CN114558170A

CN114558170A - Growth factor-containing skull repairing polyether-ether-ketone material and preparation method thereof

Info

Publication number: CN114558170A
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: 2022-05-31
Anticipated expiration: 2042-01-24
Also published as: CN114558170B

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 the method comprises the following steps: molding polyether-ether-ketone to obtain a matrix; modifying the substrate by the poly-dopamine nano-microspheres to obtain a modified substrate; mixing the mineralization auxiliary agent with the guided tissue regeneration layer solution to obtain a mineralized guided tissue regeneration layer solution; mixing the microspheres loaded with the growth factors with the mineralization leading tissue regeneration layer solution to obtain the mineralization leading tissue regeneration layer solution containing the growth factors; mixing a cross-linking agent, a modified matrix and a mineralized guided tissue regeneration layer solution containing growth factors to obtain a mixture; reacting, drying, crosslinking, resolving and sterilizing the mixture to obtain a skull repairing polyether-ether-ketone material; the poly-dopamine nano-microsphere is used for crosslinking and modifying the polyether-ether-ketone, so that the raw materials are easy to obtain, the poly-dopamine nano-microsphere is safe and environment-friendly, and the hidden danger brought to a human body in the preparation process and the use of a final product is avoided.

Description

Growth factor-containing skull repairing 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 shock injury, car accident injury, bullet injury, skull malignant tumor excision, congenital malformation, after craniotomy decompression, and the like. In principle, skull defects with the maximum diameter of more than 3cm need to be subjected to skull reconstruction surgery, and when the skull defects exceed 3cm, clinical symptoms can be generated. Successful skull reconstruction needs to meet 3 requirements: (1) maintaining the integrity of the dura mater, i.e. the protection of the brain; (2) the barrier between the cranium and the outside is protected, namely the biology and the materials are stable; (3) maintaining the normal dome-like shape of the head, i.e. aesthetic requirements.

The ideal skull defect repair material satisfies the following characteristics: (1) the acquisition is convenient; (2) the biocompatibility is high; (3) can completely match with the defect part and has good ductility; (4) good biomechanical performance, brain barrier protection and external force resistance; (5) has the potential of inducing osteogenesis; (6) the head image examination is compatible; (7) is resistant to infection.

At present, the skull repairing materials applied to clinic mainly comprise autogenous bones, allogeneic bones, xenogeneic bones and artificial materials. Autologous bone repair is the gold standard for skull reconstruction. The autologous bone tissue has good bone conductivity and tissue compatibility, no immune rejection reaction and low postoperative femoral leakage rate, but has the problems of limited supply area, difficult shaping, increased secondary trauma, higher bone absorption rate of transplanted bone and the like, and is limited in clinical application. The allogeneic bone is generally subjected to special sterilization treatment, does not have common infectious diseases and has no immunogenicity. Allogeneic bone may be surgically biologically combined with autologous tissue, allowing for vascularization of the tissue and in-growth reconstruction of autologous tissue. However, the clinical application of allogeneic bone to skull defects is limited by the high infection rate after operation, the absorption rate of transplanted bone, religion, ethics and other factors. The source of xenogenic bone is rich, but the immunogenicity is strong, the freeze-dried bone, the calcined bone and the deproteinized bone which are used clinically are prepared by respectively carrying out freeze-drying, high-temperature calcination, irradiation, decalcification and other treatments on animal bone tissues, removing organic components such as cells, collagen and the like, retaining a natural pore structure, eliminating the antigenicity, but having small mechanical strength of the tissues, looseness, frangibility, poor mechanical strength and reduced plasticity.

The clinically common artificial skull repairing materials mainly comprise hydroxyapatite, polymethyl methacrylate, titanium mesh, polyether ether ketone and the like. Polyether ether ketone (PEEK) is a wholly aromatic semi-crystalline thermoplastic special engineering plastic, has excellent physical and chemical properties, mechanical and thermal properties and the like, and can keep higher wear resistance and lower friction coefficient at a high temperature of 250 ℃. Polyetheretherketone has the characteristics of high melting point (334 ℃), low creep, high elastic modulus, excellent friction performance, high temperature resistance, chemical corrosion resistance and the like, and is increasingly used as a biological material to be applied to orthopedic implants and prostheses. Compared with the traditional hard tissue implanted metal material (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 being implanted. In addition, the polyetheretherketone as a biological material also has the advantages of radioactive ray permeability, no artifact generated by magnetic resonance scanning and the like, can better evaluate the postoperative recovery condition, and is currently used in the fields of skull, jaw bone, vertebra lumbar, artificial joint, oral defect repair and the like.

