Preparation method of bacterial cellulose-based multi-layer structure functional bionic scaffold

Abstract

The application relates to a preparation method of a bacterial cellulose-based multilayer structure functional bionic scaffold, which comprises the following steps: firstly, injecting a biomaterial solution into a silica gel model with a microchannel system to perform freeze drying to obtain a porous biological scaffold; secondly, performing crosslinking treatment on the porous biological scaffold; thirdly, inoculating a culture bacteria solution with a Xylobacter concentration of 10 9 ~ 10 15 individuals / mL on the crosslinking-treated porous biological scaffold, and performing static culture and freeze drying to obtain a bacterial cellulose-based composite scaffold; and finally, adopting a coating solution to coat the bacterial cellulose-based composite scaffold through a microchannel system, and performing demolding treatment to obtain the bacterial cellulose-based multilayer structure functional bionic scaffold. The application provides a functional coating process for the multilayer bionic scaffold by using the shrinkage space of the treated material, the micro-pore channel and the injection method of the patterned drainage network, effectively prevents the penetration of the coating solution, and realizes the perfect solidification of the coating solution on the scaffold.

Description

Technical Field

[0001] This invention belongs to the field of animal body structure biological scaffold technology, and relates to a method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold. Background Technology

[0002] Tissue engineering research mainly includes seed cells, scaffold materials, in vitro tissue construction, and in vivo tissue or organ replacement. Tissue engineering scaffold materials play a central role in tissue engineering research, not only providing structural support for specific cells but also acting as templates to guide tissue regeneration and control tissue structure. Finding suitable scaffold materials and mimicking corresponding tissue structures are current research priorities. Using biomimetic principles, by combining various natural biomaterials, it is possible to simulate the extracellular matrix components of tissues and organs to construct scaffold materials—i.e., biomimetic materials—that resemble the structure and properties of the corresponding tissues. The preparation of biomimetic materials requires a clear understanding of the corresponding tissue or organ. The extracellular matrix of most tissues contains an internal porous and loose layer and an outer dense layer structure.

[0003] For example, cartilage consists of cartilage tissue and the surrounding perichondrium. Cartilage tissue is composed of chondrocytes, matrix, and collagen fibers. A thin layer of dense connective tissue, called the perichondrium, surrounds the cartilage. The perichondrium has two layers: the outer layer, rich in fibrous components, is continuous with the surrounding tissue and primarily provides protection; the inner layer contains cells that provide essential nutrients. Bone is mainly composed of two types of bone tissue: cancellous bone and cortical bone. Cancellous bone forms the interior of bone, while cortical bone is the outermost, denser boundary. Cancellous bone is highly porous, while cortical bone has a dense structure that protects the vulnerable inner parts. Similarly, urethral tissue has a dense layer on the inner and outer walls, while the middle layer is porous and loose. Therefore, the fabrication of biomimetic scaffolds is complex, requiring multi-layered simulation to better match the structure of the original tissue.

[0004] Various scientific methods have emerged for exploring the structure and fabrication of biomimetic scaffolds. For example, Chinese patent CN 104992604 A discloses a method for constructing a segmental, individualized human urethral tissue model, and ultimately constructs the urethral tissue model using 3D printing; Chinese patent CN 105031725 A constructs a silicone model that can be filled with material by using a silicone casting process on the urethral model, and then prepares the corresponding urethral scaffold by filling it with a solution of biomaterial. Existing technologies have achieved relatively successful examples in the fabrication of porous and sparse-dense structures, but the fabrication of complex biomimetic structures still needs improvement.

[0005] To address the aforementioned issues, this invention employs bacterial cellulose as the dense layer of a complex structure. Bacterial cellulose (BC) is synthesized by microorganisms, starting with the polymerization of individual glucose units into β-1,4-glucan chains. Secreted linear glucan chains then aggregate into protofibrils, ultimately forming a membrane. While its chemical composition is similar to that of plant fibers, its physical properties differ. BC possesses high tensile strength, high water-holding capacity, high crystallinity, an ultrafine and fine pure fibrous network structure, and excellent biocompatibility. The three-dimensional nanonetwork structure of BC perfectly mimics the extracellular matrix (ECM). These superior properties of BC make it crucial for the fabrication of biomimetic scaffold materials.

[0006] Meanwhile, to further enhance the functionality of the dense layer, this invention combines coating technology with patterning technology, achieving the fabrication of a biomimetic scaffold with a functionalized complex structure through a microchannel system. Significant progress has been made in the application of coating materials in biomedical materials. Coating materials can also achieve their functionality through physical and chemical excitation response mechanisms, effectively addressing the aforementioned iatrogenic infection problems. Achieving uniform coating application on the scaffold has become one of the key issues to be solved. The existence of micro- and nano-scale patterning technology can improve the interfacial interaction with the substrate, prevent the aggregation of nanomaterials, and enable the self-assembly of nanomaterials from bottom to top or top to bottom. Therefore, the combination of patterning technology and coating application is of practical significance.

[0007] In summary, selecting appropriate materials and methods to prepare biomimetic scaffolds with porous structures and dense layers is essential for better conformation to native tissue structures. Furthermore, by imbuing biomimetic scaffolds with functional coatings, the scaffold materials can achieve both tissue structure simulation and functionality, which is of practical significance. Summary of the Invention

[0008] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold involves first injecting a biomaterial solution into a silicone mold and then freeze-drying it to obtain a porous bioscaffold. The porous bioscaffold is then cross-linked. Next, a culture solution is inoculated onto the cross-linked porous bioscaffold (the specific inoculation location is not critical; the culture solution only needs to be injected into the gap between the scaffold and the silicone mold, ensuring the entire mold is submerged). After static culture and freeze-drying, a bacterial cellulose-based composite scaffold is obtained. Finally, a coating solution is applied to the bacterial cellulose-based composite scaffold through a microchannel system on the silicone mold (utilizing the drainage effect of the microchannel system to ensure uniform coating of the bacterial cellulose-based composite scaffold). The scaffold is then demolded to obtain the bacterial cellulose-based multilayer functional biomimetic scaffold.

[0011] The biomaterial is one or more of the following: silk fibroin, gelatin, collagen and chitosan, extracellular matrix and biodegradable polymers (such as PGL, PLA, PLGA, PCL);

[0012] The silicone model has a three-dimensional tissue model cavity inside; the microchannel system on the silicone model consists of micropore channels, drainage grooves, and patterned drainage networks. The micropore channels are channels formed by the injection needle passing through the outer wall of the silicone model perpendicular to the axial direction of the silicone model (axial direction: perpendicular to the horizontal plane, along the direction of the central axis of the silicone model) to the surface of the internal three-dimensional tissue model cavity. The patterned drainage network is an ordered arrangement of grooves formed on the surface of the three-dimensional tissue model cavity through subtractive processing. The drainage grooves are grooves that pass vertically through the patterned drainage network. The drainage grooves are located on the surface of the three-dimensional tissue model cavity and are connected to the micropore channels.

