CN106075534A - A kind of chitosan alginate dressing and impregnation preparation method thereof - Google Patents

Abstract

The present invention provides a kind of chitosan alginate dressing, by being dissolved in weak acid solution by chitosan compound, prepares chitosan mixed liquor, is then infiltrated by the alginate dressing of commercialization and prepare chitosan alginate dressing in chitosan mixed liquor.Present invention incorporates chitosan and the advantage of tradition alginate dressing, while strengthening the original hemostasis of alginate dressing, moisture-absorbing moisture-keeping performance, there is again antibiotic property and promote the performances such as wound healing.Product is to human body without overt toxicity, abundant raw material, and cost of manufacture is low, can be widely applied to Wound treatment.

Description

A kind of chitosan alginate dressing and its dipping preparation method

technical field

The invention belongs to the field of medical dressings, and in particular relates to a chitosan alginate dressing and a preparation method for dipping the same.

Background technique

In the early 1960s, British scientist Winter (Winter G D. Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. [J]. Rev.ciênc.agron,1995,4(8): 293-4) put forward the theory of moist wound healing through animal wound comparison experiments, that is, the healing effect of wounds in a moist environment is better than that in an open dry environment. The emergence of this theory has led to the development, production and application of a number of wet dressings, including alginate dressings (Zhao Lin, Song Jianxing. Research status and progress of wound dressings [J]. Chinese Tissue Engineering Research and Clinical Rehabilitation, 2007 , 11(9):1724-6).

The raw material of alginate dressing is a kind of cellulose-like alginic acid extracted from brown seaweed, which is a kind of insoluble polysaccharide. Alginate fiber can be prepared by wet spinning. The alkaline concentrated solution of sodium alginate is extruded through the spinneret and sent into the acidic coagulation bath containing calcium ions. The ion exchange between sodium alginate and calcium ions forms an insoluble Alginate fibers for water. The fiber is made into an alginate dressing through non-woven technology, which is easy to fold and apply, and is also an ideal filling body. After covering the wound, this product contacts with the exudate of the wound, changes the insoluble alginate into soluble sodium alginate through ion exchange, and releases calcium ions at the same time, so it has the function of hemostasis (Wang Qinghua, Zhong Wenfei, He Meng. Clinical application of alginate dressings : Comparison with characteristics of traditional materials[J]. Chinese Tissue Engineering Research and Clinical Rehabilitation, 2010,14(3):533-6), can be used for postoperative wound packing, and play a good role in hemostasis and drainage. In addition, alginate dressings have good absorption performance and can absorb 20 times their own mass of exudate (5-7 times that of gauze) (Lin Xiaohua, Huang Zonghai, Yu Jinlong. Research and development of alginate fiber and its biomedical application[J ]. Tissue Engineering Research in China, 2013(12):2218-24). After absorbing the liquid, the dressing swells into a sodium alginate gel, forming a soft, moist, gel-like semi-solid substance on the wound (Ye Qin, Chen Jiong. Application of alginate dressings on wounds in burn donor areas[J ].Zhejiang Medicine, 2001,23(4):248-9), the wound is isolated from the outside world, which can form an airtight environment without atmospheric oxygen, accelerate the proliferation of new microvessels, and maintain a moist environment and improve the regenerative ability of epidermal cells , Accelerating the movement of epidermal cells and promoting wound healing are of great significance (Lee K Y, Mooney D J. Alginate: Properties and biomedical applications[J]. Progress in Polymer Science, 2012,37(1):106-26). Due to the formation of gel at the wound, this makes it impossible to adhere to the wound when the dressing is removed, preventing secondary trauma to the wound during removal of the dressing.

