CN105749322A - 含纳米银的胶原-壳聚糖支架 - Google Patents
含纳米银的胶原-壳聚糖支架 Download PDFInfo
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Abstract
本发明提供一种含纳米银的胶原?壳聚糖支架,通过纳米银与胶原?壳聚糖溶液混合均匀后一步法冻干并交联制备获得,其中纳米银的浓度范围为10ug/ml?50ug/ml,纳米银粒子的粒径为10?30nm。本发明支架不仅具有抗菌,促进创面愈合等作用,而且为创面提供了合理的纳米银缓释体系,为创面提供长期有效的药物缓释,保持创面的药物浓度均衡有效且不过剩。是一种新型医用敷料,实现预防和抵抗创面修复过程中的感染和炎症,促进局部组织更好的血管化。该新型敷料不仅拓展了纳米银的临床应用范围,还可进一步阐明纳米银促进伤口愈合的机制,为有效使用组织工程产品提供一种新的促进手段。
Description
技术领域
本发明属于医用敷料领域,具体涉及一种含纳米银的胶原-壳聚糖支架。
背景技术
随着大面积烧伤及难愈性创面治疗研究的逐步推进,组织工程皮肤的研发成为研究的热点,但这些产品往往缺乏抗菌性能,难以抵抗病原微生物的侵害。而感染对皮肤移植物的成活具有重要影响。目前,已经有较多方法,包括支架的改性(如在支架中引入细胞外基质(Pieper J S,van Wachem P B,van Luyn M J A,Brouwer L A,Hafmans T,Veerkamp J H,and van Kuppevelt T H:Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats.Biomaterials 2000;21:1689-99;Anderson C R,Ponce A M and Price R J:Immunohistochemical identification of an extracellular matrix scaffold that microguides capillary sprouting in vivo.J Histochem Cytochem2004;52:1063-72.)、改变支架结构、孔径(Gafni Y,Zilberman Y,Ophir Z,Abramovitch R,Jaffe M,Gazit Z,Domb A,and Gazit D:Design of a filamentous polymeric scaffold for in vivo guided angiogenesis.Tissue Eng 2006;12:3021-34;Druecke D,Langer S,Lamme E,Pieper J,Ugarkovic M,Steinau H U,and Homann H H:Neovascularization of poly(ether ester)block-copolymer scaffolds in vivo:long-term investigations using intravital fluorescent microscopy.J Biomed Mater Res A 2004;68:10-8.)等、添加抗感染的成分或药物(藻酸盐,银离子)等,来改善植入物的抗感染性能。但感染仍是组织工程皮肤替代物移植存活与否的关键问题之一,目前尚无有效的方法及途径改善组织工程产品移植后感染的问题。
目前创面外用药品种繁多,一般的外用药通常只具有单一的防止创面感染或促进创面愈合的作用,临床使用受到一定的限制。NAg敷料是采用纳米技术,将NAg超微颗粒加入医用常规敷料获得的一种新型抗感染敷料,可以加速烧伤创面的愈合,缩短住院时间,减少医疗费用,在耐药性、安全性、过敏性及促进创面愈合等方面效果显著(陈炯,韩春茂,张力成,and等:纳米银用于Ⅱ度烧伤创面的疗效及安全性评价.中华创伤杂志2009;25:451-455;王春莲,黄燕萍and董永盛:纳米银敷料与磺胺嘧啶锌软膏治疗烧伤创面的疗效观察.现代医院2010;10:46-47.),且无明显毒副作用(Thirumurugan G,Veni V S,Ramachandran S,Rao J V,and Dhanaraju M D:Superior wound healing effect of topically delivered silver nanoparticle formulation using eco-friendly potato plant pathogenic fungus:synthesis and characterization.J Biomed Nanotechnol 2011;7:659-66.)。
