CN111375067B - Chitosan scaffold and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a chitosan bracket and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) dissolving chitosan in an acid solution to prepare a solution with the concentration less than or equal to 10 percent (w/v); (2) removing insoluble substances in the step (1) to obtain a clear solution; (3) directly adding the clear solution obtained in the step (2) into a mould for freeze forming; (4) and (5) freezing and drying to obtain the chitosan scaffold. The preparation method is simple to operate, does not need cross linking, and the prepared product does not contain medicines or other antibacterial substances, realizes the sterilization and bacteriostasis effects only by using the characteristics of the chitosan bracket, is effective to gram-positive bacteria and gram-negative bacteria, has strong antibacterial action and good biocompatibility, and can be biodegraded. The antibacterial chewing gum can be prepared by using the extract as chewing gum component, thereby effectively preventing oral diseases caused by bacteria.
Description
Technical Field
The invention relates to an antibacterial product, and particularly relates to a chitosan stent and a preparation method and application thereof.
Background
In recent years, oral micro-ecological imbalance has gradually been proved to be the main cause of various oral diseases, caries and periodontitis are the most common chronic infectious diseases in the oral cavity, and the increase of opportunistic pathogenic bacteria is closely related to the occurrence and development of the oral diseases and can further influence the whole body health. The chewing gum is popular in modern life, is suitable for people of all ages, and can obviously improve the microenvironment of the oral cavity when being chewed properly in the aspect of oral health care. However, the antibacterial ingredients in the chewing gum still have insufficient efficacy, and the antibacterial efficacy of the chewing gum against the etiology is certainly enhanced in order to better exert the potential oral health effects of the chewing gum.
Chitosan (CS) is obtained by deacetylating chitin, which is widely present in the shells of crustaceans such as shrimps and crabs, fungi and plant cell walls, and is a natural polysaccharide having a second content to cellulose in nature. Chitosan is a unique renewable polymer, has good biocompatibility, biodegradability and bioadhesive property, and has been widely researched in the fields of drug delivery and tissue engineering. A large number of amino groups in the molecular structure of the chitosan are positively charged after protonation, and the chitosan shows excellent antibacterial activity when the bacteria surface is negatively charged and the bacteria surface interacts with each other.
At present, in the application research of chitosan in the medical field, a cross-linking agent is required to be added, and a drug or other active substances are loaded at the same time, and the chitosan is used as a carrier or a bracket for drug delivery, tissue engineering or antibiosis, but the research of directly using the chitosan bracket without the drug or other antibacterial substances as an antibacterial active ingredient is almost not available.
Through the literature search, reports about eliminating and inhibiting common oral cariogenic bacteria and periodontal pathogenic bacteria by preparing a chitosan scaffold without medicines or other antibacterial substances are not found at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a chitosan stent and a preparation method and application thereof, the chitosan stent does not contain medicines or other antibacterial substances, and can be used for preparing antibacterial chewing gum, so that the problems that the chewing gum has no antibacterial effect or has poor antibacterial effect and bacterial oral diseases cannot be effectively prevented by chewing the chewing gum in the prior art can be effectively solved.
In order to achieve the purpose, the invention adopts the technical scheme that: the preparation method of the chitosan scaffold comprises the following steps:
s1: dissolving chitosan with deacetylation degree of more than 70% in an acid solution to prepare a chitosan solution with mass concentration of less than or equal to 10%;
s2: transferring the chitosan solution into a mold, and freezing for 20-25 h at-45 to-90 ℃ until the chitosan solution is completely solidified and molded;
s3: and (5) carrying out freeze drying treatment on the sample obtained in the step S2 to obtain the chitosan scaffold.
According to the invention, chitosan with a deacetylation degree of more than 70% is used as a raw material, the deacetylation degree of the chitosan is in positive correlation with the number of free amino groups and electropositivity of the chitosan, the higher the deacetylation degree is, the stronger the electropositivity is, and the antibacterial activity of the chitosan is mainly derived from the electropositivity of the chitosan, so that the chitosan with a certain deacetylation degree is required to have the antibacterial activity, and the chitosan with a deacetylation degree of less than 70% has the weaker antibacterial activity. Amino groups in chitosan molecules are positively charged after being protonated, and have strong interaction with biomacromolecules with negative charges on the surfaces of bacteria, so that the cell walls of the bacteria are damaged, the metabolism of the bacteria is interfered, and finally the bacteria die.
When the chitosan solution is prepared, the concentration is controlled to be below 10%, the viscosity is moderate, the operation is convenient, and the finally obtained bracket has a rich porous honeycomb structure, is convenient for capturing bacteria, is beneficial to killing the bacteria and has a good antibacterial effect.