[0013] The culture medium is a mixture of Acetobacter xylinum and culture medium, with a concentration of 10 in the culture medium. 9 ~10 15 per mL.

[0014] As a preferred technical solution:

[0015] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above uses an injection needle with a specification of 25G to 30G, i.e., a micropore channel diameter of 0.3 to 0.5 mm.

[0016] The method for fabricating a bacterial cellulose-based multilayer functional biomimetic scaffold, as described above, involves grooves forming a patterned drainage network with a width of 30–200 μm and a depth of 30–100 μm. All grooves are uniformly arranged on the surface of the three-dimensional tissue model cavity, with an interval of 30–100 μm between adjacent grooves. The grooves can be perpendicular to the axial direction of the silicone model and parallel to the horizontal plane, uniformly arranged on the surface of the three-dimensional tissue model cavity; alternatively, the grooves can be perpendicular to the horizontal plane and parallel to the axial direction of the silicone model, uniformly arranged on the surface of the three-dimensional tissue model cavity.

[0017] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above, the culture medium formula is as follows:

[0018]

[0019] Water balance;

[0020] Substance A is one or more of glucose, fructose, sucrose, and mannitol;

[0021] The HLB value of the surfactant is 15-18.

[0022] Cultivating bacterial cellulose in a relatively small mold affects bacterial fermentation due to varying oxygen levels, thus influencing the formation of the dense layer. Therefore, the culture medium formulation is modified based on the dissolved oxygen content. Surfactants play a crucial role in the food industry, serving as solubilizers, foaming and defoaming agents, and food thickeners. Surfactants are classified into ionic, nonionic, and special surfactants. Nonionic surfactants generally have lower toxicity; therefore, this invention focuses on selecting a low-toxicity nonionic surfactant as a solubilizer for the culture medium, thereby increasing the final oxygen content of the culture medium.

[0023] The surfactant in the culture medium is added last. After preparation, the culture medium needs to be autoclaved, specifically at 121°C and 0.1 MPa for 30 minutes. Since the solubility of oxygen in water decreases with increasing temperature, the oxygen content of the culture medium decreases after autoclaving. The amount of oxygen in the culture medium significantly affects bacterial growth in the mold and the density of the generated bacterial cellulose. To increase the oxygen content, after the culture medium returns to room temperature (28–35°C) under sterile conditions, a surfactant is added. The selected surfactant has an HLB value between 15 and 18; a higher HLB value indicates stronger hydrophilicity. The amount of surfactant used in this invention is controlled based on two points: first, the toxicity of the surfactant itself can affect the growth of bacterial cellulose; therefore, referring to the GB2760-2014 standard for the use of food additives, the toxicity of the surfactant is ensured to not affect the bacterial fermentation process; second, the surfactant concentration should generally be slightly higher than its critical micelle concentration to allow it to function effectively. Based on these two considerations, this invention achieves a balance between controlling the amount of surfactant used. Ultimately, the amount is controlled between 0.05 and 0.1 wt%.

[0024] The oxygenation mechanism of surfactants: On the one hand, surfactant molecules are composed of hydrophilic and hydrophobic groups. When a small amount of nonionic surfactant is added to an aqueous solution, the hydrophobic groups point towards the air, while the hydrophilic groups attract water molecules. A small number of surfactant molecules will arrange themselves in a regular and orderly manner on the surface of the liquid. Under these circumstances, the original gas-liquid interface is transformed into a gas-hydrophobic interface, which reduces the surface tension and facilitates diffusion from the gas phase to the liquid phase. On the other hand, as the amount of surfactant increases to the critical micelle concentration (CMC), the liquid interface will not be able to arrange surfactant molecules too densely. In order to exist stably, they will arrange themselves to form micelles with hydrophobic groups facing inward and hydrophilic groups facing outward. Air is encapsulated in the micelles and dispersed in a continuous phase.

[0025] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above uses polyoxyethylene (20EO) methyl glucoside sesquioleate (HLB value 15.0), polyoxyethylene monostearate (Myrj49, Myrj51, Myrj52, Myrj53, with corresponding HLB values ​​of 15.0, 16.0, 16.9, and 17.9, respectively), Tween 20 (HLB value 16.7), or Tween 40 (HLB value 15.6) as the surfactant.

[0026] The specific steps of the preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold as described above are as follows:

[0027] (1) Preparation of silicone mold;

[0028] First, the 3D printed model is placed in a square container. Then, the silicone and curing agent are mixed and stirred (stirring time is 4-8 minutes). The resulting mixture is poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment to remove air bubbles generated during stirring. Finally, the square container is removed from the vacuum drying oven and allowed to cool and cure at room temperature (time is 3-7 days) before demolding (removing the 3D printed model). This yields a silicone mold with a three-dimensional tissue model cavity inside. The hardness of the silicone mold, measured using a Shore hardness tester, is 5-20 degrees, preferably 10-15 degrees. It is relatively soft and has good shrinkage. Therefore, tools can be used during demolding to expand the inner diameter of the silicone by 0.5-2 mm, which facilitates the complete removal of the support.

[0029] (2) A silicone model with a microchannel system is obtained by processing a silicone mold;

[0030] (3) The biomaterial solution was injected into the silicone model until the entire cavity of the three-dimensional tissue model was filled. After freezing at -20℃ for 12 to 24 hours, the porous biological scaffold was obtained by freeze-drying.

[0031] (4) Inject cross-linking agent into the silicone model to completely impregnate the porous biological scaffold. Perform room temperature cross-linking treatment on the porous biological scaffold. After cross-linking, inject 75% alcohol into the silicone model and soak for 10-20 minutes to remove excess cross-linking agent. In order to avoid the scaffold affecting bacterial growth during subsequent inoculation, place the scaffold after alcohol soaking in sterile culture medium for 10-20 minutes to replace the excess 75% alcohol.

[0032] (5) The cultured bacterial solution was inoculated onto the porous biological scaffold after the cross-linking treatment in step (4) to generate an interlaced nanoscale network structure on the outer wall of the porous biological scaffold, thus obtaining a bacterial cellulose-based composite scaffold.

[0033] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model are soaked in 75% alcohol and freeze-dried in sequence to shrink the porous biological scaffold inward by 0.3-1 mm. The coating solution is injected into the shrinkage gap to fill the entire gap. After curing and drying, the mold is removed to obtain a bacterial cellulose-based multilayer functional biomimetic scaffold.