The greatest limitation of Polyetheretherketone (PEEK) as a biomedical material is the biological inertness. The PEEK material has poor hydrophilicity and is not beneficial to the 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, and has poor associativity and poor implantation effect.

In order to realize the optimal osseointegration after implantation, a surface modification method is the preferred way to enhance the bioactivity of the PEEK surface without affecting the advantages of the PEEK material. The most common method is to functionalize PEEK by preparing a bioactive coating by physical or chemical methods. Many of the prior art coatings (including hydroxyapatite, bioactive particles, etc.) have been reported to enhance the osteointegration of PEEK implants to some extent. However, these techniques are carried out using 98% concentrated sulfuric acid or under high temperature conditions (above 360 ℃).

Disclosure of Invention

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

The embodiment of the invention provides a preparation method of a growth factor-containing skull repairing polyetheretherketone material, which comprises the following steps:

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

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

modifying the substrate by the poly-dopamine nano-microspheres to obtain a modified substrate;

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

dissolving a polymer in dichloromethane to obtain a polymer solution;

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

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

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

mixing the microspheres loaded with the growth factors with the mineralization leading tissue regeneration layer solution to obtain the mineralization leading tissue regeneration layer solution containing the 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) reacting, drying, crosslinking, resolving and sterilizing the mixture to obtain the skull repairing polyether-ether-ketone material containing the growth factors.

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

the growth factor comprises 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 for guiding the tissue regeneration layer solution is 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 at least one of the copolymers of the above substances.

Optionally, every 100mL of the mineralization leading 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 mineralization guide tissue regeneration layer solution, the mass fraction of the degradable membrane material is 0.5-20%; the ratio of the amount of the calcium ion to the amount of the phosphate ion is 1-2: 1.

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

Optionally, 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 3h to 24 h; 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 50 Pa.

Optionally, the crosslinking comprises glutaraldehyde steam crosslinking and thermal crosslinking, the crosslinking temperature of the glutaraldehyde steam crosslinking is 37-52 ℃, the concentration of glutaraldehyde steam crosslinked by the glutaraldehyde steam 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, and the crosslinking time of the thermal crosslinking is 12-48 h.

Optionally, the analysis temperature for analysis is 37 ℃ to 52 ℃, and the analysis time for analysis is 2d to 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 modifying the substrate with the polydopamine nano-microsphere to obtain a modified substrate specifically includes:

soaking the substrate in the solution of the polydopamine nano-microspheres for stirring reaction to obtain a modified substrate;

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

dissolving trihydroxymethyl aminomethane powder in a solvent to obtain a trihydroxymethyl aminomethane solution;

and dissolving dopamine hydrochloride powder in the trihydroxymethyl aminomethane solution to obtain a solution of the polydopamine nano-microspheres.

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

One or more technical solutions in the embodiments of the present invention have at least 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 polyether-ether-ketone, so that the raw material is easy to obtain, safe and environment-friendly, and hidden dangers brought to a human body in the preparation process and the use of a final product are avoided.

The above description is only an overview of the technical solutions of the present invention, and the present invention can be implemented in accordance with the content of the description so as to make the technical means of the present invention more clearly understood, and the above and other objects, features, and advantages of the present invention will be more clearly understood.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

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

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings 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. If there is a conflict, the present specification will control.

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

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the applicant finds in the course of the invention: the 3D printing titanium mesh technology solves the problems of plasticity and compatibility of a repair material in skull repair application, the processing speed is high, the implant can be manufactured within 1-2 days, and the waiting time of a patient is reduced; the porous through structure of the human-like skeleton can effectively overcome the problems of stress shielding and low biological activity commonly existing in the implant, and simultaneously can minimize the heat dissipation in the cranial cavity and maintain the normal heat conduction level. However, the single 3D printing titanium mesh belongs to a biological inert material, has no biological activity, cannot be rapidly fused with soft tissues, and cannot effectively promote the repair and regeneration of bone tissues.

