CN117427212A - Biological functional porous polyether-ether-ketone bone tissue scaffold material and preparation method thereof - Google Patents
Biological functional porous polyether-ether-ketone bone tissue scaffold material and preparation method thereof Download PDFInfo
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- CN117427212A CN117427212A CN202311434756.2A CN202311434756A CN117427212A CN 117427212 A CN117427212 A CN 117427212A CN 202311434756 A CN202311434756 A CN 202311434756A CN 117427212 A CN117427212 A CN 117427212A
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
The invention belongs to the field of medical composite materials, and particularly relates to a biological functional porous polyether-ether-ketone bone tissue scaffold material and a preparation method thereof. In addition, the PEEK material has excellent biocompatibility and is simple to machine. The invention also provides a corresponding preparation method, and the whole technical scheme can promote the development of the biological composite material and provide scientific basis and technical support for the application of the novel artificial bone repair material in the medical field.
Description
Technical Field
The invention belongs to the field of new materials, and particularly relates to a biological functional porous polyether-ether-ketone bone tissue scaffold material and a preparation method thereof.
Background
Clinically common bone repair materials are mainly divided into three main categories: metal medical material, ceramic medical material, and polymer medical material. Among them, the metal medical materials are most widely used due to their high strength, easy molding, low cost, etc., and include titanium, titanium alloy, stainless steel, etc. However, the elastic modulus of the metal materials is greatly different from that of human bones, so that a stress shielding effect is easy to generate, the tissue of the planting part is absorbed, the implant is loosened and even falls off, and meanwhile, metal ions are released, so that inflammatory reaction occurs at the wound part, and the compatibility of the materials is reduced. The ceramic medical material HAs good tissue affinity and bone binding capacity, and is widely applied clinically, such as Hydroxyapatite (HA), bioactive molecular glass, tricalcium phosphate and the like. However, the ceramic medical material has poor strength and toughness, and has low degradation rate and solubility in a certain physiological environment, and can harm the health of human bodies when serious.
High molecular medical materials (polyurethane and the like) have high specific strength and good fatigue resistance, but most of the materials are inert, and the surface of the materials is required to be modified to meet the performance requirements. In addition, some high polymer materials are easy to absorb ultraviolet rays or visible light to degrade the materials, but compared with the high polymer materials, the high polymer materials have higher cost performance in terms of service life, mechanical performance and the like. Therefore, it is urgent to find a new bone substitute material.
Polyether ether-ketone (PEEK) polymer materials are polymers formed by interconnecting ketone bonds and ether bonds repeatedly occurring in a main chain structure, and have been widely used in the fields of aerospace, mechanical manufacturing, medical treatment and the like due to their excellent characteristics since being invented, and are called as "the most promising novel artificial bone materials". However PEEK also has some unavoidable drawbacks as an implant:
(1) The PEEK material has low surface energy, and is difficult to modify by adopting a conventional treatment method;
(2) PEEK belongs to inert materials, has low surface biological activity and is not beneficial to adsorption of cells and proteins;
(3) The PEEK has poor antibacterial property, and the surface of the PEEK is easy to be infected with bacteria after being implanted as an implant, so that inflammatory reaction is caused.
Dopamine (DA) is a neurotransmitter secreted by mammals and spontaneously polymerizes in weakly alkaline solutions to form black Polydopamine (PDA). The PDA can be adhered to the surface of almost any solid material. The PDA surface has a large number of phenolic hydroxyl and amino functional groups, and can be tightly combined with the substrate material, so that a compact film is formed on the surface. In addition, because of the existence of a large amount of phenolic hydroxyl groups, the substrate material can also be combined with other substances, such as metal ions, protein active substances and the like to realize secondary reaction, so as to obtain corresponding properties. Thus, in general, the surface of the material may be modified with DA to improve the surface state of the material, as the surface of the material is rendered inert.
The osteogenic active factor BFP-1 belongs to one member of the beta superfamily of transforming factors, is an active molecular chain extracted from the immature region of bone morphogenetic protein BMP-7, and the molecular structure of BFP-1 is more stable than BMP-7 and the osteogenic activity of BFP-1 is better than that of BMP-7. BFP-1 is grafted onto the surface of the implant material, and as the implant is implanted into the body, the BFP-1 can play a positive role in osteogenic differentiation and the combination of the implant and the bone interface. In addition, the osteogenic active factor BFP-1 can also regulate the formation of blood vessels, and can improve the differentiation of mesenchymal stem cells and the activity of alkaline phosphatase.
Therefore, how to better solve the problems of low surface energy, poor osseointegration performance and the like of the PEEK material, and integrate the functional materials into an organic whole becomes a big problem to be solved in the field.
Disclosure of Invention
Aiming at a plurality of defects existing in the prior art, the invention provides a biological functional porous polyether-ether-ketone bone tissue scaffold material and a preparation method thereof, wherein the bone tissue scaffold is prepared by adhering a PEEK matrix material and an osteogenic active factor BFP by polydopamine, so that a SPEEK-PDA-BFP composite bionic configuration is realized, and the polyether-ether-ketone (PEEK) is a novel high polymer, has excellent elastic modulus and mechanical stability, and overcomes the shielding stress effect of the high elastic modulus of the traditional medical metal material. In addition, the PEEK material has excellent biocompatibility and is simple to machine. The inventors have provided the corresponding preparation methods at the same time. The technical scheme provided by the invention can promote the development of biological composite materials and provide scientific basis and technical support for the application of the novel artificial bone repair material in the medical field.
