CN113244454A - Collagen-reinforced scaffold based on cellulose nanowhiskers and preparation method thereof - Google Patents
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Abstract
The invention provides a collagen reinforced scaffold material based on cellulose nanowhiskers and a preparation method thereof, wherein the scaffold material comprises modified cellulose nanowhiskers and collagen, the cellulose nanowhiskers are oxidized by sodium periodate to obtain a dialdehyde cellulose nanowhisker solution, then the solution is mixed with the collagen to cause aldehyde groups on the surface of the collagen to have Schiff base reaction with amino groups of the collagen, and then the collagen reinforced scaffold material is obtained by dialysis and drying treatment, so that the mechanical property and the water absorption of the material are enhanced, the better biocompatibility of the material is kept, and the application range of the collagen scaffold is expanded.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a collagen-reinforced scaffold based on cellulose nanowhiskers and a preparation method thereof.
Background
Collagen is the most important structural protein of the spine animal, and accounts for about 1/3 of the total amount of protein in the human body, and the main function in the human body is to serve as a support for tissues and to give tension to the tissues. Meanwhile, molecules and fibers of collagen play important roles in the growth and development of organisms, and the differentiation, adhesion, movement and the like of cells. The collagen has good biocompatibility and low immunogenicity. Therefore, in recent years, collagen is widely used in clinic as a novel biomaterial, but simple collagen has poor mechanical strength and thermal stability, and is rapidly degraded after being implanted into a body, and cannot achieve an expected repair effect, so that the application of collagen is limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a collagen-reinforced scaffold based on cellulose nanowhiskers and a preparation method thereof.
According to a first aspect of the invention there is provided a cellulose nanowhisker-based collagen-reinforced scaffold, characterised in that the scaffold material comprises oxidatively modified cellulose nanowhiskers and collagen. The oxidation modification is to introduce aldehyde group into the surface of the cellulose nano whisker, and open C on glucose ring on cellulose molecular chain2、C3Chemical bonds between positions, followed by C2、C3Oxidizing secondary hydroxyl on the position into aldehyde group to obtain dialdehyde cellulose nano whisker, then combining the whisker and collagen to ensure that the aldehyde group on the surface of the whisker and the amino group of the collagen have Schiff base reaction to obtain the collagen reinforced bracket material
Further, the collagen-reinforced scaffold of the present invention does not contain a crosslinking agent.
According to a second aspect of the invention, the invention provides a method for preparing a collagen-reinforced scaffold based on cellulose nanowhiskers, which is characterized by comprising the following steps:
(1) dispersing cellulose nano whisker powder in purified water by ultrasonic, adding sodium periodate, performing oxidation reaction for a period of time in a dark place, stopping the reaction by using ethylene glycol, and dialyzing in the purified water to obtain dialdehyde cellulose nano whisker;
(2) mixing the dialdehyde cellulose nanowhiskers with collagen, continuously stirring for reaction, dialyzing in purified water after the reaction is finished to remove redundant collagen to obtain a dialdehyde cellulose nanowhisker/collagen solution, measuring the concentration of the solution so as to adjust the structure and the mechanical property of a final product according to different applications, and directly entering the step (4) if the concentration meets the requirement, or entering the step (3);
(3) adjusting the concentration of a dialdehyde cellulose nanowhisker/collagen solution by dilution or concentration
(4) Standing at low temperature, then pre-freezing and drying to obtain the collagen reinforced scaffold material based on the cellulose nanowhiskers.
Further, the mass fraction of the cellulose nanowhiskers dispersed in the purified water in the step (1) is 0.01% -10%.
Further, the concentration of the sodium periodate reagent in the step (1) is 0.01mol/L-0.5 mol/L.
Further, the reaction time in the step (1) is 2-12h, preferably 6 h; the reaction temperature is 30-90 deg.C, preferably 50 deg.C.
Further, the dialysis time in the step (1) and the step (2) is 6-72h, preferably 24-36 h.
Further, the mass ratio of the dialdehyde cellulose nanowhiskers to the collagen in the step (2) is 1: 1.
Further, the reaction time of the magnetic stirring in the step (2) is 12-48h, preferably 24 h;
further, the total mass concentration of the cellulose nanowhisker/collagen solution in the step (3) is 1-10%.
Further, the drying treatment in step (3) is one of freeze drying, supercritical drying and critical point drying.
