CN110464867B

CN110464867B - Piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading traditional Chinese medicine exosomes and preparation method thereof

Info

Publication number: CN110464867B
Application number: CN201910910621.6A
Authority: CN
Inventors: 彭丽华; 许学寒
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2020-07-28
Anticipated expiration: 2039-09-25
Also published as: CN110464867A

Abstract

The invention provides a piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading traditional Chinese medicine exosomes and a preparation method thereof, and the preparation method comprises the sub-step of: reacting gelatin with methacrylic anhydride to prepare methacrylic acid esterified gelatin; preparing traditional Chinese medicine exosomes through differential centrifugation and the like; compounding the gold nanorods, exosomes and methacrylic acid esterified gelatin to prepare a conductive pre-gel system; and compounding the conductive pre-gel and the polyvinylidene fluoride piezoelectric film to prepare the piezoelectric composite dressing. The dressing prepared by the invention is a novel dressing with the effect of piezoelectric stimulation composite drugs, polyvinylidene fluoride generates electric potential to stimulate a wound area through conductive gel, and the electric potential is cooperated with Chinese medicine exosomes to promote wound healing and nerve regeneration, so that the problems in the existing wound healing and nerve repairing processes can be effectively solved. Therefore, the composite dressing has wide application value in the treatment of wound and nerve repair.

Description

Piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading traditional Chinese medicine exosomes and preparation method thereof

Technical Field

The invention relates to a piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicine exosomes and a preparation method thereof.

Background

The skin is the first line of defense of the human body and also the largest organ of the human body, and has multiple functions of feeling, protection and the like. In daily life, however, the skin is affected by various external factors, often causing trauma, and bringing heavy pressure and burden to individuals and society. Wound healing, however, is a complex and dynamic process involving the co-participation of various tissues and cells in regulation. The uncontrolled adjustment of the wound repair process results in serious scars after the wound surface is healed and the auxiliary organs can not regenerate, thereby causing repair obstacles. Meanwhile, the surface of the skin is distributed with a complex neural network, which mainly comprises different nervous systems such as sensory nerves and autonomic nerves, and the neural network and the sensory nerves and the autonomic nerves jointly play various functions such as perception, physiological regulation and the like. Severe skin damage can damage the receptors of the skin and the nerve fibers associated with them, resulting in loss of sensory function in the different skin.

Peripheral nerve repair is a common clinical problem, and spontaneous peripheral nerve repair is almost always incomplete and poorly restored. Suturing nerve stumps is difficult to perform after peripheral nerve injury results in a nerve defect, especially when there is a large nerve gap (20 mm or longer in humans), often requiring some form of graft to be inserted between the nerve stumps to bridge the gap and support axonal regeneration. The current gold standard therapy for peripheral nerve gap repair is the implantation of an autologous nerve graft, which is usually the transplantation of a less functionally important nerve segment from another part of the body. However, autologous nerve transplantation has inherent disadvantages including limited supply of donor nerves, the need for a second operation, mismatch between donor nerves and recipient sites, and potential differences in tissue structure and size.

The mesenchymal stem cell is a pluripotent cell having the potential to differentiate into various cell types including adipocytes, endothelial cells, osteoblasts and chondrocytes, and nerve cells, etc. And since the mesenchymal stem cells of bone marrow are abundant in source and easy to be isolated, the mesenchymal stem cells are considered as one of the most important stem cell sources for regenerative medicine and are also widely used for cell therapy and tissue engineering. However, stem cells in the natural state cannot differentiate into specific neural lineage cell types, but under the influence of external factors such as chemical growth factors, extracellular matrix components, electrical stimulation and the like, mesenchymal stem cells can differentiate into neural cells to promote peripheral nerve repair.

