CN114146229A - Preparation method of nanofiber scaffold and method for constructing tissue engineering material by adopting nanofiber scaffold and melanocytes - Google Patents
Preparation method of nanofiber scaffold and method for constructing tissue engineering material by adopting nanofiber scaffold and melanocytes Download PDFInfo
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- CN114146229A CN114146229A CN202010936045.5A CN202010936045A CN114146229A CN 114146229 A CN114146229 A CN 114146229A CN 202010936045 A CN202010936045 A CN 202010936045A CN 114146229 A CN114146229 A CN 114146229A
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Abstract
The invention discloses a nanofiber scaffold, a method for constructing a tissue engineering material by the nanofiber scaffold and melanocytes and application of the tissue engineering material, wherein the preparation method of the nanofiber scaffold comprises the following steps: s1, dissolving chitosan and alkaline fibroblast growth factor in a solvent to obtain a spinning solution; s2, performing electrostatic spinning by using the spinning solution to obtain a nanofiber membrane; and S3, crosslinking the nanofiber membrane by using a crosslinking agent to obtain the nanofiber scaffold. The tissue engineering material constructed by the nanofiber scaffold and the melanocytes can be used for quickly, simply, conveniently, cheaply and efficiently producing the melanocytes in batches and on a large scale.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a method for constructing a tissue engineering material by using a nanofiber scaffold, the nanofiber scaffold and melanocytes and application of the constructed tissue engineering material in treating vitiligo.
Background
Vitiligo is a common and frequently occurring depigmentation skin disease. The clinical manifestations are mainly localized or generalized white spot, clear border, no subjective symptoms, and the global incidence of leucoderma is 0.1-4%. At present, the pathogenesis of vitiligo is not clear, and the functional loss of melanocytes is generally considered to be caused by some reasons, and the main pathological theories comprise a genetic theory, an autoimmune theory, an oxidative stress theory, a neuropsychiatric theory, a viral theory, a melanocyte self-destruction theory and the like.
Vitiligo rarely directly harms the physiological health of patients or poses fatal threats, but a great deal of research finds that the vitiligo can be accompanied by other immune diseases, including thyroid diseases, diabetes, pernicious anemia, psoriasis and the like, and is simultaneously related to melanoma and hearing loss.
Current methods of treatment include pharmacotherapy, photochemotherapy, phototherapy, and surgical therapy. Among them, melanocyte transplantation is the effective method for treating stable-phase vitiligo at present, and represents the development direction of vitiligo transplantation therapy. Among them, cell suspension transplantation is the most common, but a number of clinical results suggest that this method has two major problems: 1) the cell suspension has fluidity, and the cells are difficult to attach at certain moving parts; 2) after the cell suspension is transplanted, the growth environment is changed, and thus the activity of the cells is damaged. Therefore, by introducing a tissue engineering technology, a melanocyte carrier is developed, and melanocytes are cultured on the carrier and are transplanted to a focal area integrally, so that the existing problems can be effectively improved, and the possibility of improving the transplanting curative effect of the melanocytes is provided.
Application publication No. CN 103861147a discloses "a method for culturing melanocytes based on a nanofiber scaffold" and further discloses that a nanofiber scaffold in which melanocytes are cultured is prepared by electrospinning one or two biocompatible materials selected from chitosan, gelatin, collagen, fibrin, hyaluronic acid, chondroitin sulfate, polyethylene glycol, polyvinyl alcohol, dissolved in a solvent.
Compared with the traditional compact membrane, the nanofiber membrane in the method has high specific surface area and porosity, can better simulate the extracellular matrix of a human body and promote the renewal of cell culture fluid and melanocyte metabolites, but still has no great improvement on the aging problem of melanocytes.
