CN110812533B

CN110812533B - Preparation method of nanofiber gel composite matrix for constructing skin tissue

Info

Publication number: CN110812533B
Application number: CN201911123977.1A
Authority: CN
Inventors: 陈苏; 崔婷婷; 余加飞; 王格飞
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2021-10-29
Anticipated expiration: 2039-11-18
Also published as: CN110812533A

Abstract

The invention relates to a preparation method of a nanofiber gel composite matrix for constructing skin tissues, which comprises the steps of injecting a main body gel dissolving solution in a porcine fibrin adhesive into main body gel lyophilized powder to obtain a fibrin solution; adding polycaprolactone and silk fibroin into a container containing formic acid to obtain a PCL/SF spinning solution; taking the PCL/SF spinning solution as an internal phase and the fibrin solution as an external phase, and spinning to obtain a nano fabric consisting of the PCL/SF core-shell nano fibers coated by fibrin; and spraying the freeze-dried powder catalyst solution on the obtained nano fabric to form gel, thereby obtaining the nano fiber gel composite matrix for constructing the skin tissue. The method is easy to operate and universal, and can realize large-area skin regeneration at the skin defect part with the full thickness of the abdomen.

Description

Preparation method of nanofiber gel composite matrix for constructing skin tissue

Technical Field

The invention relates to a preparation method of a nano fabric, in particular to a preparation method of a nanofiber gel composite matrix for constructing skin tissues.

Background

In nature, human avoidance of external aggressions relies primarily on the skin. The skin, which is the external epithelium of the body, maintains homeostasis in the body and repairs lesions throughout life. It is highly desirable to mimic the skin to obtain artificial materials with advanced potential. To this end, a number of techniques (e.g., skin grafting, reprogramming of wound resident cells) and materials (e.g., porous foams, biocompatible membranes, biomaterials, and functional gels) have been developed. However, research progress to repair the abdomen for extensive burns and even intestinal exposure is inefficient and this is indeed a major challenge in this field. In order to rapidly construct skin tissue of a patient for regeneration, a more rapid and efficient skin regeneration method and a new artificial material, especially one having excellent biocompatibility, tissue regeneration and non-surgical intervention, must be developed, otherwise the artificial material never compares favorably with human skin.

The nanofabric provides an extracellular skin-like matrix that can better replenish cells and can readily bind bioactive molecules, enhancing penetration of nutrients and oxygen. Therefore, the preparation of the ultrathin-diameter, high-permeability, good interconnected pore structure and biocompatible ultrafine nanofiber membrane on a large scale has important significance as a biological material. However, it seems not easy to prepare large area nanofiber scaffold materials based on current fiber spinning techniques, electrostatic spinning, micro-fluidic spinning and solution air-jet spinning. At the same time, the availability of nanofiber scaffold sealants to ensure a moist skin healing environment and skin tissue regeneration remains a barrier. It is highly desirable that the sealant be synchronously coupled to the fabric scaffold, which will allow fibroblasts to easily bind to the nanofiber scaffold, accelerate tissue regeneration, and eliminate inflammation and toxic effects. Thus, rapid preparation of nanogel composite matrices using simple, efficient, low cost methods can combine the advantages of fibrous matrices (strong mechanical properties) and gels (maintaining a moist wound healing environment).

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of a nanofiber gel composite matrix for constructing skin tissues, which is easy to operate and universal and can realize large-area skin regeneration at the skin defect with full thickness of the abdomen.

The technical scheme of the invention is as follows: a preparation method of a nanofiber gel composite matrix for constructing skin tissues comprises the following specific steps:

a. injecting the main body gel dissolving solution in the pig-derived fibrin adhesive into the main body gel lyophilized powder to obtain fibrin solution; adding polycaprolactone PCL and silk fibroin SF into a container containing formic acid FA, and stirring at room temperature to a solution state, thereby obtaining a PCL/SF spinning solution;

b. b, spinning by taking the PCL/SF spinning solution obtained in the step a as an internal phase and taking a fibrin solution as an external phase, wherein a certain air pressure is set in the spinning process, the nano fibers are collected on a nylon 66 screen mesh to obtain a nano fiber membrane consisting of PCL/SF core-shell nano fibers coated by fibrin, and the screen mesh is away from a nozzle of the injector by a certain distance; then, drying the nanofiber membrane at room temperature in vacuum to remove residual FA; obtaining a nano fabric consisting of PCL/SF core-shell nano fibers coated by fibrin;

c. and c, injecting the catalyst solution in the pig-derived fibrin adhesive into the catalyst freeze-dried powder to obtain a freeze-dried powder catalyst solution, and spraying the freeze-dried powder catalyst solution on the nano fabric obtained in the step b to form gel so as to obtain the nano fiber gel composite matrix for constructing the skin tissue.

