CN110917382A

CN110917382A - Preparation method of amnion-spongy chitosan composite double-layer wound dressing

Info

Publication number: CN110917382A
Application number: CN201911213914.5A
Authority: CN
Inventors: 陈敬华; 杨漾; 纪倩; 张燕燕; 戴雨桐
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-03-27

Abstract

The invention discloses a preparation method of an amnion-spongy chitosan composite double-layer wound dressing, and belongs to the technical field of medical dressings. The amnion dressing and spongy chitosan with excellent antibacterial and swelling properties are combined by a cross-linking method to prepare the amnion-chitosan composite double-layer wound dressing, the wound healing rate of a mouse in the 8d of the dressing can reach 87.67%, and the skin thickness of a mouse wound tissue reaches 693.9233 microns; in addition, the dressing increases the enrichment concentration of hemoglobin from 72.8g/L to 685.39g/L of amniotic membrane group; the swelling coefficient is improved to 800 percent from 300 percent of the amniotic membrane group; the tensile stress and the nominal strain of the dressing are increased to 1.14-2.29 MPa and 6.34-6.97%, and the dressing can rapidly absorb wound exudate, prevent wound infection, maintain the moist environment of the wound and promote wound healing while meeting the filling effect of deeper wounds.

Description

Preparation method of amnion-spongy chitosan composite double-layer wound dressing

Technical Field

The invention relates to a preparation method of an amnion-spongy chitosan composite double-layer wound dressing, belonging to the technical field of medical dressings.

Background

The amnion is a common wound dressing, has the advantages of better covering a wound, keeping the moist environment of the wound, simultaneously being rich in effective components such as polysaccharide, collagen, growth factors and the like, and being beneficial to the proliferation and differentiation of cells and the recovery of the wound. It is difficult to fill deeper wounds and does not rapidly absorb wound exudate.

Chitosan (chitin), also known as chitosan, is obtained by deacetylation of chitin (chitin) widely existing in nature, and is widely used in various fields such as medicine, cosmetics, chemical engineering and the like due to its excellent properties such as biological functionality, compatibility, blood compatibility, safety, microbial degradability and the like. The chitosan sponge dressing has the advantages of antibiosis, high swelling rate and the like, but the sponge dressing has the defects of poor mechanical property, high brittleness and the like, is easy to be adhered to a wound surface, needs to be debrided repeatedly, and is not beneficial to wound surface recovery.

The medical dressing is used as a covering material of a wound, can replace damaged skin to play a role of temporary barrier in the healing process of the wound, provides a wound healing environment, avoids wound infection, and is a medical appliance widely used in clinic. At present, the dressings used in clinic are various and comprise amnion, animal skin, collagen, chitosan, alginic acid and the like, but the dressings are only used as a single dressing, and the dressings are difficult to achieve the effects of rapidly healing deeper wounds, absorbing wound exudate, promoting wound recovery and the like. Therefore, the search for an ideal and high-quality biological dressing has become an important part in clinical research and application of medical dressings.

Disclosure of Invention

Aiming at the problems of the prior art, the invention provides a novel amnion-chitosan composite double-layer dressing and a preparation method thereof.

The first purpose of the invention is to provide an amnion-chitosan composite double-layer dressing, which is prepared by adding chitosan and cross-linking agent solution to amnion.

In one embodiment, the amnion-chitosan composite double-layer dressing has a mixing mass ratio of amnion, chitosan and cross-linking agent of 1: (1-3): (0.2-0.6).

In one embodiment, the mixing mass ratio of the amnion, the chitosan and the cross-linking agent is 1:1: 0.2.

In one embodiment, the mixing mass ratio of the amnion, the chitosan and the cross-linking agent is 1:2: 0.4.

In one embodiment, the mixing mass ratio of the amnion, the chitosan and the cross-linking agent is 1:3: 0.6.