Different types and concentrations of ions exist inside and outside human cells. When skin is injured, different ion gradients across the wound produce a trans-epithelial potential difference, which in turn produces an endothelial electric field, and remains until the skin regeneration process is complete. The endothelial field not only regulates skin cell behavior during wound healing, but also promotes skin regeneration. It has been found that endothelial electric fields stimulate fibroblast proliferation and differentiation into myofibroblasts, keratinocyte migration and angiogenesis.

Electrical stimulation can promote differentiation of stem cells into neural cells, and can also promote healing of wounds. Therefore, conductive scaffolds, conductive dressings, and the like, are also gradually attracting attention of researchers. The conductive hydrogel has excellent biocompatibility and certain mechanical strength on one hand; on the one hand, the drug can be encapsulated and released by erosion or degradation and the like under the electric stimulation. These properties make the conductive gel an excellent drug carrier. However, the traditional conductive gel is often compounded with conductive polymers such as polypyrrole and carbon nanotubes, and is often difficult to degrade.

Conductive dressings such as conductive gels require electrical stimulation in conjunction with external devices during use. The construction of most electrical stimulation devices is complicated by the need for external energy input and wire connections. This increases the steps of treatment and the requirements of treatment on the one hand, and also places additional economic and psychological burdens on the patient on the other hand. Therefore, the electrical stimulation therapy without external instruments may be widely applied clinically.

When the piezoelectric material is deformed under the action of pressure or tension in a certain direction, the centers of positive and negative charges in the piezoelectric material are relatively transferred to generate a polarization phenomenon, and charges are generated on the surface of the piezoelectric material so as to generate a potential difference. The piezoelectric material is commonly used in the fields of sensors, generators and the like, wherein the most important material is an inorganic piezoelectric material, including piezoelectric ceramics prepared from PZT, perovskite titanium and the like. However, as research has shown, organic materials also have these properties.

Polyvinylidene fluoride (PVDF) and copolymers thereof have extremely strong piezoelectric effect compared with the traditional inorganic piezoelectric materials, are organic substances, and have good biocompatibility and mechanical properties. The existing research mainly utilizes the stimulation effect of the potential difference on cells. However, it is often difficult to achieve a good therapeutic effect simply by virtue of the properties of the material itself. In clinical use, a synergistic effect with a drug is also required, but research in this field is still blank at present.

Disclosure of Invention

The invention aims to provide a piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicine exosomes and a preparation method thereof, aiming at solving the problems that:

a. skin regeneration and nerve regeneration problems during wound healing;

b. the problem that external instruments are needed for electrical stimulation;

c. the piezoelectric stimulation acts synergistically with the drug.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention firstly discloses a preparation method of a piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicine exosomes, which comprises the following steps:

A. reacting gelatin with methacrylic anhydride to prepare methacrylic acid esterified gelatin;

B. preparing and purifying the traditional Chinese medicine exosomes by differential centrifugation and density gradient centrifugation;

C. dissolving methacrylate gelatin in ultrapure water or phosphate buffer solution, then compounding the gold nanorods and the prepared traditional Chinese medicine exosomes with the methacrylate gelatin, and doping a photosensitizer to prepare a conductive pre-gel system; D. and spin-coating the conductive pre-gel on the polyvinylidene fluoride piezoelectric film, and then preparing the composite dressing through photo-crosslinking. In a preferred embodiment of the present invention, in the step a, the grafting ratio of the methacrylated gelatin is 20 to 90%.

In a preferred embodiment of the present invention, in step B, the source of the exosome is one or more of ginseng, fructus ligustri lucidi, radix salviae miltiorrhizae, radix rehmanniae, saffron and radix paeoniae.

