CN108273130B - Three-dimensional micro-nano fiber composite support and preparation method thereof - Google Patents
Three-dimensional micro-nano fiber composite support and preparation method thereof Download PDFInfo
- Publication number
- CN108273130B CN108273130B CN201810164702.1A CN201810164702A CN108273130B CN 108273130 B CN108273130 B CN 108273130B CN 201810164702 A CN201810164702 A CN 201810164702A CN 108273130 B CN108273130 B CN 108273130B
- Authority
- CN
- China
- Prior art keywords
- micro
- scaffold
- nano
- fiber
- dimensional
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
-
- 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
- A61L27/56—Porous materials, e.g. foams or sponges
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Pharmacology & Pharmacy (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Nonwoven Fabrics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Artificial Filaments (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a three-dimensional micro-nano fiber composite scaffold which is formed by a three-dimensional mesh structure formed by mutually interweaving and penetrating uniformly distributed micro cellulose and nano bacterial cellulose. The preparation method comprises the following steps: preparing a micron fiber scaffold with the diameter of 1-5 mu m; preparing a nano-bacterial fiber scaffold with the thickness of 1-5mm by a static culture method as a substrate, and performing membrane-liquid interface culture at intervals of 4-10h according to the volume ratio of 0.01mL/cm2‑0.1mL/cm2Uniformly covering a liquid culture medium in the micrometer cellulose bracket, removing the nanometer bacterial fiber bracket, and then carrying out purification treatment to obtain the three-dimensional micro-nano fiber composite bracket with the micrometer fibers and the nanometer fibers mutually interwoven and uniformly distributed. The method has the advantages of simple and convenient operation, low cost, no pollution and easy realization of large-scale production, and the micro-nano fiber scaffold not only has a three-dimensional micro-nano fiber structure and high porosity, but also has good biological activity, biocompatibility and high bionic property, and has wide application value in the field of tissue engineering.
Description
Technical Field
The invention relates to preparation of a biomedical material, in particular to a three-dimensional micro-nano fiber composite scaffold and a preparation method thereof.
Background
The development and progress in the field of tissue engineering play a role in playing a role in human health civilization, and the main research content of the tissue engineering is to construct an implant with biological activity in vitro, implant the implant into the body to repair tissue defects and replace organ functions; or as an in vitro device, temporarily replaces the functions of organs, and achieves the purposes of improving the living quality and prolonging the life. The research field relates to materials science, engineering and life science. It is known that the regeneration and repair of tissue to be replaced need the skillful cooperation among three major elements of tissue engineering (artificial in vivo extracellular matrix-scaffold, cell and information factor), wherein the scaffold is the basis of the tissue engineering and is the key of success or failure of the tissue engineering. Therefore, the preparation or the exploration of the tissue engineering scaffold which is similar to the human extracellular matrix in structure and components has important significance. Research shows that the in vivo extracellular matrix not only comprises pores and fibers of micron (generally meaning 1-1000 mu m), but also has pores and fibers of nanometer scale (generally meaning 1-1000 nm), and the multilayer geometric structural unit has a synergistic effect which is unknown so far and directly controls the behavior and the function of cells. In order to explore the coordination mechanism of the micro-nano structure and provide a new design principle and technical support for the design and construction of regenerative medicine and tissue engineering scaffolds, the in vivo extracellular matrix needs to be constructed in a simulation way.
Electrospinning has been of interest in recent years as a simple and effective method of producing fibrous materials. The related literature reports that the diameter of the micro-nano fiber scaffold obtained by adopting a single electrostatic spinning method is too large to facilitate bionics, and a disordered layer-by-layer superposed structure cannot construct a three-dimensional network structure formed by interweaving micro-nano fibers, so that the biological requirements on scaffold biological functions cannot be met. From comprehensive analysis of the existing micro-nano fiber support preparation technology, electrostatic spinning is still the most widely and effectively applied technology at present. The diameter of the electrostatic spinning fiber can be regulated between several nanometers and tens of micrometers, and recent reports also show that a real three-dimensional bracket (with the thickness of 2-3 mm) can be obtained by the improved electrostatic spinning technology. Nevertheless, it is difficult to prepare a three-dimensional scaffold with a micro-nano structure which is not layered and mutually permeated by a single electrostatic spinning technology, and simultaneously, a nanofiber with the dimension close to 10 nm is not easy to obtain.
