CN113797393A

CN113797393A - A multi-level skin wound repair scaffold with integrated functions and preparation method thereof

Info

Publication number: CN113797393A
Application number: CN202111127416.6A
Authority: CN
Inventors: 吴桐; 周子艺; 冷向峰; 刘娜; 王元非; 张小佩; 宁胥超
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2021-09-26
Filing date: 2021-09-26
Publication date: 2021-12-17
Anticipated expiration: 2041-09-26
Also published as: CN113797393B

Abstract

The invention belongs to the technical field of medical materials, and relates to a function-integrated multi-level skin wound repair stent and a preparation method thereof.A main body structure of the repair stent is a three-layer stent deposited layer by layer, an upper-layer stent is a polycaprolactone nanofiber stent which is loaded with a centripetal gradient gelatin/epidermal growth factor bioactive coating and has centripetal orientation, a middle-layer stent is a polycaprolactone/berberine nanofiber stent, and a lower-layer stent is a reticular nanofiber stent of a coating acellular dermal matrix; the biomaterial with the gradient structure designed by the invention has better biocompatibility and degradability, especially has the potential of promoting the growth of cells, has important application in wound repair and regeneration tissue engineering, and provides a new thought for the functionalization of the nanofiber.

Description

Function-integrated multi-level skin wound repair support and preparation method thereof

The technical field is as follows:

the invention belongs to the technical field of medical materials, and particularly relates to a function-integrated multi-level skin wound repair stent and a preparation method thereof.

Background art:

wound healing is mainly composed of four stages of hemostasis, inflammation, proliferation and remodeling, but a single non-surgical treatment can only promote wound healing generally. In particular, traditional dressing management is excellent in accelerating the "hemostasis" phase; the antimicrobial dressing is irreplaceable for reducing the "inflammatory" phase, which is the rate-limiting step in burn wound healing; physical therapy promotes the "proliferation" and "remodeling" phases directly by electrons and photons. An ideal wound dressing must support all phases that require hemostasis, antimicrobial activity, cell proliferation and regeneration, respectively. The nanofiber scaffold prepared by electrostatic spinning is a key component in a tissue engineering concept, and the functional modification of the tissue engineering scaffold can enable the scaffold to simultaneously provide topological structure signals and biochemical signals, so that the migration and proliferation of cells can be promoted. The mysterious function of a functionalized scaffold is that it is capable of creating a customized microenvironment that directs cellular behavior through specific cell-substance interactions. Different topological structures of the nano-fiber are found to provide different contact clues to regulate the activities of cells by simulating the extracellular matrix, and support the proliferation, the angiogenesis and the stem cell multi-directional differentiation of the cells. However, the regulation of cellular activities by physical means alone is not sufficient to induce tissue regeneration in a timely manner, and the regeneration effect is not obvious, and physical features are combined with biochemical cues to provide cues, especially through the synergistic effect of induction of scaffold topology and chemotaxis of biochemical factors, which can help to rapidly establish a microenvironment that can mimic the structure and function of native ECM. Electrostatic spraying is an evolution of electrostatic spinning technology, provides a simple and convenient way to produce relatively uniform nanoparticles, and in recent years, electrostatic spraying is used as a main method for coating bioactive proteins, can simulate cell chemotaxis, further provides biological signals for cells and assists cell growth.

The epidermal growth factor is a good preparation for promoting wound healing, has good effects of improving and treating skin wound burn, and researches show that the epidermal growth factor has strong capacity of promoting migration and proliferation of epidermal cells, and the Acellular Dermal Matrix (ADM) mainly removes epidermis and cells of tissues by physical and chemical methods, retains the form, components and three-dimensional structure of extracellular matrix, provides a place for survival and metabolism of host cells, and further completes repair and reconstruction of defective tissues. The acellular dermal matrix and the epidermal growth factor have excellent biocompatibility and induction function of histiocytes, and are popularized and used as novel carrier materials for tissue engineering.

