CN112972763A - PEEK porous microsphere with mineralized extracellular matrix coated on surface, and preparation method and application thereof - Google Patents

PEEK porous microsphere with mineralized extracellular matrix coated on surface, and preparation method and application thereof Download PDF

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CN112972763A
CN112972763A CN202110210473.4A CN202110210473A CN112972763A CN 112972763 A CN112972763 A CN 112972763A CN 202110210473 A CN202110210473 A CN 202110210473A CN 112972763 A CN112972763 A CN 112972763A
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peek
microspheres
mineralized
porous
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章培标
孙烁
王宗良
焦自学
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Changchun Institute of Applied Chemistry of CAS
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Abstract

本发明涉及聚醚醚酮技术领域，尤其涉及一种表面包被矿化细胞外基质的PEEK多孔微球、其制备方法及应用。所述制备方法包括：PEEK微球表面多孔化处理、灭菌、浸泡于DMEM培养基，然后与传至第3代的MC3T3‑E1细胞与所述浸泡后的PEEK微球在DMEM培养基中混匀，进行培养，通过更换培养基培养以及脱细胞处理，得到表面包被矿化细胞外基质的PEEK多孔微球。本发明制备的表面包被矿化细胞外基质的PEEK多孔微球提高了细胞的培养密度与效率，有利于细胞的黏附、增殖与分化，从仿生学角度模拟了成骨过程。

Figure 202110210473

The invention relates to the technical field of polyetheretherketone, in particular to a PEEK porous microsphere coated with a mineralized extracellular matrix, a preparation method and application thereof. The preparation method includes: surface porosity treatment of PEEK microspheres, sterilization, soaking in DMEM medium, and then mixing with the MC3T3-E1 cells transferred to the third generation and the soaked PEEK microspheres in DMEM medium. homogenized, cultured, cultured by changing the medium and decellularized to obtain PEEK porous microspheres coated with mineralized extracellular matrix. The PEEK porous microspheres with surface-coated mineralized extracellular matrix prepared by the invention improve the culture density and efficiency of cells, are beneficial to the adhesion, proliferation and differentiation of cells, and simulate the osteogenesis process from the perspective of bionics.

Figure 202110210473

Description

PEEK porous microsphere with mineralized extracellular matrix coated on surface, and preparation method and application thereof
Technical Field
The invention relates to the technical field of polyether-ether-ketone, in particular to PEEK porous microspheres with mineralized extracellular matrix coated on the surface, and a preparation method and application thereof.
Background
In the fields of clinical cell therapy and bone tissue engineering, large-scale cell culture has become the core and hot spot of research. In recent years, microspheres have attracted much attention for their wide application in cell therapy and bone tissue engineering. Compared with the traditional planar cell culture system, the microsphere has great advantages in the aspects of maintaining cell differentiation phenotype, being injectable and being used as a cell carrier to repair irregular bone defects. However, the relatively small surface area of smooth surfaced microspheres limits cell adhesion, thereby reducing their cell loading capacity. The porous microspheres have interconnected pore structures and larger specific surface area on the surfaces and inside, so that the porous microspheres are more favorable for transporting nutrients and oxygen, and can support cell adhesion and growth, which is the key point for improving the cell culture density. In addition, it has been demonstrated that modification of the chemical and physical properties of the microsphere surface can affect key steps in tissue repair, such as immune response, vascularization and cell differentiation. Therefore, the cell supporting ability of the microspheres can be further enhanced by surface modification treatment.
Cell culture usually requires a large number of microspheres, but the reuse rate is not high, which causes huge resource waste. Therefore, it is very necessary to develop a recyclable cell culture microsphere, which can not only withstand the autoclaving treatment, but also have sufficient strength to support the growth of cells and withstand the shear force during 3D culture.
At present, the methods for preparing porous microspheres mainly include a solvent evaporation method, an emulsion polymerization method, a seed swelling method, a sintering method, a synthesis method, a spray drying method, a phase separation method, and the like. Such as: firstly, the Liuling will introduce the hydroxyl group and pyridine group with functionality into the polyaryletherketone molecular structure to prepare a series of polyaryletherketone polymers containing functional groups, then the micelle balling method is utilized to prepare a novel micelle which takes polyaryletherketone as a core and takes another polymer as a shell and is connected with a non-covalent bond, and finally the chemical crosslinking method is utilized to prepare the polyaryletherketone microsphere material. Zhang Chun Feng et al uses hyperbranched polyaryletherketone with rigid structure as starting point, and utilizes solvent volatilization method to prepare hyperbranched polyaryletherketone microsphere.
However, in the prior art, the polyaryletherketone microsphere with a core-shell structure or hollow and porous structure is prepared from the aspects of molecular design, functional group introduction and the like. The core-shell structure microspheres need to be further removed from the outer shell structure, and only solid microspheres can be obtained. The hollow porous microspheres need further crosslinking reaction, and the hollow microspheres can be obtained only after another polymer is removed, so that the experimental conditions are severe, and the controllability of the size and the shape of the hollow microspheres is poor.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a PEEK porous microsphere with a surface coated with a mineralized extracellular matrix, a preparation method and an application thereof.
The invention provides a preparation method of PEEK porous microspheres with mineralized extracellular matrix coated on the surface, which comprises the following steps:
A) under the condition of protective gas, the PEEK microspheres are placed in the treatment fluid to be heated and stirred, and after drying, the PEEK microspheres with porous surfaces are obtained;
the treatment liquid comprises dimethyl sulfoxide and sodium borohydride;
B) sterilizing the PEEK microspheres with porous surfaces, and then soaking the PEEK microspheres in a DMEM culture medium for 12-24 hours to obtain soaked PEEK microspheres;
C) uniformly mixing MC3T3-E1 cells which are transmitted to the 3 rd generation and the soaked porous PEEK microspheres in a DMEM culture medium, culturing, replacing the DMEM culture medium after 22-26 hours to continue culturing, replacing the DMEM culture medium with a mineralized culture medium after 22-26 hours to culture, replacing the DMEM culture medium with the mineralized culture medium after 22-26 hours, then replacing the liquid once every 46-50 hours, and performing acellular treatment on the 7 th-9 th days of replacing the mineralized culture medium to obtain the PEEK porous microspheres with the mineralized extracellular matrix coated on the surfaces;
the mineralized culture medium comprises DMEM culture medium and CaCl2、K2HPO4And polyaspartic acid.
