WO2021114005A1 - 一种羊膜-海绵状壳聚糖复合双层伤口敷料的制备方法 - Google Patents

一种羊膜-海绵状壳聚糖复合双层伤口敷料的制备方法 Download PDF

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WO2021114005A1
WO2021114005A1 PCT/CN2019/122412 CN2019122412W WO2021114005A1 WO 2021114005 A1 WO2021114005 A1 WO 2021114005A1 CN 2019122412 W CN2019122412 W CN 2019122412W WO 2021114005 A1 WO2021114005 A1 WO 2021114005A1
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chitosan
amniotic membrane
dressing
layer
double
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PCT/CN2019/122412
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French (fr)
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陈敬华
杨漾
纪倩
张燕燕
戴雨桐
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江南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/28—Polysaccharides or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments

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  • the invention relates to a preparation method of an amniotic membrane-sponge chitosan composite double-layer wound dressing, which belongs to the technical field of medical dressings.
  • Amniotic membrane is a common wound dressing. Its advantage is that it can better cover the wound and maintain a moist environment for the wound. At the same time, it is rich in polysaccharides, collagen, growth factors and other effective ingredients, which is beneficial to cell proliferation and differentiation and wound recovery. However, it is difficult to fill deep wounds and cannot quickly absorb wound exudate.
  • Chitosan also known as chitin
  • Chitosan is obtained by deacetylation of chitin, which is widely present in nature, because of its biofunctionality and compatibility, blood compatibility, and safety. As well as the excellent properties of microbial degradability, it is widely used in various fields such as medicine, cosmetics, and chemical industry.
  • Chitosan sponge dressing has some advantages such as antibacterial and high swelling rate.
  • the shortcomings of sponge dressing are poor mechanical properties and high fragility. It is prone to adhesions on the wound surface and requires repeated debridement, which is not conducive to wound recovery.
  • the medical dressing can replace the damaged skin to act as a temporary barrier during the wound healing process, provide a wound healing environment, and avoid wound infection. It is a widely used medical device in the clinic.
  • dressings used in clinical practice including amniotic membrane, animal skin, collagen, chitosan, alginic acid, etc., but only used as a single dressing, it is difficult to simultaneously heal deeper wounds, absorb wound exudate, and promote wound recovery. All aspects of utility. Therefore, finding an ideal and high-quality biological dressing has become the most important thing in the clinical research and application of medical dressings.
  • the present invention provides a novel amniotic membrane-chitosan composite double-layer dressing and a preparation method thereof.
  • the first object of the present invention is to provide an amniotic membrane-chitosan composite double-layer dressing, which is prepared by adding chitosan and a cross-linking agent solution to the amniotic membrane.
  • the mixing mass ratio of the amniotic membrane, chitosan, and cross-linking agent of the amniotic membrane-chitosan composite double-layer dressing is 1: (1-3): (0.2-0.6).
  • the mixing mass ratio of the amniotic membrane, chitosan, and crosslinking agent is 1:1:0.2.
  • the mixing mass ratio of the amniotic membrane, chitosan, and crosslinking agent is 1:2:0.4.
  • the mixing mass ratio of the amniotic membrane, chitosan, and crosslinking agent is 1:3:0.6.
  • the second object of the present invention is to provide a method for preparing an amniotic membrane-chitosan composite double-layer dressing, which includes the following steps:
  • step (2) The lyophilized double-layer membrane in step (2) is transferred to an environment of 30-80°C, and the reaction is continued for 6 hours to solid-phase cross-link the double-layer membrane.
  • the concentration of chitosan in the step (2) is 10-30 mg/mL.
  • the chitosan solution is prepared by dissolving chitosan in an acetic acid solution with a concentration of 0.5-2%, so that the concentration of chitosan reaches 10-30 mg/mL.
  • the chitosan solution is mixed with a cross-linking agent and then added to the surface of the amniotic membrane.
  • the present invention also claims the application of the double-layer dressing in surgical wound treatment.
  • the present invention also claims the application of the preparation method of the double-layer dressing in the production of medical dressings.
  • the present invention uses a cross-linking method to combine an amniotic membrane dressing that is beneficial to wounds and a sponge-like chitosan with excellent antibacterial and swelling properties to prepare an amniotic membrane-chitosan composite double-layer wound dressing.