According to an exemplary embodiment of the invention, a preparation method of a growth factor-containing skull repairing polyetheretherketone material is provided, which comprises the following steps:

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

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

in some embodiments, the matrix may be made of a two-dimensional woven material of polyetheretherketone or by 3D printing, and the following description is given only for the 3D printing:

firstly, performing CT flat scanning and enhanced scanning on a skull defect part, then performing three-dimensional reconstruction, and determining 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 bone defect model in an STL format, importing the bone defect model into a 3D printer, correcting and calibrating the skull defect model by using the software, and performing modeling by using digital equipment according to the data of the model to manufacture a polyether-ether-ketone matrix matched with the defect part. The raw material of the polyetheretherketone meets the standard of YY/T0660-2008 Standard Specification for Polyetheretherketone (PEEK) polymer for surgical implants.

S3, modifying the substrate by the poly-dopamine nano-microspheres to obtain a modified substrate;

the polydopamine is mainly secreted from the foot gland of the mussel, contains a large amount of adhesive protein, is secreted into seawater, gradually coagulates, forms byssus and firmly adheres to the surface of a substrate material. Mytilus inspired polydopamine coating technology is capable of providing strong adhesive interactions with a variety of materials and biomolecules containing amine and thiol functional groups. 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 universal functional surface modification method. The polydopamine can be used for modifying regular surfaces, and can also 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 also be directly used for connecting biomolecules and medicaments or combined with other coating technologies to prepare multifunctional composite coatings. When the polydopamine is coated on the surface of the substrate material, the thickness can be thin, the combination is firm, and the surface of the substrate material can obtain good hydrophilicity and adhesiveness. The literature reports that the polydopamine coating can promote in-vitro osteogenic differentiation and calcium mineralization, and can promote osteogenesis and increase osseointegration in-vivo experiments. The poly-dopamine nano-microsphere modification is carried out on the polyether-ether-ketone, so that the surface hydrophilicity and the bioactivity of the porous polyether-ether-ketone material can be improved, and the secondary coating modification is carried out on the surface of the porous polyether-ether-ketone material, so that the adhesion, the proliferation and the secretion of extracellular matrix of vascular endothelial cells on the surface of the material are facilitated, the rapid fusion of the repairing material and soft tissues is accelerated, and the surface morphology and the biological performance of the repairing material are more in line with the requirements of clinical application of skull repair.

In some embodiments, modifying the substrate with polydopamine nanospheres to obtain a modified substrate specifically includes:

s3.1, soaking the substrate in the solution of the polydopamine nano-microspheres for stirring reaction to obtain a modified substrate;

s3.1.1, dissolving trihydroxymethyl aminomethane powder in a solvent to obtain a trihydroxymethyl aminomethane solution;

s3.1.2, dissolving dopamine hydrochloride powder in the trihydroxymethyl aminomethane solution to obtain a solution of the polydopamine nano-microspheres.

Specifically, the preparation method of the dopamine solution comprises the following steps: dissolving trihydroxymethyl aminomethane powder in deionized water, titrating with dilute hydrochloric acid to adjust the pH value to 7.5-10, dissolving dopamine hydrochloride powder in the trihydroxymethyl aminomethane solution, mixing and stirring for 30-120 min to form a dopamine solution. The mass concentration of the dopamine in the dopamine solution is 0.1-20 mg/mL. Dopamine can undergo oxidative polymerization in an alkaline (pH is more than 7.5) aerobic environment to form polydopamine nano-microspheres. With increasing pH, dopamine gradually self-polymerizes to form polydopamine, which gradually changes from light brown to dark brown in color. And adding the prepared polyether-ether-ketone matrix into a dopamine solution, carrying out magnetic stirring reaction at room temperature for 24-48 h, 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 37-52 ℃ for 12-24 h to obtain the modified matrix.

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

specifically, mixing a calcium nitrate tetrahydrate solution, a diammonium phosphate solution and a tissue regeneration guiding layer solution to obtain a mineralized tissue regeneration guiding layer solution, which comprises the following steps:

s4.1, preparing a tissue regeneration layer solution;

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

In some embodiments, the solute that directs the 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 their copolymers.

