Multifunctional composite biological material, preparation method and application thereof
阅读说明：本技术 一种多功能复合生物材料、制备方法及其应用 (Multifunctional composite biological material, preparation method and application thereof ) 是由 毛青 胡秀林 张维 税朝毅 吴力克 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种多功能复合生物材料的制备方法及应用,属于生物医用材料技术领域。其主要由细菌纤维素即BC和水溶性壳聚糖组成,其形态为膜状,其结构为排列紧密均匀的三维多孔网状结构。其制备方法为：将BC纤维打成浆,经冷冻超微粉碎后,溶于N,N-二甲基乙酰胺复合溶剂中,密封摇匀,然后静置,加热,即得活化BC溶液；将水溶性壳聚糖粉末与活化BC溶液进行混配,加热,搅拌均匀,然后倒入模具中,并加入凝固液,制成膜状,即得多功能复合生物材料。本发明的多功能复合生物材料的结构排列紧密均匀,疏松透气,具有抗菌、抑菌、止血和促进伤口愈合的优异性能,在生物医学治疗中作为医用敷料具有极大的实用价值。(The invention discloses a preparation method and application of a multifunctional composite biological material, and belongs to the technical field of biomedical materials. The antibacterial cellulose film mainly comprises Bacterial Cellulose (BC) and water-soluble chitosan, is in a film shape, and has a three-dimensional porous network structure which is arranged closely and uniformly. The preparation method comprises the following steps: pulping BC fiber, freezing, micronizing, dissolving in N, N-dimethylacetamide composite solvent, sealing, shaking, standing, and heating to obtain activated BC solution; and mixing the water-soluble chitosan powder and the activated BC solution, heating, uniformly stirring, pouring into a mold, adding the solidification solution, and preparing into a film shape to obtain the multifunctional composite biological material. The multifunctional composite biomaterial has the advantages of compact and uniform structural arrangement, looseness and air permeability, excellent performances of antibiosis, bacteriostasis, hemostasis and wound healing promotion, and great practical value as a medical dressing in biomedical treatment.)
1. The multifunctional composite biomaterial is characterized by mainly comprising Bacterial Cellulose (BC) and water-soluble chitosan which are fused, and is in a film shape, and the structure of the multifunctional composite biomaterial is a three-dimensional porous net structure which is tightly and uniformly arranged.
2. The multifunctional composite biomaterial of claim 1, wherein the BC is an activated BC fiber powder; the method of activation comprises the steps of:
s1, sequentially decoloring the BC membrane, processing into fine fragments, grinding into slurry by a colloid mill, filtering, discharging residues, freezing, micronizing, freeze-drying, removing 50% of water, and pulping again to obtain BC pulp;
s2, adding the BC pulp into an ethylenediamine aqueous solution, soaking or stirring for 1.5-2 h, then filtering to obtain a precipitate, washing with water, and filtering; and soaking the mixture in ethanol, filtering, taking out the BC filter cake, crushing, performing vacuum drying, taking out the solid when the water content is reduced to 5-10%, crushing for 3-5 seconds, and drying again to obtain the activated BC fiber powder.
3. The multifunctional composite biomaterial of claim 2, wherein the mass percentage of the ethylenediamine in the ethylenediamine aqueous solution in the s2 is 15%.
4. The multifunctional composite biomaterial as claimed in claim 2, wherein in s2, the precipitate is washed with water, and when the filtrate is neutral, the washing is finished.
5. The multifunctional composite biomaterial of claim 2, wherein the purity of the ethanol in the s2 is 95-99%.
6. The method for preparing a multifunctional composite biomaterial as claimed in any one of claims 2 to 5, comprising the steps of:
a1, dissolving the activated BC fiber powder in an N, N-dimethylacetamide (DMAc) composite solvent, sealing and shaking up, standing for 24-30 h, and heating at 60-70 ℃ to obtain an activated BC solution; the activated BC fiber powder and DMAc composite solvent are mixed according to the weight ratio of g: l is 15: 1;
a2, crushing water-soluble chitosan into powder, adding the powder into the activated BC solution, heating and stirring to uniformly mix the water-soluble chitosan powder and the activated BC solution, then pouring the mixture into a mould, adding a solidification solution to prepare a film-shaped object, and customizing the thickness of the film to be 0.5mm to obtain the multifunctional composite biological material; the water-soluble chitosan powder and the activated BC solution are 0.5-2.5: 100 in terms of g: mL.
