CN-116214795-B - Preparation method of bacterial cellulose-based multi-layer structure functional bionic scaffold

CN116214795BCN 116214795 BCN116214795 BCN 116214795BCN-116214795-B

Abstract

The invention relates to a preparation method of a bacterial cellulose-based multi-layer structure functional bionic scaffold, which comprises the steps of firstly injecting a biological material solution into a silica gel model with a micro-channel system, freeze-drying to obtain a porous biological scaffold, then performing cross-linking treatment on the porous biological scaffold, inoculating culture bacteria liquid with the concentration of 10 9 ～10 15 bacteria/mL on the cross-linked porous biological scaffold, performing static culture and freeze-drying to obtain a bacterial cellulose-based composite scaffold, and finally coating the bacterial cellulose-based composite scaffold with a coating solution through the micro-channel system, and performing demoulding treatment to obtain the bacterial cellulose-based multi-layer structure functional bionic scaffold. The invention provides a functional coating process for a multilayer bionic bracket by utilizing a shrinkage space after material treatment, a micropore channel and a patterned drainage network injection method, so that the penetration of a coating solution is effectively prevented, and the coating solution is perfectly solidified on the bracket.

Inventors

SUN WEIHUA
YAN ZHIYONG
LI ZHE
CUI LI
Tang Xineng
WEI JIALIN
YAO YONGBO
CHEN CHIHAO
XU ZHI
SHENG JUNLU

Assignees

嘉兴学院

Dates

Publication Date: 20260512
Application Date: 20221205

Claims (11)

