CN115814166A - Preparation method of nanofiber silk fibroin hydrogel - Google Patents
Preparation method of nanofiber silk fibroin hydrogel Download PDFInfo
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Abstract
The disclosure relates to the technical field of biological materials, and provides a preparation method of a nanofiber silk fibroin hydrogel, which comprises the following steps: s1, preparing dry regenerated silk fibroin; s2, respectively dissolving the regenerated silk fibroin and the chitosan in a hexafluoroisopropanol solvent, and mixing according to a certain proportion to obtain a first mixed solution; s3, preparing first nanofibers from the first mixed solution through electrostatic spinning, drying, crosslinking and cleaning; and S4, preparing the nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin. Provides a novel nano-fiber silk fibroin hydrogel scaffold material, has a special fibrous microstructure, is beneficial to the adhesion and proliferation of chondrocytes, has high porosity, and has excellent mechanical property and biocompatibility.
Description
Technical Field
The disclosure relates to the technical field of biological materials, in particular to a preparation method of nanofiber silk fibroin hydrogel.
Background
Cartilage damage and cartilage defects caused by tumors, trauma, inflammation, developmental deformity, osteoarthritis and degenerative joint disease cause great pain to patients, affect normal life of patients, and limit endogenous self-repair capacity of cartilage. On the one hand, chondrocytes are difficult to self-proliferate after injury due to their high degree of differentiation and low aggregability, and on the other hand, cartilage lacks progenitor cell migration due to compactness and non-vascularization of the extracellular matrix
Differentiation and vasculotrophy. Thus, damaged cartilage often suffers from irreversible and persistent pathological processes. Currently, clinical treatment methods for articular cartilage defects include subchondral microfracture, arthroplasty, autologous chondrocyte transplantation, allogeneic cartilage transplantation, xenogeneic cartilage transplantation, and prosthetic joint replacement. However, these surgical procedures often fail to provide patients with durable joint function and have associated complications or problems such as secondary lesions in the donor area, progressive structural abnormalities in the joint area, early graft resorption, immune rejection and disease transmission, prosthetic joint wear, donor shortages, and the like.
Tissue engineering provides a promising tissue repair technology for cartilage defects, and bionic is applied to medical science to prepare cell constructs by simulating in-vivo microenvironment in vitro with biomaterials or to control the formation of tissues and organs in vivo. As one of the key elements of tissue engineering, the bioscaffold functions as a temporary ECM with supporting, trophic, and morphologically inducible functions.
For decades, silk fibroin has been widely used for preparing cartilage tissue engineering scaffolds due to its outstanding biocompatibility, and the electrospinning technology has also been applied to the preparation of various nanoporous scaffolds. However, electrospun nanofibers alone are not suitable for use as a scaffold for articular cartilage defect repair.
Disclosure of Invention
Object of the invention
In view of the above problems, the present application aims to provide a novel nanofiber silk fibroin hydrogel scaffold material, which has a special fibrous microstructure, is beneficial to adhesion and proliferation of chondrocytes, has high porosity, and has excellent mechanical properties and biocompatibility.
(II) technical scheme
In a first aspect of the embodiments of the present disclosure, a method for preparing a nanofiber silk fibroin hydrogel is provided, including:
s1, preparing dry regenerated silk fibroin;
s2, respectively dissolving the regenerated silk fibroin and the chitosan in a hexafluoroisopropanol solvent, and mixing according to a certain proportion to obtain a first mixed solution;
s3, preparing first nanofibers from the first mixed solution through electrostatic spinning, drying, crosslinking and cleaning;
and S4, preparing the nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin.
In a possible embodiment, the step S1 of preparing dried regenerated silk fibroin comprises:
degumming and drying certain mass of silkworm cocoon silk to generate silk fibroin fiber;
dissolving the silk fibroin fibers in a lithium bromide solution to obtain a silk fibroin aqueous solution;
dialyzing the silk fibroin aqueous solution to generate a regenerated silk fibroin solution;
and (3) carrying out vacuum drying on the regenerated silk fibroin solution to generate regenerated silk fibroin.
In one possible embodiment, the vacuum drying time is 40-60h.
In a possible embodiment, the mass ratio of the regenerated silk fibroin and the chitosan in the first mixed solution in the step S2 is 1:0.1-1.
