CN115400143B

CN115400143B - Method for modifying bacterial cellulose by maleic acid, product and application thereof

Info

Publication number: CN115400143B
Application number: CN202210875976.8A
Authority: CN
Inventors: 黄曹兴; 王许才; 勇强
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2024-01-26
Anticipated expiration: 2042-07-21
Also published as: CN115400143A

Abstract

The invention discloses a method for modifying bacterial cellulose by maleic acid, a product and application thereof. The invention takes safe food-grade maleic acid as a modifier and bacterial cellulose as a raw material to prepare nano bacterial cellulose, and the nano bacterial cellulose and gelatin are compounded to prepare the bacterial cellulose-gelatin ink. The biological scaffold is printed by a 3D printing technology and is applied to a rat skull defect model for a repair experiment, and the result shows that the prepared maleic acid-treated bacterial cellulose-gelatin scaffold has good skull defect repair promoting capability, and the repair effect reaches 2.21 times of that of a blank group. The bacterial cellulose-gelatin biological scaffold prepared by modifying the bacterial cellulose with maleic acid has remarkable effect in promoting bone defect repair, and has great application potential and economic benefit.

Description

Method for modifying bacterial cellulose by maleic acid, product and application thereof

Technical Field

The invention belongs to the technical field of biological new materials, and particularly relates to a method for modifying bacterial cellulose by maleic acid, a product and application thereof.

Background

With the development of technology, the life of human beings is prolonged, and the population has an aging trend. The prevalence of rheumatic diseases such as fractures, lumbago, scoliosis, osteoporosis, bone infection or tumor, congenital defects, oral and maxillofacial diseases, and osteoarthritis has increased year by year. Meanwhile, with the progress of industrial modernization, automobiles, high buildings and the like are gradually popularized, and factors such as bone necrosis or trauma and the like can also cause a large-scale bone defect, so that the daily life of people is seriously affected. Bone tissue engineering technology is to provide mechanical support and biological function for cells by mimicking the basic principle of natural tissue structure in bones. The bone tissue engineering scaffold commonly used at present is mainly divided into three types of natural high polymer materials, synthetic high polymer materials and inorganic materials. Bone tissue engineering scaffolds have been demonstrated to have the ability to promote bone repair, and are widely used in the field of bone repair. However, it is still limited by the influence of materials, such as: most natural high molecular materials are expensive and have poor mechanical strength; the synthetic polymer material has poor biological activity; the inorganic substance has high brittleness, is usually required to be used in combination with other materials, has poor compatibility with organic substances, is easy to generate structure change which is difficult to control in the degradation process, has complex preparation process and low operability. Therefore, the choice of a biological material with excellent performance for preparing an ideal bone tissue engineering scaffold has significant meaning. The research progress of the existing bone tissue engineering scaffold is still not ideal, and the scaffold has the characteristics of rich sources, simple preparation, good biocompatibility, good mechanical property and the like, and has good cost benefit and excellent capability of promoting bone repair. Meanwhile, the bone defect part structure in real life is always an irregular and complex structure, and the functional requirements of different positions of the bone defect part structure can be different. 3D printing technology is an emerging technology with rapid and accurate reconstruction or repair of defective organs or tissue complex structures. At present, the method has obtained extensive attention and research in the fields of tissue engineering and the like, and has potential research value in the field of bone tissue engineering scaffolds.