1. The preparation method of the bacterial cellulose-based multi-layer structure functional bionic scaffold is characterized by comprising the steps of firstly injecting a biological material solution into a silica gel model, performing freeze drying to obtain a porous biological scaffold, performing cross-linking treatment on the porous biological scaffold, inoculating culture bacteria liquid on the cross-linked porous biological scaffold, performing static culture and freeze drying to obtain a bacterial cellulose-based composite scaffold, and finally performing coating treatment on the bacterial cellulose-based composite scaffold by adopting a coating solution through a micro-channel system on the silica gel model, and demolding to obtain the bacterial cellulose-based multi-layer structure functional bionic scaffold; The biological material is more than one of silk fibroin, gelatin, collagen, chitosan, extracellular matrix and degradable synthetic polymer; the micro-channel system on the silica gel model consists of a micro-pore channel, a drainage groove and a patterning drainage network, wherein the micro-pore channel is a pore canal which is formed by crossing the outer wall of the silica gel model to the surface of the cavity of the internal three-dimensional tissue model along the axial direction perpendicular to the silica gel model by an injection needle, the patterning drainage network is an orderly arranged groove formed by material reduction processing on the surface of the cavity of the three-dimensional tissue model, the drainage groove is a groove which vertically penetrates through the patterning drainage network, the drainage groove is positioned on the surface of the cavity of the three-dimensional tissue model, and the drainage groove is connected with the micro-pore channel; The culture bacterial liquid is a mixed liquid of acetobacter xylinum and a culture liquid, and the concentration of acetobacter xylinum in the culture bacterial liquid is 10 9 ~10 15 /mL; The diameter of the micropore channel is 0.3-0.5 mm; The width of the grooves forming the patterned drainage network is 30-200 mu m, the depth of the grooves is 30-100 mu m, all the grooves forming the patterned drainage network are uniformly arranged on the surface of the cavity of the three-dimensional tissue model, and the interval between every two adjacent grooves is 30-100 mu m.
2. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 1, wherein the formula of the culture solution is as follows: 1-6wt% of a substance A; 0.05-1wt% of peptone; 0.05-1wt% of yeast extract; 0.01-0.5wt% of citric acid; 0.04-0.4wt% of disodium hydrogen phosphate; 0.02-0.2wt% of monopotassium phosphate; 0.05-0.1wt% of a surfactant; The balance of water; the substance A is more than one of glucose, fructose, sucrose and mannitol; the HLB value of the surfactant is 15-18.
3. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 2, wherein the surfactant is polyoxyethylene monostearate, tween 20 or tween 40.
4. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 3, which is characterized by comprising the following specific steps: (1) Preparing a silica gel mold; Firstly, placing a 3D printing model with a three-dimensional tissue morphology into a square container, then, injecting a mixture obtained by mixing and stirring silica gel and a curing agent into the square container to fully submerge the 3D printing model, then, transferring the square container into a vacuum drying oven for treatment, finally, taking out the square container from the vacuum drying oven, cooling and curing at room temperature, and then, demoulding to obtain a silica gel mold with a three-dimensional tissue model cavity inside, wherein the hardness of the silica gel mold is 5-20 ℃ measured by adopting a Shore hardness tester; (2) Processing a silica gel mold to obtain a silica gel model with a micro-channel system; (3) Injecting the biological material solution into a silica gel model until the whole three-dimensional tissue model cavity is filled, and performing freeze drying after freezing for 12-24 hours at the temperature of-20 ℃ to obtain a porous biological scaffold; (4) Injecting a cross-linking agent into the silica gel model to enable the cross-linking agent to fully soak the porous biological scaffold, and performing room-temperature cross-linking treatment on the porous biological scaffold; (5) Inoculating culture bacteria liquid on the porous biological scaffold subjected to the crosslinking treatment in the step (4), and generating a staggered nano-scale network structure on the outer wall of the porous biological scaffold to obtain the bacterial cellulose-based composite scaffold; (6) And (3) sequentially soaking the bacterial cellulose-based composite scaffold obtained in the step (5) and the silica gel model in 75% alcohol, freeze-drying to enable the porous biological scaffold to shrink inwards by 0.3-1 mm, injecting a coating solution into the shrinking gap to enable the whole gap to be full of the coating solution, curing and drying, and then performing demoulding treatment to obtain the bacterial cellulose-based multi-layer structure functional bionic scaffold.
5. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 4, wherein the mass ratio of silica gel to curing agent in the step (1) is 100:1-10.
6. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 4, wherein the concentration of the biological material solution in the step (3) is 2-10wt%.
7. The preparation method of the bacterial cellulose-based multi-layer structure functional bionic scaffold, which is disclosed in claim 4, is characterized in that the freeze-drying process parameters in the step (3) are that the cold trap temperature is-50 to-40 ℃, the sample temperature is-30 to-20 ℃, the vacuum degree is 0.1Pa, and the freeze-drying time is 8 to 12 hours.
8. The preparation method of the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 4, wherein the cross-linking agent in the step (4) is glutaraldehyde, acetic anhydride or diglycidyl ether dissolved in absolute ethyl alcohol to prepare a solution with the concentration of 0.2-2wt%, and the cross-linking treatment time is 5-10 min.
9. The preparation method of the bacterial cellulose-based multi-layer structure functional bionic scaffold is characterized in that the time of soaking in 75% alcohol in the step (6) is 10-20 minutes, the freeze-drying process parameters are that the cold trap temperature is-50 to-40 ℃, the sample temperature is-30 to-20 ℃, the vacuum degree is 0.1Pa, the freeze-drying time is 8-12 hours, the curing and drying temperature is room temperature, and the curing and drying time is 10-15 minutes.
10. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to claim 4, wherein the coating solution in the step (6) enters grooves of a patterned drainage network through micropore channels and drainage grooves, and forms a coating on the surface of the bacterial cellulose-based composite scaffold to obtain the bacterial cellulose-based multi-layer structure functional bionic scaffold.
11. The method for preparing the bacterial cellulose-based multi-layer structure functional bionic scaffold according to any one of claims 1-10, wherein the outer wall of the bacterial cellulose-based multi-layer structure functional bionic scaffold is provided with an ultrafine nano-network structure, the diameter of the outer wall ultrafine nano-fiber is within 100nm, and the pore diameter of the bacterial cellulose-based multi-layer structure functional bionic scaffold is 100-300 μm.

