CN117883641B - A bone-like multi-level structure composite film material and its preparation method and application - Google Patents
A bone-like multi-level structure composite film material and its preparation method and application Download PDFInfo
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- 239000000463 material Substances 0.000 title claims abstract description 120
- 239000002131 composite material Substances 0.000 title claims abstract description 114
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 80
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- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
- D01D5/0038—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion the fibre formed by solvent evaporation, i.e. dry electro-spinning
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0069—Electro-spinning characterised by the electro-spinning apparatus characterised by the spinning section, e.g. capillary tube, protrusion or pin
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0092—Electro-spinning characterised by the electro-spinning apparatus characterised by the electrical field, e.g. combined with a magnetic fields, using biased or alternating fields
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Mechanical Engineering (AREA)
- Textile Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Dispersion Chemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Composite Materials (AREA)
- Materials Engineering (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to a composite film material with a skeleton-like multi-layer structure, and a preparation method and application thereof, and the composite film material comprises the following steps: (1) Polyvinyl alcohol (PVA) and collagen are used as raw materials and mixed with a solvent to prepare spinning solution; carrying out electrostatic spinning on the spinning solution, and drying to obtain a composite film; (2) And (3) placing the composite film into a prepared calcium carbonate mineralization solution, performing biomineralization, and drying to obtain the composite film material with the skeleton-like multi-layer structure. The composite film material has good biocompatibility and excellent mechanical property, and the electrostatic spinning technology is easy to operate, low in cost and applicable to mass preparation, and the biomineralization process is carried out at normal temperature and normal pressure.
Description
Technical Field
The invention relates to the technical field of bionic thin film materials, in particular to a skeleton-like multi-layer structure composite thin film material, and a preparation method and application thereof.
Background
The natural process of bone healing in vivo following a fracture or other injury is known as bone repair. One of the most common methods of supporting bone healing is through auxiliary devices such as artificial membranes, scaffolds, etc., in order to promote cell adhesion, proliferation and differentiation to prevent non-osteogenic tissue from filling the bone defect. To date, absorbable artificial membranes made from collagen fibers are the most common biological membranes in clinic. These bioactive membranes have high biocompatibility and low risk of complications. However, the main disadvantage of these membranes is their poor mechanical properties, mismatch with the natural periosteal tissue, and inability to meet the surgical suture requirements, which severely limits their clinical application. Recent research results indicate that mechanical properties of scaffolds, including strength, stiffness and elasticity, are important factors directly affecting cell adhesion, proliferation and differentiation capabilities. Therefore, how to obtain a bioactive film material with good mechanical properties, which is beneficial to cell proliferation and differentiation, has been the focus of attention of researchers.
The nanofiber membrane scaffold prepared by electrostatic spinning is more and more focused in the field of tissue engineering scaffolds because of the advantages of easy control, strong material operability, good mechanical strength and the like. The yellow green et al prepared polylactic acid-glycolic acid (PLGA) osteogenic scaffolds by using an electrospinning method, and showed good potential in promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). The prior art adopts organic spinning to obtain a composite film material, and has the following problems: 1. the raw materials are high polymers, compared with the protein in the organism, the biological compatibility is lower, and a certain complication risk exists; 2. the film composite material obtained by spinning is a simple organic matter and has poor mechanical property; 3. there is room for improvement in both overall process flow and material performance.
In order to prepare materials with good mechanical properties, inorganic substances are generally introduced as reinforcing phases to improve the overall properties of the composite material. The Bertassoni et al prepares a protein hydrogel film carrying human bone marrow mesenchymal stem cells (hMSCs), and promotes protein induction to generate hydroxyapatite by adding Ca 2+、PO4 3- and a nucleation inhibitor, thereby not only improving the mechanical strength of the gel film, but also being beneficial to the osteogenesis and differentiation of the hMSCs. The technology has the problems that the size of the composite film material is limited by the protein self-assembly process, the requirements on the process conditions of the self-organization and gel forming processes are high, the self-assembly is long in time consumption (> 48 h) under alkaline conditions (pH is more than 11), commercialization is difficult to realize, and the requirement of large-scale production cannot be met.
