CN107233613B - 一种水生生物源交联胶原蛋白复合多层医用敷料 - Google Patents
一种水生生物源交联胶原蛋白复合多层医用敷料 Download PDFInfo
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- CN107233613B CN107233613B CN201710421775.XA CN201710421775A CN107233613B CN 107233613 B CN107233613 B CN 107233613B CN 201710421775 A CN201710421775 A CN 201710421775A CN 107233613 B CN107233613 B CN 107233613B
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Abstract
本发明公开了一种水生生物源高纯度高活性医用胶原蛋白，解决了天然胶原蛋白敷料的机械强度差、抗降解能力弱、伤口易受微生物感染的问题。采用酸提法结合胃蛋白酶去除端肽的方法提取胶原蛋白，通过超高压处理和组织捣碎的方法提高提取率，并通过脱细胞、除杂蛋白、脱脂、多次盐析、透析、除菌、脱色和除热源等一系列操作纯化，制备高纯度高活性医用胶原蛋白。通过真空冷冻干燥工艺制得孔隙均匀的网络状胶原蛋白海绵，再经物理交联和化学交联的方式对胶原蛋白海绵进行改性制备内层，然后与壳聚糖外层膜和医用无纺布基布层进行复合，最后经Co‑60灭菌后得到复合多层医用敷料成品。
Description
技术领域
本发明属于医用材料技术领域，具体涉及一种水生生物源交联胶原蛋白复合多层医用敷料。
背景技术
胶原蛋白是一种来源广泛、低免疫原性、生物相容性好的天然可降解生物大分子，其具有优良的止血性能，对凝血功能障碍的患者也具有良好的止血作用，且能够促进肉芽组织的再生和修复，能促进创伤愈合。多年来的临床实践表明，传统的物理、化学、机械的止血方法均存在着一定的局限性和不良反应，而诸如胶原蛋白类的天然高分子物质医用敷料是一种有效的外科止血方式。胶原蛋白可以适用于各种实质性脏器、体表和骨组织、血管组织以及结缔组织的止血。美国IMS公司对伤口敷料类产品进行市场调查，可吸收止血剂占据了整个敷料类市场份额的75％，而胶原蛋白基生物医用敷料是可吸收止血剂的很大组成部分。从1983年胶原止血海绵问世以来，将胶原蛋白作为一种可降解的止血、愈创材料基质的研究已成为国内外的热点。
目前，用于生物医用材料的胶原蛋白主要来源于陆生动物，例如猪皮、牛跟腱和鼠尾，而由于口蹄疫、疯牛病等疾病的传播和宗教信仰的限制，从水生生物中寻找医用胶原蛋白作为替代来源则变得越来越重要。目前国内广泛应用的止血、愈创医用敷料质量参差不齐，都有各自的不足之处，国外的医用敷料因其高昂的价格限制了其广泛应用。至今仍然没有一种理想的水生生物源胶原蛋白基止血、愈创医用敷料能够同时解决胶原蛋白的应用瓶颈。
发明内容
本发明的目的在于提供一种水生生物源交联胶原蛋白复合多层医用敷料，从而解决天然胶原蛋白敷料机械强度差、抗降解能力弱、伤口易受微生物感染的问题。
本发明首先提供一种改性胶原蛋白海绵，其制备方法如下：
1)高纯度高活性医用胶原蛋白的制备：