In the early 1990s, the UK developed the first alginate fiber wound dressing (Sorbsan) (Gilchrist T, Martin A M. Wound treatment with Sorbsan—An Alginate fibredressing[J].Biomaterials,1983,4(4): 317-20). After that, dressings based on alginate fiber continued to enter the market. At present, the main alginate fiber dressing brands on the market include Sorbsan, Kahostat, MelgisorbPlus, AlgiSiteM and Seasorb, etc. However, the ability of simple alginate dressings to promote wound healing is limited (Gu Qisheng, Wang Shuaishuai, Wang Qingsheng, et al. Application status and research progress of alginate dressings[J]. Chinese Journal of Prosthetic and Reconstructive Surgery, 2014(2): 255-8 ). The commercialized functional alginate dressing products are still relatively single, except for silver-containing alginate dressings, no other breakthrough products have been seen on the market so far. And silver-containing alginic acid dressings are used as antibacterial wound dressings. In order to achieve the purpose of effective antibacterial, generally the silver content is relatively high. At present, the main silver-containing dressings on the market include ionic dressings with a silver content of 9mg/100cm and metallic dressings with a silver content of 104rag/100cm. The silver content is 1% to 7% converted to the mass ratio of the alginate fiber. These dressings all have certain cytotoxicity (Rattanaruengsrikul V, PimphaN, Supaphol P. Development of Gelatin Hydrogel Pads as Antibacterial Wound Dressings [J]. Macromolecular Bioscience, 2009, 9(10): 1004–15), and the silver component is not only antibacterial Besides, it has no other obvious function of promoting wound healing. Alginate dressings with both functional and antibacterial properties are becoming a research hotspot.

Chitosan has good biocompatibility, biodegradability and physiological functions such as antibacterial and anti-inflammatory, hemostasis and healing, and is an excellent medical dressing and tissue engineering material. However, not a single chitosan has the above effects, but chitosans with different molecular weights have different pharmacological effects. For example, high molecular weight chitosan has anti-staphylococcus aureus effect, and low molecular weight chitosan has anti-staphylococcus aureus effect. Escherichia coli and the role of moisturizing (Yang Hu, Zheng Lijun, Huang Xinjian, etc. Chitosan skin care lotion promotes wound healing in rats [J]. Chinese Journal of Dermatology, 2011,44(12):891-3). Our research team mixed chitosan with different molecular weights and added other auxiliary materials to make a chitosan mixture to give full play to the performance of chitosan. It has obtained a national invention patent (patent number: ZL 2011100512896, authorized announcement date: 2012.11.28) (Tan Weiqiang, Yang Hu. Chitosan skin care lotion and its preparation method. Patent number: ZL 2011100512896, State Intellectual Property Office of the People's Republic of China, National Invention Patent, date of authorization announcement: 2012.11.28). The mixture has stable properties, good film-forming properties, and good antibacterial and moisturizing properties (Zhang Liyun, Ding Shili, Chen Zhang, et al. Research on moisturizing and antibacterial effects of chitosan skin care lotion[J]. Materials Bulletin, 2013, 27( 2):52-6), anti-aging and promoting wound healing (Yang Hu, Zheng Lijun, Huang Xinjian, et al. Research on Chitosan skin care lotion promoting wound healing in rats[J]. Chinese Journal of Dermatology, 2011, 44(12):891-3).

Contents of the invention

The purpose of the present invention is to provide a chitosan alginate dressing, which is prepared by the following method: a certain amount of chitosan mixture is dissolved in a weak acid solution to obtain a chitosan mixture; then commercialized alginate The dressing is soaked in the chitosan mixture for a certain period of time to make a new chitosan alginate dressing (dipping method).

Concrete preparation steps are as follows:

(1) Preparation of chitosan mixture: take a certain amount of acid solution, add a certain amount of chitosan mixture, mix evenly, and prepare a chitosan mixture with a mass concentration of 10-20%. The acid used is one of acetic acid, citric acid or sorbic acid, and the mass concentration of the acid solution is 1%. The chitosan mixture used is uniformly mixed with high molecular weight and low molecular weight chitosan, and the weight ratio range is 1:1; in extreme cases, high molecular weight or low molecular weight chitosan can also be used alone.