At present, no research report about the research method for preparing the gradient protein and the multifunctional integration of the three-layer structure in the research is found.

The invention content is as follows:

the invention aims to solve the technical problem of providing a function-integrated multi-level skin wound repair bracket and a preparation method thereof.

In order to achieve the purpose, the main structure of the functionally integrated multi-level skin wound repair scaffold provided by the invention is a three-layer scaffold deposited layer by layer, the upper-layer scaffold is a polycaprolactone nanofiber scaffold loaded with a centripetal gradient gelatin/epidermal growth factor bioactive coating and having centripetal orientation, the middle-layer scaffold is a polycaprolactone/berberine nanofiber scaffold, and the lower-layer scaffold is a reticular nanofiber scaffold coated with acellular dermal matrix.

The invention also provides a preparation method of the function-integrated multi-level skin wound repair bracket, which comprises the following specific steps:

(1) preparing an upper layer of the stent: dissolving polycaprolactone in a solvent to obtain a polycaprolactone spinning solution; taking polycaprolactone spinning solution as a raw material of the upper layer of the stent, adding a point electrode at the center of a circular electrode as a receiving device, and carrying out electrostatic spinning to obtain a centripetal-oriented polycaprolactone fiber stent; then the polycaprolactone fiber scaffold is used as a receiving device, the gelatin/epidermal growth factor mixed solution is subjected to electrostatic spraying, magnets with different diameters are replaced below the receiving device, and the polycaprolactone nanofiber scaffold which is loaded with the gelatin/epidermal growth factor bioactive coating with centripetal gradient and has centripetal orientation is obtained;

(2) preparing a middle layer of the stent: dissolving the mixture of gelatin and berberine in a solvent to obtain a gelatin/berberine spinning solution; taking gelatin/berberine spinning solution as a raw material of the middle layer of the stent, and carrying out random spinning to obtain the middle layer of the gelatin/berberine nano-fiber stent;

(3) preparing a lower layer of the bracket: dissolving a mixture of polycaprolactone and indocyanine green in a solvent to obtain polycaprolactone/indocyanine green spinning solution; taking polycaprolactone/indocyanine green spinning solution as a raw material of the lower layer of the stent, preparing nano yarn, manually preparing a grid structure, performing light welding on cross points, then randomly spinning gelatin spinning solution on the grid basis, and performing electrostatic spray coating on the acellular dermal matrix spraying solution to obtain the reticular nano fiber stent of the coating acellular dermal matrix;

(4) preparing a function-integrated multi-level skin wound repair bracket: and depositing the three layers of the supports layer by layer to obtain the function-integrated multi-layer skin wound repair support.

In the step (1), the solvent is hexafluoroisopropanol, and the mass-volume ratio of polycaprolactone to hexafluoroisopropanol is 0.12g:1 mL.

The preparation method of the gelatin/epidermal growth factor mixed solution in the step (1) comprises the following steps: dissolving gelatin and epidermal growth factor in acetic acid, wherein the mass volume ratio of the gelatin to the epidermal growth factor to the acetic acid is 0.006 g:1mL of: 1 mL.

In the step (2), the solvent is hexafluoroisopropanol, and the mass volume ratio of the gelatin to the berberine to the hexafluoroisopropanol is 0.12g: 0.0012g-0.012 g:1 mL.

In the step (3), the solvent is hexafluoroisopropanol, and the mass-volume ratio of the polycaprolactone to the acellular dermal matrix to the hexafluoroisopropanol is 0.12g: 0.001 g:1 mL.

The preparation method of the acellular dermal matrix spray liquid in the step (3) comprises the following steps: dissolving the acellular dermal matrix in hexafluoroisopropanol to obtain an acellular dermal matrix spray solution, wherein the mass-volume ratio of the acellular dermal matrix to the hexafluoroisopropanol is 0.02 g:1 mL.