将鱼皮用刀片刮去鱼鳞、残肉和脂肪，冷水洗净，用切片机切成0.5–1cm2的小块。鱼皮经超高压处理以提高胶原蛋白的提取率，压力为100–300MPa，处理时间为5–20min。将鱼皮浸泡于0.5％–10％的Triton X-100的磷酸缓冲液中，置于振荡器上震荡12–48h，60–200rpm，4℃，在蒸馏水中震荡漂洗，再将鱼皮浸泡于1％–5％的SDS磷酸缓冲液中，置于振荡器上震荡12–48h，60–200rpm，4℃，在蒸馏水中震荡漂洗，重复上述步骤3次以脱除组织中的细胞；加入0.1M NaOH溶液浸泡除去非胶原成分及色素，蒸馏水反复洗至中性；加入1 10％正丁醇溶液搅拌去除脂肪，蒸馏水反复洗涤。使用组织捣碎机充分搅碎后，加入含0.5％(w/w)胃蛋白酶的0.5M醋酸中进行提取48h，离心(9,000–15,000rpm,30min，4℃)，所得上清液为胶原蛋白粗提液，再经研细的NaCl盐析，至溶液中NaCl最终浓度为0.9M，静置一夜，9,000rpm离心30min,收集沉淀溶于0.5M醋酸中，经二次盐析后的胶原蛋白复溶于0.5M醋酸中，溶液对0.02M的磷酸氢二钠溶液透析至pH≥8进行灭酶，再对蒸馏水透析除盐，直至透析外液用硝酸银溶液检测无氯离子即为透析终点；透析后的胶原蛋白溶液用0.22或0.45μm的板式膜进行除菌，最后用活性炭进行脱色，离心后去除活性炭获得高纯度高活性的医用胶原蛋白，低温真空冷冻干燥；
2)内层多孔胶原蛋白海绵的制备：
将步骤1)制备的高纯度高活性医用胶原蛋白溶于0.1M–0.5M的醋酸溶液中，配制成质量百分比浓度为0.5％–1％的胶原蛋白溶液，采用真空脱泡和低温低速离心(3,000–5,000rpm,5–10min，4℃)并用的脱泡方式脱泡，再将处理的溶液零下40℃预冷冻24h–36h，零下38℃冷冻干燥24h–48h制成厚度为3mm的内层多孔胶原蛋白海绵；
3)内层胶原蛋白海绵交联改性：
将步骤2)所制备的内层多孔胶原蛋白海绵进行改性处理，得到改性的内层胶原蛋白海绵；
所述的改性方法，其一种方法是将内层多孔胶原蛋白海绵置于真空干燥箱中，在常温下抽真空，然后升温至110℃–130℃，加热脱水1–3d完成改性；
其再一种的改性方法，是将胶原蛋白海绵放入等离子体处理仪的反应腔内，抽真空，待压强至50–200Pa时，在功率为80–120W下，放电处理5–10min完成改性；
再一种改性方法，将胶原蛋白海绵浸泡于化学交联剂进行交联，交联结束后除去残余交联剂后，低温真空冷冻干燥完成改性；
所述的化学交联剂，为戊二醛、碳二亚胺盐酸盐/羟基琥珀酰亚胺、京尼平交联、茶多酚、去甲二氢愈创木二酸或叠氮二苯基磷。
本发明所提供的改性胶原蛋白海绵用于制备医用敷料；
本发明再一个方法提供一种水生生物源交联胶原蛋白复合多层医用敷料，其制备方法如下：
1)外层壳聚糖膜的制备：
将脱乙酰度不小于80％的壳聚糖配制成质量分数为1％–20％的醋酸溶液，真空脱泡后，于50℃真空干燥制成厚度为2mm的膜；
2)内、外层点胶复合：
在步骤1)制备的外层壳聚糖膜的一侧涂布聚丙烯酸酯类生物胶，使改性胶原蛋白海绵和外层壳聚糖膜进行复合制成内外复合层；
3)复合多层敷料的制备：
在内外复合层上贴附上基布层，灭菌后制成复合多层敷料。
所述的基布层为医用无纺布，将无纺布的一侧涂布医用压敏热熔胶，使复合敷料的内外层与基布层复合。
