CN116271182A - Electrospinning recombinant humanized type III collagen wound dressing and preparation method thereof - Google Patents

Abstract

The invention provides an electrostatic spinning recombinant humanized III type collagen wound dressing, which is a fibrous membrane with a porous structure, wherein the fibrous membrane is formed by stacking and cross-linking recombinant humanized III type collagen fibers with uniform diameters, and the diameter of the fibers in the fibrous membrane is not more than 600nm and the diameter distribution range is narrow. The invention also provides a preparation method of the wound dressing, which comprises the following steps: (1) Dissolving recombinant humanized III type collagen in an acetic acid solution with the concentration of 85-95 wt.% to obtain a spinning solution; (2) carrying out electrostatic spinning on the spinning solution; (3) Immersing the recombinant humanized III type collagen nanofiber membrane obtained by spinning into a cross-linking agent solution prepared by taking 95-100% ethanol as a solvent for cross-linking, cleaning, gradient dewatering and critical point drying to obtain the collagen nanofiber membrane. The invention can solve the problem that the existing preparation method of the animal-derived collagen fiber membrane is not suitable for recombinant humanized III type collagen, and can reduce the fiber diameter and the diameter distribution range.

Description

Electrospinning recombinant humanized type III collagen wound dressing and preparation method thereof

technical field

The invention belongs to the field of biological materials, and relates to an electrospinning recombinant humanized type III collagen wound dressing and a preparation method thereof.

Background technique

The skin is the outermost organ of the human body in contact with the surrounding environment, and is the first important barrier against external dangers, so it is extremely vulnerable to trauma. If the wound is not treated and waits for it to recover on its own, it is easy to cause adverse consequences such as wound inflammation or scar hyperplasia. The production and deposition of collagen play a very important role in the wound healing process, therefore, increasing the production and deposition of collagen in the wound is an effective means to promote wound healing.

Electrospinning is a simple and low-energy technique for preparing continuous nanofibers, and the electrospun fibrous scaffolds can fully mimic the structure of natural extracellular matrix (ECM). People first achieved the preparation of electrospun collagen nanofibers by adding polymer material polyethylene oxide (PEO) to the spinning system of collagen aqueous solution. At present, collagen nanofibers with a diameter distribution between 100 and 1000 nm can be prepared by electrospinning technology, but it is difficult to control the diameter distribution range at a low level. Fibers with different diameters have different mechanical strengths, different biological effects, and different degradation rates. A wide range of diameter distribution means that the diameter of the fiber obtained by spinning is unstable, and the fiber membrane formed on this basis will have problems of different mechanical strength, degradation rate and biological effects. If there is a large difference in the degradation rate of different parts of the fiber membrane, it will lead to a weak region of the fiber membrane that is prone to rapid degradation, which will accelerate the degradation rate of the entire fiber membrane. At the same time, in the prior art, when preparing pure collagen nanofibers, the organic solvent hexafluoroisopropanol (HFIP) is usually used to dissolve collagen. On the one hand, hexafluoroisopropanol is environmentally unfriendly and expensive, and the preparation process will cause environmental pollution. Pollution, due to high cost, it is difficult to achieve large-scale production. On the other hand, hexafluoroisopropanol will cause part of the collagen in the fiber to lose its triple helical structure, and may remain in the fiber to cause toxicity, which greatly limits the production of pure collagen. Industrial applications of protein nanofibers.

Recombinant humanized type III collagen (rhCol III) is a kind of active collagen obtained through a series of separation and purification processes through genetic engineering and fermentation technology, which is highly consistent with the amino acid sequence of human natural collagen. Recombinant humanized type III collagen has the advantages of good biological compatibility and non-immunogenicity, and has been used in the research of facial filling, artificial bone and biological valves. However, there is no report on the preparation of recombinant humanized type III collagen fiber membrane in the prior art.

Due to the good water solubility of recombinant humanized type III collagen, when the collagen fiber membrane of recombinant humanized type III collagen is used as a wound dressing, it must be cross-linked, otherwise its stability and mechanical properties cannot meet the requirements. Application requirements. However, the water solubility of animal-derived collagen is poor, and the animal-derived collagen fiber membrane can meet the application requirements as a wound dressing with or without cross-linking, and the animal-derived collagen fiber can be cross-linked in an aqueous solution system. However, this method cannot be used for the cross-linking of collagen fibers of recombinant humanized type III collagen, because it is soluble in water and cannot be cross-linked in an aqueous solution. At the same time, the traditional vacuum drying or natural drying methods suitable for animal-derived collagen fiber membranes are not suitable for drying collagen fiber membranes of recombinant humanized type III collagen, because these drying methods will cause severe swelling and deformation of the fibers And the mutual adhesion between fibers affects the morphology of the collagen fibers of the recombinant humanized type III collagen and the original pore structure of the fibrous membrane, and at the same time causes the problem that the collagen fiber membrane of the recombinant humanized type III collagen is easily broken. It can be seen from the above that although there is a relatively mature process in the prior art for the preparation of ordinary animal-derived collagen fiber membranes, there are still some challenges for the preparation of recombinant humanized type III collagen fiber membranes. challenge. If a preparation method suitable for recombinant humanized type III collagen fiber membranes can be developed, and the problem of wide fiber diameter distribution range that exists in the preparation of collagen fiber membranes in the prior art can be solved, it will be beneficial to promote recombinant humanized type III collagen The application of protein fiber membrane in the field of biomedicine will have a positive effect.