As a preferred embodiment of the present invention, the step B specifically comprises:

1) cleaning the medicinal materials, and squeezing to obtain juice; sieving the squeezed juice with 100-300 mesh sieve to remove medicinal material residue; performing differential centrifugation on the sieved juice, firstly centrifuging for 10-60 minutes under the centrifugal force of 1000-10000g, and discarding the precipitate; then centrifuging the supernatant for 10-120 minutes under the centrifugal force of 3000-30000 g; discarding the precipitate, centrifuging the supernatant for 10-480 minutes under the centrifugal force of 10000-300000 g;

2) resuspending the precipitate obtained in the last step of the step 1) by ultrapure water or phosphate buffer salt solution, and then carrying out density gradient centrifugal purification, wherein the density gradient solution is prepared from sucrose or CsCl, and the concentration of each gradient solution is 10-90%; the centrifugal force is 100000-;

3) sucking out exosomes in different concentration intervals, and centrifuging for 10-360 minutes under the centrifugal force of 50000-300000g to obtain precipitates. Resuspending with ultrapure water or phosphate buffered saline solution to obtain the Chinese medicinal exosome.

In a preferable embodiment of the present invention, the length-diameter ratio of the gold nanorods is 1 to 5.

As a preferable scheme of the invention, in the conductive pre-gel system in the step C, the mass percentage concentration of the methacrylated gelatin is 5-30%, the concentration of the nanorod is 0.1-5mg/ml, the concentration of the traditional Chinese medicine exosome is 1ng-10ug/ml, and the photosensitizer is one or two of L AP and I2959.

As a preferred scheme of the invention, the preparation method of the polyvinylidene fluoride piezoelectric film comprises the following steps: coating by a spin coating method, and then annealing and polarizing to prepare a polyvinylidene fluoride piezoelectric film; the thickness of the film after spin coating is 10-50 μm, the annealing temperature is 60-150 ℃, the polarization temperature is 50-150 ℃, the voltage is 80-200V/μm, and the polarization time is 1-8 hours.

In the step D, the photocrosslinking time in the conductive pre-gel system is 10 to 360 seconds, and the thickness of the methacrylated gelatin after spin coating is 1 to 5 mm.

The invention also discloses the composite dressing prepared by the method.

The invention also discloses application of the composite dressing in the fields of intelligent response materials, wound healing and nerve repair.

Due to the adoption of the technical scheme, the invention has the following beneficial effects:

1. the invention has wide application range, does not need to use special materials and reduces the production cost;

2. the conductive gel prepared by the invention has good conductivity and mechanical strength, is degradable, has a slow release effect, and has cell adhesion capability and biocompatibility in an in vitro experiment;

3. the traditional Chinese medicine exosome prepared by the invention is derived from traditional Chinese medicines, has wide source range, easy preparation and high yield, and has wide medical and commercial prospects. In vitro experiments prove that the method can effectively promote the proliferation and migration of the mesenchymal stem cells and promote the differentiation of the mesenchymal stem cells like neural cells;

4. the composite dressing prepared by the invention can generate electric stimulation by itself and entrap and release exosomes. On one hand, the method can induce stem cells to migrate to wounds and promote stem cell differentiation through the action of exosomes; on one hand, the electric potential generated by the polyvinylidene fluoride acts on the stem cells through the conduction of the conductive gel, provides electric stimulation, and promotes the differentiation of the stem cells to nerve cells and the healing of wounds by cooperating with exosomes.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of gelatin and methacrylated gelatin;

FIG. 2 is a graph showing the results of cell adhesion of the composite dressing;

FIG. 3 is a UV scan of gold nanorods;

FIG. 4 is a transmission electron microscope result image of gold nanorods;

FIG. 5 is a graph of live and dead staining results for composite dressings;

FIG. 6 is a particle size potential diagram of exosomes;

FIG. 7 is a transmission electron microscopy result plot of exosomes;

FIG. 8 is a graph showing the results of CCK-8 proliferation of stem cells 1 day after exosome administration;

FIG. 9 is a graph of Nestin (Nestin) immunofluorescence results 7 days after exosome administration of stem cells;

FIG. 10 is a graph of the results of wound healing in rats with composite dressings;

FIG. 11 is a graph showing the results of the weight of rats with the composite dressing;

