阅读说明：本技术 一种仿生胶原膜包被片及其制备方法 (Bionic collagen membrane-coated tablet and preparation method thereof ) 是由 翟志臣 郝丽静 石志锋 卞钲淇 薛永业 于 2021-05-14 设计创作，主要内容包括：本发明公开了一种仿生胶原膜包被片及其制备方法。该方法包括：配制胶原重组缓冲液,将胶原的醋酸溶液与胶原重组缓冲液混合均匀,得到混合液,调节所述混合液pH为中性；将载体竖立浸泡在混合液中,保温处理,随后烘干,得到所述仿生胶原膜包被片。本发明通过简单的胶原自组装制备方法得到的仿生胶原膜包被片,具有如下优点：胶原与载体片贴合紧密,平整度好,操作简便,具有纳米纤维结构,且具有胶原体内独有的67nm周期性螺纹结构。(The invention discloses a bionic collagen membrane coated tablet and a preparation method thereof. The method comprises the following steps: preparing a collagen recombination buffer solution, uniformly mixing an acetic acid solution of collagen with the collagen recombination buffer solution to obtain a mixed solution, and adjusting the pH of the mixed solution to be neutral; and vertically soaking the carrier in the mixed solution, performing heat preservation treatment, and then drying to obtain the bionic collagen membrane coated sheet. The bionic collagen membrane coating sheet prepared by the simple collagen self-assembly preparation method has the following advantages: the collagen and the carrier sheet are tightly jointed, the flatness is good, the operation is simple and convenient, and the collagen has a nanofiber structure and a unique 67nm periodic thread structure in the collagen body.)

1. A preparation method of a bionic collagen membrane coated sheet is characterized by comprising the following steps:

(1) preparing a collagen recombination buffer solution;

(2) uniformly mixing a collagen acetic acid solution with a collagen recombination buffer solution to obtain a mixed solution, and adjusting the pH of the mixed solution to be neutral to obtain a mixed solution after the pH is adjusted;

(3) and (3) vertically soaking the carrier in the mixed solution with the pH adjusted in the step (2), performing heat preservation treatment, and drying to obtain the bionic collagen membrane coated sheet.

2. The method for preparing a biomimetic collagen membrane-coated sheet according to claim 1, wherein the preparation of the collagen recombination buffer solution in the step (1) comprises: mixing NaCl, HEPES, Na₂HPO₄And adding the collagen into water, and dissolving uniformly to obtain the collagen recombination buffer solution.

3. The method for preparing a biomimetic collagen membrane-coated sheet according to claim 1, wherein the collagen-acetic acid solution in the step (2) is a mixture obtained by uniformly mixing collagen and an acetic acid solution; the collagen is type I collagen or type II collagen; the concentration of the acetic acid solution is 0.005M to 0.05M.

4. The method for preparing a biomimetic collagen membrane-coated sheet according to claim 1, wherein in the collagen acetate solution in the step (2), the concentration of collagen is 0.02-0.5 mg/ml.

5. The method of claim 1, wherein in the mixed solution of step (2), the collagen concentration is 0.01-0.25 mg/ml, the NaCl concentration is 120-150 mM, the HEPES concentration is 20-40 mM, and Na is added₂HPO₄The concentration of (B) is 20 to 40 mM.

6. The method for preparing a biomimetic collagen membrane coated sheet according to claim 1, wherein the carrier in the step (3) is one of a high molecular polymer, glass and a silicon wafer.

7. The method for preparing a bionic collagen membrane-coated sheet according to claim 1, wherein the temperature of the heat preservation treatment in the step (3) is 25-35 ℃.

8. The method for preparing a bionic collagen membrane-coated sheet according to claim 1, wherein the heat preservation treatment time in the step (3) is 1-5 h.

9. The method for preparing a biomimetic collagen membrane-coated sheet according to claim 1, wherein the drying temperature in the step (2) is below 60 ℃ and the drying time is 2h to 48 h.

10. A biomimetic collagen membrane-coated sheet prepared by the preparation method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the field of cell or tissue culture materials, and particularly relates to a bionic collagen membrane coating sheet and a preparation method thereof.

Background

Culturing the cells on an extracellular matrix helps maintain the differentiation phenotype of the cells in vitro, and serum-free media can be used to culture the cells. Extracellular matrix materials commonly used in cell culture at present are various types of collagen, fibronectin, laminin and the like. Collagen has long been known as the most abundant extracellular matrix component in human and animal bodies, and its excellent biocompatibility and bioactivity.