The bacterial cellulose is a fibrous natural nano material synthesized by acetobacter xylinum and the like, and has the characteristics of hyperfine three-dimensional network structure, high water absorption performance, higher biocompatibility, adaptability and biodegradability, simple preparation, environmental friendliness, no pollution and the like. At present, bacterial cellulose enters a practical stage in the industries of food industry, paper industry, acoustic equipment and the like. In addition, the advantages of the bacterial cellulose are not only represented by excellent biocompatibility, high strength and modulus, but also represented by natural nano-scale (the diameter of the bacterial cellulose can be regulated and controlled within the range of 10-100 nm) fibers, a three-dimensional network structure, abundant nanopores and the like, so that the bacterial cellulose has a scaffold structure similar to an extracellular matrix in vivo.
The method adopts a culture method of contacting a liquid culture medium and a membrane interface, and can thoroughly solve the problem of inconsistent geometric structures of the upper surface and the lower surface of a bacterial cellulose product. More importantly, although the traditional culture method can be used for manufacturing a micro-nano fiber scaffold (in detail, see 'a micro-nano fiber tissue engineering scaffold and a preparation method thereof CN 201310720956.4'), it is difficult to obtain a scaffold with sufficient thickness and completely uniform distribution and interweaving of micro-fibers and nano-fibers. The invention creatively adopts a membrane-liquid interface culture method to thoroughly solve the technical problems. Specifically, the bacterial cellulose prepared by a static culture method is used as a substrate, a cellulose acetate micron fiber support and a bacterial cellulose nano support are compounded by a membrane-liquid interface culture method, so that the bacterial cellulose nano support forms an ultrathin film with a layer-by-layer structure in pores of the cellulose acetate micron fiber support, and finally a micron and nano micro-nano structure support which is mutually interwoven, mutually communicated and uniformly distributed is obtained.
Disclosure of Invention
Aiming at the prior art, the invention provides a three-dimensional micro-nano fiber composite scaffold, which is a three-dimensional reticular tissue engineering scaffold with micron and nano-sized interwoven, communicated and uniformly distributed, and has a micron structure for cell migration and a nano structure required for cell growth and propagation; meanwhile, the material has high porosity, good bioactivity, biocompatibility and high bionic property, and is expected to become a substitute material for tissue repair and regeneration and a carrier for conveying various drug molecules. In addition, the preparation method of the three-dimensional micro-nano fiber composite support provided by the invention has the advantages of simplicity and convenience in operation, low cost, no harsh requirements on equipment and environmental friendliness.
In order to solve the technical problem, the invention provides a three-dimensional micro-nano fiber composite scaffold which is formed by a three-dimensional net structure formed by mutually interweaving and penetrating uniformly distributed micro fibers and nano bacterial cellulose.
The invention provides a preparation method of a three-dimensional micro-nano fiber composite bracket, which comprises the following steps:
step one, preparing a micron fiber scaffold with the diameter of 1-5 microns, drying for 12 hours in a vacuum drying oven at the temperature of 80 ℃, and sterilizing for later use;
step two, preparing a nano bacterial cellulose bracket with the thickness of 1-5mm by a static culture method for later use;
step three, placing the micrometer fiber support prepared in the step one on the nanometer bacterial cellulose support prepared in the step two, and then adopting a membrane liquid interface culture method to perform culture every 4-10h according to the volume ratio of 0.01mL/cm2-0.1 mL/cm2In an amount that liquid medium is uniformly covered in the micro fiber scaffold, repeating three times;
and step four, removing the nano bacterial cellulose bracket, and then carrying out purification treatment to obtain the three-dimensional micro-nano fiber composite bracket formed by the three-dimensional reticular structure formed by mutually interweaving and penetrating the uniformly distributed micro fibers and the nano bacterial cellulose.
