CN109381731B - 一种医用生物敷料及其制备方法 - Google Patents
一种医用生物敷料及其制备方法 Download PDFInfo
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- CN109381731B CN109381731B CN201810417695.1A CN201810417695A CN109381731B CN 109381731 B CN109381731 B CN 109381731B CN 201810417695 A CN201810417695 A CN 201810417695A CN 109381731 B CN109381731 B CN 109381731B
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Abstract
本发明公开了一种医用生物敷料及其制备方法。该医用生物敷料由大鲵皮肤制成。该医用生物敷料具体制备方法为:先剥取大鲵皮肤得到全厚皮片,并进行超低温冷冻处理;然后清洗、消毒,去除表皮层和腺体得到中间皮片,并再次用复合碱性清洗剂I、缓冲液II和表面活性剂清洗中间皮片,另使用固定剂进行固定处理,再使用复合酶对致密纤维层进行消化;最后使用漂白剂漂白,并浸泡于冷冻保护液中,冷冻保存即得。本发明通过使用特异性的生物皮加工方法,以大鲵皮肤为来源制备的生物敷料可作为真皮替代品,为烧伤创面提供支持和保护。本发明的制备方法可以达到一定的抑菌效果,并且具有抵免疫原性的特性。
Description
技术领域
本发明涉及一种医用生物敷料,特别是一种医用大鲵皮肤敷料及其制备方法。
背景技术
医用敷料主要功能是覆盖、保护破损皮肤,同时提供有助于伤口愈合的环境。传统敷料主要使用天然纤维性材料类敷料,大量文献报道指出传统敷料会出现与渗出物结痂,造成伤口粘连,换药时引发疼痛及新的创伤,以及细菌滋生、保湿及止血性能不佳等问题。使用异种非灵长类哺乳动物(如猪、牛、羊等)或者同种异体(如尸体)为供体来源的生物敷料作为一种新型医用敷料,在治疗烧伤创面领域有着良好的治疗效果,但是随着研究的深入,诸如免疫原性高、黏附性差、需要多次换药等不足也被指出。近年来随着疯牛病和口蹄疫的爆发,更是引起了人们对使用这些哺乳动物来源的产品的安全性及对人类健康状况影响的关注,因此从水产品中寻找新的敷料替代原料成为了新的方向。
中国大鲵是我国特有的有尾两栖动物,富含多种人体必需氨基酸、金属硫蛋白和丰富的胶原蛋白,肌肉含有丰富的二十二碳六烯酸(DHA)、活性钙等物质。通过对大鲵皮肤组织结构的研究发现,大鲵皮肤可制成粒面皮革,皮革的透气性和透水性能良好,并且柔软度、丰满度、弹性、延伸性都十分突出;大鲵皮肤有足够的厚度和强度,较高的收缩温度,稳定的耐腐蚀、耐酶、耐化学作用特性;对大鲵皮肤中胶原蛋白进行初步提取和纯化后,经测定为I型胶原蛋白,具有低抗原性、低过敏性、易酶解等优点,这些特点使大鲵皮肤制成一种潜在的生物敷料供体成为可能。
发明内容
针对目前传统敷料存在的伤口创面愈合速度慢,容易引起伤口感染,换药疼痛的问题,以及哺乳动物来源的生物敷料存在高免疫原性、生物安全性无法保证的问题,本发明提供一种医用生物敷料及其制备方法,其能为创面提供良好的支持作用,同时具备低抗原性、低过敏性的特点。
为解决上述技术问题,本发明提供了一种医用生物敷料,其由大鲵皮肤制成。
