CN114504407A

CN114504407A - 3D printing skull repairing titanium mesh containing growth factors and preparation method thereof

Info

Publication number: CN114504407A
Application number: CN202210024476.3A
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: 2022-01-11
Filing date: 2022-01-11
Publication date: 2022-05-17

Abstract

The invention particularly relates to a 3D printing skull repairing titanium mesh containing growth factors and a preparation method thereof, wherein the preparation method comprises the following steps: analyzing the head to be repaired to obtain skull defect part information; 3D printing is carried out according to the skull defect part information to obtain a titanium mesh skull model; adding the titanium mesh skull model into a dopamine solution to obtain a polydopamine microsphere modified titanium mesh skull model; mixing hydroxyapatite, microspheres, a collagen solution and a silk fibroin solution to obtain 3D printing biological ink; printing the 3D printing biological ink into a coating to cover the polydopamine microsphere modified titanium mesh skull model, and performing post-treatment to obtain a skull repairing titanium mesh; the application combines 3D printing of the titanium mesh and the guided tissue regeneration layer containing the growth factors, so that the surface roughness and the bioactivity of the titanium mesh material can be increased, the combination of the material and bones is promoted, the adhesion and the proliferation of cells are promoted, the osteogenesis is induced, and the repair and the healing of skull tissues are accelerated.

Description

3D printing skull repairing titanium mesh containing growth factors and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a 3D printing growth factor-containing skull repairing titanium mesh 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, calcined bone and deproteinized bone used clinically are obtained 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, a natural pore structure is maintained, the antigenicity is eliminated, but the tissue has small mechanical strength, is loose and fragile, has poor mechanical strength and reduces the plasticity.

The clinically common artificial skull repairing materials mainly comprise hydroxyapatite, polymethyl methacrylate, polyether-ether-ketone, titanium mesh and the like.

The hydroxyapatite has good biocompatibility, osteoconductivity and osteoinductivity, and after being implanted into a body, calcium and phosphorus can be liberated from the surface of the material and absorbed by body tissues, and new bone tissue growth is induced. However, the hydroxyapatite is easy to break under the action of external force after operation, the infection rate after operation is high, and in addition, the hydroxyapatite is degraded too fast in vivo and is usually used for repairing small-area bone defects left by cranial bone drilling, and the large-area bone defects need to be fixed by a titanium mesh.

The polymethyl methacrylate has light weight, low price and strong plasticity, can be instantly shaped according to the shape of the bone defect, and is firmly fixed. The polymethyl methacrylate has the main defects of brittle texture, easy brittle fracture under the action of external force, certain thermal damage to surrounding tissues in the curing process in the operation, and high probability of postoperative infection and exposure.

Polyether ether ketone (PEEK) is a wholly aromatic semi-crystalline thermoplastic polymer material, has good biocompatibility, wear resistance and stable chemical characteristics, and can be sterilized by high-temperature steam or gamma irradiation. The polyether-ether-ketone has strong plasticity, and the elasticity, strength, heat insulation property, stability and other aspects of the polyether-ether-ketone are equivalent to those of the autogenous skull, so that the rejection reaction is generally avoided. The X-ray can penetrate through the magnetic material, has no magnetism, has no artifact in CT or MRI images, and does not influence the postoperative image analysis of patients. However, the melting point of polyetheretherketone is very high (the glass transition temperature is 143 ℃ C., the melting point reaches 343 ℃ C.), which makes its processing extremely difficult. In addition, the PEEK rapid forming piece manufactured by pure 3D printing is loose in material, the mechanical property cannot meet the medical requirement, the operation cost of the PEEK personalized skull is high, and the application of the PEEK rapid forming piece in the personalized skull repairing operation is limited.

The titanium net has the advantages of good biocompatibility and physical and chemical properties, strong plasticity, no magnetism and the like, and can resist secondary trauma. After the implant, fibroblasts can grow into micropores of the titanium mesh, so that the titanium mesh and tissues are integrated, the titanium mesh has the tendency of calcification and ossification, the X-ray examination and the electroencephalogram examination of the skull are not influenced, the hand feeling is good, the uniformity and the attractiveness are realized, and the titanium mesh is widely applied to the field of clinical skull defect repair.

Titanium net that uses clinically is mostly finished product titanium net, and it can maintain stable spatial structure and mechanical properties, nevertheless need tailor again and moulding according to the defective condition of difference, needs repeat many times usually to moulding by hand often the error is great, and the prosthetic integration of preparation is relatively poor, and the precision is lower, can not reach the effect of matching completely, in addition easily forms sharp edge at moulding in-process, has increased the risk that postoperative titanium net exposes. However, the single 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. In addition, the expansion with heat and contraction with cold and the quick heat conduction of the titanium mesh are simply used, so that the problems of sensitivity, irritation, related complications and the like of the scalp, the dura mater and the surrounding skull are solved.

Disclosure of Invention

The application aims to provide a 3D printing skull repairing titanium mesh containing growth factors and a preparation method thereof, and aims to solve the problem that new bone cannot be effectively induced by singly using a finished titanium mesh at present.