Further, in the first step of the preparation method, the micro fiber scaffold is any one of a cellulose acetate micro fiber scaffold, a PLGA micro fiber scaffold, a PCL micro fiber scaffold, a polylactic acid micro fiber scaffold and a fibroin micro fiber scaffold.
In the second step, the strain adopted in the nano bacterial cellulose bracket is any one of acetobacter xylinum, acetobacter aceti, acetobacter gluconicum, acetobacter acetogenins and acetobacter pasteurianus.
Compared with the prior art, the invention has the beneficial effects that:
the process for obtaining the three-dimensional micro-nano fiber composite bracket is simple and convenient, does not need strong acid and strong alkali, and is green and pollution-free. The obtained micro-nano support has a three-dimensional network structure with uniform distribution and interpenetration of micro-fibers and nano-celluloses, and is expected to become a substitute material for tissue repair and regeneration and a carrier for conveying various drug molecules.
Drawings
FIG. 1-1 is an SEM photograph of a cellulose acetate microfiber scaffold prepared in example 1 of the present invention;
FIGS. 1-2 are SEM photographs of bacterial cellulose/cellulose acetate scaffolds prepared in example 1 of the present invention;
FIG. 2-1 is an SEM photograph of a cellulose acetate microfiber scaffold prepared in example 2 of the present invention;
FIG. 2-2 is an SEM photograph of a bacterial cellulose/cellulose acetate scaffold prepared in example 2 of the present invention;
FIG. 3-1 is an SEM photograph of a cellulose acetate microfiber scaffold prepared in example 3 of the present invention;
FIG. 3-2 is an SEM photograph of a bacterial cellulose/cellulose acetate scaffold prepared in example 3 of the present invention;
FIG. 4-1 is a fluorescent inverted microscope photograph of cells co-cultured with scaffolds for 1, 4 and 7 days in example 4 of the present invention;
FIG. 4-2 is a graph showing the results of proliferation of cells on the scaffold for 1, 4 and 7 days in example 4 of the present invention.
Detailed Description
The invention provides a three-dimensional micro-nano fiber composite bracket which is formed by a three-dimensional net structure formed by mutually interweaving and penetrating uniformly distributed micro fibers and nano bacterial cellulose. The design idea is as follows: the three-dimensional reticular tissue engineering scaffold which is mutually interwoven with the micron and the nanometer, is mutually communicated and uniformly distributed is prepared, has the micron structure of cell migration and the nanometer structure required by cell growth and propagation, and is expected to become a substitute material for tissue repair and regeneration and a carrier for conveying various drug molecules.
The preparation method of the three-dimensional micro-nano fiber composite bracket comprises the following steps:
step one, preparing a micrometer fiber scaffold with the diameter of 1-5 microns, wherein the micrometer fiber scaffold can be any one of a cellulose acetate micrometer fiber scaffold, a PLGA micrometer fiber scaffold, a PCL micrometer fiber scaffold, a polylactic acid micrometer fiber scaffold and a fibroin micrometer fiber scaffold.
For example, cellulose acetate spinning solutions with different concentrations are prepared by different solvents, electrostatic spinning is carried out under certain process parameters to prepare a micron fiber scaffold with the diameter of 1-5 μm, and then the micron fiber scaffold is placed in a vacuum drying oven to be dried for 12 hours at the temperature of 80 ℃ and then is sterilized for later use;
step two, preparing a nano bacterial cellulose bracket with the thickness of 1-5mm by a static culture method for later use; in the invention, the strain adopted in the nano bacterial cellulose bracket is any one of acetobacter xylinum, acetobacter aceti, acetobacter xylinum, acetobacter acetogenins and acetobacter pasteurianus.