Preferably, the PEEK microspheres are prepared according to the following method:
mixing polyether-ether-ketone powder with concentrated sulfuric acid to obtain a mixed solution;
preparing microspheres from the mixed solution by adopting an air flow method;
and carrying out hydrothermal reaction on the microspheres and water to obtain the PEEK microspheres.
Preferably, in step a), the protective gas is nitrogen;
the dosage ratio of the dimethyl sulfoxide to the sodium borohydride is 60-80 mL: 130-150 mg;
the treatment fluid is prepared according to the following method:
and (3) mixing dimethyl sulfoxide and sodium borohydride, and heating to 115-125 ℃ to obtain a treatment solution.
Preferably, in the step A), the heating and stirring temperature is 115-125 ℃, the time is 5-7 hours, and the heating and stirring rotating speed is 200-400 rpm.
Preferably, in the step a), after the heating and stirring, washing is further included;
the washing comprises the following steps:
and immersing the heated and stirred PEEK microspheres in methanol for 15-30 min, primary water for 10-20 min, 0.4-0.6 mol/L hydrochloric acid solution for 10-20 min, primary water for 10-20 min and ethanol with the volume concentration of 95-100% for 10-20 min in sequence.
Preferably, in step B), the sterilization comprises:
and sterilizing the PEEK microspheres with the porous surfaces by using 75% ethanol by volume concentration, washing the PEEK microspheres by using PBS buffer solution, and then performing steam sterilization at the temperature of 115-120 ℃ for 25-35 min.
Preferably, in the step C), before the MC3T3-E1 cells transferred to the 3 rd generation and the soaked porous PEEK microspheres are mixed in the DMEM medium, the method further comprises:
the MC3T3-E1 cells passed to passage 3 at 2X 104The density of each well is planted in a cell culture plate;
and uniformly mixing the MC3T3-E1 cells transferred to the 3 rd generation and the soaked porous PEEK microspheres in a DMEM culture medium for 1-2 min.
Preferably, in step C), the decellularization treatment comprises the following steps:
a) washing the porous PEEK microspheres cultured by the mineralized culture medium for 1-3 times by using PBS buffer solution, then transferring the porous PEEK microspheres into a cell removal solution, and placing the cell removal solution on a shaking table with the rotating speed of 60-100 rpm for 10min at room temperature;
the cell removal solution comprises NH4OH and Triton X-100; in the cell-removing solution, NH4The concentration of OH is 20mmol/L, and the concentration of Triton X-100 is 0.25 wt%;
b) washing the PEEK microspheres obtained in the step a) by using a PBS buffer solution, and then carrying out freeze drying.
Preferably, in step C), CaCl is added to the mineralized medium2Has a concentration of 4.5X 10-3～9×10-3mol/L，K2HPO4Has a concentration of 2.1X 10-3～4.2×10-3mol/L, and the concentration of polyaspartic acid is 0.05-0.2 mg/mL.
The invention also provides the PEEK porous microsphere with the surface coated with mineralized extracellular matrix, which is prepared by the preparation method.
The invention also provides application of the PEEK porous microspheres with the mineralized extracellular matrix coated on the surface in large-scale cell culture and preparation of injectable bone implant materials.
The invention provides a preparation method of PEEK porous microspheres with mineralized extracellular matrix coated on the surface, which comprises the following steps: A) under the condition of protective gas, the PEEK microspheres are placed in the treatment fluid to be heated and stirred, and after drying, the PEEK microspheres with porous surfaces are obtained; the treatment liquid comprises dimethyl sulfoxide and sodium borohydride; B) sterilizing the PEEK microspheres with porous surfaces, and then soaking the PEEK microspheres in a DMEM culture medium for 12-24 hours to obtain soaked PEEK porous microspheres; C) will be transmitted toUniformly mixing the 3 rd generation MC3T3-E1 cells and the soaked PEEK porous microspheres in a DMEM culture medium, culturing, replacing the DMEM culture medium after 22-26 hours to continue culturing, replacing the DMEM culture medium with a mineralized culture medium after 22-26 hours to culture, replacing the DMEM culture medium with the mineralized culture medium after 22-26 hours, replacing the liquid once every 46-50 hours, and performing decellularization treatment on 7 th-9 th days of replacing the DMEM culture medium with the mineralized culture medium to obtain the PEEK porous microspheres with the mineralized extracellular matrix coated on the surfaces; the mineralized culture medium comprises DMEM culture medium and CaCl2、K2HPO4And polyaspartic acid.
The PEEK porous microspheres with the surfaces coated with mineralized extracellular matrix improve the culture density and efficiency of cells, are beneficial to adhesion, proliferation and differentiation of the cells, and simulate an osteogenesis process from the perspective of bionics. In addition, the PEEK porous microspheres adopting the mineralized extracellular matrix can be used as injectable materials for minimally invasive surgery, and have important significance in repairing irregular bone defects.