The embodiment of the invention provides a preparation method of a 3D printing skull repairing titanium mesh containing growth factors, which comprises the following steps:

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

3D printing is carried out according to the skull defect part information to obtain a titanium mesh skull model;

adding the titanium mesh skull model into a dopamine solution to obtain a polydopamine microsphere modified titanium mesh skull model;

obtaining growth factor loaded microspheres;

obtaining a type I collagen solution;

obtaining silk fibroin solution;

mixing hydroxyapatite, the microspheres loaded with growth factors, the type I collagen solution and the silk fibroin solution to obtain 3D printing biological ink;

printing the 3D printing biological ink into a coating to cover the polydopamine microsphere modified titanium mesh skull model according to the skull defect part information to obtain a primary product;

and performing post-treatment on the primary product to obtain a 3D printed skull repairing titanium mesh, wherein the post-treatment comprises freeze drying, crosslinking, analysis technology and irradiation sterilization with cobalt 6025 kGy dosage.

Optionally, the method for obtaining the polydopamine microsphere modified titanium mesh skull model specifically comprises the following steps:

dissolving trihydroxymethyl aminomethane powder in deionized water, titrating with dilute hydrochloric acid to adjust the pH 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;

adding the titanium mesh skull model into a dopamine solution, carrying out magnetic stirring reaction for 24-48 h at room temperature, repeatedly washing the polydopamine microsphere modified titanium mesh 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 polydopamine microsphere modified titanium mesh skull model.

Optionally, the obtaining of the microsphere loaded with the growth factor specifically includes:

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;

and mixing the second emulsion and the second polyvinyl alcohol, and evaporating to obtain the microspheres loaded with 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, bone morphogenetic protein-2 and platelet-derived growth factor.

Preferably, the microsphere is poly (lactide-co-glycolide) (abbreviated PLGA) and the growth factor is bone morphogenetic protein-2 (BMP-2).

Optionally, the thickness of the titanium mesh skull model is 0.4 mm-10 mm, and the mesh diameter of the titanium mesh skull model is 0.4 mm-0.8 mm; the mass concentration of the dopamine in the dopamine solution is 0.1-20 mg/mL; every 100mL of the 3D printing biological ink contains 0.1-20 g of the microsphere, the diameter of the microsphere is 1-100 mu m, and the microsphere comprises 0.1-10% of growth factor and 90-99.9% of polymer by mass fraction; every 100mL of the 3D printing biological ink contains 0.5g to 1.5g of hydroxyapatite, the hydroxyapatite is medical-grade nano hydroxyapatite, and the particle size of the medical-grade nano hydroxyapatite is 50nm to 150 nm; 0.5-3 g of type I collagen is contained in each 100mL of the 3D printing biological ink, and 0.5-10 g of silk fibroin is contained in each 100mL of the 3D printing biological ink.

Optionally, the obtaining of the type I collagen solution specifically includes:

dissolving type I collagen in a first solvent to obtain a type I collagen solution;

wherein the first solvent is an acetic acid solution, and the concentration of the acetic acid solution is 0.03-0.07 mol/L.

The silk fibroin solution obtained specifically comprises:

dissolving silk fibroin in a second solvent, and then dialyzing to obtain a silk fibroin solution;

wherein the second solvent is a lithium bromide solution or a calcium chloride ternary system solution.

Optionally, the freeze drying includes a pre-freezing process and a freeze drying process, the pre-freezing temperature of the pre-freezing process is-80 ℃ to-20 ℃, the pre-freezing time of the pre-freezing process is 3h to 24h, the freezing temperature of the freeze drying process is-50 ℃ to 37 ℃, the pressure of the freeze drying process is 0.1Pa to 50Pa, and the freezing time of the freeze drying process is 24h to 72 h.

Optionally, the crosslinking process comprises a glutaraldehyde steam crosslinking process and a thermal crosslinking process, wherein the glutaraldehyde steam crosslinking is carried out in a closed container at a crosslinking temperature of 37-52 ℃, the volume concentration of glutaraldehyde steam crosslinked by glutaraldehyde steam is 5-25%, and the time of glutaraldehyde steam crosslinking is 2-12 h; the thermal crosslinking is carried out in a vacuum drying oven at the temperature of 100-110 ℃ and the pressure of 10-150 Pa for 12-48 h.

Optionally, the analysis process is carried out in a forced air drying oven at an analysis temperature of 37-52 ℃ for 2-4 d.

Based on the same inventive concept, the embodiment of the invention also provides a 3D printed skull repairing titanium mesh containing the growth factors, and the skull repairing titanium mesh is prepared by adopting the preparation method of the 3D printed skull repairing titanium mesh 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:

the embodiment of the invention provides a preparation method of a 3D printing skull repairing titanium mesh containing growth factors, which comprises the following steps: analyzing the head to be repaired to obtain skull defect part information; 3D printing is carried out according to the skull defect part information to obtain a titanium mesh skull model; adding the titanium mesh skull model into a dopamine solution to obtain a polydopamine microsphere modified titanium mesh skull model; mixing hydroxyapatite, microspheres, a collagen solution and a silk fibroin solution to obtain 3D printing biological ink; printing the 3D printing biological ink into a coating to cover the polydopamine microsphere modified titanium mesh skull model, and performing post-treatment to obtain a skull repairing titanium mesh; the 3D printing skull repair titanium mesh is combined with the 3D printing titanium mesh and the guided tissue regeneration layer containing the growth factors, so that the surface roughness and the bioactivity of a titanium mesh material can be increased, the combination of the material and bones is promoted, the adhesion and the proliferation of cells are promoted, the osteogenesis is induced, and the skull tissue repair and healing are accelerated.