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

preferably, the degradable membrane material is selected from type I collagen and silk fibroin. Specifically, the preparation method of the solution for guiding the tissue regeneration layer comprises the following steps: obtaining silk fibroin solution and 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 the 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 dissolved 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 to type I collagen in the solution of the guided tissue regeneration layer is (0.3-3): 1; the mass ratio of the amount of the substances added with the calcium ions in the mineralization guide 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 amount of the substances added with the calcium ions to the amount of the substances added with the phosphate ions is 1-2: 1.

the type I collagen is a main structural protein of a spine animal, is extracellular matrix secreted by osteoblasts in an osteogenesis process, is a scaffold deposited by calcium salt, an accelerant of a bone matrix double layer and a template of the double layer; can promote cell migration, adsorption and differentiation and regulate cell growth, is approved by the FDA in the United states as a biological material, 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 property and high degradation rate. The silk fibroin has excellent biocompatibility, biodegradability and better mechanical property, is easy to sterilize and shape, is widely applied to the aspects of ligament tissue repair, vascular tissue transplantation, cartilage tissue repair, skin tissue regeneration, nerve tissue engineering and the like, but has mechanical strength far lower than that of bone tissue, and the degradation speed of pure silk fibroin is too slow. The nano-hydroxyapatite has good bone conductivity and biocompatibility, but the single hydroxyapatite has larger brittleness and low toughness. Therefore, the hydroxyapatite, the type I collagen and the silk fibroin are used in a compounding way, so that the problem of insufficient performance of a single material can be solved, the advantage complementation of various materials is realized, the obtained bone repair material has good mechanical property and controllable biodegradation time, the morphological structure of the skull repair material can be maintained within a certain time or for a long time, and the bone repair material is matched with the biomechanical property of original skull bone tissues of an implanted part; the type I collagen and the silk fibroin are natural fiber proteins, have good biocompatibility and bone induction performance, are beneficial to the adhesion, proliferation and extracellular matrix secretion of seed cells on the surface of the material, accelerate the rapid fusion of the repairing material and soft tissues, and can stimulate the differentiation of chondrocytes and osteoblasts around an implanted part to form new bone tissues; has good bone inductivity, and can stimulate the differentiation of chondrocytes and osteoblasts around the implanted part to form new bone tissues.

S5, obtaining microspheres loaded with growth factors;

s5.1, dissolving a polymer in dichloromethane 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, coacervate, glycosaminoglycan, chitosan, cellulose, acrylate polymer, homopolymer of glycolic or lactic acid and copolymer derived from poly (lactide-co-glycolide) (abbreviated as PLGA). Preferably, the polymer is poly (lactide-co-glycolide) (abbreviated PLGA).

S5.2, mixing a growth factor 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 various growth factors such as transforming growth factor-beta 1 (TGF-beta 1), bone morphogenetic protein-2 (BMP-2)), or Platelet Derived Growth Factor (PDGF). Preferably, the growth factor is bone morphogenetic protein-2 (BMP-2).

Bone morphogenetic protein-2 (BMP-2) is considered to be a growth factor having the strongest bone induction ability and promoting bone regeneration, and can induce the proliferation and differentiation of undifferentiated mesenchymal stem cells in the osteoblast direction across species, thereby promoting bone repair. Bone morphogenetic protein-2 (BMP-2) is contained in poly (lactide-co-glycolide) (PLGA) microspheres, and growth factors can be released for a long time through slow diffusion and slow degradation of a microsphere carrier, so that the osteogenic activity is maintained for a long time, and the repair and healing of skull defects are accelerated.

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 then 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, by mass fraction, 0.1% to 10% growth factor and 90% to 99.9% biopolymer.

Specifically, the method of preparing the microsphere comprises: a quantity of the microsphere-prepared polymer was dissolved in Dichloromethane (DCM) to make a polymer solution. 1mL of the polymer solution was added to a glass container and a defined amount of growth factor was added. The mixture was sonicated with an ultrasound 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 above 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 methylene chloride. Finally, the microspheres were collected by filtration, washed several times with distilled water and freeze-dried 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 guided tissue regeneration layer solution to obtain the mineralized guided tissue regeneration layer solution containing the growth factors;

in some embodiments, the microspheres are present in an amount of 0.1g to 20g per 100mL of the mineralization inducing tissue regeneration layer solution comprising growth factors.

The growth factor bone morphogenetic protein-2 (BMP-2) 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 a microsphere carrier, the number of wound surface new capillary blood vessels and the number of fibroblasts are promoted, the skull tissue repair and healing are accelerated, the reason that the microsphere concentration is controlled to be 0.1-20 g/100mL is that the microspheres can release the growth factor for a long time through degradation, and the skull tissue repair and healing are accelerated, the adverse effect of overlarge concentration value is that the microsphere degradation speed is slow, the growth factor release is influenced, the skull tissue repair and healing are influenced, and the undersized adverse effect is that the microsphere degradation speed is fast, the growth factor content is small, the release speed is fast, and the skull tissue repair and healing are influenced.