The theoretical basis of the invention is as follows:
PEEK material has several advantages as an implant material:
(1) Ideal modulus of elasticity: compared with metal medical materials, the PEEK material has the density closer to that of human bones, the elastic modulus of titanium and titanium alloy is 100-116GPa, and the elastic modulus of the PEEK material is only 3-4GPa, so that the PEEK material is the material which is the most similar to the elastic modulus of human cortical bone at present, can avoid bone loss at the edge of a planting position caused by stress shielding, and is gradually becoming the first choice of human implantation materials at present;
(2) Good biosafety: the biological safety of the material is characterized by no mutagenicity, no toxicity, no sensitization and the like after the material is implanted into a human body. The PEEK material does not release any toxic ions after being implanted into a human body, is friendly to cells and organs, can promote the combination speed of the PEEK material and the bone growth, and has no adverse effect on the human body basically;
(3) Excellent mechanical stability: the PEEK material is formed by connecting ketone bonds and ether bonds which repeatedly appear in a main chain structure, the structure is very stable according to the chemical formula, the PEEK material can be repeatedly sterilized under high pressure for about 3000 times at the high temperature of 134 ℃, the performance of the material can not be changed under the conditions of irradiation of hot steam, ethylene oxide and gamma rays, and the PEEK material is only dissolved in high-concentration strong acid according to the report at present;
(4) The PEEK material is easy to process and form, and can be prepared into various shapes and sizes without complex procedures.
Therefore, the inventor decides to fix the osteogenic active substance BFP-1 on the surface of the PEEK material by taking PDA as a connector, regulate the reaction condition and the preparation process, and finally construct a composite modified layer with good soft tissue sealing and excellent osseointegration capability on the surface of the PEEK material.
Under the guidance of the theory, the specific technical scheme of the invention is as follows:
a biological functional porous polyether ether ketone bone tissue scaffold is prepared from polydopamine to adhere PEEK matrix material and osteogenic active factor BFP, so as to realize the preparation of SPEEK-PDA-BFP composite bionic configuration.
The PEEK matrix material is required to be pretreated, specifically, a PEEK sample after pretreatment is put into sulfuric acid solution for sulfonation; the sulfonated SPEEK is placed in a mixed solution for modification. The loading of BFP-1 in the finally obtained material is 0.8-1.2mg/cm 2 。
The inventor further provides a preparation method of the biological functionalized porous polyether-ether-ketone bone tissue scaffold, which comprises the following specific steps:
(1) Processing PEEK matrix material into a disc shape with the specification of 20mm multiplied by 1 mm;
(2) Sequentially polishing the sample by using silicon sand paper with the model numbers of 800#, 1000#, 1500#, and 2000 #; respectively ultrasonically oscillating and cleaning the polished PEEK sample with acetone, alcohol and deionized water for 30min; drying at 60 ℃ to obtain PEEK with smooth surface and no impurities;
(3) Immersing the PEEK in concentrated sulfuric acid with the concentration of 98%; carrying out ultrasonic treatment, wherein the ultrasonic frequency is 40Hz, and the ultrasonic treatment time is 5-15min; after ultrasonic treatment, cleaning PEEK by deionized water, and then respectively carrying out ultrasonic treatment by acetone, alcohol and deionized water for 30min; the aim is to remove residual sulfur-containing groups as much as possible; drying the cleaned sample at 60 ℃ to obtain SPEEK, wherein a plurality of pores are uniformly distributed on the surface of PEEK, and the pores are distributed at 2-8 mu m in size and are mutually connected to form a 3D network structure;
(4) Mixing 50mL of prepared dopamine solution with the concentration of 4mg/mL with 50mL of polypeptide solution with the concentration of 2mg/mL prepared by dissolving the osteogenic active factor BFP-1 in phosphate buffer solution; immersing the SPEEK sample obtained in the step (3) in the prepared mixed solution, and carrying out heat preservation and stirring treatment at the temperature of 4 ℃ for 24 hours; and washing the sample subjected to heat preservation and stirring by deionized water, and drying at room temperature to obtain a target product SPEEK-PDA-BFP.
In addition, for better contrast, the preparation method further comprises the following steps:
placing the SPEEK obtained in the step (3) into 100mL of dopamine solution with the concentration of 2mg/mL for self-polymerization reaction, and stirring for 24 hours at room temperature; washing the stirred SPEEK with deionized water to remove polydopamine remained on the surface in a physical adsorption mode, so that the surface of the SPEEK is only provided with polydopamine linked by a bond action; drying to obtain SPEEK-PDA; the SPEEK-PDA can be used as a reference substance to perform functional comparison with the SPEEK-PDA-BFP.
According to the preparation method, the PEEK material is sulfonated to prepare the 3D porous network structure, then the self-polymerization reaction of dopamine is utilized to generate polydopamine, a large number of phenolic hydroxyl functional groups contained on the surface of polydopamine are subjected to chemical grafting with amino functional groups on the osteogenic active factor BFP-1 through Michael addition reaction, the BFP-1 is fixed on the surface of the matrix material, the problems of bioinert property, low surface energy and poor biological activity of the PEEK material are solved, and a SPEEK-PDA-BFP composite bionic structure with excellent biocompatibility and osseointegration capacity is finally constructed.
Compared with the traditional method that a layer of more uniform polydopamine is loaded on the surface through the self-polymerization reaction of dopamine, and then the secondary stirring is carried out to load the modifier, the method provided by the application utilizes the self-polymerization reaction of the dopamine and rich functional groups thereof on the basis of the porous structure after sulfonation, so that the modifier BFP-1 can react with the chemical functional groups of the dopamine to generate chemical bonds in the modification process to form chemical grafts, and the dopamine slowly self-polymerization process can generate stress on the structure of the dopamine due to the reaction, and the spontaneous coating of the modifier BFP-1 is carried out, so that the result that the dopamine is integrally loaded at the pores after the dopamine is finally formed, and the modifier has the grafting reaction of the chemical bonds and is coated by the dopamine. In the traditional preparation method, the PDA is stirred and then loaded, so that a uniform PDA layer is formed on the surface of the PDA after the PDA is stirred, and BFP can only be loaded outside the PDA layer during secondary loading.