[0034] In the preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above, the mass ratio of silicone to curing agent in step (1) is 100:1 to 10.

[0035] In the preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above, the concentration of the biomaterial solution (solvent is water) in step (3) is 2-10 wt%.

[0036] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above, the process parameters for freeze drying in step (3) are: cold trap temperature -50 to -40℃, sample temperature -30 to -20℃, vacuum degree 0.1Pa, freeze drying time 8 to 12h.

[0037] In the preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above, the crosslinking agent in step (4) is a solution with a concentration of 0.2-2 wt% prepared by dissolving glutaraldehyde, acetic anhydride or diglycidyl ether in anhydrous ethanol, and the crosslinking treatment time is 5-10 min.

[0038] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above includes the following steps: in step (6), the soaking time in 75% alcohol is 10-20 minutes, and the freeze-drying process parameters are: cold trap temperature -50 to -40℃, sample temperature -30 to -20℃, vacuum degree 0.1 Pa, freeze-drying time 8-12 h, curing temperature is room temperature, and curing time is 10-15 min.

[0039] As described above, in the preparation method of a bacterial cellulose-based multilayer functional biomimetic scaffold, the coating solution in step (6) enters the groove of the patterned drainage network through microporous channels and drainage grooves, and forms a coating on the surface of the bacterial cellulose-based composite scaffold, thereby obtaining a bacterial cellulose-based multilayer functional biomimetic scaffold.

[0040] The above-described method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold features an outer wall with an ultrafine nano-network structure. The diameter of the ultrafine nanofibers on the outer wall is less than 100 nm. The pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 100–300 μm. Furthermore, the ultrafine nano-network structure of the scaffold material effectively prevents the coating solution from penetrating into the pores. The presence of microporous channels and a drainage network system ensures that the coating solution is uniformly coated onto the scaffold surface.

[0041] The preparation method of the bacterial cellulose-based multilayer functional biomimetic scaffold described above uses an improved culture medium formula, which shortens the bacterial fermentation cycle after inoculation from 8-14 days to 3-7 days. Furthermore, the dense bacterial cellulose layer can fill the shrinkage gap within a shorter growth cycle, meaning that the bacterial cellulose membrane on the surface of the multilayer functional biomimetic scaffold grows to 0.3-1 mm.

[0042] The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold as described above involves uniformly coating a functional coating solution onto a bacterial cellulose membrane on the scaffold surface via a microchannel system, ensuring continuous and effective functional performance. Compared to simple porous scaffolds, the multilayer functional biomimetic scaffold prepared by this invention can effectively increase tensile strength by 1.1 to 5 times (existing technologies prepare scaffolds with porous structures and dense layers, while this invention adds a uniform coating to the outer wall of the scaffold using the process described herein, thus improving its tensile strength to a certain extent).

[0043] Invention Mechanism:

[0044] The fabrication process of functional biomimetic scaffolds is actually carried out within a mold. The mold's sidewalls possess microchannel structures, and the inner walls of the mold cavity have patterned drainage networks. After filling the mold with one or more of materials such as silk fibroin, gelatin, collagen, and chitosan, and freeze-drying, a porous scaffold is obtained. The freeze-dried porous scaffold exhibits shrinkage gaps. If a coating is applied to the porous scaffold through the microchannels on the mold sidewall, it is difficult to prevent the coating solution from penetrating into the porous scaffold. While this method can simulate a porous structure and impart a dense, functional coating to the outer wall, the penetration of the coating solution into the porous structure affects the porous structure itself. Changes in the porous structure actually influence cell growth, adhesion, and nutrient transport. Furthermore, the coating on the scaffold is often uneven. Therefore, directly applying the coating solution to the porous scaffold through the sidewall is not ideal. Preventing the penetration of the coating solution into the porous structure and ensuring a uniform distribution of the coating solution on the surface have become crucial. This invention involves inoculating a scaffold with a high density of bacterial strains. Through the self-assembly of bacterial cellulose, an interlaced nanoscale network structure is generated on the outer wall of the scaffold (too low a bacterial strain density will result in poor compactness and fail to prevent the coating solution from penetrating). This prevents the coating solution from penetrating. Furthermore, by utilizing a microchannel system and a patterned drainage network, the coating solution is perfectly solidified on the scaffold, thus giving it functionality.

[0045] Furthermore, freeze-drying is a common drying method, and the material itself undergoes slight shrinkage after the sublimation of crystal water, thus creating shrinkage gaps within the mold. These shrinkage gaps provide conditions for coating application. Utilizing these gaps, a coating solution is injected, forming a functional coating on the outer wall. Ensuring the coating solution uniformly fills the shrinkage spaces is a key issue. In the presence of these gaps, microporous channels are used to inject the coating solution into the gaps. Guided by drainage channels on the inner wall of the three-dimensional tissue model cavity and transported by grooves in a patterned drainage network, the coating solution forms a uniform coating on the surface of the bacterial cellulose-based composite scaffold, resulting in a multi-layered functional biomimetic scaffold with sustained and effective functionality and excellent mechanical properties.

[0046] Beneficial effects:

[0047] (1) A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold of the present invention utilizes the shrinkage space after material treatment, microporous channel injection and the construction of a drainage network system to provide a process that can impart a uniform functional coating to the multilayer biomimetic scaffold.

[0048] (2) The present invention provides a method for preparing a bacterial cellulose-based multilayer structure functional biomimetic scaffold. In terms of bacterial culture and fermentation, by changing the formula of the culture medium and increasing the oxygen content, the cycle of bacterial fermentation and growth to the entire shrinkage space is shortened, while the density of the three-dimensional network structure of the outer wall of the biomimetic scaffold is improved.

[0049] (3) The present invention provides a method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, which reduces the risk of in vivo transplantation materials during surgery by introducing a functional coating. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the overall structure of the silicone model;

[0051] Figure 2 This is a schematic diagram of the cross-section of the silicone model;

[0052] Figure 3 This is a schematic diagram of the microchannel system on the silicone model in Example 1;

[0053] Figure 4 This is a schematic diagram of the microchannel system on the silicone model in Example 2;

[0054] Figure 5 Electron micrograph of the cross-section of the bacterial cellulose-based multilayer functional biomimetic scaffold prepared in Example 1;

[0055] Figure 6Electron micrograph of the outer wall of the bacterial cellulose-based multilayer functional biomimetic scaffold prepared in Example 1;

[0056] Among them, 1-micropore channel, 2-drainage groove, 3-patterned drainage network. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0058] The curing agent used in this invention is the curing agent corresponding to Dow Corning DC184 silicone.