S7, 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;

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

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

And S8, reacting, drying, crosslinking, resolving and sterilizing the mixture to obtain the skull repairing polyether-ether-ketone material.

In some embodiments, the drying is freeze-drying, the freeze-drying comprises a first freeze-drying and a 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 24 h; 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 50 Pa.

The reason for controlling the drying temperature of the first freeze drying to be-20 to-80 ℃ and the time to be 3 to 24 hours is to meet the actual freeze dryer equipment parameters and the product freeze-drying process requirements, the temperature is higher, the time can be longer, the temperature is lower, the equipment temperature parameters cannot reach, and the subsequent freeze-drying process product structure can be influenced.

The reason why the temperature of the second freeze drying is controlled to be-50-37 ℃, the pressure is 0.1-50 Pa and the time is 24-72 h is that the actual equipment parameters of the freeze dryer and the freeze-drying process requirements of the product 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, the crosslinking comprises glutaraldehyde steam crosslinking and thermal crosslinking, the crosslinking temperature of the glutaraldehyde steam crosslinking is 37-52 ℃, the concentration of glutaraldehyde steam 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, and the crosslinking time of the thermal crosslinking is 12-48 h.

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

The reason that 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 is that the thermal crosslinking temperature is low, the crosslinking time is long, and the thermal crosslinking temperature is high, so that the structural performance of the product is influenced.

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

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

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

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 full cross-linking reaction between the polydopamine microsphere modified matrix and the mineralized and guided tissue regeneration layer.

The preparation method of the growth factor-containing skull repairing polyetheretherketone material of the present application will be described in detail with reference to examples, comparative examples and experimental data.

Example 1

A preparation method of a growth factor-containing skull repairing polyetheretherketone material comprises the following steps:

obtaining or preparing the polyetheretherketone material. And obtaining the polyetheretherketone two-dimensional woven fabric material or preparing the polyetheretherketone material by 3D printing. The method for preparing the polyether-ether-ketone material by 3D printing comprises the following steps: firstly, performing CT flat scanning and enhanced scanning on a skull defect part, then performing three-dimensional reconstruction, and determining 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 bone defect model in an STL format, importing the bone defect model into a 3D printer, correcting and calibrating the skull defect model by using the software, and performing modeling by using digital equipment according to the data of the model to manufacture polyether-ether-ketone matched with the defect part. The raw material of the polyetheretherketone meets the standard of YY/T0660-2008 Standard Specification of Polyetheretherketone (PEEK) polymer for surgical implants;

the poly-dopamine microspheres modify polyether-ether-ketone. Dopamine can undergo oxidative polymerization in an alkaline (pH is more than 7.5) aerobic environment to form polydopamine nano-microspheres. Preparing a dopamine solution: dissolving 0.121g of tris (hydroxymethyl) aminomethane powder in 100mL of deionized water, titrating with dilute hydrochloric acid to adjust the pH value to 8.5, dissolving 200mg of dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring 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 used for modifying the poly ether ketone, and a mineralization guide tissue regeneration layer containing growth factors is chemically crosslinked. The preparation method comprises the following steps:

preparing a solution for guiding a tissue regeneration layer; the preparation method comprises the following steps: obtaining silk fibroin solution and I type 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 the type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1 percent; dissolving fibroin in the fibroin solution in a lithium bromide solution or a calcium chloride ternary system solution, wherein the mass fraction is 5%; the mass ratio of silk fibroin and type I collagen in the solution of the guided tissue regeneration layer is 7: 3;

preparing a mineralized tissue regeneration layer guiding solution; dripping a tetrahydrate calcium nitrate solution and a diammonium hydrogen phosphate solution into the guided tissue regeneration layer solution, adjusting the pH value to 7 by ammonia water, uniformly mixing, standing the solution, separating out precipitates, and washing away impurity ions to obtain a mineralized guided tissue regeneration layer solution; the mass ratio of the amount of the substances added with calcium ions in the mineralization guide tissue regeneration layer to the protein in the protein solution is 0.01mol/g, and the molar ratio of the amount of the substances added with calcium ions to the amount of the substances added with phosphate ions is 1.67;

preparing microspheres loaded with growth factors;