本发明的优点和有益效果如下：
1.本发明制备的复合多层医用敷料的主要原料胶原蛋白来源广泛，由陆生动物变为水生生物，提高了鱼类下脚料的综合利用率，实现了水产品资源的高值化利用。
2.本发明中胶原蛋白的提取纯化工艺成熟，可以制备出高纯度高活性的具有完整三螺旋结构的I型医用胶原蛋白。
3.本发明中制备的内层胶原蛋白海绵色泽洁白，具有孔隙均匀的网络状结构，有利于组织渗出液的吸收并提高止血性能。
4.本发明中使用物理和化学交联的方法对胶原蛋白海绵进行改性，使其机械性能和抗酶降解能力显著提高。
5.本发明将胶原蛋白与壳聚糖复合，实现了复合材料的优势互补作用。
6.本发明制备的复合多层医用敷料兼具了内层胶原蛋白多孔海绵的止血、促进伤口愈合、吸收组织渗出液、促进细胞生长的功效和外层壳聚糖膜的抵御细菌侵入、保持伤口的湿润环境等功效以及无纺布基布层的固定和支撑作用，是一种新型的高性能的医用敷料，相比于传统医用敷料，其具有优良的止血效果，有利于促进伤口愈合，解决了单一胶原蛋白敷料机械强度差、抗降解能力弱、伤口易受微生物感染、易引起炎症反应等一系列问题。
附图说明
图1为实施例1制备的医用胶原蛋白的SDS-PAGE图谱。泳道1：蛋白质标准品；泳道2：加β-巯基乙醇；泳道3：不加β-巯基乙醇；泳道4：Sykes还原法；
图2为实施例1制备的医用胶原蛋白的傅里叶红外光谱图；
图3为实施例1制备的医用胶原蛋白的圆二色谱图；
图4为内层胶原蛋白海绵的表观图(A)和扫描电镜图：表面(B)、横截面(C)、纵截面(D)。
具体实施方式
下面通过具体实施例对本发明作进一步详述，以下实施例只是描述性的，不是限定性的，不能以此限定本发明的保护范围。
实施例1
水生生物源交联胶原蛋白复合多层医用敷料的制备方法，步骤如下：
(1)高纯度高活性医用胶原蛋白的制备：罗非鱼皮用刀片刮去鱼鳞、残肉和脂肪，冷水洗净，用切片机切成0.5–1cm2的小块。鱼皮经超高压处理以提高胶原蛋白的提取率，压力为200MPa，处理时间为5min。将鱼皮浸泡于2％的Triton X-100的磷酸缓冲液中，置于振荡器上震荡48h，100rpm，4℃，在蒸馏水中震荡漂洗，再将鱼皮浸泡于1％的SDS磷酸缓冲液中，置于振荡器上震荡48h，100rpm，4℃，在蒸馏水中震荡漂洗，重复上述步骤3次以脱除组织中的细胞。加入0.1M NaOH溶液(1:20,w/v)浸泡24h除去非胶原成分及色素，蒸馏水反复洗至中性。加入10倍体积10％正丁醇溶液搅拌48h，以去除脂肪，蒸馏水反复洗涤。使用组织捣碎机充分搅碎后，加入含0.5％(w/w)胃蛋白酶的0.5M醋酸(1:50,w/v)提取48h，离心(10,000rpm,30min，4℃)，所得上清液为胶原蛋白粗提液，再经研细的NaCl盐析，至溶液中NaCl最终浓度为0.9M，静置一夜，9,000rpm离心30min,收集沉淀溶于0.5M醋酸中，经二次盐析后的胶原蛋白复溶于0.5M醋酸中，溶液对0.02M的磷酸氢二钠溶液透析至pH≥8进行灭酶，再对蒸馏水透析除盐，直至透析外液用硝酸银溶液检测无氯离子即为透析终点。透析后的胶原蛋白溶液用0.22μm的板式膜进行除菌，最后经1％(w/v)的药用活性炭脱色和除热源，离心后去除活性炭获得高纯度高活性的医用胶原蛋白，低温真空冷冻干燥。