Description

Preparation method of bacterial cellulose-based multi-layer structure functional bionic scaffold Technical Field The invention belongs to the technical field of animal body structure biological scaffolds, and relates to a preparation method of a bacterial cellulose-based multi-layer structure functional bionic scaffold. Background The tissue engineering research content mainly comprises seed cells, scaffold materials, in-vitro tissue construction and replacement of in-vivo tissues or organs, and the tissue engineering scaffold materials play a central role in the tissue engineering research, not only provide structural support for specific cells, but also play a role of a template, guide tissue regeneration and control tissue structure. Finding suitable scaffold materials and how to simulate the corresponding tissue structures are all the key points of current research. By applying the principle of bionics, extracellular matrix components of tissues and organs can be simulated by compounding various natural biological materials to construct a scaffold material which is a bionic material similar to the corresponding tissue structure and performance. The preparation of the bionic material firstly needs to have clear knowledge on corresponding tissues or organs, and the extracellular matrix of most tissues comprises an inner porous loose layer and a compact layer structure of the outer wall. For example, cartilage is composed of cartilage tissue and surrounding cartilage membranes, and cartilage tissue is composed of chondrocytes, stroma and collagen fibers. The cartilage is surrounded by a thin layer of dense connective tissue called perichondrium. The perichondrium is divided into two layers, the outer layer has multiple fiber components, and is continuous with successor tissue outside the perichondrium, and mainly has protective effect, and the inner layer contains cells to provide necessary nutrition conditions. Bone is mainly composed of two types of bone tissue, cancellous bone, which constitutes the interior of the bone, and cortical bone, which is the higher density boundary of the outermost layer. Cancellous bone is highly porous, while cortical bone has a dense structure that protects the delicate parts of the interior. The urethral tissue also has a dense layer on the inner and outer wall sides and a porous and loose structure in the middle. Therefore, the preparation of the bionic scaffold is complex, and multiple layers of the bionic scaffold are required to be simulated to be more fit with the structure of the original tissue. For the exploration and preparation of the structure of the bionic scaffold, various scientific methods emerge. For example, chinese patent CN 104992604A discloses a method for constructing segmental individuation human urinary tract tissue entity model, and finally constructs urinary tract tissue model through 3D printing, chinese patent CN 105031725A constructs a silica gel model for filling materials after performing a membrane pouring process on the urinary tract model through a silica gel membrane pouring process, and prepares a corresponding urinary tract stent by filling a solution of biological materials. The prior art has been a successful example in the preparation of porous structures and dense structures, but the preparation of complex biomimetic structures still needs to be improved. In order to solve the above problems, the present invention adopts Bacterial Cellulose (BC) as a dense layer of a complex structure, bacterial Cellulose (BC) is synthesized by microorganisms, and the polymerization of single glucose units into β -1, 4-glucan chains starts, and secreted linear glucan chains are aggregated into fibrils, and finally film is formed. It is similar to the chemical composition of plant fiber, but has different physical properties, BC has high tensile strength, high water holding capacity, high crystallinity, superfine and fine pure fiber network structure, and good biocompatibility, and the three-dimensional nano network structure of BC can perfectly simulate extracellular matrix (ECM). The excellent properties of BC in many aspects also make the BC play an important role in the preparation of the bionic scaffold material. Meanwhile, in order to further improve the functionality of the compact layer, the invention combines a coating technology and a patterning technology, and realizes the preparation of the bionic scaffold with a functionalized complex structure through a micro-channel system. The application of the coating material on biomedical materials has great progress, the coating material can realize the functionality through a physical and chemical excitation response mechanism, and the medical infection problem can be effectively solved. And how to achieve uniform application of the coating to the stent is also one of the key issues to be addressed. The micro-nano patterning technology can improve the interface effect with the matrix, prevent th