temperature of the first freeze drying is-80 ℃ to-20 ℃, and the drying time of the first freeze drying is 3-24 hours; the drying temperature of the second freeze drying is-50 ℃ to 37 ℃, the drying time of the second freeze drying is 24h to 72h, and the drying pressure of the second freeze drying is 0.1Pa to 50Pa.

5. The method for preparing the growth factor-containing skull repair polyether-ether-ketone material according to claim 1, wherein the modification of the substrate by the polydopamine nanospheres is performed to obtain a modified substrate, and specifically comprises the following steps:

6. A skull repairing polyether-ether-ketone material, characterized in that the polyether-ether-ketone material is prepared by the preparation method of the growth factor-containing skull repairing polyether-ether-ketone material according to any one of claims 1 to 5.