FIG. 12 is a graph showing the immunohistochemical results of rat skin neurofilament protein (NF-L) with composite dressing.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Example 1: a piezoelectric-conductive gel for promoting peripheral nerve repair and a composite dressing for loading Chinese medicine exosomes and a preparation method thereof comprise the following steps:

1) reacting gelatin with methacrylic anhydride, dialyzing with 8k-14kDa dialysis bag at room temperature for 1 week, and lyophilizing to obtain methacrylated gelatin;

2) firstly, cleaning privet, juicing, and screening by a 100-mesh sieve; after centrifugation at 1000g and 12000g by differential centrifugation, the supernatant was centrifuged at 100000g with an ultracentrifuge, and the pellet was resuspended in PBS. Preparing a density gradient solution by using sucrose, and then performing ultracentrifugation for 60 minutes at 200000 g; centrifuging exosomes of different components at 50000g for 60 minutes to prepare traditional Chinese medicine exosomes;

3) by HAuCl₄With CTAB, NaBH₄Preparing into seed solution, HAuCl₄、CTAB、AgNO₃AA is prepared into a growth solution, and then the growth solution is grown at 25 ℃ overnight by a seed growth method to prepare gold nanorods;

4) dissolving methacrylate gelatin with PBS to make its concentration reach 5%, adding gold nanorod to make its concentration reach 1mg/ml, adding Chinese medicinal exosome to make its final concentration reach 10 μ g/ml, and mixing with photosensitizer L AP to obtain hydrogel pre-gel system;

5) dissolving polyvinylidene fluoride powder monomer with anhydrous DMF (dimethyl formamide), rotationally coating the monomer into a film, annealing at 120 ℃, polarizing for 8 hours at 100 ℃ under an electric field of 120V/mum after annealing, and preparing polyvinylidene fluoride with the thickness of 10μm;

6) spin-coating a hydrogel pre-gelling system on polyvinylidene fluoride, and then carrying out photo-crosslinking for 60s to prepare the composite dressing, wherein the thickness of the hydrogel is 2 mm;

after preparing the methacrylated gelatin, dissolving the methacrylated gelatin in deuterated water,then testing with NMR instrument to obtain relevant material¹H-NMR spectrogram, determining the chemical structure of the methacrylated gelatin; as shown in FIG. 1, compared with gelatin, the H spectrum of methacrylated gelatin has a peak (shown in a black dotted line box) with a chemical shift between 5.2 and 5.6ppm, which represents a hydrogen atom on a double-bond carbon. And are affected by local shielding effects and remote shielding effects, the two hydrogen atoms on the double bond carbon are at different chemical shifts.

Preparing composite dressing, ultraviolet irradiating the composite dressing in a super clean bench for 0.5 hr, washing with complete culture solution of mesenchymal stem cells to remove alcohol and soaking in hydrogel, balancing at 37 deg.C, and mixing with 1 × 10⁵Cell density of/m L was seeded on gel and placed at 37 ℃ in 5% CO₂After culturing for 24 hours, the medium was aspirated, washed twice with PBS, fixed by soaking in 4% paraformaldehyde for 10 minutes, and washed 2 times with PBS containing 0.1% Triton X-100 for about 5 minutes each. The Actin-Tracker Green staining solution was diluted with PBS containing 5% BSA and 0.1% Triton X-100 at a ratio of 1:100, added dropwise to the cells, incubated at room temperature in the dark for 1 hour, and washed twice with PBS. Add 500. mu.l/ml DAPI solution, incubate for 15 minutes at room temperature, remove the DAPI solution, wash twice with PBS, and immediately place under confocal microscope for observation. As shown in FIG. 2, after 24h of culture, the cells were normally adhered to the hydrogel, and the morphology of the cells was normal, with fusiform extension.