In vivo, after procollagen molecules are synthesized, the telopeptides at two ends are cut off by enzyme to form collagen molecules with the length of about 300nm and the diameter of 1.5nm, and then a plurality of collagen molecules are self-assembled to form nano collagen fibers with a typical D-band structure of 67 nm. The generation of 67nm D-band is due to overlap and gap generated when collagen molecules self-assemble in the first-to-last phase crosslinking. The Overlap segment is about 30nm long and the gap segment is about 37nm long. The depth of the thread structure is between 3nm and 5 nm. As shown in fig. 1. Under the environment of proper pH, temperature and ion concentration and under the action of static electricity and the like, collagen molecules obtained by acid extraction can be self-assembled in vitro to form collagen fibers with a periodic thread structure similar to that in vivo.

Although many companies (BD company, etc.) of various countries have collagen-coated surfaces on commercial culture vessels and products are on the market, the collagen-coated well plates are coated only by adsorbing collagen molecules on the well plates themselves, and do not have a fiber structure and a typical D-band periodic thread structure peculiar to collagen in vivo. While the cells may respond to the geometry of the substrate surface and may be as small as nanoscale in size. Therefore, the preparation of the collagen coating with the nano structure has important significance for the culture of special cells and the research of the biological function of the natural fiber structure in the collagen body.

The national institute of standards and technology (USA) prepares a collagen fiber coating on the surface of a non-TC treated cell culture pore plate (polystyrene), but most of collagen is washed away, so that the concentration requirement of the collagen used for coating the pore plate is high, and the coating rate of the collagen is low.

Earlier stage CN103060192B reports a culture vessel coated by a bionic collagen membrane and a preparation method thereof, but the horizontal coating method on the surface of the vessel needs a plurality of washing processes, the operation is more complicated, the coating form is only limited to the culture vessel, and the method has certain limitations.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a bionic collagen membrane coated sheet and a preparation method thereof.

The preparation method provided by the invention can prepare the uniform and smooth bionic collagen membrane coating sheet with a natural fiber structure and a periodic thread structure, and the coating sheet can be used for enhancing cell adhesion, cell proliferation and cell function and the cytological function of collagen. The collagen membrane is tightly combined with the surface of the carrier and is not easy to fall off.

The purpose of the invention is realized by at least one of the following technical solutions.

The preparation method of the bionic collagen membrane coated sheet provided by the invention comprises the following steps:

(1) preparing a collagen recombination buffer solution;

(2) uniformly mixing a collagen acetic acid solution with a collagen recombination buffer solution to obtain a mixed solution, and adjusting the pH of the mixed solution to be neutral to obtain a mixed solution after the pH is adjusted;

(3) and (3) vertically soaking the carrier in the mixed solution with the pH adjusted in the step (2), performing heat preservation treatment, and drying to obtain the bionic collagen membrane coated sheet.

Further, the preparation of the collagen recombination buffer solution in the step (1) comprises the following steps: mixing NaCl, HEPES, Na₂HPO₄And adding NaOH into water (preferably deionized water), and dissolving uniformly to obtain the collagen recombination buffer solution.

Further, in the collagen recombination buffer solution, the concentration of NaCl is 270-540 mM, the concentration of HEPES is 60-120 mM, and Na₂HPO₄The concentration of (B) is 60 to 120 mM.

Preferably, in the collagen recombination buffer, the concentration of NaCl is 270mM, the concentration of HEPES is 60mM, Na₂HPO₄Has a concentration of 60 mM.

Further, the collagen acetic acid solution in the step (2) is a mixture obtained by uniformly mixing collagen and an acetic acid solution; the collagen is type I collagen or type II collagen; the concentration of the acetic acid solution is 0.005M to 0.05M.

Further, in the collagen acetic acid solution in the step (2), the concentration of the collagen is 0.02-0.5 mg/ml.

Further, the volume ratio of the acetic acid solution of collagen to the buffer solution in the step (2) varies according to the preparation concentration of the collagen recombination buffer solution.

Preferably, the volume ratio of the collagen acetic acid solution to the collagen recombination buffer solution in the step (2) is 1: 1-3: 1.

Further preferably, the volume ratio of the collagen acetate solution to the collagen recombination buffer solution in the step (2) is 1: 1.

Further, in the mixed solution of the step (2), the concentration of collagen is 0.01-0.25 mg/ml, the concentration of NaCl is 120-150 mM, the concentration of HEPES is 20-40 mM, and Na₂HPO₄The concentration of (B) is 20 to 40 mM.

Preferably, in the mixed solution of the step (2), the concentration of NaCl is 135mM, the concentration of HEPES is 30mM, and Na₂HPO₄Is 30 mM.

Further, in the step (3), the carrier is a sheet material.

Preferably, the carrier in the step (3) is one of a high molecular polymer, glass and a silicon wafer. The angle of the carrier soaked in the mixed solution can be adjusted.