The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.

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 that: the 3D printing titanium mesh technology is applied to skull repair, so that the problems of plasticity and compatibility of a repair material are solved, the processing speed is high, 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 3D printing skull repairing titanium mesh containing growth factors is provided, and the method comprises the following steps:

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

specifically, first, CT scout and enhancement scans are performed on the head defect site, and then three-dimensional reconstruction is performed to determine the position, size, shape, etc. of the head skull defect.

S2, performing 3D printing according to the skull defect part information to obtain a titanium mesh skull model;

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 bone defect model by using the software, molding by using digital equipment according to the data of the model, and manufacturing a titanium mesh matched with the defect part, wherein the edge of the titanium mesh is 1cm higher than the edge of the defect. The chemical components of the pure titanium prosthesis accord with the GB/T13810-2017 Standard of titanium and titanium alloy processing materials for surgical implants.

In some embodiments, the 3D printing titanium net is integrally formed, the edge of the titanium net is provided with 3-4 integrated protruding blocks, and the end of each protruding block is provided with a titanium nail hole. The thickness of the titanium net is 0.4 mm-10 mm, and the diameter of the mesh is 0.4 mm-0.8 mm.

The thickness of the titanium mesh is controlled to be 0.4-10 mm so as to better meet the requirement of clinical application of skull repair, the too large thickness value can not effectively promote the combination of materials and soft and hard tissues and can not effectively induce osteogenesis, and the too small thickness value can easily form sharp edges, thereby increasing the risk of exposure of the titanium mesh after operation.

Can increase the surface roughness and the bioactivity of the titanium mesh material, promote the combination of the material and the bone, promote the adhesion and the proliferation of cells, induce osteogenesis and accelerate the repair and the healing of skull tissues.

The diameter of the mesh is controlled to be 0.4-0.8 mm, which is beneficial to the adhesion of osteoblasts and the growth of new bones, and the repair and healing of cranial tissues are accelerated, the problems of leakage of soft tissue fluid and related complications caused by overlarge diameter are caused, and the growth of soft and hard tissues, particularly new bones, is not facilitated by undersize.

S3, obtaining the polydopamine microsphere modified titanium mesh skull model, which specifically comprises the following steps:

dissolving trihydroxymethyl aminomethane powder in deionized water, titrating with dilute hydrochloric acid to adjust the pH 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, the dopamine gradually undergoes self-polymerization to form polydopamine along with the increase of the pH, and the color gradually changes from light brown to dark brown;

S4, obtaining microspheres loaded with growth factors;

in some embodiments, growth factor-loaded microspheres are obtained, specifically comprising:

s4.1, dissolving a polymer in dichloromethane to obtain a polymer solution;

s4.2, mixing a growth factor with the polymer solution to obtain a first emulsion;

s4.3, mixing the first emulsion with first polyvinyl alcohol to obtain a second emulsion;

and S4.4, mixing the second emulsion with second polyvinyl alcohol, and then evaporating to obtain the microspheres loaded with the growth factors.

Specifically, a certain amount of the polymer for preparing microspheres was dissolved in Dichloromethane (DCM) to prepare 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 second 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 freeze-dried in a freeze-dryer. The dried microspheres were stored at 4 ℃ until use.

In some embodiments, the polymer is a biocompatible and biodegradable polymer, and in particular, may be 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 acid or lactic acid, and copolymer derived from poly (lactide-co-glycolide) (abbreviated as PLGA).

In some embodiments, the growth factor comprises at least one of vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor, transforming growth factor-beta, bone morphogenetic protein-2, and platelet-derived growth factor.

S5, obtaining a type I collagen solution;

in some embodiments, the obtaining of the type I collagen solution specifically comprises:

S6, obtaining a silk fibroin solution;

in some embodiments, a silk fibroin solution is obtained, specifically comprising:

in this embodiment, the molar ratio of calcium chloride/water/ethanol in the calcium chloride ternary system solution is 1:8: 2.

S7, mixing hydroxyapatite, the microspheres loaded with growth factors, the type I collagen solution and the silk fibroin solution to obtain 3D printing biological ink;

in some embodiments, the bio-ink comprises microspheres with a diameter of 1-100 μm and a concentration of 0.1-20 g/100mL, the microspheres comprising 0.1-10% growth factor and 90-99.9% biopolymer by weight; the particle size of the medical grade nano hydroxyapatite is 50-150 nm, and the concentration of the hydroxyapatite is 0.5-1.5 g/100 mL; the concentration of the type I collagen is 0.5-3 g/100mL, the concentration of the silk fibroin is 0.5-10 g/100mL, and the balance is water.

