CN116082453A - 一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架 - Google Patents
一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架 Download PDFInfo
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- CN116082453A CN116082453A CN202310198944.3A CN202310198944A CN116082453A CN 116082453 A CN116082453 A CN 116082453A CN 202310198944 A CN202310198944 A CN 202310198944A CN 116082453 A CN116082453 A CN 116082453A
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- gelatin
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- bone defect
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Abstract
本发明涉及生物医学材料领域,具体涉及一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架。所述多肽G‑1,其氨基酸序列如下:Arg‑Gly‑Asp‑Gly‑Pro‑Leu‑Gly‑Met‑Arg‑Gly‑Asp。本发明还涉及含多肽G‑1的骨缺损修复材料,包括以下成分:明胶微球支架、明胶微球和多肽G‑1。该材料中的明胶微球支架和明胶微球之间通过具有酶切响应的多肽G‑1连接,在细菌感染的微环境中,细菌会分泌大量的明胶酶,当该微球支架植入骨感染缺损区域时,多肽中的酶切位点会被明胶酶特异性切割,明胶微球从支架表面释放出来,微球可以进一步装载药物或功能性生长因子作为缓释的载体来抗菌和成骨。
Description
技术领域
本发明涉及生物医学材料领域,具体涉及一种用于明胶酶酶切响应的多肽及含多肽的骨缺损修复支架。
背景技术
骨髓炎是由于微生物入侵或自身炎症过程而发生的,会严重损害骨组织的功能和再生能力。骨髓炎感染后容易造成骨缺损,不利于局部感染控制,这些缺损中的血肿形成又为细菌定植创造了机会。全身应用抗生素有助于减少感染;然而,局部缺血条件和“血-骨”屏障的存在会导致抗生素在感染部位渗透不良。临床对骨髓炎的治疗通常包括手术清创、坏死骨切除,以及随后的自体骨移植来填补骨缺损区域。
明胶/纳米羟基磷灰石复合材料是近几十年里迅速发展起来的一种较为理想的骨缺损修复材料,在组成上与人骨组织一致,具有良好的骨相容性、骨传导性等生物学特性。
其中:
明胶(gelatin)是一种天然聚合物,由胶原蛋白水解而来,具有促进细胞粘附的Arg-Gly-Asp(RGD)活性实体,与细胞外基质非常相似。明胶的优点包括:可与胶原相媲美的生物学性能,易加工成微球材料,凝胶过程没有剧烈的化学反应,可通过调节交联强度而控制降解速率,以及明胶分子链上丰富的功能基团使其容易进行化学改性。这些性能使明胶微球成为药物和蛋白的理想载体。
纳米羟基磷灰石(n-HA,Ca10(PO4)6(OH)2),是脊椎动物的骨骼和牙齿中的主要无机物成分,生物相容性好,化学稳定性高,降解后不产生有害物质,已被广泛用于骨组织修复。选择人工制备的羟基磷灰石晶体作为明胶微球的无机增强相制备有机-无机复合微球,可以使其具有明胶微球的可塑性和易加工特性,又具有羟基磷灰石颗粒的刚性和成骨性。
现有多肽产品如:FAM-AMP抗菌剂(201911017237.X,公开号为CN110669147A),或抗菌肽AMP(201911016699.X,公开号为CN110692651A),但是,这些抗菌剂都是单独或通过激光发挥作用,没有在微球或支架上负载。