Bacterial cellulose is an extracellular polysaccharide produced by microbial fermentation, which consists of glucose monomers only, is simple to extract and has a high purity. Bacterial cellulose has a chemical composition and structure similar to that of plant cellulose, and is a natural high molecular polymer formed by connecting a plurality of beta-1, 4-glycosidic bonds. However, they have a large difference in properties from plant cellulose and have many excellent properties. For example, ultra-fine 3D network structures; the polymerization degree and the crystallinity are higher; has higher Young's modulus and tensile strength; has good air permeability and hydrophilicity; has good biodegradability and excellent biocompatibility; has the characteristics of structural controllability in the biosynthesis process and the like. At present, bacterial cellulose is increasingly researched and applied in the field of tissue engineering, and a plurality of good results are obtained. However, bacterial cellulose is stable in structure and difficult to process due to more hydrogen bond connection, and has a plurality of surface functional groups, namely hydroxyl groups, and single in structure function, so that the application field of the bacterial cellulose is limited. Meanwhile, bacterial cellulose has limited its application in the field of 3D printing due to its large fibrous structure and lack of stable self-support. The existing modification method uses reagents which are often toxic and have potential threat to cell growth. Thus, exploring a suitable way to modify bacterial cellulose has potential research value for expanding its field of application.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for modifying bacterial cellulose by maleic acid, so that the prepared nanocellulose has the capability of promoting bone repair. Another technical problem to be solved by the present invention is to provide a product of the method for modifying bacterial cellulose with maleic acid. A further technical problem to be solved by the present invention is to provide the use of said cellulose in bone defect repair.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

use of maleic acid modified bacterial cellulose in cell proliferation or bone repair.

Use of maleic acid modified bacterial cellulose in 3D printing.

The application of maleic acid modified bacterial cellulose and gel in printing bone defect site repairing scaffold.

A method of modifying bacterial cellulose with maleic acid comprising the steps of:

(1) Purifying the prepared bacterial cellulose, freeze-drying and shearing into fine fragments for later use;

(2) Adding the sheared bacterial cellulose fragments into a maleic acid solution with constant temperature, and stirring; after the reaction is finished, adding deionized water, stopping the reaction, and centrifugally washing to be neutral to obtain a precipitate which is maleic acid modified bacterial cellulose;

(3) Crushing the modified bacterial cellulose by using a wall breaking machine to prepare bacterial cellulose dispersion; homogenizing by a high-pressure homogenizer to obtain nano bacterial cellulose dispersion.

In the method for modifying bacterial cellulose by maleic acid, the reaction temperature is 100 ℃, the reaction time is 120min, and the stirring speed is 300rpm.

In the method for modifying bacterial cellulose by maleic acid, the concentration of maleic acid solution in a reaction system is 60%, and the solid-liquid ratio of the sheared bacterial cellulose and the acid solution is 1:10.

according to the method for modifying the bacterial cellulose by the maleic acid, the bacterial cellulose is homogenized for 3 times under 600bar, so that the nano bacterial cellulose is obtained.

Bacterial cellulose obtainable by the method of modifying bacterial cellulose with maleic acid.

The bacterial cellulose is used as an accelerant in cell proliferation or bone repair or 3D printing.

A method of preparing a bone defect site repair scaffold comprising the steps of:

(1) Preparing nano bacterial cellulose dispersion liquid and concentrating for later use;

(2) Preparing bacterial cellulose-gelatin biological ink by blending bacterial cellulose with concentration of 3% and gelatin with concentration of 10%; stirring bacterial cellulose and gelatin at 60 ℃ and 300rpm for 60min, and then incubating at 37 ℃ and 300rpm for 120min to obtain bacterial cellulose-gelatin bio-ink;

(3) Printing by adopting a squeeze type 3D printer, and cross-linking and solidifying by using EDC/NHS as a cross-linking agent to obtain the bacterial cellulose-based bracket.

The beneficial effects are that: compared with the prior art, the invention has the advantages that:

1) The invention takes bacterial cellulose as raw material, and is prepared by simple purification and shearing. The preparation process of the raw materials is simple and convenient.

2) The invention selects the food grade solid acid-maleic acid modified bacterial fiber which is green and mild to prepare the nano bacterial cellulose, and the preparation method is simple. The cell experiment result shows that the cell has good capability of promoting proliferation and differentiation of cells.

3) The invention combines maleic acid modified bacterial cellulose with gelatin to prepare the biological ink, thereby successfully improving the printability of the bacterial cellulose. The printing result shows that the method can meet the requirement of the bone tissue engineering on the complex structure of the defect part.

4) According to the invention, the bacterial cellulose-based gel scaffold is prepared through 3D printing, and in vivo animal experiments show that the bacterial cellulose-based gel scaffold has good capability of promoting bone defect part repair and has potential application value in the field of bone tissue engineering.