Therefore, the existing absorbable artificial membrane generally cannot have mechanical properties and biological activities, or has high process requirements, and is difficult to realize large-scale production.
Disclosure of Invention
The invention aims to overcome the technical defects, and provides a skeleton-like multi-layer structure composite film material, a preparation method and application thereof, and solves the technical problems that an absorbable artificial film in the prior art cannot have mechanical properties, biological activity and long preparation time.
In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:
In a first aspect, the invention provides a method for preparing a composite film material with a skeleton-like multi-layer structure, which is characterized by comprising the following steps: (1) Polyvinyl alcohol and collagen are used as raw materials, and are mixed with a solvent to prepare spinning stock solution; carrying out electrostatic spinning on the spinning solution, and drying to obtain a composite film; (2) And (3) placing the composite film into a prepared calcium carbonate mineralization solution, performing biomineralization, and drying to obtain the composite film material with the skeleton-like multi-layer structure.
In a second aspect, the invention provides a composite film material with a skeleton-like multi-layer structure, which is prepared by the preparation method.
In a third aspect, the present invention provides the use of the above-described composite film material of a bone-like multi-layered structure as a bioactive material.
Compared with the prior art, the invention has the beneficial effects that:
1. According to the invention, the mixed aqueous solution of polyvinyl alcohol (PVA)/collagen is used as the spinning solution, and the polyvinyl alcohol (PVA) and the collagen have excellent performance, good biocompatibility, no toxicity or harm, mild conditions and environmental protection compared with other electrostatic spinning materials;
2. The calcium carbonate directionally mineralizes and grows in the nano fiber, the simulated biomineralization reduces the reaction conditions, the interaction between the organic/inorganic materials is fully utilized, the mechanical property and the biocompatibility of the composite material are further improved, and the obtained composite film material has excellent performance;
3. the invention adopts the electrostatic spinning technology, and the technology has the advantages of easy operation and low cost in the process of preparing the nanofiber membrane material, and the preparation method and the technology are simple, the product performance is stable, the size is controllable, the preparation method is suitable for mass preparation, and the post-treatment technology is simple.
Therefore, the composite film material has good biocompatibility and excellent mechanical property, and the electrostatic spinning technology is easy to operate and low in cost, the biomineralization process is carried out at normal temperature and normal pressure, and the composite film material is suitable for mass preparation and has wide application prospect in the field of biological materials.
Drawings
FIG. 1 is a scanning electron microscope image of the present invention, wherein A is a single polyvinyl alcohol/collagen film prepared in comparative example 1, B is a composite film material of a skeleton-like multi-layered structure of intra-fiber mineralized calcium carbonate prepared in example 1, and C is a non-intra-fiber mineralized calcium carbonate-loaded polyvinyl alcohol/collagen film material prepared in comparative example 2;
FIG. 2 is a transmission electron microscope image of the composite film material of the skeleton-like multi-layer structure prepared in example 1 of the present invention; wherein A-C are transmission electron microscope pictures under different magnifications;
FIG. 3 is a graph showing the hardness and Young's modulus of a composite film material having a skeleton-like multi-layered structure prepared in example 1 of the present invention, compared with that of a single polyvinyl alcohol/collagen film prepared in comparative example 1;
FIG. 4 is a nanoindentation graph of the calcium carbonate-loaded polyvinyl alcohol/collagen composite film material prepared in comparative example 2 of the present invention;
FIG. 5 is a comparison of the crystal violet staining of cells of a composite film material of a bone-like multi-layered structure prepared in example 1, a single polyvinyl alcohol/collagen film prepared in comparative example 1, and a non-intra-fiber mineralized calcium carbonate polyvinyl alcohol/collagen film prepared in comparative example 2 according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention aims to provide a composite film material with a skeleton-like multi-layer structure, a preparation method thereof and application thereof in the field of biological materials, wherein the composite film material with the skeleton-like multi-layer structure is prepared by selecting low-cost commercial polyvinyl alcohol, collagen and the like as raw materials and adopting an electrostatic spinning technology, and then inorganic materials are deposited under mild conditions in a simulated biomineralization process, so that the composite film material has mechanical properties and biological activity and potential economic value.