For example, Acetobacter xylinum 2 (naturally, the inoculated strain may be Acetobacter xylinum 1, and the amount of the strain in the culture medium after inoculation is small compared to Acetobacter xylinum 2, and the culture time is long for the strain to proliferate and secrete the bacterial cellulose) is inoculated into the culture medium in a sterile super clean bench, and the culture medium is transferred as a liquid culture medium after 3 days of culture. Inoculating with the volume of the bacterial liquid being 10% of the volume of the culture medium, placing in a constant-temperature incubator at 30 ℃ for standing culture for 2-3 d, almost exhausting the culture medium in the pore plate, and obtaining a bacterial cellulose bracket with the thickness of 1-5mm as a substrate, thereby providing strains for the micro-nano fiber composite bracket in the subsequent treatment process.
Step (ii) ofThirdly, the bacterial fiber scaffold with the thickness of 1-5mm prepared by the static culture method in the second step is taken as a substrate, and the micron fiber scaffold prepared in the first step is placed on the substrate; then, adopting membrane liquid interface culture method to perform membrane liquid interface culture every 4-10h according to 0.01mL/cm2-0.1 mL/cm2The liquid culture medium is uniformly covered in the micron fiber scaffold, bacteria on the surface of the substrate are proliferated at a gas-liquid interface by using oxygen in pores of the micron fiber scaffold and continuously secrete bacterial cellulose, so that the bacterial cellulose nano-cellulose is uniformly distributed in the pores of the micron fiber scaffold, and the three-dimensional micro-nano fiber scaffold with the micron fibers and the nano-cellulose interwoven is formed, wherein the operation is repeated for a plurality of times according to the thickness of the micron fiber scaffold, and the thickness of the nano-bacterial cellulose scaffold is equivalent to that of the micron fiber scaffold after the operation is usually repeated for three times.
And step four, after removing the nano bacterial cellulose scaffold, carrying out purification treatment (namely washing with deionized water and NaOH, soaking in tert-butyl alcohol, and carrying out vacuum freeze drying) to obtain the three-dimensional micro-nano fiber composite scaffold formed by the three-dimensional reticular structures which are formed by mutually interweaving and penetrating uniformly distributed micro fibers and nano bacterial cellulose.
The technical scheme of the invention is further described in detail with reference to the accompanying drawings and specific embodiments, wherein the specific embodiments are that firstly a cellulose acetate micron fiber scaffold is prepared by an electrostatic spinning method, a bacterial fiber scaffold prepared by a strain static culture method by using 2-ring acetobacter xylinum is used as a substrate, and finally a three-dimensional bacterial cellulose/cellulose acetate micron-nano fiber composite scaffold is prepared by a membrane liquid interface culture method. The described embodiments are merely illustrative of the invention and are not intended to be limiting.
Examples 1,
The method for preparing the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber composite scaffold by electrospinning cellulose acetate with the diameter of about 1 mu m comprises the following steps:
step one, using a mixture of 1: 1, dissolving cellulose acetate by using acetone and glacial acetic acid as solvents, preparing 10 wt% of cellulose acetate spinning solution, performing electrostatic spinning under the conditions of 10 kV and 5.00 mL/h to prepare a cellulose acetate micron fiber support with the diameter of about 1 mu m, placing the support in a vacuum drying oven, drying for 12 h at the temperature of 80 ℃, and performing sterilization treatment for later use; FIG. 1-1 is an SEM photograph of the cellulose acetate microfiber scaffold produced;
and step two, inoculating 2-ring acetobacter xylinum into the culture medium in a sterile super-clean workbench, culturing for 3 d, and transferring as a liquid culture medium. Inoculating with 10% of the culture medium, transferring 0.5 mL of the inoculated culture medium to 24-well plate (the diameter of the plate is about 15 mm, and the bottom area is about 1.77 cm)2) Placing the mixture in a constant temperature incubator at 30 ℃ for static culture for 2-3 d, almost exhausting the culture medium in a pore plate to obtain a bacterial cellulose bracket with the thickness of about 3 mm as a substrate, and providing strains for the micro-nano bracket;
step three, placing the cellulose acetate micro-fiber bracket obtained in the step one on the bacterial cellulose bracket obtained in the step two, and taking 0.1 mL of liquid culture medium, namely, 0.056 mL/cm2The amount of the culture medium is uniformly covered in the cellulose acetate micrometer fiber scaffold obtained in the step one, the culture medium with the same volume is covered at intervals of about 6 hours, and the operation is repeated for three times to obtain the composite scaffold with the thickness of the bacterial cellulose scaffold being equal to that of the cellulose acetate micrometer fiber scaffold.