The hydroxyapatite is used as a main component of natural bone inorganic salt, has good bone conductivity and biocompatibility, is considered as an ideal material for bone defect repair, particularly the nano-scale hydroxyapatite is similar to inorganic components in natural bone, can be introduced into a skull repair material to have great superiority in the aspects of mechanics and biology, is beneficial to the growth of new bone tissues and vascular tissues, the reason for controlling the particle size of the hydroxyapatite to be 50-150 nm is that the material is easy to obtain, has good mechanical property and bone conductivity, better meets the requirements of clinical application of skull repair, the overlarge particle size is not beneficial to the adhesion of the hydroxyapatite on a guide tissue regeneration layer, the 3D printing molding of 3D printing biological ink on a titanium mesh is influenced, the mechanical property of the guide tissue regeneration layer is influenced, and the undersize adverse effect is that the hydroxyapatite is easy to agglomerate, the mechanical property of the guided tissue regeneration layer is influenced; the reason for controlling the concentration of the hydroxyapatite to be 0.5-1.5 g/100mL is that the material better meets the requirements of clinical application of skull restoration in the aspects of mechanics and biology, the too small concentration value can affect the osteoconductivity of a guide tissue regeneration layer and is not beneficial to the growth of new bone tissues, and the too large concentration value is not beneficial to 3D printing and forming of 3D printing biological ink on a titanium mesh, so that the formed guide tissue regeneration layer material has high brittleness, and the mechanical strength is difficult to meet the requirements.

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, the problem of insufficient performance of a single material can be solved, the advantage complementation of various materials is realized, the obtained skull repairing film has good mechanical property and controllable biodegradation time, the reason for controlling the concentration of the type I collagen to be 0.5-3 g/100mL is that the solution is easy to prepare, the material is more in line with the requirement of clinical application of skull repairing, the adverse effect of overlarge concentration value is that the type I collagen is slow to dissolve, the aperture of the prepared guide tissue regeneration layer material is small, the growth of soft and hard tissues is not facilitated, the repair and healing of skull tissues are influenced, the adverse effect of undersize is that the prepared guide tissue regeneration layer material is low in mechanical strength and high in degradation speed, and the requirement of clinical application of skull repairing is not satisfied.

Collagen and silk fibroin are both natural fibrous proteins, have good biocompatibility and bone induction performance, can stimulate differentiation of chondrocytes and osteoblasts around an implanted part to form new bone tissues, and control the concentration of the silk fibroin to be 0.5-10 g/100mL, wherein the adverse effect of overlarge concentration value is that the silk fibroin is slow to dissolve, the aperture of the prepared guide tissue regeneration layer material is small, the mechanical property is high, the degradation time is slow, soft and hard tissues are not favorable to grow in, the repair and healing of skull tissues are influenced, and the adverse effect of undersize is that the prepared guide tissue regeneration layer material is low in mechanical strength and high in degradation speed, and the requirement of clinical application of skull repair is difficult to meet.

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.

S8, printing the 3D printing biological ink into a coating to cover the titanium mesh skull model according to the skull defect part information to obtain a primary product;

specifically, the confirmed skull defect three-dimensional reconstruction model is corrected, printing parameters are set, and the obtained 3D printing biological ink is used for 3D printing a coating on the printed titanium mesh.

In some embodiments, the set printing parameters are: material deposition rate: 10mm/s, extrusion speed: 0.09mm/min, print layer thickness: 0.5mm, platform forming temperature: -20 to 4 ℃, air pressure: 100-300 kpa, mesh diameter 0.4-0.8 mm, printing thickness: 2-4 mm, and the diameter of the equipment needle is 0.4 mm.

And S9, carrying out post-treatment on the primary product to obtain the 3D printed skull repairing titanium mesh.

Specifically, the prepared printing product is subjected to post-treatment, wherein the post-treatment comprises freeze drying, crosslinking and analysis processes and irradiation sterilization with cobalt 6025 kGy dose, and the 3D printing skull repairing titanium mesh is obtained.

The freeze-drying includes a pre-freezing process and a freeze-drying process, and in some embodiments, the freeze-drying process conditions are as follows: pre-freezing for 3-24 h at-20 to-80 ℃, and then drying the frozen body for 24-72 h at-50 to 37 ℃ and under the pressure of 0.1 to 50 Pa.

The reason that the pre-freezing temperature is controlled to be-20 to-80 ℃ and the time is controlled to be 3 to 24 hours is that the actual equipment parameters of the freeze dryer and the freeze-drying process requirements of products are met, the pre-freezing temperature is higher, the pre-freezing time is longer, the pre-freezing temperature is lower, the equipment temperature parameters cannot reach, and the product structure of the subsequent freeze-drying process is influenced.

The reason for controlling the freeze-drying temperature to be-50-37 ℃, the pressure to be 0.1-50 Pa and the time to be 24-72 h is that the actual freeze-drying machine equipment parameters and the product freeze-drying process requirements 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.