In a first aspect, the invention provides a method for preparing a composite film material with a skeleton-like multi-layer structure, comprising the following steps:
(1) Polyvinyl alcohol (PVA) and collagen are used as raw materials and mixed with a solvent to prepare spinning solution; carrying out electrostatic spinning on the spinning solution, and drying to obtain a composite film;
(2) And (3) placing the composite film in a prepared calcium carbonate mineralization solution for biomineralization, ensuring that the composite film is completely immersed in the mineralization solution in the biomineralization process, and drying to obtain the composite film material with the skeleton-like multi-layer structure.
Preferably, the solvent in step (1) is water.
Preferably, in the step (1), the mass ratio of the polyvinyl alcohol, the collagen and the solvent is 1: (1-2): (10-20).
Preferably, in the step (1), the preparation process of the spinning solution specifically includes: mixing and stirring polyvinyl alcohol and a solvent for 3-4 hours under the water bath condition of 70-90 ℃; and adding collagen under the condition of room temperature, mixing and stirring for 2-4 hours to obtain spinning solution.
Preferably, in step (1), the conditions of electrospinning include: the voltage is 15-21 kV, the distance between the spray head and the roll shaft is 10-15 cm, and the temperature is 20-30 ℃.
Preferably, in the step (1), the drying temperature is 30-40 ℃ and the drying time is 20-24 h.
Preferably, in the step (2), the mineralized solution is formed by mixing (by mass ratio) 1 (0.5-1): 2-3): 0.5-1): 7-8): 1750-1850, and calcium chloride, polyacrylic acid (PAA), hydroxyethyl piperazine ethane sulfur sulfonic acid (HEPES), sodium carbonate, sodium chloride and solvent.
Preferably, in step (2), the biomineralization is performed at 30-40℃for 24-48 hours.
Preferably, in the step (2), the drying is naturally dried for 2-4 hours.
In a second aspect, the present invention provides a skeletal multi-level structured composite film material.
Preferably, the raw materials of the composite film material with the skeleton-like multi-layer structure are polyvinyl alcohol (PVA), collagen and solvent water, the composite film is obtained by electrostatic spinning, and then the composite film material is prepared by biomineralization calcium carbonate, the mineralized composite material takes the polyvinyl alcohol/collagen as a template, the calcium carbonate directionally grows in the fibers to form the composite film material with the skeleton-like multi-layer structure, the diameter of the mineralized fibers is 500-1000 nm, and the calcium carbonate uniformly grows in the fibers along the long axes of the fibers.
In a third aspect, the present invention provides the use of a skeletal multi-layered structured composite film material as a bioactive material.
Preferably, the hardness of the bioactive material is 200-250 MPa, the Young's modulus is 5-6 GPa, and the bioactive material does not show obvious cytotoxicity within 7 days, and can promote cell proliferation and inhibit apoptosis.
The invention innovatively utilizes PVA/collagen mixed solution to obtain the composite film through an electrostatic spinning technology, and simulates a biomineralization process to prepare the composite film material with a skeleton-like multi-layer structure. The basic working principle is that a mixed solution of PVA/collagen is subjected to electrospinning by adopting an electrospinning method to obtain a composite fiber film, and mineralization is carried out in a prepared mineralization solution at a certain temperature to realize mineralization of calcium carbonate in PVA/collagen nanofibers, so that a composite film material with a skeleton-like multi-layer structure is obtained; the main action mechanism and the advantages are as follows:
(1) Due to the demands for film size, two-dimensional composite film materials are widely used in various biological stent fields. The electrostatic spinning has the advantages of easy operation and low cost in the process of preparing the two-dimensional film material, and the material prepared by the electrostatic spinning has the advantages of controllable size and the like, can effectively improve the adhesiveness of cells, and promotes the proliferation and differentiation of the cells, thereby improving the biomedical activity of the film bracket.