  • the wound healing rate in mice can reach 87.67%, and the skin thickness of the wound tissue in mice reaches 693.9233 ⁇ m, which is significantly higher than that of the control group and the drug group.
  • the dressing increases the concentration of hemoglobin from 72.8g/L in the amniotic membrane group to 685.39 g/L;
  • the swelling coefficient of the dressing is increased from 300% of the amniotic membrane group to 800%;
  • the tensile stress and nominal strain of the unilateral chitosan sponge dressing are only 0.29MPa and 2.6%, while the tensile stress of the composite double-layer membrane is only 0.29MPa and 2.6%.
  • the tensile stress and the nominal strain are 2.29 MPa and 6.97% (M1-1), 1.14 MPa and 6.34% (M1-3), respectively, which satisfies the filling effect of deeper wounds and can quickly absorb wound exudate.
  • To prevent wound infection maintain a moist environment on the wound surface and promote wound healing.
  • Figure 1 shows the results of DAPI staining of acellular amniotic membrane
  • Figure 2 is a schematic diagram of the synthesis process of a double-layer dressing
  • Figure 3 is a schematic diagram of a double-layer dressing product
  • Figure 4 is a schematic diagram of scanning electron microscope characterization of double-layer dressing and double-layer accessories: (1) scanning electron micrograph of the surface of amniotic membrane; (2) scanning electron micrograph of chitosan surface; (3) scanning electron micrograph of M1-1 section; 4) Scanning electron microscope atlas of M1-3 section;
  • Figure 5 is a comparison of wound healing in each group of mice: (1) a visual graph of wound healing in mice; (2) a statistical graph of wound healing rate in mice; (3) a statistical graph of mouse skin thickness;
  • Figure 6 is a comparison of the procoagulant function, hemostatic ability and swelling performance of each dressing: (1) the procoagulant function of each dressing; (2) the hemostatic ability of each dressing; (3) the comparison of the swelling performance of each dressing from 0 to 60 minutes; (4) Comparison of the swelling performance of each dressing at 0 ⁇ 720min.
  • Figure 7 shows the comparison of tensile stress and nominal strain of each dressing.
  • the concentration (m/v) involved in the embodiment refers to the mass volume concentration, that is, every 100 mL of the solution contains the compound by mass (g), for example, 2% (m/v). 2g/100mL.
  • the synthesis flow chart of the double-layer dressing is shown in Figure 2.
  • the specific steps are as follows: Cut the decellularized amniotic membrane into a 3cm ⁇ 10cm rectangle and place it in a mold. Dissolve chitosan in a 1% acetic acid aqueous solution to prepare a 20mg/ml solution.
  • the linking agent PEGDGE is 1:1:0.2 (m/m/m) by adding the chitosan and the crosslinking agent solution to the amniotic membrane to prepare double-layer membranes M1-1 and M1-3 with different thicknesses. After freezing at -80°C, freeze-dry. After lyophilization, the mold was transferred to an oven at 40°C, and the reaction was continued for 6 hours.
  • the cross-linked double-layer dressing M1-1 is finally obtained.
  • the double-layer dressings prepared in Example 2 and this example were characterized by scanning electron microscopy. The results are shown in Figure 4. It can be clearly seen that the dense layer of M1-1 and M1-3 has a thickness of about 20-30 ⁇ m, and the lower layer Depending on the amount of chitosan added in the preparation, different thicknesses of hollow and loose texture are formed. The thickness of the M1-1 and M1-3 lower layer dressings are respectively 200 ⁇ m and 600 ⁇ m.
  • 0.1mol/L sodium citrate buffer solution add 2.10g of citric acid to 100ml of double-distilled water to prepare citric acid mother liquor, called A solution; add 2.94g of trisodium citrate to 100ml of double-distilled water to make sodium citrate mother liquor, Called B liquid; mix A and B liquids in a ratio of 1:1.32 (v/v) to adjust the pH of the solution to 4.0.
  • Streptozotocin (STZ) solution Dissolve STZ in 0.1 mol/L sodium citrate buffer, prepare a 10 mg/mL STZ solution, and filter and sterilize it with a 0.22 ⁇ m filter. Pay attention to the preparation in the dark, and use it now.