Contents of the invention

The method for preparing animal-derived collagen fiber membranes in the prior art is not suitable for the preparation of recombinant humanized type III collagen fiber membranes, and the fiber diameter distribution range in the collagen fiber membranes prepared by the prior art is difficult to control at a low level Problem, the present invention provides an electrospinning recombinant humanized type III collagen wound dressing and its preparation method to solve the problem that the existing animal source collagen fiber membrane preparation method is difficult to apply to the recombinant humanized type III collagen fiber membrane manufacturing problems, and reduce the diameter and diameter distribution range of the fibers in the wound dressing.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention adopts is as follows:

An electrospun recombinant humanized type III collagen wound dressing, the wound dressing is a fibrous membrane with a porous structure formed by stacking and cross-linking recombinant humanized type III collagen fibers with uniform diameters, the recombinant The diameter of the humanized type III collagen fiber is not more than 550nm, and the fiber diameter of the recombined humanized type III collagen fiber crosslinked is not more than 600nm.

Further, in the above-mentioned technical scheme of electrospinning recombinant humanized type III collagen wound dressing, the average diameter of the recombinant humanized type III collagen fibers is 100-450 nm, and the recombinant humanized type III collagen fibers The standard deviation of the diameter distribution does not exceed 55nm. The average diameter of the cross-linked fibers of the recombinant humanized type III collagen fibers is between 150-500 nm, and the standard deviation of the diameter distribution of the cross-linked fibers of the recombinant humanized type III collagen fibers is not more than 55 nm .

The present invention also provides a preparation method for the above-mentioned electrospinning recombinant humanized type III collagen wound dressing, comprising the following steps:

(1) The recombinant humanized type III collagen was dissolved in an acetic acid solution with a concentration of 85wt.% to 95wt.% to obtain a spinning solution, and the concentration of the recombinant humanized type III collagen in the spinning solution was 45wt. %～50wt.%;

(2) injecting the spinning solution into an electrospinning device for electrospinning to obtain a recombinant humanized type III collagen nanofiber membrane with uniform fiber diameter;

(3) Immerse the recombinant humanized type III collagen nanofiber membrane in the cross-linking agent solution for cross-linking, wash and remove the unreacted cross-linking agent after cross-linking, then gradient dehydration, and then dry at the critical point to obtain the recombinant human Type III collagen wound dressing; the cross-linking agent solution is prepared by using ethanol with a volume percentage of 95%-100% as a solvent.

In the technical scheme of the above preparation method, during electrospinning in step (2), it is preferable to control the positive voltage to be 10-27kV, the negative voltage to be -1.5--10kV, the flow rate to be 0.1-1mL/h, and the receiving distance to be 5-20cm .

In the technical scheme of the above-mentioned preparation method, the size of the needle used in step (2) during electrospinning is comprehensively determined according to the size of the fiber diameter and the above-mentioned electrospinning parameters in actual application. Usually, the size of the needle used in electrospinning is It is 15 ~ 30G.

In the technical solution of the above preparation method, the concentration of the cross-linking agent in the cross-linking agent solution is 0.5wt.%-5wt.%.

In the technical scheme of the method prepared above, the crosslinking agent is glutaraldehyde, genipin, diepoxy compound, transglutaminase or 1-(3-dimethylaminopropyl)-3-ethyl Composition of carbodiimide and N-hydroxysuccinimide.

Further, when the cross-linking agent is glutaraldehyde, genipin, diepoxy compound or transglutaminase, the cross-linking agent is dissolved in the solvent to obtain the cross-linking agent solution; when the cross-linking agent is When combining 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide with N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethyl Dissolve carbodiimide and N-hydroxysuccinimide in a solvent and adjust the pH value to 5.4 to 5.8 to obtain a crosslinking agent solution. Further, 1-(3-dimethylaminopropyl)-3- The molar ratio of ethyl carbodiimide to N-hydroxysuccinimide is (0.25~4):1, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide in the crosslinking agent solution The concentration of amine is 0.5wt.%～5wt.%.

In the above-mentioned technical scheme, the gradient dehydration described in step (3) refers to using different concentrations of ethanol to cross-link and wash the recombinant humanized type III collagen nanofiber membrane in sequence from low to high in ethanol volume percentage Soak and dehydrate. Further, it is preferable to dehydrate the cross-linked and washed recombinant humanized type III collagen nanofiber membrane with gradient dehydration with 30%-100% ethanol by volume.

In the above technical solution, the ethanol with a volume percentage of 95% to 100% and ethanol with a volume percentage of 30% to 100% means that the volume fraction of ethanol in the ethanol-water solution is 95% to 100% or 30% to 100% %.

In the above technical solution, in step (3), at least one of PBS buffer solution or Na ₂ HPO ₄ solution, and water are used to wash to remove unreacted cross-linking agent.

The present invention proves through experiments: (1) the recombinant humanized type III collagen wound dressing provided by the present invention has good flexibility, can be curled and folded without damage, has good mechanical properties, and can satisfy the application as a wound dressing (2) The recombinant humanized type III collagen wound dressing provided by the present invention has good air permeability and water permeability, which is conducive to wound breathing, avoids cell hypoxia at the wound, promotes wound healing, and is beneficial to water Steam permeation will not cause excessive accumulation of moisture in the wound due to impermeability, which is conducive to the evaporation of sweat on the skin surface; (3) The recombinant humanized type III collagen wound dressing provided by the present invention has good hydrophilicity and water absorption Good hydrophilicity is good for cell adhesion, spreading and proliferation, good water absorption is good for the absorption of wound exudate, at the same time, dry wound dressings will physically adhere to wet wounds, Therefore, the wound dressing is not easy to fall from the wound; (4) the recombinant humanized type III collagen wound dressing provided by the present invention, co-cultured with cells for 7 days, has good structural stability and can be provided within a long period of time. A structure similar to the extracellular matrix for cell spreading and proliferation; (5) the recombinant humanized type III collagen wound dressing provided by the present invention has good biocompatibility and cell adhesion, and cells can spread and grow therein .