(2)内层多孔胶原蛋白海绵的制备：将上述高纯度高活性医用胶原蛋白溶于0.5M的醋酸溶液中，按质量百分比，配制成浓度为1％的胶原蛋白溶液，磁力搅拌器搅拌至完全溶解，采用真空脱泡和低温低速离心(5,000rpm,5min，4℃)并用的脱泡方式脱泡，倒入六孔板模具中，厚度为3mm，低温预冷冻24h，冷冻干燥48h。
(3)内层胶原蛋白海绵交联改性：将胶原蛋白海绵浸入磷酸盐缓冲液(0.054MNa2HPO4,0.013M NaH2PO4,pH 7.4)，加入GTA使其浓度为1％，于室温下交联3d，用蒸馏水冲洗，后用新配制的1.2mg/mL NaBH4磷酸盐缓冲液(pH 7.4)清洗60min，再用0.2M甘氨酸溶液处理24h，以处理未反应的醛基，最后用4M NaCl溶液冲洗60min，蒸馏水反复清洗。低温预冷冻24h，冷冻干燥48h。
所述的改性方法，其一种方法是将内层多孔胶原蛋白海绵置于真空干燥箱中，在常温下抽真空，然后升温至110℃–130℃，加热脱水1–3d完成改性；
其再一种的改性方法，是将胶原蛋白海绵放入等离子体处理仪的反应腔内，抽真空，待压强至50–200Pa时，在功率为80–120W下，放电处理5–10min完成改性；
再一种改性方法，将胶原蛋白海绵浸泡于化学交联剂进行交联，交联结束后除去残余交联剂后，低温真空冷冻干燥完成改性；
具体如下：
1、戊二醛(GTA)交联：将胶原蛋白海绵浸入磷酸盐缓冲液(0.054M Na2HPO4,0.013MNaH2PO4,pH 7.4)，加入GTA使其浓度为0.25％–2％，于室温下交联1–3d，用蒸馏水冲洗，后用新配制的1.2mg/mL NaBH4磷酸盐缓冲液(pH 7.4)清洗60min，再用0.2M甘氨酸溶液处理24h，以处理未反应的醛基，最后用4M NaCl溶液冲洗60min，蒸馏水反复清洗。
2、碳二亚胺盐酸盐/羟基琥珀酰亚胺(EDC/NHS)交联：将胶原蛋白海绵浸泡于pH为5.5的含50mM 2-N-吗啉已烷磺酸(MES)的40％乙醇中60min，再将其浸泡在含50mM MES、20–100mM EDC、8–40mM NHS(nEDC:nNHS＝2.5:1)的40％乙醇(pH 5.5)中，室温下交联2–24h，用0.1M Na2HPO4清洗，再分别用1M、2M、4M NaCl溶液清洗，最后用蒸馏水反复清洗。
3、京尼平交联：将胶原蛋白海绵浸入磷酸盐缓冲液(0.054M Na2HPO4,0.013MNaH2PO4,pH 7.4)或30％乙醇(pH 5.5)中，加入京尼平使其浓度为0.25％–2％，于室温下交联1–3d，用蒸馏水反复清洗。
4、茶多酚(TP)交联：将胶原蛋白海绵浸入含0.25％–2％的TP水溶液中，于室温下交联1–3d，用蒸馏水反复清洗。
5、去甲二氢愈创木二酸(NDGA)交联：将胶原蛋白海绵浸入0.1M磷酸盐缓冲液(pH7.0)30min，将NDGA加入到1mL 0.1M NaOH及9mL磷酸盐缓冲液中配制成浓度为1–5mg/mL的NDGA溶液，再将胶原蛋白海绵浸泡于上述NDGA溶液中，交联1–3d，用蒸馏水冲洗，后用70％乙醇浸泡，再用磷酸盐缓冲液浸泡，最后用蒸馏水反复清洗。
6、叠氮二苯基磷(DPPA)交联：将胶原蛋白海绵浸入含0.25％–2％DPPA的二甲基甲酰胺(DMF)溶液中，交联1–3d，用硼酸盐缓冲液(0.04M硼砂、0.04M硼酸，pH 8.9)清洗过夜，再用70％乙醇清洗，最后用蒸馏水反复清洗。