Drawings
FIG. 1 is an SEM image of PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces thereof, which are prepared in example 1 of the invention, at 200 μm;
FIG. 2 is an SEM image of PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces thereof, which are prepared in example 1 of the invention, at a position of 10 μm;
FIG. 3 is an inverted fluorescence microscope photograph of the PEEK porous microspheres coated with mineralized extracellular matrix on the surface, which are obtained by staining with calcein after culturing for 7 days using MC3T3-E1 cell line, according to example 1 of the present invention;
FIG. 4 is an inverted fluorescence microscope image of the smooth PEEK microspheres of example 1 of the present invention after calcein staining after 7 days of culture using the MC3T3-E1 cell line;
FIG. 5 is a graph showing the effect of smooth PEEK microspheres and PEEK porous microspheres of the bread-mineralized extracellular matrix on the proliferation of MC3T3-E1 cells in example 3 of the present invention;
FIG. 6 is a graph showing the effect of PEEK porous microspheres having their surface coated with mineralized extracellular matrix on the production of cellular alkaline phosphatase in example 4 of the present invention;
FIG. 7 is a graph showing the effect of smooth PEEK microspheres on the production of cell alkaline phosphatase in example 4 of the present invention;
FIG. 8 is a graph of the effect of smooth PEEK microspheres and PEEK porous microspheres of the bread-mineralized extracellular matrix on the alkaline phosphatase activity of cells in example 5 of the present invention;
FIG. 9 is a graph of the effect of smooth PEEK microspheres and PEEK porous microspheres of the inventive example 5 having a bread mineralized extracellular matrix on intracellular calcium deposition;
FIG. 10 is a graph showing the evaluation of the effect of smooth PEEK microspheres and PEEK porous microspheres coated with mineralized extracellular matrix on bone repair in example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of PEEK porous microspheres with mineralized extracellular matrix coated on the surface, which comprises the following steps:
A) under the condition of protective gas, the PEEK microspheres are placed in the treatment fluid to be heated and stirred, and after drying, the PEEK microspheres with porous surfaces are obtained;
the treatment liquid comprises dimethyl sulfoxide and sodium borohydride;
B) sterilizing the PEEK microspheres with porous surfaces, and then soaking the PEEK microspheres in a DMEM culture medium for 12-24 hours to obtain soaked PEEK porous microspheres;
C) uniformly mixing MC3T3-E1 cells which are transmitted to the 3 rd generation and the soaked PEEK porous microspheres in a DMEM culture medium, culturing, replacing the DMEM culture medium after 22-26 hours to continue culturing, replacing the DMEM culture medium with a mineralized culture medium after 22-26 hours to culture, replacing the DMEM culture medium with the mineralized culture medium after 22-26 hours, then replacing the liquid once every 46-50 hours, and performing decellularization treatment on the 7 th-9 th day of replacing the mineralized culture medium to obtain the PEEK porous microspheres with the mineralized extracellular matrix coated on the surfaces;
the mineralized culture medium comprises DMEM culture medium and CaCl2、K2HPO4And polyaspartic acid.
In certain embodiments of the invention, the PEEK microspheres are prepared according to the following method:
mixing polyether-ether-ketone powder with concentrated sulfuric acid to obtain a mixed solution;
preparing microspheres from the mixed solution by adopting an air flow method;
and carrying out hydrothermal reaction on the microspheres and water to obtain the PEEK microspheres.
The PEEK microspheres with smooth surfaces and porous interiors are prepared by a phase separation method. Further, after hydroxylation treatment in the step A), a uniform porous structure is generated on the surface of the PEEK microsphere and is communicated with the internal pores. Meanwhile, the chemical property of the surface of the PEEK microsphere is changed by hydroxylation treatment, and the hydrophilic property of the PEEK is improved. And the surface porous morphology is cooperated, so that cell proliferation and adhesion are facilitated.
In certain embodiments of the present invention, the concentrated sulfuric acid has a mass concentration of 95% to 98%.
In certain embodiments of the invention, the ratio of polyetheretherketone powder to concentrated sulfuric acid is 1.8 g: 30 mL.
In certain embodiments of the present invention, the polyetheretherketone powder is mixed with concentrated sulfuric acid at a temperature of 26 ℃ for a period of 2 hours. In certain embodiments of the invention, the mixing of the polyetheretherketone powder with the concentrated sulfuric acid is a stirring mixing. The stirring method for the stirring and mixing is not particularly limited in the present invention, and a stirring method known to those skilled in the art may be employed.
In some embodiments of the present invention, after mixing the peek powder with the concentrated sulfuric acid, the method further comprises: standing for 10 min.
After the mixed solution is obtained, the microspheres are prepared from the mixed solution by adopting an air flow method.
Preferably, the method specifically comprises the following steps:
and adding the mixed solution into a 50mL needle cylinder, preparing 1000mL of ethanol sedimentation liquid with the volume concentration of 30% by using the needle cylinder provided with a Teflon needle head with the diameter of 27G, carrying out ice bath, and preparing microspheres by adopting an air flow method, wherein the distance between the Teflon needle head and the liquid level of the ethanol sedimentation liquid is 14 cm. The gas adopted in the gas flow method is nitrogen, and the flow rate of the nitrogen is 4.5L/min.
In some embodiments of the present invention, before hydrothermally reacting the microspheres with water, the hydrothermal reaction further comprises:
and soaking the microspheres in primary water for 24 hours, changing water every 8 hours, and filtering by using a screen to obtain the microspheres with the diameter of 500-600 microns.
In certain embodiments of the present invention, the water used for the hydrothermal reaction is primary water.
In certain embodiments of the present invention, the hydrothermal reaction is carried out at a temperature of 170 ℃ for a period of 8 hours.
In some embodiments of the present invention, after the hydrothermal reaction, the method further comprises:
and cooling to room temperature, and performing vacuum drying to obtain the PEEK microspheres.
The method and parameters of the vacuum drying are not particularly limited in the present invention, and those known to those skilled in the art can be used.
In the invention, the PEEK microspheres are smooth PEEK microspheres.
After the PEEK microspheres are obtained, the PEEK microspheres are placed in treatment fluid under the condition of protective gas, heated and stirred, and dried to obtain the PEEK microspheres with porous surfaces (namely hydroxylated PEEK microspheres).
In certain embodiments of the invention, the shielding gas is nitrogen.
In the invention, the treatment liquid comprises dimethyl sulfoxide and sodium borohydride.
In some embodiments of the present invention, the ratio of the dimethyl sulfoxide to the sodium borohydride is 60-80 mL: 130-150 mg. In certain embodiments, the ratio of dimethyl sulfoxide to sodium borohydride is 70 mL: 140 mg.
In certain embodiments of the invention, the treatment fluid is prepared according to the following method:
and (3) mixing dimethyl sulfoxide and sodium borohydride, and heating to 115-125 ℃ to obtain a treatment solution.
In some embodiments of the invention, the PEEK microspheres are placed in the treatment fluid, heated and stirred at the temperature of 115-125 ℃ for 5-7 hours. In some embodiments of the invention, the PEEK microspheres are placed in the treatment fluid and heated and stirred at the rotating speed of 200-400 rpm.
In certain embodiments of the invention, after the heating and stirring, further comprising washing.
In certain embodiments of the invention, the washing comprises:
and immersing the heated and stirred PEEK microspheres in methanol for 15-30 min, primary water for 10-20 min, 0.4-0.6 mol/L hydrochloric acid solution for 10-20 min, primary water for 10-20 min and ethanol with the volume concentration of 95-100% for 10-20 min in sequence.