Preferably, the ultrasonic time in the step (3) is 10min, because the inventor finds that the contact angle of the material reaches the maximum when being subjected to ultrasonic treatment for 10min, the contact angle is 123 DEG + -3.7 DEG, the surface of the material is in a hydrophobic state, and because innumerable micro-scale small holes are formed on the surface of the material after being treated by concentrated sulfuric acid, the holes are mutually connected to form a 3D porous network structure, and the structure can provide growth active sites for the growth, attachment and proliferation of osteoblasts during the subsequent in vitro cell combined culture.
In summary, the technical scheme provided by the invention provides a novel biological functional porous polyether-ether-ketone bone tissue scaffold material, which can promote the development of biological composite materials and provide scientific basis and technical support for the application of novel artificial bone repair materials in the medical field.
Drawings
FIG. 1 is a graph comparing water contact angles of different ultrasound times SPEEK prepared in the present invention.
FIG. 2 is a graph comparing water contact angles of different final samples prepared in the present invention.
FIG. 3 is an infrared spectrum of SPEEK, SPEEK-PDA, and SPEEK-PDA-BFP;
the top is SPEEK-PDA-BFP, the middle is SPEEK-PDA, the bottom is SPEEK, and the top is 3349cm -1 The peak is caused by the stretching vibration of the phenolic hydroxyl group contained in the dopamine, and only the SPEEK has no characteristic peak, so that the dopamine can be proved to be successfully coated on the surface of the SPEEK sample; at 928cm -1 、1593cm -1 The peak at (c=o) represents the stretching vibration peak of carbonyl (c=o) because the phenolic hydroxyl group in dopamine is oxidized to a phthalquinone functional group under aerobic conditions, i.e. the product occurring during the self-polymerization of dopamine to polydopamine; at 1491cm -1 The peak at the peak represents the N-H bending vibration peak in BFP-1, indicating that BFP-1 was successfully loaded into the PEEK matrix materialAnd (5) feeding.
FIG. 4 is an SEM image of the surface of a different sample prepared in the present invention;
FIG. 4a is a surface image of PEEK; FIG. 4b shows a SPEEK treated surface with a number of voids providing sites for loading BFP and PDA; FIG. 4c shows that the pore structure of the sulfonated PEEK surface is substantially uniformly wrapped by polydopamine after the SPEEK is modified by dopamine, which is equivalent to covering the sample surface with a uniform PDA membrane layer, and the membrane layer has a certain fold shape; FIG. 4d shows that after grafting the material with the osteogenic active factor BFP-1, a plurality of uniform micron-sized agglomerated small particles are attached to the surface or at the surface pore structure, and these small particles are the chemically grafted osteogenic active factor BFP-1.
FIG. 5 shows the mineralization effect of the bone tissue scaffold prepared according to the present invention on 1.5 times SBF simulated body fluid immersion for 7 days and 14 days;
FIGS. 5a, b are graphs of SPEEK simulating body fluid mineralization effects on days 7 and 14, respectively;
FIGS. 5c, d are graphs of SPEEK-PDA simulated body fluid mineralization effects on days 7 and 14, respectively;
FIGS. 5e, f are SPEEK-PDA-BFP mimics body fluid mineralization effects on days 7 and 14, respectively;
after the material is soaked in SBF simulated body fluid for 7 days, the SPEEK and the SPEEK-PDA hardly have precipitation, but have smaller spherical particles, and the size is about 400 nm; however, after soaking for 14 days, the surface of the material has a large number of clustered particles, and the sediment size is about 5 mu m and is distributed uniformly; in sharp contrast, after 7 days of soaking, more agglomerates had appeared on the surface of SPEEK-PDA-BFP, and the number of calcium-phosphorus precipitates increased dramatically with prolonged soaking time, while the appearance of a large amount of calcium-phosphorus precipitates meant that the implant material had osteogenic activity.
FIG. 6 shows cell adhesion rates of surfaces of different materials prepared according to the present invention;
the surface of SPEEK after concentrated sulfuric acid treatment belongs to an inert surface, the content of hydrophilic groups is low, so that the capacity of cell adhesion is limited, and the surface of SPEEK after dopamine coating and BFP-1 grafting is provided with a large number of hydrophilic phenolic hydroxyl groups and amino groups, and the existence of the hydrophilic groups can promote the growth and adhesion of osteoblasts. From this, it can be demonstrated that the composite modified layer PDA-BFP has the effect of promoting cell adhesion and proliferation.
FIG. 7 shows the bacteriostasis rates of the surfaces of different materials prepared according to the invention;
the dopamine can inhibit the growth of bacteria, on the other hand, the BFP-1 has the functions of sterilization and anti-inflammation, and can react with organic matters in bacteria, so that bacteria are decomposed to destroy the morphological structure, and under the synergistic effect of the two, the bacteriostatic effect of a SPEEK-PDA-BFP sample is optimal.
FIG. 8 is a graph showing statistics of ALP activity values of surfaces of different materials prepared according to the present invention;
alkaline phosphatase is an important constituent of human osteoblasts, and is mainly involved in the proliferation and differentiation of cells. The alkaline phosphatase activity of each group was significantly improved (above 7 days) by day 14, and the alkaline phosphatase activity value of the SPEEK-PDA-BFP group was still higher than that of the other two groups (p < 0.05); the data above is sufficient to demonstrate that the SPEEK-PDA-BFP composite bionic configuration has obvious promotion effect on proliferation and differentiation of osteoblasts under the synergistic effect of PDA and BFP-1.