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

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

and (3) performing freeze drying, glutaraldehyde steam crosslinking, thermal crosslinking, resolution process and cobalt 60 irradiation sterilization to obtain the skull repairing polyetheretherketone material. Specifically, the freeze-drying process conditions are as follows: pre-freezing at-60 deg.C for 12h, and freeze-drying at 10 deg.C under 10Pa for 48 h; the technological conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 12 hours at the temperature of 40 ℃ and the concentration of glutaraldehyde steam of 10 percent; the technological conditions of the thermal crosslinking are as follows: crosslinking for 48h in a vacuum drying oven at 100 ℃ and 100 Pa; the resolving process conditions are as follows: the temperature and time for the analysis were 37 ℃ and 2d in the air-drying oven.

Example 2

the poly-dopamine microspheres modify polyether-ether-ketone. Dopamine can undergo oxidative polymerization in an alkaline (pH is more than 7.5) aerobic environment to form polydopamine nano-microspheres. Preparing a dopamine solution: dissolving 0.121g of tris (hydroxymethyl) aminomethane powder in 100mL of deionized water, titrating with dilute hydrochloric acid to adjust the pH value to 8.5, dissolving 250mg of dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring for 80min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, carrying out magnetic stirring reaction for 24 hours at room temperature, 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 50 ℃ for 12 hours;

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

preparing a mineralized tissue regeneration layer guiding solution; dropwise adding a calcium nitrate tetrahydrate solution and a diammonium hydrogen phosphate solution into the guided tissue regeneration layer solution, adjusting the pH value to 7.5 by using ammonia water, uniformly mixing, standing the solution, separating out a precipitate, washing away impurity ions, and obtaining a mineralized guided tissue regeneration layer solution; the mass ratio of the amount of the substances added with calcium ions in the mineralization guide tissue regeneration layer to the protein in the protein solution is 0.015mol/g, and the molar ratio of the amount of the substances added with calcium ions to the amount of the substances added with phosphate ions is 1.67;

preparing growth factor-loaded microspheres;

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

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

and (3) carrying out freeze drying, glutaraldehyde steam crosslinking, thermal crosslinking, an analytic process and cobalt 60 irradiation sterilization to obtain the skull repairing polyether-ether-ketone material. Specifically, the freeze-drying process conditions are as follows: pre-freezing at-50 deg.C for 12h, and freeze-drying at 20 deg.C under 20Pa for 48 h; the technological conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 6 hours at the temperature of 40 ℃ and the concentration of glutaraldehyde steam of 20 percent; the technological conditions of the thermal crosslinking are as follows: crosslinking for 24 hours in a vacuum drying oven under the conditions of 105 ℃ and 50 Pa; the resolving process conditions are as follows: the analysis temperature was 50 ℃ for 3d in a forced air drying oven.

Example 3

the poly-dopamine microspheres modify polyether-ether-ketone. Dopamine can undergo oxidative polymerization in an alkaline (pH is more than 7.5) aerobic environment to form polydopamine nano-microspheres. Preparing a dopamine solution: dissolving 0.121g of tris (hydroxymethyl) aminomethane powder in 100mL of deionized water, titrating with dilute hydrochloric acid to adjust the pH to 9.0, dissolving 300mg of dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring for 90min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, carrying out magnetic stirring reaction for 48 hours at room temperature, 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;

preparing a solution for guiding a tissue regeneration layer; the preparation method comprises the following steps: obtaining silk fibroin solution and 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 the type I collagen in 0.05mol/L acetic acid solution, and the mass fraction is 1.5%; dissolving fibroin in the silk fibroin solution in a lithium bromide solution or a calcium chloride ternary system solution, wherein the mass fraction is 7.5%; the mass ratio of silk fibroin and type I collagen in the solution of the guided tissue regeneration layer is 1: 1;

preparing a mineralized tissue regeneration layer guiding solution; dropwise adding a calcium nitrate tetrahydrate solution and a diammonium hydrogen phosphate solution into the guided tissue regeneration layer solution, adjusting the pH to 8.0 by using ammonia water, uniformly mixing, standing the solution, separating out a precipitate, washing away impurity ions, and obtaining a mineralized guided tissue regeneration layer solution; the mass ratio of the amount of the substances added with calcium ions in the mineralization guide tissue regeneration layer to the protein in the protein solution is 0.02mol/g, and the molar ratio of the amount of the substances added with calcium ions to the amount of the substances added with phosphate ions is 1.67;

preparing growth factor-loaded microspheres;