The preparation method of the gelatin spinning solution in the step (3) comprises the following steps: dissolving gelatin in hexafluoroisopropanol to obtain a gelatin spinning solution, wherein the mass volume ratio of the gelatin to the hexafluoroisopropanol is 0.12g:1 mL.

The voltage of the PCL spinning solution for electrostatic spinning in the step (1) is 10KV, the flow rate is 1mL/h, and the duration is 10 min.

The voltage of the gelatin/epidermal growth factor spray liquid electrostatically sprayed in the step (1) is 18KV, the flow rate is 0.5mL/h, and the duration of each gradient is 30 s.

And (3) in the step (2), the voltage of the electrostatic spinning gelatin/berberine spinning solution is 15KV, the flow rate is 1mL/h, the duration is 1h-2h, a roller is used as a receiving device, and the rotating speed is 900 rmp.

And (3) in the step (3), the electrostatic spinning polycaprolactone/indocyanine green mixed solution has a positive voltage of 9.6KV, a negative voltage of 3.5KV, a flow rate of 1mL/h and a duration of 1h-2h, is received by a collecting roller, is manually placed into a grid-shaped structure (with the length of 3mm), and is subjected to photosolder intersection points.

And (3) the voltage of the spraying liquid of the electrostatic spraying ADM in the step (3) is 18KV, the flow rate is 0.5mL/h, and the duration is 3 min.

And (3) in the step (3), the voltage of the electrostatic spinning gelatin spinning solution is 10KV, the flow rate is 1mL/h, the duration is 2min, and the grid fiber support subjected to the photosolder is used as a receiving device.

The invention also provides application of the function-integrated multi-level skin wound repair scaffold in preparation of materials for promoting wound healing.

The invention relates to a function-integrated multi-level skin wound repair bracket, which is a multi-level bioactive bracket penetrating through all healing processes of cells, wherein the upper layer of the bracket is a centripetally oriented nanofiber bracket, and gradient deposition is carried out by a simple method for changing magnetic field force distribution, a bioactive factor (epidermal growth factor) forms gradient in an electrostatic spraying manner, so that healing speed of the damaged wound in different directions is accelerated, cell migration in skin is guided, and skin tissues are pulled to be gradually reduced from the periphery to the central wound defect; the middle layer of the bracket consists of a nano fiber bracket of antibacterial traditional Chinese medicine berberine and a bioactive substance gelatin, and aims to solve the problem that the wound is not healed for a long time due to bacterial infection in the process of healing the wound; the lower layer of the scaffold is prepared into a reticular topological structure and coated with ADM, so that a three-dimensional framework for growth and metabolism is provided for tissue cells, and the reticular fibrous scaffold regulates and controls the growth factor substance paracrine by adipose-derived stem cells to accelerate the remodeling of tissues, thereby completing the physiological repair of the tissues. The invention provides a conceptual model of a function-integrated multi-level skin wound repair bracket, mainly aims at further research on healing of chronic wounds and burn wounds at present, and has important research significance and application value in tissue engineering repair.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention selects natural materials similar to human body such as epidermal growth factor, acellular dermal matrix, gelatin and polymer material polycaprolactone with good biocompatibility as basic materials for the first time to prepare the multi-layer bioactive skin tissue repair bracket with gradient bioactive protein. Different structures and biological functions of the multi-level scaffold are designed aiming at the regeneration of epidermis and dermis tissues, the multi-level scaffold is suitable for the healing of large-area wound surfaces, and the provided physical clues and biochemical signals cooperate to guide the migration of cells from the edge part to the central part.

(2) The biomaterial with the gradient structure designed by the invention has better biocompatibility and degradability, particularly has the potential of promoting the growth of cells, has important application in wound repair and regeneration tissue engineering, and provides a new thought for functionalization of nano fibers.

Description of the drawings:

FIG. 1 is a schematic diagram of the structural principle of the device for preparing the polycaprolactone fiber scaffold on the upper layer of the scaffold.