Example 2: a piezoelectric-conductive gel for promoting peripheral nerve repair and a composite dressing for loading Chinese medicine exosomes and a preparation method thereof comprise the following steps:

1) reacting gelatin with methacrylic anhydride, dialyzing with 8k-14kDa dialysis bag at room temperature for 2 weeks, and lyophilizing to obtain methacrylated gelatin;

2) firstly, cleaning and juicing salvia miltiorrhiza, and sieving with a 200-mesh sieve; after centrifugation at 2000g followed by 10000g by differential centrifugation, the supernatant was centrifuged at 120000g using an ultracentrifuge, and the pellet was resuspended in PBS. Gradient with CsCl followed by centrifugation at 100000g for 60 min; centrifuging exosomes of different components at 100000g for 60 minutes to prepare traditional Chinese medicine exosomes;

3) by HAuCl₄With CTAB, NaBH₄Preparing into seed solution, HAuCl₄、CTAB、AgNO₃AA is prepared into a growth solution, and then the growth solution is grown overnight at 28 ℃ by a seed growth method to prepare gold nanorods;

4) dissolving the methacrylate gelatin by PBS to make the concentration reach 10%; then adding gold nanorods to enable the concentration of the gold nanorods to reach 2 mg/ml; then adding Chinese medicinal exosome to make the final concentration reach 1 mug/ml.

Then mixing with a photosensitizer L AP to obtain a water gel pre-gel system;

5) dissolving polyvinylidene fluoride powder monomer with anhydrous DMF (dimethyl formamide), rotationally coating the monomer into a film, annealing at 100 ℃, polarizing for 4 hours at 130 ℃ under an electric field of 120V/mum after annealing, and preparing polyvinylidene fluoride with the thickness of 20 μm;

6) spin-coating a hydrogel pre-gelling system on polyvinylidene fluoride, and then carrying out photo-crosslinking for 30s to prepare the composite dressing, wherein the thickness of the hydrogel is 5 mm;

firstly, testing the prepared gold nanorods by using an ultraviolet visible light absorption spectrometer, and determining the absorption peak of the gold nanorods at 350-900 nm; as shown in fig. 3, the gold nanorods have two absorption peaks corresponding to the major and minor diameters of the gold nanorods, respectively.

And then dripping the gold nanorod solution on a copper net plated with a carbon film, naturally drying, dripping the gold nanorod solution on the copper net, and observing the appearance of the gold nanorod solution through a transmission electron microscope. As shown in fig. 4: the gold nanorods are of a standard rod-shaped structure, uniform in appearance and good in dispersibility in water, and the length-diameter ratio of the gold nanorods is about 4.2 through measurement.

After preparing the composite dressing, using a complete culture solution of the mesenchymal stem cells to wash out alcohol and soak hydrogel, balancing at 37 ℃, inoculating the digested mesenchymal stem cells on the gel with the cell density of 1 × 105/M L, placing the gel in an incubator with 5% CO2 at 37 ℃ to culture for 48h, removing the culture solution, washing 3 times by PBS, adding Calcein AM and PI solutions which are respectively diluted to 2 mu M and 8M Mm by PBS, incubating for 30-45 min in a dark place, finally washing three times by PBS, removing residual reagents, immediately placing a sample to be tested under a laser confocal microscope to observe, wherein living cells show green, nucleic acid substances in dead cells are dyed red, and after culturing for 48h, the cells in the composite dressing basically show green and do not see red, thereby proving that the composite dressing has good biocompatibility.

Example 3: a piezoelectric-conductive gel for promoting peripheral nerve repair and a composite dressing for loading Chinese medicine exosomes and a preparation method thereof comprise the following steps:

1) reacting gelatin with methacrylic anhydride, dialyzing in a dialysis bag with 8k-14kDa at room temperature for 1 week, and freeze-drying to prepare methacrylated gelatin;

2) cleaning rehmannia root, juicing and sieving by a 150-mesh sieve; after centrifugation at 2000g followed by 10000g by differential centrifugation, the supernatant was centrifuged at 150000g using an ultracentrifuge and the pellet was resuspended in PBS. Gradient solution with sucrose, then ultracentrifugation at 150000g for 120 min; centrifuging exosomes of different components at 150000g for 60 minutes to prepare traditional Chinese medicine exosomes;