And step four, removing the basement membrane of the scaffold obtained in the step three to obtain a three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber scaffold, cleaning with deionized water and NaOH, soaking with tert-butyl alcohol, and freeze-drying in vacuum to prepare the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber composite scaffold. And FIGS. 1-2 are SEM pictures of the prepared micro-nano fiber composite scaffold.
Examples 2,
The method for preparing the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber scaffold by electrospinning cellulose acetate with the diameter of about 2 mu m comprises the following steps:
step one, using a mixture of 1: 1, dissolving cellulose acetate by using acetone and glacial acetic acid as solvents, preparing 15 wt% of cellulose acetate spinning solution, performing electrostatic spinning under the conditions of 10 kV and 5.00 mL/h to prepare a cellulose acetate micron fiber support with the diameter of about 2 mu m, placing the support in a vacuum drying oven, drying for 12 h at the temperature of 80 ℃, and performing sterilization treatment for later use; FIG. 2-1 is an SEM photograph of the cellulose acetate microfiber scaffold produced;
and step two, inoculating 2-ring acetobacter xylinum into the culture medium in a sterile super-clean workbench, culturing for 3 d, and transferring as a liquid culture medium. Inoculating with 10% of the culture medium, transferring 0.5 mL of the inoculated culture medium to 24-well plate (the diameter of the plate is about 15 mm, and the bottom area is about 1.77 cm)2) Placing the mixture in a constant temperature incubator at 30 ℃ for static culture for 2-3 d, almost exhausting the culture medium in a pore plate to obtain a bacterial cellulose bracket with the thickness of about 3 mm as a substrate, and providing strains for the micro-nano bracket;
step three, placing the fiber support obtained in the step one on the bacterial cellulose obtained in the step two, and taking 0.1 mL of liquid culture medium, namely, 0.056 mL/cm2The amount of the culture medium is uniformly covered in the cellulose acetate micrometer fiber scaffold obtained in the step one, the culture medium with the same volume is covered at intervals of about 6 hours, and the operation is repeated for three times to obtain the composite scaffold with the thickness of the bacterial cellulose scaffold being equal to that of the cellulose acetate micrometer fiber scaffold.
And step four, removing the basement membrane from the support obtained in the step three to obtain a three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber support, washing with deionized water and NaOH, soaking in tert-butyl alcohol, and freeze-drying in vacuum, thereby preparing the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber support. And 2-2 is an SEM photograph of the prepared micro-nano fiber composite bracket.
