CN103691005B - A kind of micro--Na fibrous tissue engineering rack and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biomaterials, in particular to a micro-nano fiber tissue engineering scaffold and a preparation method thereof. The micro-nanofibrous tissue engineering scaffold includes micron fibers, bacterial cellulose nanofibers, micropores and bacterial cellulose nanopores; the diameter of the micron fibers is 5-500 microns, and the diameter of the bacterial cellulose nanofibers is 10 -100 nanometers; the diameter of the micro-pores is 100-500 microns, and the diameter of the bacterial cellulose nano-pores is 10-100 nanometers. Aiming at the above-mentioned prior art, the present invention provides a micro-nano fiber tissue engineering scaffold with a high degree of bionicity, stable scaffold structure, suitable porosity and pore size, which can provide a suitable external environment for cells; its preparation method is simple and easy It is feasible, low in cost, wide in sources of raw materials, and the preparation process is environmentally friendly and pollution-free.

Description

A kind of micro-nano fiber tissue engineering scaffold and preparation method thereof

technical field

The invention relates to the technical field of biomaterials, in particular to a micro-nano fiber tissue engineering scaffold and a preparation method thereof.

Background technique

Whether it is tissue engineering or regenerative medicine, the performance of scaffold materials determines the ultimate success or failure. From the perspective of geometric characteristics, the extracellular matrix in vivo (hereinafter referred to as ECM) is a three-dimensional fibrous structure, which not only contains micro-scale pores and fibers, but also has nano-scale pores and fibers, that is, ECM has multi-scale fibers (50-500 mm in diameter). Nano) and porous structures, these nanoscale structures work in concert with microstructures to control the behavior of cells and the function of tissues and organs. Therefore, from the perspective of bionics, it is necessary to design and manufacture fibrous biomimetic scaffolds with appropriate geometric properties from both the micron and nanometer levels, that is, to imitate the geometric structure of the natural ECM at multiple scales. Therefore, researchers began to prepare scaffolds composed of both micron and nanofibers (hereinafter referred to as micro-nanofibrous scaffolds) several years ago.

In recent years, many researchers have used wet spinning and electrospinning techniques to prepare biomimetic fiber scaffolds. However, wet spinning technology cannot prepare nanoscale fibers, and the scaffold has large pores; electrospinning technology can prepare submicron-scale fibers, but it is not easy to achieve true nanoscale (ie, less than 100 nanometers), and it is even more difficult to prepare micro-nano fibers structure, and the preparation method is complicated, the cost is high, and the strength of the scaffold is low. A large number of literatures have reported a layer-like scaffold structure, that is, layers of microfibers and layers of nanofibers are alternately stacked. Therefore, the preparation of a low-cost, high-strength, and good biocompatibility micro-nano fiber scaffold has become a hot topic in the field of biomaterials at home and abroad.

Bacterial cellulose is a fibrous natural nano-material synthesized by Acetobacter xylinum. It has high strength, Young's modulus up to 15-30 GPa, high purity, high crystallinity, high water holding capacity and good biocompatibility. The diameter is just between 10-100 nanometers, and it is an ideal scaffold material for nanofibrous tissue engineering. Therefore, bacterial cellulose nanofibers can become nanofiber components in micro-nanofibrous scaffolds. However, there is no relevant disclosed technology for combining bacterial cellulose nanofibers and microfibers into micro-nanofiber scaffolds so far.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention provides a micro-nano fiber tissue engineering scaffold with a high degree of bionicity, stable scaffold structure, suitable porosity and pore size, which can provide a suitable external environment for cells; its preparation method is simple and easy It is feasible, low in cost, wide in sources of raw materials, and the preparation process is environmentally friendly and pollution-free.

The present invention is achieved through the following technical solutions:

A micro-nanofibrous tissue engineering scaffold, comprising micron fibers, bacterial cellulose nanofibers, micropores and bacterial cellulose nanopores; the diameter of the micron fibers is 5-500 microns, and the diameter of the bacterial cellulose nanofibers is 10-100 nanometers; the diameter of the micron pores is 100-500 microns, and the diameter of the bacterial cellulose nanopores is 10-100 nanometers.

The material of the micron fibers is a degradable material with good biocompatibility, which is one of cellulose, collagen, gelatin, polylactic acid, chitosan, and sodium alginate.