The washing can remove sodium borohydride or dimethyl sulfoxide that may remain on the microspheres.
In certain embodiments of the invention, the drying is vacuum drying. The method and parameters of the vacuum drying are not particularly limited in the present invention, and those known to those skilled in the art can be used.
And after obtaining the PEEK microspheres with porous surfaces, sterilizing the PEEK microspheres with porous surfaces, and then soaking the PEEK microspheres in a DMEM culture medium for 12-24 hours to obtain the soaked PEEK porous microspheres.
In certain embodiments of the invention, the sterilizing comprises:
and sterilizing the PEEK microspheres with the porous surfaces by using 75% ethanol by volume concentration, washing the PEEK microspheres by using PBS buffer solution, and then performing steam sterilization at the temperature of 115-120 ℃ for 25-35 min.
In certain embodiments of the invention, the steam sterilization pressure is 0.1 MPa.
After the soaked PEEK porous microspheres are obtained, uniformly mixing MC3T3-E1 cells which are transmitted to the 3 rd generation with the soaked PEEK porous microspheres in a DMEM culture medium, culturing, replacing the DMEM culture medium after 22-26 h for continuous culture, replacing the DMEM culture medium with a mineralization culture medium after 22-26 h for culture, replacing the DMEM culture medium with the mineralization culture medium after 22-26 h, then replacing the DMEM culture medium once every 46-50 h, and performing decellularization treatment on the 7 th-9 th cells which are replaced with the mineralization culture medium to obtain the PEEK porous microspheres with the surface coated with the mineralization extracellular matrix.
In some embodiments of the present invention, before mixing the MC3T3-E1 cells passed to the 3 rd generation and the soaked PEEK porous microspheres in the DMEM medium, the method further comprises:
the MC3T3-E1 cells passed to passage 3 at 2X 104The density of each well was seeded in a cell culture plate.
In certain embodiments of the invention, the cell culture plate is a 48-well cell culture plate.
In some embodiments of the invention, the MC3T3-E1 cells passing to the 3 rd generation and the soaked PEEK porous microspheres are mixed uniformly in a DMEM medium for 1-2 min.
In some embodiments of the invention, the MC3T3-E1 cells transferred to the 3 rd generation and the soaked PEEK porous microspheres are uniformly mixed in a DMEM culture medium, and the culture temperature is 36-37 ℃.
In the invention, the continuous culture by replacing the DMEM medium after 22-26 h refers to that: and (4) after 22-26 h, replacing the used DMEM medium with a new DMEM medium to continue culturing, wherein the components and the content of the new DMEM medium are the same as those of the DMEM medium before culturing.
In certain embodiments of the invention, the DMEM medium is replaced after 24 hours. In certain embodiments of the invention, the temperature for the further incubation is 36-37 ℃.
And (3) replacing the used DMEM medium with a new DMEM medium, and replacing the DMEM medium with a mineralized medium for culturing after 22-26 hours.
In certain embodiments of the invention, the DMEM medium is replaced with mineralized medium after 24 h.
In certain embodiments of the invention, the temperature for culturing after 22-26 hours by replacing the DMEM medium with the mineralized medium is 36-37 ℃.
In the present invention, the mineralized medium comprises CaCl2、K2HPO4And polyaspartic acid。
In certain embodiments of the invention, the mineralized medium comprises CaCl2Has a concentration of 4.5X 10-3～9×10- 3mol/L，K2HPO4Has a concentration of 2.1X 10-3～4.2×10-3mol/L, and the concentration of polyaspartic acid is 0.05-0.2 mg/mL. In certain embodiments, the mineralized medium comprises CaCl2Has a concentration of 9X 10-3mol/L，K2HPO4Has a concentration of 4.2X 10-3mol/L, the concentration of polyaspartic acid is 0.2 mg/mL.
In certain embodiments of the invention, the mineralized medium is prepared according to the following method:
addition of CaCl to DMEM Medium2、K2HPO4And polyaspartic acid to obtain the mineralized culture medium.
And changing the culture medium into a mineralized culture medium for culturing for 22-26 h, and then changing the culture medium.
In certain embodiments of the invention, the medium is changed to mineralized medium and the medium is changed after 24 hours.
In the invention, the liquid changing means: and replacing the used mineralized culture medium with a new mineralized culture medium, wherein the components and the content of the new mineralized culture medium are the same as those of the mineralized culture medium before culture.
And after the liquid is changed, the liquid is changed every 46-50 hours. In certain embodiments of the invention, the liquid change is performed every 48 hours after the liquid change. The liquid change is the same as the above, and is not described again.
And 7 th to 9 th days after the culture medium is changed into a mineralized culture medium, and performing cell removal treatment.
In certain embodiments of the invention, the decellularization process comprises the steps of:
a) washing PEEK porous microspheres cultured by a mineralized culture medium for 1-3 times by using a PBS buffer solution, then transferring the PEEK porous microspheres into a cell removal solution, and placing the PEEK porous microspheres on a shaking table with the rotating speed of 60-100 rpm for 10min at room temperature;
the cell removal solution comprises NH4OH and Triton X-100; in the cell-removing solution, NH4Concentration of OH20mmol/L, the concentration of Triton X-100 is 0.25 wt%;
b) washing the PEEK microspheres obtained in the step a) by using a PBS buffer solution, and then carrying out freeze drying.
In certain embodiments of the invention, the decellularization process further comprises drying.
The method and parameters for drying are not particularly limited in the present invention, and those known to those skilled in the art can be used.
The source of the above-mentioned raw materials is not particularly limited in the present invention, and may be generally commercially available.
The preparation method provided by the invention does not need to add inorganic components for pore forming.
The invention utilizes the mineralization process of the liquid-based precursor initiated by the polymer to carry out biomineralization, and the biomineralization can be formed on the surface of the collagen and in the collagen. Meanwhile, the prepared mineralized cell culture medium is utilized, the mineralized extracellular matrix can be formed while cell culture is carried out, and the process of mineralization is not required to be additionally carried out, so that time and labor are saved. The mineralized extracellular matrix coated on the surface of the PEEK porous microcarrier has larger density and more uniform distribution by matching with repeated decellularization and recellularization processes, thereby enhancing the osteoinductive performance of the microcarrier and being beneficial to the adhesion, proliferation and differentiation of cells. Meanwhile, the microspheres can resist high-temperature and high-pressure sterilization, can be recycled, and are beneficial to large-scale cell culture. Therefore, the invention has important significance in tissue engineering large-scale cell culture and injectable bone repair materials.