(4)外层壳聚糖膜的制备：将壳聚糖配制成质量分数为10％的醋酸溶液，真空脱泡，倒入六孔板模具中，厚度为2mm，于50℃真空干燥箱中烘干成膜。
(5)内、外层点胶复合：在上述外层壳聚糖膜的一侧涂布聚丙烯酸酯类生物胶，使内层胶原蛋白海绵和外层壳聚糖膜进行复合。
(6)复合多层敷料的制备：复合敷料的基布层为医用无纺布，将无纺布的一侧涂布医用压敏热熔胶，使复合敷料的内外层与基布层复合。
(7)包装、灭菌：交联胶原蛋白复合多层医用敷料包装后经Co-60灭菌。
实施例2
水生生物源交联胶原蛋白复合多层医用敷料的制备方法，步骤如下：
(1)高纯度高活性医用胶原蛋白的制备：鳕鱼皮用刀片刮去鱼鳞、残肉和脂肪，冷水洗净，用切片机切成0.5–1cm2的小块。鱼皮经超高压处理以提高胶原蛋白的提取率，压力为150MPa，处理时间为10min。将鱼皮浸泡于1％的Triton X-100的磷酸缓冲液中，置于振荡器上震荡48h，120rpm，4℃，在蒸馏水中震荡漂洗，再将鱼皮浸泡于1％的SDS磷酸缓冲液中，置于振荡器上震荡48h，120rpm，4℃，在蒸馏水中震荡漂洗，重复上述步骤3次以脱除组织中的细胞。加入0.1M NaOH溶液(1:20,w/v)浸泡24h除去非胶原成分及色素，蒸馏水反复洗至中性。加入10倍体积10％正丁醇溶液搅拌48h，以去除脂肪，蒸馏水反复洗涤。使用组织捣碎机充分搅碎后，加入含0.5％(w/w)胃蛋白酶的0.5M醋酸(1:60,w/v)提取48h，离心(12,000rpm,30min，4℃)，所得上清液为胶原蛋白粗提液，再经研细的NaCl盐析，至溶液中NaCl最终浓度为0.9M，静置一夜，9,000rpm离心30min,收集沉淀溶于0.5M醋酸中，经二次盐析后的胶原蛋白复溶于0.5M醋酸中，溶液对0.02M的磷酸氢二钠溶液透析至pH≥8进行灭酶，再对蒸馏水透析除盐，直至透析外液用硝酸银溶液检测无氯离子即为透析终点。透析后的胶原蛋白溶液用0.45μm的板式膜进行除菌，最后经2％(w/v)的药用活性炭脱色和除热源，离心后去除活性炭获得高纯度高活性的医用胶原蛋白，低温真空冷冻干燥。
(2)内层多孔胶原蛋白海绵的制备：将上述高纯度高活性医用胶原蛋白溶于0.5M的醋酸溶液中，按质量百分比，配制成浓度为0.8％的胶原蛋白溶液，磁力搅拌器搅拌至完全溶解，采用真空脱泡和低温低速离心(5,000rpm,8min，4℃)并用的脱泡方式脱泡，倒入六孔板模具中，厚度为3mm，低温预冷冻24h，冷冻干燥36h。
(3)内层胶原蛋白海绵交联改性：将胶原蛋白海绵浸泡于pH为5.5的含50mM 2-N-吗啉已烷磺酸(MES)的40％乙醇中60min，再将其浸泡在含50mM MES、100mM EDC、40mM NHS的40％乙醇(pH 5.5)中，室温下交联4h，用0.1M Na2HPO4清洗，再分别用1M、2M、4M NaCl溶液清洗，最后用蒸馏水反复清洗。低温预冷冻24h，冷冻干燥36h。
(4)外层壳聚糖膜的制备：将壳聚糖配制成质量分数为5％的醋酸溶液，真空脱泡，倒入六孔板模具中，厚度为2mm，于50℃真空干燥箱中烘干成膜。
(5)内、外层点胶复合：在上述外层壳聚糖膜的一侧涂布聚丙烯酸酯类生物胶，使内层胶原蛋白海绵和外层壳聚糖膜进行复合。