3) by HAuCl₄With CTAB, NaBH₄Preparing into seed solution, HAuCl₄、CTAB、AgNO₃AA is prepared into a growth solution, and then the growth solution is grown overnight at 30 ℃ by a seed growth method to prepare gold nanorods

4) Dissolving the methacrylated gelatin by using ultrapure water to enable the concentration of the methacrylated gelatin to reach 20 percent; then adding gold nanorods to enable the concentration of the gold nanorods to reach 2 mg/ml; then adding Chinese medicinal exosome to make the final concentration reach 1 mug/ml. Then mixing with a photosensitizer I2959 to obtain a water gel pre-gel system;

5) dissolving polyvinylidene fluoride powder monomer with anhydrous DMF (dimethyl formamide), rotationally coating to form a film, annealing at 120 ℃, polarizing for 8 hours at 115 ℃ under an electric field of 90V/mum after annealing, and preparing polyvinylidene fluoride with the thickness of 20μm;

6) spin-coating a hydrogel pre-gelling system on polyvinylidene fluoride, and then carrying out photo-crosslinking for 60s to prepare the composite dressing, wherein the thickness of the hydrogel is 3 mm;

and (3) analyzing the particle size potential of the prepared exosome in a Malvern particle size analyzer. As shown in FIG. 6, the exosomes have uniform particle size and potential distribution, wherein the particle size is 144.1 + -2.762 nm, the PDI is 0.218, and the surface potential is-27.4 + -0.451 mV.

Then the exosome solution is dripped on a copper net plated with a carbon film, and then counterstaining is carried out by uranyl acetate, and observation is carried out by a transmission electron microscope. As shown in fig. 7, exosomes are in a standard cake-like structure.

Collecting the third generation bone marrow mesenchymal stem cells, and adding 5 × 10³The exosomes with different concentrations are cultured on a 96-well plate for 24 hours, then exosomes with different concentrations are added into the 96-well plate, the 96-well plate is put into a cell culture box to be incubated for 6 hours, then culture solution is sucked out and washed twice by PBS, the culture solution is replaced by fresh culture solution containing 10% serum, after the culture is continued for 18 hours, the culture solution is removed, DMEM low-sugar culture solution 100 mu L containing CCK-8 is added, the cell culture box is put into the cell culture box to be cultured for 4 hours, the absorbance value is measured at 475nm of a microplate reader, the cell proliferation percentage is calculated, as shown in figure 8, the exosomes have good proliferation effect on the mesenchymal stem cells under the culture of the exosomes with three different groups from low concentration to high concentration, and the proliferation rate is gradually increased.

Example 4: a piezoelectric-conductive gel for promoting peripheral nerve repair and a composite dressing for loading Chinese medicine exosomes and a preparation method thereof comprise the following steps:

1) reacting gelatin with methacrylic anhydride, dialyzing in a dialysis bag with 8k-14kDa at room temperature for 2 weeks, and freeze-drying to prepare methacrylated gelatin;

2) firstly, washing ginseng, juicing and sieving by a 200-mesh sieve; after centrifugation at 3000g followed by 10000g by differential centrifugation, the supernatant was centrifuged at 150000g using an ultracentrifuge and the pellet was resuspended in PBS. Gradient solution with sucrose, then ultracentrifugation at 100000g for 120 min; centrifuging exosomes of different components at 30000g for 60 minutes to prepare traditional Chinese medicine exosomes;

3) by HAuCl₄With CTAB, NaBH₄Preparing into seed solution, HAuCl₄、CTAB、AgNO₃、AA is prepared into a growth solution, and then the growth solution is grown overnight at 25 ℃ by a seed growth method to prepare gold nanorods

4) Dissolving methacrylated gelatin with ultrapure water to make the concentration reach 10%, then adding gold nanorod to make the concentration reach 3mg/ml, then adding Chinese medicine exosome to make the final concentration reach 2 mug/ml, and mixing with photosensitizer L AP to obtain hydrogel pre-gel system;