(2) Polyvinyl alcohol (PVA) is used as a synthetic polymer, has good biocompatibility, is nontoxic and harmless, and has low price. The collagen fiber is derived from the living body and has good biocompatibility. PVA can play a role of a surfactant in the mixed solution, so that collagen fibers are uniformly dispersed in the solution, an organic template is played in the spinning process, and the collagen fibers are wrapped to form a nanofiber composite film. But also has good physical properties and certain mechanical strength.
(3) And carrying out electrostatic spinning on the mixed solution to obtain a composite film, and further mineralizing the composite film to obtain the composite film material with the skeleton-like multi-layer structure. Calcium carbonate is a common mineral in living bodies in nature, and has good biological activity and excellent mechanical properties. Calcium chloride, polyacrylic acid (PAA), hydroxyethyl piperazine ethylsulfanilic acid (HEPES), sodium carbonate, sodium chloride and solvent water are mixed to prepare a mineralized solution, which is favorable for simulating a biological mineralization process, calcium carbonate grows in the nano fiber of the composite film in an mineralized manner, and the organic/inorganic combination effectively improves the biological activity and mechanical property of the composite film material.
(4) Biomineralization is one way to produce materials of superior properties in nature. Under the condition of low temperature and normal pressure, mineral ions mineralize and grow along the long axis inside the collagen fiber under the in-vivo regulation and control, and the oriented mineral structure and the interaction between organic and inorganic enable bones/shells and the like to have excellent mechanical strength. Therefore, the invention not only utilizes electrostatic spinning to prepare the composite film with controllable size; and calcium carbonate is mineralized and grown in the composite film fiber by successfully simulating the biomineralization process, so that the bioactivity and mechanical property of the composite film material are improved, and the composite film material has a huge application prospect when being used for biological materials. The preparation method is simple, can react at low temperature and normal pressure, is suitable for mass production, and has important significance for developing high-performance biological film materials.
The present invention will be described in further detail by way of the following specific examples, which are not intended to limit the scope of the invention.
The collagen adopted in the invention is type I collagen (bovine Achilles tendon) which is obtained from Shanghai Biotechnology service Limited of China.
Example 1
The preparation method of the skeleton-like multi-layer structure composite film material comprises the following steps:
(1) Preparing a composite film by an electrostatic spinning method: mixing and stirring 2g of polyvinyl alcohol and 20g of water for 4 hours at 90 ℃, adding 2g of collagen for mixing and stirring for 4 hours at room temperature to obtain an electrostatic spinning dope, electrostatic spinning the dope at the voltage of 21kV, the distance between a spray head and a roll shaft of 15cm, the temperature of 30 ℃, and finally drying at 40 ℃ for 24 hours to obtain a polyvinyl alcohol/collagen composite film (PVA/Col);
(2) Preparing a composite film material with a skeleton-like multi-layer structure from biomineralization calcium carbonate: placing the composite film in the step (1) into a prepared calcium carbonate mineralization solution, wherein the mineralization solution is prepared by mixing and titrating 0.0555g of calcium chloride, 0.04g of polyacrylic acid (PAA), 0.1192g of hydroxyethyl piperazine ethylene sulfate (HEPES), 0.0535g of sodium carbonate, 0.4382g of sodium chloride and 100ml of water by mass, and naturally drying for 4 hours after mineralization at 37 ℃ to obtain the composite film material PVA/Col-CaCO 3 with the skeleton-like multi-layer structure.
Comparative example 1
In order to study the influence of calcium carbonate on the performance of the composite film material, a single non-mineralized polyvinyl alcohol/collagen film material was prepared, and the specific steps not specifically described in the specific steps were the same as those of the preparation method of the composite film material of the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen of the above-mentioned example 1.
The difference from example 1 is that: the polyvinyl alcohol/collagen film material (PVA/Col) obtained in step (1) of example 1 was used as it is without performing the treatment of step (2).