Examples 3,
The method for preparing the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber scaffold by electrospinning cellulose acetate with the diameter of about 5 mu m comprises the following steps:
step one, using a mixture of 1: 1, dissolving cellulose acetate by using acetone and glacial acetic acid as solvents, preparing 20 wt% of cellulose acetate spinning solution, performing electrostatic spinning under the conditions of 10 kV and 5.00 mL/h to prepare a micron cellulose acetate micron fiber support with the diameter of about 5 microns, placing the support in a vacuum drying oven, drying for 12 hours at the temperature of 80 ℃, and performing sterilization treatment for later use; FIG. 3-1 is an SEM photograph of the cellulose acetate microfiber scaffold produced;
and step two, inoculating 2-ring acetobacter xylinum into the culture medium in a sterile super-clean workbench, culturing for 3 d, and transferring as a liquid culture medium. Inoculating with 10% of the culture medium, transferring 0.5 mL of the inoculated culture medium to 24-well plate (the diameter of the plate is about 15 mm, and the bottom area is about 1.77 cm)2) Placing the mixture in a constant temperature incubator at 30 ℃ for static culture for 2-3 d, almost exhausting the culture medium in a pore plate to obtain a bacterial cellulose bracket with the thickness of about 3 mm as a substrate, and providing strains for the micro-nano bracket;
step three, placing the fiber support obtained in the step one on the bacterial cellulose obtained in the step two, and taking 0.1 mL of liquid culture medium, namely, 0.056 mL/cm2The amount of the culture medium is uniformly covered on the cellulose acetate micrometer fiber scaffold obtained in the step one, the culture medium with the same volume is covered at intervals of about 6 hours, and the operation is repeated for three times to obtain the composite scaffold with the thickness of the bacterial cellulose scaffold being equal to that of the cellulose acetate micrometer fiber scaffold.
And step four, removing the basement membrane from the support obtained in the step three to obtain a three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber support, washing with deionized water and NaOH, soaking in tert-butyl alcohol, and freeze-drying in vacuum, thereby preparing the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber composite support. And 3-2 is an SEM photograph of the prepared micro-nano fiber composite bracket.
Examples 4,
The method for evaluating the biocompatibility of the three-dimensional bacterial cellulose/cellulose acetate micro-nano fiber scaffold prepared in the embodiment 2 comprises the following specific steps:
step one, placing three bracket materials (wherein the three-dimensional bacterial cellulose/cellulose acetate micro-nano bracket is an experimental group, and the bacterial cellulose bracket and the cellulose acetate micro-nano fiber bracket are control groups) with the diameter of 15 mm and the thickness of 1 mm in a high-temperature sterilization box at 121 ℃, and sterilizing for 30 min for later use.
Secondly, selecting preosteoblasts for resuscitation and passageAfter reaching a steady state, the cell concentration was 1X 105Cells/ml were seeded onto three fibrous scaffolds, which were then placed at 37 ℃ in 5% CO2And (3) carrying out static co-culture in an incubator, and changing the culture solution every other day, wherein the culture periods are 1 d, 4 d and 7 d respectively.
Step three, observing the growth conditions of the cells cultured on the bracket for 1 d, 4 d and 7 d by using an inverted microscope, wherein a figure 4-1 is a fluorescence inverted microscope photograph of the cells cultured on the bracket for 1, 4 and 7 days in the embodiment 4 of the invention.
And step four, removing the culture solution of the tissue cells cultured for 1 d, 4 d and 7 d (during the period of maintaining and changing the culture solution every other day), washing the tissue cells for 2 times by HBSS, adding 500 mu L of serum-free culture solution into each hole, and adding 50 mu L of MTT into each hole under the condition of keeping away from light. The plates were then placed in the incubator for 4 h, the MTT was decanted, DMSO (450. mu.L) was added, the shaker shaken for 5 min, and the solution was then transferred rapidly to a 96-well plate (150. mu.L per well) for testing. Absorbance (OD) was measured with a microplate reader at 490 nm wavelength, 3 replicates per group, and the assay was repeated 3 times. FIG. 4-2 is a graph showing the results of proliferation of cells on the scaffold for 1, 4 and 7 days in example 4 of the present invention.