这些敷料主要是将纳米银或者银离子负载于敷料内部,创面敷料释放出来一定量的纳米银或者银离子维持创面的菌群平衡。但在这些方法里,敷料只起到了载体的作用,承载的纳米银很快会释放出来,通常会在创面渗液中突释出来发挥抗菌作用(Toy L W and Macera L:Evidence-based review of silver dressing use on chronic wounds.J Am Acad Nurse Pract 2011;23:183-92.)。由于银的高生物活性,释放到创面或者目标器官时,它会优先被创面渗液或组织中的相关蛋白、氯离子及基团捕获,导致银颗粒在某些部位高浓度异常聚集,虽然能有效参与抗菌,但这些过剩的银颗粒或者银离子将在表皮细胞、巨噬细胞、成纤维细胞内聚集,导致细胞的凋亡,最终导致创面的不愈合或者延迟愈合(Atiyeh B S,Costagliola M,Hayek S N,and Dibo S A:Effect of silver on burn wound infection control and healing:review of the literature.Burns 2007;33:139-48;Trop M,Novak M,Rodl S,Hellbom B,Kroell W,and Goessler W:Silver-coated dressing acticoat caused raised liver enzymes and argyria-like symptoms in burn patient.J Trauma 2006;60:648-52.)。也有相关报道称烧伤病人过量的使用纳米银相关产品会导致肝功能的下降,有轻微银中毒的表现(Trop M,Novak M,Rodl S,Hellbom B,Kroell W,and Goessler W:Silver-coated dressing acticoat caused raised liver enzymes and argyria-like symptoms in burn patient.J Trauma 2006;60:648-52.)。而且纳米银的快速释放将会导致敷料的频繁更换,这将给病人带来高昂的治疗费用。
总的来说,纳米银的确是有效的抗菌药物,但是无节制的使用或者无控制的释放势必会适得其反,对创面修复没有益处(Cho Lee A R,Leem H,Lee J,and Park K C:Reversal of silver sulfadiazine-impaired wound healing by epidermal growth factor.Biomaterials2005;26:4670-6;Mintz E M,George D E and Hsu S:Silver sulfadiazine therapy in widespread bullous disorders:potential for toxicity.Dermatol Online J 2008;14:19.)。因此,安全有效的使用纳米银需要有合理的缓释体系,为创面提供长期有效的药物缓释,保持创面的药物浓度均衡有效且不过剩。
发明内容
本发明的目的是提供一种含纳米银的胶原-壳聚糖支架(NAg-CCS),是通过纳米银与胶原-壳聚糖(CCS)溶液混合均匀后一步法冻干并交联制备成为目标支架,制备出具有抗菌、安全,促进伤口愈合功能且保持创面纳米银药物浓度均衡有效且不过剩的新型医用敷料。
本发明提供的支架采用以下技术方案制备获得:将牛跟腱来源的Ⅰ型胶原蛋白与壳聚糖按照9:1的质量比溶解到0.5mol/L的乙酸溶液中,以10ppm为目标浓度加入一定量到纳米银溶液后混匀4h,然后加入总质量的0.5%的戊二醛并混匀,得到质量分数为0.5%的含纳米银胶原- 壳聚糖溶液,将纳米银,胶原-壳聚糖溶液注入4cm×4cm的模具,于-20℃下冷冻2小时,然后冻干24小时得到厚度为2mm的多孔NAg-CCS支架。纳米银的浓度范围为10ug/ml-50ug/ml,纳米银粒子的粒径为10-30nm。