Comparative example 2
In order to study the influence of mineralized and grown calcium carbonate and supported calcium carbonate on the performance of the composite film material with the skeleton-like multi-layer structure of polyvinyl alcohol/collagen, namely, the influence of calcium carbonate added into the film material in different forms, other raw materials are controlled to be identical, only the components of mineralized solution are changed, and the composite film material with the skeleton-like multi-layer structure of polyvinyl alcohol/collagen of the supported calcium carbonate is prepared, wherein the steps which are not specifically described in the specific steps are the same as the preparation method of the composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen of the embodiment 1.
The difference from example 1 is that: the mineralization solution in step (2) was not added with 0.04g polyacrylic acid (PAA), and the other steps and conditions were the same as in example 1, and the resulting material was designated PVA/Col@CaCO 3.
Performance testing
1. The thin film materials obtained in example 1 and comparative examples 1 to 3 were each subjected to electron microscopic scanning, and the results are shown in FIG. 1.
From the graph a in fig. 1 (microscopic morphology of the unmineralized single polyvinyl alcohol/collagen film material of comparative example 1 tested by scanning electron microscopy), it can be seen that only a simple nanofiber structure exists; from the graph B in FIG. 1 (the microscopic morphology of the composite film material with the skeleton-like multi-layer structure, which is prepared in example 1, is obtained through scanning electron microscope test), calcium carbonate crystals can be seen to uniformly grow in the fibers of the composite film material; from the graph C in FIG. 1 (the microstructure obtained by scanning electron microscope testing of the calcium carbonate-loaded polyvinyl alcohol/collagen composite film material prepared in comparative example 2), it can be seen that the microstructure of the material obtained in comparative example 2 is calcium carbonate-loaded crystal particles, and that calcium carbonate is not mineralized and grown in the nanofiber.
2. The transmission electron microscope test of example 1 was conducted at various magnifications, and as shown in A-C of FIG. 2, it can be seen that calcium carbonate crystals were grown in a direction oriented in the long axis direction inside the fiber.
3. Mechanical properties of the composite film material of skeleton-like multi-layered structure (PVA/Col-CaCO 3) prepared in example 1, PVA/Col prepared in comparative example 1 and the polyvinyl alcohol/collagen composite film material loaded with calcium carbonate of comparative example 2 were respectively tested, wherein the results of nanoindentation test of example 1 and comparative example 1 (using nanoindenter TI-980, bruker-Hysite, germany) are shown in FIG. 3, and it is known that: the hardness of the composite film material PVA/Col-CaCO 3 obtained in example 1 was 211.6MPa, and the Young's modulus was 5.6GPa; comparative example 1 unmineralized single polyvinyl alcohol/collagen film material PVA/Col hardness was 113.5MPa and young's modulus was 2.1GPa.
From the above, the mechanical properties of the composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen of the embodiment 1 of the invention are obviously higher than those of the single non-mineralized polyvinyl alcohol/collagen film material of the comparative example 1, the hardness and Young modulus of the composite film material are improved by more than 2 times, which indicates that the mechanical properties of the composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen are obviously improved compared with those of the single non-mineralized polyvinyl alcohol/collagen film material, and the composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen has excellent properties.
When the polyvinyl alcohol/collagen composite film material loaded with calcium carbonate obtained in comparative example 2 is subjected to nano indentation test, the results obtained by selecting different areas are different, the difference is large, and as shown in fig. 4, the mechanical properties are poor.
Therefore, the calcium carbonate growth mode has obvious influence on the performance of the obtained material, and the mechanical performance of the mineralized and grown calcium carbonate composite film material is far higher than that of the supported calcium carbonate composite film material, so that the mineralized and grown calcium carbonate mode directly obtains the technical effect of obviously improving the mechanical performance of the composite material.
4. Cytotoxicity test was performed on the composite film material of the skeleton-like multi-layered structure obtained in example 1 and PVA/Col and the like of comparative example 1 as samples. The method comprises the following steps:
BMSCs (bone marrow mesenchymal stem cells) were cultured in an alpha-MEM medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin at 37℃under partial pressure of 5% CO 2 in an incubator.