FIG. 2 is a schematic diagram of the principle of the method for preparing the centripetal gradient step of loading the epidermal growth factor in the upper layer of the stent according to the present invention.

Fig. 3 is a schematic diagram of a gradient characterization result of the function-integrated multi-level skin wound repair scaffold.

FIG. 4 is a graph showing the experimental results of the effect of gradient signals of bioactive proteins on epidermal cell migration in example 2, wherein A-C are fluorescence micrographs of the migration status of fibroblasts of the present invention after growing on the surface of a circular gradient coating for 3 days; D-E statistics of total cell number and cell number in three regions.

FIG. 5 is a graph showing the results of the antibiotic test of different ratios of gelatin/berberine in the layer of the scaffold of example 3 after incubation for 24h in a uniform Staphylococcus aureus agarose plate.

Fig. 6 is a fluorescent microscope photograph of cytoskeleton after adipose-derived stem cells are seeded on the multi-level skin wound repair scaffold with function integration in example 4 according to the invention and grow for 3 days.

Fig. 7 is a diagram of a three-layer stent of a function-integrated multi-layer skin wound repair stent, wherein a is a stent upper layer (a centripetal orientation stent), B is a stent middle layer (a traditional Chinese medicine berberine random stent), and C is a stent lower layer (a reticular fiber stent).

Fig. 8 is a schematic diagram of animal experimental results of the function-integrated multi-level skin wound repair scaffold.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples and figures.

Example 1:

the embodiment relates to a preparation method of a function-integrated multi-level skin wound repair bracket, which comprises the following specific steps:

(1) preparing an upper layer of the stent: dissolving 0.24g of polycaprolactone in 2mL of hexafluoroisopropanol, and magnetically stirring until the polycaprolactone is completely dissolved to obtain a polycaprolactone spinning solution; taking 2mL of polycaprolactone spinning solution, adding a point electrode at the center of a ring electrode as a receiving device, sucking the polycaprolactone spinning solution into a 5mL syringe, connecting a No. 20 stainless steel needle and connecting with 10KV high voltage, wherein the flow rate is 1mL/h, and the duration is 10min, so as to obtain a centripetal-oriented nanofiber scaffold; then taking 2mL of gelatin/epidermal growth factor mixed solution, sucking the mixed solution into a 5mL syringe, connecting a No. 20 stainless steel needle and connecting with 18KV high pressure, and controlling a micro-injection pump to adjust the flow rate (mL/h) of the mixed solution to be as follows: 0.3mL/h, adopting an electrostatic spraying method, taking a centripetal orientation support as a receiving device, placing round magnets with the diameters of 4/8/12 below the support to prepare a gradient, adding no magnet to the last layer, and keeping the duration of each layer for 30s to obtain the centripetal orientation nanofiber support loaded with the centripetal gradient of the epidermal growth factor as the upper layer of the support;

the preparation method of the gelatin/epidermal growth factor mixed solution comprises the following steps: dissolving 0.006g of gelatin in 1mL of acetic acid, mixing with 1mL of epidermal growth factor mother liquor, and magnetically stirring until the gelatin is completely dissolved to obtain a gelatin/epidermal growth factor mixed solution;

the fluorescence intensity test below proves that this example generates a circular gradient of active protein on the centripetally arranged nanofibers,

in order to visualize the gradient, rhodamine B is used as a dye, and the diameter and the number of the round magnets (figure 2) are changed according to the method of the step (1) to respectively obtain the nanofiber scaffold covered with the rhodamine coating with low gradient, high gradient and uniformity. FIG. 3 shows the relative fluorescence intensity at different positions on the nanofiber scaffold, with the relative fluorescence intensity in the central region being nearly 20 times that in the peripheral region (FIG. 3, low gradient). The slope of this gradient can also be increased by using a mold whose dimensions are exactly the same as the underlying magnet (fig. 3, high gradient), in which case there is a 40-fold difference in relative fluorescence intensity between the center and the periphery.