A method for preparing a micro-nano fiber tissue engineering scaffold, characterized in that it comprises the following steps:

⑴ Prepare bacterial fermentation medium: use deionized water as solvent, and the mass fractions of each solute are: glucose 2.5%, yeast powder 0.75%, peptone 1.0%, Na2HPO 41.0%, stir in a beaker at room temperature until the solute is completely dissolved, Adjust the pH of the medium to 4-5 by adding acetic acid dropwise;

(2) Autoclaving: Put the pre-prepared micron fiber scaffold into the medium prepared in step (1), then autoclave the medium at 115°C for 30 minutes, take out the medium and cool it to room temperature under the irradiation of ultraviolet light;

(3) Bacteria inoculation: inoculate a certain amount of three-day-old bacterial strains in the sterilized medium in step (2);

⑷ Preparation of micro-nano fiber scaffold: Put the culture medium inoculated in step (3) into a shaker to implement dynamic and static alternate culture method, that is, first dynamic culture for 30-60 minutes, then static culture for 60-120 minutes, so Repeatedly, the rotation speed of the shaker during dynamic culture is 140-500rpm, and the total culture time is 24-168 hours; the temperature of the medium is 30-35°C, and the pH of the medium is 4-5; Boil and wash with L sodium hydroxide for 20 minutes, then wash with deionized water until the solution becomes neutral, freeze and dry the scaffold in liquid nitrogen to obtain a micro-nano fiber tissue engineering scaffold.

The micron pores of the micron fiber support are greater than 300 microns, and the micron fiber support is prepared by wet spinning or electrospinning.

The beneficial effects brought by the present invention are:

The preparation method is simple and easy, the cost is low, and the source of raw materials is wide; the preparation process is green and environmentally friendly without any pollution, and the prepared micro-nano fiber tissue engineering scaffold has a high degree of bionicity, the scaffold structure is stable, and has suitable porosity and pore size , can provide a suitable external environment for cells. The present invention combines wet spinning and electrospinning technology with bacterial fiber nano-cellulose cultivation technology, first adopts traditional wet spinning or electrospinning technology to prepare micron fiber support, and then places the micron support on bacterial fiber In-situ co-cultivation was carried out in the culture medium of cellulose, and through the method of dynamic and static culture, the nanofibers of bacterial cellulose penetrated between the microfibers, and the interpenetrating network micro-nanofiber tissue engineering scaffold was prepared. The interpenetrating network type micro-nano fiber tissue engineering scaffold prepared by the present invention can overcome the defects of the existing micro-nano fiber tissue engineering scaffold, is beneficial to the adhesion, proliferation and differentiation of cells, and provides a suitable external environment for cell growth, and The preparation method is simple, it is a green manufacturing process, and the process is highly controllable.

Description of drawings

The present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is the SEM photo of the cellulose microfiber-bacterial cellulose nanofiber scaffold;

Figure 2 is the SEM photograph of the gelatin microfiber-bacterial cellulose nanofiber scaffold.

Detailed ways

The content of the present invention is further described below by way of specific embodiments, but these embodiments do not limit protection scope of the present invention:

Embodiment one:

As an embodiment of the micro-nanofibrous tissue engineering scaffold of the present invention, it includes micron fibers, bacterial cellulose nanofibers, micropores and bacterial cellulose nanopores; the diameter of the micron fibers is 5 microns, and the bacterial cellulose The diameter of the nanofiber is 10 nanometers; the diameter of the micropore is 100 micrometers, and the diameter of the bacterial cellulose nanopore is 10 nanometers.

A preparation method for preparing micro-nanofibrous tissue engineering scaffolds using cellulose as microfiber scaffolds, cutting the cellulose microfiber scaffolds into square pieces with a size of 20×20×0.5 mm, and preparing Acetobacter xylinum culture medium, that is, using deionized Water is the solvent, and the mass fractions of each solute are: 2.5% glucose, 0.75% yeast powder, 1.0% peptone, 1.0% Na2HPO4, stir in a beaker at room temperature until the solute is completely dissolved, and adjust the pH of the medium to 4 by adding acetic acid dropwise; Put the cellulose microfiber scaffold into the culture medium, seal it, and then sterilize the culture medium at 115°C for 30 min, take out the culture medium and cool it to room temperature under the irradiation of ultraviolet light; inoculate 40 ml three Day-old Acetobacter xylinum strains; put the inoculated medium into a shaker, and perform dynamic-static alternate culture at 30 °C and 160 rpm: dynamic 30 minutes → static 60 minutes → dynamic 30 minutes → static 60 minutes After 30 hours of co-cultivation, the scaffolds were taken out, first boiled and washed with 0.1 mol/L sodium hydroxide for 20 min, and then washed with deionized water until the solution was neutral, and the scaffolds were frozen and dried in liquid nitrogen to obtain micro-nano fiber scaffolds.