The chitosan mixture is composed of high-molecular-weight chitosan and low-molecular-weight chitosan. The chitosan with a molecular weight greater than 100,000 Daltons is a high-molecular-weight chitosan, and the chitosan with a molecular weight lower than 100,000 Daltons is a low-molecular-weight chitosan.

(2) Prepare chitosan alginate dressing: immerse the commercial alginate dressing in the chitosan mixture for a certain period of time (15 minutes-2 hours), take it out and dry it (3-12 hours in a freeze dryer or drying oven for 30 minutes - 1 hour).

(3) Disinfection treatment: use ultraviolet light for 30 minutes.

The invention combines the advantages of chitosan and traditional alginate dressings to prepare a novel chitosan alginate dressing with the functions of antibacterial, moisturizing, hemostasis and promoting wound healing, which is a novel medical dressing. It has the following advantages: (1) No obvious cytotoxicity: a large number of studies have confirmed that chitosan is a polysaccharide linked by glucosamine with glycosidic bonds, which can be degraded into Oligosaccharides, glucosamine and N-acetyl glucosamine can be absorbed by body tissues and organs, have no cytotoxicity, no hemolysis, no sensitization, good biocompatibility, and can meet the basic requirements of medical hygiene and cosmetic raw materials. In 1998, Suzuki et al. used fibroblasts (L929) to conduct in vitro cytotoxicity experiments of alginic acid and found that alginic acid had no significant effect on the proliferation of fibroblasts and had good cytocompatibility. (2) Antibacterial properties: (1) The chitosan mixture used is uniformly mixed with high molecular weight and low molecular weight chitosan, which can inhibit bacteria from different mechanisms; (2) Alginate encounters sodium ions After the exudate, sodium ion-calcium ion exchanged to form sodium alginate gel, which played a slow-release effect of chitosan; (3) the antibacterial property of chitosan was enhanced with the increase of deacetylation degree. The degree of deacetylation of chitosan used reaches 90-95%, which increases the antibacterial performance of chitosan; (4) the antibacterial activity of chitosan is strengthened along with the increase of chitosan concentration, and this dressing adopts 15% chitosan The chitosan concentration is higher when the mixture is made; (5) The antibacterial property of chitosan is inversely proportional to the pH value. This experiment uses 1% weak acid solution to dissolve chitosan, providing an acidic environment with a low pH value. (3) Hygroscopicity and moisture retention: After chitosan is degraded, the hydrogen bond in the chitosan molecule is weakened, the crystal structure is destroyed, and the molecule is in a loose state, which is conducive to the approach of water molecules and the hydrophilic groups in the molecules. Groups such as hydroxyl and amino groups form hydrogen bonds, greatly improving the moisture absorption and moisturizing properties, especially chitosan oligosaccharides (molecular weight below 10kD) have better moisture absorption and moisturizing functions than glycerin and hyaluronic acid, and within a certain molecular weight range, As the molecular weight decreases, the moisturizing and moisturizing properties gradually increase. When the alginate dressing is in contact with wound exudate containing sodium ions, the alginate fibers are converted into sodium alginate fibers by ion exchange to form a fibrous gel, which can absorb 20 times its own weight in exudate. Liquid volume (5-7 times of gauze). And the sodium alginate fiber swells into a sodium alginate gel after absorbing liquid, forming a soft, moist, gel-like semi-solid substance on the wound surface, forming a moist environment suitable for wound healing on the wound surface. (4) Hemostatic property: When the alginate dressing is in contact with blood, it will undergo ion exchange with Na+ to produce part of sodium alginate. On the one hand, this reaction breaks the ionization balance in the blood and activates the release of coagulation factors. On the other hand, the sodium alginate solution is viscous and prevents the flow of blood. Moreover, alginate swells with water, absorbs water in the blood, increases blood concentration and viscosity, slows down the flow rate, and accelerates blood coagulation. (5) Promote wound tissue regeneration: Chitosan is a chemotactic agent for macrophages, and there are receptors for bacterial polysaccharides on the surface of macrophages, and chitosan, as an analog of bacterial polysaccharides, can stimulate the activation of macrophages. While phagocytizing foreign debris and bacteria, macrophages produce a large number of growth factors such as fibroblast growth factor and vascular endothelial growth factor, which induce fibroblast proliferation, migration, extracellular matrix precipitation, and stimulate endothelial cells to form new blood vessels. Provides oxygen and essential nutrients for tissue repair. (6) Ease of removal: After the alginate fiber contacts with the exudate, it can form a soft hydrogel to prevent secondary trauma to the wound during the removal of the gauze. (7) The dressing requires simple materials, no complicated technical requirements and high-end equipment requirements, and is easy to promote.