Further, the temperature of the heat preservation treatment in the step (3) is 25-35 ℃.

Preferably, the incubation temperature in step (3) is 30 ℃.

Further, the time of the heat preservation treatment in the step (3) is 1-5 h.

Further, the drying temperature in the step (2) is below 60 ℃, and the drying time is 2-48 h.

The invention provides a bionic collagen membrane coating sheet prepared by the preparation method.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the preparation method provided by the invention has the advantages that through the self-assembly process which is simple and convenient to operate, the bonding force between collagen and the surface of the carrier is increased through the surface tension in the solution evaporation process, the problem of poor bonding force of the collagen on the surface of the carrier is solved, and the non-woven fabric type nanofiber bionic collagen film coating with a uniform and flat structure is prepared.

(2) Compared with horizontal coating on the surface of a vessel, the preparation method provided by the invention omits the process of washing and drying for many times, and is greatly and simply operated. And the vessel surface needs to be kept strictly still in the horizontal coating process, otherwise the coating effect is influenced, and even if the carrier is vertically coated and has vibration in the coating process, the obtained collagen fiber membrane is still uniform and flat.

(3) The collagen film attached to the surface of the bionic collagen film coating sheet provided by the invention exists in a nano fiber form, and when the recombination temperature is 25-35 ℃, the formed fiber has a typical 67nm periodic thread structure.

(4) According to the preparation method provided by the invention, the collagen film on the coating sheet can control the coating rate and the film thickness of the collagen fibers by adjusting the concentration of collagen.

(5) The bionic collagen membrane coated sheet provided by the invention has good activity and good cell affinity, and is superior to a commercial tissue-treated cell pore plate in cell adhesion.

Drawings

FIGS. 1a and 1b are SEM images of bionic collagen membrane coated sheet prepared in example 1 at different magnifications;

FIGS. 2a and 2b are SEM images of bionic collagen membrane coated sheet prepared in example 2 at different magnifications;

FIG. 3a is a scanning electron micrograph of ATDC5 cells adhering to the bionic collagen membrane-coated sheet prepared in example 1 after being cultured;

FIG. 3b is a scanning electron micrograph of ATDC5 cells cultured in a commercial tissue-treated cell well plate;

FIG. 3c is a scanning electron micrograph of ATDC5 cells adhering to the bionic collagen membrane-coated sheet prepared in example 2;

FIG. 4 is a scanning electron micrograph of a bionic collagen membrane coated sheet prepared in example 3;

fig. 5 is a scanning electron microscope image of the collagen membrane-coated sheet prepared in comparative example 1.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Example 1

A method for coating a bionic collagen membrane comprises the following steps:

(1) adding the type I collagen into 0.02M acetic acid solution to prepare collagen acetic acid solution with the type I collagen concentration of 0.2 mg/ml.

(2) Preparing a collagen recombination buffer solution; the buffer contained 270mM NaCl, 60mM HEPES, 60mM Na₂HPO₄。

(3) Uniformly mixing the collagen acetic acid solution obtained in the step (1) and the collagen recombination buffer solution obtained in the step (2) in a ratio of 1:1 to obtain a mixed solution, adjusting the pH of the mixed solution to be neutral by using 1M NaOH, adding 1ml of the mixed solution after the pH is adjusted into a 12-hole plate, vertically erecting a polystyrene sheet in the hole plate, covering the hole plate with a cover, and keeping the temperature of an oven at 30 ℃ for 1 h.

(4) And (4) opening the cover of the 12-hole plate containing the recombinant collagen obtained in the step (3), and drying in an oven at 30 ℃ for 2h to obtain the bionic collagen membrane coated sheet (the bionic collagen membrane coated polystyrene sheet).

As can be seen from fig. 1a and 1b, the collagen film attached to the surface of the polystyrene sheet is in the form of nanofibers having a diameter of about 200nm and having a typical periodic thread structure of 67 nm. The collagen film is evenly distributed on the surface of the polystyrene sheet.

ATDC5 cells (mouse model cells) were cultured on the bionic collagen membrane-coated sheet prepared in example 1 and a commercial tissue-treated cell-well plate (Corning3512) for 24 hours, respectively, and then fixed using glutaraldehyde, dehydrated with gradient alcohol and air-dried, and observed with a scanning electron microscope after plating with gold film, respectively. The results are shown in fig. 3a and 3b, respectively. FIG. 3a is a scanning electron micrograph of ATDC5 cells adhering to the bionic collagen membrane-coated sheet prepared in example 1 after being cultured; FIG. 3b is a scanning electron micrograph of ATDC5 cells after culturing on a common cell well plate.