  • mice All mice were randomly divided into control group and model group, and fasted for 12 hours before operation. Mice in the model group were injected intraperitoneally with STZ solution at a dose of 70 mg/kg, and the control group was given the same dose of 0.1 mol/L sodium citrate buffer for 4 consecutive days. One week after STZ injection, fasting blood glucose was measured, and mice with blood glucose higher than 13.5 mmol/L were selected for the formal experiment.
  • mice were randomly divided into negative group, drug group, M1-1 group, and M1-3 group.
  • the negative group was treated with 3M membrane; the drug group was given Yunnan Baiyao to smear the wound; the M1-1 group and M1-3 group were treated with double-layer membranes M1-1 and M1-3 respectively, and the dressing was changed every 3 days.
  • Observation and statistics Wound healing The mice were sacrificed 14 days later, the wound tissues were taken, immersed in formalin solution, and the skin thickness and wound conditions were statistically analyzed.
  • mice in each group is shown in Table 1 and Figure 5 (1) and (2).
  • the results showed that after 14 days of treatment, the wound healing rate of each treatment group was similar, but the wound healing rate of the M1-3 group on the 3rd, 5th, and 8th day was much higher than that of the other groups, and the wound healing rate was better than that of the negative group and the negative group.
  • the drug group shows that the M1-3 double-layer dressing constructed in the present invention has a better healing rate in wound recovery.
  • the skin thickness of the wound tissue of each group of mice is shown in Table 2 and Figure 5 (1) and (3). The results showed that the skin thickness of the wound tissue in the M1-1 and M1-3 groups on the 3rd, 5th, and 14th days was significantly higher than that of the negative group and the drug group, and the difference between the M1-1 and M1-3 groups was not significant.
  • Comparative example 1 Comparison of procoagulant performance of amniotic membrane, M1-1 and M1-3
  • Amniotic membrane is good for wound recovery, but it cannot stop bleeding quickly for bleeding wounds.
  • This experiment compared the procoagulant performance of amniotic membrane, M1-1 and M1-3 through in vitro coagulation experiments. Cut medical gauze (control), dBAM amniotic membrane, M1-1, and M1-3 into circles (diameter 15mm) and place them in a petri dish. Instill 100uL of blood containing 10% sodium citrate respectively. Incubate the blood-containing membrane at 37°C for 5 minutes, and then slowly add 50 ml of distilled water from the edge to dissolve the free red blood cells in the water. The results are shown in Figure 6 (1).
  • the water in the medical gauze group and dBAM turned red, indicating that the platelets in the medical gauze and dBAM are difficult to coagulate; the aqueous solution of M1-1 is slightly red, and the aqueous solution of M-3 is almost transparent.
  • the hemostatic properties of amniotic membrane, M1-1 and M1-3 were evaluated by detecting the hemoglobin content in the dressing.
  • the specific steps are as follows: add 2.5mL hemoglobin test solution to the sample in the petri dish, mix well, let stand for 5 minutes, adjust the zero with double distilled water, measure the absorbance value of each tube, and quantitatively determine 540nm in different dressings with a UV detector The absorbance value of each red blood cell solution at the wavelength, repeat 6 times for each group.
  • Hemoglobin content (g/L) (OD-OD 0 ) ⁇ 367.7 (OD 0 represents the blank absorbance value, OD represents the absorbance value of the sample). Take the hemoglobin concentration of 100 ⁇ L blood as the reference value.
  • the results are shown in Figure 6(2).
  • the hemoglobin concentrations of 100 ⁇ L of blood, gauze, dBAM amniotic membrane, M1-1, and M1-3 are 919.25g/L, 113.99g/L, 72.80g/L, 293.42g/L, and 682.39g, respectively.
  • /L the hemoglobin concentration of M1-1, M1-3 is significantly higher than the gauze and dBAM amniotic membrane group
  • the hemoglobin concentration of M1-3 is closer to the reference concentration of 100 ⁇ L blood, indicating that the double-layer dressing with spongy chitosan is easier to aggregate Hemoglobin, thereby improving the hemostatic ability of the wound dressing.
  • Comparative example 2 Comparison of swelling performance of amniotic membrane, M1-1 and M1-3
  • Amniotic membrane is conducive to wound recovery, but it cannot fill deeper wounds and can only cover the wound; for wounds with more wound exudate, it cannot effectively absorb the exudate, which increases the risk of wound infection. risk.