7. The preparation method of the multifunctional composite biomaterial according to claim 6, wherein in the a1, the DMAc composite solvent is prepared by heating and mixing LiCl and DMAc at a ratio of 9:100 in g: mL at a temperature of 60-70 ℃.
8. The method for preparing the multifunctional composite biomaterial of claim 6, wherein in the a2, the mixing proportion of the water-soluble chitosan powder and the activated BC solution is 2:100 in g: mL.
9. The method for preparing the multifunctional composite biomaterial as claimed in claim 6, wherein the coagulating liquid added in the step a2 is glycerol.
10. Use of a multifunctional composite biomaterial as claimed in any one of claims 1 to 5 as a medical dressing in biomedical therapy.
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method and application of a multifunctional composite biomaterial.
Background
The BC has a porous three-dimensional network structure, the size of the microfiber is about 50-80nm, the nanofiber range has a large specific surface area, and the nanofiber contains a large number of hydroxyl groups and has a plurality of effective reactive sites. Therefore, the BC hyperfine three-dimensional nano-network structure can be used as a matrix to control or synthesize a novel nano-composite material with a specific morphological function, and inorganic substances or polymer molecules which are easy to disperse and stable in state can be introduced into the BC network structure, so that BC-based composite materials with different functional characteristics can be prepared, and the requirements of different fields can be met. The excellent structural characteristics and template performance of the BC provide enough space for the preparation of the composite material, and simultaneously the size, shape, structure and properties of the nano material can be controlled, and the dense three-dimensional network can uniformly disperse the nano material therein without a large amount of accumulation, so that the nano composite material with expected shape and size is obtained.
At present, the preparation method of BC composite materials reported in domestic and foreign research mainly comprises the following steps: biological compounding, solution soaking and solution blending.
The biocomposite method is to mix other reinforcing molecules or units in the culture medium before BC biosynthesis, and the reinforcing materials grow in BC microfibril to become part of BC fibril network structure during BC growth to form bacterial cellulose-based composite. Adding different micro-nano particles into a BC fermentation medium by Serafica and the like to prepare a BC-based composite material, and compounding iron oxide particles into a BC network to enable BC to have magnetism; calcium carbonate and talcum powder are introduced into BC, so that the prepared composite material has good flexibility and reaction sensitivity, and the mechanical property strength is increased by 1-2 times; the paper fiber micro-particles are added into the BC biological fermentation liquid, and the BC fibers grow together to form a film which mutually penetrates to form a network, so that the mechanical strength and the toughness are improved. Saibuatong et al added aloe gel to BC fermentation medium, and static culture prepared a novel fiber reinforced biological composite membrane, and found that adding 30 wt% aloe gel to the medium significantly enhanced the mechanical strength, crystallinity, water absorption and water vapor permeability of the composite membrane, and further narrowed the pore size distribution, and the average pore size was reduced 4/5 compared with unmodified BC. Yan and the like add multi-walled carbon nanotubes (MWNTs) into a culture medium to prepare a bacterial cellulose-carbon nanotube composite material, the carbon nanotubes are embedded into a network of BC fibers to construct a bacterial cellulose fiber-carbon nanotube three-dimensional network structure, and test results show that the crystallization index, the grain size and the cellulose I alpha content of the material are all reduced. Hyaluronic Acid (HA) with different molecular weights is added into a BC culture medium by virtue of Zhuqingmei and the like, a BC/HA composite membrane is prepared by fermentation, and the characterization shows that the HA is attached to microfibrils of the BC in a cross-linked manner, so that the yield of the composite is improved, and the thermal stability and the tensile strength of the BC are also enhanced. Chen et al added pectin to the BC media, which affected the BC membrane structure, yield and growth rate, the BC filaments became thin, and the pectin filled the BC lattice causing the structure to become tight. Esra and the like compound different nano-particles into a BC three-dimensional network structure, such as cellulose microfibrils, exfoliated graphene nano-sheets and clay, by a biological compounding method, and test results show that the nano-particles can be embedded in BC gaps and microfibrils, the dispersion is good, and the thermal stability and residue amount of the BC composite membrane are obviously increased. Ana and the like add Polycaprolactone (PCL) powder into a BC culture medium, then successfully prepare the bacterial cellulose/polycaprolactone nano composite membrane by a hot pressing method, the PCL powder is uniformly dispersed in a BC grid, the network structure of the BC is not changed, the thermal stability and the mechanical property are obviously enhanced, and meanwhile, the material shows good biocompatibility and biodegradability and has great application potential in the fields of biomedicine and food packaging.