Natural Cellulose is one of the most abundant biomasses in nature, and Cellulose Nanowhiskers (CNWs) are rod-like crystals obtained by removing the inter-sequence crystal regions in Cellulose by acid hydrolysis, and are nanostructures with an aspect ratio of several tens to one hundred. The inventor believes that the cellulose nanowhisker retains the original properties of cellulose, has high crystallinity, high specific surface area, high strength, biodegradability and good biocompatibility, and is suitable for use as a reinforcing material. The cellulose nano whisker after being oxidized has the characteristics of light weight, good biocompatibility, degradability and the like, is easy to fully contact with a wound surface, and is an ideal filler as a biological composite material.
According to the invention, the cellulose nanowhiskers are modified, aldehyde groups are introduced to the surfaces of the cellulose nanowhiskers, and then the oxidized cellulose nanowhiskers are combined with collagen, so that the aldehyde groups on the surfaces of the cellulose nanowhiskers and the amino groups of the collagen undergo Schiff base reaction, thereby obtaining the collagen-reinforced scaffold material. The reaction avoids the use of a cross-linking agent, the biocompatibility of collagen and cellulose is kept, and the modifiability and the enhancement function of the cellulose are utilized, so that the material has better mechanical property and degradation resistance; and the scaffold materials with different mechanical strength, water absorption and degradation resistance can be obtained by adjusting the oxidation degree and the drying mode of the dialdehyde cellulose nanowhisker. Therefore, the reinforced bracket can be used as wound dressing and tissue engineering material for skin repair.
Drawings
Figure 1 is a picture of a solution of dialdehyde cellulose nanowhiskers of the invention.
Figure 2 is a picture of a cellulose nanowhisker-based collagen-reinforced scaffold made in accordance with the present invention.
Fig. 3 is a graph of the enzymolysis resistance quality of the collagen-reinforced scaffold based on the cellulose nanowhiskers of the present invention.
Detailed Description
For further understanding of the present invention, the cellulose nanowhisker-based collagen-reinforced scaffold provided by the present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples, and those skilled in the art can make insubstantial modifications and adjustments within the spirit of the core teachings of the present invention and still fall within the scope of the present invention.
Preparation of collagen reinforced scaffold material based on cellulose nanowhiskers
Example 1
(1) Preparing the dialdehyde cellulose nano whisker: dispersing 1g of cellulose nanowhisker powder in 100ml of purified water by ultrasonic; under the condition of keeping out of the sun, 0.05mol/L of sodium periodate is prepared, added into purified water containing cellulose nanowhiskers, the pH value of the system is adjusted to 3.0, the mixture is stirred and reacts for 6 hours under the condition of 50 ℃, then 1g of ethylene glycol is added to stop the reaction, and the mixture is dialyzed for 48 hours in the purified water to remove the redundant sodium periodate, so that the dialdehyde cellulose nanowhisker solution (shown in figure 1) is obtained.
(2) Preparing a collagen reinforced scaffold material based on the cellulose nanowhiskers: transferring the obtained dialdehyde cellulose nanowhiskers into a 100ml beaker, reconfiguring the concentration to be 0.5%, adding collagen according to the mass ratio of 1:1, continuously stirring at normal temperature for 24h, then placing the mixture into purified water for dialysis for 48h, placing the dialyzed solution into the beaker, adjusting the total mass concentration to be 1%, placing 20ml of the dialyzed solution into a freeze-drying tray, pre-freezing the solution at the temperature of-20 ℃, and then freeze-drying the solution at the temperature of-80 ℃ for 48h to obtain the collagen reinforced scaffold. (sample 1, shown in FIG. 2)
Example 2
The procedure was repeated in the same manner as in example 1 except that the concentration of sodium periodate was adjusted to 0.1mol/L (sample 2).
Example 3
The procedure was repeated in the same manner as in example 1 except that the concentration of sodium periodate was adjusted to 0.15mol/L (sample 3).
Example 4
Dissolving collagen in 0.01mol/L acetic acid solution to make its mass concentration be 1%, placing 20ml in a freeze-drying tray, pre-freezing at-20 deg.C, and freeze-drying at-80 deg.C for 48 hr to obtain pure collagen scaffold material (sample 4 control group).