The present invention also proves through experiments that the preparation of recombinant humanized type III collagen nanofiber membrane by electrospinning according to the method of the present invention can solve the problem of using hexafluoroisopropanol as a solvent to prepare spinning solution to prepare recombinant humanized collagen nanofiber membrane. Type III collagen nanofiber membranes are prone to clogging, difficult to achieve continuous spinning, and have a wide range of fiber diameter distribution. Cross-linking and drying the recombinant humanized type III collagen nanofiber membrane obtained by electrospinning by the method of the present invention can highly retain the morphology of the fiber and the morphology of the fiber membrane, and achieve a high degree of simulating the natural extracellular matrix ( The purpose of the ECM) structure can solve the problems that the existing method of drying will cause serious swelling and deformation of the fiber and the destruction of the fiber membrane morphology.

Compared with the prior art, the technical solution provided by the present invention has produced the following beneficial technical effects:

1. The present invention provides an electrospun recombinant humanized type III collagen wound dressing, which is composed of recombinant humanized type III collagen fibers with uniform diameters stacked and cross-linked to form fibers with a porous structure Membrane, the diameter of the recombinant humanized type III collagen fibers is no more than 550nm and the diameter distribution range is narrow, and the fiber diameter of the recombinant humanized type III collagen fibers is only slightly increased after crosslinking (crosslinking The fiber diameter after crosslinking is not more than 600nm) and the diameter distribution range of the crosslinked fiber is also very narrow. The invention solves the problem of the wide diameter distribution range of the animal-derived collagen fiber membrane prepared in the prior art, is beneficial to improving the mechanical strength, degradation rate and uniformity of biological effects of different parts of the fiber membrane, and improves the use performance of the fiber membrane .

2. The present invention proves through experiments that the recombinant humanized type III collagen wound dressing provided by the present invention has good flexibility, can be curled and folded without damage, has good mechanical properties, and has good air permeability and water permeability It is good for wound breathing, avoids hypoxia of cells in the wound, and promotes wound healing. It is also conducive to the penetration of water vapor, which can avoid excessive accumulation of water in the wound due to imperviousness. It also has good hydrophilicity and water absorption. Facilitate cell adhesion, spreading and proliferation, and facilitate the absorption of wound exudate. The above physical properties are conducive to its application as a wound dressing in the field of biomedical materials.

3. The present invention also confirms through cell co-cultivation experiments that the recombinant humanized type III collagen wound dressing provided by the present invention has good structural stability and can provide a structure similar to extracellular matrix for cells in a relatively long period of time. It also has good biocompatibility and cell adhesion, and cells can spread and grow in it. These properties are beneficial to wound repair.

4. The present invention also provides a preparation method for the above-mentioned recombinant humanized type III collagen wound dressing, which solves the problem of wide fiber diameter distribution range and water-soluble bands in the preparation of cross-linked recombinant humanized type III collagen fiber membranes. Due to the problems of cross-linking and the severe deformation of fibers caused by the existing drying methods, the preparation of cross-linked collagen fiber membranes of pure recombinant humanized type III collagen has been realized for the first time. The preparation method is simple and the preparation process is pollution-free. Facilitate the realization of large-scale production.

Description of drawings

Fig. 1 is a scanning electron microscope image and a fiber diameter frequency histogram (N=100) of the rhCol III nanofiber membrane prepared in Examples 1-3.

2 is a digital photograph of the electrospinning process of Comparative Example 1 and Example 4.

Fig. 3 is a scanning electron micrograph and a fiber diameter frequency histogram (N=120) of rhCol III nanofiber membranes prepared in Comparative Example 1 and Example 4.

Fig. 4 is a scanning electron micrograph and a fiber diameter frequency histogram (N=120) of the rhCol III wound dressing prepared in Example 5 and Example 6.

Fig. 5 is a photograph of the rhCol III wound dressings prepared in Example 5 and Example 6 in curled and folded states.

6 is a scanning electron micrograph of the rhCol III wound dressings prepared in Comparative Example 2 and Comparative Example 3.

7 is a photo of the rhCol III wound dressing prepared in Comparative Example 2 after vacuum drying.

Fig. 8 is the stress-strain curve and Young's modulus test result of the rhCol III wound dressing prepared in Examples 5 and 6.

A and B two figures of Fig. 9 are the water permeability and gas permeability test results of Tegaderm commercial dressing, GA prepared by embodiment 5, EN prepared by embodiment; C and D two figures of Fig. 9 are GA, D prepared by embodiment 5 The test results of the water absorption capacity and water contact angle of the EN prepared in the examples.

Fig. 10 is a scanning electron microscope image of GA and EN co-cultured with cells for 1 day, 4 days and 7 days.

Panel A of Figure 11 is the live/dead staining results of GA and EN co-cultured with cells for 4 days, and Figure 11 is the results of phalloidin/DAPI staining of GA and EN co-cultured with cells for 1 day and 4 days.

Figure 12 is the scanning electron micrographs of GA and EN co-cultured with cells for 1 day, 3 days and 7 days.

13 is a scanning electron micrograph of the rhCol III nanofiber membrane prepared in Example 11.

14 is a scanning electron micrograph of the rhCol III nanofiber membrane prepared in Comparative Example 4.

