An asymmetric structure chitosan gel, chitosan film and a preparation method thereof

Abstract

The application discloses an asymmetric structure chitosan gel, a chitosan film and a preparation method thereof. Chitosan powder is added into an acidic aqueous solution to be dissolved, and an acidic chitosan aqueous solution is obtained; the obtained acidic chitosan aqueous solution is poured into a mold, and after standing, the mold is placed in an anionic surfactant solution to be coagulated; then the mold containing the chitosan solution is transferred to a basic solution to be coagulated again or eluted, and an asymmetric structure chitosan gel is obtained; after deionized washing and freeze-drying, a chitosan film with an asymmetric structure is obtained. The film prepared by the application comprises a dense layer and a loose layer, and the film has good biocompatibility, biodegradability, mechanical properties, softness, adhesion, water absorption, air permeability, and the like; meanwhile, the preparation method has the advantages of simple process, short time consumption, easy large-scale production, great application potential and market prospect in absorbable guiding tissue regeneration films.

Description

Technical Field

[0001] This invention belongs to the field of chitosan materials and relates to a technology for preparing asymmetric chitosan film materials, specifically to a chitosan film with controllable asymmetric structure and its preparation method. Background Technology

[0002] Chitin is widely available and abundant in nature, making it the most abundant nitrogen-containing natural organic polymer besides protein. It is widely found in the epidermis of crustaceans such as shrimp and crabs, various insects, the skeletons of mollusks, and the cell walls of mushrooms and fungi. Chitosan, a deacetylated derivative of chitin, is the only positively charged basic polysaccharide found in nature. Due to its excellent biodegradability and biocompatibility, chitosan has been widely used in drug delivery, hemostatic materials, wound dressings, and regenerative tissue engineering.

[0003] Clinically, guided tissue regeneration technology is often used to assist osteogenic repair and address issues such as bone defects and insufficient bone mass. Guided tissue regeneration membranes act as a physical barrier, preventing surrounding fibrous and connective tissue from invading the bone defect, creating space for osteoblasts to grow bone, and effectively promoting new bone formation. Induced bone regeneration membranes are mainly made of non-degradable or biodegradable materials. Polytetrafluoroethylene (ePTFE) is a typical non-degradable guided tissue regeneration membrane that has achieved good clinical results; however, it requires a second surgery to remove the membrane, which undoubtedly causes additional trauma to patients and increases treatment costs. Currently, guided tissue regeneration membranes made of biodegradable materials, such as polylactic acid, polyethylene glycol, collagen, and chitosan, have been developed, avoiding the need for a second surgery due to their biodegradability. Among them, the commercially available collagen membrane Bio-Guide has been used in clinical practice and has achieved excellent clinical results.

[0004] Studies have shown that guided tissue regeneration membranes with asymmetric structures exhibit excellent osteogenic repair functions. The dense layer effectively isolates bone defects from the invasion of surrounding connective fibrous tissue, while the loose layer facilitates osteoblast adhesion, migration, and thrombus stabilization. Currently, techniques for constructing asymmetric membranes mainly include drying-assisted phase inversion, electrospinning, and electrodeposition. These methods suffer from problems such as cumbersome processes, long processing times, high equipment costs, and difficulty in scaling up production. Therefore, researching a simple, low-cost, and easily scalable technique to achieve the economical and efficient production of guided tissue regeneration membranes with asymmetric structures is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the problems of high production cost, complex process, and introduction of crosslinking agents in current tissue regeneration film production, and to provide a biocompatible, absorbable, low-cost, time-saving, and scalable asymmetric chitosan film preparation technology.

[0006] A method for preparing an asymmetric chitosan gel includes the following steps:

[0007] Step 1: Add chitosan powder to an acidic aqueous solution and stir until the chitosan powder is completely dissolved to obtain a chitosan aqueous solution;

[0008] Step 2: Pour the obtained chitosan aqueous solution into the mold, let it stand, and remove air bubbles;

[0009] Step 3: Transfer the mold containing the chitosan solution from Step 2 to a primary coagulation bath containing anionic surfactants to react and obtain a dense lamellar gel precursor.