Description of drawings

Fig. 1 is the chitosan alginate dressing finished product (containing 15% chitosan) prepared by dipping method.

Fig. 2 is the bacteriostatic experiment result of chitosan alginate dressing to Staphylococcus aureus (a) and Escherichia coli (b).

Fig. 3 is the percent increase of rat skin moisture after topical chitosan alginate dressing.

Figure 4 is a rat wound model in the experimental research on promoting wound healing.

Figure 5 shows the wound healing of rats on day 3 (left), day 7 (middle) and day 14 (right).

Figure 6 shows the statistics of the wound area of the rats on the 3rd day, 7th day, and 14th day.

Fig. 7 is the HE staining pictures of wound tissue of the chitosan alginate dressing group and the alginate dressing group on the 3rd day, 7th day and 14th day.

detailed description

The present invention is further described in conjunction with drawings and embodiments.

Embodiment 1 The making of novel chitosan alginate dressing

7.5g high molecular weight chitosan (molecular weight is 800,000), 7.5g low molecular weight chitosan (molecular weight is 3,000) is added in 85g weakly acidic solution, makes 15% chitosan mixed solution after stirring; The commercial alginate dressing was immersed in the chitosan solution for 1 hour, the dressing was taken out, dried in a drying oven at 60°C for 30 minutes, and then sterilized by ultraviolet light for 30 minutes to obtain a 15% novel chitosan alginate dressing (Fig. 1 ).

Embodiment 2 The making of novel chitosan alginate dressing

5.0g high molecular weight chitosan (molecular weight is 800,000), 5.0g low molecular weight chitosan (molecular weight is 3 thousand) is added in 90g weakly acidic solution, makes 10% chitosan mixed liquor after stirring; The commercial alginate dressing was immersed in the chitosan solution for 15 minutes, the dressing was taken out, dried in a freeze dryer for 12 hours, and then sterilized by ultraviolet light for 30 minutes to obtain a 10% novel chitosan alginate dressing.

Embodiment 3 The making of novel chitosan alginate dressing

10.0g high molecular weight chitosan (molecular weight is 800,000), 10.0g low molecular weight chitosan (molecular weight is 3 thousand) is added in 80g weakly acidic solution, after stirring, make 20% chitosan mixed liquor; The commercial alginate dressing was immersed in the chitosan solution for 2 hours, the dressing was taken out, dried in a drying oven at 60° C. for 1 hour, and then sterilized by ultraviolet light for 30 minutes to obtain a new 20% chitosan alginate dressing.

Embodiment 4 The making of novel chitosan alginate dressing

15g high molecular weight chitosan (molecular weight is 800,000) is added in 85g weakly acidic solution, makes 15% chitosan mixed solution after stirring; Commercial alginate dressing is immersed in chitosan solution 1 hour, The dressing was taken out, dried in a freeze dryer for 3 hours, and sterilized by ultraviolet light for 30 minutes to obtain a 15% novel chitosan alginate dressing.