Detailed ways

The electrospun recombinant humanized type III collagen wound dressing provided by the present invention and its preparation method will be further described through examples below. It is necessary to point out that the following examples are only used to further illustrate the present invention, and cannot be interpreted as limiting the protection scope of the present invention. Those skilled in the art make some non-essential improvements and adjustments to the present invention according to the above-mentioned content of the invention and carry out specific implementation. Belong to the protection scope of the present invention.

Example 1

In this example, the preparation of recombinant humanized type III collagen (rhCol III) nanofibrous membrane is as follows:

(1) adding rhCol III to an acetic acid (HAC) solution with a concentration of 90wt.%, and stirring at room temperature until the rhColIII is completely dissolved to obtain a uniform and transparent spinning solution. The concentration of rhCol III in the spinning solution is 42.5wt.%.

(2) Inject the spinning solution into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 10kV, negative high voltage -1.5kV, flow rate 0.5mL/h, receiving distance 10cm, needle 27G, to obtain uniform diameter rhCol III nanofibrous membrane.

The scanning electron microscope image of the rhCol III nanofiber membrane prepared in this example is shown in Figure A1 of Figure 1, from which it can be seen that the diameter of the nanofibers in the rhCol III nanofiber membrane is very uniform. The fiber diameter of the rhCol III nanofibrous membrane prepared in this embodiment is counted and the fiber diameter frequency histogram (N=100) is drawn. The diameters of the fibers in the nanofibrous membrane are mainly distributed between 100 and 150 nm and the distribution range of the fiber diameters is narrow. Calculate the average diameter of the fibers and the standard deviation (SD) of the fiber diameters, and express the fiber diameters as the form of the average diameter ± SD, The result was 126nm±31nm.

Example 2

In the present embodiment, the rhCol III nanofiber membrane is prepared, and the steps are as follows:

(1) adding rhCol III to an acetic acid solution with a concentration of 90wt.%, and stirring at room temperature until rhCol III is completely dissolved to obtain a uniform and transparent spinning solution, the concentration of rhCol III in the spinning solution is 42.5wt.%.

(2) The spinning solution is injected into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 10kV, negative high voltage -1.5kV, flow rate 0.5mL/h, receiving distance 13cm, needle 30G, to obtain uniform diameter rhCol III nanofibrous membrane.

The scanning electron microscope image of the rhCol III nanofiber membrane prepared in this example is shown in Figure 1, B1, from which it can be seen that the diameter of the nanofibers in the rhCol III nanofiber membrane is very uniform. The fiber diameter of the rhCol III nanofibrous membrane prepared in this embodiment is counted and the fiber diameter frequency histogram (N=100) is drawn, the result is shown in Figure 1 B2, as can be seen from this figure, the rhCol III The diameters of the fibers in the nanofibrous membrane are mainly distributed between 220 and 300 nm and the distribution range of the fiber diameters is narrow. Calculate the average diameter of the fibers and the standard deviation (SD) of the fiber diameters, and express the fiber diameters as the form of the average diameter ± SD, The result was 261nm±40nm.

Example 3

In the present embodiment, the rhCol III nanofiber membrane is prepared, and the steps are as follows:

(1) adding rhCol III to an acetic acid solution with a concentration of 90wt.%, and stirring at room temperature until rhCol III is completely dissolved to obtain a uniform and transparent spinning solution, the concentration of rhCol III in the spinning solution is 42.5wt.%.

(2) Inject the spinning solution into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 16kV, negative high voltage -1.5kV, flow rate 0.5mL/h, receiving distance 10cm, needle 22G, to obtain uniform diameter rhCol III nanofibrous membrane.

The scanning electron microscope image of the rhCol III nanofiber membrane prepared in this example is shown in Figure 1, C1. From this figure, it can be seen that the diameter of the nanofibers in the rhCol III nanofiber membrane is very uniform. The fiber diameter of the rhCol III nanofibrous membrane prepared in this embodiment is counted and the fiber diameter frequency histogram (N=100) is drawn, the result is shown in Figure 1 C2, as can be seen from this figure, the rhCol III The diameters of the fibers in the nanofibrous membrane are mainly distributed between 370 and 500 nm and the distribution range of the fiber diameters is narrow, the average diameter of the fibers and the standard deviation (SD) of the fiber diameters are calculated, and the fiber diameters are expressed in the form of the average diameter ± SD, The result was 426nm±51nm.

In combination with Examples 1 to 3, it can be seen that under the condition of a certain concentration of the spinning solution, the fiber diameter of the rhCol III nanofiber membrane obtained by spinning can be adjusted by adjusting the electrospinning parameters. The rhCol III nanofiber membrane with uniform fiber diameter and narrow diameter distribution range can be obtained. The fiber diameter distribution range is narrower, which is beneficial to improve the quality stability of the rhCol III nanofiber membrane.

Example 4

In the present embodiment, the rhCol III nanofiber membrane is prepared, and the steps are as follows:

(1) adding rhCol III to an acetic acid solution with a concentration of 90wt.%, and stirring at room temperature until rhCol III is completely dissolved to obtain a uniform and transparent spinning solution, the concentration of rhCol III in the spinning solution is 42.5wt.%.

(2) Inject the spinning solution into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 23kV, negative high voltage -5kV, flow rate 0.8mL/h, receiving distance 10cm, needle 27G, and rhCol with uniform diameter III Nanofibrous Membranes.