In one possible embodiment, the crosslinking is ethanol crosslinking.
In a possible embodiment, the step S4 of preparing a nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin includes:
preparing a regenerated silk fibroin solution based on the regenerated silk fibroin;
adding the first nano-fibers into the regenerated silk fibroin solution, uniformly mixing, and adding a cross-linking agent for chemical cross-linking to obtain a suspension;
and (4) freezing and shaping the suspension to generate the nanofiber silk fibroin hydrogel.
In one possible embodiment, the cross-linking agent comprises one or more of 1,4-butanediol diglycidyl ether, 1,3-diglycidyl ether glycerol, bisphenol a diglycidyl ether derivatives, resorcinol diglycidyl ether, 3,4-hydroxyphenyl methane triglycidyl ether, neopentyl glycol diglycidyl ether.
In one possible embodiment, the crosslinking agent is 1,4-butanediol diglycidyl ether.
In a second aspect of the embodiments of the present disclosure, a nanofiber silk fibroin hydrogel prepared by the above preparation method is provided.
In a possible embodiment, the nanofiber silk fibroin hydrogel comprises a plurality of fibers therein, each of the fibers has an average diameter of 130-150mm, and the nanofiber silk fibroin hydrogel has a porosity of greater than 90%.
(III) advantageous effects
Compared with the prior art, the embodiment of the disclosure has the following beneficial effects: provides a novel nano-fiber silk fibroin hydrogel scaffold material, has a special fibrous microstructure, is beneficial to the adhesion and proliferation of chondrocytes, has high porosity, and has excellent mechanical property and biocompatibility.
Drawings
To more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed for the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without inventive efforts.
Fig. 1 is a flow diagram of some embodiments of a method of making a nanofiber silk fibroin hydrogel according to the present disclosure.
Detailed Description
Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present disclosure are shown in the drawings, it is to be understood that the disclosure may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided for a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the disclosure are for illustration purposes only and are not intended to limit the scope of the disclosure.
It should be noted that, for convenience of description, only the portions related to the related invention are shown in the drawings. The embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict.
It should be noted that the terms "first", "second", and the like in the present disclosure are only used for distinguishing different devices, modules or units, and are not used for limiting the order or interdependence relationship of the functions performed by the devices, modules or units.
It is noted that references to "a", "an", and "the" modifications in this disclosure are intended to be illustrative rather than limiting, and that those skilled in the art will recognize that "one or more" may be used unless the context clearly dictates otherwise.
The names of messages or information exchanged between devices in the embodiments of the present disclosure are for illustrative purposes only, and are not intended to limit the scope of the messages or information.
The preparation method of the nanofiber silk fibroin hydrogel of the present disclosure is described in detail below with reference to fig. 1, and comprises the following steps:
step S1, preparing dry regenerated silk fibroin.
The step S1 includes:
and S11, degumming and drying certain mass of cocoon silks to generate silk fibroin fibers.
And S12, dissolving the silk fibroin fibers in a lithium bromide solution to obtain a silk fibroin aqueous solution.
And S13, dialyzing the silk fibroin aqueous solution to generate a regenerated silk fibroin solution.
And S14, performing vacuum drying on the regenerated silk fibroin solution to generate regenerated silk fibroin.
The steps are as follows: degumming and drying certain mass of silkworm cocoon silk to generate silk fibroin fiber; and (2) putting the silkworm cocoon silk into a sodium carbonate aqueous solution with the mass percent of 0.5-1%, heating and boiling for 30min at 100 ℃, repeatedly boiling for three times, rinsing with deionized water after each boiling to remove sericin in raw silk, and drying the degummed silk in an oven to obtain the silk fibroin fiber. And dissolving the silk fibroin fibers in a lithium bromide aqueous solution with the molar concentration of 9.0mol/L, and then filtering to remove impurities and LiBr to obtain the silk fibroin aqueous solution. The dialysis is to pour the cooled silk fibroin solution into a dialysis bag (the solution accounts for 1/3-2/3 of the volume of the dialysis bag), put the dialysis bag into deionized water at the temperature of 6-12 ℃ for dialysis for 3-5 days and concentrate the solution to obtain a regenerated silk fibroin aqueous solution, and then dry the regenerated silk fibroin aqueous solution in vacuum for 40-60h at the temperature of-40-45 ℃ to generate dry regenerated silk fibroin.