Example 5 CCK-8 method detects the proliferative toxicity of new chitosan alginate dressing to cells

principle:

Cell counting kit-8 is referred to as CCK-8 reagent for short. The method of using CCK-8 reagent to detect cell activity is usually called cck-8 method. It is to overcome the poor stability of MTT method to detect cell activity and the repeated experimental results are easy to appear. Insufficient such as large differences and new cell viability detection methods that have emerged in recent years. Based on WST-8, it is widely used in the rapid and high-sensitivity detection of cell proliferation and cytotoxicity. Similar to MTT, WST-8 can be reduced by dehydrogenase in mitochondria to generate highly water-soluble orange-yellow formazan in the presence of electron coupler. The amount of formazan produced is directly proportional to the number of viable cells, so this property can be used directly for cell proliferation and toxicity assays. Use a microplate reader to measure the OD value at a wavelength of 450 mM, which reflects the number of living cells.

experimental method:

1) Preparation of material extract: According to the extract test in "GB/T 16886.5-2003/ISO 10993-5:1999 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test". The new chitosan alginate dressing and the alginate dressing ^were respectively made into 1mm thick, 1cm2 square pieces, added 10ml of culture medium and placed in a 37°C thermostat for 24 hours to obtain the material extract.

2) Preparation of cell suspension: Take NIH3T3 mouse embryonic fibroblast cells in the logarithmic growth phase (Cell Bank of Chinese Academy of Sciences), dilute with DMEM cell culture medium by centrifugation and pipette to form a single cell suspension with a concentration of 10×10 ⁴ /ml .

3) Inoculation of 96-well culture plate: Inoculate the prepared single cell suspension in a 96-well culture plate, the volume of cell suspension in each well is 100ul, duplicate wells of 5 wells, add 100ul of PBS around the well plate to maintain humidity. Place them in a 37°C, 5% CO ₂ incubator.

4) Extraction solution exchange: After 24 hours, the cells have completely adhered to the wall. Discard the original culture medium, get the DMEM high-sugar culture fluid (experimental group) containing volume fraction 10%, 50%, 100% novel chitosan alginate dressing extract, containing volume fraction 10%, 50%, 100% %alginate dressing extract DMEM high-sugar culture solution (negative control group), DMEM high-sugar culture solution containing 10% fetal bovine serum (blank control group), DMEM containing 5% dimethyl sulfoxide High-sugar culture solution (positive control group), 100ul per well, was placed in an incubator to continue culturing for 24h.

5) Absorbance measurement: Take out the 96-well plate, add 10uL of CCK-8 detection reagent to each well, place in a _CO2 incubator and incubate for 2 hours, select a wavelength of 450nm to measure the absorbance value of each well on an enzyme-linked immunosorbent assay instrument, and repeat the experiment 3 times Take the average and record the result.

6) Calculate the relative growth rate (RGR) of cells according to the formula: RGR=experiment A value/blank control group A value×100%. Then according to the RGR value according to "GB/T 16175-1996. The relationship between the relative proliferation rate of cells and the grading of cytotoxicity in "Biological Evaluation Test Method of Medical Silicone Materials" evaluates the cytotoxicity grade of the material. RGR≥100%, toxicity grade 0; between 75%-99%, toxicity grade 1; between 50%-74%, toxicity grade 2; between 25%-49%, toxicity grade 3; in 1 %-24%, toxicity grade 4; 0, toxicity grade 5. Cytotoxicity evaluation within 0, 1 level can be considered as meeting the standard of non-cytotoxicity.

Experimental results: See Table 1 for the absorbance value, relative proliferation rate and cytotoxicity test results of NIH3T3 cells cultured with each dressing extract for 24 hours.

Table 1. The absorbance value, relative proliferation rate and cytotoxicity detection of NIH3T3 cells cultured in each dressing extract for 24 hours (n=6)

Table 1 shows that the new chitosan alginate dressing extracts with volume fractions of 10%, 50%, and 100% have no obvious inhibitory effect on cell growth, and the toxicity is 0 or 1, that is, the new chitosan alginate The dressing had no apparent cytotoxicity.