Comparative example 1

In this comparative example, using hexafluoroisopropanol as a solvent to prepare a spinning solution to prepare a rhCol III nanofiber membrane, the steps are as follows:

(1) adding rhCol III to hexafluoroisopropanol, and stirring at room temperature until rhCol III is completely dissolved to obtain a uniform and transparent spinning solution, the concentration of rhCol III in the spinning solution is 20wt.%;

(2) Inject the spinning solution into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 15kV, negative high voltage -5kV, flow rate 0.5mL/h, receiving distance 10cm, needle 27G, to obtain rhCol III nanofibers membrane.

2 is a digital photograph of the electrospinning process of Comparative Example 1 and Example 4. (a) and (b) of Fig. 2 are the state of continuous electrospinning 0h and 5min of the spinning solution prepared by comparative example 1 using hexafluoroisopropanol as solvent, as can be seen from the figure, continuous spinning of comparative example 1 for 5min That is to say, clogging occurs. This is because hexafluoroisopropanol has a low boiling point and can be completely evaporated at room temperature. Although this is beneficial to spinning, too fast volatilization of hexafluoroisopropanol will cause clogging, resulting in poor spinning process. continuous. (c) and (d) of Fig. 2 are the states of continuous electrospinning 0h and 5h of the spinning solution prepared by embodiment 4 with 90% acetic acid solution as a solvent. As can be seen from the figure, continuous electrospinning in embodiment 4 5h, still no clogging phenomenon. It shows that the method of the present invention uses acetic acid solution as a solvent to prepare the spinning solution, which can effectively solve the problems of easy clogging and inability to continue electrospinning in the spinning solution prepared with hexafluoroisopropanol as a solvent.

The scanning electron micrographs of the rhCol III nanofiber membranes prepared in Comparative Example 1 and Example 4 are shown in Fig. 3 A1 and B1 respectively. As can be seen from this figure, compared with Example 4, the rhCol III nanofiber membranes prepared in Comparative Example 1 The diameter of the fibers in the fibrous membrane is larger, and the diameter difference between the fibers is also relatively larger. Further, the fiber diameters of the rhColIII nanofibrous membranes prepared in Example 4 and Comparative Example 1 were counted and the fiber diameter frequency histogram (N=120) was drawn. The results are shown in Figure 3 A2 and B2. As can be seen from the figure, the diameters of the fibers in the rhCol III nanofibrous membrane prepared in Comparative Example 1 are mainly distributed between 100 and 500 nm, and the average diameter of the fibers and the standard deviation (SD) of the fiber diameters are calculated, and the fiber diameters are expressed as the average diameter ± In the form of SD, the result is 296nm±106nm. And the diameter of the fiber in the rhCol III nanofibrous membrane prepared in embodiment 4 is mainly distributed between 150～250nm, calculate the average diameter of fiber and the standard deviation (SD) of fiber diameter, the fiber diameter is expressed as mean diameter ± SD form, the result is 194nm ± 44nm. Compared with Comparative Example 1, the fiber diameter in Example 4 is significantly smaller, and the fiber diameter distribution range is also significantly narrower.

Example 5

In this example, an electrospun rhColIII wound dressing was prepared based on the rhColIII nanofiber membrane in Example 4, and the steps were as follows:

The rhCol III nanofiber membrane prepared in Example 4 was cut into a size of 5cm×4cm, immersed in 2wt.% glutaraldehyde ethanol solution for crosslinking, and crosslinked in a shaker at 37°C and 40rpm for 24h, after the reaction Repeatedly wash with PBS buffer to remove unreacted glutaraldehyde, soak in PBS buffer for 10 minutes after washing, repeat the above-mentioned operation of washing and soaking in PBS buffer 3 times, and then use the ethanol volume concentration in order from low to high. Gradient dehydration was performed with an ethanol solution with an ethanol volume concentration of 30% to 100%, followed by critical point drying to prepare a cross-linked rhCol III nanofibrous membrane, namely rhCol III wound dressing, denoted as GA.

Example 6

In this example, in this example, an electrospun rhCol III wound dressing is prepared on the basis of the rhCol III nanofiber membrane in Example 4, and the steps are as follows:

The rhCol III nanofiber membrane prepared in Example 4 was cut into a size of 5 cm × 4 cm, immersed in a _crosslinking agent solution, and crosslinked at ₄ ° C for 24 hours. Repeatedly wash with ultrapure water, soak in PBS buffer solution for 10min after cleaning, repeat the above-mentioned 0.1mol/L Na ₂ HPO ₄ solution soaking, ultrapure water washing and PBS buffer soaking operation 3 times, and then according to the volume concentration of ethanol by From low to high, gradient dehydration was carried out with ethanol solutions with ethanol volume concentrations of 30% to 100%, followed by critical point drying to obtain cross-linked rhCol III nanofibrous membranes, namely rhCol III wound dressings, denoted as EN.

The cross-linking agent solution is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the molar ratio of EDC and NHS: The ratio of 4:1 was dissolved in 95% ethanol, and then the pH value was adjusted to 5.6 with MES buffer solution, and the concentration of EDC in the cross-linking agent solution was 1wt.%.