研究发现当细菌感染宿主时,感染微环境凝固酶、磷脂酶、明胶酶、毒素等致病因子表达显著增高形成特殊的细菌感染微环境。基于此原因,为了将明胶微球支架和明胶微球进行连接并实现酶响应性释放,发明人还设计合成两端为RGD,中间具有明胶酶酶切位点的多肽,该多肽对感染微环境具有特异性响应,被设计用来连接微球支架和明胶/羟基磷灰石微球。在感染发生时,明胶酶表达,多肽被切割,微球可以进一步释放作为药物或功能性生长因子的负载及缓释的载体。
发明内容
为了达到上述技术目的,本发明具体通过以下技术方案实现。
第一方面,本发明提供了一种用于明胶酶酶切响应水解的多肽G-1。
一种用于明胶酶酶切响应水解的多肽G-1,其氨基酸序列如下:Arg-Gly-Asp-Gly-Pro-Leu-Gly-Met-Arg-Gly-Asp(缩写为RGD-G-P-L-G-M-RGD);
一种用于明胶酶酶切响应水解的多肽G-1的制备方法,其包括以下步骤:
1)称二氯树脂放入多肽合成仪中,加入DCM(二氯甲烷)溶胀半小时,然后去除DCM,加入序列中第一个氨基酸天冬氨酸Asp,及DIEA(N,N-二异丙基乙胺),DMF(N,N-二甲基甲酰胺)、DCM溶液,反应1个小时;
2)加入甲醇,反应半小时,抽掉反应液,用DMF、甲醇洗净。
3)加入哌啶去除Fmoc(9-芴甲氧羰基)保护基,洗净,取树脂用茚三酮检测为蓝色;
4)依照前面的方式依次加入序列中R-M-G-L-P-G-D-G-R氨基酸,直至最后一个氨基酸精氨酸Arg反应完成,用哌啶去除Fmoc,取树脂用茚三酮检测为蓝色,最后将树脂用甲醇洗干净并抽干。
RGD由精氨酸、甘氨酸和天冬氨酸组成的三肽(Arg-Gly-Asp),也是最常应用于种植体表面的一种生物活性短肽,是各种细胞外基质中最常见的基本结构部分,也是广泛存在于细胞识别系统的基本单位。所设计的G-1中细胞粘附RGD能够与细胞整合素结合并促进细胞粘附和扩散,该RGD修饰的肽表面与整合素的亲和相互作用在细胞粘附过程中起着至关重要的作用。此外多肽G-1对细菌的感染微环境具有特异性响应,这种具有模块化分子自组装策略的创新方法可能为组织工程中的多功能生物界面设计开辟了可能性。
第二方面,本发明提供了一种含多肽G-1的骨缺损修复材料,包括以下成分:明胶微球支架、明胶微球和多肽G-1。
具体的,骨缺损修复材料,包括以下配比的原料:1g明胶微球支架、150-300mg明胶微球和2-3mM多肽G-1。
所述明胶微球可以市售购买,也可以由以下方法制备:
1)将明胶与水混合,纳米羟基磷灰石与水混合,然后将明胶水溶液加入到纳米羟基磷灰石浆料中,制备得到8~15%明胶/纳米羟基磷灰石溶液;
2)将步骤1)得到的浆料,缓慢加入到40~50℃的油中,搅拌,然后边搅拌边冰浴冷却,再加入丙酮溶解,过滤,干燥,得到明胶微球。
上述明胶微球制备方法中:
所述步骤1)中:
所述明胶、羟基磷灰石的重量比是2-6:1。
所述明胶与水混合,制备成浓度为12-17%的明胶水溶液。以15%为例,1.5mg明胶溶于10ml水中,制得。
所述羟基磷灰石浆料,质量分数为0.265g/ml。可市售购买,或根据文献方法进行制备。
所述步骤2)中:所述油为橄榄油,在水油乳液法制备明胶/磷灰石微球中,橄榄油的使用更能增加所制备的微球的稳定性;并且橄榄油本身无毒,易被丙酮清洗掉,能够提高微球的生物相容性。
所述油、丙酮与明胶的含量分别为:80ml、90~140ml和1.0~2.0g;
搅拌速度为700rpm。
搅拌时间为10~20分钟。
冷却到5℃,然后保温25~35分钟;
丙酮分两次加入,先加入80~100ml,搅拌速度增加至1000rpm,搅拌10~20分钟,然后加入剩余丙酮,过滤。
所述明胶微球支架是由以下方法制备:将明胶微球加入到香草醛乙醇溶液中交联,然后丙酮清洗,过滤,干燥,再加入去离子水润湿后,干燥,得到明胶微球支架。
上述方法中:
所述香草醛乙醇溶液中香草醛和乙醇的重量体积比为1:8~12;