  • This experiment compared the swelling properties of amniotic membrane, M1-1 and M1-3 through in vitro coagulation experiments.
  • swelling coefficient (%) (W t -W 0 )/W 0 , where W t is the weight of the dressing at time t, and W 0 is the initial weight of the dressing.
  • W t is the weight of the dressing at time t
  • W 0 is the initial weight of the dressing.
  • the results are shown in Figure 6(3).
  • the data shows that the three dressings of dBAM, M1-1 and M1-3 can reach the swelling equilibrium in a relatively fast time, and the swelling ratios are 305%, 366%, and 880%, respectively.
  • the chitosan layer can effectively absorb wound exudate and promote the repair of wound microenvironment.
  • Comparative Example 3 Comparison of mechanical properties of amniotic membrane, M1-1, M1-3 and monolayer chitosan membrane
  • Chitosan sponge dressing alone as a wound dressing has poor mechanical properties and is prone to stick to the wound.
  • the debridement process is not conducive to wound recovery.
  • Compounding it with amniotic membrane can effectively increase its mechanical properties, reduce the adhesion of wounds, and facilitate the growth of its wounds.

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Abstract

一种羊膜-海绵状壳聚糖复合双层伤口敷料的制备方法，属于医用敷料技术领域。利用交联法将羊膜敷料及具有抗菌、溶胀性能优异的海绵状壳聚糖进行组合，制备羊膜-壳聚糖复合双层创面敷料，该敷料在第8d的小鼠创伤愈合率可达87.67％，小鼠伤口组织皮肤厚度达到693.9233μm；此外，该敷料将血红蛋白的富集浓度由羊膜组的72.8g/L提升至685.39g/L；将溶胀系数由羊膜组的300％提升至800％；将敷料的拉伸应力及标称应变增加至1.14～2.29MPa和6.34～6.97％，在满足较深伤口填充效果的同时，快速吸收创面渗出液，防止伤口感染，保持创面的湿润环境并促进创面愈合。

Description

一种羊膜-海绵状壳聚糖复合双层伤口敷料的制备方法 技术领域
本发明涉及一种羊膜-海绵状壳聚糖复合双层伤口敷料的制备方法，属于医用敷料技术领域。
背景技术
羊膜是一种常见的伤口敷料，其优点在于能够较好地覆盖伤口，保持伤口湿润环境，同时富含多糖、胶原蛋白、生长因子等有效成分，有益于细胞的增殖分化和伤口的恢复。但是其对于较深的伤口难以进行填充，同时不能快速的吸收伤口渗出液。