[0010] Step 4: Transfer the mold from Step 3 to a secondary coagulation bath or elution bath containing an alkaline solution to form a loose layer on the dense layer gel precursor, thereby obtaining an asymmetric chitosan gel containing both a dense and a loose layer.

[0011] Preferably, in step 1, the acidic aqueous solution is any one or a mixture of several of hydrochloric acid, acetic acid, citric acid, and oxalic acid.

[0012] More preferably, in step 1, the pH of the chitosan aqueous solution is 3 to 6, and the dissolution rate is accelerated by using a suitable pH.

[0013] More preferably, in step 1, the chitosan solution has a mass concentration of 0.5-5% and a molecular weight of 10,000-1,000,000 g / mol.

[0014] Preferably, in step 2, for ease of operation, the mold is a flat-bottomed mold with one end open.

[0015] Preferably, in step 2, the degassing treatment is natural settling, negative pressure degassing, or microwave degassing.

[0016] Preferably, in step 3, the anionic surfactant is selected from any one or a mixture of several of alkylbenzene sulfonates, alkyl sulfonates, alkyl sulfonates, alkyl sulfates, fatty alcohol sulfates, fatty alcohol polyoxyethylene ether sulfates, α-alkenyl sulfonates, alkyl sulfonate acetamides, alkyl succinate sulfonates, and alkanolamine alkylbenzene sulfonates.

[0017] More preferably, in step 3, the concentration of the primary coagulation bath is 0.3-20%, the temperature is 20-50℃, and the gelation time is 1-30 min.

[0018] Preferably, in step 4, the alkaline solution is any one or a mixture of several of potassium hydroxide, sodium hydroxide, lithium hydroxide, and ammonia.

[0019] More preferably, in step 4, the concentration of the alkaline solution is 0.4-20%; the reaction time of the secondary coagulation bath or elution bath is 1-60 min, and the reaction temperature is 20-50℃.

[0020] On the other hand, the present invention also provides a method for preparing an asymmetric chitosan film, comprising the following steps:

[0021] Step 5: After washing the asymmetric chitosan gel prepared by the above method with deionized water, transfer it to a humectant solution;

[0022] Step 6: Freeze-dry the asymmetric chitosan gel obtained in Step 5 to obtain a chitosan film with an asymmetric structure.

[0023] The asymmetric chitosan film obtained by the above method has a dense upper layer and a loose lower layer. The thickness of the dense and loose layers and the pore size are controllable, which has a natural technical advantage in the field of guiding tissue regeneration.

[0024] Preferably, in step 5, the humectant is any one or a mixture of several of the following: glycerol, butylene glycol, polyethylene glycol, propylene glycol, ethylene glycol, xylitol, polypropylene glycol, sorbitol, and petrolatum.

[0025] More preferably, in step 5, the concentration of the humectant is 1-20%, and the humectant is evenly distributed in the gel by direct addition or by spraying.

[0026] Preferably, in step 6, the freeze-drying time is 2 to 12 hours.

[0027] More preferably, in step 6, the freezing temperature for freeze drying is -20 to -50°C.

[0028] Preferably, a functional factor is added in step 5 or step 1 of preparing the asymmetric chitosan gel to obtain an asymmetric chitosan film with the desired function.

[0029] Further preferably, the functional factors include, but are not limited to, any one or more combinations of growth factors, antibacterial agents, etc.; specifically, they may be selected from one or more of hydroxyapatite, calcium phosphate, bioactive short peptides, heparin, bone morphogenetic protein 2, bone morphogenetic protein-4, bone morphogenetic protein 9, bone morphogenetic protein, transforming growth factor-β, vascular endothelial growth factor, platelet-derived growth factor, macrolide antibiotics, aminoglycoside antibiotics, β-lactam antibiotics, and tetracycline antibiotics.

[0030] This invention also protects an asymmetric chitosan film prepared using the above method.

[0031] The present invention also protects an asymmetric chitosan film comprising a dense layer and a loose layer on the dense layer.

[0032] Preferably, the thickness of the dense layer is 0.1 to 60 micrometers, and the thickness of the loose layer is 10 to 400 micrometers.