5) dissolving polyvinylidene fluoride powder monomer with anhydrous DMF (dimethyl formamide), rotationally coating to form a film, annealing at 120 ℃, polarizing for 8 hours at 100 ℃ under an electric field of 120V/mum after annealing, and preparing polyvinylidene fluoride with the thickness of 30μm;

6) spin-coating a hydrogel pre-gel system on polyvinylidene fluoride, and then carrying out photo-crosslinking for 120s to prepare the composite dressing, wherein the thickness of the hydrogel is 5 mm;

placing the composite adjuvants in a super clean bench, ultraviolet irradiating for 0.5 hr, washing with complete culture solution of mesenchymal stem cells to remove alcohol and soaking hydrogel, balancing at 37 deg.C, mixing the digested mesenchymal stem cells with a mixture of 1 × 10⁵Cell density of/m L was seeded on gel and placed at 37 ℃ in 5% CO₂The culture solution is changed every other day in the incubator, after the culture is carried out for 14 days, a gel sample is taken out for nidogen (Nestin) staining. The method comprises the following specific steps: soaking and cleaning a hydrogel sample twice by PBS, adding 4% paraformaldehyde to cover cells, fixing at room temperature for 15 minutes, soaking and cleaning three times by PBS, permeabilizing by 0.1% Triton X-100 for 5 minutes, adding goat serum to seal for 30 minutes after cleaning, removing liquid, cleaning, adding primary antibody, incubating at 4 ℃ overnight, fully soaking and cleaning five times by PBS, adding fluorescent secondary antibody, incubating at 37 ℃ in the dark for 1 hour, fully soaking and cleaning three times by PBS, adding DAPI staining solution to cover cells, staining at room temperature for 10 minutes, soaking and cleaning three times by PBS, adding PBS to soak, and detecting and observing by a confocal microscope. As shown in FIG. 9, after the cells are stained, strong green fluorescence is shown, which indicates that the stem cells on the composite dressing are highly expressed in nestin, and the mesenchymal stem cells of the bone marrow show a neural differentiation tendency.

Example 5: a piezoelectric-conductive gel for promoting peripheral nerve repair and a composite dressing for loading Chinese medicine exosomes and a preparation method thereof comprise the following steps:

2) firstly, cleaning saffron, juicing, and sieving with a 150-mesh sieve; centrifuging with 3000g and 5000g by differential centrifugation, collecting supernatant, centrifuging with ultracentrifuge at 100000g, and resuspending the precipitate with ultrapure water. Gradient solution with sucrose, then ultracentrifugation at 100000g for 60 min; centrifuging exosomes of different components at 10000g for 60 minutes to prepare traditional Chinese medicine exosomes;

3) by HAuCl₄With CTAB, NaBH₄Preparing into seed solution, HAuCl₄、CTAB、AgNO₃AA, preparing growth solution, and then growing at 25 deg.C overnight by seed growth method to prepare gold nanorods

4) Dissolving methacrylated gelatin with ultrapure water to make the concentration reach 15%, then adding gold nanorod to make the concentration reach 2mg/ml, then adding Chinese medicine exosome to make the final concentration reach 0.1 microgram/ml, then mixing photosensitizer L AP to obtain water gel pre-gel system;

5) dissolving polyvinylidene fluoride powder monomer with anhydrous DMF (dimethyl formamide), rotationally coating to form a film, annealing at 120 ℃, polarizing for 8 hours at 60 ℃ under an electric field of 120V/mum after annealing, and preparing polyvinylidene fluoride with the thickness of 40μm;

6) spin-coating a hydrogel pre-gelling system on polyvinylidene fluoride, and then carrying out photo-crosslinking for 80s to prepare the composite dressing, wherein the thickness of the hydrogel is 3 mm;

an acute wound model was established by dividing 48 clean SD male rats (weight 150-.