The biological composite method is the most common method for preparing BC composite materials and is green and environment-friendly, but the technology has certain limitations, such as: some particles with biological antibacterial activity, such as silver nanoparticles, nano-zinc oxide, titanium dioxide, etc., cannot be directly added to the BC medium because they are toxic to BC fermenting microorganisms; further, if the particles are added to the BC culture medium, flocculation and sedimentation are likely to occur, and it is difficult to form a BC film by dynamic agitation fermentation culture.
The solution immersion method is a method in which the BC is immersed in a solution containing nanoparticles, and the particles are reinforced by physical adsorption, hydrogen bonding, or the like to be bonded to the BC. Due to the large amount of gaps in the inner structure of the BC, the solution and the small particles can easily shuttle among the gaps, and meanwhile, the BC contains a large amount of hydroxyl groups, so that hydrogen bonds can be easily formed with the nanoparticles, and the combination between the BC and the nanoparticles is reinforced. The nano particles used in the solution soaking method can be polymers, inorganic non-metallic materials, metal materials and the like, the application range is wide, and the method is simple and easy to operate. Collagen is the most important extracellular fibrin insoluble in water, and is the skeleton constituting the extracellular matrix, and is capable of forming semi-crystalline fibers in the extracellular matrix to provide tension and elasticity to cells. The Cai and the like prepare the bacterial cellulose/collagen composite material by soaking the BC wet film in a collagen solution, the collagen is simultaneously dispersed on the surface of the BC and inside the network and is connected with the BC through hydrogen bonds, the thermal stability of the composite material is obviously improved, the Young modulus and the tensile strength are greatly improved, the biocompatibility of the bacterial cellulose/collagen composite material is evaluated through a cell adhesion experiment, cell adhesion and proliferation are formed after 48 hours, good biocompatibility is shown, and the bacterial cellulose/collagen composite material is expected to be applied to wound dressings and tissue engineering scaffolds. Saska and the like firstly carry out esterification modification on BC through glycine, then 1-ethyl- (3-dimethylaminopropyl) carbodiimide is used as a cross-linking agent to cross-link I-type glue and the modified BC to prepare the BC-COL composite material, characterization of an osteoblast adhesion proliferation experiment shows that after 17 days, the total protein content in cells and the activity of alkaline phosphatase in the cells are higher than those of pure BC, and the BC-COL composite material is possibly used as a biomedical material to be applied to bone tissue engineering. Kim et al soaked the dried BC film in a chloroform solution of poly-l-lactic acid (PLLA) and naturally volatilized the solvent at room temperature to prepare a BC/PLLA nanocomposite with biocompatibility. As the diameter of the microfiber in BC is about 50-80nm and is smaller than the wavelength of visible light, the transparent property of the PLLA is kept, and simultaneously, the mechanical property of the material is improved due to the addition of the PLLA, and the tensile strength and the Young modulus are respectively improved by about 2 times and 1.5 times compared with the pure PLLA. The bacterial cellulose can also be compounded with some inorganic materials, such as silver nanoparticles, nano-silica, montmorillonite, hydroxyapatite and the like, and the prepared composite material has improved antibacterial and mechanical properties. Katepatch and the like successively immerse the BC film in a zinc nitrate solution and an ammonium hydroxide solution, and simultaneously, through ultrasonic treatment, the nano zinc oxide (ZnO) is successfully introduced into a BC matrix, the size of the ZnO is 54-63nm and is close to the diameter of BC fiber filaments, and antibacterial tests show that the material has extremely strong bactericidal property on gram-negative bacteria and gram-positive bacteria, so that the material is a potential material used as a medical antibacterial dressing. The study shows that the MMT particles can be uniformly dispersed on the surface of bacterial cellulose and in a three-dimensional network structure, the crystallinity is reduced from 63.22 percent of pure BC to 49.68 percent, the mechanical property and the thermal stability of the composite material are obviously improved, the water holding capacity is reduced to some extent, but the water release rate is increased.