FIG. 3a shows that ATDC5 adheres well to a collagen-coated fibrous membrane sheet (biomimetic collagen membrane-coated sheet prepared in example 1), showing good cell affinity; the spreading area of the cells cultured in the well plate is much smaller than that of the cells cultured on the collagen fiber membrane (fig. 3b), which shows that the collagen membrane on the bionic collagen membrane-coated sheet prepared in example 1 has good activity and is superior to the commercialized tissue-treated cell well plate in terms of cell adhesion.

Example 2

A method for coating a bionic collagen membrane comprises the following steps:

(1) adding the type I collagen into 0.005M acetic acid solution to prepare collagen acetic acid solution with the type I collagen concentration of 0.05 mg/ml.

(2) Preparing a collagen recombination buffer solution; the buffer contained 270mM NaCl, 60mM HEPES, 60mM Na₂HPO₄。

(3) Uniformly mixing the collagen acetic acid solution obtained in the step (1) and the buffer solution obtained in the step (2) in a ratio of 1:1 to obtain a mixed solution, adjusting the pH of the mixed solution to be neutral by using 1M NaOH, adding 1ml of the mixed solution after the pH is adjusted into a 12-hole plate, vertically erecting a polyethylene sheet in the hole plate, covering the hole plate with a cover, and keeping the temperature of an oven at 30 ℃ for 2 hours.

(4) And (4) opening the cover of the 12-hole plate containing the recombinant collagen obtained in the step (3), and drying in an oven at 30 ℃ for 12h to obtain the bionic collagen membrane coated sheet (the polyethylene sheet coated by the bionic collagen membrane).

As can be seen from fig. 2a and 2b, the prepared collagen film is uniformly distributed on the surface of the carrier, and is flat and uniform, and has a typical 67nm periodic thread structure.

Fig. 3c shows that ATDC5 adhered well to the collagen-coated membrane sheet with fibrous structure (the biomimetic collagen membrane-coated sheet prepared in example 2) and showed good cell affinity, indicating that the collagen membrane activity of the biomimetic collagen membrane-coated sheet prepared in example 2 was good and superior to that of the commercial tissue-treated cell-well plate in terms of cell adhesion.

Example 3

A method for coating a bionic collagen membrane comprises the following steps:

(1) adding the type I collagen into 0.05M acetic acid solution to prepare collagen acetic acid solution with the type I collagen concentration of 0.5 mg/ml.

(2) Preparing a collagen recombination buffer solution; the buffer contained 540mM NaCl, 120mM HEPES, 120mM Na₂HPO₄。

(3) Uniformly mixing the collagen acetic acid solution obtained in the step (1) and the buffer solution obtained in the step (2) in a ratio of 3:1 to obtain a mixed solution, adjusting the pH of the mixed solution to be neutral by using 1MNaOH, adding 1ml of the mixed solution after the pH is adjusted into a 12-hole plate, vertically erecting a silicon wafer in the hole plate, covering a cover, and keeping the temperature of an oven at 30 ℃ for 5 hours.

(4) And (4) opening the cover of the 12-hole plate containing the recombinant collagen obtained in the step (3), and drying in a 35 ℃ oven for 48 hours until the solution is dried to obtain the bionic collagen membrane coated sheet (the silicon wafer coated by the bionic collagen membrane).

As can be seen from FIG. 4, the collagen film prepared was uniformly distributed on the surface of the carrier, and was flat and uniform, with a typical 67nm periodic thread structure.

Comparative example 1

A method for coating a glass sheet with a collagen film comprises the following steps:

(1) adding the type I collagen into 0.005M acetic acid solution to prepare collagen acetic acid solution with the type I collagen concentration of 0.02 mg/ml.

(2) Preparing a collagen recombination buffer solution; the buffer contained 270mM NaCl, 60mM HEPES, 60mM Na₂HPO₄。

(3) Uniformly mixing the collagen acetic acid solution obtained in the step (1) and the buffer solution obtained in the step (2) in a ratio of 1:1 to obtain a mixed solution, adjusting the pH of the mixed solution to be neutral by using 1MNaOH, adding 1ml of the mixed solution after the pH is adjusted into a 12-hole plate, vertically erecting a glass sheet in the hole plate, covering the hole plate with a cover, and keeping the temperature of an oven at 37 ℃ for 2 hours.

(4) And (4) opening the cover of the 12-hole plate containing the recombinant collagen obtained in the step (3), and drying in an oven at 37 ℃ for 4h to obtain the bionic collagen membrane coated sheet (the glass sheet coated by the bionic collagen membrane).

As can be seen from fig. 5, the collagen film prepared in comparative example 1 was uniformly distributed on the surface of the carrier, but many fibers were entangled with each other due to the higher incubation temperature, and did not have the typical 67nm periodic thread structure.

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.