Preparation method and application of layer-by-layer self-assembled tissue engineering membrane

Abstract

The application discloses a preparation method of a layer-by-layer self-assembly tissue engineering membrane and application thereof, and belongs to the technical field of medical preparations. The glass slide is taken out after being placed in the SA / glycerin / gelatin solution; the glass slide is taken out after being immersed in the CS / NaCl / glycerin / gelatin solution; the glass slide is immersed in a CaCl2 solution, taken out, dried, and then immersed in ethanol; and the membrane taken off from the glass slide is dried and stored. The application uses chitosan and sodium alginate as main raw materials, and prepares a deformable tissue engineering membrane through a self-assembly and ion replacement method. 2+ and Na + alternating treatment to realize control deformation. The membrane in a diastolic state is used to culture cells until the cells on the membrane surface form a dense monolayer of cells, and then the monolayer of cells is separated from the lower self-assembly membrane through CaCl2 solution treatment, so that the monolayer of cells on the membrane is separated from the lower self-assembly membrane. Thus, a monolayer of cells for tissue engineering is obtained.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and more specifically to a method for preparing a layer-by-layer self-assembled tissue engineering membrane and its application. Background Technology

[0002] Chitosan (CS) is the deacetylated product of chitin and is a natural, biodegradable polycationic polysaccharide. Chitosan possesses advantages such as non-toxicity, good biocompatibility, and biodegradability, making it widely used in biomedicine and pharmaceuticals. CS molecules are rich in amino and hydroxyl groups, which can form hydrogen bonds; therefore, CS is insoluble in water and organic solvents, but soluble in (dilute) acid solutions.

[0003] Sodium alginate (SA) is an anionic polysaccharide extracted from marine brown algae, mainly composed of 1-4 linked α-L-mannuronic acid and β-D-mannuronic acid residues. Due to its biocompatibility, biodegradability, low cost, non-toxicity, and good film-forming properties, sodium alginate has been widely used and studied. However, the large number of free hydroxyl groups (-OH) and carboxylates (-COO-) distributed along the sodium alginate backbone leads to its high hydrophilicity, resulting in poor mechanical properties and thermal stability due to its rigidity and brittleness. Therefore, it is necessary to interact sodium alginate with other materials to improve its water sensitivity and mechanical properties.

[0004] The amino group gives CS a positive charge, while under neutral and weakly alkaline conditions, the carboxyl group gives SA a large negative charge. This is the basis for the electrostatic adsorption self-assembly of CS and SA. Layer-by-layer self-assembly is an effective technique for improving membrane mechanical properties, using electrostatic interactions between ions as the driving force for film formation. This method allows control over the structure and thickness of the self-assembled membrane, and due to the non-specificity of electrostatic interactions, biofunctional macromolecules can be easily introduced into the membrane.

[0005] Therefore, how to provide a specific method for preparing layer-by-layer self-assembled tissue engineering membranes and its applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a layer-by-layer self-assembled tissue engineering membrane and its application, such as the use of the membrane in obtaining tissue engineering monolayer cells in vitro.

[0007] The layer-by-layer self-assembled CS / SA membrane is water-soluble, when Ca... 2+Converting the SA in the CS / SA membrane to water-insoluble calcium alginate (CA) allows the membrane to maintain a stable morphology in water. Compared to the previous CS / SA membrane, the CS / CA membrane exhibits significant shrinkage. However, if the CS / CA membrane is re-soaked in a NaCl solution of a certain concentration, some of the Ca in the membrane... 2+ Then it can be Na + The replacement causes the membrane to expand and increase in volume. Therefore, the volume of the chitosan / sodium alginate / calcium alginate self-assembled membrane is related to the Na... + Ca 2+ Ion concentration exhibits a significant responsiveness.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a layer-by-layer self-assembled tissue engineering membrane includes the following steps:

[0010] (1) Under magnetic stirring, SA, glycerin and gelatin are dissolved in distilled water to obtain SA / glycerin / gelatin solution for later use;

[0011] (2) Dissolve CS, sodium chloride, glycerol and gelatin in HAc solution and stir to obtain CS / NaCl / glycerol / gelatin solution;

[0012] (3) After placing the glass slide in the SA / glycerol / gelatin solution prepared in step (1), remove it and dry it to obtain a glass slide with an SA film.