The invention is frozen at-45 to-90 ℃, the temperature is in a lower range, the low temperature is kept in the process of transferring the sample to the freeze dryer, and the shape of the final bracket is better.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the deacetylation degree of the chitosan is 80-95%.
According to the invention, chitosan with deacetylation degree of 80-95% is preferably selected, and within the deacetylation degree range, the chitosan has high electropositivity, so that the obtained scaffold has a good bacteriostatic effect, free amino groups repel each other, a more abundant and stable honeycomb structure is formed in the subsequent freezing process, and the bacteriostatic effect is better.
The acid solution has a volume concentration of 0.03-3%, and the solute is at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, taurine, fatty acid, adipic acid, citric acid, malic acid, gluconic acid, lactic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, and ascorbic acid.
Further, the mass concentration of the chitosan solution is 1-2%.
Further, the freezing temperature of the chitosan solution in S2 is-80 ℃, and the freezing time is 24 h.
Further, in S3, the temperature for lyophilization is-85 ℃ and the time is 24 h.
Further, the chitosan solution is subjected to impurity removal treatment before freezing, wherein the impurity removal method is centrifugation, filtration, suction filtration or ultrafiltration.
The chitosan scaffold provided by the invention is simple in preparation method, and the antibacterial chitosan scaffold with bacteriostasis and antibiosis can be obtained without loading antibacterial substances in modes of crosslinking and the like.
After the chitosan scaffold is prepared, the product has strong antibacterial effect, so the chitosan scaffold has wide application value in the aspect of antibiosis, and for example, the chitosan scaffold can be used as a chewing gum component to prepare antibacterial chewing gum, so that oral diseases caused by bacteria can be effectively prevented.
The invention has the beneficial effects that: the chitosan scaffold preparation method is simple and easy to operate, does not need cross-linking, and the prepared product does not contain medicines or other antibacterial substances, realizes the sterilization and bacteriostasis effects only by using the characteristics of the chitosan scaffold, is effective to gram-positive bacteria and gram-negative bacteria, has strong antibacterial action and good biocompatibility, can be biodegraded, and solves the problem that the blank chitosan scaffold in the prior art can not be directly used as an antibacterial active ingredient.
Drawings
FIG. 1 is an appearance diagram of a chitosan stent;
FIG. 2 is a microstructure diagram of a chitosan scaffold (0.5 mg/piece);
FIG. 3 is a microstructure diagram of a chitosan scaffold (1 mg/piece);
FIG. 4 is a graph showing the effect of chitosan scaffold and cross-linked chitosan scaffold on resisting Streptococcus mutans;
FIG. 5 is a diagram showing the effect of chitosan scaffolds and cross-linked chitosan scaffolds against Porphyromonas gingivalis;
FIG. 6 is a graph showing the results of physical stability of chitosan scaffolds in bacterial culture medium.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example one
A preparation method of a chitosan scaffold comprises the following steps:
s1: adding chitosan powder with deacetylation degree of 80% into butyric acid solution with concentration of 2% (v/v), stirring for dissolving, and preparing chitosan solution with concentration of 1% (w/v);
s2: centrifuging the chitosan solution obtained in the step S1 at 20000RPM for 10min to remove impurities such as insoluble substances;
s3: adding the chitosan solution obtained in the step S2 into a 96-hole cell culture plate, and freezing for 24 hours at the temperature of minus 45 ℃ to completely solidify and form the solution;
s4: carrying out freeze drying treatment on the frozen sample in the 96-well plate obtained in the step S3, wherein the freeze drying temperature is-50 ℃ and the time is 24 h; obtaining the chitosan scaffold product with the mass of 0.5 mg/piece.
The chitosan solution before the freeze forming and the chitosan scaffold after the freeze drying are respectively taken to take pictures, and the result is shown in figure 1. The chitosan scaffold was taken for line scanning electron microscopy and the results are shown in FIG. 2.
As can be seen from FIG. 1, the chitosan solution was a clear solution and the chitosan scaffold was a white solid. As can be seen from FIG. 2, the chitosan scaffold had a porous cellular structure on a microscopic scale.
Example two
A preparation method of a chitosan scaffold comprises the following steps:
s1: adding chitosan powder with deacetylation degree of 85% into 0.01M hydrochloric acid solution, stirring and dissolving to obtain chitosan solution with concentration of 2% (w/v);
s2: centrifuging the chitosan solution obtained in the step S1 at 20000RPM for 10min to remove impurities such as insoluble substances;
s3: adding the chitosan solution obtained in the step S2 into a 96-hole cell culture plate, and freezing for 24 hours at the temperature of minus 80 ℃ to completely solidify and form the solution;
s4: carrying out freeze drying treatment on the frozen sample in the 96-well plate obtained in the step S3, wherein the freeze drying temperature is-85 ℃, and the time is 24 h; obtaining the chitosan scaffold product with the mass of 1 mg/piece.