These results confirm that nanofibers of active proteins aligned along radial directions successfully produced circular gradients.

(2) Preparing a middle layer of the stent: 5mL of gelatin/berberine spinning solution is sucked into a 5mL injector, a random electrostatic spinning mode is adopted, a No. 20 stainless steel needle head is connected and is connected with 10KV high voltage, the flow rate of an injection pump is controlled to be 1mL/h, the duration is 2h, and random nano fibers are received by a roller at the rotating speed of 900 rmp; namely the middle layer of the bracket;

the preparation method of the gelatin/berberine spinning solution comprises the following steps: respectively dissolving 0.6g of a mixture of gelatin and berberine in 5mL of hexafluoroisopropanol, and magnetically stirring until the mixture is completely dissolved; wherein the mass ratio of gelatin to berberine is 1: 1;

(3) preparing a lower layer of the bracket: respectively sucking 10mL of polycaprolactone/indocyanine green spinning solution into 5mL of injectors, connecting a No. 20 stainless steel needle in a nano yarn pair spinning mode, connecting the needle with a 9.6KV positive voltage and a 3.5KV negative voltage, controlling the flow rate of an injection pump to be 1mL/h, receiving yarns by a collecting roller, manually placing the yarns to form a grid structure, and performing light welding on four corners of a grid; filling 2mL of gelatin spinning solution into a 5mL injector, connecting a No. 20 stainless steel needle and connecting with 10KV high voltage, controlling the flow rate of a micro injection pump to be 1mL/h, and receiving by the grid fiber support to obtain a support lower layer; dissolving ADM in hexafluoroisopropanol to obtain ADM spraying liquid, wherein the mass volume ratio of ADM to hexafluoroisopropanol is 0.02 g:1mL, performing ADM electrostatic spraying on the surface of the lower layer of the support, wherein the voltage is 18KV, the flow rate is 0.5mL/h, and the duration is 3min, so as to obtain the lower layer of the support containing ADM bioactivity.

The preparation method of the gelatin/berberine spinning solution comprises the following steps: dissolving 1.2g of gelatin and 0.12g of berberine in 10mL of hexafluoroisopropanol, and magnetically stirring until the gelatin and the berberine are completely dissolved to obtain a gelatin/berberine spinning solution;

the preparation method of the polycaprolactone/indocyanine green spinning solution comprises the following steps: dissolving 1.2g of polycaprolactone/indocyanine green mixture in 10mL of hexafluoroisopropanol, and magnetically stirring until the polycaprolactone/indocyanine green mixture is completely dissolved to obtain polycaprolactone/indocyanine green spinning solution; wherein the mass ratio of the polycaprolactone to the indocyanine green is 120: 1;

Example 2:

this example is an experiment to investigate the effect of bioactive protein gradient signals on epidermal cell migration. Firstly, according to the method in the step (1) of the embodiment 1, a centripetal gradient coating fiber sheet, a uniform coating fiber sheet and a pure glass sheet are respectively prepared, the pure glass sheet is used as a blank control group and is respectively placed in holes of a 24-hole plate, a Polydimethylsiloxane (PDMS) cylinder with the diameter of 6mm is placed at the central part of a fiber scaffold in each hole, epidermal cells are respectively and uniformly planted in the peripheral area of the PDMS cylinder which is not covered on the fiber scaffold, and the cell planting concentration is 5 multiplied by 10⁵cells/mL. After 6h of cell adhesion, the PDMS cylinder was removed and the cells started to migrate.