Drawings

FIG. 1 is an optical image (a), birefringence image (b), AFM and SEM image (c) of pure bacterial cellulose and modified bacterial fiber dispersion fibers;

FIG. 2 is a letter printed with bacterial cellulose dispersion as bioink (a), pure bacterial cellulose and modified bacterial cellulose-gelatin gel pattern printed (A linear print, B spiral print, C Hilbert print, D hexagonal print, E bone stick model print) (B), ear model print (C);

FIG. 3 shows MC3T3-E1 cell viability (a) at various bacterial cellulose addition concentrations, MC3T3-E1 cell viability (b) at various incubation times, staining (c) of live cells (green fluorescence) and dead cells (red fluorescence) (. P < 0.05; p < 0.01; p < 0.005);

FIG. 4 shows ALP, COL1 and RUNX2 mRNA gene expression levels at various incubation times of MC3T3-E1 cells (a), 7d alkaline phosphatase staining and ALP activity (b), alizarin red staining and mineralization nodule promotion rate in cells 28d (c) (. P < 0.05; p < 0.01; p < 0.005);

FIG. 5 is a schematic illustration of the experimental procedure (a) for preparing intra-gel osteogenesis, photographs of a bone repair scaffold before implantation (b) and after implantation (c); pure bacterial cellulose and modified bacterial cellulose-gelatin gel were implanted 28 days later SD-rat skull reconstructed Micro-CT image (d), bone density analysis (E), H & E staining and Masson trichromatic staining (f);

FIG. 6 shows the results of biochemical examination (a), histological examination (b) of SD rat serum treated with pure bacterial cellulose and modified bacterial cellulose-gelatin gel.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific examples thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1

The method for modifying bacterial cellulose by maleic acid comprises the following steps:

1) Bacterial cellulose (commercially available from Hainan coconut food Co., ltd.) was freeze-dried at a temperature below-75℃for more than 24 hours until it was completely dried. And sheared with scissors to fragments of about 1.5 x 1.5mm in size.

2) 18g of maleic acid and 12g of deionized water were weighed into a 100mL three-necked flask and heated in an oil bath until the maleic acid dissolved into a clear solution. Continuously heating to 100 ℃, adding 3g of chopped absolute dry bacterial cellulose, and reacting for 2 hours under the conditions of 100 ℃ and 300rpm mechanical stirring. Then, 60mL of deionized water at 80℃was added to stop the reaction. Finally, the sample is centrifugally washed for more than 7 times by distilled water under the conditions of 8000rpm and 5min until the pH value of the supernatant reaches neutrality, and the maleic acid modified bacterial cellulose is obtained.

3) Adding maleic acid modified bacterial cellulose into a wall breaking machine, adding 400mL deionized water, homogenizing for 90s (twice), and removing bubbles by ultrasonic treatment for 30 min. Finally, the bacterial cellulose liquid is poured into a high-pressure homogenizer and homogenized for 3 times under the pressure of 600bar, and the nano bacterial cellulose dispersion liquid is obtained.

The bacterial cellulose fiber microscopic morphology size was analyzed by Atomic Force Microscopy (AFM). After diluting the bacterial cellulose dispersion with distilled water to 0.01% (w/v), the sample was uniformly dispersed by ultrasonic, 10. Mu.L of the sample was dropped on a mica sheet, and dried in a vacuum oven at 30℃for 12 hours. Microscopic images of bacterial cellulose fibers were obtained using AFM scanning. Simultaneously, the sample is freeze-dried for 24 hours in advance, fixed on a sample table by double sided adhesive tape and subjected to metal spraying treatment. The bacterial cellulose fiber microstructure was again observed using a field emission Scanning Electron Microscope (SEM).

FIG. 1a is an apparent graph of a prepared bacterial cellulose dispersion, and it can be found that the maleic acid-modified bacterial cellulose dispersion has better light transmittance, and at the same time, the maleic acid-modified bacterial cellulose dispersion exhibits a remarkable birefringence phenomenon (FIG. 1 b). The atomic force microscope and the field emission scanning electron microscope show that (figure 1 c) the fiber size of the maleic acid modified bacterial cellulose is obviously reduced, but the natural three-dimensional network structure is obvious. Meanwhile, the maleic acid modified bacterial cellulose fiber exhibits a short rod-like structure.