The crosslinking process includes a glutaraldehyde steam crosslinking process and a thermal crosslinking process, and in some embodiments, the process conditions of glutaraldehyde steam crosslinking are as follows: crosslinking for 2-12 h at the temperature of 37-52 ℃ and the concentration of glutaraldehyde steam of 10-30%; the thermal crosslinking is carried out in a vacuum drying oven at the temperature of 100-110 ℃ and the pressure of 10-150 Pa for 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 heat crosslinking temperature is controlled to be 100-110 ℃, the pressure is 10-150 Pa, and the crosslinking time is 12-48 h is that the heat crosslinking temperature is low, the crosslinking time is long, and the heat crosslinking temperature is high, so that the structural performance of the product is influenced.

In some embodiments, the desorption process is carried out in a forced air drying oven at a desorption temperature of 37-52 ℃ for 2-4 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.

The 3D printed growth factor-containing skull repairing titanium mesh and the preparation method thereof according to the present application will be described in detail below with reference to examples, comparative examples and experimental data.

Example 1

A preparation method of a 3D printing skull repairing titanium mesh containing growth factors comprises the following steps:

s1, preparing a 3D printing titanium mesh. Firstly, performing CT flat scanning and enhanced scanning on a head defect part, then performing three-dimensional reconstruction, and determining the position, size, shape and the like of the head skull defect; 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 bone defect model by using the software, molding by using digital equipment according to the data of the model, and manufacturing a titanium mesh matched with the defect part, wherein the edge of the titanium mesh is 1cm higher than the edge of the defect. The chemical components of the pure titanium prosthesis accord with the standard of GB/T13810-2017 titanium and titanium alloy processing materials for surgical implants; 3D prints titanium net and is integrated into one piece, and the titanium net edge sets up 4 pieces of integration protrusion pieces, and protrusion piece end is equipped with titanium nail hole. The thickness of the titanium mesh is 0.4mm, and the diameter of the mesh is 0.6 mm.

S2, preparing the polydopamine microsphere modified titanium mesh. 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 titanium mesh obtained or prepared in advance into a dopamine solution, carrying out magnetic stirring reaction for 36 hours at room temperature, repeatedly washing the polydopamine microsphere modified titanium mesh for 2-3 times by using pure water, and drying the polydopamine microsphere modified titanium mesh for 24 hours in a forced air drying oven at 40 ℃.

S3, low-temperature 3D printing guided tissue regeneration coating film. And mixing the microspheres loaded with the growth factors, the medical nano hydroxyapatite, the silk fibroin solution and the type I collagen solution to prepare the 3D printing biological ink. Correcting the skull defect three-dimensional reconstruction model confirmed in the step S1, setting printing parameters, and performing 3D printing on the obtained biological ink on the titanium mesh printed in the step S23D to obtain a tissue regeneration guiding coating film; the diameter of microspheres in the biological ink is 1-100 mu m, the concentration of the microspheres is 10g/100mL, and the microspheres comprise 0.5% of growth factors and 99.5% of biopolymers in mass fraction; the average particle size of the medical grade nano hydroxyapatite is 100nm, and the concentration of the hydroxyapatite is 0.75g/100 mL; the concentration of the type I collagen is 1g/100mL, the concentration of the silk fibroin is 3g/100mL, and the balance is water.

The printing parameters are as follows: material deposition rate: 10mm/s, extrusion speed: 0.09mm/min, print layer thickness: 0.5mm, platform forming temperature: -10 ℃, gas pressure: 200kpa, mesh diameter 0.4mm, print thickness: 3mm and the diameter of the needle of the equipment is 0.4 mm.

And S4, performing post-treatment on the printing product prepared in the step S3, wherein the post-treatment comprises freeze drying, crosslinking and analyzing processes and irradiation sterilization with cobalt 6025 kGy dosage to obtain the 3D printing skull repairing titanium mesh. 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

s1, preparing a 3D printing titanium mesh. Firstly, performing CT flat scanning and enhanced scanning on a head defect part, then performing three-dimensional reconstruction, and determining the position, size, shape and the like of the head skull defect; 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 bone defect model by using the software, molding by using digital equipment according to the data of the model, and manufacturing a titanium mesh matched with the defect part, wherein the edge of the titanium mesh is 1cm higher than the edge of the defect. The chemical components of the pure titanium prosthesis accord with the standard of GB/T13810-2017 titanium and titanium alloy processing materials for surgical implants; 3D prints the titanium net and is integrated into one piece, and the titanium net edge sets up 3 pieces of integration protrusion pieces, and protrusion piece end is equipped with titanium nail hole. The thickness of the titanium mesh is 0.8mm, and the diameter of the mesh is 0.6 mm.

S2, preparing the polydopamine microsphere modified titanium mesh. 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 titanium mesh obtained or prepared in advance into a dopamine solution, carrying out magnetic stirring reaction for 24 hours at room temperature, repeatedly washing the polydopamine microsphere modified titanium mesh for 2-3 times by using pure water, and drying the polydopamine microsphere modified titanium mesh for 12 hours in a forced air drying oven at 50 ℃.