Example 6 Antibacterial properties of novel chitosan alginate dressings detected by zone of inhibition method

experimental method:

1) Dressing production: cut the commercial alginate dressing into a number of circular dressings with a diameter of 6 mm, and then immerse them in 15% chitosan solution, 1% weak acid solution, penicillin injection (1.6 million U dissolved in 20ml normal saline), streptomycin injection (1 million U dissolved in 20ml normal saline) for 1 hour, dried in a 60°C drying oven for 30 minutes, and sterilized by ultraviolet light for 30 minutes, respectively to prepare novel chitosan alginate dressings, Alginate dressing, penicillin dressing, streptomycin dressing.

2) Bacterial inhibition zone test: take Staphylococcus aureus and Escherichia coli bacteria suspension with a turbidity of 0.5mcf, and evenly spread the bacteria on the plate with a medical cotton swab, and repeat 3 plates for each bacteria. Use sterile tweezers to paste the prepared circular dressing on the bacteria-containing dish, and paste three different dressings on each dish (respectively new chitosan alginate dressing, alginate dressing, and penicillin dressing or streptomycin dressing ), with a certain distance between dressings. The above operations were all carried out on a clean bench, and the diameter of the antibacterial zone of the dressing was measured after incubation at 37°C for 24 hours.

If the dressing is bacteriostatic, a transparent zone of sterile growth, the bacteriostatic zone, will appear around the area where the dressing is applied. The diameter of the antibacterial zone is used as the evaluation index. The larger the diameter of the antibacterial zone, the better the inhibition effect of the dressing on the tested bacteria, and vice versa.

Experimental results: See Figure 2 for the diameter of the inhibition zone of each dressing against Staphylococcus aureus and Escherichia coli, and see Table 2 for the statistical results.

Table 2. The diameter of the inhibition zone of each dressing sample to Staphylococcus aureus and E. coli (mm, n=3)

Note: The blank diameter of the dressing is 6mm, and each data in the table is the value after subtracting the blank diameter.

The experimental results of Staphylococcus aureus (Figure 2a): the diameter of the inhibition zone of penicillin dressing against Staphylococcus aureus is the largest, reaching an average of 36 mm; the average diameter of the inhibition zone of new chitosan alginate dressing against Staphylococcus aureus is 12mm, it has a certain antibacterial effect; alginate dressing has no obvious antibacterial zone on Staphylococcus aureus, and has no antibacterial effect.

The experimental results of Escherichia coli (Fig. 2b): the diameter of streptomycin dressing against Escherichia coli was the largest, with an average of 35 mm; the average diameter of the new chitosan alginate dressing against E. coli was 5 mm, with a certain Bacteriostasis: Alginate dressing has no obvious bacteriostatic zone and no bacteriostatic effect on Escherichia coli.

The experimental results showed that the chitosan alginate dressing had significant antibacterial effects on both Staphylococcus aureus and Escherichia coli.

Example 7 Detection of moisturizing performance of novel chitosan alginate dressing

experimental method:

Take 6 SD rats (provided by the animal laboratory of the First Affiliated Hospital of Zhejiang University, weighing 200-250g, all male), after depilation on the back, wash them with a mild detergent, and let them stand in a constant environment for 30min. Draw a 2cm×2cm square on the left and right backs. The test site was detected with a digital skin moisture detector, repeated 3 times, and the average value was obtained as the initial humidity value of the back skin of the rat, which was recorded as A ₀ . The new chitosan alginate dressing and the alginate dressing were applied to the corresponding test site, and the humidity values of the test site were measured 2 hours, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours after the dressing, and the tests were repeated for 3 hours. times, take the average value and record it as A _n (n=2, 6, 12, 24, 48, 72). Humidity increase percentage of the test site %P _n = (A _{n -} A ₀ )/A ₀ (n = 2, 6, 12, 24, 48, 72) × 100%. Data are expressed as mean ± standard deviation, all data are processed with SPSS19.0 software, p<0.05 is considered statistically significant. The comparison between two groups was performed by paired sample T test.