The scanning electron micrographs of the rhCol III wound dressings prepared in Example 5 and Example 6 are shown in Figure 4 A1 and B1, respectively, as can be seen from this figure, compared to the uncrosslinked rhCol III nanofibrous membrane in Example 4 In Examples 5-6, after cross-linking to form the rhCol III wound dressing, the diameter of the fibers in the rhCol III wound dressing is still relatively uniform. Further, the fiber diameters of the rhCol III wound dressings prepared in Examples 5-6 were counted and a fiber diameter frequency histogram (N=120) was drawn. The results are shown in Figure 4 A2 and B2, from which it can be seen that , the diameter of the fiber of the rhCol III wound dressing prepared in Example 5 is mainly distributed between 170～300nm, the average diameter of the fiber and the standard deviation (SD) of the fiber diameter are calculated, and the fiber diameter is expressed as the form of the average diameter ± SD, The result is 227nm ± 46nm; The diameter of the fiber of the rhCol III wound dressing prepared by embodiment 6 is mainly distributed between 200～320nm, calculate the average diameter of the fiber and the standard deviation (SD) of the fiber diameter, the fiber diameter is expressed as the average diameter In the form of ±SD, the result is 250nm±44nm. After crosslinking, the diameter of the fiber increases relative to the diameter of the fiber in the rhCol III nanofiber membrane prepared in Example 4, but the fiber diameter distribution range is still very narrow, while maintaining the fiber structure of the rhCol III nanofiber membrane and rich porous morphology.

The rhCol III wound dressing prepared in Example 5 and Example 6 has good flexibility, can be curled and folded without damage, as shown in Figure 5, and the left and right two groups of pictures in Figure 5 show Example 5 and Photos of the rhColIII wound dressing prepared in Example 6 in curled and folded states.

Comparative example 2

The operation of this comparative example is basically the same as that of Example 5, except that the cross-linked rhCol III nanofibrous membrane after cross-linking and washing was vacuum-dried at room temperature (25° C.) to obtain a rhCol III wound dressing.

Comparative example 3

The operation of this comparative example is basically the same as that of Example 6, except that the cross-linked rhCol III nanofibrous membrane after cross-linking and washing was vacuum-dried at room temperature (25° C.) to obtain a rhCol III wound dressing.

The scanning electron micrographs of the rhCol III wound dressings prepared in Comparative Example 2 and Comparative Example 3 are shown in Figure 6 (A) and (B) respectively, as can be seen from this figure, the cross-linked rhCol III of Comparative Example 4 and Comparative Example 5 After the nanofibrous membrane was dried in vacuum, severe swelling and deformation occurred, the diameter of the fibers became larger, and serious adhesion occurred between the fibers. The above reasons caused the pore size of the rhCol III wound dressing to become significantly smaller and severely damaged the original fibers. shape. The rhCol III wound dressing whose morphology is destroyed cannot effectively biomime the extracellular matrix, which is unfavorable for its biological effect. Simultaneously, vacuum drying can cause the mechanical properties of the rhCol III wound dressings prepared in Comparative Example and Comparative Example 3 to deteriorate, and broken after vacuum drying, for example, Figure 7 shows that the rhCol III wound dressings prepared in Comparative Example 2 were After drying, it can be seen from the picture that the rhCol III wound dressing is obviously broken. Poor mechanical properties will also affect its performance as a wound dressing.

Example 7

In this example, the mechanical properties of the rhCol III wound dressings prepared in Examples 5 and 6 were tested.

The rhCol III wound dressings prepared in Examples 5 and 6 were subjected to stress-strain curve and Young's modulus tests. Specifically, the two rhCol III wound dressings were soaked in PBS for 10 minutes, cut into rectangular samples with a size of 6.5mm*6.5mm*0.3mm, fixed on a dynamic mechanical analyzer, and stretched longitudinally at a speed of 2000 μm/min , until it is broken, the stress-strain curves and Young's modulus of the two rhCol III wound dressings can be obtained respectively. Each group set 4 parallel samples. The results are shown in (A) and (B) of Figure 8. It can be seen from Figure 8 that GA and EN have good mechanical properties, especially GA, which has relatively better mechanical properties.

Example 8

In this example, the rhCol III wound dressing prepared in Examples 5 and 6 and the Tegaderm commercial dressing were tested for air and water permeability analysis, water absorption rate and water contact angle.

Tegaderm Commercial Dressing consists of a polyurethane film backing, acrylate adhesive, urethane/silicone backcoat, polyester and polyethylene film absorbent pad, and is a waterproof, highly breathable wound dressing. Here, the rhCol III wound dressing prepared in Examples 5 and 6 and the Tegaderm commercial dressing were tested for air and water permeability, so as to compare their differences in water and air permeability.

Water permeability test: Tegaderm commercial dressings and rhCol III wound dressings prepared in Examples 5 and 6 were cut into rectangular samples of 1cm*1cm, and 20 color-changing silica gel particles were added to multiple 5mL sample bottles, and the cut rectangular Sample Cap the vial and seal the rim with parafilm. Observe the changes of the silica gel particles in the sample bottle, and take pictures and record them on the 0th, 1st, and 3rd day. Three parallel samples were set up for each group.

Water permeability test: Tegaderm commercial dressings, rhCol III wound dressings prepared in Examples 5 and 6 were cut into rectangular samples of 1cm*1cm, respectively, and 2mL ferrous chloride solution was added to multiple 5mL sample bottles, and the cut Rectangular samples were capped and the edges were sealed with parafilm. Observe the change situation of the ferrous chloride solution in the sample bottle, take pictures and record on the 0th, 1st, and 6th day. Three parallel samples were set up for each group.

Water absorption capacity test: cut the rhCol III wound dressings prepared in Examples 5 and 6 into rectangular samples of 1 cm*1 cm, weigh them, and record them as W0. Immerse each cut rectangular sample in ultrapure water at 37°C, completely immerse it, take it out after 30 minutes, drain off the excess water, weigh it, and record it as Wt. Calculate the water absorption ratio, water absorption ratio=(Wt-W0)/W0. Three parallel samples were set up for each group.