[0059] The hardness in this invention is measured using a Shore hardness tester.

[0060] The three-dimensional tissue morphology in the embodiments of the present invention is the three-dimensional tissue morphology of the human urethra, human ear, human meniscus, or human finger bone.

[0061] Example 1

[0062] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0063] (1) Preparation of silicone mold;

[0064] First, a 3D printed model with a three-dimensional tissue morphology (the three-dimensional tissue morphology of the human urethra) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:1 is stirred for 6 minutes and poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 40℃, drying time of 12h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 5 days before demolding, resulting in a silicone mold with a hardness of 5 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0065] (2) A silicone model with a microchannel system is obtained by processing a silicone mold, such as... Figure 1 , 2 As shown; specifically, after the silicone has cured and molded, a microchannel system is constructed, such as... Figure 3As shown, the microchannel system consists of a microporous channel 1, a drainage groove 2, and a patterned drainage network 3. First, a 25G injection needle is inserted transversely through the outer wall of the silicone model along a direction perpendicular to the axial direction of the silicone model until it reaches the surface of the cavity of the internal three-dimensional tissue model, thereby forming a microporous channel with a diameter of 0.5 mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model through subtractive processing, thus constructing the patterned drainage network. The grooves are parallel to the axial direction of the silicone model and perpendicular to the horizontal plane, and are evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 200 μm, the groove depth is 30 μm, and the interval between two adjacent grooves is 30 μm. Then, grooves perpendicular to the patterned drainage network are constructed on the surface of the cavity of the three-dimensional tissue model, namely drainage grooves. The drainage grooves are connected to the microporous channel. The groove width of the drainage groove is 200 μm and the groove depth is 30 μm.

[0066] (3) A 10wt% silk fibroin solution (solvent is water) was injected into a silicone model with microporous channels until the entire groove was filled. After freezing at -20℃ for 18h, it was freeze-dried for 8h under the conditions of cold trap temperature of -45℃, sample temperature of -20℃ and vacuum degree of 0.1Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0067] (4) Inject crosslinking agent (a 1 wt% solution prepared by dissolving glutaraldehyde in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 8 min.

[0068] (5) The concentration of Acetobacter xylinum was 10. 10 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0069] The culture medium was formulated as follows: 3.5 wt% glucose, 0.5 wt% peptone, 0.6 wt% yeast extract, 0.5 wt% citric acid, 0.04 wt% disodium hydrogen phosphate, 0.2 wt% potassium dihydrogen phosphate, 0.06 wt% polyoxyethylene (20EO) methyl glucoside sesquioleate, and water as the remainder. The growth cycle of the bacterial cellulose dense layer, from fermentation to full growth and filling the entire shrinkage space, was 3 days.

[0070] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 15 minutes. Then, they were freeze-dried for 12 hours under the conditions of cold trap temperature of -45℃, sample temperature of -20℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 1 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove to fill the entire gap. After curing and drying at room temperature for 10 minutes, the scaffold was demolded to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0071] The preparation process of the coating solution is as follows: Polyhexamethylene biguanide hydrochloride (PHMB) and sodium dodecanoate are dissolved in water at a concentration of 0.01 g / mL. Then, sodium stearate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is dried by freeze dryer. Finally, 1.0 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.0 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, and forms a uniform coating with a thickness of 10 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be continuously exerted, and the uniform coating can kill 99.1% of Staphylococcus aureus.

[0072] like Figure 5 , 6 As shown, the outer wall of the prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 43±28nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 108±59μm; compared with the simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 1.2 times.

[0073] Example 2

[0074] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0075] (1) Preparation of silicone mold;

[0076] First, a 3D printed model with a three-dimensional tissue morphology (human ear three-dimensional tissue morphology) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:10 is poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 50℃, drying time of 10h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 7 days before demolding, resulting in a silicone mold with a hardness of 20 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0077] (2) A silicone model with a microchannel system is obtained by processing a silicone mold, such as... Figure 1 , 2 As shown; specifically, after the silicone has cured and molded, a microchannel system is constructed, such as... Figure 4 As shown, the microchannel system consists of micropore channels 1, drainage grooves 2, and patterned drainage networks 3. First, a 30G injection needle is inserted transversely through the outer wall of the silicone model along a direction perpendicular to the axial direction of the silicone model until it reaches the surface of the cavity of the internal three-dimensional tissue model, thereby forming micropore channels with a diameter of 0.3 mm. Then, through subtractive processing, orderly arranged grooves are formed on the surface of the cavity of the three-dimensional tissue model, thus constructing the patterned drainage network. These grooves are perpendicular to the axial direction of the silicone model and parallel to the horizontal plane, and are evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 150 μm, the groove depth is 100 μm, and the interval between two adjacent grooves is 100 μm. Then, on the surface of the cavity of the three-dimensional tissue model, grooves perpendicular to the patterned drainage network are constructed, namely drainage grooves. The drainage grooves are connected to the micropore channels. The groove width is 150 μm and the groove depth is 100 μm.

[0078] (3) A 2wt% gelatin solution (solvent is water) was injected into a silicone model with microporous channels until the entire groove was filled. After freezing at -20℃ for 12h, it was freeze-dried for 12h under the conditions of cold trap temperature of -40℃, sample temperature of -30℃ and vacuum degree of 0.1Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0079] (4) Inject crosslinking agent (a 2wt% solution prepared by dissolving acetic anhydride in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 10 min.

[0080] (5) The concentration of Acetobacter xylinum was 10. 9 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0081] The culture medium formula is as follows: fructose 1wt%, peptone 0.05wt%, yeast extract 1wt%, citric acid 0.01wt%, disodium hydrogen phosphate 0.4wt%, potassium dihydrogen phosphate 0.02wt%, Myrj49 0.1wt%, and water as the remainder; the growth cycle of the bacterial cellulose dense layer from fermentation to complete growth and filling the entire shrinkage space is 7 days.

[0082] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 10 minutes. Then, they were freeze-dried for 8 hours under the conditions of cold trap temperature of -40℃, sample temperature of -30℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.4 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove, so that the coating solution filled the entire gap. After curing and drying at room temperature for 15 minutes, the mold was removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0083] The preparation process of the coating solution is as follows: dioxanone ethylene oxide and sodium dodecanoate are dissolved in water at a concentration of 0.02 g / mL, respectively. Then, sodium stearate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried by freeze dryer. Finally, 1.2 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.2 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, forming a uniform coating with a thickness of 4.5 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be continuously exerted, and the uniform coating can kill 99.1% of Staphylococcus aureus and 99.3% of Escherichia coli.