Experimental results: See Table 3 for the test results of the moisture increase in the skin of rats after applying the dressing, and see Figure 3 for the curve of the humidity increase percentage over time.

Table 3. Rat skin moisture increase after external dressing (%, x±SD, n=6)

Table 3 and Fig. 3 are the ratio of skin moisture increase on the back of each sample at 2h, 6h, 12h, 24h, and 48h and the curve of the increase ratio of skin humidity over time. It can be seen from Table 3 and Figure 3 that the ratio of the new chitosan-alginate dressing to the increase in rat skin humidity is higher than that of the alginate dressing. After paired sample T test, at 2h, 6h, 12h, 24h, and 48h, the humidity increase percentage of the new chitosan alginate dressing group was higher than that of the alginate dressing group, and there was a significant difference (p<0.05). Therefore, compared with the traditional alginate dressing, the new chitosan alginate dressing has better moisture retention when used for 2 days or less.

Example 8 Effect of novel chitosan alginate dressing on wound healing in rats

experimental method:

1) Experimental animals and grouping:

16 clean-grade rats (provided by the Animal Experiment Center of the First Affiliated Hospital of Zhejiang University School of Medicine), weighing 200-250g, male, were randomly divided into two groups, A and B, with 8 rats in each group. A new type of chitosan alginate dressing, and an alginate dressing was applied to the right side of the wound. The rats in group B were covered with new-type chitosan alginate dressing on the right wound, and alginate dressing on the left wound.

2) Establishment of animal trauma model (Figure 4):

After the rats were reared routinely for 3 days, they were intraperitoneally injected with 2% pentobarbital sodium 30-50ml/1000g, and the skin was prepared locally on the back after successful anesthesia. A symmetrical circular incision with a diameter of 2 cm was designed on both sides of the midline of the back of the rat. After disinfection, the full layer of the rat skin including the sarcolemma was excised along the incision line to form an open wound that reached deep into the fascia.

3) Administration method:

Immediately after the establishment of the trauma model, physiological saline cotton balls were used to stop the bleeding of the wound. A new chitosan alginate dressing was applied to the left wound of the rats in group A, and an alginate dressing was applied to the right wound. The rats in group B were covered with new-type chitosan alginate dressing on the right wound, and alginate dressing on the left wound. The dressing covers the entire wound. After being bandaged, they were reared in single cages, and the dressing was changed every 3 days until healed.

4) Assess wound healing

(1) Observe the wound healing with naked eyes.

(2) Calculation of the wound area: using transparent sulfuric acid film tracing method, 2 rats were randomly selected in each group, a total of 4 rats. 0, 3, 7, and 14 days after administration, the wound surface was drawn with translucent paper, and then the figure was traced on the small graph paper, the number of grids on the small graph paper was measured to indicate the wound area, and the final wound healing time was recorded . Wound healing rate=(original wound area-unhealed wound area)/original wound area×100%. Healing time: After the wound surface is made, the wound healing time is defined as the number of days when the wound healing rate is greater than or equal to 95%. The time required for wound healing after modeling was recorded.

(3) Wound histological staining: 4 rats (2 random rats in group A and 2 random rats in group B) were sacrificed at 3, 7 and 14 days after dressing application. A 3mm wide repair tissue was taken through the center of the unhealed wound, and the length included 5mm normal skin tissue for HE staining. Under a microscope, the new tissue was observed, including the distribution of new blood vessels and collagen fibers.

5) Statistical analysis: SPSS19.0 statistical software was used to perform paired sample T test for significance test, and P<0.05 was considered statistically significant.