The invention also provides the PEEK porous microsphere with the surface coated with mineralized extracellular matrix, which is prepared by the preparation method.
The PEEK porous microsphere with the surface coated with mineralized extracellular matrix provided by the invention comprises:
hydroxylated PEEK microspheres;
and a mineralized extracellular matrix attached to the hydroxylated PEEK microspheres.
In embodiments of the invention, the hydroxylated PEEK microspheres have an internal and external interconnected microporous structure. The hydroxylated PEEK microspheres are obtained by processing the PEEK microspheres in the step A).
In embodiments of the invention, a mineralized extracellular matrix is attached to the outer surface of the hydroxylated PEEK microspheres and in the internal pores of the hydroxylated PEEK microspheres. Mineralized extracellular matrix is uniformly attached to the outer surface of the hydroxylated PEEK microspheres.
In certain embodiments of the invention, the mineralized extracellular matrix is in the form of a mesh.
The PEEK porous microspheres with the mineralized extracellular matrix coated on the surface are prepared for the first time, the preparation method is simple, and the surface chemical modification and the formation of the porous morphology are simultaneously realized by utilizing one-step reaction. The generation of extracellular matrix on the surface of the microsphere and biomineralization are carried out synchronously, and the microsphere can be sterilized at high temperature and high pressure and recycled.
When large-scale cell culture is carried out, the PEEK microspheres play a supporting role, and the porous structure on the surface is favorable for anchoring and growing cells. Meanwhile, the surface of the hydroxylated PEEK microsphere improves the hydrophilic property of the PEEK material, thereby being beneficial to cell adhesion and proliferation. The extracellular matrix is an autocrine extracellular matrix, is an autocrine complex entity of cells, and mainly consists of structural proteins mainly comprising collagen, proteoglycan and various adhesion proteins. The extracellular matrix mainly based on collagen provides a temporary basis for mineralized deposition in osteogenic development, and simultaneously contains a large number of signal molecules to regulate the proliferation and differentiation of cells and the interaction among cells. Therefore, the extracellular matrix coated on the surface of the microsphere can enhance the interaction between cells during cell culture and promote the proliferation and osteogenic differentiation of the cells. Meanwhile, from the viewpoint of bionics, the tissue engineering material should be similar to the growth and development process of natural bones. To this end, we synchronized the extracellular matrix production on the microsphere surface with biomineralization.
The PEEK porous microspheres with the surfaces coated with mineralized extracellular matrix improve the culture density and efficiency of cells, are beneficial to adhesion, proliferation and differentiation of the cells, and simulate an osteogenesis process from the perspective of bionics. In addition, the PEEK porous microspheres adopting the mineralized extracellular matrix can be used as injectable materials for minimally invasive surgery, and have important significance in repairing irregular bone defects.
Therefore, the invention also claims the application of the PEEK porous microspheres with the mineralized extracellular matrix coated on the surface in scale cell culture and preparation of injectable bone implant materials.
In order to further illustrate the present invention, the following examples are provided to describe a PEEK porous microsphere with a mineralized extracellular matrix coated on the surface and a preparation method thereof in detail, but should not be construed as limiting the scope of the present invention.
The starting materials used in the following examples are all commercially available.
Example 1
Preparing smooth PEEK microspheres:
weighing 1.8g of PEEK powder, adding the PEEK powder into 30mL of concentrated sulfuric acid (the mass concentration is 96 percent), placing the mixture into a water bath kettle at 26 ℃, mechanically stirring the mixture for two hours, and standing the mixture for 10 minutes after the PEEK powder is completely dissolved. And (3) adding the mixed liquid into a 50mL syringe, preparing 1000mL of 30% ethanol settling liquid with a Teflon needle head diameter of 27G, carrying out ice bath, wherein the height of the needle head from the liquid level of the settling liquid is 14cm, and preparing smooth PEEK microspheres by adopting an air flow method at the nitrogen flow rate of 4.5L/min. After obtaining the PEEK balls, soaking the PEEK balls in water for 24 hours once, and changing the water for 1 time every 8 hours. Filtering the mixture by using a screen to obtain PEEK balls with the diameter of 500-600 mu m. Transferring the PEEK balls into a 75mL reaction kettle, adding 20mL primary water, and reacting for 8h at 170 ℃; cooled to room temperature and dried in vacuo.
And (3) carrying out surface porous treatment on the PEEK microspheres:
70mL of freshly distilled dimethyl sulfoxide (DMSO) and 140mg of sodium borohydride (NaBH)4) Adding into a 100mL conical flask, heating to 120 ℃ in an oil bath to enable NaBH4And completely dissolving. 0.1g of smooth PEEK microspheres were added to a conical flask to be completely immersed, and magnetic stirring was performed at 120 ℃ and 300rpm for 6 hours under the protection of nitrogen atmosphere. After removing the PEEK microspheres from the DMSO, they were washed by sequentially immersing in the following stirred solutions: methanol 20min, primary water 15min, 0.5mol/L hydrochloric acid 15min, primary water 15min, and 95% ethanol 15 min. And (3) carrying out vacuum drying on the washed PEEK microspheres and storing the PEEK microspheres in a nitrogen atmosphere.