[0084] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an ultrafine nano-network structure on its outer wall, with the diameter of the ultrafine nanofibers on the outer wall being 26±32nm. The pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 120±34μm. Compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 2 times.

[0085] Example 3

[0086] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0087] (1) Preparation of silicone mold;

[0088] First, a 3D printed model with a three-dimensional tissue morphology (three-dimensional tissue morphology of human finger bone) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:8 is stirred for 8 minutes and poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 55℃, drying time of 9h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 3 days before demolding, resulting in a silicone mold with a hardness of 16 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0089] (2) A silicone model with a microchannel system is obtained by processing a silicone mold; specifically, after the silicone is cured and molded, a microchannel system is constructed. The microchannel system consists of a micropore channel 1, a drainage groove 2 and a patterned drainage network 3. First, a 29G injection needle is used to penetrate the outer wall of the silicone model along the direction perpendicular to the axial direction of the silicone model until the surface of the cavity of the internal three-dimensional tissue model, thereby forming a micropore channel with a diameter of 0.33mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model by subtractive processing, that is, a patterned drainage network is constructed. The groove is perpendicular to the axial direction of the silicone model and parallel to the horizontal plane. It is evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 30μm, the groove depth is 60μm, and the interval between two adjacent grooves is 70μm. Then, on the surface of the cavity of the three-dimensional tissue model, a groove that passes vertically through the patterned drainage network is constructed, that is, a drainage groove. The drainage groove is connected to the micropore channel. The groove width of the drainage groove is 30μm and the groove depth is 60μm.

[0090] (3) A collagen solution with a concentration of 4 wt% (solvent is water) was injected into a silicone model with microporous channels until the entire groove was filled. After freezing at -20℃ for 24 h, it was freeze-dried for 10 h under the conditions of cold trap temperature of -50℃, sample temperature of -25℃ and vacuum degree of 0.1 Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0091] (4) Inject crosslinking agent (a 0.2 wt% solution prepared by dissolving diglycidyl ether in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 5 min.

[0092] (5) The concentration of Acetobacter xylinum was 10. 9 The mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0093] The culture medium formula is as follows: sucrose 6wt%, peptone 1wt%, yeast extract 0.05wt%, citric acid 0.05wt%, disodium hydrogen phosphate 0.2wt%, potassium dihydrogen phosphate 0.18wt%, Myrj51 0.05wt%, and water as the remainder; the growth cycle of the bacterial cellulose dense layer from fermentation to complete growth and filling the entire shrinkage space is 6 days.

[0094] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 20 minutes. Then, they were freeze-dried for 10 hours under the conditions of cold trap temperature of -50℃, sample temperature of -25℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.7 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove to fill the entire gap. After curing and drying at room temperature for 14 minutes, the scaffold was demolded to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0095] The preparation process of the coating solution is as follows: dioxanone ethylene oxide and sodium dodecanoate are dissolved in water at a concentration of 0.02 g / mL, respectively. Then, sodium stearate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried by freeze dryer. Finally, 1.3 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.3 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, forming a uniform coating with a thickness of 9.1 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be sustained, and the uniform coating can kill 99.4% of Staphylococcus aureus and 99.5% of Escherichia coli.

[0096] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an outer wall with an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 30±36nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 132±48μm; compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 1.8 times.

[0097] Example 4

[0098] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0099] (1) Preparation of silicone mold;

[0100] First, a 3D printed model with a three-dimensional tissue morphology (the three-dimensional tissue morphology of the human meniscus) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:2 is poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 50℃, drying time of 10h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 4 days before demolding, resulting in a silicone mold with a hardness of 6 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0101] (2) A silicone model with a microchannel system is obtained by processing a silicone mold; specifically, after the silicone is cured and molded, a microchannel system is constructed. The microchannel system consists of a micropore channel 1, a drainage groove 2 and a patterned drainage network 3. First, a 27G injection needle is used to penetrate the outer wall of the silicone model along the direction perpendicular to the axial direction of the silicone model until the surface of the cavity of the internal three-dimensional tissue model, thereby forming a micropore channel with a diameter of 0.4 mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model by subtractive processing, that is, a patterned drainage network is constructed. The groove is perpendicular to the axial direction of the silicone model and parallel to the horizontal plane. It is evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 100 μm, the groove depth is 50 μm, and the interval between two adjacent grooves is 40 μm. Then, on the surface of the cavity of the three-dimensional tissue model, a groove that passes vertically through the patterned drainage network is constructed, that is, a drainage groove. The drainage groove is connected to the micropore channel. The groove width of the drainage groove is 100 μm and the groove depth is 50 μm.

[0102] (3) Inject an 8 wt% chitosan solution (water as solvent) into a silicone model with microporous channels until the entire groove is filled. After freezing at -20℃ for 15 h, freeze-dry for 8 h under the conditions of cold trap temperature of -48℃, sample temperature of -22℃ and vacuum degree of 0.1 Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0103] (4) Inject crosslinking agent (a 0.8 wt% solution prepared by dissolving glutaraldehyde in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 6 min.

[0104] (5) The concentration of Acetobacter xylinum was 10. 12 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0105] The culture medium formula is as follows: mannitol 5wt%, peptone 0.1wt%, yeast extract 0.1wt%, citric acid 0.1wt%, disodium hydrogen phosphate 0.3wt%, potassium dihydrogen phosphate 0.15wt%, Myrj52 with an HLB value of 16.9 0.1wt%, and water as the balance. The growth cycle of the bacterial cellulose dense layer from fermentation to complete growth and filling the entire shrinkage space is 6 days.

[0106] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 12 minutes. Then, they were freeze-dried for 11 hours under the conditions of cold trap temperature of -48℃, sample temperature of -22℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.5 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove, so that the coating solution filled the entire gap. After curing and drying at room temperature for 11 minutes, the mold was removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0107] The preparation process of the coating solution is as follows: Dioxanone ethylene oxide and sodium stearate are dissolved in water at a concentration of 0.03 g / mL, respectively. Then, the sodium stearate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried by a freeze dryer. Finally, 1.1 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.1 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, forming a uniform coating with a thickness of 5.3 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be continuously exerted, and the uniform coating can kill 99.7% of Staphylococcus aureus and 99.8% of Escherichia coli.

[0108] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an outer wall with an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 34±43nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 128±40μm; compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 3 times.