Preferably, the mass concentration of the main gel lyophilized powder in the fibrin solution in the step a is 1.9-4.8%.

Preferably, the mass concentration of the formic acid solution in the step a is 85-95%.

Preferably, the mass concentration of the PCL/SF spinning solution in the step a is 14-25%; wherein the mass ratio of PCL to SF is 0.25-2.5.

Preferably, the air pressure in the step b is in the range of 0.01-0.5 MPa; the distance between the screen and the nozzle of the injector in the step b ranges from 23 cm to 37 cm; in step b, the flow rate of the inner phase and the flow rate of the outer phase are both 0.1-5 mL/h.

Preferably, the fiber diameter of the nanofabric in step b is 44-250 nm.

Preferably, the area of the nano fabric in the step b is 4X 4-40X 140cm²。

Preferably, the temperature of vacuum drying in the step b is 25-35 ℃; the vacuum drying time is 6-12 h.

Preferably, the tensile strength of the nanofabric in step b is in the range of 2.3-6.2 MPa.

Preferably, the tensile strength of the nanofiber gel composite matrix obtained in the step c is 6.8-8.2 MPa.

Preferably, the weight average molecular weight of the polycaprolactone PCL is 80000; the concentration of the catalyst freeze-dried powder in the freeze-dried powder catalyst solution obtained in the step c is preferably 0.06-0.1g/ml (the volume ratio of the mass of the catalyst freeze-dried powder to the freeze-dried powder catalyst solution).

We developed a microfluidic air-jet spinning process to realize the degradable fibrin-coated polycaprolactone/silk fibroin (PCL/SF) large-area nano fabric (40 x 140 cm)²) The minimum fiber diameter can reach 44 nm. The prepared nano flocculant is prepared from materials which are biodegradable, have good biocompatibility and are easy to combine with fibrin glue, such as PCL, SF, fibrin and the like. And the nanofiber fabric gel composite matrix successfully realizes the regeneration of abdominal skin tissues and goes through three stages of the preparation of the nanofiber fabric, the reconstruction of the skin tissues and the formation of artificial skin. The artificial skin is based on the reaction of fibrin catalyst and nano fibrin, and the final product can be used as sealant, and can maintain moist wound healing environment and certain tissue adhesion force in the process of skin tissue regeneration.

Has the advantages that:

1. the nano gel composite matrix prepared by the invention is defined as the fiber of the nano fabric of the artificial skin, and has the characteristics of adjustable diameter and controllable appearance.

2. The preparation method of the nano-gel composite matrix defined as the nano-fabric of the artificial skin has the advantages of simple equipment and convenient operation, and can realize large-scale preparation.

3. The tensile strength of the nano-fabric defined as the artificial skin by the nano-gel composite matrix prepared by the invention can be regulated and controlled by the ratio of PCL to SF.

4. The nanogel composite matrix prepared by the invention can combine the advantages of a fiber matrix (strong mechanical property) and a gel (maintaining a moist wound healing environment).

5. The nano-gel composite matrix prepared by the invention has excellent biocompatibility and histocompatibility.

6. The nano fabric in the nano gel composite matrix prepared by the invention has abundant porous structure and high surface area, and can promote recruitment of cells.

7. The artificial skin in the nano gel composite matrix prepared by the invention can promote angiogenesis, collagen deposition and granulation tissue formation, and effectively prevent full-layer skin defect wound infection.

Drawings

FIG. 1 is an SEM image of a nanofabric of a nanogel composite matrix prepared in example 1;

FIG. 2 is a graph of the particle size distribution of the nanofabric of the nanogel composite matrix prepared in example 1;

FIG. 3 is a schematic representation of a large area nanofabric of the nanogel composite matrix prepared in example 1;

fig. 4 is a diagram of quantitative analysis for verifying the cytocompatibility of the nanogel composite matrix prepared in example 1.

Detailed Description

The present invention is illustrated below by way of specific examples, but the present invention is not limited to the following examples, and the porcine fibrin adhesive described in the following examples is purchased from general hospitals in the military region of Nanjing.