[0033] Preferably, the water absorption rate of the loose layer is 1000% to 5000%, more preferably 1500% to 2000%.

[0034] The present invention also protects the use of an asymmetric chitosan film for guiding tissue regeneration.

[0035] The principle of this invention is as follows:

[0036] Chitosan powder, when dissolved in an acidic solution, exhibits polycationic electrolyte properties and can electrostatically interact with anionic surfactants in the primary coagulation bath, undergoing a sol-gel transition to obtain a dense chitosan gel precursor. Upon transfer to an alkaline secondary coagulation / elution bath, the electrostatic interaction is disrupted, and the anionic surfactants in the dense chitosan gel precursor are removed in situ as sacrificial templates. Meanwhile, the positively charged chitosan molecular chains are neutralized by the alkali, forming a dense chitosan hydrogel layer. After passing through the dense chitosan gel precursor layer, the alkaline solution continues to diffuse downwards into the lower acidic chitosan solution, causing the acidic chitosan solution to gel due to deprotonation, forming a loose and porous chitosan hydrogel layer. After freeze-drying, a pure chitosan film with an asymmetric structure is obtained. The resulting film is biodegradable and has good mechanical properties, softness, conformability, water absorption, and air permeability. At the same time, the complete elution of the anionic surfactant ensures that the asymmetric structure pure chitosan film of this invention maintains the same good biocompatibility as the raw chitosan.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The asymmetric chitosan film prepared by this invention includes a dense layer and a porous layer. The thickness and pore structure of the dense and porous layers can be precisely controlled. The method is simple, time-saving, and easy to scale up for production. It also reduces production costs and improves the product quality of pure chitosan films. It has great application and market prospects in absorbable tissue regeneration films.

[0039] Instruction manual illustrations

[0040] Figure 1 This is an electron microscope image of the asymmetric chitosan gel film obtained in Example 1 of the present invention.

[0041] Figure labels: 1-dense layer, 2-loose layer. Detailed Implementation

[0042] The present invention is further illustrated below by way of examples. It should be understood that the examples of the present invention are for illustrative purposes and not for limiting the present invention. Simple modifications made to the present invention based on the essence of the present invention are all within the scope of protection claimed by the present invention.

[0043] Example 1

[0044] 0.8 g of chitosan powder was dissolved in dilute hydrochloric acid of a certain concentration, and the pH was adjusted to 5.5 to obtain a 0.8% (w / w) chitosan aqueous solution. The resulting solution was poured into a petri dish and allowed to stand to eliminate air bubbles. The petri dish containing the chitosan solution was placed in a 5% anionic surfactant solution at 20°C for 5 min. The petri dish was then removed and transferred to a 2% sodium hydroxide solution at 20°C for 10 min. The resulting asymmetric chitosan hydrogel was removed, washed with deionized water, and then placed in a 10% glycerol aqueous solution for 30 min. The hydrogel was then frozen with liquid nitrogen and dried in a vacuum at -40°C for 12 h to obtain an asymmetric chitosan gel film. The electron micrograph is shown below. Figure 1 As shown, it can be clearly seen that it consists of a dense layer 1 and a loose layer 2 located on the lower surface of the dense layer 1.

[0045] Example 2

[0046] 2g of chitosan powder was dissolved in dilute hydrochloric acid of a certain concentration, and the pH was adjusted to 5 to obtain a 2% (w / w) chitosan aqueous solution. Hydroxyapatite was added to the solution and mixed thoroughly. The resulting mixture was poured into a petri dish and allowed to stand to eliminate air bubbles. The petri dish containing the solution was placed in a 15% anionic surfactant solution at 20°C for 2 min. The petri dish was then removed and transferred to a 1% sodium hydroxide solution at 20°C for 5 min. The resulting asymmetric chitosan hydrogel was removed, washed with deionized water, and placed in a 5% glycerol aqueous solution for 30 min. The hydrogel was then frozen with liquid nitrogen and dried in a vacuum at -40°C for 8 h to obtain an asymmetric chitosan gel film.