The solution soaking method is simple and easy to operate, and is applicable to both solution and suspension of the reinforced material, and the prepared composite material basically keeps the excellent performance of BC. However, the size requirement of the method for the reinforced material is higher, and because the aperture of the network structure in the BC is about 0.5-1.5 μm, the particles with the particle size of submicron and nanometer can permeate into the BC ultramicro fiber net, for some hydrophobic materials, the BC compound is difficult to prepare by a solution soaking method; in addition, the distribution of the BC fibril structure is not uniform, the penetrated material is difficult to be dispersed in BC completely and uniformly, and the content of the added material in the composite is not determined, so that a new preparation method of the BC composite is needed to solve the problems.
The blending compounding method is to dissolve BC in solvent or pulp BC intoAnd (4) homogenizing the slurry suspension, then uniformly mixing with a reinforcing material, and preparing a membrane and drying to obtain the BC composite material. The blending compounding method is a very good method for preparing BC composite materials, is suitable for various ranges of added materials, is easy to control the content and distribution of the added materials, has low requirements on the properties of the materials, but can damage the original structure of the BC and has certain influence on the performance of the BC. The method comprises the following steps of uniformly mixing bacterial cellulose and acrylic acid by a mechanical method, treating the mixture by electron beam radiation to obtain the BC/AA composite hydrogel, and testing the BC/AA composite hydrogel to show that the BC/AA composite hydrogel is in a porous network structure, the water swelling rate reaches 4000-6000%, and the water evaporation degree is 2175-2280g/
Scholars at home and abroad have a great deal of research on modification, modification and compounding of BC, and BC derivatives and compounds with different properties are prepared, but the research in the aspect is still in the initial stage, although some technologies have been clinically tested and converted into industrial production and practical application, most of the research still stays at the primary level of laboratories and physicochemical property research, and a great deal of research work is needed in order to fully exert the excellent properties of BC, meet the application requirements of different industrial fields and realize the real value of BC.
Chitosan is a biological polymer with antibacterial property and biocompatibility, can be used as a drug carrier and a stent material in the field of biomedicine, and is known as an excellent natural biological material. However, the chitosan and BC are prepared into the composite biomaterial by a melting and blending method, and further prepared into the medical dressing with multiple functions of inhibiting bacteria, stopping bleeding and promoting healing, and the reports are not found yet.
Disclosure of Invention
In view of the above, the present invention aims to provide a multifunctional composite biomaterial, a preparation method and an application thereof.
In order to achieve the above purpose, the inventor of the present invention provides a technical solution of the present invention through long-term research and a great deal of practice, and the specific implementation process is as follows:
1. a multifunctional composite biomaterial is mainly formed by fusing Bacterial Cellulose (BC) and water-soluble chitosan, is in a film shape, and has a three-dimensional porous network structure which is arranged closely and uniformly.
Preferably, the BC is activated BC fiber powder; the method of activation comprises the steps of:
s1, sequentially decoloring the BC membrane, processing into fine fragments, grinding into slurry by a colloid mill, filtering, discharging residues, freezing, micronizing, freeze-drying, removing 50% of water, and pulping again to obtain BC pulp;
s2, adding the BC pulp into an ethylenediamine aqueous solution, soaking or stirring for 1.5-2 h, then filtering to obtain a precipitate, washing with water, and filtering; and soaking the mixture in ethanol, filtering, taking out the BC filter cake, crushing, performing vacuum drying, taking out the solid when the water content is reduced to 5-10%, crushing for 3-5 seconds, and drying again to obtain the activated BC fiber powder.
In s1, the BC membrane needs to be sufficiently decolorized so as not to generate colored impurities; pulping again, preferably grinding the BC film into pulp by using a colloid mill, wherein the working environment of the colloid mill is lower than 15 ℃.
Preferably, in the s2, the ethylene diamine content in the ethylene diamine aqueous solution is 15% by mass. Wherein the addition amount of ethylenediamine in the aqueous solution is sufficient to stabilize the bacterial cellulose and prevent further decomposition.