Example 2

Cell viability was detected using a cell proliferation and toxicity detection kit (CCK-8). MC3T3-E1 cells were grown at 5X 10 ³ Density of individual cells/wells was seeded in 96-well plates at 37 ℃ with 5% co ₂ The culture was carried out under the condition for 12 hours, and then incubated with sterile BC dispersion solutions of different concentrations (0-400. Mu.g/mL) for 1 day and 5 days, respectively. Subsequently, the cultured cells were removed, and washed 3 times with PBS. CCK-8 reagent (10. Mu.L/well) was added to each well and incubated for 1h at 37℃in a cell incubator. Cell concentration absorbance was determined using an enzyme-labeled instrument at a wavelength of 450 nm.

After incubating the cells with bacterial cellulose dispersion obtained under optimal treatment conditions for 1 day, the cells were washed 3 times with PBS buffer and MC3T3-E1 cells were stained with Calcein-AM/PI using live/dead cell staining kit for 15min (37 ℃/5% CO) ₂ ) Cell morphology was observed under a fluorescence microscope.

The addition of the bacterial fibre dispersion treated in a different way, from the analysis of FIG. 2a, resulted in 10-200. Mu.g/mL, was not significantly toxic to MC3T3-E1 preosteoblasts. Second, the cells were also not significantly toxic by incubation in the medium containing the bacterial fiber dispersion for 5 days. The Calcein-AM and propidium iodide solutions can stain living and dead cells, respectively. From the analysis of fig. 2b, most cells survived, and the number of cell death was small, indicating that bacterial cellulose was not significantly toxic to cells. Meanwhile, the AM fluorescence spectrum of the maleic acid treated bacterial cellulose has brighter green fluorescence, which indicates that the AM fluorescence spectrum has better bioactivity.

Example 3

(1) Cell-associated mRNA gene expression profiling

The effect of bacterial cellulose on osteoblast proliferation and differentiation was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) technique. MC3T3-E1 cells and sterile BC, MA-BC dispersion (100. Mu.g/mL) were incubated in GM medium containing 0.1. Mu.M dexamethasone, 50. Mu.g/mL ascorbic acid and 10mM beta-glycerophosphate. During osteogenic differentiation, the medium was changed every 3 days. The expression of osteogenic related genes including alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx 2) and collagen type 1 (COL 1) was detected by q-PCR. The primers used for PCR are shown in Table 1.

TABLE 1 primer sequences

Osteogenic differentiation is a complex biological process involving the expression of multiple genes and distinct phases of specific marker genes. Among them, ALP is expressed as a cell membrane-associated enzyme protein and is considered as another early bone marker for osteoblast differentiation. COL-1 is an early osteoblast marker and is associated with reduced expression levels with prolonged days of cell proliferation. RUNX2 is a key transcription factor, and is considered as an early marker of osteogenic differentiation. From the analysis of FIG. 3a, it was found that the mRNA gene expression level of maleic acid-treated bacterial cellulose was significantly increased as compared with untreated bacterial cellulose, and no decrease occurred with the lapse of time.

(2) Determination of cell proliferation differentiation Capacity

3X 10 in 12-well plate ⁴ Individual/well MC3T3-E1 cells, followed by 100. Mu.g/mL of BC and MA-BC dispersion at 37℃with 5% CO ₂ Incubate under conditions for 12h. The culture solution is replaced every 3 daysAnd twice. After 14 days, the cells were washed 3 times with PBS solution and collected, and insoluble material was removed by centrifugation. Adding the supernatant into a 96-well plate, uniformly mixing with p-nitrophenyl phosphate solution of an alkaline phosphatase detection kit, incubating at 37 ℃ for 20min, and adding a stop solution. Absorbance was measured at 405nm using a microplate reader. In addition, cells after 14 days of incubation were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15min, ALP stained with BCIP/NBT alkaline phosphatase chromogenic kit and photographed.