FIG. 9 is a graph comparing the analysis of example 3 8 weeks post-operative material with surrounding bone tissue Micro-CT;
FIGS. 9a, b, c and d are CT images of PEEK, SPEEK, SPEEK-PDA and SPEEK-PDA-BFP, respectively; FIGS. 9e, f are CT diagrams of trabecular bone structures employing SPEEK-PDA-BFP;
compared with the SPEEK sample in the control group, the volume of the new bone of the SPEEK-PDA-BFP sample has a significant difference with the repair area of the defect. As can be seen from the graph, no new bone tissue is observed at the bone defect part of the SPEEK group sample, the bone repair effect is not obvious, and the wound closure rate is 0; the repair effect of the SPEEK-PDA-BFP group sample on the bone defect part is obvious, the wound closure rate is 93%, and the new bone tissue is relatively complete; the new bone tissue in the circle (d) - (f) of SPEEK-PDA-BFP is a trabecular bone structure formed around the material after being implanted into the body, so that the tissue is continuous and compact, and the bone maturity is high; the implant material PEEK can play a role of a 'bone connecting bridge' and a supporting role in the bone reconstruction process, PDA plays a role in promoting growth and proliferation of bone cells, BFP-1 plays a role in bone tissue formation and differentiation, and the synergistic effect of the three plays a role in repairing a bone defect part, so that the SPEEK-PDA-BFP has excellent bone tissue compatibility.
FIG. 10 is a scanning electron microscope result of a SPEEK-PDA-BFP set of materials 8 weeks after the procedure of example 3;
FIG. 10a is a 1000X view of the implant material and new bone interface;
fig. 10b is a graph of osteoblasts 3000×;
FIGS. 10c, d are 2500X and 5000X, respectively, of osteoblasts;
when the implant material is observed under 1000 times, the surface of the implant material is covered with a layer of thick bone trabecula and is arranged in a honeycomb shape, which shows that the bone maturity of the new bone tissue on the surface of the implant material after double modification of PDA and BFP-1 is high (figure 10 a), and the implant is almost completely fused with the original bone tissue; the fact that the osteogenic fibers criss-cross at 3000 times the surface of the material and form osseous bonds with the implant material demonstrates that the PDA has the effect of guiding bone tissue growth (fig. 10b and c). At 5000 times, a plurality of osteoblasts can be seen to be distributed on the surface of the matrix material and are tightly combined with the fibroblasts, and the matrix material has a certain attaching capability and is in an active state, so that the effect of BFP-1 on proliferation and differentiation of the osteoblasts is demonstrated.
FIG. 11 is a view of HE staining of a SPEEK-PDA-BFP matrix and surrounding bone tissue pathological sections 8 weeks after implantation into animal bone; wherein fig. 11a is a bone trabecular structure around an implant; FIG. 11b is a Harvard system; FIG. 11c is cartilage tissue surrounding an implant; FIG. 11d is a new blood vessel;
as can be seen in FIG. 11a, the trabecular bone structure around the SPEEK-PDA-BFP implant is much coarser, has been nearly mature, has substantially all of the bone defect area covered, and even a portion of the bone tissue is covered onto the implant and grows anteriorly along the material boundary. The trabecular structure of the new bone in fig. 11b forms a connection with the original cortical bone, creating a new haverse system, indicating that the bone tissue reconstruction is essentially complete. As can be seen from FIG. 11c, the cartilage tissue surrounding the SPEEK-PDA-BFP implant appears in a large area, indicating that bone tissue growth is vigorous in this area. In addition, coarse new blood vessels appear around the SPEEK-PDA-BFP implant (FIG. 11 d), demonstrating that BFP-1 can promote revascularization during the bone remodeling stage.
Detailed Description
The present invention will be further described in connection with the following examples which will enable those skilled in the art to more fully understand the invention and are not intended to limit the invention in any way; the various materials used in the examples below, including biological materials, are commercially available. PEEK is purchased from Nanjing first plastic special engineering plastic products Co., ltd; BFP-1 is purchased from Shanghai Yao Biotechnology Co., ltd. And other reagents used are all commercially available.
Example 1
A preparation method of a biological functional porous polyether-ether-ketone bone tissue scaffold material comprises the following specific steps:
(1) Processing PEEK samples into disc shapes with the specification of 20mm multiplied by 1 mm;
(2) Sequentially polishing the sample by using silicon sand paper with the model numbers of 800#, 1000#, 1500#, and 2000 #; respectively ultrasonically oscillating and cleaning the polished PEEK sample with acetone, alcohol and deionized water for 30min; and then drying at 60 ℃ to obtain PEEK with smooth surface and no impurities.
(3) Immersing the PEEK in concentrated sulfuric acid with the concentration of 98%; carrying out ultrasound with the ultrasound frequency of 40Hz and the ultrasound time of 0min, 5min, 10min and 15min respectively;
after ultrasonic treatment, cleaning PEEK by deionized water, and then sequentially carrying out ultrasonic treatment on the PEEK by acetone, alcohol and deionized water for 30 minutes respectively, so as to remove residual sulfur groups as much as possible; drying the cleaned sample at 60 ℃ to obtain SPEEK, wherein a plurality of pores are uniformly distributed on the surface of PEEK, and the pores are distributed at 2-8 mu m in size and are mutually connected to form a 3D network structure;
(4) Placing the treated SPEEK in 100mL of dopamine solution with the concentration of 2mg/mL for self-polymerization reaction, and stirring for 24 hours at room temperature; washing the stirred SPEEK with deionized water to remove polydopamine remained on the surface in a physical adsorption mode, so that the surface of the SPEEK is only provided with polydopamine linked by a bond action; drying to obtain SPEEK-PDA;
(5) Mixing 50mL of prepared dopamine solution with the concentration of 4mg/mL with 50mL of polypeptide solution with the concentration of 2mg/mL prepared by dissolving the osteogenic active factor BFP-1 in phosphate buffer solution; immersing the SPEEK sample obtained in the step (3) in the prepared mixed solution, and carrying out heat preservation and stirring treatment at the temperature of 4 ℃ for 24 hours; and washing the sample subjected to heat preservation and stirring by deionized water, and drying at room temperature to obtain a target product SPEEK-PDA-BFP.