As can be seen from fig. 1-2, 2-2 and 3-2, compared with the layered micro-nano structure scaffold reported in the related literature, the micro-nano fiber composite scaffold prepared by the invention is a three-dimensional reticular tissue engineering scaffold with micron and nano interweaved, interpenetration and uniform distribution, and has a three-dimensional micro-nano fiber structure, a very large porosity (the porosity of the micro-nano fiber composite scaffold measured by a mercury porosimeter is more than 90%, the porosities of the three embodiments are respectively 90.2%, 91.3% and 93.4%) and a high imitative property, as can be seen from fig. 4-1 and 4-2, the three-dimensional micro-nano fiber composite scaffold prepared by the invention is non-toxic to cells and the cells can normally grow and proliferate, and is calculated according to the cell MTT result: compared with a control group BC (BC) scaffold, the cell activities of the 1 st, the 4 th and the 7 th cells of the micro-nano fiber composite scaffold are respectively 98%, 150% and 176%, which shows that the micro-nano fiber composite scaffold has good biological activity and biocompatibility. Therefore, the three-dimensional micro-nano fiber composite scaffold prepared by the invention is expected to become a substitute material for tissue repair and regeneration and a carrier for conveying various drug molecules.
Although the present invention has been described in detail with reference to the drawings, the present invention is not limited to the above embodiments, which are only illustrative and not restrictive, and those skilled in the art can make many modifications without departing from the spirit of the present invention, which falls within the protection of the present invention.
Claims (3)
1. A preparation method of a three-dimensional micro-nano fiber composite scaffold is provided, the three-dimensional micro-nano fiber composite scaffold is composed of a three-dimensional mesh structure formed by mutually interweaving and penetrating uniformly distributed micro fibers and nano bacterial cellulose, and the preparation method is characterized by comprising the following steps:
step one, preparing a micron fiber scaffold with the diameter of 1-5 microns, drying for 12 hours in a vacuum drying oven at the temperature of 80 ℃, and sterilizing for later use;
step two, preparing a nano bacterial cellulose bracket with the thickness of 1-5mm by a static culture method for later use;
step three, placing the micrometer fiber support prepared in the step one on the nanometer bacterial cellulose support prepared in the step two, and then adopting a membrane liquid interface culture method to perform culture every 4-10h according to the volume ratio of 0.01mL/cm2-0.1 mL/cm2In an amount that liquid medium is uniformly covered in the micro fiber scaffold, repeating three times;
and step four, removing the nano bacterial cellulose bracket, and then carrying out purification treatment to obtain the three-dimensional micro-nano fiber composite bracket formed by the three-dimensional reticular structure formed by mutually interweaving and penetrating the uniformly distributed micro fibers and the nano bacterial cellulose.
2. The method for preparing the three-dimensional micro-nano fiber composite scaffold according to claim 1, wherein in the first step, the micro fiber scaffold is any one of a cellulose acetate micro fiber scaffold, a PLGA micro fiber scaffold, a PCL micro fiber scaffold, a polylactic acid micro fiber scaffold and a fibroin micro fiber scaffold.
3. The method for preparing the three-dimensional micro-nano fiber composite scaffold according to claim 1, wherein in the second step, the strain adopted in the nano bacterial cellulose scaffold is any one of acetobacter xylinum, acetobacter aceti, acetobacter gluconicum, acetobacter acetogenium and acetobacter pasteurianus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810164702.1A CN108273130B (en) | 2018-02-27 | 2018-02-27 | Three-dimensional micro-nano fiber composite support and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810164702.1A CN108273130B (en) | 2018-02-27 | 2018-02-27 | Three-dimensional micro-nano fiber composite support and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108273130A CN108273130A (en) | 2018-07-13 |
| CN108273130B true CN108273130B (en) | 2021-02-09 |
Family
ID=62808835