Preferably, in s2, the filtration all adopts the suction filtration mode, and the ethylenediamine aqueous solution recovered through suction filtration is preserved in a brown bottle and can be reused for 3-4 times. When the water-soluble ethylene diamine is used later, the percentage of the ethylene diamine in the water solution is supplemented to reach 15%.
Preferably, in s2, washing with water is completed when the filtrate is neutral. During washing, water is needed to be added for immersion precipitation each time, and the BC pulp is washed. During suction filtration, the pH value of water drops at the water outlet is checked by adopting pH test paper to judge the washing end point. The water is distilled water.
Preferably, in the s2, the purity of the ethanol is 95-99%.
Preferably, in s2, the vacuum drying mode is that the raw materials are placed in a vacuum drying oven and dried at the temperature of 45-55 ℃. Wherein, too high temperature can affect the activity of the BC fiber powder.
Preferably, in s2, the BC is pulverized to a particle size of about 100 mesh by a low-temperature pulverizer of a colloid mill, and the pulverizing time cannot be too long. Because the crushing time is too long, the fiber powder is attached to the inner wall of the container in a static way, and is inconvenient to pour.
2. A preparation method of a multifunctional composite biological material comprises the following steps:
a1, dissolving the activated BC fiber powder in an N, N-dimethylacetamide (DMAc) composite solvent, sealing and shaking up, standing for 24-30 h, and heating at 60-70 ℃ to obtain an activated BC solution; the activated BC fiber powder and DMAc composite solvent are mixed according to the weight ratio of g: l is 15: 1;
a2, crushing water-soluble chitosan into powder, adding the powder into the activated BC solution, heating and stirring to uniformly mix the water-soluble chitosan powder and the activated BC solution, then pouring the mixture into a mould, adding a solidification solution to prepare a film, and customizing the thickness of the film to be 0.5mm to obtain the multifunctional composite biological material; the water-soluble chitosan powder and the activated BC solution are 0.5-2.5: 100 in terms of g: mL.
Preferably, in the a1, the DMAc composite solvent is prepared by heating and mixing LiCl and DMAc at a ratio of 9:100 in g: mL at a temperature of 60-70 ℃.
Preferably, in the a1, if the ambient temperature is too low during the standing process, external heating can be used for assisting.
Wherein the heating at 60-70 ℃ in the a1 aims to enhance the solution fluidity to fully dissolve the BC, and if a small amount of BC particles are not dissolved, a small amount of DMAc can be supplemented, and the dissolved BC solution is a light yellow or beige uniform viscous liquid.
Preferably, in the a2, the mixing proportion of the water-soluble chitosan powder and the activated BC solution is 2:100 in g: mL.
Preferably, the coagulating liquid added in the a2 is glycerol.
3. An application of multifunctional composite biological material as medical dressing in biomedical treatment.
The invention has the beneficial effects that:
1) the multifunctional composite biological material prepared from BC and chitosan is in a film shape, has a three-dimensional porous reticular structure, is closely and uniformly arranged, is loose and breathable, has excellent performances of resisting bacteria, inhibiting bacteria, stopping bleeding and promoting wound healing, and has great practical value as a medical dressing in a biomedical material.
2) The preparation method of the multifunctional composite biomaterial comprises the steps of activating and dissolving BC, adding water-soluble chitosan powder, and melting and blending, so that the multifunctional composite biomaterial is prepared.
Drawings
FIG. 1 is a scanning electron microscope image of the multifunctional composite biomaterial of the present invention;
FIG. 2 is a Fourier transform infrared spectrum of the multifunctional composite biomaterial of the present invention;
FIG. 3 is a cut-out shape of the multi-functional composite biomaterial sample prepared in this example 1;
FIG. 4 is a graph showing load-displacement curves of the multifunctional composite biomaterial produced in this example 1;
FIG. 5 is a microscope image of wound tissue of the multifunctional composite biomaterial prepared in this example 1 on
FIG. 6 is a microscope photograph of wound tissue of the multifunctional composite biomaterial prepared in this example 1, on
FIG. 7 is a microscope photograph of wound tissue of the multifunctional composite biomaterial prepared in this example 1, on
Detailed Description
The present invention is further illustrated by the following specific examples so that those skilled in the art can better understand the present invention and can practice it, but the examples are not intended to limit the present invention.
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