Soaking and sterilizing the PEEK microspheres with porous surfaces by using ethanol with the volume concentration of 75%, fully washing the PEEK microspheres for 3 times by using PBS buffer solution, performing high-temperature high-pressure steam sterilization for 30min at the temperature of 120 ℃ and under the pressure of 0.1MPa, and then soaking the PEEK microspheres in a conventional DMEM culture medium for 24h to obtain the soaked PEEK porous microspheres. The MC3T3-E1 cells passed to passage 3 at 2X 104And planting the cells in 48-hole cell culture plates according to the density of each hole, fully and uniformly mixing the cells and the soaked PEEK porous microspheres by using conventional DMEM, culturing at 36-37 ℃, replacing the used DMEM medium by using a new conventional DMEM medium after 24 hours (the component and the content of the new DMEM medium are the same as those of the DMEM medium before culture), and continuously culturing at 36-37 ℃. After another 24h, the medium was changed to mineralized medium (supplemented with 9X 10 in conventional DMEM medium)-3mol/L CaCl2，4.2×10-3mol/L K2HPO4And 0.2mg/mL polyaspartic acid) at 36-37 ℃, changing the culture solution after 24 hours (replacing a used mineralized culture medium with a new mineralized culture medium, wherein the components and the content of the new mineralized culture medium are the same as those of the mineralized culture medium before culture), and changing the culture solution every 48 hours later.
And (3) performing cell removal treatment at the 8 th d of changing the culture medium into a mineralized culture medium:
a) washing PEEK porous microspheres cultured by a mineralized culture medium for 2 times by using a PBS buffer solution, then transferring the PEEK porous microspheres into a cell removal solution, and placing the PEEK porous microspheres on a shaking table with the rotating speed of 80rpm for 10min at room temperature;
the cell removal solution comprises NH4OH and Triton X-100; in the cell-removing solution, NH4The concentration of OH is 20mmol/L, and the concentration of Triton X-100 is 0.25 wt%;
b) washing the PEEK porous microspheres obtained in the step a) by using PBS buffer solution, and freeze-drying to prepare the PEEK porous microspheres with the surfaces coated with mineralized extracellular matrix.
Scanning electron microscope scanning analysis is carried out on the prepared PEEK porous microspheres with the surface coated with mineralized extracellular matrix, and the results are shown in fig. 1 and fig. 2. FIG. 1 is an SEM image of PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces thereof, which are prepared in example 1 of the invention, at 200 μm. As shown in fig. 1, the porous PEEK microsphere has a dense mineralized cell matrix covering the surface and is distributed uniformly. FIG. 2 is an SEM image of PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces thereof, which are prepared in example 1 of the invention, at 10 μm. As can be seen from FIG. 2, the surface of the porous microspheres has a thick mineralized extracellular matrix, and a silk-like extracellular matrix can be observed in the pores. The results show that the mineralized extracellular matrix is successfully coated on the surface of the porous PEEK microsphere. The matrix is compact and is distributed uniformly, and can penetrate into the pore structure, thereby further enriching the topological appearance of the porous microsphere.
Example 2
MC3T3-E1 cells at 2X 104Density of wells and PEEK porous microspheres of example 1 surface-coated mineralized extracellular matrix were seeded in 24 wells of 48-well cell culture plates, and MC3T3-E1 cells were plated at 2X 104Density per well and smooth PEEK microspheres of example 1 were planted in another 24 wells of a 48-well cell culture plate, cultured at 37 ℃, changed every 2d with a new DMEM medium (the new DMEM medium had the same composition and content as the DMEM medium before culturing as described above), transferred to a new 48-well cell culture plate after 7d culturing, washed 1 time with PBS, added 500 μ L of calcein staining solution diluted to 2-5 μmol/L per well, incubated in a 37 ℃ oven for 10min, then aspirated and discarded, added PBS and soaked for 5min, then 500 μ L PBS was added to the wells, and cells were observed and photographed with a fluorescence microscope with a filter having an excitation wavelength of 490nm and an emission wavelength of 515nm, with the results shown in fig. 3 and 4.
FIG. 3 is an inverted fluorescence microscope photograph of the PEEK porous microspheres coated with mineralized extracellular matrix on the surface, which are obtained by staining with calcein after culturing for 7 days using MC3T3-E1 cell line, in example 1 of the present invention. FIG. 4 is an inverted fluorescence microscope image of the smooth PEEK microspheres of example 1 of the present invention after calcein staining after 7 days of culture using the MC3T3-E1 cell line.
As can be seen from fig. 3 and 4, compared with the PEEK microsphere with smooth surface, the porous microsphere with mineralized extracellular matrix coated on the surface has more cell adhesion, which significantly promotes the effective adhesion of cells.
Example 3
MC3T3-E1 cells at 2X 104Density of wells and PEEK porous microspheres of example 1 surface-coated mineralized extracellular matrix were seeded in 24 wells of 48-well cell culture plates, and MC3T3-E1 cells were plated at 2X 104Density of wells and smooth PEEK microspheres of example 1 were seeded in another 24 wells of 48-well cell culture plate, and cultured at 37 ℃, with replacement every 2d with new DMEM medium (the composition and content of the new DMEM medium are the same as those of the DMEM medium before culturing described above). After culturing for 1, 3 and 7 days respectively, absorbing and removing the original culture medium in the holes, then adding 30 mu L of CCK-8 reagent and 500 mu L of DMEM cell culture medium into each hole, incubating for 2h in a 37 ℃ incubator, then sucking 200 mu L of liquid from each hole, transferring the liquid to a new 96-hole plate, and detecting the absorbance at 450nm by using an enzyme-labeling instrument. The results are shown in the figure. FIG. 5 is a graph showing the effect of smooth PEEK microspheres and PEEK porous microspheres that included a melanized extracellular matrix on the proliferation of MC3T3-E1 cells in example 3 of the present invention.
As can be seen from FIG. 5, the absorbance values of the PEEK microsphere sets with smooth surfaces at 1, 3 and 7d were 0.319. + -. 0.016, 0.387. + -. 0.075 and 0.589. + -. 0.041, respectively. The absorbance values of PEEK porous microsphere groups coated with mineralized extracellular matrix at 1 d, 3d and 7d are respectively 0.369 +/-0.033, 0.460 +/-0.061 and 0.994 +/-0.028. Comparing the two groups of data, the absorbance value of the PEEK porous microsphere group with the mineralized extracellular matrix coated on the surface is obviously higher than that of the PEEK microsphere group with smooth surface on the 7 th day. Therefore, compared with the PEEK microsphere group with smooth surfaces, the PEEK porous microsphere group with the mineralized extracellular matrix coated on the surfaces obviously promotes the proliferation of cells when being cultured for 7 days.