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810164702.1A Expired - Fee Related CN108273130B (en) | 2018-02-27 | 2018-02-27 | Three-dimensional micro-nano fiber composite support and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108273130B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750553B (en) | 2019-01-24 | 2022-01-28 | 北京京东方技术开发有限公司 | Container for food preservation and preparation method thereof |
| CN110559103B (en) * | 2019-08-05 | 2021-08-31 | 华东交通大学 | Through-membrane stent and preparation method thereof |
| CN115414532B (en) * | 2022-09-13 | 2023-12-19 | 华东交通大学 | Bacterial cellulose-based integrated osteochondral scaffold with interface barrier layer and preparation method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250378B (en) * | 2011-03-30 | 2012-12-05 | 东华大学 | Bacterial cellulose/polymer composite film and preparation method thereof |
| CN103483606B (en) * | 2012-06-11 | 2015-11-18 | 钟春燕 | A kind of preparation method of bacterial cellulose composite polymer superfine fiber material |
| CN103638560A (en) * | 2013-12-04 | 2014-03-19 | 天津工业大学 | Preparation method of bracket with micro-nano structure |
| CN103691005B (en) * | 2013-12-24 | 2015-10-28 | 华东交通大学 | A kind of micro--Na fibrous tissue engineering rack and preparation method thereof |
| CN103893825B (en) * | 2014-02-24 | 2015-06-10 | 钟春燕 | Method for preparing bacterial cellulose compounded amnion extracellular matrix material containing collagen |
| CN105132273A (en) * | 2015-09-18 | 2015-12-09 | 南京荣之盛生物科技有限公司 | Culture reactor for multiple bacterial cellulose membranes and application of culture reactor |
-
2018
- 2018-02-27 CN CN201810164702.1A patent/CN108273130B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN108273130A (en) | 2018-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Halib et al. | The remarkable three-dimensional network structure of bacterial cellulose for tissue engineering applications | |
| Sheikh et al. | 3D electrospun silk fibroin nanofibers for fabrication of artificial skin | |
| Fu et al. | Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation | |
| Fu et al. | Skin tissue repair materials from bacterial cellulose by a multilayer fermentation method | |
| Bian et al. | The construction and performance of multi-level hierarchical hydroxyapatite (HA)/collagen composite implant based on biomimetic bone Haversian motif | |
| Yang et al. | A novel electrospun-aligned nanoyarn-reinforced nanofibrous scaffold for tendon tissue engineering | |
| CN108273130B (en) | Three-dimensional micro-nano fiber composite support and preparation method thereof | |
| Hong et al. | Three-dimensional scaffold of electrosprayed fibers with large pore size for tissue regeneration | |
| US7285637B2 (en) | Method for the preparation of a non-woven silk fibroin fabrics | |
| Mandal et al. | Biospinning by silkworms: silk fiber matrices for tissue engineering applications | |
| Rajasekaran et al. | Role of nanofibers on MSCs fate: Influence of fiber morphologies, compositions and external stimuli | |
| CN101302486B (en) | Acetobacter xylinum and method for preparing nano-cellulose skin tissue repair material by using the same | |
| US20070275458A1 (en) | Three dimensional-BIO-mimicking active scaffolds | |
| CN107988158B (en) | Three-dimensional tumor model acellular porous scaffold, construction method and application thereof | |
| WO2020237785A1 (en) | Gradient material for guiding regeneration of periodontal hard and soft tissues and preparation method therefor | |
| CN109529123B (en) | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof | |
| Liu et al. | Effect of the porous microstructures of poly (lactic-co-glycolic acid)/carbon nanotube composites on the growth of fibroblast cells | |
| CN107349475B (en) | The artificial organ engineering skin and preparation method thereof that nano fibrous membrane is layering with stem cell | |
| CN103007344B (en) | Hollow heterotype bacteria cellulose artificial blood vessel stent material with gradient structure and preparation method thereof | |
| Luo et al. | Interpenetrated nano-and submicro-fibrous biomimetic scaffolds towards enhanced mechanical and biological performances | |
| Luo et al. | Fabrication of a novel hierarchical fibrous scaffold for breast cancer cell culture | |
| Li et al. | Mechanically-reinforced 3D scaffold constructed by silk nonwoven fabric and silk fibroin sponge | |
| Wan et al. | Submicrofiber‐Incorporated 3D Bacterial Cellulose Nanofibrous Scaffolds with Enhanced Cell Performance | |
| WO2018107573A1 (en) | Fibroin fiber frame and manufacturing method thereof | |
| Meng et al. | Recent advances of electrospun nanofiber-enhanced hydrogel composite scaffolds in tissue engineering |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210209 |