The chitosan scaffold was taken and examined by line scanning electron microscopy, the results are shown in FIG. 3.
As can be seen from FIG. 3, 1 mg/chitosan scaffold still has a micro-porous cellular structure, which indicates that the content of chitosan in the scaffold does not affect the micro-structure.
EXAMPLE III
A preparation method of a chitosan scaffold comprises the following steps:
s1: adding chitosan powder with the deacetylation degree of 95% into acetic acid solution with the concentration of 2% (v/v), stirring and dissolving to obtain chitosan solutions with the concentrations of 1% and 2% (w/v), respectively;
s2: centrifuging the chitosan solution obtained in the step S1 at 20000RPM for 10min to remove impurities such as insoluble substances;
s3: adding the chitosan solution obtained in the step S2 into a 96-hole cell culture plate, and freezing for 24 hours at the temperature of minus 80 ℃ to completely solidify and form the solution;
s4: carrying out freeze drying treatment on the frozen sample in the 96-well plate obtained in the step S3, wherein the freeze drying temperature is-85 ℃, and the time is 24 h; obtaining chitosan scaffold products with the mass of 0.5 mg/piece and 1 mg/piece respectively.
Inoculating gram-positive bacteria Streptococcus mutans (UA159) and gram-negative bacteria Porphyromonas gingivalis (W83) into sterile Brain heart infusion Broth (BHI) culture medium for subculture, respectively sucking bacterial liquid and adding into 96-well plate (Costar 3599; Corning Inc., NY) when the bacterial strain concentration is moderate and the activity is strong, respectively adding into 0.5 mg/and 1 mg/chitosan scaffold, and anaerobically (10% H) at 37 deg.C2,10％CO2，80％N2) Culturing for 2h and 6h, then taking out a 96-well plate, respectively sucking 100 mu l of bacterium liquid in each well, after gradient dilution to proper concentration, uniformly coating on a BHI agar solid medium plate, culturing for 24-48 h at 37 ℃, then counting colony forming numbers (CFU) on the plate to observe the concentration of bacteria, and further quantitatively analyzing the killing effect of the chitosan bracket on the bacteria. The killing effect on Streptococcus mutans is shown in FIG. 4, and the killing effect on Porphyromonas gingivalisThe result is shown in FIG. 5.
As can be seen from FIG. 4, 0.5 mg/chitosan scaffold prepared by the method of the present invention can significantly inhibit the growth of cariogenic bacteria Streptococcus mutans within 2 hours, has significant bactericidal effect within 6 hours, and can kill 80% of bacteria; in addition, 1 mg/chitosan stent prepared by the method has certain effect of killing cariogenic bacteria streptococcus mutans within 2 hours, can kill 32% of bacteria, and can kill 100% of bacteria within 6 hours.
As can be seen from figure 5, 0.5 mg/chitosan scaffold prepared by the method can obviously kill periodontal pathogenic bacteria porphyromonas gingivalis in 2 hours, 53% of bacteria in 6 hours and 94% of bacteria in 6 hours; in addition, 1 mg/chitosan stent prepared by the method has strong effect of killing periodontal pathogenic bacteria porphyromonas gingivalis within 2 hours, can kill 87% of bacteria, and can kill 100% of bacteria within 6 hours.
Example four
A preparation method of a chitosan scaffold comprises the following steps:
s1: adding chitosan powder with the deacetylation degree of 95% into acetic acid solution with the concentration of 2% (v/v), and stirring for dissolving to obtain chitosan solution with the concentration of 1% (w/v);
s2: centrifuging the chitosan solution obtained in the step S1 at 20000RPM for 10min to remove impurities such as insoluble substances;
s3: adding the chitosan solution obtained in the step S2 into a 96-hole cell culture plate, and freezing for 24 hours at the temperature of minus 80 ℃ to completely solidify and form the solution;
s4: carrying out freeze drying treatment on the frozen sample in the 96-well plate obtained in the step S3, wherein the freeze drying temperature is-85 ℃, and the time is 24 h; obtaining the chitosan scaffold product with the mass of 0.5 mg/piece.
The chitosan scaffold and the bacterial culture medium BHI are incubated for 7 days at 37 ℃, the chitosan scaffold is taken out at different time points, washed three times by PBS, fixed by 2.5% glutaraldehyde, kept overnight at 4 ℃, dehydrated in an ethanol gradient manner, dried and detected by a scanning electron microscope, and the physical stability of the chitosan scaffold in the bacterial culture medium is observed, and the result is shown in figure 6.