(6)复合多层敷料的制备：复合敷料的基布层为医用无纺布，将无纺布的一侧涂布医用压敏热熔胶，使复合敷料的内外层与基布层复合。
(7)包装、灭菌：交联胶原蛋白复合多层医用敷料包装后经Co-60灭菌。
本发明通过控制胶原蛋白的提取纯化工艺，得到了高纯度高活性的医用胶原蛋白。SDS-PAGE图谱(图1)显示实施例中制备的胶原蛋白的α链组成为(α1)2α2，傅里叶红外光谱图(图2)显示其具有胶原蛋白的各伸缩振动峰，圆二色谱图(图3)表明其在221nm处有一正吸收峰，在196nm处有一负吸收峰，可判定提取的胶原蛋白为具有完整三螺旋结构的I型胶原蛋白。
内层胶原蛋白海绵色泽洁白，具有孔隙均匀的网络状结构，孔隙率≥98％，有利于组织渗出液的吸收(图4)。
同时，本发明对未改性和改性的胶原蛋白海绵的特性进行了分析。
试验和测试部分：
抗张强度的测定：参照GB/T 1040.3-2006，将胶原蛋白海绵裁成适合的大小，两端固定在质构仪上，设置参数如下：速度为60mm/min；标距为10mm；力量为300N。抗张强度TS＝F/S×1000，其中F为样品断裂时承受的最大张力(N)；S为样品的横截面积(mm2)。
液体吸收性的测定：参照YY/T 0471.1-2004，将已知质量(Wd)的胶原蛋白海绵样品浸润在预热至37℃的试验液中，并在37℃水浴中孵育5h，用钝头镊子夹持样品一端，悬垂30s后称重(Ww)，液体吸收量＝(Ww-Wd)/Wd。其中，试验液为由氯化钠和氯化钙的溶液组成，该溶液含142mM Na+和2.5mM Ca2+。根据EN 13726-1，该溶液的离子含量相当于人体血清或创面渗出液。
酶降解性的测定：将胶原蛋白海绵裁成适当大小，精确称重，浸润在1mL含50mMCaCl2的Tris-HCl溶液(0.1M,pH 7.4)中，在37℃水浴1h，然后添加200U细菌胶原酶，溶解于1mL的0.1M Tris-HCl溶液中，37℃孵育24h。通过添加0.2mL的0.25M EDTA溶液并在冰中迅速冷却来结束酶的消化。随后，混合物在5000rpm、4℃下离心15min，上清液用于测定羟脯氨酸含量。
实验结果：
对胶原蛋白海绵进行改性处理，机械强度、液体吸收性和抗酶降解能力均显著提高，具体数据如表1所示。
表1改性对胶原蛋白海绵机械性能、液体吸收性和抗酶降解能力的影响
采用本发明制备的医用级鱼皮胶原蛋白，可充分有效的去除鱼皮的非胶原成分，获得高活性高纯度的胶原蛋白，提高了鱼类下脚料的综合利用率，实现了水产品资源的高值化利用，并且溶血率为1％–3％，具有良好的生物相容性。
同时，制备的复合多层医用敷料兼具了内层胶原蛋白多孔海绵的止血、促进伤口愈合、吸收组织渗出液、促进细胞生长的功效和外层壳聚糖膜的抵御细菌侵入、保持伤口的湿润环境等功效以及无纺布基布层的固定和支撑作用，是一种新型的高性能的医用敷料，解决了单一胶原蛋白敷料机械强度差、抗降解能力弱、伤口易受微生物感染、易引起炎症反应等一系列问题。
上述实施例仅是为了更清楚地说明而作的举例，并非是对实施方式的限定。对于本领域的普通技术人员来说，在不脱离本发明原理的情况下还可以在上述基础上做出变化或替换，仍处于本发明创造的保护范围之内。
Claims (4)
1.一种水生生物源交联胶原蛋白复合多层医用敷料，其特征在于，所述的敷料的制备方法如下：
1）外层壳聚糖膜的制备：