[0047] Example 3

[0048] 1g of chitosan powder was dissolved in a certain concentration of dilute hydrochloric acid, and the pH was adjusted to 5.5 to obtain a 1% (w / w) chitosan aqueous solution. The resulting solution was poured into a petri dish and allowed to stand to eliminate air bubbles. The petri dish containing the chitosan solution was placed in a 10% anionic surfactant solution at 20℃ for 1 min. The petri dish was then removed and transferred to a 4% sodium hydroxide solution at 20℃ for 5 min. The resulting asymmetric chitosan hydrogel was removed, washed with deionized water, and then sprayed with a 20% glycerol aqueous solution. The resulting hydrogel was frozen with liquid nitrogen and then dried in a vacuum at -40℃ for 12 h to obtain an asymmetric chitosan gel film.

[0049] The above embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Although the invention has been described in detail with reference to the embodiments, those skilled in the art should understand that various combinations, modifications, or equivalent substitutions of the technical solutions of the invention do not depart from the spirit and scope of the invention and should be covered within the scope of the claims of the invention.

Claims

1. A method for preparing an asymmetric chitosan gel, characterized in that: Includes the following steps: Step 1: Add chitosan powder to an acidic aqueous solution and stir until the chitosan powder is completely dissolved to obtain a chitosan aqueous solution; Step 2: Pour the obtained chitosan aqueous solution into the mold, let it stand, and remove air bubbles; Step 3: Transfer the mold containing the chitosan solution from Step 2 to a primary coagulation bath containing anionic surfactants to react and obtain a dense lamellar gel precursor. Step 4: Transfer the mold from Step 3 to a secondary coagulation bath or elution bath containing an alkaline solution to form a loose layer on the dense layer gel precursor, thereby obtaining an asymmetric chitosan gel containing both a dense and a loose layer.

2. The method for preparing the asymmetric chitosan gel according to claim 1, characterized in that: In step 1, the acidic aqueous solution is any one or a mixture of several of hydrochloric acid, acetic acid, citric acid, and oxalic acid, and the pH of the resulting chitosan aqueous solution is 3 to 6.

3. The method for preparing the asymmetric chitosan gel according to claim 1, characterized in that: In step 2, the degassing process includes natural settling, negative pressure degassing, and microwave degassing.

4. The method for preparing the asymmetric chitosan gel according to claim 1, characterized in that: In step 3, the anionic surfactant is selected from any one or a mixture of several of alkylbenzene sulfonates, alkyl sulfonates, alkyl sulfonates, alkyl sulfates, fatty alcohol sulfates, fatty alcohol polyoxyethylene ether sulfates, α-alkenyl sulfonates, alkyl sulfonate acetamides, alkyl succinate sulfonates, and alkanolamine alkylbenzene sulfonates.

5. The method for preparing the asymmetric chitosan gel according to claim 1, characterized in that: In step 4, the alkaline solution is any one or a mixture of several of potassium hydroxide, sodium hydroxide, lithium hydroxide, and ammonia water.

6. A method for preparing an asymmetric chitosan film, characterized in that, Includes the following steps: Step 5: After washing the asymmetric chitosan gel prepared by the preparation method according to any one of claims 1 to 5 with deionized water, transfer it to a humectant solution; Step 6: Freeze-dry the asymmetric chitosan gel obtained in Step 5 to obtain a chitosan film with an asymmetric structure.

7. The method for preparing the asymmetric chitosan film according to claim 6, characterized in that: In step 5, the humectant is any one or a mixture of several of the following: glycerol, butylene glycol, polyethylene glycol, propylene glycol, hexanediol, xylitol, polypropylene glycol, sorbitol, and petrolatum.

8. The method for preparing the asymmetric chitosan film according to claim 6, characterized in that: In step 6, the freeze-drying time is 2 to 12 hours.

9. The method for preparing the asymmetric chitosan film according to claim 6, characterized in that: In step 5 or step 1 of preparing asymmetric chitosan gel, a functional factor is added to obtain an asymmetric chitosan film with the desired function.

10. An asymmetric chitosan film, prepared by the preparation method according to any one of claims 6 to 9, for guiding tissue regeneration.

Images