壳聚糖(chitosan)又称脱乙酰甲壳素，是由自然界广泛存在的几丁质(chitin)经过脱乙酰作用得到的，因其具有生物官能性和相容性、血液相容性、安全性以及微生物降解性等优良性能，被广泛用于医药、化妆品、化工等各领域中。壳聚糖海绵敷料具有抗菌，溶胀率高等有点，但海绵状敷料的缺点为机械性能差、脆性大等缺点，易于创面发生黏连，需要反复清创，不利于创面恢复。
医用敷料作为伤口处的覆盖物，在伤口的愈合过程中，可代替受损的皮肤起到暂时性屏障作用，提供创面愈合环境，避免伤口感染，是临床中广泛使用的医疗器械用品。目前临床中使用的敷料种类繁多，包括羊膜、动物皮、胶原、壳聚糖、海藻酸等，但仅仅作为单一敷料进行使用，难以兼顾快速愈合较深伤口、吸收创面渗液、促进创面恢复等各方面的效用。因此，寻找一种理想、优质的生物敷料已成为医用敷料临床研究和应用中的重中之重。
发明内容
针对现有的技术难点及存在的问题，本发明提供了一种新型羊膜-壳聚糖复合双层敷料及其制备方法。
本发明的第一个目的是提供一种羊膜-壳聚糖复合双层敷料，所述敷料是将壳聚糖与交联剂溶液加入到羊膜上制备而成。
在一种实施例中，所述羊膜-壳聚糖复合双层敷料的羊膜、壳聚糖、交联剂的混合质量比为1：(1～3)：(0.2～0.6)。
在一种实施方式中，所述羊膜、壳聚糖、交联剂的混合质量比为1:1:0.2。
在一种实施方式中，所述羊膜、壳聚糖、交联剂的混合质量比为1:2:0.4。
在一种实施方式中，所述羊膜、壳聚糖、交联剂的混合质量比为1:3:0.6。
本发明的第二个目的是提供一种羊膜-壳聚糖复合双层敷料的制备方法，包括如下步骤：
(1)将羊膜进行脱细胞处理：新鲜羊膜用去离子水漂洗干净，然后浸于0.25％～0.5％胰酶溶液中，搅拌1～3小时，用去离子水洗净后，在1％的SDS水溶液处理12小时～24小时，获得下层敷料；
(2)将脱细胞羊膜：壳聚糖：交联剂PEGDGE按照1：(1～3)：(0.2～0.6)，(m/m/m)的比例，将壳聚糖与交联剂溶液加入至步骤(1)制备的脱细胞处理后的羊膜上，冻干；
(3)将步骤(2)冻干后的双层膜转移至30-80℃环境中，继续反应6小时，使双层膜固相交联。
在一种实施方式中，所述步骤(2)中壳聚糖的浓度为10～30mg/mL。
在一种实施方式中，所述壳聚糖溶液是将壳聚糖溶于浓度为0.5～2％的醋酸溶液中，使壳聚糖的浓度达10～30mg/mL。
在一种实施方式中，所述壳聚糖溶液与交联剂混合后加入至羊膜表面。
本发明还要求保护所述双层敷料在外科伤口处理中的应用。
本发明还要求保护所述双层敷料的制备方法在生产医用敷料中的应用。
本发明的有益效果：
本发明利用交联法将对伤口有益的羊膜敷料及具有抗菌、溶胀性能优异的海绵状壳聚糖进行组合，制备一种述羊膜-壳聚糖复合双层创面敷料，该敷料在第8d的小鼠创伤愈合率可达87.67％，小鼠伤口组织皮肤厚度达到693.9233μm，显著高于对照组和药物组；此外，该敷料将血红蛋白的富集浓度由羊膜组的72.8g/L提升至685.39g/L；将敷料的溶胀系数由羊膜组的300％提升至800％；单侧壳聚糖海绵敷料的拉伸应力及标称应变仅为0.29MPa及2.6％，而复合双层膜的拉伸应力及标称应变分别为2.29MPa及6.97％(M1-1)，1.14MPa及6.34％(M1-3)，在较好的满足较深伤口填充效果的同时，能够快速吸收创面渗出液，防止伤口感染，保持创面的湿润环境并促进创面愈合。
附图说明
图1为脱细胞羊膜DAPI染色结果；
图2为双层敷料合成流程示意图；
图3为双层敷料产品示意图；
图4为双层敷料双层辅料电子显微镜扫描表征示意图：其中(1)羊膜表面的扫描电镜图谱；(2)壳聚糖表面的扫描电镜图谱；(3)M1-1截面扫描电镜图谱；(4)M1-3截面扫描电镜图谱；
图5为各组小鼠伤口愈合比较：其中(1)小鼠伤口愈合情况直观图；(2)小鼠伤口愈合 率统计图；(3)小鼠皮肤厚度统计图；
图6为各敷料促凝血功能、止血能力和溶胀性能比较：其中(1)各敷料促凝血功能比较；(2)各敷料止血能力比较；(3)各敷料在0～60min的溶胀性能比较；(4)各敷料在0～720min的溶胀性能比较。