将脱乙酰度不小于80%的壳聚糖配制成质量分数为1%–20%的醋酸溶液，真空脱泡后，于50℃真空干燥制成厚度为2mm的膜；
2）内、外层点胶复合：
在步骤1）制备的外层壳聚糖膜的一侧涂布聚丙烯酸酯类生物胶，使改性胶原蛋白海绵和外层壳聚糖膜进行复合制成内外复合层；
3）复合多层敷料的制备：
在内外复合层上贴附上基布层，灭菌后制成复合多层敷料；
所述的改性胶原蛋白海绵的制备步骤如下：
1）高纯度高活性医用胶原蛋白的制备：
将鱼皮用刀片刮去鱼鳞、残肉和脂肪，洗净后切成小块；鱼皮经超高压处理后浸泡于0.5%–10%的Triton X-100的磷酸缓冲液中，置于振荡器上震荡12–48h，60–200rpm，4℃，在蒸馏水中震荡漂洗，再将鱼皮浸泡于1%–5%的SDS磷酸缓冲液中，置于振荡器上震荡12–48h，60–200rpm，4℃，在蒸馏水中震荡漂洗脱除组织中的细胞；加入0.1M NaOH溶液浸泡除去非胶原成分及色素，蒸馏水反复洗至中性；加入正丁醇溶液搅拌去除脂肪，蒸馏水反复洗涤；使用组织捣碎机充分搅碎后，加入含胃蛋白酶的醋酸中进行提取，离心得上清液为胶原蛋白粗提液，再经的NaCl盐析，至溶液中NaCl最终浓度为0.9M，静置后，9,000rpm离心30min,收集沉淀溶于0.5M醋酸中，经二次盐析后的胶原蛋白复溶于0.5M醋酸中，溶液用0.02M的磷酸氢二钠溶液透析至pH≥8进行灭酶，再用蒸馏水透析除盐，直至透析外液用硝酸银溶液检测无氯离子即为透析终点；透析后的胶原蛋白溶液用0.22或0.45μm的板式膜进行除菌，最后用活性炭进行脱色，离心后去除活性炭获得胶原蛋白，低温真空冷冻干燥；
2）内层多孔胶原蛋白海绵的制备：
将步骤1）制备的胶原蛋白溶于0.1M–0.5M的醋酸溶液中，配制成质量百分比浓度为0.5%–1%的胶原蛋白溶液，采用真空脱泡和低温低速离心并用的脱泡方式脱泡，再将处理的溶液零下40℃预冷冻24h–36h，零下38℃冷冻干燥24h–48h制成厚度为3mm的内层多孔胶原蛋白海绵；
3）内层胶原蛋白海绵交联改性：
将步骤2）所制备的内层多孔胶原蛋白海绵进行改性处理，得到改性的内层胶原蛋白海绵；
其中改性处理，其一种方式是将内层多孔胶原蛋白海绵置于真空干燥箱中，在常温下抽真空，然后升温至110℃–130℃，加热脱水1–3d完成改性；
或是将胶原蛋白海绵放入等离子体处理仪的反应腔内，抽真空，待压强至50–200Pa时，在功率为80–120W下，放电处理5–10min完成改性；
或是将胶原蛋白海绵浸泡于化学交联剂进行交联，交联结束后除去残余交联剂后，低温真空冷冻干燥完成改性。
2.如权利要求1所述的水生生物源交联胶原蛋白复合多层医用敷料，其特征在于，所述的低温低速离心，是在4℃，3,000–5,000rpm离心5–10min。
3.如权利要求1所述的水生生物源交联胶原蛋白复合多层医用敷料，其特征在于，所述的化学交联剂，为戊二醛、碳二亚胺盐酸盐/羟基琥珀酰亚胺、京尼平、茶多酚、去甲二氢愈创木二酸或叠氮二苯基磷。
4.如权利要求1所述的水生生物源交联胶原蛋白复合多层医用敷料，其特征在于，所述的基布层为医用无纺布，将无纺布的一侧涂布医用压敏热熔胶，使复合敷料的内外层与基布层复合。
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