[0109] Example 5

[0110] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0111] (1) Preparation of silicone mold;

[0112] First, a 3D printed model with a three-dimensional tissue morphology (the three-dimensional tissue morphology of the human urethra) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:6 is stirred for 7 minutes and poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 45℃, drying time of 11h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 6 days before demolding, resulting in a silicone mold with a hardness of 10 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0113] (2) A silicone model with a microchannel system is obtained by processing a silicone mold; specifically, after the silicone is cured and molded, a microchannel system is constructed. The microchannel system consists of a micropore channel 1, a drainage groove 2 and a patterned drainage network 3. First, a 26G injection needle is used to penetrate the outer wall of the silicone model along the direction perpendicular to the axial direction of the silicone model until it reaches the surface of the cavity of the internal three-dimensional tissue model, thereby forming a micropore channel with a diameter of 0.45mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model by subtractive processing, that is, a patterned drainage network is constructed. The groove is perpendicular to the axial direction of the silicone model and parallel to the horizontal plane. It is evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 80μm, the groove depth is 50μm, and the interval between two adjacent grooves is 40μm. Then, a groove that passes vertically through the patterned drainage network is constructed on the surface of the cavity of the three-dimensional tissue model, that is, a drainage groove. The drainage groove is connected to the micropore channel. The groove width of the drainage groove is 80μm and the groove depth is 50μm.

[0114] (3) A mixture of gelatin and chitosan with a mass ratio of 1:1 and a concentration of 6 wt% (using water as the solvent) was injected into a silicone mold with microporous channels until the entire groove was filled. After freezing at -20℃ for 18 h, it was freeze-dried for 10 h under the conditions of cold trap temperature of -42℃, sample temperature of -25℃ and vacuum degree of 0.1 Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0115] (4) Inject crosslinking agent (a 1.2 wt% solution prepared by dissolving acetic anhydride in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 7 min.

[0116] (5) The concentration of Acetobacter xylinum was 10. 15 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0117] The culture medium was formulated as follows: 2 wt% of a 1:1 mixture of glucose and fructose, 0.8 wt% of peptone, 0.2 wt% of yeast extract, 0.2 wt% of citric acid, 0.1 wt% of disodium hydrogen phosphate, 0.1 wt% of potassium dihydrogen phosphate, 0.09% of Myrj53, and water as the remainder. The growth cycle of the bacterial cellulose dense layer, from fermentation to full growth and filling of the entire shrinkage space, was 5 days.

[0118] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 18 minutes. Then, they were freeze-dried for 9 hours under the conditions of cold trap temperature of -42℃, sample temperature of -25℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.85mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove, so that the coating solution filled the entire gap. After curing and drying at room temperature for 13 minutes, the mold was removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0119] The preparation process of the coating solution is as follows: Polyhexamethylene biguanide hydrochloride (PHMB) and sodium stearate are dissolved in water at a concentration of 0.02 g / mL. Then, the sodium stearate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried by freeze dryer. Finally, 1.0 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.0 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, forming a uniform coating with a thickness of 8 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be continuously exerted, and the uniform coating can kill 99.4% of Staphylococcus aureus.

[0120] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an outer wall with an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 37±28nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 146±52μm; compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 2.5 times.

[0121] Example 6

[0122] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0123] (1) Preparation of silicone mold;

[0124] First, a 3D printed model with a three-dimensional tissue morphology (the three-dimensional tissue morphology of a human finger bone) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:4 is poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 60℃, drying time of 8h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 5 days before demolding, resulting in a silicone mold with a hardness of 8 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0125] (2) A silicone model with a microchannel system is obtained by processing a silicone mold; specifically, after the silicone is cured and molded, a microchannel system is constructed. The microchannel system consists of a micropore channel 1, a drainage groove 2 and a patterned drainage network 3. First, a 28G injection needle is used to penetrate the outer wall of the silicone model along the direction perpendicular to the axial direction of the silicone model until it reaches the surface of the cavity of the internal three-dimensional tissue model, thereby forming a micropore channel with a diameter of 0.36mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model by subtractive processing, that is, a patterned drainage network is constructed. The groove is perpendicular to the axial direction of the silicone model and parallel to the horizontal plane. It is evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 90μm, the groove depth is 70μm, and the interval between two adjacent grooves is 50μm. Then, on the surface of the cavity of the three-dimensional tissue model, a groove that passes vertically through the patterned drainage network is constructed, that is, a drainage groove. The drainage groove is connected to the micropore channel. The groove width of the drainage groove is 90μm and the groove depth is 70μm.

[0126] (3) A 5 wt% collagen solution (solvent is water) was injected into a silicone model with microporous channels until the entire groove was filled. After freezing at -20℃ for 20 h, it was freeze-dried for 12 h under the conditions of cold trap temperature of -46℃, sample temperature of -28℃ and vacuum degree of 0.1 Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0127] (4) Inject crosslinking agent (a 1.5 wt% solution prepared by dissolving diglycidyl ether in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 9 min.

[0128] (5) The concentration of Acetobacter xylinum was 10. 13 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0129] The culture medium was formulated as follows: 3 wt% glucose, 0.2 wt% peptone, 0.8 wt% yeast extract, 0.3 wt% citric acid, 0.25 wt% disodium hydrogen phosphate, 0.08 wt% potassium dihydrogen phosphate, 0.08 wt% Tween 20 with an HLB value of 16.7, and water as the remainder. The growth cycle of the bacterial cellulose dense layer, from fermentation to full growth and filling the entire shrinkage space, was 4 days.

[0130] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 15 minutes. Then, they were freeze-dried for 10 hours under the conditions of cold trap temperature of -46℃, sample temperature of -28℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.8 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove, so that the coating solution filled the entire gap. After curing and drying at room temperature for 12 minutes, the mold was removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0131] The preparation process of the coating solution is as follows: Polyhexamethylene biguanide hydrochloride (PHMB) and sodium dodecanoate are dissolved in water at a concentration of 0.01 g / ml. Then, the sodium dodecanoate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried by freeze dryer. Finally, 1.2 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.2 wt% coating solution. The coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, forming a uniform coating with a thickness of 9.2 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be sustained, and the uniform coating can kill 99.2% of Staphylococcus aureus.

[0132] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an outer wall with an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 39±23nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 189±46μm; compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 3.2 times.

[0133] Example 7

[0134] A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, the specific steps of which are as follows:

[0135] (1) Preparation of silicone mold;

[0136] First, a 3D printed model with a three-dimensional tissue morphology (the three-dimensional tissue morphology of a human meniscus) is placed in a square container. Then, a mixture of silicone and curing agent at a mass ratio of 100:5 is stirred for 6 minutes and poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for treatment (vacuum degree of 0.01MPa, drying temperature of 40℃, drying time of 12h). Finally, the square container is removed from the vacuum drying oven and placed at room temperature to cool and cure for 6 days before demolding, resulting in a silicone mold with a hardness of 9 degrees and a three-dimensional tissue model cavity inside the silicone mold.