The second purpose of the invention is to provide a preparation method of the amnion-chitosan composite double-layer dressing, which comprises the following steps:

(1) carrying out decellularization treatment on the amnion: rinsing fresh amnion with deionized water, then soaking in 0.25% -0.5% pancreatin solution, stirring for 1-3 hours, washing with deionized water, and treating with 1% SDS water solution for 12-24 hours to obtain lower dressing;

(2) and (3) preparing the acellular amniotic membrane: and (3) chitosan: crosslinker pegdgge was as follows 1: (1-3): (0.2-0.6) and (m/m/m), adding chitosan and a cross-linking agent solution to the acellular processed amnion prepared in the step (1) to form a double-layer membrane with the thickness of 200-600 microns, and freeze-drying;

(3) and (3) transferring the double-layer membrane freeze-dried in the step (2) to an environment with the temperature of 30-80 ℃, and continuously reacting for 6 hours to enable the double-layer membrane to be in solid phase crosslinking.

In one embodiment, the concentration of chitosan in step (2) is 10-30 mg/mL.

In one embodiment, the chitosan solution is prepared by dissolving chitosan in 0.5-2% acetic acid solution to make the concentration of chitosan reach 10-30 mg/mL.

In one embodiment, the chitosan solution is mixed with a crosslinking agent and added to the amniotic membrane surface.

The invention also claims the use of the bi-layer dressing in the treatment of surgical wounds.

The invention also claims the application of the preparation method of the double-layer dressing in the production of medical dressings.

The invention has the beneficial effects that:

the amnion dressing which is beneficial to wounds and spongy chitosan with excellent antibacterial and swelling properties are combined by a cross-linking method to prepare the amnion-chitosan composite double-layer wound dressing, the wound healing rate of the dressing on a mouse at the 8 th day can reach 87.67%, the skin thickness of a mouse wound tissue reaches 693.9233 mu m, and the wound healing rate is obviously higher than that of a control group and a drug group; in addition, the dressing increases the enrichment concentration of hemoglobin from 72.8g/L to 685.39g/L of amniotic membrane group; the swelling coefficient of the dressing is improved to 800 percent from 300 percent of the amniotic membrane group; the tensile stress and the nominal strain of the single-side chitosan sponge dressing are only 0.29MPa and 2.6 percent, while the tensile stress and the nominal strain of the composite double-layer film are respectively 2.29MPa and 6.97 percent (M1-1) and 1.14MPa and 6.34 percent (M1-3), so that the wound dressing can rapidly absorb wound exudate, prevent wound infection, maintain the moist environment of the wound and promote wound healing while better meeting the filling effect of deeper wounds.

Drawings

FIG. 1 shows DAPI staining of decellularized amniotic membrane.

Fig. 2 is a schematic diagram of a bilayer dressing synthesis scheme.

Figure 3 is a schematic view of a two-layer casting product.

FIG. 4 is a schematic representation of scanning electron microscopy of a bilayer dressing bilayer adjuvant: wherein (1) the scanning electron microscope atlas of the amnion surface; (2) scanning electron microscope atlas of chitosan surface; (3) m1-1 section scanning electron microscope atlas; (4) m1-3 section scanning electron microscope atlas.

FIG. 5 is a comparison of wound healing in mice of each group: wherein (1) a visual graph of the wound healing condition of the mouse; (2) a statistical graph of the wound healing rate of the mice; (3) statistical skin thickness in mice.

Fig. 6 compares the procoagulant function, hemostatic ability, and swelling performance of each dressing: wherein (1) the procoagulant functions of the dressings are compared; (2) comparing the hemostatic abilities of the dressings; (3) comparing the swelling performance of the dressings in 0-60 min; (4) the swelling performance of the dressings is compared at 0-720 min.

Figure 7 is a comparison of the tensile stress and nominal strain for each dressing.

Detailed Description

Unless otherwise specified, the concentrations (m/v) referred to in the examples refer to mass-to-volume concentrations, i.e., the compounds are contained in mass (g) per 100mL of solution, e.g., 2% (m/v) means 2g/100 mL.