本发明提供一种新的纳米银缓释系统来规避上述普通纳米银敷料的不足,通过将纳米银与胶原-壳聚糖(脱乙酰甲壳素)溶液(CCS溶液)混合均匀后一步法冻干并交联制备成为目标支架,预防和抵抗创面修复过程中的感染和炎症,以促进局部组织更好的血管化。该新型敷料不仅拓展了纳米银的临床应用范围,还可进一步阐明纳米银促进伤口愈合的机制,为有效使用组织工程产品提供一种新的促进手段。
本发明具有以下优点:1)在CCS溶液中添加NAg颗粒可以明显抑制致病菌的生长,且在一定程度上提供了合理的缓释体系,为创面提供长期有效的药物缓释,保持创面的药物浓度均衡有效且不过剩。2)纳米银单体颗粒,与CCS溶液的简单的物理混合,会稳定颗粒的化学形态,并包裹颗粒,既可达到有效的缓释作用,也可以有效的降低其细胞毒性。3)磺胺嘧啶银一直是国内外公认的一种较好的创面抗感染用药,但它除Ag+外还含有磺胺嘧啶,具有一定的毒副反应,发明的支架保留了Ag+高效抗感染,促进创面愈合的优点,剔除了磺胺成分过敏和Ag+过度沉积的缺点。4)本发明的支架所需材料简单,无需复杂的技术要求和高端的设备需求,成本低,易于推广。5)本发明支架为创面提供了合理的纳米银缓释体系,为创面提供长期有效的药物缓释,保持创面的药物浓度均衡有效且不过剩。
附图说明
图1为NAg-CCS真皮支架制备流程示意图。
图2为左CCS和右NAg-CCS两组支架在电镜下的微观结构。
图3为两种支架在干湿两种状态下的杨氏模量。
图4为CCS(A)和NAg-CCS(B)两组支架的孔隙率测定。
图5为纳米银的在室温和37℃下的缓释实验。
图6为NAg-CCS的体外抗菌研究.A为对大肠杆菌的抗菌作用,B为对金黄色葡萄球菌的抗菌作用。(a)CCS组(b)CCS+0.001%纳米银(c)CCS+0.005%纳米银(d)CCS+0.01%纳米银。
图7为CCS和NAg-CCS支架与细胞相容性实验。
图8为NAg-CCS细胞粘附实验代表图。
图9为NNAg-CCS细胞粘附实验代表图细胞统计图。
图10为CCS(0ppm),Nag-CCS(10ppm)和#NAg-CCS(10ppm#)的CCK-8检测。
具体实施方式
本发明结合附图和实施例作进一步的描述。
实施例1 含纳米银的胶原-壳聚糖支架(NAg-CCS)的制备
将Ⅰ型胶原与壳聚糖按照9:1的质量比溶解到0.5mol/L的乙酸溶液中,加入一定量的纳米银溶液后混匀4H,然后加入0.5%的戊二醛并混匀,得到不同纳米银浓度的,质量分数为0.5%的胶原-壳聚糖溶液。将胶原-壳聚糖溶液,纳米银胶原-壳聚糖溶液注入4cm×4cm的模具,于-20℃下冷冻2小时,然后冻干24小时得到厚度为2mm的多孔NAg-CCS支架;用去离子水反复漂洗6次,每次10分钟,支架经二次冷冻-冻干得到交联后的CCS支架和NAg-CCS支架。上述交联后的支架经Co-60辐照消毒灭菌,备用(图1)。
实施例2 物理性能的评价
2.1微结构表征
实验方法:
将CCS与NAg-CCS根据实验要求制备成合适的扫描样品,经表面喷金后,置于XL30扫描电镜下分别观察上述两种支架的微观形貌特征。
实验结果:
CCS和NAg-CCS组支架的微观结构如图2所示。图2右是含纳米银的胶原壳聚糖支架表面纳米银的电镜下的形态,如图显示纳米银均匀分布在支架的内部及表面,被胶原包括的纳米银颗粒,其粒径(50-60纳米)大于原始的纳米银粒径(图2)。
2.2机械强度的测定
实验方法
CCS组及NAg-CCS组在干湿性状态下的机械强度用万能材料力学试验机(Instron,model5543,UK)进行测定。在进行测定之前,各组样品被制成4.0cm×1.0cm大小,每组样品分成两个亚组,每个亚组3个平行样品,其中一个亚组用于干性状态下的测定,另一个亚组的样品经室温下PBS浸泡1h后行机械强度的测定。测定条件为20±2℃at,速率为1mm/min。最后,杨氏模量(Young’s modulus)按照公式进行计算(Dai W,Kawazoe N,Lin X,Dong J,and Chen G:The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering.Biomaterials 2010;31:2141-52.)。
EM=(F/A0)/(ΔL/L0)
实验结果
弹性模量检测结果显示(图3),干性状态的的杨氏模量明显高于湿性状态。两种支架(NAg-CCS(a,b),CCS(c,d))在干湿两种状态下的杨氏模量,没有明显的区别,没有统计学差异(p>0.05)。
2.3支架的孔隙率测定
实验方法