Water contact angle test: cut the rhCol III wound dressings prepared in Examples 5 and 6 into rectangular samples of 2cm*2cm, and drop a drop of water on each cut rectangular sample. A water droplet was caught with a water contact angle tester and the contact angle was measured. 3 parallel samples in each group.

Figures A and B of Figure 9 are the test results of water permeability and air permeability of Tegaderm commercial dressing, GA prepared in Example 5, and EN prepared in Example 9. It can be seen from the figure that compared with Tegaderm commercial dressings, GA and EN have better air permeability, which is conducive to wound breathing, avoiding the hypoxia of cells in the wound, and promoting wound healing; at the same time, Tegaderm commercial dressings have better water permeability, which means It is conducive to the penetration of water vapor, and will not cause excessive accumulation of moisture in the wound due to impermeability, such as conducive to the evaporation of sweat on the skin surface.

Figures C and D of Figure 9 are the test results of water absorption ratio and water contact angle of GA prepared in Example 5 and EN prepared in Example 5. It can be seen from the figure that GA and EN have good hydrophilicity and water absorption. Good hydrophilicity is good for cell adhesion, spreading and proliferation. Good water absorption is good for the absorption of wound exudate. At the same time, The dry wound dressing will physically adhere to the wet wound, so the wound dressing will not easily fall off from the wound.

Example 9

In this example, the stability of the rhCol III wound dressing after the rhCol III wound dressing prepared in Examples 5 and 6 were co-cultured with cells for a certain period of time was tested.

The GA prepared in Example 5 and the EN prepared in Example 6 were co-cultured with NIH 3T3 cells respectively. Specifically, the GA and EN were cut into circular samples with a diameter of 6 mm, put into a low-adhesion 24-well plate, and used The PBS buffer was pre-soaked for 10 minutes, the PBS buffer was aspirated, 100,000 NIH 3T3 cells were added to the well plate, and the well plate was placed in a cell culture incubator at 37°C for co-cultivation. 3 parallel samples in each group. After 1 day, 4 days and 7 days of co-cultivation, GA and EN were taken out for scanning electron microscope test, and the results are shown in Figure 10. At the same time, based on the scanning electron microscope image in Figure 10, the diameter data of the fiber was analyzed, and the results are shown in Table 1.

Table 1 The change of fiber diameter with co-culture time

It can be seen from Table 1 that with the increase of culture time, the diameter of the fibers in the GA group gradually increased, while the diameter of the fibers in the EN group gradually decreased. This may be due to the slow degradation of the wound dressing in the EN group during the co-culture process, while Compared with the EN group, the wound dressings in the GA group were not easy to degrade, and the fibers swelled to a certain extent as the co-culture time increased. But overall, the fibers in the wound dressings of the GA and EN groups have good stability and will not degrade rapidly, which can provide an extracellular matrix-like structure for cell spreading and proliferation over a longer period of time .

Example 10

In this example, the growth state of cells in the rhCol III wound dressing prepared in Examples 5 and 6 was investigated.

The GA prepared in Example 5 and the EN prepared in Example 6 were co-cultured with NIH 3T3 cells respectively. Specifically, the GA and EN were cut into circular samples with a diameter of 6 mm, put into a low-adhesion 24-well plate, and used The PBS buffer was pre-soaked for 10 minutes, the PBS buffer was aspirated, 100,000 NIH 3T3 cells were added to the well plate, and the well plate was placed in a cell culture incubator at 37°C for co-cultivation. 3 parallel samples in each group. Live/dead staining was performed after 4 days of co-cultivation, and the results are shown in panel A of Figure 11 , and phalloid pen/DAPI staining was performed after 1 and 4 days of co-cultivation, and the results are shown in panel B of Figure 11 . After 1 day, 3 days and 7 days of co-cultivation, scanning electron microscope analysis was performed to observe the cell morphology, and the results are shown in FIG. 12 .

It can be seen from Figure 11 that the wound dressings of the GA group and the EN group have good biocompatibility, almost no dead cells, and the cells proliferate normally as the co-culture time increases. It can be seen from Figure 12 that the wound dressings of the GA group and the EN group have good cell adhesion, and the cells can spread and grow in it and have cell pseudopodia protruding out.

Example 11

In the present embodiment, rhCol III nanofiber membranes are prepared by preparing spinning solutions of different concentrations with 90wt.% acetic acid (HAC) solution, and the steps are as follows:

(1) Add rhCol III to an acetic acid solution with a concentration of 90wt.%, stir at room temperature until rhCol III is completely dissolved, and obtain a uniform and transparent spinning solution; prepare 6 groups of spinning solutions with different concentrations of rhCol III, and each group spins The concentrations of rhCol III in the silk liquid were 35wt.%, 40wt.%, 42.5wt.%, 45wt.%, 47.5wt.% and 50wt.% respectively;

(2) Inject the spinning solutions with different rhCol III concentrations prepared in step (1) into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 23kV, negative high voltage -1.5kV, flow rate 0.5mL/h , the receiving distance is 10cm, the needle is 27G, and the rhCol III nanofiber membrane is obtained.

The scanning electron micrograph of the rhCol III nanofiber membrane prepared in this embodiment is shown in Figure 13, as can be seen from Figure 13, when the spinning solution is prepared as a solvent in 90wt.% acetic acid solution, when the rhCol III in the spinning solution When the concentration is 45wt.%-50wt.%, the rhCol III nanofiber membrane with uniform fiber diameter can be prepared.