Example 1

15g of the main gel hydrolysate in the pig-derived fibrin adhesive was injected into 0.3g of the main gel lyophilized powder to obtain a 1.96 wt% fibrin solution. Then, 0.5g of PCL (weight average molecular weight 80000) and 2.0g of dry SF were dissolved in a solution containing 15g of formic acid (the mass concentration of the formic acid solution is 85%), and the solution was sufficiently stirred at room temperature for 6 hours to obtain a PCL/SF spinning solution with a mass fraction of 14.2 wt% (the mass ratio of PCL to SF is 0.25). The obtained PCL/SF spinning solution is used as an internal phase, the fibrin solution is used as an external phase, and the two solutions are used as a reservoir of a precursor and are filled into an injector. The two injectors are respectively connected to two inlets of the T-shaped microfluidic chip and used as a reactor for preparing the fibrin-coated PCL/SF nano-fiber. The prepared spinning solution was fed using a stainless steel blunting needle (24G) at a fixed feed rate (0.1 mL/h, 0.5mL/h inside and outside, respectively). During MBS, the air pressure was maintained at 0.01 MPa. The nanofibers were collected on a nylon 66 screen 23 cm from the injector nozzle. Preparing a PCL/SF core-shell nanofiber membrane coated by fibrin, and performing vacuum drying at 25 ℃ for 6h to remove residual FA to obtain a nano fabric, wherein the fiber diameter of the nano fabric is between 44 and 96nm, and the nano fabric is shown in an SEM image of the nano fabric shown in figure 1 and a particle size diagram shown in figure 2; the obtained nanometer fabric has tensile strength of 2.3MP and area size of 40 × 140cm². And finally, injecting a catalyst solution in the pig-derived fibrin adhesive into the freeze-dried catalyst powder to obtain a freeze-dried catalyst solution with the concentration of the freeze-dried catalyst powder of 0.06g/mL, and spraying the freeze-dried catalyst solution on the nano fabric to enable the shell fibrin and the catalyst thereof to carry out gelation reaction by gas spraying so as to prepare the fiber gel composite matrix as the artificial skin. The physical diagram of the large-area nano fabric of the prepared nano gel composite matrix is shown in figure 3. The tensile strength of the obtained fiber-gel composite matrix was 8.2 MPa. Then the biocompatibility and the non-toxic property of the artificial skin material are verified through a cell experiment. Finally, the histocompatibility of the artificial skin is verified through in vivo dying staining experiment, as shown in figure 4, the nanofiber gel composite membrane is shown on the 3 rd dayThe surface cell activity can reach 90%, and the product has good cell compatibility and no toxicity. Thereby promoting a series of functions of skin regeneration, such as promoting angiogenesis, collagen deposition and granulation tissue formation, and effectively preventing wound infection of full-layer skin defect.

Example 2

15g of the main gel hydrolysate in the pig-derived fibrin adhesive was injected into 0.5g of the main gel lyophilized powder to obtain a fibrin solution of 3.33 wt%. Then, 0.9g of PCL (weight average molecular weight 80000) and 2.1g of dry SF were dissolved in 15g of formic acid, and they were sufficiently stirred at room temperature for 6 hours to obtain a PCL/SF spinning solution (0.428, w/w) having a mass fraction of 16.7% by weight. The obtained PCL/SF spinning solution is used as an internal phase, the fibrin solution is used as an external phase, and the two solutions are used as a reservoir of a precursor and are filled into an injector. The two injectors are respectively connected to two inlets of the T-shaped microfluidic chip and used as a reactor for preparing the fibrin-coated PCL/SF nano-fiber. The prepared spinning solution was fed using a stainless steel blunting needle (24G) at a fixed feed rate (1.5 mL/h for inside and 5mL/h for outside, respectively). During MBS, the air pressure was maintained at 0.35 MPa. The nanofibers were collected on a nylon 66 screen, which was 30 cm from the injector nozzle. A nanofabric consisting of fibrin-coated PCL/SF core-shell nanofibers was prepared, with fiber diameters between 60-160 nm. The obtained nanometer fabric has tensile strength of 5.5MPa and area size of 4 × 4cm². And vacuum-dried at 30 ℃ for 8h to remove residual FA. And finally, injecting a catalyst solution in the pig-derived fibrin adhesive into the freeze-dried catalyst powder to obtain a freeze-dried catalyst solution with the concentration of the freeze-dried catalyst powder of 0.08g/ml, and spraying the freeze-dried catalyst solution on the nano fabric to enable the shell fibrin and the catalyst thereof to carry out gelation reaction by gas spraying so as to prepare the fiber gel composite matrix as the artificial skin. The tensile strength of the obtained fiber-gel composite matrix was 7.1 MPa. The biocompatibility and non-toxic properties of the artificial skin material were then verified by cell experiments as well. Finally, the in vivo living and dead staining experiment shows that the artificial skin has excellent histocompatibilityAnd the cell activity on the surface of the nanofiber gel composite membrane can reach 85%, and the nanofiber gel composite membrane has good cell compatibility and no toxicity. Thereby promoting a series of functions of skin regeneration, such as promoting angiogenesis, collagen deposition and granulation tissue formation, and effectively preventing wound infection of full-layer skin defect.