As shown in fig. 10 and 11, the body weight and the healing rate of the compound adjuvant group were significantly increased compared to the blank control group, with a significant difference at the sixth day. Where the body weight results objectively demonstrate that the composite dressing has no deleterious systemic effects on SD rats, while a significant increase in healing rate demonstrates that the composite dressing is effective in enhancing the healing rate of wounds.

Placing the tissue slices in a repair box filled with EDTA antigen repair buffer solution (PH8.0) for antigen repair, dropwise adding 3% BSA to uniformly cover the tissues, sealing at room temperature for 30 minutes, gently throwing off the sealing solution, dropwise adding a neurofilament protein (NF-L) primary antibody prepared by PBS according to a certain proportion on the slices, placing the slices in a wet box, incubating overnight at 4 ℃, placing the slides in PBS, shaking and washing 3 times on a decoloring shaking table for 5 minutes each time, dropwise adding a secondary antibody covering tissue of a corresponding species with the primary antibody, incubating at dark room temperature for 50 minutes, after fully washing 3 times by PBS, dropwise adding DAPI staining solution, incubating at dark room temperature for 10 minutes, fully soaking and washing for five times by PBS, soaking and washing the slices, observing and acquiring images under a confocal microscope, as shown in figure 12, in the skin tissue slices of the composite dressing group, highly expressing red fluorescence, forming silks, and showing the distribution of a large number of neurofilaments.

Claims

1. A preparation method of a piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading traditional Chinese medicine exosomes is characterized by comprising the following steps:

C. dissolving methacrylate gelatin in ultrapure water or phosphate buffer salt solution, then compounding the gold nanorods and the prepared traditional Chinese medicine exosomes with the methacrylate gelatin, and doping a photosensitizer to prepare a conductive pre-gel system, wherein in the conductive pre-gel system, the mass percentage concentration of the methacrylate gelatin is 5-30%, the concentration of the nanorods is 0.1-5mg/ml, the concentration of the traditional Chinese medicine exosomes is 1ng-10ug/ml, and the photosensitizer is one or two of L AP and I2959;

D. and spin-coating the conductive pre-gel on the polyvinylidene fluoride piezoelectric film, and then preparing the composite dressing through photo-crosslinking.

2. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: in the step A, the grafting ratio of the methacrylated gelatin is 20-90%.

3. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: in the step B, the source medicinal materials of the exosome are one or more of ginseng, glossy privet fruit, salvia miltiorrhiza, rehmannia, saffron and Chinese herbaceous peony.

4. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: the step B specifically comprises the following steps:

2) resuspending the precipitate obtained in the last step of the step 1) by using ultrapure water or phosphate buffer salt solution, and then performing centrifugal purification by density gradient, wherein the density gradient solution is prepared from sucrose or CsCl; the centrifugal force is 100000-;

3) sucking the exosomes in different concentration intervals out, and centrifuging for 10-360 minutes under the centrifugal force of 50000-300000g to obtain precipitates;

resuspending with ultrapure water or phosphate buffered saline solution to obtain the Chinese medicinal exosome.

5. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: the length-diameter ratio of the gold nanorods is 1-5.

6. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: the preparation method of the polyvinylidene fluoride piezoelectric film comprises the following steps: coating by a spin coating method, and then annealing and polarizing to prepare a polyvinylidene fluoride piezoelectric film; the thickness of the film after spin coating is 10-50 μm, the annealing temperature is 60-150 ℃, the polarization temperature is 50-150 ℃, the voltage is 80-200V/μm, and the polarization time is 1-8 hours.

7. The method for preparing the piezoelectric composite dressing for promoting peripheral nerve repair and wound healing and loading Chinese medicinal exosomes according to claim 1, which is characterized in that: in the step D, in the conductive pre-gel system, the photocrosslinking time is 10-360 seconds, and the thickness of the methacrylate esterified gelatin after spin coating is 1-5 mm.

8. A composite dressing made according to the method of any one of claims 1 to 7.