This example studies the effect of gradient coating of bioactive protein on human skin fibroblast migration and uses uniform fibronectin coating and blank slides as controls to promote full-scale wound healing. FIGS. 4A-C show migration of fibroblasts after 3 days of culture on gradient protein coating and uniform protein coating covered fiber slides and show migration on blank slides. More and more cells tend to migrate throughout the migration zone of the gradient set. As shown in fig. 4D, the total number of cells in the gradient across the migrated zone was significantly increased relative to the homogeneous group (P <0.05) and the blank slide (P < 0.01). It is clear that chemotaxis provided by the bioactive protein gradient induces migration of cells to regions with higher protein content. This example also equally divides the migration zone into three regions, and measures the number of migrated cells in the different regions. In the region closest to the seeding zone (region I), the gradient group had more migrating cells (P <0.05) than the blank slide. As shown in fig. 4E, cells tended to migrate to a position farther on the gradient set relative to the homogeneous and blank slides. The number of migrated cells was significantly increased in the gradient group II and III regions compared to the homogeneous group and the blank slide (P <0.05 and P < 0.01).

Example 3:

in this example, an experiment on antibacterial performance of berberine in the middle layer of the scaffold was performed, and a berberine/gelatin nanofiber scaffold containing 1%, 5% and 10% berberine was prepared according to the preparation method of the middle layer of the scaffold prepared in the step (2) of example 1, and the pure gelatin nanofiber scaffold was used as a control group. The antibacterial performance of the antibacterial agent is measured by a disc agar diffusion method. First, it will be approximately 1 × 10⁸The E.coli suspension was inoculated per mL onto nutrient agar plates 9cm in diameter. The middle layer of the berberine scaffold with different proportions is cut into a circle with the diameter of 1.5 cm. The nanofiber mat was soaked with Phosphate Buffered Saline (PBS) and then covered in the center of the agar plate. The whole process is carried out under aseptic conditions. The agar plates were then incubated at 37 ℃ for 24h and further observed.

The preparation method of the 1%, 5% and 10% berberine/gelatin spinning solution comprises the following steps: 1% berberine/gelatin spinning solution: dissolving 0.6g of gelatin and 0.006g of berberine in 5mL of hexafluoroisopropanol, and magnetically stirring until the gelatin and the berberine are completely dissolved to obtain 1% of a berberine/gelatin spinning solution; 5% berberine/gelatin spinning solution: dissolving 0.6g of gelatin and 0.03g of berberine in 5mL of hexafluoroisopropanol, and magnetically stirring until the mixture is completely dissolved to obtain 5% berberine/gelatin spinning solution; 10% berberine/gelatin spinning solution: 0.6g of gelatin and 0.06g of berberine were dissolved in 5mL of hexafluoroisopropanol and magnetically stirred until completely dissolved, yielding a 10% berberine/gelatin spinning solution.

The antibacterial effect of different amounts of berberine was evaluated by measuring the size of the E.coli growth inhibitory antibacterial zone formed by the middle layer of the scaffold on the agar plate. As shown in fig. 5, the nanofiber scaffolds of pure gelatin and 1% berberine were substantially free of growth inhibition. And when the berberine content is increased to 5% and 10%, the diameter of the growth inhibition zone is increased. The size of the growth inhibition zone of the nanofiber dressing depends on the diffusion distance that the berberine portion of the middle layer fibers generates throughout the inner fiber pores. Bacteria in wound infections are generally derived from bacteria that survive wound disinfection and those that invade from the external environment. The antibacterial dressing in the middle layer of the bracket designed by the technical scheme aims to establish an effective area with the highest antibacterial concentration so that external invasive microorganisms are inactivated in the process of passing through the middle layer.

Example 4:

the embodiment is a morphological test of a multi-level skin wound repair bracket with integrated functions on adipose-derived stem cells: the adipose-derived stem cells are inoculated on a multi-level skin wound repair support with integrated functions, cultured for 3 days, fixed by 4% paraformaldehyde for 20min, permeated by 0.1% TritonX-100 solution for 5min, sealed by 1% bovine serum albumin solution for 1h, and then stained with pharloid-iFluor 488 and DAPI for actin cytoskeletons and cell nuclei. Between each run, samples were washed three times with PBS. The microscope was obtained using a fluorescence upright microscope (nikon, tokyo, japan).