[0013] (4) Immerse the glass slide with the SA film in the CS / NaCl / glycerol / gelatin solution, then remove and dry it;

[0014] (5) Repeat steps (1) to (4) for assembly and drying until the designed number of layers is reached to obtain a glass slide of water-soluble layer-by-layer self-assembled tissue engineering membrane;

[0015] (6) Immerse the glass slide containing the water-soluble layer-by-layer self-assembled tissue engineering membrane in CaCl2 solution, remove it, dry it, and then immerse it in ethanol.

[0016] (7) The membrane removed from the glass slide is dried and stored to obtain a layer-by-layer self-assembled tissue engineering membrane.

[0017] Preferred: Step (1) Magnetic stirring conditions: 150-250 rpm; mass-volume ratio of SA, glycerol, gelatin and distilled water: 1.5g:1mL:1mL:100mL, gelatin concentration: 0.1g / mL, stirring continuously for 4h.

[0018] Preferred: Step (2) CS, sodium chloride, glycerol, gelatin and HAc solution mass-volume ratio: 1.5g:1g:1mL:0.1mL:100mL, gelatin concentration: 0.1g / mL, HAc solution: 1%, v / v; stirring; temperature: 20~30℃ 4h.

[0019] Preferred: Step (3) The slide is placed in the SA / glycerol / gelatin solution prepared in step (1) for 10 min; Drying: Placed in a 37℃ biochemical incubator;

[0020] Step (4): Immerse the glass slide with the SA film in the CS / NaCl / glycerol / gelatin solution for 10 min; Drying: Place in a biochemical incubator at 37℃.

[0021] Preferred steps: (6) CaCl2 solution: 1.5%, w / w, time: 30 min; drying: 20-30℃ ethanol: 75% v / v, time: 5 min.

[0022] The present invention also provides layer-by-layer self-assembled tissue engineering membranes prepared by any of the above methods.

[0023] The present invention also provides the application of the above-mentioned layer-by-layer self-assembly tissue engineering membrane in the culture and separation of monolayer cells.

[0024] Preferred method includes the following steps: when cells adhere to and grow on the membrane, a treatment cycle is performed every 16 hours, for a total of 4 to 6 treatment cycles. In each cycle, the culture medium containing different concentrations of salt ions is alternately changed in the order of treatment with 0.9% NaCl followed by treatment with 0.5% CaCl2.

[0025] Preferred treatment cycles: 6 times; treatment time with 0.9% NaCl: 4 hours; treatment time with CaCl2: 12 hours.

[0026] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for preparing a layer-by-layer self-assembled tissue engineering membrane and its application. The technical effect achieved is that the present invention uses chitosan (CS) and sodium alginate (SA) as main raw materials to prepare a deformable tissue engineering membrane through self-assembly and ion substitution methods, which can be used to prepare a deformable tissue engineering membrane through Ca... 2+ and Na + Alternating treatments allow for controlled deformation. Cells are cultured using a membrane in a relaxed state until a dense monolayer of cells forms on the membrane surface. Treatment with CaCl2 solution then causes the membrane to contract rapidly, separating the monolayer of cells from the underlying self-assembled membrane. This allows for the production of monolayers of cells for tissue engineering in vitro using these deformable, layer-by-layer self-assembling tissue engineering membranes. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The attached figure is a schematic diagram of the layer-by-layer self-assembly tissue engineering membrane provided by the present invention.

[0029] Figure 2 The attached figure is a flowchart illustrating the preparation process of the layer-by-layer self-assembled tissue engineering membrane provided by the present invention.

[0030] Figure 3 The attached figure is a line graph showing the expansion and contraction properties of the layer-by-layer self-assembled tissue engineering membrane provided by the present invention, wherein 2+14: NaCl treatment for 2h and CaCl2 treatment for 12h; 4+12: NaCl treatment for 4h and CaCl2 treatment for 12h; 8+8: NaCl treatment for 8h and CaCl2 treatment for 8h.