As can be seen from FIG. 6, the chitosan scaffold prepared by the method of the present invention is slightly degraded after being incubated in the bacterial culture medium for 1 day, and a small amount of cavities are formed, but the chitosan scaffold still maintains an intact form until the 7 th day when the structure is obviously damaged, which indicates that the chitosan scaffold has good physical stability.
EXAMPLE five
A preparation method of a chitosan scaffold comprises the following steps:
s1: adding chitosan powder with the deacetylation degree of 95% into acetic acid solution with the concentration of 2% (v/v), and stirring for dissolving to obtain chitosan solution with the concentration of 2% (w/v);
s2: centrifuging the chitosan solution obtained in the step S1 at 20000RPM for 10min to remove impurities such as insoluble substances;
s3: adding 6% sodium glycerophosphate cross-linked chitosan molecules into the chitosan solution obtained in the step S2 under magnetic stirring, adding the cross-linked chitosan into a 96-hole cell culture plate, and freezing for 24 hours at the temperature of minus 80 ℃ to completely solidify and form the solution;
s4: carrying out freeze drying treatment on the frozen sample in the 96-well plate obtained in the step S3, wherein the freeze drying temperature is-85 ℃, and the time is 24 h; obtaining the cross-linked chitosan scaffold product with the mass of 1 mg/piece.
Inoculating gram-positive bacteria streptococcus mutans (UA159) and gram-negative bacteria porphyromonas gingivalis (W83) into a sterile Brain heart infusion Broth (BHI) culture medium for subculture, respectively sucking bacterial liquid and adding the bacterial liquid into a 96-well plate (Costar 3599; Corning Inc., NY) when the bacterial concentration is moderate and the vitality is strong, then respectively adding 1 mg/cross-linked chitosan scaffold, and carrying out anaerobic treatment (10% H) at 37 ℃2,10％CO2，80％N2) Culturing for 2h and 6h, taking out a 96-well plate, respectively sucking 100ul bacterial liquid in each well, diluting to proper concentration in a gradient manner, uniformly coating on a BHI agar solid medium plate, culturing for 24-48 h at 37 ℃, then counting colony forming numbers (CFU) on the plate to observe the bacterial concentration,further quantitatively analyzing the bacterial killing effect of the chitosan scaffold. The killing effect on Streptococcus mutans is shown in FIG. 4, and the killing effect on Porphyromonas gingivalis is shown in FIG. 5.
As can be seen from FIG. 4, 1 mg/cross-linked chitosan scaffold had neither bactericidal nor bacteriostatic effects on cariogenic Streptococcus mutans for 2h and 6 h.
As can be seen from FIG. 5, 1 mg/cross-linked chitosan scaffold had neither bactericidal nor bacteriostatic effects on the periodontal pathogen Porphyromonas gingivalis at 2h and 6 h.
Through the above embodiments, it can be proved that the chitosan scaffold obtained by the invention has good bactericidal and bacteriostatic effects and good physical stability, and can be added into chewing gum as a bacteriostatic agent, thereby having good prevention and treatment effects on oral diseases.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (8)
1. The preparation method of the chitosan bracket is characterized in that the prepared chitosan bracket is used for preparing antibacterial chewing gum, and the preparation method of the chitosan bracket comprises the following steps:
s1: dissolving chitosan with deacetylation degree of 80-95% in an acid solution to prepare a chitosan solution with mass concentration of less than or equal to 10%;
s2: transferring the chitosan solution into a mold, and freezing for 20-25 h at-45 to-90 ℃ until the chitosan solution is completely solidified and molded;
s3: and (5) carrying out freeze drying treatment on the sample obtained in the step S2 to obtain the chitosan scaffold.
2. The method for preparing a chitosan scaffold according to claim 1, wherein: the volume concentration of the acidic solution is 0.03-3%, and the solute is at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, taurine, fatty acid, adipic acid, citric acid, malic acid, gluconic acid, lactic acid, maleic acid, fumaric acid, tartaric acid, succinic acid and ascorbic acid.
3. The method for preparing a chitosan scaffold according to claim 1, wherein: the mass concentration of the chitosan solution is 1% -2%.
4. The method for preparing a chitosan scaffold according to claim 1, wherein: the freezing temperature of the chitosan solution in the S2 is-80 ℃, and the freezing time is 24 h.
5. The method for preparing a chitosan scaffold according to claim 1, wherein: in S3, the freeze-drying temperature is-85 ℃ and the time is 24 h.
6. The method for preparing a chitosan scaffold according to claim 1, wherein: the chitosan solution is also subjected to impurity removal treatment before being frozen, wherein the impurity removal method is centrifugation, filtration or ultrafiltration.
7. The chitosan scaffold prepared by the method for preparing the chitosan scaffold of any one of claims 1 to 6.
8. An antibacterial chewing gum, which is characterized in that: comprising the chitosan scaffold of claim 7.
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