裁取1cm×1cm的NAg-CCS及CCS支架,用游标卡尺精确测量支架的厚度,计算出支架的表观体积V,用分析天平称量支架的干重为W。将支架吸满无水乙醇,用滤纸吸去表面多余的液体,称重为Q,无水乙醇的密度记为ρ,支架的孔隙率按下公式来计算。重复6次。
实验结果
CCS(图4A)和NAg-CCS(图4B)支架在电镜下观察没有结构上的明显差异,孔径为136±5μm,孔隙率为93.6%,其微观结构十分相似。
2.4纳米银缓释实验
实验方法
采用蒸馏水作为释药介质,分别取直径为2cm的CCS支架(物理吸附等量的纳米银溶液,烘干机干燥),NAg-CCS支架,置于含100mlPBS的离心管中,37℃恒温恒速振荡,每隔24h高速离心后吸取释药介质上清300μl,放入的EP管中用于检测。离心管内加入蒸馏水300μl,使释放介质体积保持恒定。至无药物释放时,分别加入50单位胶原酶降解。采用电感耦合等离子体质谱仪(icp-ms、ELAN DRC-e PerkinElmer,美国)检测纳米银浓度。每组设3个平行样本。
实验结果
含纳米银的胶原壳聚糖支架分别在室温及37℃条件下对纳米银的缓释作用如图5所示。我们发现在前9天两组实验的纳米银浓度均稳定升高,37℃组的纳米银浓度在各时间点均高于室温组。在两组实验早期均没有出现纳米银的突释现象,1-6天两组中纳米银的浓度均稳定升高。9天时纳米银的释放量达最高值,即61.6%。9天以后纳米银的浓度逐渐降低,我们分析可能的原因是释放后的纳米银颗粒不稳定导致颗粒沉淀(图5)。15天时加入50单位/ml胶原酶降解CCS支架后,累计释放80.45±1.7%纳米银。
实施例3 NAg-CCS的体外抗菌研究
实验方法
将金黄色葡萄球菌及大肠杆菌种植于营养培养基上,CO2培养箱中培养24小时。将CCS及NAg-CCS经环氧乙烷灭菌后,切割成直径为1CM大小的圆片,备用。将该支架圆片用生理盐水湿润后平铺于培养皿中,培养24小时候观察抑菌圈的大小。
实验分组:实验组纳米银浓度分别为10,50,100ppm的NAg-CCS支架。
对照组:空白CCS支架。
实验结果
NAg-CCS的体外抗菌研究结果如图6所示。结果通过抑菌圈的大小来对比含不同浓度的纳米银已经与空白支架的对比来研究目标支架的抗菌性能。结果显示空白支架没有明显的抑菌圈,而含纳米银的支架的抑菌圈随着浓度的升高而增大。在大肠杆菌组当浓度为0.001%时其抑菌圈平均宽度为1mm,0.005%时其抑菌圈平均宽度为2mm,0.01%时平均宽度为2.5mm。而在金黄色葡萄球菌组,当浓度为0.001%时其抑菌圈平均宽度为0.5mm,0.005%时其抑菌圈平均宽度为2.5mm,0.01%时平均宽度为3mm。统计学分析结果显示,在纳米银对金黄色葡萄球菌及大肠埃希菌的抑制作用没有明显区别(p>0.05),尽管结果中我们发现纳米银对金黄色葡萄球菌的抑菌圈略大于大肠埃希菌。
实施例4 支架与细胞相容性实验
实验方法:
1)支架准备:
空白组支架:CCS支架;
实验组支架:NAg-CCS;
用12孔板模具制各组支架,每组取3个支架,实验前支架正反面各紫外线取灭菌30min。
2)支架上种植细胞
对数期生长的成纤维细胞经PBS洗涤,消化,用含0.5%FBS的DMEM培养基制成细胞悬液。经细胞记数,按每孔106/2ml接种与12孔板支架上。细胞在支架上培养3天和7天后采用荧光素异硫氰酸酯(FITC)+4,6-二脒基-2-苯基吲哚(DAPI)进行支架-细胞复合物的荧光双染色及HE染色。方法:将细胞与支架共培养的混合材料从培养基中取出,首先在PBS中清洗两次去除残留在支架中的培养基,然后放入4%的多聚甲醛中固定过夜,再用PBS多次清洗除去多聚甲醛,用100μg/mL DAPI于37℃下荧光标记成纤维细胞核30分钟,FITC染支架,然后再采用激光共聚焦显微镜(20倍)下观察支架中细胞的生长情况。
3)细胞粘附实验
细胞粘附实验室根据Mould等报道的实验稍作修改完成[26]。将支架置于24孔板中,室温下加入无血清样培养基孵育1小时,PBS漂洗。无菌滤纸将支架内部多余的水分充分吸干,备用。通过胰蛋白酶及EDTA将成纤维细胞重悬并计数,使细胞浓度在1×105cells/500μL。每孔滴入1ml细胞悬液,滴加时注意将细胞悬液充分吸入支架内部,然后加入1mlDMEM培养基,37℃孵育2小时。将孵育后的支架用PBS漂洗3次将未粘附的细胞洗脱,福尔马林1小时固定,石蜡包埋后切片做HE染色,具体步骤如下:
(1)切片烤片并脱蜡至水:将切片放入60℃恒温箱烘烤1h左右;二甲苯Ⅰ,10min;二甲苯Ⅱ,10min;100%乙醇Ⅰ,3min;100%乙醇Ⅱ,3min;95%乙醇,3min;85%乙醇,3min;75%乙醇,3min;自来水漂洗,5min;蒸馏水漂洗,3min。