为解决上述技术问题,本发明还提供了一种医用生物敷料制备方法,其包括以下步骤:
(1)对大鲵皮肤进行剥离,得到全厚皮片;
(2)对所述全厚皮片进行超低温冷冻处理;
(3)解冻后在缓冲液I中浸泡清洗1-3次,缓冲液I的有效成分为:L-抗坏血酸10-200mM,青链霉素混合液500ppm,无菌磷酸盐缓冲盐水(sPBS);
(4)去除清洗后全厚皮片的表皮层和部分真皮疏松结缔组织,得到中间皮片;
(5)使用复合碱性清洗剂I和缓冲液II对中间皮片清洗1-3次,其中复合碱性清洗剂I的有效成分为:氢氧化钠15-25mg/mL,助剂为:偏硅酸钠30-60mg/mL,碳酸钠15-30mg/mL;
(6)使用表面活性剂处理清洗后的中间皮片;
(7)使用固定剂I和固定剂II对经步骤(6)处理的中间皮片进行固定处理,固定剂I有效成分为:含谷氨酰胺0.1-4mM的Hank's平衡盐溶液;
(8)使用复合酶对经步骤(7)处理的中间皮片的致密纤维层进行消化,复合酶的主要成分为:氯化钠100-150mM,氯化钾3-6mM,磷酸氢二钠0.1-1.5mM,葡萄糖3-10mM,EDTA0.5-3mM,胰蛋白酶0.01-5g/L,链霉蛋白酶0.01-5g/L,木瓜凝乳蛋白酶0.01-5g/L;
(9)使用漂白剂对经步骤(8)处理的中间皮片进行漂白处理;
(10)将经步骤(9)处理的中间皮片浸泡于含有葡聚糖5-15mM、蔗糖2-10mM、EDTA0.5-3mM的冷冻保护液中,冷冻保存,即得。
在上述制备方法中,所述步骤(1)对大鲵皮肤剥离后得到全厚皮片的操作为:对大鲵的皮肤进行初步清洗,去除表面分泌抗菌肽,使用取皮刀对大鲵躯干背侧区域进行取皮操作,取皮厚度约为1300-1500μm,包含全部表皮层、真皮层及皮下少量脂肪,然后将取下的皮片置入4℃的PBS中浸泡5-12分钟,重复3次后吸去多余水分备用。
所述步骤(2)超低温冷冻处理的降温速度为0.5-10℃/分钟,优选为5℃/分钟;冷冻温度为-80至-196℃,优选为-196℃;冷冻时间为1-3小时,优选为2小时。
所述步骤(3)解冻的温度为60-100℃,优选为60℃;所述浸泡清洗的温度为2-8℃,优选为4℃;每次浸泡清洗时间为30-90分钟,每次浸泡清洗时间优选为60分钟。
所述步骤(5)的缓冲液II有效成分为:氯化钠100-150mM,氯化钾3-6mM,硫酸镁0.1-1.5mM,氯化镁0.1-1.5mM,磷酸氢二钠0.1-1.5mM,磷酸二氢钾0.1-1.5mM,碳酸氢钠2-6Mm。
步骤(5)的复合碱性清洗剂I的有效成分浓度优选为:氢氧化钠20mg/mL。助剂为:偏硅酸钠40mg/mL,碳酸钠20mg/mL。缓冲液II有效成分浓度优选为:氯化钠125mM,氯化钾4mM,硫酸镁1mM,氯化镁1mM,磷酸氢二钠1mM,磷酸二氢钾1mM,碳酸氢钠5mM。所述步骤(6)的表面活性剂为含有0.1-1%X-100的杜氏磷酸盐缓冲液,优选为含有0.5%X-100的杜氏磷酸盐缓冲液。
所述步骤(7)的固定剂II有效成分为:含十二烷基硫酸钠(SDS)0.01-1%的Hank's平衡盐溶液,其中的十二烷基硫酸钠(SDS)可替换为α-烯基磺酸钠,或者烷基苯磺酸钠。
所述步骤(7)的固定剂I有效成分浓度优选为:含谷氨酰胺2mM的Hank's平衡盐溶液。固定剂II有效成分浓度优选为:含十二烷基硫酸钠(SDS)1%的Hank's平衡盐溶液。