The inventors conducted contact angle tests on different samples obtained by ultrasonic treatment of concentrated sulfuric acid of example 1 for different times to investigate the contact effect with an electrolyte, and the results are shown in fig. 1 and 2;
as can be seen from fig. 1, the PEEK sample surface water molecule contact angle without surface modification treatment is 85 ° ± 1.6 °, that is, the PEEK material itself is low in hydrophilicity and is close to a hydrophobic state. After the treatment of high-concentration concentrated sulfuric acid, the contact angle of water molecules on the surface of the SPEEK sample is obviously improved and reaches the maximum when the ultrasonic treatment is carried out for 10 minutes, the contact angle measured at the moment is 123+/-3.7 degrees, and the surface of the material is in a hydrophobic state, because countless micro-scale pores are generated on the surface of the material after the treatment of the concentrated sulfuric acid, the pores are mutually connected to form a 3D porous network structure, and the structure can provide growth active sites for growth, attachment and proliferation of osteoblasts during the subsequent in-vitro cell combined culture. In addition, the porous network structure can also reduce the free energy of the surface of the material, thereby reducing the wettability and obviously reducing the hydrophilicity; therefore, the ultrasonic time in the step (3) is 10min, and all samples prepared by the ultrasonic time of 10min are adopted in the following experiments.
Untreated PEEK surfaces are in a hydrophobic state; the surface of the sample subjected to sulfonation treatment has a porous network structure, the free energy of the surface of the material is reduced, and the wettability is also reduced; the surface of the PDA modified material contains a large number of hydrophilic groups, namely phenolic hydroxyl groups and amino groups, so that the hydrophilicity of the material is greatly increased; after BFP-1 is grafted, the surface hydrophilicity of the material is slightly improved. The hydrophilic-hydrophobic change of the surface of the material indirectly proves that the material is successfully modified, and the surface energy of the material is improved.
FIG. 3 is an infrared spectrum of different samples prepared in the above example (10 min), showing that the PDA modified SPEEK material has a new expansion vibration peak of phenolic hydroxyl O-H and a C=O expansion vibration peak of carbonyl after sulfonation, and that the DA completes self-polymerization reaction to generate polydopamine and loads the polydopamine onto the surface of the SPEEK material; after grafting the osteogenic active factor BFP-1, a bending vibration peak of N-H and a stretching vibration peak of C-N of the representative BFP-1 are newly appeared.
FIG. 4 is an SEM image of the surface of various materials prepared in the above example (10 min), and micro-scale pores appear on the surface of PEEK material after sulfonation, which exist to further reduce the surface energy of the material; after the combination with dopamine, the pore structure of the material surface is basically completely covered, and a typical PDA film with a fold shape appears; after grafting the osteogenic active factor BFP-1, the surface of the material is uniformly distributed with agglomerated particles.
Example 2
In vitro biological evaluation of the biological functionalized porous polyether-ether-ketone bone tissue scaffold comprises the following specific steps:
700mL of deionized water was first placed in a plastic beaker that was not scratched, then the beaker was placed in a water bath and heated to 36.5C uniformly, and stirring was continued. Sequentially add 11.994g NaCl,0.525g NaHCO 3 ，0.336g KCl，0.342g K 2 HPO 4 ·3H 2 O，0.458g MgCl 2 ·6H 2 O，0.417g CaCl 2 ，0.107g Na 2 SO 4 9.086g of Tris is added into a plastic beaker to be fully dissolved, then the pH of the mixed solution is adjusted to 7.4 by using 1mmol/L of HCl solution, finally the solution is transferred to a long-neck flask, residual liquid in the plastic beaker is flushed out and poured into the flask, deionized water is added to the scale mark of the flask, and the preparation of the SBF solution is completed at 1.5 times.
Care should be taken in the configuration process: (1) Avoiding adding several reagents for dissolution at the same time, and continuing adding the next compound after the last compound is completely dissolved; (2) In the preparation process, the solution is ensured to be colorless and transparent, no sediment is generated on the surface of the container, and if the sediment is generated, the instrument is required to be cleaned for reconstitution; (3) The prepared SBF solution needs to be stored in a plastic bottle at the temperature of 5-10 ℃ in a refrigerator, and the effective period is 30 days.
In this example, MC3T3-E1 mouse embryo osteoblasts were selected as experimental cells, and cultured using DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, and recovery of osteoblasts was performed on an ultra-clean bench irradiated with ultraviolet light for more than half an hour, and specific experimental procedures were as follows:
(1) Taking out frozen MC3T3-E1 osteoblast embryo cells from a liquid nitrogen tank at the temperature of minus 40 ℃, and thawing the frozen MC3T3-E1 osteoblast embryo cells in a water bath at the temperature of 37 ℃ to ensure that the MC3T3-E1 osteoblast embryo cells are completely thawed in a short time;
(2) Sucking MC3T3-E1 cells by using a pipette which has reached sterilization conditions, transferring the MC3T3-E1 cells into a 50mL centrifuge tube, adding 5mL of culture medium, and then placing the mixture into a centrifuge for centrifugation for 5min, wherein the rotating speed is set to be 1000r/min;
(3) The supernatant after centrifugation was discarded, 5mL of medium was added again, and then the cells were gently blown off with a pipette and transferred to a flask at 37℃with 5% CO 2 Is cultured under the condition of (2). The culture medium is changed every two days, and 1:2 passage inoculation is carried out after the cell density reaches 80% -90%.