And S2, respectively dissolving the regenerated silk fibroin and the chitosan in a hexafluoroisopropanol solvent, and mixing according to a certain proportion to obtain a first mixed solution.
In some embodiments, the mass ratio of the regenerated silk fibroin and the chitosan in the first mixed solution in the step S2 is 1:0.1-1.
And S3, preparing the first nanofiber from the first mixed solution through electrostatic spinning, drying, crosslinking and cleaning.
In some embodiments, the electrospinning conditions are a voltage of 12 to 15KV, a flow rate of 0.4mL/h to 0.6mL/h, an aluminum foil plate receiver spaced 12cm to 15cm from the needle, a relative temperature and relative humidity of 25 ℃ and 50%, respectively, and a drying time of 3 to 5 days.
In some embodiments, the crosslinking is gradient crosslinking using ethanol, specifically, 100% ethanol soaking for 8-12 minutes, 90% ethanol soaking for 8-12 minutes, 70% ethanol soaking for 8-12 minutes, and pure water washing for 3 times to remove ethanol, to obtain water-insoluble silk fibroin/chitosan nanofibers, i.e., the first nanofibers.
And S4, preparing the nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin.
In some embodiments, the step S4 includes: firstly, preparing a regenerated silk fibroin solution based on the regenerated silk fibroin; secondly, adding the first nano-fibers into the regenerated silk fibroin solution, uniformly mixing, and adding a cross-linking agent for chemical cross-linking to obtain a suspension; and thirdly, freezing and shaping the suspension to generate the nanofiber silk fibroin hydrogel.
Specifically, the preparation steps are as follows:
ball-milling the mixed fibroin protein/chitosan nano-fiber into short fiber by a ball mill, quickly freezing by liquid nitrogen, and continuously lyophilizing at-40 deg.C for 25-30 hr to obtain fibroin protein/chitosan nano-short fiber; according to the regeneration of silk fibroin: the regenerated silk fibroin and the short fibers are weighed according to the proportion of the short fibers =3-5:1, and a prepared cross-linking agent is added, wherein the cross-linking agent can be one or more of 1,4-butanediol diglycidyl ether, 1,3-diglycidyl ether glycerol, bisphenol A diglycidyl ether derivatives, resorcinol diglycidyl ether, 3,4-hydroxyphenyl methane triglycidyl ether and neopentyl glycol diglycidyl ether. Preferably, 1,4-butanediol diglycidyl ether is selected as a cross-linking agent, sufficiently and uniformly mixed by vortex, and injected into a mold. Immediately freezing at-25 ℃ to-30 ℃ for 20-25h, unfreezing at room temperature, adding absolute ethyl alcohol with the same volume, standing for a period of time, and taking out the bracket to obtain the nanofiber silk fibroin hydrogel, wherein the average diameter of each fiber of the obtained nanofiber silk fibroin hydrogel is 130-150mm, and the porosity of the nanofiber silk fibroin hydrogel is more than 90%.
The nanofiber silk fibroin hydrogel prepared according to the above preparation method is described in detail below.
In some embodiments, the nanofiber silk fibroin hydrogel comprises a plurality of fibers therein, each of the fibers has an average diameter of 130-150mm, and the nanofiber silk fibroin hydrogel has a porosity of greater than 90%.
The nanofiber silk fibroin hydrogel prepared by the present disclosure and the preparation method thereof are illustrated by specific examples below.
Example 1
Degumming and drying certain mass of silkworm cocoon silk to generate silk fibroin fiber; and (2) putting the silkworm cocoon silk into a sodium carbonate aqueous solution with the mass percentage of 0.5%, heating and boiling for 30min at the temperature of 100 ℃, repeatedly boiling for three times, rinsing with deionized water after each boiling to remove sericin in raw silk, and drying the degummed silk in an oven to obtain the silk fibroin fiber. And dissolving the silk fibroin fibers in a lithium bromide aqueous solution with the molar concentration of 9.0mol/L, and then filtering to remove impurities and LiBr to obtain the silk fibroin aqueous solution. And pouring the cooled silk fibroin solution into a dialysis bag (the solution accounts for 1/3 of the volume of the dialysis bag), putting the dialysis bag into deionized water at the temperature of 6 ℃, dialyzing for 3 days, concentrating to obtain a regenerated silk fibroin aqueous solution, and carrying out vacuum drying on the regenerated silk fibroin aqueous solution at the temperature of-40 ℃ for 40 hours to generate regenerated silk fibroin.