图7为各敷料拉伸应力及标称应变比较。
具体实施方式
在不做特别说明的情况下，实施例中涉及的浓度(m/v)均指质量体积浓度，即每100mL溶液中含有按质量(g)计的化合物，例如2％(m/v)表示2g/100mL。
实施例1 羊膜的脱细胞条件的优化
采用四种条件分别对羊膜进行脱细胞处理。将新鲜羊膜用去离子水漂洗干净，然后将羊膜浸于0.25％或0.5％胰酶溶液中，搅拌2小时，用去离子水洗净后，在1％的SDS水溶液处理12小时或24小时。处理后用去离子水充分清洗羊膜，自然风干后，利用DAPI进行染色，DAPI染色结果见图1。通过对DNA残留量的观察可以发现，当采用0.5％胰酶处理2小时后，再用1％SDS处理24小时，羊膜中的细胞被高效彻底地去除，因此选择0.5％胰酶处理2小时结合1％SDS处理24小时作为羊膜的脱细胞条件。
实施例2 双层敷料的合成
双层敷料合成流程图见图2。具体步骤如下：将脱细胞后羊膜剪成3cm×10cm的长方形置于模具中，将壳聚糖溶于1％的醋酸水溶液中配制成20mg/ml的溶液，分别按照羊膜：壳聚糖：交联剂PEGDGE以1:1:0.2(m/m/m)的质量比例，将壳聚糖与交联剂溶液加入羊膜上，制备厚度不同的双层膜M1-1,M1-3，放于-80℃冷冻后，冻干。冻干后将模具转移至40℃烘箱中，继续反应6小时。最终得到交联的双层敷料M1-1。
实施例3 双层敷料的合成
将脱细胞后羊膜剪成3cm×10cm的长方形置于模具中，将壳聚糖溶于1％的醋酸水溶液中配制成20mg/ml的溶液，分别按照羊膜：壳聚糖：交联剂PEGDGE以1:2:0.4(m/m/m)的质量比例，将壳聚糖与交联剂溶液加入羊膜上，制备厚度不同的双层膜M1-1,M1-3，放于-80℃冷冻后，冻干。冻干后将模具转移至40℃烘箱中，继续反应6小时。最终得到交联的双层敷料M1-2。
实施例4 双层敷料的合成
将脱细胞后羊膜剪成3cm×10cm的长方形置于模具中，将壳聚糖溶于1％的醋酸水溶液中配制成20mg/ml的溶液，分别按照羊膜：壳聚糖：交联剂PEGDGE以1:3:0.6(m/m/m)的 质量比例，将壳聚糖与交联剂溶液加入羊膜上，制备厚度不同的双层膜M1-1,M1-3，放于-80℃冷冻后，冻干。冻干后将模具转移至40℃烘箱中，继续反应6小时。最终得到交联的双层敷料M1-3。
对实施例2和本实施例制备得到的双层敷料进行扫描电子显微镜表征，结果如图4所示，可以清楚的看到M1-1,M1-3上层的致密层厚度约20-30μm，下层则根据制备中壳聚糖添加量的不同，形成多空的、疏松质地的不同厚度，M1-1,M1-3下层敷料的厚度分别为200μm和600μm。
实施例5 不同双层敷料对小鼠伤口愈合的影响
0.1mol/L柠檬酸钠缓冲液：将2.10g柠檬酸加入双蒸水100ml配成柠檬酸母液，称为A液；将2.94g柠檬酸三钠加入双蒸水100ml配成柠檬酸钠母液，称为B液；将A、B液按1:1.32(v/v)比例混合，调定溶液pH＝4.0。
链脲佐菌素(STZ)溶液：将STZ溶于0.1mol/L柠檬酸钠缓冲液中，配制成10mg/mL浓度的STZ溶液，并用0.22μm滤菌器过滤除菌。注意避光配制，现用现配。
将所有小鼠随机分为对照组和模型组，术前12h禁食。模型组小鼠按照70mg/kg的剂量腹腔注射的STZ溶液，对照组给予相同剂量的0.1mol/L柠檬酸钠缓冲液，连续注射4天。STZ注射结束一周后，测空腹血糖，选择血糖高于13.5mmol/L的小鼠纳入正式实验。
在纳入小鼠的背部剪开直径为1cm的创伤，将小鼠随机分为阴性组、药物组、M1-1组、M1-3组。阴性组采用3M膜处理创面；药物组给予云南白药涂抹创面；M1-1组、M1-3组分别采用双层膜M1-1及M1-3处理创面，每隔3天换药，观察并统计创伤愈合情况。14天后处死小鼠，取伤口组织，浸入福尔马林溶液中，对皮肤厚度及伤口情况进行统计学分析。
各组小鼠创伤愈合情况见表1和图5(1)和(2)。结果表明，在处理14天后，各治疗组的创伤愈合率相近，但M1-3组在第3d、第5d、第8d的创伤愈合率远远高于其他组，创伤愈合速率优于阴性组和药物组，表明本发明中构建的M1-3双层敷料在创面恢复中具有更好的愈合速率。