The cell material culture is then carried out, in particular as follows:
(1) Firstly, placing a sample into a new 6-hole plate, and then irradiating with ultraviolet light for 1h to perform sterilization treatment;
(2) Taking out preserved and cultured MC3T3-E1 cells, pouring out the culture medium, repeatedly washing with PBS buffer solution for three times, then dripping pancreatin containing 0.25% EDTA (ethylenediamine tetraacetic acid), stopping digestion when the periphery of the cells is observed to be shiny by a light mirror, and finally centrifuging for 5min to obtain osteoblasts;
(3) Two cell droplets are counted on a cell counting plate and a suitable amount of liquid is addedWhen the cell culture medium is subjected to concentration control, the concentration is about 2×10 4 Each mL was then added to the 6-well plate containing the sample using a pipette and incubated at 37℃with 5% CO 2 Culturing was performed under the conditions for 48 hours. The cells used in the subsequent experiments were osteoblasts cultured in this manner.
A simulated body fluid soaking experiment, a cell adhesion proliferation experiment, an antibacterial test experiment and an alkaline phosphatase activity test experiment are respectively carried out by taking a pure SPEEK as a blank control group and taking a SPEEK-PDA as a positive control group, so that comprehensive in-vitro biological performance evaluation is carried out on the composite bionic SPEEK-PDA-BFP. The experiments are all standardized routine biological experimental procedures, and finally the following conclusions are obtained:
(1) As can be seen from FIG. 5, after 7 days of soaking in 1.5 times SBF simulated body fluid, the amount of calcium and phosphorus precipitation of the SPEEK-PDA-BFP group material is higher than that of the SPEEK group and the SPEEK-PDA group, and the amount of calcium and phosphorus precipitation also increases with the prolonged soaking time. Therefore, the osteogenic active factor BFP-1 can actively promote the formation of the bone-like apatite precipitate, and the composite bionic SPEEK-PDA-BFP has excellent in-vitro osteogenic activity.
(2) FIG. 6 shows cell adhesion rate detection, wherein osteoblasts were first co-cultured on three sample surfaces in 96-well plates for 24h, and after removal, repeatedly washed with PBS buffer to remove cells that did not adhere successfully; under the condition of avoiding light, 30 mu L of CCK-8 solution and 300 mu L of culture medium are added into each hole, the culture is continued for 12 hours, 100 mu L of supernatant is sucked, the absorbance value (OD) is measured by an enzyme-labeling instrument, the wavelength is set to 450nm, the average value is calculated three times for each sample, and a histogram is drawn according to the data.
Cell adhesion rate calculation formula:
as can be seen from FIG. 6, the osteoblast MC3T3-E1 has the least adhesion amount to the surface of SPEEK (blank control) material, and the adhesion rate thereof can be calculated to be 8.67.+ -. 0.36% according to the formula. The adhesion effect on the surface of the SPEEK-PDA material is better than that of the SPEEK-PDA material, and the adhesion rate reaches 27.65+/-0.43%. Whereas the adhesion rate of the surface of the SPEEK-PDA-BFP (experimental group) material is somewhat lower than that of the SPEEK-PDA (positive control group), the adhesion rate is 25.62 + -0.27%, which is consistent with the previous contact angle characterization result. The surface of SPEEK after being treated by concentrated sulfuric acid belongs to an inert surface, the content of hydrophilic groups is low, so that the capacity of cell adhesion is limited, and the surface of SPEEK after being coated by dopamine and grafted with BFP-1 is provided with a large number of hydrophilic phenolic hydroxyl groups and amino groups, so that the growth and adhesion of osteoblasts are promoted by the hydrophilic groups. From this, it can be demonstrated that the composite modified layer PDA-BFP has the effect of promoting cell adhesion and proliferation. Therefore, the in vitro cell compatibility of the composite bionic SPEEK-PDA-BFP is excellent.
(3) Fig. 7 shows the results of the material bacteriostasis study. Streaking on LB plates to resuscitate gram-positive bacteria, and resuscitating on BHI plates to resuscitate gram-negative bacteria. Absorbing a proper volume of bacterial liquid, inoculating the bacterial liquid into a liquid culture medium, adding a SPEEK material, a SPEEK-PDA material and a SPEEK-PDA-BDP material into the liquid culture medium, and placing the liquid culture medium at 37 ℃ for 2500rmp shake culture for 24 hours; the supernatant was discarded, and 20mL of PBS buffer was added to wash three times to remove bacteria that did not adhere successfully. Then, the materials are placed into a sterile centrifuge tube, a little NaCl is added, vortex centrifugation and shaking are carried out for 1min at the maximum rotation speed, bacterial suspension is collected, and the bacteriostasis rate is calculated.
The calculation formula of the bacteriostasis rate is as follows:
after the bacteriostasis test, the surface of the SPEEK-PDA-BFP material has the best bacteriostasis effect, and the bacteriostasis rate reaches 100 percent; the SPEEK-PDA material has a bacteriostasis rate of more than 80%, and compared with a pure SPEEK group material, the modified material has bacteriostasis with certain capacity, and can also show that the PDA and the BFP-1 have good bacteriostasis effects.