Example 1 optimization of the Decellularization conditions of amniotic Membrane

Four conditions were used to perform decellularization of the amniotic membrane. Rinsing fresh amnion with deionized water, soaking amnion in 0.25% or 0.5% pancreatin solution, stirring for 2 hr, washing with deionized water, and treating with 1% SDS water solution for 12 hr or 24 hr. After treatment, the amnion was thoroughly washed with deionized water, and naturally dried, and then stained with DAPI, and the DAPI staining results are shown in fig. 1. As a result of observation of the residual amount of DNA, it was found that, when the amnion was treated with 0.5% trypsin for 2 hours and then with 1% SDS for 24 hours, the cells in the amnion were efficiently and completely removed, and therefore, the acellular condition of the amnion was selected from the combination of 2 hours of treatment with 0.5% trypsin and 24 hours of treatment with 1% SDS.

EXAMPLE 2 Synthesis of bilayer dressings

The flow chart for the two-layer dressing synthesis is shown in figure 2. The method comprises the following specific steps: cutting the acellular amnion into a rectangle of 3cm multiplied by 10cm, placing the rectangle in a mould, dissolving chitosan in 1% acetic acid water solution to prepare a solution of 20mg/ml, and respectively preparing the amnion according to the following ratio: and (3) chitosan: adding chitosan and cross-linking agent solution onto amnion at mass ratio of 1:1:0.2 or 1:3:0.6(M/M/M) to obtain double-layer membranes M1-1 and M1-3 with different thicknesses, freezing at-80 deg.C, and lyophilizing. After lyophilization, the molds were transferred to a 40 ℃ oven and the reaction was continued for 6 hours. Finally, the cross-linked double-layer dressing is obtained, and the finished double-layer dressing is shown in figure 3. The prepared two-layer dressing is characterized by a scanning electron microscope, and as a result, as shown in figure 4, the thicknesses of the dense layer of the upper layer of M1-1 and M1-3 can be clearly seen to be about 20-30 μ M, the thickness of the lower layer of the dressing is different to form a porous and loose texture according to the addition amount of chitosan in the preparation, and the thicknesses of the dressings of the lower layers of M1-1 and M1-3 are respectively 200 μ M and 600 μ M.

Example 3 Effect of different bilayer dressings on wound healing in mice

0.1mol/L sodium citrate buffer: adding 2.10g of citric acid into 100ml of double distilled water to prepare a citric acid mother solution, namely solution A; adding 2.94g of trisodium citrate into 100ml of double distilled water to prepare a sodium citrate mother solution, namely solution B; the A, B solutions were mixed at a ratio of 1:1.32(v/v), and the pH of the solution was adjusted to 4.0.

Streptozotocin (STZ) solution: STZ was dissolved in 0.1mol/L sodium citrate buffer to prepare a 10mg/mL STZ solution, which was sterilized by filtration using a 0.22 μm filter. Prepared in dark place, and prepared immediately before use.

All mice were randomly divided into control and model groups and fasted 12h prior to surgery. The model group mice were administered with 70mg/kg of the intraperitoneal injection of STZ solution, and the control group was administered with the same dose of 0.1mol/L sodium citrate buffer solution for 4 consecutive days. One week after the end of STZ injection, fasting plasma glucose was measured and mice with plasma glucose above 13.5mmol/L were selected for inclusion in the study.

Wounds of 1cm in diameter were cut on the backs of the mice housed, and the mice were randomly divided into a negative group, a drug group, M1-1 group, and M1-3 group. The negative group adopts 3M membrane to treat the wound surface; the drug group is administered with Yunnan white drug powder to smear the wound surface; the M1-1 group and M1-3 group adopt double-layer films M1-1 and M1-3 to treat the wound surface respectively, the dressing change is carried out every 3 days, and the wound healing condition is observed and counted. After 14 days, the mice were sacrificed and wound tissue was taken and immersed in formalin solution for statistical analysis of skin thickness and wound condition.