表1 各组小鼠创伤愈合率比较(％)
Figure PCTCN2019122412-appb-000001
各组小鼠伤口组织皮肤厚度见表2和图5(1)和(3)。结果表明，M1-1和M1-3组在第3d、第5d和第14d的伤口组织皮肤厚度较阴性组和药物组显著增加，M1-1和M1-3组之间 的差异并不明显。
表2：各组小鼠伤口组织皮肤厚度比较(μm)
Figure PCTCN2019122412-appb-000002
对比例1：羊膜、M1-1和M1-3促凝血性能比较
羊膜有利于伤口恢复，但是对于出血的伤口无法快速止血。本实验通过体外凝血实验比较了羊膜，M1-1及M1-3的促凝血性能。将医用纱布(对照)、dBAM羊膜、M1-1、M1-3切成圆形(直径15mm)，置于培养皿中。分别滴入100uL含10％柠檬酸钠的血液。将含血的膜在37℃下孵育5分钟，然后缓慢地从边缘加入50毫升蒸馏水，将游离的红细胞溶解在水中。结果见图6(1)，医用纱布组和dBAM的水变成了红色，说明医用纱布和dBAM中血小板很难凝固；M1-1的水溶液微红，M-3的水溶液几乎透明。漂洗水的颜色越浅，凝血越快，这说明海绵样层的厚度对凝血速度有重要影响。
此外，通过对敷料中血红蛋白含量的检测评价羊膜，M1-1及M1-3的止血性能。具体步骤如下：将上述培养皿中的样品中加入2.5mL血红蛋白测试液，充分混匀，静置5min,通过双蒸水调零，测定各管的吸光度值，紫外检测器定量测定不同敷料中540nm波长下各红细胞溶液的吸光度值，每组重复6次。血红蛋白含量(g/L)＝(OD-OD 0)×367.7(OD 0表示空白吸光度值，OD表示样品的吸光度值)。以100μL血液的血红蛋白浓度为参考值。结果见图6(2)，100μL血液、纱布、dBAM羊膜、M1-1、M1-3的血红蛋白浓度分别为919.25g/L、113.99g/L、72.80g/L、293.42g/L、682.39g/L，M1-1、M1-3的血红蛋白浓度显著高于纱布和dBAM羊膜组，M1-3的血红蛋白浓度更加接近100μL血液的参考浓度，说明增加海绵状壳聚糖的双层敷料更容易聚集血红蛋白，从而提高创面敷料的止血能力。
对比例2：羊膜、M1-1和M1-3溶胀性能比较
羊膜有利于伤口恢复，但是对于较深的伤口无法起到填充作用，只能起到伤口的覆盖作用；对于伤口渗出液较多的伤口，其无法有效吸收渗出液，加大伤口感染的风险。本实验通过体外凝血实验比较了羊膜，M1-1及M1-3的溶胀性能。将dBAM羊膜、M1-1及M1-3分别称重后浸泡于PBS中，分别于0、0.5、1、3、10、30、60、180、360、540及720min对样品重量进行测定，取后用滤纸吸取表面多余水分，分别对敷料重量进行测定(n＝3)。根据敷料重量的变化计算相应的溶胀系数：溶胀系数(％)＝(W t-W 0)/W 0,其中W t为t时间点敷料重量，W 0为敷料的起始重量。结果见图6(3)，数据表明，dBAM,M1-1及M1-3的三种 敷料均能在较快的时间内达到溶胀平衡，溶胀倍数分别为305％、366％、880％，表明壳聚糖层能够有效吸收伤口渗出液，促进伤口微环境的修复。
对比例3：羊膜、M1-1、M1-3及单层壳聚糖膜机械性能比较
壳聚糖海绵敷料单独作为伤口敷料，机械性能较差，易出现黏附在伤口上的情况，而清创过程则不利于伤口的恢复。将其与羊膜进行复合可以有效的增加其机械性能，减少创面的黏附，有利于其伤口的生长。本实验通过万能拉力机对脱细胞羊膜、M1-1、M1-3及单层壳聚糖膜(参考M1-3的条件合成，区别在于，不添加羊膜)的机械性能进行比较，发现脱细胞羊膜、M1-1、M1-3及单层壳聚糖膜的拉伸应力分别为12.39MPa、2.29MPa、1.15MPa、0.29MPa，标称应变分别为7.09％、6.97％、6.31％、2.6％。从图7可以看出，含有羊膜的双层膜M1-1及M1-3，其拉伸应力及标称应变较壳聚糖膜显著提高。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种制备羊膜-壳聚糖复合双层敷料的方法，其特征在于，包括如下步骤：