S3, low-temperature 3D printing guided tissue regeneration coating film. And mixing the microspheres loaded with the growth factors, the medical nano hydroxyapatite, the silk fibroin solution and the type I collagen solution to prepare the 3D printing biological ink. Correcting the skull defect three-dimensional reconstruction model confirmed in the step S1, setting printing parameters, and performing 3D printing on the obtained biological ink on the titanium mesh printed in the step S23D to obtain a tissue regeneration guiding coating film; the diameter of microspheres in the biological ink is 1-100 mu m, the concentration of the microspheres is 6g/100mL, and the microspheres comprise 0.5% of growth factors and 99.5% of biopolymers in percentage by mass; the average particle size of the medical grade nano hydroxyapatite is 120nm, and the concentration of the hydroxyapatite is 0.5g/100 mL; the concentration of the type I collagen is 1.5g/100mL, the concentration of the silk fibroin is 3g/100mL, and the balance is water.

The printing parameters are as follows: material deposition rate: 10mm/s, extrusion speed: 0.09mm/min, print layer thickness: 0.5mm, platform forming temperature: -4 ℃, gas pressure: 250kpa, mesh diameter 0.4mm, print thickness: 2mm and the diameter of the needle of the device is 0.4 mm.

And S4, performing post-treatment on the printing product prepared in the step S3, wherein the post-treatment comprises freeze drying, crosslinking and analyzing processes and irradiation sterilization with cobalt 6025 kGy dosage to obtain the 3D printing skull repairing titanium mesh. 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

s1, preparing a 3D printing titanium mesh. Firstly, performing CT flat scanning and enhanced scanning on a head defect part, then performing three-dimensional reconstruction, and determining the position, size, shape and the like of the head skull defect; 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 bone defect model by using the software, molding by using digital equipment according to the data of the model, and manufacturing a titanium mesh matched with the defect part, wherein the edge of the titanium mesh is 1cm higher than the edge of the defect. The chemical components of the pure titanium prosthesis accord with the standard of GB/T13810-2017 titanium and titanium alloy processing materials for surgical implants; 3D prints the titanium net and is integrated into one piece, and the titanium net edge sets up 3 pieces of integration protrusion pieces, and protrusion piece end is equipped with titanium nail hole. The thickness of the titanium mesh is 1mm, and the diameter of the mesh is 0.6 mm.

S2, preparing the polydopamine microsphere modified titanium mesh. 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. And (4) adding the titanium mesh prepared in the step (S1) into a dopamine solution, carrying out magnetic stirring reaction for 48 hours at room temperature, repeatedly washing the poly-dopamine microsphere modified titanium mesh with pure water for 2-3 times, and drying in a forced air drying oven for 24 hours at 40 ℃.

S3, low-temperature 3D printing guided tissue regeneration coating film. And mixing the microspheres loaded with the growth factors, the medical nano hydroxyapatite, the silk fibroin solution and the type I collagen solution to prepare the 3D printing biological ink. Correcting the skull defect three-dimensional reconstruction model confirmed in the step S1, setting printing parameters, and performing 3D printing on the obtained biological ink on the titanium mesh printed in the step S23D to obtain a tissue regeneration guiding coating film; the diameter of microspheres in the biological ink is 1-100 mu m, the concentration of the microspheres is 15g/100mL, and the microspheres comprise 0.6% of growth factors and 99.4% of biopolymers in mass fraction; the average particle size of the medical grade nano hydroxyapatite is 150nm, and the concentration of the hydroxyapatite is 1g/100 mL; the concentration of the type I collagen is 2g/100mL, the concentration of the silk fibroin is 3g/100mL, and the balance is water.

The printing parameters are as follows: material deposition rate: 10mm/s, extrusion speed: 0.09mm/min, print layer thickness: 0.5mm, platform forming temperature: 0 ℃, air pressure: 250kpa, mesh diameter 0.4mm, print thickness: 3mm, and the diameter of the needle of the equipment is 0.4 mm.

And S4, performing post-treatment on the printing product prepared in the step S3, wherein the post-treatment comprises freeze drying, crosslinking, analysis technology and irradiation sterilization with cobalt 6025 kGy dosage, and obtaining the 3D printing skull repairing titanium mesh. 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 the concentration of glutaraldehyde steam of 25 percent; 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 resolving process conditions are as follows: in a forced air drying oven, the analysis temperature is 45 ℃ and the analysis time is 3 d.

Example 4

s1, preparing a 3D printing titanium mesh. Firstly, performing CT flat scanning and enhanced scanning on a head defect part, then performing three-dimensional reconstruction, and determining the position, size, shape and the like of the head skull defect; 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 bone defect model by using the software, molding by using digital equipment according to the data of the model, and manufacturing a titanium mesh matched with the defect part, wherein the edge of the titanium mesh is 1cm higher than the edge of the defect. The chemical components of the pure titanium prosthesis accord with the standard of GB/T13810-2017 titanium and titanium alloy processing material for surgical implants; 3D prints the titanium net and is integrated into one piece, and the titanium net edge sets up 3 pieces of integration protrusion pieces, and protrusion piece end is equipped with titanium nail hole. The thickness of the titanium mesh is 0.6mm, and the diameter of the mesh is 0.6 mm.