Prior to the cell compatibility experiments, the samples were soaked in 75% ethanol and sterilized with uv light irradiation for 2 hours. Then, after BMSCs were adhered to the cell culture plate for 12 hours, each group of samples was washed twice with Phosphate Buffered Saline (PBS) and placed in an orifice plate at a ratio of 0.1g/10mL of medium, and cells of passage 2-5 were used.
Cells after 7 days of culture were stained with crystal violet to characterize cell viability: after removal of the medium, cells were fixed with 4% PFA for 15 min at room temperature, then stained with 0.1% crystal violet for 15 min and washed twice with PBS. Cell morphology was observed under confocal laser scanning microscopy.
And a blank group was set, which means the result obtained by culturing cells without using a thin film material.
After staining with cell crystal violet, as shown in fig. 5, it can be seen that: after 7 days of cell culture, the composite film material with the skeleton-like multi-layer structure of the example 1 does not show significant cytotoxicity; comparative example 1 the composite film material did not exhibit significant cytotoxicity.
Whereas the calcium carbonate-loaded polyvinyl alcohol/collagen composite film material obtained in comparative example 2 showed no significant cytotoxicity after 7 days of cell culture, but the number of cells was small compared with example 1.
Therefore, the composite material prepared by the specific preparation method provided by the invention simulates the biomineralization process, so that calcium carbonate grows in the film nanofiber along the long axis in an oriented manner, and excellent mechanical properties can be realized.
In summary, the raw materials of the composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen are polyvinyl alcohol (PVA), collagen and solvent water, the composite film is obtained by electrostatic spinning, and then the composite film material is prepared by biomineralization of calcium carbonate, wherein the diameter of mineralized fibers is 500-1000nm, and the calcium carbonate uniformly grows in the fibers along the long axes of the fibers, and the preparation method comprises the following steps: 1) Preparing a polyvinyl alcohol/collagen film material by an electrostatic spinning method; 2) And simulating the biomineralization process to prepare the composite film material with the skeleton-like multi-layer structure. The experimental detection shows that the following results (which can be realized within the scope of the preparation method of the invention and specific examples are not listed here):
The composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen is tested by a scanning electron microscope and a transmission electron microscope, and calcium carbonate crystals well grow in the fiber in a mineralized manner along the long axis direction.
The composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen has the hardness of 200-250MPa and the Young modulus of 5-6GPa through nano indentation test.
The composite film material with the skeleton-like multi-layer structure of the polyvinyl alcohol/collagen has no obvious cytotoxicity in 7 days after being subjected to a biocompatibility test, and can promote cell proliferation and inhibit apoptosis.
The simple polyvinyl alcohol/collagen film material is subjected to nanoindentation test, the hardness is 113.5MPa, and the Young modulus is 2.1GPa. The mechanical properties of the mineralized and grown calcium carbonate-like composite film material with the skeleton-like multi-layer structure are obviously higher than those of a single polyvinyl alcohol/collagen film material, the hardness and Young modulus of the mineralized and grown calcium carbonate-like composite film material are improved by more than 2 times, and the mineralized calcium carbonate obviously improves the mechanical properties of the material, so that the mineralized calcium carbonate-like composite film material with the skeleton-like multi-layer structure has good biocompatibility and excellent mechanical properties.
The application of the composite film material with the skeleton-like multi-layer structure as the bioactive film material has the hardness of 200-250MPa and the Young modulus of 5-6GPa, does not show obvious cytotoxicity within 7 days, and can promote cell proliferation and inhibit apoptosis. The composite film material has the advantages of good biocompatibility, low toxicity, mild preparation conditions and environmental protection; and the electrostatic spinning technology is easy to operate, low in cost, the biomineralization process is carried out at normal temperature and normal pressure, and the method is suitable for large-batch preparation and has wide application prospect in bioactive film materials.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.
Claims (9)
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