(4) FIG. 8 shows the results of alkaline phosphatase activity assay using alkaline phosphatase assay kit, the principle being that ALP catalyzes the hydrolysis of disodium phosphate to produce phenol and disodium hydrogen phosphate in an environment of pH=10, then phenol reacts with 4-aminoantipyrine in an alkaline solution, potassium ferricyanide oxidizes to red quinone derivatives, and finally enzyme activity is measured by the shade of color. The specific operation steps are as follows:
(1) Cells were incubated on the surface of each material for 7 days, 14 days, washed 3 times with PBS, scraped off the material surface with cell scrapers, aspirated into 1.5mL EP tubes, and lysed by addition of cell lysate until no cell clumps were apparent;
(2) Sequentially adding 30 mu L of deionized water into blank holes, adding 30 mu L of phenol standard solution into standard holes, adding 30 mu L X-100 into measurement holes, adding 50 mu L of buffer solution and 50 mu L of culture medium into each hole, uniformly mixing, putting into a 37 ℃ water bath, adding 150 mu L of color development solution into each hole after a period of time, and measuring the OD value of each hole according to an enzyme-labeled instrument;
(3) Mixing 20 mu L X-100 with 200 mu L BAC working solution, uniformly mixing at 37 ℃, colorizing at 562nm, and recording OD value;
(4) Calculating ALP vitality values according to the formula:
the ALP activity value of SPEEK-PDA-BFP is higher than that of other two groups of materials and is improved to a certain extent along with the extension of time. The composite bionic SPEEK-PDA-BFP promotes proliferation and differentiation of osteoblasts under the dual actions of polydopamine PDA and osteogenic active factor BFP-1.
Example 3
The inventor constructs a SPEEK-PDA-BFP composite bionic configuration on the basis of a preparation method of an early-stage experimental material, and implants the SPEEK-PDA-BFP composite bionic configuration into the femur of an experimental rat. The in vivo biocompatibility and the osseointegration performance of the implant material and the bone tissue microscopic morphology and pathological section of the implant material are comprehensively evaluated through observation of the physiological health condition of rats.
The inventor chooses male SD rats (Vetolihua) with weight below 300g and week 3-8. Rats were placed 5 per cage in ivc animal cages and raised in water using conventional sterilized feed.
The method of completely random grouping is adopted, and the method is randomly divided into 7 groups of three groups. Material preparation:
(1) Processing the SPEEK-PDA-BFP material obtained in the step 5 of the example 1 into a cylindrical shape with the diameter of 1-2mm and the thickness of 2cm, then soaking the material in 75% ethanol solution for a period of time, and then sterilizing by ultraviolet irradiation;
(2) Transferring the sterilized material into a beaker, repeatedly washing with PBS buffer solution to remove residual ethanol on the surface of the material, and drying for later use.
Surgical implementation:
(1) The SD rat is required to be fasted and water-forbidden for 8 hours before operation;
(2) Firstly, rats are anesthetized, inhalation anesthesia (isoflurane is adopted as anesthetic), induced gas flow is set to be 3L/min, and gas flow is maintained to be 1L/min. After the cornea of the rat is retarded in reflection, the knee at one side is routinely prepared, sterilized by iodophor and spread. Cutting the inner skin and subcutaneous tissue of the knee with a scalpel, freeing the muscles and ligaments, and exposing the inner condyle of the femur;
(3) A drill bit is used for vertically drilling the middle of the medial condyle of the femur by using a Kirschner wire drill with the diameter of 3mm and the depth of about 5mm, so that a circular bone defect is formed. After the drilling is formed, an ophthalmic forceps is used for insertion to confirm whether the drilling structure is qualified or not, and tissue fragments in the drilling are cleaned to form a bone defect model;
(4) And then placing sterilized SPEEK and modified implants thereof in bone defect drill holes according to groups, checking the operation field, suturing layer by layer, and closing the incision. Then the knee at the other side is subjected to the same operation to finish the bilateral bone defect model and material implantation;
(5) After the operation is finished, the rat is separated from the anesthesia respirator and placed in a rearing cage for close observation, and after the rat is anesthetized and revived. Recovering the drinking water after waking up. Penicillin (8 ten thousand units/day) is administered prophylactically on the same day of surgery and the first two days after surgery, and is injected intraperitoneally to prevent postoperative infection.
Drawing materials after operation:
(1) Part of the rats were anesthetized and sacrificed at 4 and 8 weeks after the operation, and the femoral part tissues were obtained and specimens were prepared. Firstly, preparing a 10% aqueous solution by using chloral, sucking 1-1.5mL of the aqueous solution for intraperitoneal injection to anesthetize rats;
(2) Then, the abdominal wall was cut with scissors to free the abdominal viscera and peritoneum, exposing the abdominal aorta. Blood was collected in the abdominal aorta using a disposable lancet in combination with a 20mL syringe. After blood collection, the animals die after blood loss;
(3) Then the skin and subcutaneous tissues of the knees at both sides of the rat are cut off by a surgical knife, the patellar ligament is cut off, the patella and quadriceps femoris are turned up, other deep soft tissues are cut off, and the lower end of the femur is fully exposed. Cutting femoral condyle and small part of femoral diaphysis by using a pendulum saw, and immediately placing the femoral condyle and small part of femoral diaphysis into 4% paraformaldehyde fixing solution and 2.5% alkaline glutaraldehyde fixing solution for fixing according to grouping and the required treatment of the next step;
(4) Animal carcasses were treated as required by the laboratory. And (5) calibrating and numbering each specimen, and carrying out the next processing according to the requirement.
The inventor comprehensively evaluates the osseointegration capability of the material through Micro-CT analysis, microscopic morphology analysis, pathological section analysis and the like. The following conclusions were drawn:
(1) FIG. 9 shows the results of a Micro-CT test, wherein a femur part sample is soaked in 4% paraformaldehyde solution for 24 hours, then dried, fixed on a sample table, and scanned and three-dimensionally reconstructed at high resolution, the scanning parameters are 20KV,7 muA, and 360 DEG rotation scanning is performed. Subsequent data were analyzed by software Dragonfly for wound closure rate of the samples.
The Micro-CT contrast graph of the postoperative 8-week material and the surrounding bone tissue can be obtained: SPEEK substrate (blank) had the lowest degree of bone repair, with a wound closure rate of 0. The SPEEK-PDA-BFP composite bionic structure is formed by continuous and compact bone trabecular structures with higher maturity, and the wound closure rate is 93%. The PDA-BFP modified layer can promote the generation and differentiation of bone tissue, and the SPEEK-PDA-BFP composite bionic structure has excellent bone tissue compatibility.