S2, preparing the polydopamine microsphere modified titanium mesh. 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 9.5, dissolving 200mg of dopamine hydrochloride powder in tris (hydroxymethyl) aminomethane solution, mixing and stirring for 60min to form dopamine solution. Adding the titanium mesh prepared in the step S1 into a dopamine solution, carrying out magnetic stirring reaction for 36 hours at room temperature, repeatedly washing the polydopamine microsphere modified titanium mesh for 2-3 times by using pure water, and drying the polydopamine microsphere modified titanium mesh for 12 hours in a forced air drying oven at 45 ℃;

s3, low-temperature 3D printing guided tissue regeneration coating film. And mixing the microspheres loaded with the growth factors, the medical nano hydroxyapatite, the silk fibroin solution and the type I collagen solution to prepare the 3D printing biological ink. Correcting the skull defect three-dimensional reconstruction model confirmed in the step S1, setting printing parameters, and performing 3D printing on the obtained biological ink on the titanium mesh printed in the step S23D to obtain a tissue regeneration guiding coating film; the diameter of microspheres in the biological ink is 1-100 mu m, the concentration of the microspheres is 20g/100mL, and the microspheres comprise 1% of growth factors and 90% of biological polymers in percentage by mass; the average particle size of the medical grade nano hydroxyapatite is 120nm, and the concentration of the hydroxyapatite is 1.5g/100 mL; the concentration of the type I collagen is 3g/100mL, the concentration of the silk fibroin is 3g/100mL, and the balance is water.

The printing parameters are as follows: material deposition rate: 10mm/s, extrusion speed: 0.09mm/min, print layer thickness: 0.5mm, platform forming temperature: 4 ℃, air pressure: 300kpa, mesh diameter 0.4mm, print thickness: 3mm, and the diameter of the needle of the equipment is 0.4 mm.

And S4, performing post-treatment on the printing product prepared in the step S3, wherein the post-treatment comprises freeze drying, crosslinking and analyzing processes and irradiation sterilization with cobalt 6025 kGy dosage to obtain the 3D printing skull repairing titanium mesh. Specifically, the freeze-drying process conditions are as follows: pre-freezing for 24h at-50 ℃, and then drying the frozen body 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: the temperature of the analysis in the air-drying oven was 45 ℃ and the time of analysis was 4 d.

Comparative example 1

And (4) obtaining a finished product titanium mesh in the market, and cutting and shaping to obtain the skull repairing titanium mesh.

Experimental example 1

The titanium meshes obtained in example 1 and comparative example 1 were used for a skull defect model of a New Zealand rabbit. 40 New Zealand rabbit skull defect repair models are taken for the test and divided into a control group and a test group, wherein each group comprises 20 cases. The test group is the example 1, the skull repairing titanium mesh is printed by 3D, the comparison group is the comparative example 1, the comparison group is a commercially available finished product titanium mesh, and the skull repairing titanium mesh is prepared by cutting and shaping. The test period is 3-18 months, and the conditions of complications (incisional infection, titanium net leakage, bone leakage, titanium net infection and asymmetric skull) after the skull defect repair of the New Zealand rabbits are observed. The results are shown in the following table:

the skull repairing titanium mesh prepared by the method is used for a skull defect model of a New Zealand rabbit, and complications are obviously lower than those of a control group, because the titanium mesh is prepared by a 3D printing method, the titanium mesh can be molded according to different defect conditions, the manufactured titanium mesh has high precision, no sharp edge can be formed in the molding process, the compatibility with a defect part is good, and the matching effect can be completely achieved; in addition, the 3D printing titanium mesh and the guided tissue regeneration layer containing growth factors are adopted, so that the surface roughness and the bioactivity of the titanium mesh material can be increased, and the risks of postoperative infection and exposure are avoided.

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

(1) the raw materials are easy to obtain, safe and environment-friendly, and hidden danger brought to human bodies in the preparation process and the use of final products is avoided;

(2) the titanium mesh is prepared by adopting a 3D printing method, the shaping can be carried out according to different defect conditions, the precision of the prepared titanium mesh is higher, sharp edges cannot be formed in the shaping process, the compatibility with defect parts is better, the matching effect can be completely achieved, and the risk of post-operation titanium mesh exposure is avoided;

(3) 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. 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 titanium mesh, so that the biocompatibility and the bioactivity of the porous titanium mesh can be improved, and the secondary coating modification is carried out on the surface of the porous titanium mesh, so that the adhesion, the proliferation and the secretion of extracellular matrix of seed cells on the surface of the material are facilitated, and the rapid fusion of the repairing material and soft tissues is accelerated;

(4) good bone conductivity, and is beneficial to the growth of new bone tissues and vascular tissues. Hydroxyapatite is the main component of inorganic salt of natural bone, has good bone conductivity and biocompatibility, is considered as an ideal material for bone defect repair, and particularly, the nano-grade hydroxyapatite is similar to the inorganic component of the natural bone, and can be introduced into the bone repair material to ensure that the material has great superiority in the aspects of mechanics and biology, thereby being beneficial to the growth of new bone tissues and vascular tissues;