The mass ratio of the regenerated silk fibroin to the regenerated silk fibroin is 0.1:1, respectively dissolving the chitosan and the regenerated silk fibroin in a hexafluoroisopropanol solvent, and mixing to obtain a first mixed solution.
And (3) drying the first mixed solution in a vacuum drying oven for 3 days at the voltage of 12KV, the flow rate of 0.4mL/h, the distance between an aluminum foil flat plate receiver and a needle head of 12cm and the relative temperature and the relative humidity of 25 ℃ and 50% respectively. After the gradient crosslinking of ethanol, the water insolubility is increased, and the gradient ethanol crosslinking conditions are as follows: 100% ethanol for 8 minutes, 90% ethanol for 8 minutes, and 70% ethanol for 8 minutes. Washing with pure water for 3 times to remove ethanol to obtain water insoluble fibroin/chitosan nanofiber.
Ball milling the mixed fibroin protein/chitosan nano fiber into short fiber with a ball mill, quickly freezing with liquid nitrogen, and continuously lyophilizing at-40 deg.C for 30 hr to obtain the fibroin protein/chitosan nano short fiber.
According to the regeneration silk fibroin: weighing regenerated silk fibroin and short fibers according to the proportion of the short fibers =3:1, namely weighing 30mg of extracted silk fibroin to prepare silk fibroin solution, adding 10mg of freeze-dried nano short fibers, and adding 1mmol/mL1, 4-butanediol diglycidyl ether solution. Vortex, mix well and inject into mould. Immediately freezing at-30 ℃ for 20h, unfreezing at room temperature, adding equal volume of absolute ethyl alcohol, standing for 10min, and taking out the scaffold to obtain the nanofiber silk fibroin hydrogel, wherein the average diameter of each fiber of the obtained nanofiber silk fibroin hydrogel is 130mm, and the porosity of the nanofiber silk fibroin hydrogel is 92.5%.
Example 2
(1) Preparing dry regenerated silk fibroin;
degumming and drying certain mass of silkworm cocoon silk to generate silk fibroin fiber; and (2) putting the silkworm cocoon silk into a sodium carbonate aqueous solution with the mass percentage of 0.5%, heating and boiling for 30min at the temperature of 100 ℃, repeatedly boiling for three times, rinsing with deionized water after each boiling to remove sericin in raw silk, and drying the degummed silk in an oven to obtain the silk fibroin fiber. And dissolving the silk fibroin fibers in a lithium bromide aqueous solution with the molar concentration of 9.0mol/L, and then filtering to remove impurities and LiBr to obtain the silk fibroin aqueous solution. And pouring the cooled silk fibroin solution into a dialysis bag (the solution accounts for 2/3 of the volume of the dialysis bag), putting the dialysis bag into deionized water at the temperature of 6 ℃, dialyzing for 3 days, concentrating to obtain a regenerated silk fibroin aqueous solution, and carrying out vacuum drying on the regenerated silk fibroin aqueous solution at the temperature of-45 ℃ for 60 hours to generate regenerated silk fibroin.
Mixing the regenerated silk fibroin with the mixed solution at a mass ratio of 1:1, respectively dissolving the chitosan and the regenerated silk fibroin in a hexafluoroisopropanol solvent, and mixing to obtain a first mixed solution.
And (3) drying the first mixed solution in a vacuum drying oven for 5 days at a voltage of 15KV and a flow rate of 0.6mL/h, with a distance of 15cm between an aluminum foil flat receiver and a needle head, and relative temperatures and relative humidities of 25 ℃ and 50% respectively. After the gradient crosslinking of ethanol, the water insolubility is increased, and the gradient ethanol crosslinking conditions are as follows: 100% ethanol for 12 minutes, 90% ethanol for 12 minutes, and 70% ethanol for 12 minutes. Washing with pure water for 3 times to remove ethanol, and making into water insoluble fibroin/chitosan nanofiber.