Experimental results:

1) Visual observation showed that: 3 days after modeling, the wounds on both sides of the rats contracted, and the side of the new chitosan alginate dressing shrank better, and the wound area was reduced by naked eyes. Seven days after modeling, the wound on the side of the new chitosan-alginate dressing shrank significantly, and the degree of shrinkage was significantly greater than that on the alginate side. 14 days after modeling, the wound area on the side of the new chitosan-alginate dressing was significantly smaller than that on the side of the alginate dressing. (Figure 5).

2) Calculation of wound area (Figure 6): the wound area of the new chitosan alginate dressing side was smaller than that of the alginate dressing side at 3d, 7d, and 14d, and the difference was statistically significant (117±11mm at 3d) ² vs 178±7mm ² , P<0.05; 71±13mm ² vs 136±5mm ² at 7 days, P<0.05; 24±4mm ² vs 64±9mm ² at 14 days, P<0.05). The wound healing time on both sides was 17±1.80 days for the new chitosan alginate dressing side and 21±1.45 days for the alginate dressing side, and the difference was statistically significant (P<0.05). Therefore, compared with the alginate dressing, the new chitosan alginate dressing has a better wound healing effect.

3) Histological observation results: Tissue repair could be observed 3 days after operation, the wounds on both sides were mainly infiltrated by leukocytes, capillary buds and new blood vessels formed on the side of the new chitosan alginate dressing (experimental side) (Fig. 7d); while a large number of inflammatory cells appeared on the alginate dressing side (control side), with few endothelial cells (Fig. 7a). Seven days after operation, the number of new blood vessels on the side of the new chitosan alginate dressing (experimental side) increased significantly, a capillary network was initially formed, inflammatory cells were sparser than before, fibroblasts increased, and collagen fibers formed (Fig. 7e); On the alginate dressing side (control side), neovascularization was also formed, but the number was less, and the inflammatory cells were denser than the experimental group, and fibroblasts were scattered, and no obvious collagen fiber formation was seen (Fig. 7b). On the 14th day after operation, there were more capillaries on the side of the new chitosan alginate dressing (experimental side), and the structure of the capillary network was normal. It can be seen that some new blood vessels differentiated into mature blood vessels, and the collagen fibers were abundant and arranged in an orderly manner, basically parallel to the skin Wound (Fig. 7f); the alginate dressing side (control side) is still dominated by new blood vessels, the amount is more than before, undifferentiated, fibroblast proliferation is disordered, the shape is irregular, the number of collagen fibers is also less and the arrangement is irregular (Fig. 7c).

To sum up, visual observation, wound area statistics, and histological observation all suggest that the new chitosan-alginate dressing side (experimental side) wound heals faster than the alginate dressing side. That is to say, the new chitosan alginate dressing has a better wound healing effect than the alginate dressing on the market.

Claims (4)

1. a chitosan alginate dressing, is characterized in that, realizes through the following steps: (1) Prepare chitosan mixed solution: take acid solution, add chitosan mixture, mix evenly, make mass concentration be 10-20% chitosan mixed solution, the mass concentration of acid solution is 1%; (2) Preparation of chitosan alginate dressing: immerse the commercial alginate dressing in the chitosan mixture for 15 minutes to 2 hours, take it out and dry it, dry it in a freeze dryer for 3-12 hours or dry it in a drying oven 30 minutes - 1 hour, get chitosan alginate dressing; (3) Disinfection treatment: use ultraviolet light for 30 minutes. 2. a kind of chitosan alginate dressing according to claim 1, is characterized in that, the acid used in step (1) is the one in acetic acid, citric acid or sorbic acid. 3. a kind of chitosan alginate dressing according to claim 1 is characterized in that, the chitosan mixture used in step (1) is uniformly mixed by high molecular weight and low molecular weight chitosan, and the weight ratio scope is 1 : 1; In extreme cases, high molecular weight or low molecular weight chitosan can also be used alone. 4. a kind of chitosan alginate dressing according to claim 3 is characterized in that, what molecular weight is greater than 100,000 daltons is high molecular weight chitosan, and what is lower than 100,000 daltons is low molecular weight shell polysaccharides.