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

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

Example 4

the poly-dopamine microspheres modify polyether-ether-ketone. Dopamine can undergo oxidative polymerization reaction in an alkaline (pH > 7.5) aerobic environment to form polydopamine nano-microspheres. Preparing a dopamine solution: dissolving 0.121g of tris (hydroxymethyl) aminomethane powder in 100mL of deionized water, titrating with dilute hydrochloric acid to adjust the pH to 9.5, dissolving 200mg of dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring for 60min to form dopamine solution. Adding the prepared polyether-ether-ketone into a dopamine solution, carrying out magnetic stirring reaction for 36 hours at room temperature, 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;

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

preparing a mineralized tissue regeneration layer guiding solution; dropwise adding a calcium nitrate tetrahydrate solution and a diammonium hydrogen phosphate solution into the guided tissue regeneration layer solution, adjusting the pH value to 7.5 by using ammonia water, uniformly mixing, standing the solution, separating out a precipitate, washing away impurity ions, and obtaining a mineralized guided tissue regeneration layer solution; the mass ratio of the amount of the substances added with calcium ions in the mineralization guide tissue regeneration layer to the protein in the protein solution is 0.01mol/g, and the molar ratio of the amount of the substances added with calcium ions to the amount of the substances added with phosphate ions is 1.67;

preparing growth factor-loaded microspheres;

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

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

and (3) carrying out freeze drying, glutaraldehyde steam crosslinking, thermal crosslinking, an analytic process and cobalt 60 irradiation sterilization to obtain the skull repairing polyether-ether-ketone material. Specifically, the freeze-drying process conditions are as follows: pre-freezing for 24h at-50 ℃, and then freezing and drying for 72h at 25 ℃ and 15 Pa; the technological conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 6 hours at the temperature of 40 ℃ and the concentration of glutaraldehyde steam of 20 percent; the technological conditions of the thermal crosslinking are as follows: crosslinking for 48h in a vacuum drying oven at the temperature of 110 ℃ and under the condition of 100 Pa; the resolving process conditions are as follows: in a forced air drying oven, the analysis temperature is 45 ℃ and the analysis time is 4 d.

Comparative example 1

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

Comparative example 2

In the comparative example, 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) serving as cross-linking agents are not added in the process of compounding the growth factor-containing mineralization guide tissue regeneration layer on the poly-dopamine microsphere modified polyether-ether-ketone; the rest of the procedure was the same as in example 1.

Comparative example 3

In the comparative example, 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) serving as cross-linking agents are not added in the process of compounding the growth factor-containing mineralization guide tissue regeneration layer on the poly-dopamine microsphere modified polyether-ether-ketone; the rest of the procedure was the same as in example 4.

Examples of the experiments

The skull repairing polyether-ether-ketone materials obtained in example 1 and comparative example 1 are subjected to surface contact angle detection, cell adhesion and proliferation capacity detection and alkaline phosphatase (ALP) activity detection.

Surface contact angle detection: and (3) measuring the surface contact angle of each group by using a contact angle tester at room temperature, testing 3 samples by using each group of samples, testing 2 positions by using each sample, and calculating the average value.

Cell adhesion and proliferation capacity assay: MG-63 osteoblasts were inoculated on the surface of a sample in a 24-well plate and cultured, and after the 1 st and 7 th days of culture, human cholecystokinin/cholecystokinin octapeptide (CCK-8) reagent was added and the absorbance value (OD) was measured at a wavelength of 450nm using a microplate reader.

Alkaline phosphatase (ALP) activity assay: MG-63 osteoblasts were seeded on the surface of the samples in 24-well plates for culture, and ALP activity was measured at day 7 and 14 of culture, respectively: the rinsing was repeated 3 times with PBS, 0.1% Triton-X was added, the mixture was placed in a refrigerator and lysed at 4 ℃ for 40min, after which the procedure was followed according to the bicohedral acid (BCA) kit instructions and ALP activity was detected.