所述步骤(7)的固定方法为:先将经步骤(6)处理的中间皮片放入固定剂I中,以20-80rpm的速度水平摇床孵育5-15分钟,倒去固定剂I,然后放入固定剂II中,以20-80rpm的速度摇床孵育30-90分钟,固定温度为15-25℃。其中,优选摇床速度为40rpm,优选孵育时间为10分钟,优选固定温度为20℃。
所述步骤(8)的消化温度为15-25℃,消化时间为15-20小时。
所述步骤(8)的复合酶的主要成分优选浓度为:氯化钠125mM,氯化钾4mM,磷酸氢二钠1mM,葡萄糖7mM,EDTA 2mM,胰蛋白酶0.05g/L,链霉蛋白酶2g/L,木瓜凝乳蛋白酶1g/L。消化温度优选为20℃,消化时间优选为18小时。
所述步骤(9)所使用的漂白剂的主要成分为:过氧化氢1-5%,氢氧化钠5-25g/L,EDTA0.5-3mM。步骤(9)所使用的漂白剂的主要成分优选浓度为:过氧化氢3%,氢氧化钠15g/L,EDTA 2mM。
所述步骤(9)的漂白处理时间为10-60分钟,优选为30分钟;漂白温度为2-8℃,优选为4℃。
所述步骤(10)的冷冻保护液成分优选浓度为:葡聚糖7mM,蔗糖6mM,EDTA 1mM。
本发明利用大鲵皮肤含有极高I型胶原蛋白,且I型胶原蛋白能够为创面提供良好的支持作用,并通过特殊的酶解体系,可以完全去除皮肤内的肥大细胞,使制备的敷料具备了低抗原性、低过敏性的特点。
本发明属于医用敷料领域,是一种非哺乳动物来源的异种皮敷料。本发明通过使用特异性的生物皮加工方法,以大鲵皮肤为来源制备的生物敷料可作为真皮替代品,为烧伤创面提供支持和保护。本发明的制备方法具有以下优点:
(1)大鲵的皮肤中含有大量的丝氨酸、苏氨酸、酪氨酸,本发明的取皮方法通过切取部分皮下脂肪层能够极大地减轻大鲵皮肤内重要氨基酸成分在制备过程中的流失;
(2)通过超低温快速冷冻的方法可以形成大量的胞外结晶,使大鲵皮肤真皮上层疏松的海绵层结构破坏,可以初步去除大鲵皮肤表皮生发层的细胞成分与多细胞腺体、色素细胞等成分,有利于表皮层与真皮致密层的分离;
(3)通过复合酶中链霉蛋白酶的使用,能水解真皮层的纤维迁连蛋白、黏蛋白;通过木瓜凝乳蛋白酶对皮肤的处理,能够分解精氨酸,有效去除皮肤真皮致密层的肥大细胞,极大地降低了材料的抗原性;
(4)通过使用过氧化氢复合漂白剂,能够有效杀灭细菌,而且去除残留的色素细胞,使材料更加安全、美观;
(5)通过冷冻保护液的孵育处理,能够保护敷料结构在低温保存时的完整性不被破坏,可以解冻后立即使用;
(6)本方法制备的敷料主要成分为I型胶原蛋白,并且通过特殊的酶解步骤保留了胶原蛋白的天然结构不被破坏,能够为细胞的迁移提供良好的支持;
(7)本发明制备的敷料主要成分为I型胶原蛋白,具有良好的生物相容性和生物可降解性,可以明显的促进细胞的增殖和血小板凝聚,促进创面细胞的粘附、趋化和分化。
附图说明
图1是实施例1制得的医用生物敷料的成品图。如图所见,产品为白色,无肉眼可见杂质,表面光滑,有微小孔状结构。
图2是实施例1制得的医用生物敷料经苏木精-伊红染色(HE)后的结果图,其中a、b分别为实施例1的两个不同样品结果。如图所见胶原纤维排列整齐,无残留细胞及细胞碎片,说明材料细胞脱除干净。
图3是实施例1制得的医用生物敷料正面的扫描电子显微镜照片,其中a、b、c、d分别是扫描倍数为5um、10um、100um、500um时的照片。从图片中可见胶原纤维上无细胞层覆盖,与HE观察结果一致,纤维层排列规律,胶原纤维保存完好,纤维键有孔隙存在。
图4是实施例1制得的医用生物敷料截面的扫描电子显微镜照片,其中a、b分别是扫描倍数为500um、50um时的照片。