In the figure, 6 fluorescence micrographs show the cell morphology of the adipose-derived stem cells on a multi-level scaffold. The cell skeleton of the adipose-derived stem cells is in a shuttle shape and is in a stretched shape. The overall result shows that the function-integrated multi-level skin wound repair scaffold has positive influence on the expansion of adipose stem cells.

Example 5:

the embodiment is an animal experiment of a function-integrated multi-level skin wound repair bracket, and specifically comprises the following steps: 9 SD rats (280 +/-10 g) are taken and adaptively fed for 2 days, and then the back is preserved for 24 hours before operation. Rats were anesthetized using a 10% chloral hydrate injection of 0.35ml/100g abdominal cavity. The iodophor enlarges the sterilization area, designs 4 circles with the diameter of 1.5cm on the back by using a ruler, uniformly removes the whole skin by using sterile tissue scissors, manufactures a whole skin defect model, observes the condition of the wound surface every other day, and sterilizes the wound surface by a conventional method.

After the preparation of the wound surface is finished, dividing the rats into 4 groups, namely a blank group, a gauze group, a multi-level repair scaffold group and a multi-level repair scaffold loaded adipose-derived stem cell group, wherein the blank group is not treated, and the gauze group is used for covering the wound surface of the rats with gauze; the multi-level repairing bracket group is used for treating a multi-level skin wound repairing bracket with integrated rat wound functions; the multi-level repairing scaffold loaded adipose-derived stem cell group provides the rat wound surface with the multi-level skin wound surface repairing scaffold treatment with the function integration of the adipose-derived stem cells; the wound healing was gross on

days

0, 4, 7 and 14 post-surgery, respectively, photographed with a digital camera. The Image-Pro Plus 6.0 software analyzes the healing condition of the wound surface. The results are shown in FIG. 8.

As shown in fig. 8, the wound sites treated with the multi-layered scaffold group and the multi-layered scaffold adipose-loaded stem cell group were almost completely closed by epithelialization, while the wound sites of the gauze group and the blank control group remained unchanged. On day 4, the blank group and the gauze group were clearly in the inflammatory infection stage, while the wounds of rats in the multi-layered scaffold group and the multi-layered scaffold adipose-loaded stem cell group were scabbed, and the scabs formed prevented invasion of foreign microorganisms, and on day 14, the wounds of the multi-layered scaffold-loaded adipose-loaded stem cell group were significantly smaller than those of the other three groups. Therefore, the function-integrated multi-level skin wound repair scaffold can regulate and control the secretion of adipose stem cells to accelerate the healing of wounds.

Claims

1. a multi-level skin wound repair support of functional integration, it is characterized in that, the main structure is the three-layer support of layer-by-layer deposition, and the upper support is the gelatin/epidermal growth factor bioactive coating of the load centripetal gradient with the orientation. Heart-oriented polycaprolactone nanofiber scaffold, the middle layer is a polycaprolactone/berberine nanofiber scaffold, and the lower layer is a mesh nanofiber scaffold coated with acellular dermal matrix.

2. the preparation method of the multi-level skin wound repairing support of functional integration as claimed in claim 1, is characterized in that, concrete steps comprise:

(1) Preparation of the upper layer of the stent: dissolving polycaprolactone in a solvent to obtain a polycaprolactone spinning solution; using the polycaprolactone spinning solution as a raw material for the upper layer of the stent, and adding a point electrode to the center of the ring electrode as a receiving Then, the polycaprolactone fiber scaffold was used as a receiving device, and the gelatin/epidermal growth factor mixture was electrostatically sprayed, and different diameters were replaced under the receiving device. The magnet, that is, a polycaprolactone nanofiber scaffold with centripetal orientation loaded with a centripetal gradient gelatin/epidermal growth factor bioactive coating;