The wound healing profile of each group of mice is shown in table 1 and fig. 5(1) and (2). The results show that the wound healing rate of each treatment group is similar after 14 days of treatment, but the wound healing rate of the M1-3 group at 3d, 5d and 8d is far higher than that of other groups, and the wound healing rate is better than that of the negative group and the drug group, which indicates that the M1-3 double-layer dressing constructed in the invention has better healing rate in wound recovery.

TABLE 1 comparison of wound healing rates (%)

The skin thickness of the wound tissue of each group of mice is shown in Table 2 and FIGS. 5(1) and (3). The results showed that the skin thickness of the wound tissues of

groups

3d, 5d and 14d was significantly increased in groups M1-1 and M1-3 compared to the negative group and the drug group, and the difference between groups M1-1 and M1-3 was not significant.

Table 2: comparison of wound tissue skin thickness (μm) for groups of mice

Comparative example 1: comparison of the procoagulant Performance of amnion, M1-1 and M1-3

Amniotic membrane is beneficial for wound healing, but does not provide rapid hemostasis for bleeding wounds. This experiment compared the procoagulant properties of amnion, M1-1 and M1-3 by in vitro coagulation. The gauze for medical use (control), the amniotic membrane of dBA, M1-1, M1-3 were cut into a circle (diameter 15mm) and placed in a petri dish. 100uL of blood containing 10% sodium citrate was added dropwise. The blood-containing membranes were incubated at 37 ℃ for 5 minutes, then 50 ml of distilled water was slowly added edgewise and the free erythrocytes were dissolved in water. The results are shown in fig. 6(1), the water in the gauze group and dBAM turned red, indicating that the platelets in the gauze and dBAM were difficult to coagulate; the aqueous solution of M1-1 was reddish and the aqueous solution of M-3 was almost transparent. The lighter the color of the rinse water, the faster the clotting time, indicating that the thickness of the sponge-like layer has a significant effect on the clotting rate.

In addition, the hemostatic performance of amnion, M1-1 and M1-3 was evaluated by measuring the hemoglobin content of the dressing. The method comprises the following specific steps: adding 2.5mL of hemoglobin test solution into the sample in the culture dish, fully and uniformly mixing, standing for 5min, adjusting zero by double distilled water, measuring the absorbance value of each tube, quantitatively measuring the absorbance value of each erythrocyte solution under the wavelength of 540nm in different dressings by an ultraviolet detector, and repeating each group for 6 times. Hemoglobin content (g/L) ═ OD-OD₀)×367.7(OD₀Indicates a blankAbsorbance value, OD denotes absorbance value of the sample). The hemoglobin concentration of 100. mu.L of blood was used as a reference value. The results are shown in figure 6(2), the hemoglobin concentrations of 100 mu L of blood, gauze, dBA amniotic membrane, M1-1 and M1-3 are 919.25g/L, 113.99g/L, 72.80g/L, 293.42g/L and 682.39g/L respectively, the hemoglobin concentrations of M1-1 and M1-3 are obviously higher than those of the gauze and the dBA amniotic membrane group, and the hemoglobin concentration of M1-3 is closer to the reference concentration of 100 mu L of blood, so that the double-layer dressing added with spongy chitosan is easier to gather hemoglobin, and the hemostatic capacity of the wound dressing is improved.

Comparative example 2: comparison of swelling Performance of amniotic membrane, M1-1 and M1-3