Example 3

15g of the main gel hydrolysate in the pig-derived fibrin adhesive was injected into 0.75g of the main gel lyophilized powder to obtain a 4.76 wt% fibrin solution. Then, 3.5g of PCL (weight average molecular weight 80000) and 1.5g of dry SF were dissolved in a 25mL beaker containing 15g of formic acid, and sufficiently stirred at room temperature for 6 hours to obtain a PCL/SF spinning solution (2.33, w/w) with a mass fraction of 25 wt%. The obtained PCL/SF spinning solution is used as an internal phase, the fibrin solution is used as an external phase, and the two solutions are used as a reservoir of a precursor and are filled into an injector. The two injectors are respectively connected to two inlets of the T-shaped microfluidic chip and used as a reactor for preparing the fibrin-coated PCL/SF nano-fiber. The prepared spinning solution was fed using a stainless steel blunting needle (24G) at a fixed feed rate (5 mL/h for inside and 0.1mL/h for outside, respectively). During MBS, the air pressure was maintained at 0.5 MPa. The nanofibers were collected on a nylon 66 screen, which was 37cm from the injector nozzle. A nanofabric consisting of fibrin-coated PCL/SF core-shell nanofibers was prepared, with fiber diameters between 150nm and 250 nm. The obtained nanometer fabric has tensile strength of 6.2MPa and area size of 10 × 40cm². And dried in vacuum at 35 ℃ for 12h to remove residual FA. And finally, injecting a catalyst solution in the pig-derived fibrin adhesive into the freeze-dried catalyst powder to obtain a freeze-dried catalyst solution with the concentration of the freeze-dried catalyst powder of 0.1g/ml, and spraying the freeze-dried catalyst solution on the nano fabric to enable the shell fibrin and the catalyst thereof to carry out gelation reaction by gas spraying so as to prepare the fiber gel composite matrix as the artificial skin. The tensile strength of the obtained fiber-gel composite matrix was 6.8 MPa. Then the biocompatibility and the non-toxic property of the artificial skin material are verified through a cell experiment. Finally, the in vivo dying and alive staining experiment shows that the artificial skinHas excellent histocompatibility, and the cell activity on the surface of the nanofiber gel composite membrane can reach 80%, so that the nanofiber gel composite membrane has good cell compatibility and nontoxicity. Thereby promoting a series of functions of skin regeneration, such as promoting angiogenesis, collagen deposition and granulation tissue formation, and effectively preventing wound infection of full-layer skin defect.

Claims

1. A preparation method of a nanofiber gel composite matrix for constructing skin tissues comprises the following specific steps:

a. injecting a main body gel dissolving solution in the pig-derived fibrin adhesive into main body gel freeze-dried powder to obtain a fibrin solution, wherein the mass concentration of the main body gel freeze-dried powder in the fibrin solution is 1.9-4.8%; adding polycaprolactone PCL and silk fibroin SF into a container containing formic acid FA, and stirring to a solution state to obtain a PCL/SF spinning solution; wherein the mass concentration of the PCL/SF spinning solution is 14-25%; wherein the mass ratio of PCL to SF is 0.25-2.5;

b. b, spinning by taking the PCL/SF spinning solution obtained in the step a as an internal phase and taking a fibrin solution as an external phase, wherein in the spinning process, a certain air pressure is set, the nanofibers are collected on a nylon 66 screen, and the screen is away from a nozzle of an injector by a certain distance to obtain a nanofiber membrane consisting of PCL/SF core-shell nanofibers coated by fibrin; then drying the nanofiber membrane in vacuum to obtain a nano fabric consisting of PCL/SF core-shell nanofibers coated by fibrin; wherein the air pressure range is 0.01-0.5 MPa; the distance between the screen and the nozzle of the injector in the step b ranges from 23 cm to 37 cm; in the step b, the flow rates of the inner phase and the outer phase are both 0.1-5 mL/h;

2. The method according to claim 1, wherein the formic acid solution in step a has a mass concentration of 85 to 95%.

3. The method of claim 1, wherein the fiber diameter of the nanofabric of step b is 44-250 nm.

4. The method of claim 1, wherein the area of the nanofabric in step b is 4 x 4 to 40 x 140cm²。

5. The method according to claim 1, wherein the temperature of vacuum drying in step b is 25-35 ℃; the vacuum drying time is 6-12 h.

6. The method of claim 1, wherein the nanofabric in step b has a tensile strength in the range of 2.3 to 6.2 MPa.

7. The method according to claim 1, wherein the nanofiber gel composite matrix obtained in step c has a tensile strength of 6.8 to 8.2 MPa.