[0031] Figure 4 The attached diagram shows the expansion and contraction at different stages provided by this invention.

[0032] Figure 5 The attached figure shows the swelling ratio of the CA membrane, CS membrane, and layer-by-layer self-assembled tissue engineering membrane provided by the present invention.

[0033] Figure 6 The attached figure shows the water vapor permeability of the CA membrane, CS membrane, and layer-by-layer self-assembled tissue engineering membrane provided by the present invention.

[0034] Figure 7 The attached figure shows the activity of renal epithelial cells measured by CCK-8 on days 1, 3, and 5, as provided by this invention.

[0035] Figure 8 The attached figure is a fluorescence image of AM / PI stained renal epithelial cells provided by the present invention.

[0036] Figure 9 The attached figure shows PCNA measurements of renal epithelial cells at different times in different groups, provided by the present invention.

[0037] Figure 10 The attached figure shows the β-Catenin assay results of renal epithelial cells at different time points provided by this invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] This invention discloses a method for preparing layer-by-layer self-assembled tissue engineering membranes and their applications. Experimental materials and procedures not mentioned in the embodiments are all commercially available materials or conventional experimental procedures, and their manufacturers are not limited.

[0040] Example 1

[0041] Preparation of layer-by-layer self-assembled tissue engineering membranes (see Figure 1 , 2 )

[0042] (1) Dissolve 1.5g SA, 1mL glycerol and 1mL gelatin (0.1g / mL) in 100mL distilled water under magnetic stirring (200rpm) and stir continuously for 4h to obtain SA / glycerol / gelatin solution for later use.

[0043] (2) Dissolve 1.5g CS, 1g sodium chloride, 1mL glycerol and 0.1mL gelatin (0.1g / mL) in 100mL HAc solution (1%, v / v) and stir at room temperature for 4h;

[0044] (3) After placing the glass slide into the SA / glycerol / gelatin solution prepared in step (1) for 10 min, take it out and put it into a biochemical incubator at 37°C to dry it, and obtain a glass slide with an SA film.

[0045] (4) Immerse the glass slide with SA film in CS / NaCl / glycerol / gelatin solution for 10 min, then take it out and place it in a biochemical incubator at 37°C to dry;

[0046] (5) Repeat steps (1) to (4) for assembly and drying until the designed number of layers is reached to obtain a glass slide of water-soluble layer-by-layer self-assembled tissue engineering membrane;

[0047] (6) Immerse the glass slide containing the water-soluble layer-by-layer self-assembled tissue engineering membrane in a 1.5% (w / w) CaCl2 solution for 30 min, remove it, dry it at room temperature, and then immerse it in 75% (v / v) ethanol for 5 min.

[0048] (7) The membrane removed from the glass slide is dried and stored to obtain a layer-by-layer self-assembled tissue engineering membrane (final material).

[0049] Example 2

[0050] The method for culturing and isolating monolayer epithelial cells on a layer-by-layer self-assembled tissue engineering membrane prepared in Example 1 includes the following steps:

[0051] (1) After the prepared layer-by-layer self-assembled tissue engineering membrane was subjected to moist heat sterilization (121℃, 0.10MPa, 20min), the membrane was placed in sterile distilled water for later use.

[0052] (2) Normally growing bovine kidney epithelial cells were selected for passage. After approximately 48 hours of treatment and when the cell adhesion rate was above 80%, the cells were used for experiments. The cells were then digested to adjust the cell concentration to 1×10⁻⁶. 5 Count / mL, prepare 3 transfer plates.

[0053] (3) First, place the membrane in a 48-well or 96-well plate, then add 200 μL of bovine kidney epithelial cell suspension to each well of the membrane, and incubate in an incubator containing 5% CO2 at 37°C.

[0054] (4) The cells adhered to the membrane and grew. During this period, each treatment cycle was 16 hours. In each cycle, the culture medium containing different concentrations of salt ions was alternately changed in the order of treatment with 0.9% NaCl for a certain time and then treatment with 0.5% CaCl2 for a certain time.