[0137] (2) A silicone model with a microchannel system is obtained by processing a silicone mold; specifically, after the silicone is cured and molded, a microchannel system is constructed. The microchannel system consists of a micropore channel 1, a drainage groove 2 and a patterned drainage network 3. First, a 25G injection needle is used to penetrate the outer wall of the silicone model along the direction perpendicular to the axial direction of the silicone model until the surface of the cavity of the internal three-dimensional tissue model, thereby forming a micropore channel with a diameter of 0.5mm. Then, an ordered arrangement of grooves is formed on the surface of the cavity of the three-dimensional tissue model by subtractive processing, that is, a patterned drainage network is constructed. The groove is perpendicular to the axial direction of the silicone model and parallel to the horizontal plane. It is evenly arranged on the surface of the cavity of the three-dimensional tissue model. The groove width is 160μm, the groove depth is 50μm, and the interval between two adjacent grooves is 90μm. Then, on the surface of the cavity of the three-dimensional tissue model, a groove that passes vertically through the patterned drainage network is constructed, that is, a drainage groove. The drainage groove is connected to the micropore channel. The groove width of the drainage groove is 160μm and the groove depth is 50μm.

[0138] (3) A 7wt% gelatin and silk fibroin solution (solvent is water) with a mass ratio of 1:1 was injected into a silicone model with microporous channels until the entire groove was filled. After freezing at -20℃ for 22h, it was freeze-dried for 8h under the conditions of cold trap temperature of -45℃, sample temperature of -26℃ and vacuum degree of 0.1Pa (the freeze-drying process will form a shrinkage space) to obtain a porous biological scaffold.

[0139] (4) Inject crosslinking agent (a 1.8 wt% solution prepared by dissolving glutaraldehyde in anhydrous ethanol) into the silicone model with microchannels, so that the crosslinking agent completely permeates the porous biological scaffold, and perform room temperature crosslinking treatment on the porous biological scaffold for 8 min.

[0140] (5) The concentration of Acetobacter xylinum was 10. 12 A mixture of Acetobacter xylinum and culture medium was inoculated onto the porous biological scaffold after cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold.

[0141] The culture medium formula is as follows: fructose 4wt%, peptone 0.6wt%, yeast extract 0.5wt%, citric acid 0.4wt%, disodium hydrogen phosphate 0.35wt%, potassium dihydrogen phosphate 0.05wt%, Tween 40 with an HLB value of 15.6 0.05wt%, and water as the balance. The growth cycle of the bacterial cellulose dense layer from fermentation to complete growth and filling the entire shrinkage space is 5 days.

[0142] (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model with microchannels were soaked in 75% alcohol for 18 minutes. Then, they were freeze-dried for 10 hours under the conditions of cold trap temperature of -45℃, sample temperature of -26℃ and vacuum degree of 0.1Pa. This caused the porous biological scaffold to no longer adhere tightly to the inner wall of the silicone model with microchannels, but to shrink inward by 0.3 mm. The coating solution was injected into the shrinkage gap through microporous channel injection, drainage network and drainage groove, so that the coating solution filled the entire gap. After curing and drying at room temperature for 12 minutes, the mold was removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

[0143] The preparation process of the coating solution is as follows: Dioxanone ethylene oxide and sodium octanoate are dissolved in water at a concentration of 0.03 g / mL, respectively. Then, the sodium octanoate aqueous solution is added dropwise to the former solution to make the concentration of anionic groups (millimolar) reach the same level as that of cationic groups (millimolar). After the reaction is completed, the solution is placed in a 50 ml centrifuge tube, centrifuged, and the supernatant is removed. The substrate is then dried using a freeze dryer. Finally, 1.5 g of the dried sample is weighed and dissolved in anhydrous ethanol to prepare a 1.5 wt% coating solution. The coating solution is passed through microporous channels and drainage grooves into the grooves of the patterned drainage network, forming a uniform coating with a thickness of 4.5 μm on the surface of the bacterial cellulose-based composite scaffold. The uniform coating allows its functionality to be sustained, and the uniform coating can kill 99.9% of Staphylococcus aureus and 99.9% of Escherichia coli.

[0144] The prepared bacterial cellulose-based multilayer functional biomimetic scaffold has an outer wall with an ultrafine nano-network structure, and the diameter of the ultrafine nanofibers on the outer wall is 46±19nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 172±54μm; compared with a simple porous scaffold, the tensile strength of the bacterial cellulose-based multilayer functional biomimetic scaffold is increased by 5 times.

[0145] The bacterial cellulose-based multilayer functional biomimetic scaffold prepared in this invention requires 8 to 14 days to grow normally under laboratory conditions, corresponding to the above-mentioned culture temperature and culture medium formulation. After adding a surfactant, this time is shortened to 3 to 7 days.

[0146] In Example 1, without the surfactant polyoxyethylene (20EO) methyl glucoside sesquioleate, the growth was relatively slow, and it took 13 days for the bacterial cellulose to fully grow. The thickness was 0.6 mm and could not fill the entire shrinkage space. After adding the surfactant, the growth of bacterial cellulose was accelerated due to the increased oxygen content in the culture medium. It took 7 days for the bacterial cellulose to fully grow, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 1 mm.

[0147] In Example 2, without the surfactant Myrj49, growth was relatively slow, requiring 8 days for complete bacterial cellulose growth, and the thickness was 0.2 mm, which did not fill the entire shrinkage space. After adding the surfactant, the bacterial cellulose growth was accelerated due to the increased oxygen content in the culture medium, requiring 3 days for complete growth, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.4 mm.

[0148] In Example 3, without the surfactant Myrj51, growth was relatively slow, requiring 9 days for complete bacterial cellulose growth, and the thickness was 0.4 mm, which did not fill the entire shrinkage space. After adding the surfactant, the bacterial cellulose growth was accelerated due to the increased oxygen content in the culture medium, requiring 4 days for complete growth, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.7 mm.

[0149] In Example 4, without the surfactant Myrj52, growth was relatively slow, requiring 8 days for complete bacterial cellulose growth, and the thickness was 0.3 mm, which did not fill the entire shrinkage space. After adding the surfactant, the bacterial cellulose growth was accelerated due to the increased oxygen content in the culture medium, requiring 4 days for complete growth, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.5 mm.