(2)苏木精染液染色10min;
(3)流水稍微漂洗后,1%盐酸酒精分化3秒后,流水稍洗,返蓝液返蓝15min,蒸馏水洗3min;
(4)伊红染液染色3至5min;自来水洗5min;蒸馏水洗3min;
(5)常规脱水、透明、封片、观察:70%乙醇,3min;80%乙醇,3min;95%乙醇,3min;100%乙醇Ⅰ,3min;100%乙醇Ⅱ,3min;二甲苯Ⅰ,3min二甲苯Ⅱ,3min;中性树脂封片,显微镜下观察。
4)支架内细胞培养CCK-8实验
该实验分三组:空白CCS组,NAg-CCS组,用空白支架以物理方式吸附等量浓度的纳米银再冻干后的支架作为阳性对照组(#NAg-CCS)。将制备好的支架置于紫外线下照射1h;每孔加入3mLPBS,置于37℃温箱内2h,洗掉残余交联剂(无菌台内操作);吸除PBS,适当挤压,挤出支架内的PBS液;再用培养基浸泡2h,轻柔挤压,吸除培养基;将50μL的细胞悬液加到支架中心部位,4.6×104个/孔,置于37℃细胞培养箱内2h,再加入1mL含10%FBS的DMEM培养基;24小时后换液一次,以后每48h换液一次。分别在24h、48h、72h、5d、7d、10d,检测细胞活性:吸除培养基,加入500μL含10%CCK-8的无血清DMEM培养基(检测24小时时间点时,在加入CCK-8前先加入1mL含血清培养基清洗一次),置于37℃培养箱内培养2~4h(2.5h,4h),吸取100μL培养基于96孔板内450nm酶标仪测O.D.值。
5)统计学分析:数据采用SPSS16.0(USA)进行处理,计量资料采用均数士标准差表示,p<0.05认为有统计学意义。
实验结果
1)支架与细胞相容性实验
皮肤创伤和缺损均对造成不同程度的细胞变性、坏死和组织缺损,必须通过细胞增生和细胞基质的形成来进行修复。因此我们通过检测支架中成纤维细胞细胞的活性,以及负载有纳米银的CCS支架对成纤维细胞的影响。
细胞种植到支架中第三天和第七天的细胞形貌如图7所示,成纤维细胞在CCS支架上能良好的粘附、增殖,并向支架孔内生长。随着培养时间的延长,更多的细胞出现在支架中。两组CCS支架在3天时支架内细胞均较少。3天时纳米银组支架内细胞略少于空白组支架内细胞,比空白组细胞少4.38%,但没有明显统计学意义。7天时空白组支架内细胞增加明显,明显多余纳米银组。通过计数统计发现没有明显的差异。以上所有结果均表明,采用冷冻干 燥法制备的含纳米银的胶原壳聚糖支架具有良好的生物相容性,跟空白支架比较没有明显的抑制作用(p>0.05)。
2)细胞粘附实验
细胞粘附实验结果如图8所示。图8是粘附于支架上的细胞的HE图片,从图片上我们直观的发现,随着纳米银浓度的升高,细胞粘附的数量并没有明显的降低。为了进一步证实这个现象,我们对六组所有的粘附细胞做了计数并统计,统计结果如图9,结果显示在0-20μg/ml时各组之间没有明显的统计学差异(P>0.05),而50μg/ml的纳米银浓度组的支架细胞粘附要明显少空白支架(P<0.05)。说明在低浓度的纳米银对细胞在CCS支架上的粘附没有明显影响。
3)CCK-8检测
如图10所示,三种支架对成纤维细胞增殖作用与剂量和时间成负相关。在24h时三组支架对成纤维细胞的增殖影响没有明显差别。在36h和72h三组支架的毒性差异凸显出来。在两个时间点,以物理方式吸附的胶原壳聚糖对成纤维细胞的抑制作用较其他两组更加明显,统计学分析P<0.05。而NAg-CCS组与空白CCS组的Fb增殖没有明显的统计学差异(P>0.05)。该结果说明被胶原包裹的纳米银颗粒,其细胞毒性较原始的纳米银颗粒有所降低。
Claims (2)
1.一种含纳米银的胶原-壳聚糖支架,其特征在于,通过以下制备步骤实现:将牛Ⅰ型胶原蛋白与壳聚糖按照9:1的质量比溶解到0.5mol/L的乙酸溶液中,以10ppm为目标浓度加入到纳米银溶液后混匀4h,然后加入总质量的0.5%的戊二醛并混匀,得到质量分数为0.5%的含纳米银胶原-壳聚糖溶液,将纳米银,胶原-壳聚糖溶液注入4cm×4cm的模具,于-20℃下冷冻2小时,然后冻干24小时得到厚度为2mm的多孔胶原-壳聚糖支架。
2.根据权利要求1所述的一种含纳米银的胶原-壳聚糖支架,其特征在于,纳米银的浓度范围为10ug/ml-50ug/ml,纳米银粒子的粒径为10-30nm。
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