[0055] (5) When the cell fusion rate reaches 95% or more, discard the original DMEM culture medium and wash each membrane with PBS buffer at least 3 times.

[0056] (6) Add 200 μL of sterile CaCl2 solution with a mass fraction of 0.5% to the culture well containing the membrane and incubate for 10 min.

[0057] (7) Observe the membrane and cells under a microscope. After the membrane shrinks and the monolayer cells separate from the membrane, discard the CaCl2 solution and incubate the monolayer cells again with DMEM medium.

[0058] Comparative Experiment 1

[0059] During repeated treatment with NaCl and CaCl2, some alginate polymers in layer-by-layer self-assembled tissue engineering membranes can react with Na+. + and Ca 2+ Alternating interactions cause the membrane surface area to expand or contract. This regular alternation in membrane surface area promotes the growth of surface cells. This experiment provides a standardized investigation of changes in membrane surface area.

[0060] First, layer-by-layer self-assembled tissue engineering membranes were cut into 10 mm diameter circular pieces and immersed in a 0.9% NaCl solution at room temperature for several hours, at which point the membrane diameter was measured. Then, the layer-by-layer self-assembled tissue engineering membranes were immersed in a 0.5% CaCl2 solution for several hours, and the membrane diameter was measured again. These two steps were repeated for six treatment cycles. To avoid error, all samples were prepared in triplicate. The effect of treatment time in NaCl and CaCl2 solutions on membrane deformation was investigated to determine the maximum volume difference of the membrane in these two solutions.

[0061] The results showed that after treatment with 0.5% CaCl2, the layer-by-layer self-assembled tissue engineering membranes became insoluble in water due to the conversion of sodium alginate (SA) to calcium alginate (CA). However, in treatment with 0.9% NaCl, the layer-by-layer self-assembled tissue engineering membranes exhibited slight expansion. Treatment with NaCl for 4 hours and with CaCl2 for 12 hours promoted the maximum expansion / contraction ratio of the layer-by-layer self-assembled tissue engineering membranes. In the first four treatment cycles, after 4 hours of NaCl treatment, the membrane surface expanded to 120% and 110% of its initial area, respectively; after 12 hours of CaCl2 treatment, the membrane surface contracted to 80% of its initial area. Figure 3 ).

[0062] Furthermore, the initial network structure of the layer-by-layer self-assembled tissue-engineered membrane is relatively loose, therefore in Na... + In the first four processing cycles of the substitution, more Ca 2+ The site was replaced. Na + After treatment, the network structure of the layered self-assembled tissue-engineered membranes tends to be denser and more stable, and the surface area tends to stabilize, only recovering to the initial surface area. However, since the membrane design is updated daily in clinical treatment, the deformation during the first four treatment cycles should be given more attention.

[0063] Figure 4 The changes in membrane surface area after cyclic treatment with 0.9% NaCl and 0.5% CaCl2 are shown, compared with... Figure 3 Consistent.

[0064] Comparative Experiment 2

[0065] When culturing cells in vitro, the water absorption capacity and water vapor permeability of tissue-engineered membranes both affect cell growth. Therefore, evaluating the water absorption and swelling capacity and water vapor permeability of membranes will help assess their cell growth capacity.

[0066] CA, CS membranes, and the layer-by-layer self-assembled tissue engineering membrane (LBLSM) prepared in Example 1 were cut into samples of equal mass. These samples were immersed in PBS at 25°C for 24 hours, then the free water on the membrane surface was removed, and the samples were weighed. The initial weight of the membrane was W0. The weight of the membrane after PBS immersion was W1. The swelling rate of the membrane in PBS was calculated using the following formula: Swelling rate (%) = (W1 - W0) / W0 × 100%. To avoid error, all samples were performed in triplicate.