[0150] In Example 5, without Myrj53, growth was relatively slow, requiring 12 days for complete bacterial cellulose growth, and the thickness was 0.6 mm, which did not fill the entire shrinkage space. After adding the surfactant, the bacterial cellulose growth was accelerated due to the increased oxygen content in the culture medium, requiring 7 days for complete growth, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.85 mm.

[0151] In Example 6, without the addition of surfactant Tween 20, growth was relatively slow, and it took 10 days for the bacterial cellulose to fully grow. The thickness was 0.3 mm and could not fill the entire shrinkage space. After adding the surfactant, the growth of bacterial cellulose was accelerated due to the increased oxygen content in the culture medium. It took 5 days for the bacterial cellulose to fully grow, and the dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.8 mm.

[0152] In Example 7, without the addition of surfactant Tween 40, growth was relatively slow, requiring 7 days for complete bacterial cellulose growth, and the thickness was 0.2 mm, which did not fill the entire shrinkage space. After adding surfactant, the growth of bacterial cellulose was accelerated due to the increased oxygen content in the culture medium, requiring 3 days for complete growth, and a dense bacterial cellulose layer filled the entire shrinkage space with a thickness of 0.3 mm.

Claims

1. A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold, characterized in that: First, the biomaterial solution is injected into a silicone model and freeze-dried to obtain a porous bioscaffold. Then, the porous bioscaffold is cross-linked. Next, bacterial culture is inoculated onto the cross-linked porous bioscaffold. After static culture and freeze-drying, a bacterial cellulose-based composite scaffold is obtained. Finally, a coating solution is applied to the bacterial cellulose-based composite scaffold through a microchannel system on the silicone model, and the scaffold is demolded to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold. The biomaterial is one or more of the following: silk fibroin, gelatin, collagen, chitosan, extracellular matrix, and biodegradable synthetic polymer. The silicone model has a three-dimensional tissue model cavity inside; the microchannel system on the silicone model consists of micropore channels, drainage grooves and patterned drainage networks. The micropore channels are channels formed by the injection needle penetrating the outer wall of the silicone model along a direction perpendicular to the axial direction of the silicone model until reaching the surface of the internal three-dimensional tissue model cavity. The patterned drainage network is an ordered arrangement of grooves formed on the surface of the three-dimensional tissue model cavity through subtractive processing. The drainage grooves are grooves that pass vertically through the patterned drainage network. The drainage grooves are located on the surface of the three-dimensional tissue model cavity and are connected to the micropore channels. The culture medium is a mixture of Acetobacter xylinum and culture medium, with a concentration of 10 in the culture medium. 9 ~10 15 cells / mL; The diameter of the micropore channel is 0.3~0.5mm; The grooves constituting the patterned drainage network have a width of 30~200μm and a depth of 30~100μm. All the grooves constituting the patterned drainage network are evenly arranged on the surface of the cavity of the three-dimensional tissue model, and the interval between two adjacent grooves is 30~100μm.

2. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 1, characterized in that, The culture medium formula is as follows: Substance A 1-6 wt%; Peptone 0.05–1 wt%; Yeast extract 0.05–1 wt%; Citric acid 0.01–0.5 wt%; Disodium hydrogen phosphate 0.04–0.4 wt%; Potassium dihydrogen phosphate 0.02–0.2 wt%; Surfactant 0.05~0.1wt%; Water balance; Substance A is one or more of glucose, fructose, sucrose, and mannitol; The surfactant has an HLB value of 15-18.

3. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 2, characterized in that, The surfactant is polyoxyethylene monostearate, Tween 20, or Tween 40.

4. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 3, characterized in that, The specific steps are as follows: (1) Preparation of silicone mold; First, a 3D printed model with a three-dimensional tissue morphology is placed in a square container. Then, a mixture of silicone and curing agent is poured into the square container to completely immerse the 3D printed model. The square container is then transferred to a vacuum drying oven for processing. Finally, the square container is removed from the vacuum drying oven and allowed to cool and solidify at room temperature before demolding, thus obtaining a silicone mold with a three-dimensional tissue model cavity inside. The hardness of the silicone mold, measured using a Shore hardness tester, is 5~20 degrees. (2) A silicone model with a microchannel system is obtained by processing a silicone mold; (3) Inject the biomaterial solution into the silicone model until the entire cavity of the three-dimensional tissue model is filled, freeze at -20℃ for 12~24h and then freeze-dry to obtain a porous bioscaffold; (4) Inject crosslinking agent into the silicone model to completely impregnate the porous biological scaffold and perform room temperature crosslinking treatment on the porous biological scaffold. (5) The cultured bacterial solution was used to inoculate the porous biological scaffold after the cross-linking treatment in step (4), and an interlaced nanoscale network structure was generated on the outer wall of the porous biological scaffold to obtain a bacterial cellulose-based composite scaffold. (6) The bacterial cellulose-based composite scaffold obtained in step (5) and the silicone model are soaked in 75% alcohol and freeze-dried in sequence to shrink the porous biological scaffold inward by 0.3~1mm. The coating solution is injected into the shrinkage gap to fill the entire gap. After curing and drying, the mold is removed to obtain a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

5. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, In step (1), the mass ratio of silicone to curing agent is 100:1~10.

6. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, The concentration of the biomaterial solution in step (3) is 2~10wt%.

7. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, The process parameters for freeze drying in step (3) are: cold trap temperature -50~-40℃, sample temperature -30~-20℃, vacuum degree 0.1Pa, and freeze drying time 8~12h.

8. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, In step (4), the crosslinking agent is a solution with a concentration of 0.2-2 wt% prepared by dissolving glutaraldehyde, acetic anhydride or diglycidyl ether in anhydrous ethanol, and the crosslinking treatment time is 5-10 min.

9. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, In step (6), the soaking time of 75% alcohol is 10~20 minutes; the process parameters of freeze drying are: cold trap temperature -50~-40℃, sample temperature -30~-20℃, vacuum degree 0.1Pa, freeze drying time 8~12h; the curing drying temperature is room temperature, and the curing drying time is 10~15min.

10. The method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to claim 4, characterized in that, In step (6), the coating solution enters the groove of the patterned drainage network through microporous channels and drainage grooves, and forms a coating on the surface of the bacterial cellulose-based composite scaffold, thus obtaining a bacterial cellulose-based multilayer structure functional biomimetic scaffold.

11. A method for preparing a bacterial cellulose-based multilayer functional biomimetic scaffold according to any one of claims 1 to 10, characterized in that, The outer wall of the bacterial cellulose-based multilayer functional biomimetic scaffold has an ultrafine nano-network structure, with the diameter of the ultrafine nanofibers on the outer wall being less than 100 nm; the pore size of the bacterial cellulose-based multilayer functional biomimetic scaffold is 100~300 μm.

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