Example 4
MC3T3-E1 cells at 2X 104Density of wells and PEEK porous microspheres of example 1 surface-coated mineralized extracellular matrix were seeded in 24 wells of 48-well cell culture plates, and MC3T3-E1 cells were plated at 2X 104Per well Density and smooth PEEK microspheres from example 1 were seeded into another 24 wells of a 48-well cell culture plate and cultured at 37 deg.CAfter 7d, the cells were fixed with 4 wt% paraformaldehyde solution, washed 3 times with phosphate buffer and incubated with BCIP/NBT solution in alkaline phosphatase kit for 24h at room temperature. After the incubation time, the cells were washed 2 times with phosphate buffer, and then observed and photographed with a stereoscopic microscope. The results are shown in FIGS. 6 and 7. FIG. 6 is a graph showing the effect of PEEK porous microspheres having their surface coated with mineralized extracellular matrix on the production of cellular alkaline phosphatase in example 4 of the present invention. FIG. 7 is a graph showing the effect of smooth PEEK microspheres on the production of cell alkaline phosphatase in example 4 of the present invention.
Comparing fig. 6 and 7, it can be seen that the PEEK porous microspheres coated with mineralized extracellular matrix significantly promote the generation of cellular alkaline phosphatase, i.e., have an important effect on the early differentiation of bone, compared to the smooth PEEK microspheres.
Example 5
MC3T3-E1 cells at 2X 104Density of wells and PEEK porous microspheres of example 1 surface-coated mineralized extracellular matrix were seeded in 24 wells of 48-well cell culture plates, and MC3T3-E1 cells were plated at 2X 104Density of wells and smooth PEEK microspheres of example 1 were seeded in another 24 wells of a 48-well cell culture plate, and after culturing at 37 ℃ for 7d each, the cells were washed with phosphate buffered saline and then lysed with Western and IP cell lysates containing 1mM/L PMSF. After the cell lysate is repeatedly frozen and thawed for 2 times, cell debris is removed by centrifugation for 5min at 12000g and 4 ℃. After centrifugation, 50. mu.L of the supernatant was incubated with 50. mu.L of pNPP solution at 37 ℃ for 30min in the absence of light. After incubation time 100. mu.L of stop solution was added. And absorbance measurements were performed at 450 nm. Meanwhile, the BCA protein kit measures absorbance values at 562 nm. Finally, the ratio of the two is used as the result of alkaline phosphatase quantitative analysis. The results are shown in FIG. 8. FIG. 8 is a graph showing the effect of PEEK porous microspheres and smooth PEEK microspheres having their surface coated with mineralized extracellular matrix on the alkaline phosphatase activity of cells in example 5 of the present invention. As can be seen in fig. 8, the alkaline phosphatase activity measured for the smooth PEEK microsphere set after 7d incubation was: 0.291 +/-0.020; alkali measured after 7d culture of PEEK porous microsphere group with mineralized extracellular matrix coated on surfaceThe sexual phosphatase activity was: 0.633 +/-0.054. The two have significant difference, and obviously, compared with smooth PEEK microspheres, the PEEK porous microspheres with the surfaces coated with mineralized extracellular matrix obviously improve the activity of cell alkaline phosphatase.
MC3T3-E1 cells at 2X 104Density of wells and PEEK porous microspheres of example 1 surface-coated mineralized extracellular matrix were seeded in 24 wells of 48-well cell culture plates, and MC3T3-E1 cells were plated at 2X 104Density of wells and smooth PEEK microspheres of example 1 were planted in another 24 wells of a 48-well cell culture plate, and after culturing at 37 ℃ for 14d, respectively, the cells were washed 2 times with phosphate buffer, then fixed with 4 wt% paraformaldehyde solution, and then washed with phosphate buffer. Then 500. mu.L of a 10 wt% cetylpyridinium chloride solution was added to each well, incubated at 37 ℃ for 1h, and then the absorbance value was measured at 540nm, as shown in FIG. 9. FIG. 9 is a graph of the effect of smooth PEEK microspheres and PEEK porous microspheres of the inventive example 5 having a bread with mineralized extracellular matrix on intracellular calcium deposition. As can be seen from FIG. 9, the absorbance value of the smooth PEEK microsphere group is 1.303 + -0.355, the absorbance value of the PEEK porous microsphere group with mineralized extracellular matrix coated on the surface is 2.418 + -0.092, and the absorbance value of the PEEK porous microsphere group with mineralized extracellular matrix coated on the surface is obviously higher than that of the PEEK porous microsphere group with mineralized extracellular matrix coated on the surface, which indicates that the PEEK porous microspheres with mineralized extracellular matrix coated on the surface are beneficial to calcium deposition and promote the osteogenic mineralization.
Therefore, compared with smooth PEEK microspheres, the PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces obviously improve the activity of cell alkaline phosphatase and calcium deposition in cells, promote osteogenic differentiation of the cells, and have important influence on the differentiation and growth of bones.