(2) Preparation of the middle layer of the stent: the mixture of gelatin and berberine is dissolved in a solvent to obtain a gelatin/berberine spinning solution; the gelatin/berberine spinning solution is used as the raw material of the middle layer of the stent, and random spinning is performed to obtain gelatin /Berberin nanofiber scaffold middle layer;

(3) Prepare the lower layer of the scaffold: dissolve the mixture of polycaprolactone and indocyanine green in a solvent to obtain a polycaprolactone/indocyanine green spinning solution; mix the polycaprolactone/indocyanine green spinning solution As the raw material for the lower layer of the scaffold, nano-yarns were prepared, mesh structures were manually prepared, intersections were photo-welded, and then the gelatin spinning solution was randomly spun on the mesh basis, and the acellular dermal matrix spray solution was electrostatically spray-coated , that is, the reticulated nanofiber scaffold coated with acellular dermal matrix is obtained;

(4) Preparation of a multi-level skin wound repair scaffold with integrated functions: the above-mentioned three-layer scaffold is deposited layer by layer to obtain a multi-level skin wound repair scaffold with integrated functions.

3. The preparation method of the multi-level skin wound repair stent with integrated functions according to claim 2, characterized in that, in step (1), the solvent is hexafluoroisopropanol, polycaprolactone and hexafluoroisopropanol The mass-volume ratio of 0.12g:1mL.

4. The preparation method of the multi-level skin wound repair scaffold with integrated functions according to claim 2, wherein the preparation method of the gelatin/epidermal growth factor mixed solution in the step (1) is: The growth factor was dissolved in acetic acid, and the mass-volume ratio of gelatin, epidermal growth factor and acetic acid was 0.006 g: 1 mL: 1 mL.

5. the preparation method of the multi-level skin wound repair support of functional integration according to claim 2, is characterized in that, in step (1), the voltage of electrostatic spray gelatin/epidermal growth factor spray liquid is 18KV, and flow velocity is 0.5mL /h, the duration of each gradient is 30s.

6. The preparation method of the multi-level skin wound repair scaffold with integrated functions according to claim 2, wherein the solvent in step (2) is hexafluoroisopropanol, gelatin, berberine and hexafluoroisopropanol The mass-volume ratio of 0.12g:0.0012g-0.012g:1mL.

7. the preparation method of the multi-level skin wound repairing support of functional integration according to claim 2, is characterized in that, in step (2), the voltage of electrospinning gelatin/berberine spinning solution is 15KV, and the flow rate is 1mL /h, the duration is 1h-2h, the drum is used as the receiving device, and the speed is 900rmp.

8. The method for preparing a multi-level skin wound repair scaffold with integrated functions according to claim 2, wherein in step (3), the solvent is hexafluoroisopropanol, polycaprolactone, acellular dermal matrix and The mass-to-volume ratio of hexafluoroisopropanol is 0.12g:0.001g:1mL; the preparation method of the acellular dermal matrix spray liquid is as follows: the acellular dermal matrix is dissolved in hexafluoroisopropanol to obtain the acellular dermal matrix spray liquid, The mass-volume ratio of acellular dermal matrix to hexafluoroisopropanol was 0.02 g: 1 mL.

9 . The method for preparing a multi-level skin wound repair scaffold with integrated functions according to claim 2 , wherein the positive voltage of the electrospinning polycaprolactone/indocyanine green mixed solution in step (3) is: 10 . 9.6KV, the negative voltage is 3.5KV, the flow rate is 1mL/h, the duration is 1h-2h, the collecting roller is received, manually placed in a grid-like structure, and the intersection point of light welding; the voltage of the electrostatic spray ADM spray liquid is 18KV, The flow rate was 0.5mL/h, and the duration was 3min; the voltage of the electrospinning gelatin spinning solution was 10KV, the flow rate was 1mL/h, and the duration was 2min, and the light-welded mesh fiber scaffold was used as the receiving device.

10 . The application of the functionally integrated multi-level skin wound repair scaffold according to claim 1 in the preparation of materials for promoting wound healing. 11 .