阅读说明：本技术 一种构建皮肤组织的纳米纤维凝胶复合基质的制备方法 (Preparation method of nanofiber gel composite matrix for constructing skin tissue ) 是由 陈苏 崔婷婷 余加飞 王格飞 于 2019-11-18 设计创作，主要内容包括：本发明涉及一种构建皮肤组织的纳米纤维凝胶复合基质的制备方法,将猪源纤维蛋白粘合剂中的主体胶溶解液注入主体胶冻干粉中,得到纤维蛋白溶液；将聚己内酯和丝素蛋白加入到含有甲酸的容器中得到PCL/SF的纺丝液；将PCL/SF纺丝液作为内相,纤维蛋白溶液作为外相,进行纺丝,得到由纤维蛋白包覆的PCL/SF核-壳纳米纤维组成的纳米织物；将冻干粉催化剂溶液喷在得到的纳米织物上形成凝胶,得到构建皮肤组织的纳米纤维凝胶复合基质。该方法易于操作和通用,使腹部全层厚度皮肤缺损处可以实现大面积皮肤再生。(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.)

1. 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 to a solution state to obtain 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 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;

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.

2. The method according to claim 1, wherein the mass concentration of the main lyophilized powder in the fibrin solution in step a is 1.9-4.8%.

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

4. The method according to claim 1, wherein the PCL/SF spinning solution in step a has a mass concentration of 14-25%; wherein the mass ratio of PCL to SF is 0.25-2.5.

5. The method according to claim 1, wherein the air pressure in step b is in the range of 0.01 to 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.

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

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

8. 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.

9. 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.

10. 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.

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 the three steps are carried outThe preparation of nano fabric, the reconstruction of skin tissue 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.