FIG. 1 is an SEM photo of the micro-nano fiber scaffold prepared by using cellulose as the microfiber scaffold in Example 1. FIG. It can be seen from the figure that nano-scale bacterial cellulose is deposited on the cellulose microfiber scaffold, and the micro-nano fibers are well combined.

Embodiment two:

As an embodiment of the micro-nanofibrous tissue engineering scaffold of the present invention, the difference from Embodiment 1 is that in this embodiment, the diameter of the micron fibers is 500 microns, and the diameter of the bacterial cellulose nanofibers is 100 microns. nanometer; the diameter of the micrometer pore is 500 microns, and the diameter of the bacterial cellulose nanopore is 100 nanometers.

A method for preparing micro-nanofibrous tissue engineering scaffolds using gelatin as a microfiber scaffold, cutting the gelatin microfiber scaffold into square pieces with a size of 20×20×2mm, and preparing Acetobacter xylinum culture medium (that is, using deionized water as a solvent , the mass fractions of each solute are: 2.5% glucose, 0.75% yeast powder, 1.0% peptone, 1.0% Na2HPO4), stir in a beaker at room temperature until the solute is completely dissolved, adjust the pH of the medium to 5 by adding acetic acid dropwise; Put the plain microfiber scaffold into the medium, seal it, and then sterilize the medium at 115°C for 30 min, take out the medium and cool it to room temperature under the irradiation of ultraviolet light; inoculate 40 ml three-day-old Acetobacter xylinum strains; put the inoculated medium into a shaker, and carry out dynamic-static alternate culture at 30 ℃ and 400 rpm: dynamic 60 minutes → static 60 minutes → dynamic 60 minutes → static 60 minutes, a total of After culturing for 48 hours, the scaffolds were taken out, first boiled and washed with 0.1 mol/L sodium hydroxide for 20 min, and then washed with deionized water until the solution was neutral, then the scaffolds were frozen in liquid nitrogen and dried to obtain micro-nano fiber scaffolds.

FIG. 2 is an SEM photo of the micro-nano fiber scaffold prepared by using gelatin as the microfiber scaffold in Example 2. FIG. It can be seen from the figure that nanoscale bacterial cellulose is deposited on the gelatin microfiber scaffold, and the micro-nanofibers are well combined.

Embodiment three:

As an embodiment of the micro-nanofibrous tissue engineering scaffold of the present invention, the difference from Embodiment 1 is that in this embodiment, the diameter of the micron fibers is 250 microns, and the diameter of the bacterial cellulose nanofibers is 50 microns. nanometer; the diameter of the micrometer pore is 300 microns, and the diameter of the bacterial cellulose nanopore is 60 nanometers.

Step 1 in the preparation method of micro-nanofibrous tissue engineering scaffold: prepare bacterial fermentation medium: use deionized water as solvent, and the mass fractions of each solute are: glucose 2.5%, yeast powder 0.75%, peptone 1.0%, Na2HPO41 .0%, stir in a beaker at room temperature until the solute is completely dissolved, and adjust the pH of the medium to 4.5 by adding acetic acid dropwise.

Claims (2)

1. A method for preparing a micro-nano fiber tissue engineering support, characterized in that it comprises the steps: ⑴ Prepare bacterial fermentation medium: use deionized water as solvent, the mass fractions of each solute are: glucose 2.5%, yeast powder 0.75%, peptone 1.0%, Na ₂ HPO ₄ 1.0%, stir in a beaker at room temperature until the solute is completely Dissolve, and adjust the pH of the medium to 4-5 by adding acetic acid dropwise; (2) Autoclaving: Put the pre-prepared micron fiber scaffold into the medium prepared in step (1), then autoclave the medium at 115°C for 30 minutes, take out the medium and cool it to room temperature under the irradiation of ultraviolet light; (3) Bacteria inoculation: inoculate a certain amount of three-day-old bacterial strains in the sterilized medium in step (2); ⑷ Preparation of micro-nano fiber scaffold: Put the culture medium inoculated in step (3) into a shaker to implement dynamic and static alternate culture method, that is, first dynamic culture for 30-60 minutes, then static culture for 60-120 minutes, so Repeatedly, the rotation speed of the shaker during dynamic culture is 140-500rpm, and the total culture time is 24-168 hours; the temperature of the medium is 30-35°C, and the pH of the medium is 4-5; Boil and wash with L sodium hydroxide for 20 minutes, then wash with deionized water until the solution becomes neutral, freeze and dry the scaffold in liquid nitrogen to obtain a micro-nano fiber tissue engineering scaffold. 2. The preparation method of micro-nano fiber tissue engineering support as claimed in claim 1, characterized in that the micron pores of the micron fiber support are greater than 300 microns, and the micron fiber support is formed by wet spinning or electrospinning method. be made of.