    (1)将羊膜进行脱细胞处理：将羊膜漂洗后浸于0.25％～0.5％胰酶溶液中，搅拌1～3小时，再将羊膜洗净，于SDS水溶液中处理12～24小时；
    (2)将10～30mg/mL壳聚糖和交联剂以混合溶液的形式加入至步骤(1)制备的脱细胞处理后的羊膜上，形成厚度为200μm～600μm的双层膜，冻干；其中，脱细胞羊膜、壳聚糖和交联剂的质量比为1：(1～3)：(0.2～0.6)；
    (3)将步骤(2)冻干后的双层膜转移至30-80℃环境中静置至少6h。
  2. 一种制备羊膜-壳聚糖复合双层敷料的方法，其特征在于，包括如下步骤：
    (1)将羊膜进行脱细胞处理；
    (2)将壳聚糖和交联剂以溶液形式加入至步骤(1)制备的脱细胞处理后的羊膜上，形成厚度为200μm～600μm的双层膜，冻干；其中，脱细胞羊膜、壳聚糖和交联剂的质量比为1：(1～3)：(0.2～0.6)。
    (3)将步骤(2)冻干后的双层膜转移至30-80℃环境中静置至少6h。
  3. 根据权利要求2所述的方法，其特征在于，所述羊膜、壳聚糖、交联剂的混合质量比为1:1:0.2。
  4. 根据权利要求2所述的方法，其特征在于，所述羊膜、壳聚糖、交联剂的混合质量比为1:2:0.4。
  5. 根据权利要求2所述的方法，其特征在于，所述羊膜、壳聚糖、交联剂的混合质量比为1:3:0.6。
  6. 根据权利要求2所述的方法，其特征在于，所述步骤(1)是将羊膜漂洗后浸于0.25％～0.5％胰酶溶液中，搅拌1～3小时，再将羊膜洗净，于SDS水溶液中处理12～24小时。
  7. 根据权利要求6所述的方法，其特征在于，所述交联剂为聚乙二醇二缩水甘油醚。
  8. 根据权利要求6所述的方法，其特征在于，所述步骤(2)中壳聚糖的浓度为10～30mg/mL。
  9. 根据权利要求8所述的方法，其特征在于，将壳聚糖溶于浓度为0.5～2％的醋酸溶液中，使壳聚糖的浓度达10～30mg/mL。
  10. 根据权利要求2～9任一所述的方法，其特征在于，所述壳聚糖溶液与交联剂混合后加入至羊膜表面。
  11. 一种羊膜-壳聚糖复合双层敷料，其特征在于，所述敷料是由壳聚糖与羊膜经交联剂交联形成；所述羊膜和壳聚糖的质量比为1：(1～3)。
  12. 根据权利要求11所述的羊膜-壳聚糖复合双层敷料，其特征在于，是将壳聚糖与交 联剂按照(1～3)：(0.2～0.6)的质量比混合，冻干固定，再于30-80℃固相交联后获得。
  13. 权利要求11或12所述的羊膜-壳聚糖复合双层敷料在非治疗用途下改善皮肤状态方面的应用。
  14. 权利要求11或12所述的羊膜-壳聚糖复合双层敷料在外科伤口处理中的应用。
  15. 权利要求1～10任一所述双层敷料的制备方法在生产医用敷料中的应用。
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CN109718392A (zh) * 2019-01-18 2019-05-07 广州润虹医药科技股份有限公司 复合型医用敷料及其制备方法
CN109847088A (zh) * 2019-01-18 2019-06-07 广州润虹医药科技股份有限公司 复合型脱细胞真皮基质生物敷料及其制备方法

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CN115068661A (zh) * 2022-06-21 2022-09-20 杭州倍荣生物科技有限公司 一种海藻酸钙复合多孔生物基质敷料、其制备方法和应用
CN115887735A (zh) * 2022-11-01 2023-04-04 湖南德普斯医疗科技有限公司 一种菌类杆茎脱细胞止血海绵的制备方法
CN116407671A (zh) * 2023-03-31 2023-07-11 西南大学 一种用于凝血功能障碍患者出血的Janus结构止血海绵及其制备方法

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