Comparative example 4

In this comparative example, rhCol III nanofiber membranes were prepared with 70wt.% acetic acid solution and pure acetic acid to prepare rhCol III nanofiber membranes with different concentrations, and the steps were as follows:

(1) Add rhCol III to acetic acid solution with a concentration of 70wt.%, stir at room temperature until rhCol III is completely dissolved, and obtain a uniform and transparent spinning solution; prepare 3 groups of spinning solutions with different concentrations of rhCol III, and each group spins The concentrations of rhCol III in the silk liquid are 35wt.%, 40wt.%, 45wt.% respectively;

Add rhCol III into pure acetic acid, stir at room temperature until rhCol III is completely dissolved, and obtain a uniform and transparent spinning solution; prepare 3 groups of spinning solutions with different concentrations of rhCol III, and the concentrations of rhCol III in each group of spinning solutions are respectively 35wt.%, 40wt.%, 45wt.%;

(2) Inject the three groups of spinning solutions prepared by 70wt.% acetic acid solution in step (1) into the electrospinning device for electrospinning. The spinning parameters are: positive high voltage 23kV, negative high voltage -1.5kV, flow rate 0.5mL/h, receiving distance 10cm, needle 27G, get rhCol III nanofiber membrane.

The three groups of spinning solutions prepared from pure acetic acid solution in step (1) were respectively injected into the electrospinning device for electrospinning. The spinning parameters were: positive high voltage 27kV, negative high voltage -1.5kV, flow rate 0.5mL/h, receiving The distance is 10cm, the needle is 27G, and the rhCol III nanofiber membrane is obtained.

The scanning electron microscope image of the rhCol III nanofiber membrane prepared in this comparative example is shown in Figure 14. It can be seen from Figure 14 that when the spinning solution is prepared as a solvent in 70wt.% acetic acid solution, the fiber cannot be prepared by electrospinning RhCol III nanofiber membranes with uniform diameter; when pure acetic acid is used as a solvent to prepare spinning solution, although when the concentration of rhColIII in the spinning solution is 35wt.%～40wt.%, rhColIII nanofibers with uniform fiber diameter can be prepared However, the required voltage is high, which affects the safety of the preparation process; and we found during the experiment that when the positive high voltage of electrospinning is lower than 27kV, the spinning solution prepared with pure acetic acid as a solvent is in the process of electrospinning Continuous non-beaded fibers cannot be obtained at this time. In contrast, the method of the present invention can realize electrospinning with a positive voltage of 10-27kV.

Claims (9)

1. The electrostatic spinning recombinant humanized III type collagen wound dressing is characterized in that the wound dressing is a fibrous membrane which is formed by stacking and cross-linking recombinant humanized III type collagen fibers with uniform diameters and has a porous structure, the diameter of the recombinant humanized III type collagen fibers is not more than 550nm, and the diameter of the fibers after cross-linking of the recombinant humanized III type collagen fibers is not more than 600nm.

2. The electrospun recombinant humanized type iii collagen wound dressing of claim 1, wherein the recombinant humanized type iii collagen fibers have an average diameter of 100-450 nm and a standard deviation of the diameter distribution of the recombinant humanized type iii collagen fibers is no more than 55nm.

3. The method for preparing the electrostatic spinning recombinant humanized type III collagen wound dressing according to claim 1 or 2, which is characterized by comprising the following steps:

(1) Dissolving the recombinant humanized III type collagen into an acetic acid solution with the concentration of 85-95 wt.% to obtain a spinning solution, wherein the concentration of the recombinant humanized III type collagen in the spinning solution is 45-50 wt.%;

(2) Injecting the spinning solution into an electrostatic spinning device for electrostatic spinning to obtain a recombinant humanized III type collagen nanofiber membrane with uniform fiber diameter;

(3) Immersing the recombinant humanized III type collagen nanofiber membrane into a cross-linking agent solution for cross-linking, cleaning after cross-linking to remove unreacted cross-linking agent, then carrying out gradient dehydration, and drying at a critical point to obtain the recombinant humanized III type collagen wound dressing; the cross-linking agent solution is prepared by taking 95-100% ethanol by volume percentage as a solvent.

4. The method for preparing the electrostatic spinning recombinant humanized III type collagen wound dressing according to claim 3, wherein in the step (2), during electrostatic spinning, the positive voltage is controlled to be 10-27 kV, the negative voltage is controlled to be minus 1.5-minus 10kV, the flow rate is controlled to be 0.1-1 mL/h, and the receiving distance is controlled to be 5-20 cm.

5. The method for preparing an electrospun recombinant humanized III type collagen wound dressing according to claim 4, wherein the needle size adopted in the step (2) is 15-30G.

6. The method of preparing an electrospun recombinant humanized type iii collagen wound dressing according to any one of claims 3 to 5, wherein the concentration of the cross-linking agent in the cross-linking agent solution is 0.5wt.% to 5wt.%.

7. The method for preparing an electrospun recombinant humanized type iii collagen wound dressing according to claim 6, wherein the cross-linking agent is glutaraldehyde, genipin, a diepoxide, transglutaminase or a combination of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide.

8. The method for preparing an electrostatic spinning recombinant humanized type iii collagen wound dressing according to any one of claims 3 to 5, wherein the gradient dehydration in the step (3) means that the cross-linked and washed recombinant humanized type iii collagen nanofiber membrane is soaked and dehydrated with ethanol of different concentrations in sequence from low to high volume percentage of ethanol.

9. The method for preparing the electrostatic spinning recombinant humanized type III collagen wound dressing according to claim 8, wherein the recombinant humanized type III collagen nanofiber membrane after crosslinking and washing is subjected to gradient dehydration by using 30-100% ethanol by volume percent.

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