(II) measuring the oxidation degree of the dialdehyde cellulose nano whisker
The aldehyde group content in the dialdehyde cellulose nanowhisker reflects the degree of oxidation, and the determination of the aldehyde group content is based on the Schiff base reaction of the aldehyde group with hydroxylamine hydrochloride. The method comprises the following specific steps: the dialdehyde cellulose nanowhisker solutions prepared in examples 1-3, and unoxidized 1% cellulose nanowhisker solutions were reacted in a water bath at 40 ℃ for 4h in acetic acid buffer solution of hydroxylamine hydrochloride (0.2mol/L, pH 5), respectively, and then slowly titrated with 0.1mol/L NaOH solution, and the volume of NaOH solution consumed when pH 5 was recorded, respectively, was calculated according to the following formula:
aldehyde group content%1-V2)×16.2/M
Wherein C is the concentration of NaOH solution, mol/L; v1Volume of NaOH solution consumed for dialdehyde cellulose nanowhiskers, V2The volume of NaOH solution consumed by unoxidized cellulose nanowhiskers, and M the mass of dialdehyde cellulose nanowhisker solution. The results show that the concentration of the sodium periodate solution determines the degree of oxidation of the cellulose nanowhiskers in a positive correlation, as shown in table 1.
| Sample numbering | Aldehyde group content (%) |
| Example 1 | 7.85 |
| Example 2 | 23.77 |
| Example 3 | 37.95 |
(III) measuring the tensile strength of the collagen reinforced scaffold material
The method comprises the following steps: the collagen-reinforced scaffold materials obtained in examples 1 to 4 were cut into small strips of 3cm × 1cm, and the tensile properties of the materials were measured by a tensile machine.
The results show that the tensile strength of the scaffold material with the added cellulose nanowhiskers is improved to different degrees, and the higher the degree of oxidation of the cellulose nanowhiskers, the higher the mechanical strength of the obtained scaffold material, as shown in table 2.
Table 2: examples 1-4 cellulose nanowhiskers obtained to enhance the tensile strength of cell scaffolds
| Sample numbering | Tensile Strength (N) |
| Sample 1 | 3.667N |
| Sample 2 | 4.125N |
| Sample 3 | 4.639N |
| Sample No. 4 | 2.984N |
(IV) measuring the water absorption rate of the collagen-reinforced scaffold material
The water absorption test method comprises the following steps: the crosslinked collagen scaffolds prepared in examples 1-4 were cut into 2X 3cm pieces, weighed on an analytical balance, and recorded as w1. Clamping a section of the sample by using a pair of tweezers, immersing the sample into purified water, immediately taking out the sample after complete infiltration, vertically standing for 1 minute, weighing, and recording as w2. The water absorption rate is that the bracket is supported within a certain timeThe ratio of the weight of the material absorbing moisture to its own weight. Namely, the following formula is calculated:
water absorption rate ═ w2-w1)/w1
The results show that the addition of the dialdehyde cellulose nanowhiskers did not reduce the water absorption of the scaffold material, but rather increased the water absorption of the scaffold.
Table 3: water absorption of cellulose nanowhisker-reinforced cell scaffolds obtained in examples 1 to 4
(V) determining the cell compatibility of the collagen-reinforced scaffold material based on the cellulose nanowhiskers
Mouse fibroblast cells L929 were used to evaluate the cytotoxicity of the material. According to the biological evaluation of medical instruments, part 5, in vitro cytotoxicity test, the scaffold materials of examples 1-4 were cut into pieces, 1g of the material was added to 1mL of cell culture solution, left at 37 ℃ for 24 hours, and the leaching solution was diluted with culture medium to obtain a series of leaching dilutions as test solutions. Subsequently, qualitative assessment of the material cytotoxicity was performed using agar diffusion experiments.
The result shows that the collagen reinforced bracket material bracket based on the cellulose nanowhiskers retains the better biocompatibility of the collagen and has no cytotoxicity.