Cell culture and treatment during alizarin red staining experiments were identical to ALP staining. After 21 days of incubation, 5% alizarin red staining solution (ARS) was used for 5min and unbound ARS was washed with water. Subsequently, the staining of the cells was observed by an inverted light microscope, and photographed. The stained cells were desorbed with 10% (w/v) cetylpyridinium chloride and ARS was quantitatively analyzed by measuring absorbance at 562 nm.

It is well known that alkaline phosphatase activity and mineralized nodule formation represent markers of early (7-14 days) and late (28 days) stages of osteoblast differentiation, respectively. From the analysis of fig. 3b, it can be seen that the modified bacterial cellulose showed higher ALP expression levels compared to the untreated bacterial cellulose. Meanwhile, the detection result of cell mineralization nodule formation shows (figure 3 c), and calcium nodule formation of maleic acid modified bacterial cellulose is more than that of unmodified bacterial cellulose, so that the maleic acid treated bacterial cellulose has better capability of promoting proliferation and differentiation of bone cells.

Example 4

The bacterial cellulose dispersion was fed into a printing tube and subjected to simple letter printing by extrusion.

The printability of the bacterial cellulose-gelatin gel was evaluated using a 3D printer. Four basic patterns were printed at room temperature using a 3D printer with a 0.26mm needle, 1mm pitch, spiral fill, hilbert fill, hexagonal fill, and the printed scaffolds were soaked in EDC/NHS (15 mM:6 mM) solution for crosslinking for 12h. Meanwhile, an ear model was printed using a 3D printer, and the ear structure was cross-linked by EDC/NHS.

From the analysis of fig. 4a, it is clear that the untreated bacterial cellulose had broken filaments and lines were uneven and had no printability. And the maleic acid treated bacterial cellulose can smoothly produce silk and can print out complete letters. However, the modified cellulose still has the defect of uneven filament output, which indicates that the maleic acid modification leads the bacterial cellulose to have certain printability, but still cannot meet the stable filament output line required by 3D printing. The homogenized bacterial cellulose solution was concentrated to a concentration of 3.5% and added in a 50mL flask in a ratio of 3% concentration (w/w) of bacterial cellulose and 10% concentration (w/w) of gelatin (commercially available from the company Ara Ding Yaopin), followed by stirring at 300rpm in a 60℃water bath for 1 hour (8.57 g of bacterial cellulose solution and 1g of gelatin and 0.43g of water were weighed in the case of a 10mL system). Finally, the temperature was reduced to 37 ℃ and stirring was continued for 2 hours to prepare a bio-ink, which was subjected to four basic printing filling model printing using a fine 3D printer (fig. 4 b). The results show that the prepared biological ink is successfully printed out to the required structure through an extrusion printer. However, pure gelatin and untreated bacterial cellulose-gelatin are poor in printing, and have disadvantages of discontinuous filament formation, low shape fidelity, and the like. And the printability of the bacterial cellulose-gelatin treated by the maleic acid is good, the uniform silk outlet grid spacing is clear, and the lines are uniform. Further, the ear structure model is printed, and the result shows that the modified bacterial cellulose-gelatin bio-ink successfully prints out the ear model and shows excellent printability. After the printed ear model was crosslinked, a bending test was performed, and as shown in fig. 4c, the ear model printed with the maleic acid-treated bacterial cellulose-gelatin bio-ink had a good bending resistance, which could be bent at 90 ° without breaking, and exhibited good toughness.

Example 5

Animals were divided into five groups: defect group, GEL group, BC group, and MA-BC group. The defect group is a blank group which is not filled with any gel scaffold; the GEL group is a printing bracket group filled with pure gelatin; the BC group is a filled untreated bacterial cellulose-gelatin gel scaffold group; the MA-BC group is a filled maleic acid modified bacterial cellulose-gelatin gel scaffold group.