Disclosure of Invention
In order to solve the problem that melanocytes are seriously aged when the melanocytes are cultured based on a nanofiber scaffold at present, the invention aims to provide the nanofiber scaffold, the nanofiber scaffold and the melanocytes are adopted to construct a tissue engineering material, the nanofiber scaffold has high specific surface area and porosity due to the adoption of an electrostatic spinning process, is similar to an extracellular matrix structure, can better support the attachment, growth and proliferation of the melanocytes, meanwhile, the addition of basic fibroblast growth factor (bFGF) in the nanofiber scaffold promotes the dendritic growth of the melanocytes and inhibits the aging problem of the melanocytes, and the tissue engineering material constructed by the nanofiber scaffold and the melanocytes can rapidly, simply, conveniently, cheaply, efficiently, in batches and on a large scale.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, a method for preparing a nanofiber scaffold is provided, which comprises the following steps:
s1, dissolving chitosan and alkaline fibroblast growth factor in a solvent to obtain a spinning solution;
s2, performing electrostatic spinning by using the spinning solution to obtain a nanofiber membrane;
and S3, crosslinking the nanofiber membrane by using a crosslinking agent to obtain the nanofiber scaffold.
Wherein the mass concentration of the chitosan in the S1 is 2-8%.
Wherein the mass ratio of the basic fibroblast growth factor to the chitosan in the S1 is 1:50-1: 100.
Wherein the solvent in the S1 is acetic acid with the mass concentration of 80-90%.
The specification of the electrostatic spinning injector in the S2 is 5mL, the inner diameter of a needle is 0.4-0.7mm, the receiving screen adopts aluminum foil grounding for receiving, the distance between the needle and the receiving screen is 10-20cm, the spinning voltage is 10-30KV, and the jet flow rate in the electrostatic spinning process is 0.2-1 mL/h.
Wherein the cross-linking agent in the S3 is glutaraldehyde, the mass concentration of the glutaraldehyde is 10-30%, the volume of the glutaraldehyde is 8-20mL, and the cross-linking time is 3-25 h.
In a second aspect of the present invention, there is provided a nanofiber scaffold prepared by the above-mentioned preparation method.
In a third aspect of the present invention, there is provided a method for constructing a tissue engineering material using a nanofiber scaffold and melanocytes, comprising the steps of:
s1, alkali treatment is carried out on the nanofiber scaffold;
s2, sterilizing the nano-fiber scaffold after alkali treatment;
s3, placing the sterilized nanofiber scaffold into a cell culture plate, adding cell culture solution, and placing the cell culture plate into a cell culture box for incubation;
s4, discarding the cell culture medium for hatching, and making melanocyte 2 × 103Per cm2-10×103Per cm2Inoculating the cell density of the cells on the incubated nano-fiber scaffold, simultaneously adding cell culture solution, and placing the cell culture solution into the cell for cultureCulturing in a box, replacing cell culture solution every two days, and culturing for 1-10 days to obtain the tissue engineering material.
In a fourth aspect of the invention, there is provided a tissue engineering material obtained by the above method.
In a fifth aspect of the present invention, the application of the above tissue engineering material in the preparation of a vitiligo treatment agent is provided.
Compared with the prior art, the invention has the following beneficial effects: the tissue engineering material constructed by the nanofiber scaffold and the melanocytes can be used for quickly, simply, conveniently, cheaply and efficiently producing the melanocytes in batches and on a large scale.