[0067] In addition, the water vapor permeability of the samples was determined by gravimetric analysis. 10 mL of deionized water was added to 12 15 mL centrifuge tubes, which were randomly divided into four groups: a blank control group, a CA group, a CS group, and a layer-by-layer self-assembled tissue engineering membrane group. For the CA group, CS group, and layer-by-layer self-assembled tissue engineering membrane group, the centrifuge tube openings were sealed with CA membrane, CS membrane, and layer-by-layer self-assembled tissue engineering membrane, respectively. Then, all centrifuge tubes were placed in a 37℃ biochemical incubator, and the weight change of each centrifuge tube was accurately recorded after 24 hours. The water vapor permeability was calculated using the following formula: WVP = (Wx - Wx') / (W1 - W1') × 100%. Where Wx is the initial mass of the CA, SA, and layer-by-layer self-assembled tissue engineering membrane-sealed centrifuge tubes; Wx' is the mass of the CA, SA, and layer-by-layer self-assembled tissue engineering membrane-sealed centrifuge tubes after 24 hours; W1 is the initial mass of the control centrifuge tubes; and W1' is the mass of the control centrifuge tubes after 24 hours.

[0068] The results showed that the layer-by-layer self-assembled tissue-engineered membrane had superior water absorption capacity compared to CA and CS membranes, while maintaining the same water vapor transport capacity. Figures 5-6 This indicates that the layered self-assembled tissue engineering membranes can not only better absorb excess water and maintain a moist environment for cells, but also promote the evaporation of excess water and other secretions, providing a good environment for cell growth.

[0069] Comparative Experiment 3

[0070] Kidney epithelial cells were selected to evaluate the effects of alternating ion activation and membrane-controlled deformation on cell proliferation grown on layer-by-layer self-assembling tissue-engineered membranes.

[0071] Renal epithelial cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a CO2 incubator (37°C, 5% CO2). The cytotoxicity of layer-by-layer self-assembled tissue-engineered membranes was evaluated using the CCK-8 standard method. The layer-by-layer self-assembled tissue-engineered membranes were cut into circular pieces, autoclaved, and then transferred to 96-well plates. The membranes were divided into three groups: a blank control, a layer-by-layer self-assembled tissue-engineered membrane, and a layer-by-layer self-assembled tissue-engineered membrane + NaCl + CaCl2 group. Among them, the layer-by-layer self-assembly tissue engineering membrane group uses the layer-by-layer self-assembly tissue engineering membrane directly for cell culture without any other treatment. Therefore, the final study of this group is the effect of the material's own composition on cell growth. The layer-by-layer self-assembly tissue engineering membrane + NaCl + CaCl2 group involves cyclically treating cells during the cell culture process using the layer-by-layer self-assembly tissue engineering membrane by culturing in a medium solution containing 0.9% NaCl for 4 hours and then in a medium solution containing 0.5% CaCl2 for 12 hours. Therefore, the final study of this group is the combined effect of the material's own composition and its deformation capacity on cell growth.

[0072] Specific steps: Prepare 100 μL of cell suspension (1×10⁻⁶ cells / mL). 4 Cells / mL were added to 96-well plates and incubated in a CO2 incubator (37℃, 5% CO2) for 1, 3, and 5 days, respectively. During cell culture, the layer-by-layer self-assembled tissue engineering membrane + NaCl + CaCl2 group was treated twice daily as follows: (1) After cell adhesion, the culture medium was discarded, and 100 μL of physiological saline (0.9% NaCl solution) was added and incubated for 4 h; (2) The 0.9% NaCl solution was discarded, and 100 μL of 0.5% CaCl2 was added to the culture dish and incubated for 12 h; (3) The CaCl2 solution was removed, and 100 μL of culture medium was added to culture the cells. After incubation for 1, 3, and 5 days, 10 μL of CCK-8 was added to each well and incubated for 4 h. The absorbance at 450 nm was recorded using a microplate reader to evaluate the effects of alternating ion activation and membrane-controlled deformation on cell proliferation growing on the layer-by-layer self-assembled tissue engineering membrane.

[0073] In addition, the cell viability and morphology of renal epithelial cells cultured on layer-by-layer self-assembled tissue-engineered membranes were analyzed using a calcein (AM) / propidium iodide (PI) double staining kit.