The results are shown in the following table:

note: compared with comparative example 1, p is less than 0.05; p < 0.05 in comparison with comparative example 1

From the data in the table, compared with the pure polyetheretherketone material in the comparative example 1, after the chemical crosslinking of the growth factor-containing mineralization guiding tissue regeneration layer on the poly-dopamine microsphere modified polyetheretherketone in the example 1, the contact angle of the polyetheretherketone material is obviously reduced, which indicates that the hydrophilicity is improved, and the cell adhesion is facilitated.

Compared with the pure polyether-ether-ketone material in the comparative example 1, after the poly-dopamine microsphere is used for modifying the polyether-ether-ketone and chemically crosslinking the mineralization guiding tissue regeneration layer containing the growth factors, the CCK test result shows that the chemically modified polyether-ether-ketone material has good biocompatibility, the adhesion and proliferation of the modified cells are obviously improved, and the surface activity is effectively improved.

Compared with the pure polyether-ether-ketone material in the comparative example 1, the ALP activity test result shows that the ALP activity of the chemically modified polyether-ether-ketone material is effectively improved, the surface osteogenic activity is improved and the surface biological activity of the chemically modified polyether-ether-ketone material is effectively improved after the poly-dopamine microsphere is used for modifying the chemically crosslinked mineralized guided tissue regeneration layer containing the growth factors in the polyether-ether-ketone.

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

(1) the method provided by the embodiment of the invention has the advantages that the adopted raw materials are easy to obtain, safe and environment-friendly, and the hidden danger brought 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 mineralization leading 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 mineralization leading tissue regeneration layer can be slowly degraded, the bone induction effect is fully played in the formation of new bones, and the bone formation capability of a skull defect part is improved.

Finally, it should also be 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. Therefore, it is intended that the appended claims be interpreted as including 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 changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A preparation method of a growth factor-containing skull repairing polyetheretherketone material is characterized by comprising the following steps:

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

dissolving a polymer in dichloromethane to obtain a polymer solution;

mixing a 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;

2. The method for preparing a growth factor-containing skull repairing polyetheretherketone material of claim 1, wherein the polymer comprises at least one of polycaprolactone, polyhydroxyaliphatic carboxylic acid, polyhydroxybutyrate-polyhydroxyvalerate interpolymer, polylactic acid-polycaprolactone interpolymer, polyanhydride, polysaccharide, coacervate, glycosaminoglycan, chitosan, cellulose, acrylate polymer, homopolymer of glycolic acid, homopolymer of lactic acid, and copolymer derived from poly (lactide-co-glycolide);

3. The method for preparing the growth factor-containing skull repairing PEEK material of claim 2, wherein each 100mL of the growth factor-containing mineralized guided tissue regeneration layer solution contains 0.1g to 20g of the microspheres, and the diameter of the microspheres is 1 μm to 100 μm;

4. the preparation method of the growth factor-containing skull repairing polyetheretherketone material of claim 2, wherein the mass concentration of the 1-ethyl-3- (3-dimethylamino) carbodiimide hydrochloride in the mixture is 0.1g/mL to 0.3g/mL, and the mass concentration of the N-hydroxysuccinimide is 0.01g/mL to 0.05 g/mL.

5. The preparation method of the growth factor-containing skull repairing polyetheretherketone material according to claim 1, wherein the drying is freeze drying, the freeze drying comprises a first freeze drying and a 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 24 h; 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 50 Pa.

6. The preparation method of the growth factor-containing skull repairing polyetheretherketone material according to claim 1, wherein 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, and the crosslinking time of the thermal crosslinking is 12-48 h.

7. The preparation method of the growth factor-containing skull repairing polyetheretherketone material according to claim 1, wherein the temperature of the analysis is 37 ℃ to 52 ℃ and the time of the analysis is 2d to 4 d.

8. The preparation method of the growth factor-containing skull repairing polyetheretherketone material according to claim 1, wherein the reaction temperature of the reaction is 4 ℃ to 6 ℃ and the reaction time of the reaction is 40h to 60 h.

9. The preparation method of the growth factor-containing skull repairing polyetheretherketone material according to claim 1, wherein the modifying the matrix with the poly-dopamine nano-microspheres to obtain a modified matrix specifically comprises:

10. A skull repairing polyetheretherketone material, characterized in that the polyetheretherketone material is prepared by the method of any one of claims 1 to 9, wherein the method comprises the step of preparing the growth factor-containing skull repairing polyetheretherketone material.