所述明胶微球与香草醛的质量比为1:0.8~1.2。
解释说明:香草醛交联的明胶-羟基磷灰石微球组装成微球支架,使其既具有明胶微球的可塑性和易加工特性,又具有无机羟基磷灰石颗粒的刚性和成骨性。香草素(4-羟基-3-甲氧基苯甲醛)是香兰豆提取物的主要成分,无毒,目前被广泛用作食品、饮料、化妆品和药品的调味剂。香兰素的醛基和明胶分子的氨基能形成席夫碱键,其动态重建通常在自组装过程中发生。一个香兰素分子含有一个醛基与明胶分子形成希夫碱键。需要注意的是,一个香草素分子的羟基也能够与另一个明胶分子中的羟基或氨基形成氢键,这为构建可逆杂化网络提供了基础。
本申请提供了一种含多肽G-1的骨缺损修复材料的制备方法,该方法包括以下步骤:将明胶微球和明胶微球支架在EDC/NHS中活化,再浸入多肽G-1中,用去离子水清洗,干燥,即得。
所述方法中:
EDC/NHS溶液的浓度为0.1w/v%;
加入多肽G-1和G-ctrl的浓度都为2.4mM。
所述EDC/NHS溶液中EDC和NHS的比例为1:0.8~1.2。
第四方面,本发明还提供了含多肽G-1的骨缺损修复材料在用于制备治疗骨髓炎的材料中的应用。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供了一种含多肽G-1的骨缺损修复材料,明胶微球支架和明胶微球之间通过具有酶切响应的多肽G-1连接,在细菌感染的微环境中,细菌会分泌大量的明胶酶,当该微球支架植入骨感染缺损区域时,多肽中的酶切位点会被明胶酶特异性切割,明胶微球从支架表面释放出来,微球可以进一步装载药物或功能性生长因子作为缓释的载体来抗菌和成骨。
2、本发明的明胶/纳米羟基磷灰石微球基骨修复支架原料配方简单,成分安全,使用多肽将微球支架和复合微球两者进行接枝,在感染微环境中,致病菌金黄色葡萄球菌大量表达明胶酶,酶切水解多肽,支架表面装载的复合微球释放出来,复合微球可进一步负载药物和生长因子进行局部给药用于抗菌。同时具有羟基磷灰石的明胶微球基支架为骨组织再生提供有利的多孔结构,具有良好的生物相容性、生物降解性,以及促进成骨和血管化功能,该骨修复支架最终能实现抗感染和修复骨缺损的双重功效。
3、与传统的骨髓炎治疗方法相比,本申请提供的骨缺损修复支架能够实现细菌感染微环境的响应治疗和感染性骨缺损的分级治疗,能够有效促进骨组织的再生重建,在临床上对于骨髓炎的治疗具有良好的应用前景。
4、本发明设计和拟采取的响应性酶切水解微球基骨修复支架治疗骨髓炎的策略,是适应国际生物医学材料发展趋势的前沿性科学探索,也符合国家医学发展战略需求,有望为今后临床骨髓炎治疗带来全新的实践方法。
附图说明
图1:酶切水解微球基骨修复支架的制备过程;
图2:多肽G-1两端羧基接枝支架和微球表面氨基形成酰胺键示意图;
图3:多肽接枝的明胶微球支架/明胶微球系统的前后对比;
图4:多肽接枝的明胶微球支架/明胶微球系统在细菌培养基中的响应性释放;
图5:多肽接枝的明胶微球支架/明胶微球系统对兔骨髓炎模型治疗8周后的X光图。
图3到图5中,涉及的G-1为多肽G-1,G-ctrl为杂乱肽G-ctrl。
具体实施方式
以下实施例用于说明本发明,但不限制本发明的范围。
明胶,本发明使用的明胶为A型(阿拉丁,美国);
纳米羟基磷灰石的浆料由实验室自己合成。
实施例1:多肽的制备
多肽G-1和杂乱肽G-ctrl由上海昕浩生物科技有限公司合成并纯化,其碱基序列分别为Arg GlyAsp Gly Pro Leu Gly MetArg GlyAsp(Seq_1)(缩写为RGD-G-P-L-G-M-RGD)和Arg GlyAsp Gly Pro Met Gly MetArg GlyAsp(Seq_2)(缩写为RGD-G-P-M-G-M-RGD)。
(一)多肽G-1的制备方法如下:
1、根据多肽序列长度、重量、纯度等情况,设计合成方法、步骤(合成顺序:从C端到N端)。