Example 6
Evaluation of bone repair effect using rat skull defect as a model for the smooth PEEK microspheres of example 1 and the PEEK porous microspheres of example 1 whose surfaces are coated with mineralized extracellular matrix:
the animal experiment uses SD female rats with the weight of 200-220 g, and a bone defect with the diameter of 5mm is milled on the skull of the female rats by using a milling drill as a critical bone defect. Rats were fasted 12h before surgery and water was excluded 2h before surgery. Rats were weighed before anesthesia and 0.3mL of anesthetic per 100g of body weight was administered using 10% (w/v) chloral hydrate as anesthetic. An electric skin preparation knife is used to prepare the skin of the skull area before the operation. After the limbs and the upper anterior teeth are fixed exactly, powerful iodine is used for disinfecting the operation area. The skin of about 3.5cm is cut at the middle of the skull from the back of the eye to the front of the ear by a dermatome, subcutaneous tissues are separated to periosteum by instruments such as tissue scissors, vascular forceps and the like, and the periosteum on the surface of the skull is continuously scraped by the dermatome, so that the wide and flat skull area on the two sides is fully exposed. Holding a carborundum flat head high-speed electric abrasive drill with the diameter of 5mm by two hands, controlling a pedal to operate, and continuously flushing the abrasive drill with sterile physiological saline to cool down so as to prevent the fragile tissue from being burnt. When the outer and inner plates of the skull are completely abraded away, the operation is stopped and 50 μ L of microspheres are aspirated by a pipette and injected into the bone defect area. The contralateral operation was as described above. Finally, the fascia and the like are carefully covered on the surface of the microsphere, so that the displacement of the material is prevented as much as possible, and the skin of the incision is carefully sutured. After intramuscular injection of antibiotics, rats were placed on the heat-insulating pad to accelerate the recovery from anesthesia. Each rat was intramuscularly infused with 8 ten thousand IU of penicillin sodium daily for 7 consecutive days after surgery. Rats were sacrificed at 4 weeks and 8 weeks, fixed with 4 wt% paraformaldehyde solution, then scanned by Micro CT, and the proportion of new bone in the defect area was calculated and counted using machine-compatible software, the results of which are shown in fig. 10. FIG. 10 is a graph showing the evaluation of the effect of smooth PEEK microspheres and PEEK porous microspheres coated with mineralized extracellular matrix on bone repair in example 6 of the present invention. As can be seen from FIG. 10, the neogenetic bone tissue fraction of the smooth PEEK microspheres at 4 weeks and 8 weeks was (29.514. + -. 1.553)% and (39.02. + -. 2.722)%, respectively. The PEEK porous microspheres coated with mineralized extracellular matrix have new bone tissue fractions of (42.186 +/-2.797)% and (65.190 +/-1.625)% at 4 weeks and 8 weeks, wherein the new bone tissue fractions are respectively higher than those of the PEEK porous microspheres coated with mineralized extracellular matrix. The result shows that the prepared porous PEEK microspheres with the surface coated with mineralized extracellular matrix can be injected into defect parts, and meanwhile, the repair effect on irregular critical bone defects is improved by accelerating the regeneration of bone tissues.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (11)

1. A preparation method of PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces comprises the following steps:
A) under the condition of protective gas, the PEEK microspheres are placed in the treatment fluid to be heated and stirred, and after drying, the PEEK microspheres with porous surfaces are obtained;
the treatment liquid comprises dimethyl sulfoxide and sodium borohydride;
B) sterilizing the PEEK microspheres with porous surfaces, and then soaking the PEEK microspheres in a DMEM culture medium for 12-24 hours to obtain soaked PEEK porous microspheres;
C) uniformly mixing MC3T3-E1 cells which are transmitted to the 3 rd generation and the soaked PEEK porous microspheres in a DMEM culture medium, culturing, replacing the DMEM culture medium after 22-26 hours to continue culturing, replacing the DMEM culture medium with a mineralized culture medium after 22-26 hours to culture, replacing the DMEM culture medium with the mineralized culture medium after 22-26 hours, then replacing the liquid once every 46-50 hours, and performing decellularization treatment on the 7 th-9 th day of replacing the mineralized culture medium to obtain the PEEK porous microspheres with the mineralized extracellular matrix coated on the surfaces;
the mineralized culture medium comprises DMEM culture medium and CaCl2、K2HPO4And polyaspartic acid.
2. The preparation method according to claim 1, wherein the PEEK microspheres are prepared according to the following method:
mixing polyether-ether-ketone powder with concentrated sulfuric acid to obtain a mixed solution;
preparing microspheres from the mixed solution by adopting an air flow method;
and carrying out hydrothermal reaction on the microspheres and water to obtain the PEEK microspheres.
3. The method according to claim 1, wherein in step a), the shielding gas is nitrogen;
the dosage ratio of the dimethyl sulfoxide to the sodium borohydride is 60-80 mL: 130-150 mg;
the treatment fluid is prepared according to the following method:
and (3) mixing dimethyl sulfoxide and sodium borohydride, and heating to 115-125 ℃ to obtain a treatment solution.
4. The preparation method according to claim 1, wherein in the step A), the heating and stirring temperature is 115-125 ℃, the time is 5-7 h, and the heating and stirring speed is 200-400 rpm.
5. The method according to claim 1, wherein the step a) further comprises washing after the heating and stirring;
the washing comprises the following steps:
and immersing the heated and stirred PEEK microspheres in methanol for 15-30 min, primary water for 10-20 min, 0.4-0.6 mol/L hydrochloric acid solution for 10-20 min, primary water for 10-20 min and ethanol with the volume concentration of 95-100% for 10-20 min in sequence.
6. The method for preparing according to claim 1, wherein in step B), the sterilization comprises:
and sterilizing the PEEK microspheres with the porous surfaces by using 75% ethanol by volume concentration, washing the PEEK microspheres by using PBS buffer solution, and then performing steam sterilization at the temperature of 115-120 ℃ for 25-35 min.
7. The method according to claim 1, wherein the step C) of mixing the MC3T3-E1 cells transferred to the 3 rd generation and the soaked PEEK porous microspheres in DMEM medium further comprises:
the MC3T3-E1 cells passed to passage 3 at 2X 104The density of each well is planted in a cell culture plate;
and uniformly mixing the MC3T3-E1 cells which are transmitted to the 3 rd generation and the soaked PEEK porous microspheres in a DMEM culture medium for 1-2 min.
8. The method for preparing a drug according to claim 1, wherein in step C), the decellularization treatment comprises the steps of:
a) washing the PEEK microspheres cultured by the mineralized culture medium for 1-3 times by using a PBS buffer solution, then transferring the PEEK microspheres into a cell removal solution, and placing the PEEK microspheres on a shaking table with the rotating speed of 60-100 rpm for 10min at room temperature;
the cell removal solution comprises NH4OH and Triton X-100; in the cell-removing solution, NH4The concentration of OH is 20mmol/L, and the concentration of Triton X-100 is 0.25 wt%;
b) washing the PEEK microspheres obtained in the step a) by using a PBS buffer solution, and then carrying out freeze drying.
9. The method according to claim 1, wherein in step C), CaCl is added to the mineralized medium2Has a concentration of 4.5X 10-3～9×10-3mol/L，K2HPO4Has a concentration of 2.1X 10-3～4.2×10-3mol/L, and the concentration of polyaspartic acid is 0.05-0.2 mg/mL.
10. PEEK porous microspheres with mineralized extracellular matrix coated on the surfaces, prepared by the preparation method of any one of claims 1 to 9.
11. Use of PEEK porous microspheres coated with mineralized extracellular matrix according to claim 10 for scale cell culture and preparation of injectable bone implant materials.
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