Table 4 cytotoxicity of collagen-reinforced scaffold materials based on cellulose nanowhiskers
| Sample numbering | Cytotoxic reactions |
| Sample 1 | |
| Sample 2 | |
| Sample 3 | |
| Sample No. 4 | |
(VI) determining the enzymolysis resistance of the collagen reinforced scaffold material based on the cellulose nanowhiskers
The method comprises the following steps: cutting the scaffold materials of examples 1-4 into pieces, respectively weighing 0.1g, placing the pieces into a disposable culture dish, adding 10mL of prepared Tris-HCL buffer solution containing collagenase to fully soak sponge into the buffer solution, placing the buffer solution in a thermostatic water bath with 37 ℃ for magnetic stirring, reacting for a period of time, observing the crushing condition of the collagen reinforced scaffold material, freeze-drying the degraded collagen at different time points, and weighing to obtain a mass weight loss curve.
As a result, as shown in fig. 3, the scaffold can be gradually cracked along with the increase of time, the addition of the modified cellulose nanowhiskers improves the degradation resistance of the scaffold material, and the higher the oxidation degree of cellulose, the stronger the degradation resistance of the scaffold material, so that the degradation time of the material can be controlled to a certain extent by adjusting the oxidation degree of the cellulose nanowhiskers.
The results show that the collagen reinforced scaffold material based on the cellulose nanowhiskers prepared by the method of the invention keeps good biocompatibility of collagen, has excellent water absorption, and the mechanical property and the degradation resistance of the scaffold are obviously improved due to the addition of the modified cellulose nanowhiskers. The collagen reinforced scaffold material can be applied to the fields of wound dressing and tissue engineering.
The above-described embodiments are merely illustrative of the principles of the present invention and are not to be construed as limiting the invention. Many changes, modifications and variations may be made therein without departing from the spirit and scope of the invention as defined in the following claims.
Claims (10)
1. A collagen-reinforced scaffold material based on cellulose nanowhiskers, characterized in that the scaffold material comprises oxidatively modified cellulose nanowhiskers and collagen.
2. A cellulose nanowhisker-based collagen reinforced scaffold material according to claim 1, wherein said scaffold material does not contain a cross-linking agent.
3. A preparation method of a collagen reinforced scaffold material based on cellulose nanowhiskers is characterized by comprising the following steps:
(1) dispersing cellulose nano whisker powder in purified water by ultrasonic, adding sodium periodate, performing oxidation reaction for a period of time in a dark place, stopping the reaction by using ethylene glycol, and dialyzing in the purified water to obtain dialdehyde cellulose nano whisker;
(2) mixing the dialdehyde cellulose nanowhiskers with collagen, continuously stirring for reaction, dialyzing in purified water after the reaction is finished to remove redundant collagen to obtain a dialdehyde cellulose nanowhisker/collagen solution, measuring the concentration of the solution, directly entering the step (4) if the concentration meets the requirement, or entering the step (3);
(3) adjusting the concentration of the dialdehyde cellulose nanowhisker/collagen solution by dilution or concentration;
(4) standing at low temperature, then pre-freezing and drying to obtain the collagen reinforced scaffold material based on the cellulose nanowhiskers.
4. The preparation method according to claim 3, wherein the cellulose nanowhiskers in step (1) are dispersed in purified water at a mass fraction of 0.01% to 10%.
5. The method according to claim 3, wherein the concentration of the sodium periodate reagent in the step (1) is 0.01mol/L to 0.5 mol/L.
6. The process according to claim 3, wherein the reaction time in step (1) is 2 to 12 hours, preferably 6 hours; the reaction temperature is 30-90 deg.C, preferably 50 deg.C.
7. The method according to claim 3, wherein the dialysis time in step (1) and step (2) is 6-72 hours, preferably 24-36 hours.
8. The method according to claim 3, wherein the mass ratio of the dialdehyde cellulose nanowhiskers to the collagen in the step (2) is 1: 1.
9. The method according to claim 3, wherein the reaction time of step (2) is 12 to 48 hours, preferably 24 hours, under magnetic stirring.
10. The method according to claim 3, wherein in the step (2), the concentration is required to be: the total mass concentration of the cellulose nano whisker/collagen solution is 1-10%.
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| CN114874980A (en) * | 2022-05-23 | 2022-08-09 | 北京化工大学 | Application of cholesteric cellulose nanocrystalline film in enhancing cell adhesion |
| CN115998941A (en) * | 2022-12-09 | 2023-04-25 | 陕西科技大学 | Medical dressing based on CNCs-COL and preparation and application methods thereof |
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| CN115998941A (en) * | 2022-12-09 | 2023-04-25 | 陕西科技大学 | Medical dressing based on CNCs-COL and preparation and application methods thereof |
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