具体实施方式
实施例1
(1)选取3岁,体重约1.5千克健康大鲵,使用麻醉安定后,清水洗净。使用取皮刀于大鲵躯干背侧进行取皮操作,刀片长度为8cm,去皮厚度约为1500μm,得到带有少量皮下脂肪的全厚皮片,将皮片置入4℃的PBS中浸泡5分钟,以洗去附着在皮肤上的游离脂肪、血液、体液等物质,重复浸泡3次后吸去多余水分备用。
(2)将皮片放入程序降温仪内,以5℃/分钟的降温速度,降至-80℃,放入超低温冰箱冷冻2小时。
(3)将冷冻的皮片取出,置于60℃纯化水中解冻,解冻后置于含有L-抗坏血酸50mM,青链霉素混合液500ppm的sPBS中浸泡60分钟,浸泡温度为4℃,重复3次。
(4)使用取皮刀将表皮150μm去除,制成厚度为1200mm的真皮皮片,将皮片置入含氢氧化钠20mg/mL,偏硅酸钠40mg/mL,碳酸钠20mg/mL的碱性清洗剂中浸泡30分钟,温度为4℃,再将皮片置入含有氯化钠125mM,氯化钾4mM,硫酸镁1mM,氯化镁1mM,磷酸氢二钠1mM,磷酸二氢钾1mM,碳酸氢钠5mM的缓冲液中浸泡30分钟,温度为4摄氏度,重复此步骤3次。
(6)将皮片置入含有谷氨酰胺2mM的Hank's平衡盐溶液中,室温摇床孵育20分钟,摇床速度为40rpm。
(7)将皮片置入含有十二烷基硫酸钠(SDS)0.5%的Hank's平衡盐溶液中,室温摇床孵育60分钟,摇床速度为40rpm。
(8)用生理盐水将皮片冲洗后,将皮片放入含有氯化钠125mM,氯化钾4mM,磷酸氢二钠1mM,葡萄糖7mM,EDTA 2mM,胰蛋白酶0.05g/L,链霉蛋白酶2g/L,木瓜凝乳蛋白酶1g/L的复合酶溶液中孵育18小时,孵育温度为20℃。
(9)将皮片用生理盐水清洗后,放入含有过氧化氢3%,氢氧化钠15g/L,EDTA 2mM的漂白液中漂白30分钟,温度为4℃。
(10)将皮片放入含葡聚糖7mM,蔗糖6mM,EDTA 1mM的冷冻保护液中,4℃孵育20分钟,放入医用包装袋内冷冻干燥保存。
(11)将制备好的敷料进行环氧乙烷灭菌5个循环。
实施例2
(1)选取4岁,体重约2千克健康大鲵,使用麻醉安定后,清水洗净。使用取皮刀于大鲵躯干背侧进行取皮操作,刀片长度为10cm,去皮厚度约为1600μm,得到带有少量皮下脂肪的全厚皮片,将皮片置入4℃的PBS中浸泡10分钟,重复3次后吸去多余水分备用。
(2)将皮片放入程序降温仪内,以0.5℃/分钟的降温速度,降至-80℃,放入超低温冰箱冷冻3小时。
(3)将冷冻的皮片取出,置于80℃纯化水中解冻,解冻后置于含有L-抗坏血酸200mM,青链霉素混合液500ppm的sPBS中浸泡90分钟,浸泡温度为2℃,重复1次。
(4)使用取皮刀将表皮200μm去除,制成厚度为1400mm的真皮皮片,将皮片置入含氢氧化钠25mg/mL,偏硅酸钠60mg/mL,碳酸钠30mg/mL的碱性清洗剂中浸泡30分钟,温度为4℃,再将皮片置入含有氯化钠150mM,氯化钾3mM,硫酸镁0.1mM,氯化镁1.5mM,磷酸氢二钠0.1mM,磷酸二氢钾1.5mM,碳酸氢钠2mM的缓冲液中浸泡30分钟,温度为4℃,重复此步骤2次。
(6)将皮片置入含有谷氨酰胺0.1mM的Hank's平衡盐溶液中,室温摇床孵育15分钟,摇床速度为20rpm。
(7)将皮片置入含有十二烷基硫酸钠(SDS)1%的Hank's平衡盐溶液中,室温摇床孵育30分钟,摇床速度为80rpm。