(5) 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 and the type I collagen andor the silk fibroin are compounded for use, 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 obtained bone repair material is matched with the biomechanical property of original skull bone tissues of an implanted part;

(6) good osteoinductivity, and can stimulate the differentiation of chondrocytes and osteoblasts around the implanted part to form new bone tissue. 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;

(7) bone morphogenetic protein-2 (BMP-2) is considered to be a growth factor having the strongest bone induction ability and promoting bone regeneration, and is capable of inducing proliferation and differentiation of undifferentiated mesenchymal stem cells into osteoblasts 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 number of wound surface new capillaries and the number of fibroblasts are promoted, and the repair and healing of skull tissues are accelerated;

(8) the poly dopamine microspheres and the tissue regeneration guiding layer containing the growth factors are compounded on the titanium mesh, so that the problems of thermal expansion and cold contraction, high heat conduction speed, susceptibility to cold and heat of scalp, dura mater and surrounding skull, irritation, related complications and the like caused by the fact that the titanium mesh is only used can be solved.

(9) The pore diameter and porosity of micropores communicated with the bone repair material can be accurately controlled by a 3D printing method, and the method is favorable for adhesion of seed cells and growth factors and exchange of nutrient substances and metabolites. The low-temperature rapid prototyping manufacturing technology (LDM) is a novel rapid prototyping technology based on the principle of rapid prototyping technology and combined with a phase separation method, and is different from other rapid prototyping technologies in that the temperature in a prototyping cavity is controlled to be about-30 ℃, a solution extruded by a nozzle is rapidly condensed at low temperature, the nozzle moves according to a program under the control of a computer, a printing layer is finally molded into a three-dimensional porous framework through layer-by-layer superposition, the properties and the structure of a material are not damaged in the material processing process, and the LDM belongs to the range of green manufacturing.

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 3D printed skull repairing titanium mesh containing growth factors is characterized by comprising the following steps:

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

obtaining growth factor loaded microspheres;

obtaining a type I collagen solution;

obtaining silk fibroin solution;

2. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the obtaining of the polydopamine microsphere modified titanium mesh skull model specifically comprises:

3. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the obtaining of growth factor-loaded microspheres specifically comprises:

dissolving a polymer in dichloromethane to obtain a polymer solution;

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

4. The method for 3D printing of a growth factor containing cranial repair titanium mesh according to claim 3, 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);

5. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the thickness of the titanium mesh skull model is 0.4mm to 10mm, and the mesh diameter of the titanium mesh skull model is 0.4mm to 0.8 mm; the mass concentration of the dopamine in the dopamine solution is 0.1-20 mg/mL; every 100mL of the 3D printing biological ink contains 0.1-20 g of the microsphere, the diameter of the microsphere is 1-100 mu m, and the microsphere comprises 0.1-10% of growth factor and 90-99.9% of polymer by mass fraction; every 100mL of the 3D printing biological ink contains 0.5g to 1.5g of hydroxyapatite, the hydroxyapatite is medical-grade nano hydroxyapatite, and the particle size of the medical-grade nano hydroxyapatite is 50nm to 150 nm; 0.5-3 g of type I collagen is contained in each 100mL of the 3D printing biological ink, and 0.5-10 g of silk fibroin is contained in each 100mL of the 3D printing biological ink.

6. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the obtaining of the type I collagen solution specifically comprises:

wherein the first solvent is an acetic acid solution, and the concentration of the acetic acid solution is 0.03-0.07 mol/L;

the silk fibroin solution obtained specifically comprises:

7. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the freeze drying comprises a pre-freezing process and a freeze drying process, the freezing temperature of the pre-freezing process is-80 ℃ to-20 ℃, the pre-freezing time of the pre-freezing process is 3h to 24h, the freezing temperature of the freeze drying process is-50 ℃ to 37 ℃, the pressure of the freeze drying process is 0.1Pa to 50Pa, and the freezing time of the freeze drying process is 24h to 72 h.

8. The preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to claim 1, wherein the crosslinking comprises a glutaraldehyde steam crosslinking process and a thermal crosslinking process, the glutaraldehyde steam crosslinking has a crosslinking temperature of 37-52 ℃ in a closed container, the volume concentration of glutaraldehyde steam for the glutaraldehyde steam crosslinking is 5-25%, and the time for the glutaraldehyde steam crosslinking is 2-12 h; the thermal crosslinking is carried out in a vacuum drying oven at the temperature of 100-110 ℃ and the pressure of 10-150 Pa for 12-48 h.

9. The preparation method of the 3D printed growth factor-containing skull repairing titanium mesh is characterized in that the resolving process is that the resolving temperature is 37-52 ℃ and the resolving time is 2-4D in a blast drying oven.

10. The 3D printing growth factor-containing skull repairing titanium mesh is characterized by being prepared by the preparation method of the 3D printing growth factor-containing skull repairing titanium mesh according to any one of claims 1 to 9.