Ball milling the mixed fibroin protein/chitosan nano fiber into short fiber with a ball mill, quickly freezing with liquid nitrogen, and continuously lyophilizing at-40 deg.C for 25 hr to obtain the fibroin protein/chitosan nano short fiber.
According to the regeneration of silk fibroin: weighing regenerated silk fibroin and short fibers according to the proportion of the short fibers =5:1, namely weighing 50mg of extracted silk fibroin to prepare silk fibroin solution, adding 10mg of freeze-dried nano short fibers, and adding 1mmol/mL1, 4-butanediol diglycidyl ether solution. Vortex, mix well and inject into mould. Immediately freezing at-25 deg.C for 25h, thawing at room temperature, adding equal volume of anhydrous ethanol, standing for 12min, and taking out the scaffold to obtain nanofiber silk fibroin hydrogel with average diameter of each fiber of 150mm and porosity of 95%.
The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention in the embodiments of the present disclosure is not limited to the specific combination of the above-mentioned features, but also encompasses other embodiments in which any combination of the above-mentioned features or their equivalents is made without departing from the inventive concept as defined above. For example, the above features and (but not limited to) technical features with similar functions disclosed in the embodiments of the present disclosure are mutually replaced to form the technical solution.
Claims (10)
1. A preparation method of nanofiber silk fibroin hydrogel is characterized by comprising the following steps:
s1, preparing dry regenerated silk fibroin;
s2, respectively dissolving the regenerated silk fibroin and the chitosan in a hexafluoroisopropanol solvent, and mixing according to a certain proportion to obtain a first mixed solution;
s3, preparing first nanofibers from the first mixed solution through electrostatic spinning, drying, crosslinking and cleaning;
and S4, preparing the nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin.
2. The method for preparing nanofiber silk fibroin hydrogel according to claim 1, wherein the step S1 of preparing dried regenerated silk fibroin comprises:
degumming and drying certain mass of silkworm cocoon silk to generate silk fibroin fiber;
dissolving the silk fibroin fibers in a lithium bromide solution to obtain a silk fibroin aqueous solution;
dialyzing the silk fibroin aqueous solution to generate a regenerated silk fibroin solution;
and carrying out vacuum drying on the regenerated silk fibroin solution to generate regenerated silk fibroin.
3. The method for preparing nanofiber silk fibroin hydrogel according to claim 2, wherein the vacuum drying time is 40-60h.
4. The method for preparing nanofiber silk fibroin hydrogel according to claim 1, wherein the mass ratio of the regenerated silk fibroin and chitosan in the first mixed solution in the step S2 is 1:0.1-1.
5. The method for preparing nanofiber silk fibroin hydrogel according to claim 1, wherein the crosslinking is ethanol crosslinking.
6. The method for preparing nanofiber silk fibroin hydrogel according to claim 1, wherein the step S4 of preparing nanofiber silk fibroin hydrogel based on the first nanofibers and the regenerated silk fibroin comprises:
preparing a regenerated silk fibroin solution based on the regenerated silk fibroin;
adding the first nano-fibers into the regenerated silk fibroin solution, uniformly mixing, adding a cross-linking agent, and carrying out chemical cross-linking to obtain a suspension;
and freezing and shaping the suspension to generate the nanofiber silk fibroin hydrogel.
7. The method of claim 6, wherein the cross-linking agent comprises one or more of 1,4-butanediol diglycidyl ether, 1,3-diglycidyl ether glycerol, bisphenol A diglycidyl ether derivatives, resorcinol diglycidyl ether, 3,4-hydroxyphenyl methane triglycidyl ether, and neopentyl glycol diglycidyl ether.
8. The method for preparing the nanofiber silk fibroin hydrogel of claim 6, wherein the cross-linking agent is 1,4-butanediol diglycidyl ether.
9. A nanofiber silk fibroin hydrogel, which is prepared by the preparation method of any one of claims 1-8.
10. The nanofiber silk fibroin hydrogel of claim 9, wherein the nanofiber silk fibroin hydrogel comprises a plurality of fibers therein, each of the fibers has an average diameter of 130-150mm, and the nanofiber silk fibroin hydrogel has a porosity of greater than 90%.
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