(8)用生理盐水将皮片冲洗后,将皮片放入含有氯化钠150mM,氯化钾3mM,磷酸氢二钠1.5mM,葡萄糖3mM,EDTA 3mM,胰蛋白酶0.01g/L,链霉蛋白酶5g/L,木瓜凝乳蛋白酶5g/L的复合酶溶液中孵育15小时,孵育温度为25℃。
(9)将皮片用生理盐水清洗后,放入含有过氧化氢1%,氢氧化钠5g/L,EDTA 3mM的漂白液中漂白60分钟,温度为2℃。
(10)将皮片放入含葡聚糖15mM,蔗糖2mM,EDTA 0.5mM的冷冻保护液中,4℃孵育30分钟,放入医用包装袋内冷冻干燥保存。
(11)将制备好的敷料进行环氧乙烷灭菌5个循环。
实施例3
(1)选取2岁,体重约1千克健康大鲵,使用麻醉安定后,清水洗净。使用取皮刀于大鲵躯干背侧进行取皮操作,刀片长度为7cm,去皮厚度约为1300μm,得到带有少量皮下脂肪的全厚皮片,将皮片置入4℃的PBS中浸泡12分钟,重复3次后吸去多余水分备用。
(2)将皮片放入程序降温仪内,以10℃/分钟的降温速度,降至-80℃,放入液氮中冷冻1小时。
(3)将冷冻的皮片取出,置于100℃纯化水中解冻,解冻后置于含有L-抗坏血酸10mM,青链霉素混合液500ppm的sPBS中浸泡30分钟,浸泡温度为8℃,重复4次。
(4)使用取皮刀将表皮100μm去除,制成厚度为1200mm的真皮皮片,将皮片置入含氢氧化钠15mg/mL,偏硅酸钠30mg/mL,碳酸钠15mg/mL的碱性清洗剂中浸泡90分钟,温度为2℃,再将皮片置入含有氯化钠100mM,氯化钾6mM,硫酸镁1.5mM,氯化镁0.1mM,磷酸氢二钠1.5mM,磷酸二氢钾0.1mM,碳酸氢钠6mM的缓冲液中浸泡30分钟,温度为4℃,重复此步骤2次。
(6)将皮片置入含有谷氨酰胺4mM的Hank's平衡盐溶液中,室温摇床孵育5分钟,摇床速度为80rpm。
(7)将皮片置入含有十二烷基硫酸钠(SDS)0.01%的Hank's平衡盐溶液中,室温摇床孵育90分钟,摇床速度为20rpm。
(8)用生理盐水将皮片冲洗后,将皮片放入含有氯化钠100mM,氯化钾6mM,磷酸氢二钠0.1mM,葡萄糖10mM,EDTA 0.5mM,胰蛋白酶5g/L,链霉蛋白酶0.01g/L,木瓜凝乳蛋白酶0.01g/L的复合酶溶液中孵育20小时,孵育温度为15℃。
(9)将皮片用生理盐水清洗后,放入含有过氧化氢5%,氢氧化钠25g/L,EDTA0.5mM的漂白液中漂白10分钟,温度为8℃。
(10)将皮片放入含葡聚糖5mM,蔗糖15mM,EDTA 3mM的冷冻保护液中,4℃孵育20分钟,放入医用包装袋内冷冻干燥保存。
(11)将制备好的敷料进行环氧乙烷灭菌5个循环。
Claims (12)
1.一种医用生物敷料的制备方法,其特征在于包括以下步骤:
(1)对大鲵皮肤进行剥离,得到全厚皮片;
(2)对所述全厚皮片进行超低温冷冻处理;
(3)解冻后在缓冲液I中浸泡清洗1-3次,缓冲液I的有效成分为:L-抗坏血酸10-200mM,青链霉素混合液500ppm,无菌磷酸盐缓冲盐水;
(4)去除清洗后全厚皮片的表皮层和外周的真皮疏松结缔组织,得到中间皮片;
(5)使用复合碱性清洗剂I和缓冲液II对中间皮片清洗1-3次,其中复合碱性清洗剂I的有效成分为:氢氧化钠15-25mg/mL,助剂为:偏硅酸钠30-60mg/mL,碳酸钠15-30mg/mL;
(6)使用表面活性剂处理清洗后的中间皮片;
(7)使用固定剂I和固定剂II对经步骤(6)处理的中间皮片进行固定处理,固定剂I有效成分为:含谷氨酰胺0.1-4mM的Hank's平衡盐溶液;