Drawings
The invention is described in further detail below with reference to the following figures and detailed description:
FIG. 1 is a comparison of MTS absorbance values of melanocytes cultured for 1, 3 and 5 days on control and nanofiber scaffolds, respectively;
FIG. 2 is a comparison of the melanin content values of individual cells after melanocytes were cultured on control and nanofiber scaffolds for 1, 3, and 5 days, respectively;
FIG. 3 shows melanocyte proliferation under different conditions.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Example 1 preparation of a nanofiber scaffold
1. Preparation of bFGF solution: 20mL of bFGF solution with the mass concentration of 0.5% is prepared, the solvent is acetic acid with the mass concentration of 80% -90%, and the mixture is uniformly stirred for later use;
2. preparing a spinning solution: adding 1.2g of chitosan into the solution to ensure that the mass fraction of the chitosan is 6 percent, wherein the mass ratio of the basic fibroblast growth factor to the chitosan is 1: 12, stirring for 3 hours until the chitosan is completely swelled; then stirring for 12 hours at room temperature until the polymer is completely dissolved to obtain a transparent polymer solution;
3. extracting the spinning solution by using a 5mL injector (the inner diameter of a needle head is 0.4-0.7mm), fixing the spinning solution on an electrostatic spinning device, fixing the electrostatic voltage to 12kv, the receiving distance to 12cm and the jet flow speed to 0.6mL/h, and carrying out electrostatic spinning to obtain a chitosan/bFGF nanofiber membrane;
4. placing a culture dish containing 10mL of 25% glutaraldehyde solution at the bottom of the dryer, placing the chitosan/bFGF nanofiber membrane into the dryer, crosslinking at 25 ℃, taking out after crosslinking for 10h, placing the membrane in an oven at 60 ℃ for heating for 5h, further crosslinking, and removing residual crosslinking agent to obtain the nanofiber scaffold.
Example 2 preparation of a nanofiber scaffold
1. Preparation of bFGF solution: 20mL of bFGF solution with the mass concentration of 0.5% is prepared, the solvent is acetic acid with the mass concentration of 80% -90%, and the mixture is uniformly stirred for later use;
2. preparing a spinning solution: adding 1.5g of chitosan into the solution to ensure that the mass fraction of the chitosan is 7.5%, wherein the mass ratio of the basic fibroblast growth factor to the chitosan is 1: stirring for 3 hours until the chitosan is completely swelled; then stirring for 12 hours at room temperature until the polymer is completely dissolved to obtain a transparent polymer solution;
3. extracting the spinning solution by using a 5mL injector (the inner diameter of a needle head is 0.4-0.7mm), fixing the spinning solution on an electrostatic spinning device, fixing the electrostatic voltage to 12kv, the receiving distance to 12cm and the jet flow speed to 0.6mL/h, and carrying out electrostatic spinning to obtain a chitosan/bFGF nanofiber membrane;
4. placing a culture dish containing 10mL of 25% glutaraldehyde solution at the bottom of the dryer, placing the chitosan/bFGF nanofiber membrane into the dryer, crosslinking at 25 ℃, taking out after crosslinking for 10h, placing the membrane in an oven at 60 ℃ for heating for 5h, further crosslinking, and removing residual crosslinking agent to obtain the nanofiber scaffold.
Example 3 preparation of nanofiber scaffolds
1. Preparation of bFGF solution: 20mL of bFGF solution with the mass concentration of 0.5% is prepared, the solvent is acetic acid with the mass concentration of 80% -90%, and the mixture is uniformly stirred for later use;
2. preparing a spinning solution: adding 1.0g of chitosan into the solution to ensure that the mass fraction of the chitosan is 5%, wherein the mass ratio of the basic fibroblast growth factor to the chitosan is 1:10, stirring for 3 hours until the chitosan is completely swelled; then stirring for 12 hours at room temperature until the polymer is completely dissolved to obtain a transparent polymer solution;
3. extracting the spinning solution by using a 5mL injector (the inner diameter of a needle head is 0.4-0.7mm), fixing the spinning solution on an electrostatic spinning device, fixing the electrostatic voltage to 12kv, the receiving distance to 12cm and the jet flow speed to 0.6mL/h, and carrying out electrostatic spinning to obtain a chitosan/bFGF nanofiber membrane;
4. placing a culture dish containing 10mL of 25% glutaraldehyde solution at the bottom of the dryer, placing the chitosan/bFGF nanofiber membrane into the dryer, crosslinking at 25 ℃, taking out after crosslinking for 10h, placing the membrane in an oven at 60 ℃ for heating for 5h, further crosslinking, and removing residual crosslinking agent to obtain the nanofiber scaffold.