(2) Fig. 10 shows the result of scanning electron microscope observation, the femur specimen is soaked in glutaraldehyde solution for fixation for 24 hours, then placed in PBS buffer solution, washed by shaking in an ultrasonic cleaner for 10 minutes, dehydrated step by gradient alcohol, finally replaced by diethyl ether, and freeze-dried. The specimen was observed under an electron scanning microscope after vacuum plating.
The microscopic observation result of the bone tissue growth around the implant after 8 weeks of operation is that: the SPEEK-PDA-BFP composite bionic structure has highly developed honeycomb bone trabeculae and is tightly combined with the original cortical bone, so that the implant material has excellent bone tissue conduction and osseointegration performance.
(3) Fig. 11 shows the results of pathological histology, a sample of the femoral part of a rat was taken, immersed in 4% paraformaldehyde solution for fixation for 24 hours, washed with PBS buffer, decalcified with 15% EDTA (ethylenediamine tetraacetic acid solution) for 7 days, and dehydrated with gradient ethanol. The dehydrated sample was embedded in paraffin to prepare a tissue slice having a thickness of 5. Mu.m. HE staining was then performed: dewaxing and dewatering, hematoxylin staining for 5min, differentiating with 5% acetic acid for 1min, washing, staining with eosin for 1min, dewatering, air drying, dripping neutral resin sealing, and observing with light microscope.
The pathological section analysis of the implant and the surrounding bone tissue after 8 weeks of operation can be obtained: the bone trabecular structure around the SPEEK-PDA-BFP composite bionic structure is close to maturity, the bone defect part is completely covered, and thicker blood vessels appear, which indicates that bone reconstruction is basically completed. Therefore, the SPEEK-PDA-BFP composite bionic configuration can promote bone tissue growth and differentiation and blood circulation reconstruction.
Therefore, the SPEEK-PDA-BFP composite bionic structure prepared by the invention can exert the effects of promoting bone tissue growth and proliferation and BFP-1 accelerating osteoblast differentiation and blood circulation reconstruction of the PDA, has excellent biological bone tissue compatibility and osseointegration performance, and can become an ideal bone defect replacement material.
The technical features of the above embodiments may be combined in any desired manner, and for brevity, all of the possible combinations of the technical features of the above embodiments may not be described, however, as long as there is no contradiction between the combinations of the technical features, all of which should be considered as being within the scope of the description, the description of the above embodiments may be used to help understand the principles and methods of the present invention. The above embodiments are not intended to be exclusive and should not be construed as limiting the invention.
Claims (6)
1. A biological functional porous polyether-ether-ketone bone tissue scaffold material is characterized in that: the bone tissue scaffold material consists of a polydopamine for adhering a PEEK matrix material and an osteogenic active factor BFP, wherein the BFP loading amount is 0.8-1.2mg/cm 2 。
2. The biofunctionalized porous polyetheretherketone bone tissue scaffold material according to claim 1, wherein: the PEEK matrix material is subjected to pretreatment, wherein the pretreatment is sulfonation treatment.
3. The method for preparing the biological functional porous polyether-ether-ketone bone tissue scaffold material as claimed in claim 1, which is characterized by comprising the following specific steps:
(1) Processing PEEK matrix material into a disc shape with the specification of 20mm multiplied by 1 mm;
(2) Sequentially polishing the sample by using silicon sand paper with the model numbers of 800#, 1000#, 1500#, and 2000 #; respectively ultrasonically oscillating and cleaning the polished PEEK sample with acetone, alcohol and deionized water for 30min; drying at 60 ℃ to obtain PEEK with smooth surface and no impurities;
(3) Immersing the PEEK in concentrated sulfuric acid with the concentration of 98%, and carrying out ultrasonic treatment, wherein the ultrasonic frequency is 40Hz, and the ultrasonic treatment time is 5-15min; after ultrasonic treatment, cleaning PEEK by deionized water, and then respectively carrying out ultrasonic treatment by acetone, alcohol and deionized water for 30min; drying the cleaned sample at 60 ℃ to obtain SPEEK;
(4) Mixing 50mL of prepared dopamine solution with the concentration of 4mg/mL with 50mL of polypeptide solution with the concentration of 2mg/mL prepared by dissolving the osteogenic active factor BFP-1 in phosphate buffer solution; immersing the SPEEK sample obtained in the step (3) in the prepared mixed solution, and carrying out heat preservation and stirring treatment at the temperature of 4 ℃ for 24 hours; and washing the sample subjected to heat preservation and stirring by deionized water, and drying at room temperature to obtain a target product SPEEK-PDA-BFP.
4. The method for preparing a biofunctionalized porous polyetheretherketone bone tissue scaffold material according to claim 3, further comprising the preparation of SPEEK-PDA, specifically comprising the steps of:
placing the SPEEK obtained in the step (3) into 100mL of dopamine solution with the concentration of 2mg/mL for self-polymerization reaction, and stirring for 24 hours at room temperature; washing the stirred SPEEK with deionized water to remove polydopamine remained on the surface in a physical adsorption mode, so that the surface of the SPEEK is only provided with polydopamine linked by a bond action; after drying, SPEEK-PDA is obtained.
5. The method for preparing the biological functionalized porous polyether-ether-ketone bone tissue scaffold material according to claim 3, wherein pores are uniformly distributed on the surface of the PEEK obtained in the step (3), and the pores are distributed at 2-8 μm in size and are mutually connected to form a 3D network structure.
6. The method for preparing a biofunctionalized porous polyether-ether-ketone bone tissue scaffold material according to claim 3, wherein the ultrasonic time in the step (3) is 10min.
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