The amnion is beneficial to wound recovery, but cannot play a role in filling deeper wounds and only can play a role in covering the wounds; for wounds with more wound exudates, the wound exudate cannot be effectively absorbed, and the risk of wound infection is increased. In the experiment, the swelling performance of amnion, M1-1 and M1-3 is compared through an in vitro coagulation experiment. The dBAM amniotic membrane, M1-1 and M1-3 were weighed and soaked in PBS, the sample weights were measured at 0, 0.5, 1, 3, 10, 30, 60, 180, 360, 540 and 720min, respectively, and the excess surface water was absorbed by filter paper after the sample was taken, and the dressing weights were measured, respectively (n ═ 3). The corresponding swelling factor was calculated from the change in dressing weight: swelling factor (%) - (W)_t-W₀)/W₀Wherein W is_tWeight of dressing at time t, W₀Is the initial weight of the dressing. The results are shown in fig. 6(3), and the data show that all three dressings of dBAM, M1-1 and M1-3 can reach swelling equilibrium in a relatively short time, the swelling times are respectively 305%, 366% and 880%, and the chitosan layer can effectively absorb wound exudate and promote the repair of wound microenvironment.

Comparative example 3: comparison of mechanical properties of amnion, M1-1, M1-3 and single-layer chitosan membrane

The chitosan sponge dressing is independently used as a wound dressing, the mechanical property is poor, the condition of adhesion to a wound is easy to occur, and the debridement process is not beneficial to the recovery of the wound. The wound dressing is compounded with the amnion, so that the mechanical property of the amnion can be effectively improved, the adhesion of the wound surface is reduced, and the wound can grow favorably. In the experiment, mechanical properties of the decellularized amnion, the M1-1, the M1-3 and the single-layer chitosan membrane (which is synthesized under the condition of reference M1-3 and is different from the condition that no amnion is added) are compared by a universal tensile machine, and the tensile stress of the decellularized amnion, the M1-1, the M1-3 and the single-layer chitosan membrane is respectively 12.39MPa, 2.29MPa, 1.15MPa and 0.29MPa, and the nominal strain is respectively 7.09%, 6.97%, 6.31% and 2.6%. As can be seen from FIG. 7, the tensile stress and nominal strain of the double-layer membrane containing amnion M1-1 and M1-3 are significantly improved compared with chitosan membrane.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The amnion-chitosan composite double-layer dressing is characterized in that the dressing is formed by crosslinking chitosan and amnion through a crosslinking agent; the mass ratio of the amnion to the chitosan is 1: (1-3).

2. The amnion-chitosan composite double-layer dressing according to claim 1, wherein chitosan and a cross-linking agent are mixed according to the ratio of (1-3): (0.2-0.6), freeze-drying and fixing, and then carrying out solid phase crosslinking at 30-80 ℃ to obtain the product.

3. A method for preparing an amnion-chitosan composite double-layer dressing is characterized by comprising the following steps:

(1) carrying out decellularization treatment on the amnion;

(2) adding chitosan and a cross-linking agent to the acellular processed amnion prepared in the step (1) in the form of solution, and freeze-drying; wherein the mass ratio of the acellular amniotic membrane to the chitosan to the cross-linking agent is 1: (1-3): (0.2-0.6).

(3) And (3) transferring the double-layer membrane subjected to freeze drying in the step (2) to an environment with the temperature of 30-80 ℃ and standing for at least 6 h.

4. The method according to claim 3, wherein the step (1) comprises washing amnion, immersing the washed amnion in 0.25-0.5% pancreatin solution, stirring for 1-3 hours, washing amnion, and treating the amnion in SDS aqueous solution for 12-24 hours.

5. The method of claim 3, wherein the crosslinking agent is polyethylene glycol diglycidyl ether.

6. The method according to claim 3, wherein the concentration of chitosan in the step (2) is 10-30 mg/mL.

7. The method of claim 6, wherein the chitosan is dissolved in 0.5-2% acetic acid solution to make the concentration of chitosan 10-30 mg/mL.

8. The method according to any one of claims 3 to 7, wherein the chitosan solution is mixed with a cross-linking agent and then added to the surface of the amniotic membrane.

9. Use of the amniotic membrane-chitosan composite double-layered dressing according to claim 1 or 2 for improving the skin condition in a non-therapeutic application.

10. Use of a method of manufacturing a bi-layer dressing according to any one of claims 3 to 8 in the manufacture of a medical dressing.