(8)使用复合酶对经步骤(7)处理的中间皮片的致密纤维层进行消化,复合酶的主要成分为:氯化钠100-150mM,氯化钾3-6mM,磷酸氢二钠0.1-1.5mM,葡萄糖3-10mM,EDTA 0.5-3mM,胰蛋白酶0.01-5g/L,链霉蛋白酶0.01-5g/L,木瓜凝乳蛋白酶0.01-5g/L;
(9)使用漂白剂对经步骤(8)处理的中间皮片进行漂白处理;
(10)将经步骤(9)处理的中间皮片浸泡于含有葡聚糖5-15mM、蔗糖2-10mM、EDTA 0.5-3mM的冷冻保护液中,冷冻保存,即得。
2.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(1)对大鲵皮肤剥离得到全厚皮片的方法为:对大鲵的皮肤进行初步清洗,去除表面分泌抗菌肽,使用取皮刀对大鲵躯干背侧区域进行取皮操作,取皮厚度为1300-1500μm,包含全部表皮层、真皮层及皮下少量脂肪,然后将取下的皮片置入4℃的PBS中浸泡5-12分钟,重复3次后吸去多余水分备用。
3.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(2)超低温冷冻处理的降温速度为0.5-10℃/分钟,冷冻温度为-80至-196℃,冷冻时间为1-3小时。
4.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(3)解冻的温度为60-100℃;所述浸泡清洗的温度为2-8℃,每次浸泡清洗时间为30-90分钟。
5.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(5)的缓冲液II有效成分为:氯化钠100-150mM,氯化钾3-6mM,硫酸镁0.1-1.5mM,氯化镁0.1-1.5mM,磷酸氢二钠0.1-1.5mM,磷酸二氢钾0.1-1.5mM,碳酸氢钠2-6Mm。
7.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(7)的固定剂II有效成分为:含十二烷基硫酸钠0.01-1%的Hank's平衡盐溶液。
8.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(7)的固定方法为:先将经步骤(6)处理的中间皮片放入固定剂I中,以20-80rpm的速度水平摇床孵育5-15分钟,倒去固定剂I,然后放入固定剂II中,以20-80rpm的速度摇床孵育30-90分钟,固定温度为15-25℃。
9.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(8)的消化温度为15-25℃,消化时间为15-20小时。
10.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(9)所使用的漂白剂的主要成分为:过氧化氢1-5%,氢氧化钠5-25g/L,EDTA 0.5-3mM。
11.根据权利要求1所述医用生物敷料的制备方法,其特征在于,所述步骤(9)的漂白处理时间为10-60分钟,漂白温度为2-8℃。
12.一种医用生物敷料,其特征在于,所述医用生物敷料由权利要求1-11中任一项所述方法制备而成。
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