[0074] Layer-by-layer self-assembly (LAS) tissue engineering membranes were cut into circular pieces, autoclaved, and then transferred to 6-well plates. The membranes were divided into three groups: a blank control, LAS tissue engineering membranes, and LAS tissue engineering membranes + NaCl + CaCl2. In the LAS tissue engineering membrane group, the membranes were used directly for cell culture without any other treatment; therefore, the study focused on the effect of the material's components on cell growth. In the LAS tissue engineering membrane + NaCl + CaCl2 group, cells were cyclically cultured in a 0.9% NaCl medium for 4 hours, followed by a 12-hour culture in a 0.5% CaCl2 medium.

[0075] Specific steps: Cells (1×10) 5 Cells ( / mL) were seeded in 6-well plates and incubated at 37°C in 5% CO2 for 1, 3, and 5 days. During this process, all layer-by-layer self-assembling tissue-engineered membranes were treated twice daily.

[0076] The treatment method is as follows: (1) After the cells adhere to the culture dish, discard the culture medium, add 100 μL of physiological saline, and incubate for 4 h; (2) Discard the 0.9% NaCl solution, add 100 μL of 0.5% CaCl2 to the culture dish, and incubate for 12 h; (3) Remove the CaCl2 solution, add 100 μL of culture medium to culture the cells. Then, remove the culture medium and rinse the cells with PBS. Then, add 1 mL of AM solution (5 μL of AM dissolved in 5 mL of PBS buffer) to each well and stain in the dark for 20 minutes. Finally, observe the cells using a confocal laser scanning microscope.

[0077] The results show that: CCK-8 detection results ( Figure 7 ) and AM / PI staining ( Figure 8 The results showed that, compared with the control group, cells treated with the undeformed, layer-by-layer self-assembled tissue engineering membrane exhibited stronger green fluorescence and faster cell growth, indicating that the membrane itself can significantly promote cell growth. Most epithelial cells are spindle-shaped, making them highly suitable for spreading and growing on oriented surfaces. Therefore, the high hydrophilicity, high orientation, and appropriate surface roughness of the layer-by-layer self-assembled tissue engineering membrane can provide better environmental conditions for epithelial cell growth. Furthermore, after regular deformation treatment with NaCl / CaCl2, the cells on the layer-by-layer self-assembled tissue engineering membrane had higher cell density, faster growth rate, and greater cell fusion than other groups, indicating that regular mechanical deformation is beneficial for cell division and proliferation.

[0078] Comparative Experiment 4

[0079] Proliferating cell nuclear antigen (PCNA) is closely related to cellular DNA synthesis and plays a crucial role in cell proliferation. β-catenin, a multifunctional protein widely found in various cell types, is also essential for cell proliferation, differentiation, and apoptosis. This study analyzes the expression of PCNA and β-catenin in different treatment groups to further demonstrate the promoting effect of layered self-assembled tissue-engineered sheets on cell proliferation.

[0080] The effects of layer-by-layer self-assembled tissue-engineered membranes on cell proliferation were detected using PCNA ELISA and β-Catenin ELISA kits. Layer-by-layer self-assembled tissue-engineered membranes were cut into circular pieces, autoclaved, and then transferred to 96-well plates. The membranes were divided into three groups: a blank control, layer-by-layer self-assembled tissue-engineered membranes, and layer-by-layer self-assembled tissue-engineered membranes + NaCl + CaCl2. In the layer-by-layer self-assembled tissue-engineered membrane group, the membranes were directly used for cell culture without any other treatment; therefore, the study focused on the effect of the material's own components on cell growth. In the layer-by-layer self-assembled tissue-engineered membrane + NaCl + CaCl2 group, cells were cyclically cultured in a 0.9% NaCl medium solution for 4 hours, followed by a 12-hour cycle in a 0.5% CaCl2 medium solution; therefore, the study focused on the combined effect of the material's own components and its deformation capacity on cell growth.

[0081] Specific steps: Add 100 μL of cell suspension (1 × 10⁶ cells / mL) to each 96 wells. 4 Cells ( / mL) were incubated in a CO2 incubator (37℃, 5% CO2) for 1, 3, and 5 days, respectively. During cell culture, the treatment of the layer-by-layer self-assembled tissue engineering membrane + NaCl + CaCl2 group was exactly the same as the layer-by-layer self-assembled tissue engineering membrane + NaCl + CaCl2 group treatment in the AM / PI analysis of Example 3. Finally, the OD value was obtained at 450 nm using a microplate reader.