SD rats (males) from the national commercial hospital animal center of Drum-building, university of Nanjing, medical college, for 8 weeks, were selected. An abrasive drilling method is used for establishing a 5mm diameter full thickness skull defect model, and a 3D printing bone repair bracket is implanted (figure 5 a). All rats were euthanized 4 weeks after surgery and the rat cranium was collected for histological and micro-computed tomography (micro-CT) analysis. The collected skull was scanned on a living CT80 system using a micro-CT instrument at a voltage of 55KeV, a current of 145 μA, a field of view of 32mm, and an integration time of 200ms. The three-dimensional model of the harvested skull was reconstructed using MIMICS19.0 and imported into Micro-CT software. The decalcified 5 μm cranium sections were stained with hematoxylin and eosin (H & E) and Masson's trichrome (Masson).

From the analysis of fig. 5b-d, the control and pure gelatin groups showed less amount of new bone, while the defective areas of the maleic acid-modified bacterial cellulose group showed good repairability, which had more new bone tissue. Furthermore, it was found from the bone density analysis based on the Micro-CT data (FIG. 5 e) that the maleic acid-modified bacterial cellulose-gelatin scaffold had a good bone regeneration promoting ability, and the bone density reached 0.223g/cm ³ The repair effect reached 2.21 times that of the blank group. At the same time through H&Analysis of osteogenic Performance of E and Masson trichromatic staining on rat skull defect sites (FIG. 5 f) was obtained from H&The newly formed bone, fibrous tissue and scaffold structure can be clearly seen in the E staining, with the most new bone tissue formed by the maleic acid modified bacterial cellulose-gelatin scaffold. In addition, masson's trichromatic staining can stain collagen tissue blue, while other fibrous tissue red, which can be an effective indication of the formation of new bone tissue due to the enrichment of collagen in the bone matrix. From the figure, it can be seen that the blue color of the collagen tissue formed by the maleic acid modified bacterial cellulose-gelatin scaffold is most pronounced, showing good osteogenic potential.

Example 6

All rat blood was subjected to serum biochemical analysis, and major organs (heart, liver, spleen, lung, kidney) were subjected to hematoxylin staining to evaluate the biosafety of the printed stent.

As can be seen from the analysis in FIG. 6a, all blood analysis indices (alanine aminotransferase, aspartate aminotransferase, albumin, cholesterol, c-reactive protein, urea nitrogen) were within the normal range. The histological section detection result (fig. 6 b) shows that the prepared gel scaffold has no obvious injury or inflammatory reaction to main organs (heart, liver, spleen, lung and kidney) of the SD rat, and has good in vivo biocompatibility and biosafety.

In addition, all experiments were repeated at least three times. The correlation data were analyzed using one-way anova, expressed as mean ± standard deviation. Data analysis was performed using GraphPad Prism 8, with statistical significance when P < 0.05. P < 0.05, P < 0.01, P < 0.001).

Claims

1. A method of modifying bacterial cellulose with maleic acid, comprising the steps of:

(1) Freeze-drying bacterial cellulose at below-75deg.C for more than 24 hr until completely drying, and shearing with scissors to pieces with size of about 1.5X1.5 mm;

(2) Weighing 18g of maleic acid and 12g of deionized water in a 100mL three-necked flask, heating an oil bath pot until the maleic acid is dissolved into transparent solution, continuously heating to 100 ℃, adding 3g of sheared absolute dry bacterial cellulose, reacting for 2 hours at 100 ℃ under the condition of mechanical stirring at 300rpm, then adding 60mL of deionized water at 80 ℃ to stop the reaction, and finally centrifugally washing a sample for more than 7 times under the condition of 8000rpm and 5min by using distilled water until the pH value of supernatant reaches neutrality to obtain maleic acid modified bacterial cellulose;

(3) Adding maleic acid modified bacterial cellulose into a wall breaking machine, adding 400mL of deionized water, homogenizing for 90s twice, and removing bubbles by ultrasonic treatment for 30min again; finally, the bacterial cellulose liquid is poured into a high-pressure homogenizer and homogenized for 3 times under the pressure of 600bar, and the nano bacterial cellulose dispersion liquid is obtained.

2. A bacterial cellulose obtainable by the method of maleic acid modified bacterial cellulose of claim 1.