EXAMPLE 4 preparation of tissue engineering materials
1. And (3) processing the nanofiber scaffold: soaking the nanofiber bracket in 1mol/L sodium carbonate solution for 30 minutes, and then soaking and washing the nanofiber bracket in PBS for multiple times to remove residual sodium carbonate; soaking in 75% alcohol for 12h, washing with PBS, and sterilizing by ultraviolet irradiation for 12 h; finally, the nanofiber scaffold was placed in a 12-well plate, 1mL of cell culture solution was injected into each well, and the plate was placed at 37 ℃ with 5 vol% CO2Incubating in a cell incubator for 2 h; wherein the cell culture solution is commercially available culture solution, and comprises fetal calf serum, cholera toxin, isobutyrylmethylxanthine, glutamine and gentamicin.
2. Taking healthy human foreskin tissues, digesting, separating and purifying to extract human melanocytes; melanocytes were added to cell culture medium at 37 ℃ with 5 vol% CO2Culturing in a cell culture box, replacing culture solution every 2 days, and subculturing when cultured melanocytes are observed to be approximately 80% fused; discarding the incubation cell culture medium, and mixing 8 × 103Per cm2The melanocytes are inoculated on the nanofiber bracket, and the cell culture plate is shaken lightly to ensure the uniform distribution of the cells; the cell culture plate was placed at 37 ℃ in 5 vol% CO2Culturing in a cell culture box, and replacing the cell culture solution every 2 days to obtain the tissue engineering material.
Example 5 detection of melanocyte number by MTS method
MTS method detection principle: dehydrogenase in mitochondria of living cells can reduce MTS (Chinese name: 2-p-sulfophenyl-3- (4, 5-dimethylthiazole) -5- (3-carboxymethoxyphenyl) -dihydrotetrazolium salt) into water-soluble formazan salt, formazan salt can be directly dissolved in a culture medium, formazan salt is in direct proportion to the number of cells in a certain cell number range, and the absorbance of formazan salt is measured by an enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay) at 490nm wavelength, so that the number of living cells can be indirectly reflected.
When the measurement is carried out by the MTS method, the original cell culture solution and floating dead cells are removed first.
The determination method comprises the following steps: 1mL of a cell culture solution containing 10% (by mass) MTS was added to each well of a cell culture plate, and the plate was placed in a cell culture chamber at 37 ℃ and 5 vol% CO2Incubation was carried out for 3 hours under the conditions, 100. mu.L of the melanocyte-containing solution was placed in a 96-well cell culture plate, and the absorbance of the solution at a wavelength of 490nm was measured with a microplate reader.
Melanocytes were cultured in the same manner using a cell culture plate without a nanofiber scaffold and a cell culture solution as a control, and proliferation of melanocytes in the control group and the nanofiber scaffold was compared and characterized by the MTS method, and the results are shown in fig. 1. As shown in fig. 1, when melanocytes were cultured on control group and nanofiber scaffolds for 3d to 5d, MTS value increased greatly, while when melanocytes were cultured for 1d to 3d, only a small increase was observed, indicating that melanocytes proliferated most rapidly and were most active when cultured for 3d, and melanocytes were suitable for transplantation after 3d culture. In addition, the number of melanocytes on the nanofiber scaffold was greater than that of the control group in each time period, indicating that the nanofiber scaffold was more favorable for melanocyte proliferation.
EXAMPLE 6 determination of the melanin content in Individual cells
The determination method comprises the following steps: removing the original cell culture solution, adding 0.25% pancreatin to separate melanocyte from nanofiber scaffold, and adding cell culture solution to stop digestion; and transferring the solution containing the melanocytes into a centrifuge tube, fully and uniformly beating, counting the cell density of a small amount of the solution by using a platelet counter, and calculating by combining the volume of the solution to obtain the number of the melanocytes. Centrifuging the obtained solution at 1000rpm for 5min, discarding supernatant, adding 1mL of 1mol/L NaOH solution, oscillating with vortex instrument to fully lyse cells, measuring absorbance of the solution at 475nm with enzyme-labeling instrument, wherein the absorbance has the following relationship with melanin content of melanocyte:
m(ng)＝Aλ/N×10-3×156.25
wherein m is the melanin content of a single melanocyte, AλFor absorbance values, N is the number of melanocytes in 1mL of suspension.