[0082] The results show that: Figure 9 and Figure 10 The results showed that the expression of PCNA and β-Catenin in the layered self-assembled tissue engineering membrane group and the layered self-assembled tissue engineering membrane + NaCl + CaCl2 group was stronger than that in the control group, and the expression of PCNA and β-Catenin in the layered self-assembled tissue engineering membrane + NaCl + CaCl2 group was the highest, indicating that the mechanical deformation of the layered self-assembled tissue engineering membrane is beneficial to cell proliferation.

[0083] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a layer-by-layer self-assembled tissue engineering membrane, characterized in that, Comprising the following steps: (1) Dissolve sodium alginate, glycerol and gelatin in distilled water under magnetic stirring to obtain a sodium alginate / glycerol / gelatin solution, for standby; (2) Dissolve chitosan, sodium chloride, glycerol and gelatin in HAc solution, and stir to obtain a chitosan / NaCl / glycerol / gelatin solution; (3) Place a glass slide in the sodium alginate / glycerol / gelatin solution prepared in step (1), and take it out after drying to obtain a glass slide with a sodium alginate film; (4) Immerse the glass slide with the sodium alginate film in the chitosan / NaCl / glycerol / gelatin solution, and take it out after drying; (5) Repeat the assembly and drying steps of steps (1)-(4) until the designed number of layers is reached to obtain a glass slide with a water-soluble layer-by-layer self-assembled tissue engineering membrane sheet; (6) Immerse the glass slide with the water-soluble layer-by-layer self-assembled tissue engineering membrane sheet in a CaCl2 solution, take it out after drying, and immerse it in ethanol; (7) Dry and store the membrane sheet taken from the glass slide to obtain a layer-by-layer self-assembled tissue engineering membrane sheet.

2. The production method according to claim 1, wherein The magnetic stirring condition in step (1) is 150-250 rpm; the mass / volume ratio of sodium alginate, glycerol, gelatin, distilled water is 1.5 g:1 mL:1 mL:100 mL, and the gelatin concentration is 0.1 g / mL, with continuous stirring for 4 h.

3. The production method according to claim 2, wherein In step (2), the mass / volume ratio of chitosan, sodium chloride, glycerol, gelatin and HAc solution is 1.5 g:1 g:1 mL:0.1 mL:100 mL, the gelatin concentration is 0.1 g / mL, the HAc solution is 1% v / v, and the stirring is at a temperature of 20-30°C for 4 h. In step (3), the glass slide is placed in the sodium alginate / glycerol / gelatin solution prepared in step (1) for 10 min, and the drying is performed in a 37°C biochemical incubator.

4. The production method according to claim 3, wherein In step (4), the glass slide with the sodium alginate film is immersed in the chitosan / NaCl / glycerol / gelatin solution for 10 min, and the drying is performed in a 37°C biochemical incubator. In step (6), the CaCl2 solution is 1.5% w / w, and the time is 30 min; the drying is performed at 20-30°C; and the ethanol is 75% v / v, and the time is 5 min.

5. The production method according to claim 4, wherein 6. The layer-by-layer self-assembled tissue engineering membrane sheet prepared by the method of any one of claims 1-5.

7. The use of the layer-by-layer self-assembled tissue engineering membrane sheet of claim 6 in culturing and isolating single-layer cells. Comprising the following steps:

8. The use according to claim 7, characterized in that, When the cells adhere to and grow on the membrane sheet, a treatment cycle is performed every 16 h, for a total of 4-6 treatment cycles, and in each cycle, the culture solution containing different concentrations of salt ions is alternately replaced in the order of treatment with 0.9% NaCl and then treatment with 0.5% CaCl2.

9. The use of claim 8, wherein the treatment cycle is 6 times; the treatment time with 0.9% NaCl is 4 h; and the treatment time with CaCl2 is 12 h. ​

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