Melanocytes were cultured in the same manner using a cell culture plate without a nanofiber scaffold and a cell culture solution as a control, and the melanin content in individual cells was compared after the melanocytes were cultured for the same time on the control and the nanofiber scaffold.
The melanin content of individual melanocytes after 1, 3 and 5 days of culture of melanocytes in nanofiber scaffolds and control groups, respectively, is shown in fig. 2. As can be seen from fig. 2, the melanocytes cultured on the nanofiber scaffolds always have a lower melanin content than the control group, indicating that the melanocytes on the nanofiber scaffolds are aged less than the melanocytes on the control group.
Example 7
Melanocytes were cultured in four ways, i.e., a nanofiber scaffold was constructed without adding a nanofiber scaffold as a control, and a nanofiber scaffold prepared in example 3 was prepared by adding bFGF to a cell culture solution, and the proliferation of melanocytes was compared under different conditions, and the results were represented by the MTS method, as shown in fig. 3. As can be seen from fig. 3, the melanocyte culture using the nanofiber scaffolds prepared in example 3 was significantly higher in the number of melanocytes than the other three ways.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a nanofiber scaffold is characterized by comprising the following steps:
s1, dissolving chitosan and alkaline fibroblast growth factor in a solvent to obtain a spinning solution;
s2, performing electrostatic spinning by using the spinning solution to obtain a nanofiber membrane;
and S3, crosslinking the nanofiber membrane by using a crosslinking agent to obtain the nanofiber scaffold.
2. The method for preparing a nanofiber scaffold according to claim 1, wherein the mass concentration of chitosan in S1 is 2-8%.
3. The method for preparing a nanofiber scaffold according to claim 1, wherein the mass ratio of basic fibroblast growth factor to chitosan in S1 is 1:8-1: 20.
4. The method for preparing the nanofiber scaffold as claimed in claim 1, wherein the solvent in S1 is acetic acid with a mass concentration of 80-90%.
5. The method for preparing a nanofiber scaffold as claimed in claim 1, wherein the size of the syringe for electrospinning in S2 is 5mL, the inner diameter of the needle is 0.4-0.7mm, the receiving screen is grounded by using aluminum foil, the distance between the needle and the receiving screen is 10-20cm, the spinning voltage is 10-30KV, and the jet flow rate in the electrospinning process is 0.2-1 mL/h.
6. The preparation method of the nanofiber scaffold as claimed in claim 1, wherein the cross-linking agent in S3 is selected from glutaraldehyde, the mass concentration of glutaraldehyde is 10-30%, the volume is 8-20mL, and the cross-linking time is 3-25 h.
7. A nanofiber scaffold prepared by the preparation method as set forth in any one of claims 1 to 6.
8. The method for constructing tissue engineering material using the nanofiber scaffold of claim 7 and melanocytes, comprising the steps of:
s1, alkali treatment is carried out on the nanofiber scaffold;
s2, sterilizing the nano-fiber scaffold after alkali treatment;
s3, placing the sterilized nanofiber scaffold into a cell culture plate, adding cell culture solution, and placing the cell culture plate into a cell culture box for incubation;
s4, discarding the cell culture medium for hatching, and making melanocyte 2 × 103Per cm2-10×103Per cm2Inoculating the cells to the incubated nano fiber scaffold, adding cell culture solution, putting the nano fiber scaffold into a cell culture box for culture, replacing the cell culture solution every two days, and culturing for 1-10 days to obtain the tissue engineering material.
9. Tissue engineering material obtainable by the method of claim 8.
10. Use of the tissue engineering material according to claim 9 for the preparation of a medicament for the treatment of vitiligo.
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