2、称取100mg的二氯树脂放入多肽合成仪中,加入5ml的DCM(二氯甲烷)溶胀半小时,然后抽掉DCM,加入4eq天冬氨酸Asp(序列中第一个氨基酸),及6eq DIEA(N,N-二异丙基乙胺),5ml DMF(N,N-二甲基甲酰胺)、5ml DCM溶液,反应1个小时。然后加入10ml甲醇,反应半小时,抽掉反应液,用DMF,甲醇洗净。
3、用哌啶去除Fmoc(9-芴甲氧羰基)保护基,洗净,取树酯用茚三酮检测为蓝色。
4、往多肽合成仪中加入4eq甘氨酸Gly(序列中第二个氨基酸),4eq的HBTU及6eq的DIEA,反应1个小时,抽掉液体,用DMF,甲醇洗净,取树酯用茚三酮检测为无色。
5、用哌啶去除Fmoc(9-芴甲氧羰基)保护基,洗净,取树酯用茚三酮检测为蓝色。
6、按照步骤3、4的方式依次加入序列中R-M-G-L-P-G-D-G-R氨基酸,直至最后一个氨基酸精氨酸Arg反应完成并用哌啶去除Fmoc,茚三酮检测为蓝色。
7、最后将树脂用甲醇洗干净并抽干,将树脂从合成仪中取下并倒入烧瓶中,然后往烧瓶中加一定量的TFA(三氟乙酸)切割液,震荡反应2h,将多肽从树脂载体上裂解下来并去除氨基酸的保护基团。收集滤液,然后向滤液中加入乙醚,离心,清洗即可得到该序列的粗品。
8、分析提纯和质谱检测:用ESI(电喷雾电离)离子源质谱仪检测该序列分子量的正确性;用HPLC液相色谱仪将粗品提纯至95%以上的纯度。收集纯化好的目标多肽(RGD-G-P-L-G-M-RGD)放入冻干机中进行冷冻,干燥成白色粉末。
样品冻干后再次进行HPLC和MS分析检测合格即可,HPLC分析方法流动相为含0.1%TFA的水和乙腈溶剂,检测波长为220nm。
(二)杂乱肽G-ctrl的制备方法:
如(一)方法合成,区别是步骤6中,引入的序列是R-M-G-M-P-G-D-G-R氨基酸。
实施例2:明胶微球及明胶微球支架的制备:
1、备料
纳米羟基磷灰石浆料的制备方法,是采用已知方法制备,或直接购买,或采用下面方法制备:是采用湿化学方法合成的,简而言之,将Na3PO4水溶液以1.67的Ca/P摩尔比滴入Ca(NO3)2水溶液中,两者在70℃下持续搅拌2小时,随后用NaOH溶液将pH值调节至约10。室温陈化,不断用去离子水反复洗涤沉淀,至pH约为7,离心收集磷灰石浆料。经冷冻干燥后得到磷灰石浆料的质量分数为0.265g/ml。
2、明胶/纳米羟基磷灰石微球的制备
1)15wt%明胶/纳米羟基磷灰石溶液的制备:
明胶溶液(15%,w/v)的制备:准确称取1.5g明胶溶于10ml 45℃的去离子水中得到浓度为15%的明胶溶液;
向10ml明胶溶液中加入600μL纳米羟基磷灰石浆料(159mg纳米羟基磷灰石),得到15wt%明胶/纳米羟基磷灰石溶液;
2)明胶/纳米羟基磷灰石微球的制备:
将200ml橄榄油加入圆底三颈瓶中并加热到45℃,然后向其中用注射器缓慢(1ml/min)滴入10ml浓度为15wt%的明胶/纳米羟基磷灰石混合溶液,保持温度45℃,并以700rpm的搅拌速度搅拌15分钟;在保持搅拌的情况下,将整个体系放入冰浴使整个体系冷却到4℃,保温30分钟后加入100ml丙酮,并保持在低温环境下以1000rpm的搅拌速度搅拌15分钟;再往整个体系中加入15ml丙酮后,用离心管对乳液进行分装后离心过滤,丙酮(30ml)多次洗涤,在50℃烘箱中烘干干燥,得到产物即为明胶/纳米羟基磷灰石微球(以下简称明胶微球)。
3、香草醛交联的明胶微球支架
1)将香兰素(又称香兰醛)10g溶于100ml无水乙醇得到10w/v%的香草醛/乙醇溶液;
2)称取1g烘干的明胶微球加入到10w/v%香草醛溶液中交联10h,交联后的微球用丙酮反复清洗离心过滤,放置50℃烘箱内烘干。
3)称取一定量烘干后的明胶微球,逐滴加入去离子水润湿,然后紧密填充到空心圆柱体(Φ=6mm)中,放入真空冷冻干燥机中干燥,将圆柱体中的支架取出得到的产物即为香草醛交联的明胶微球支架。
制备流程图见图1。
实施例3:多肽接枝明胶微球支架/明胶微球系统的制备:
1、多肽G-1(实施例1制备)粘接的微球支架:将1g明胶微球支架(实施例2制备)与200mg的明胶微球(实施例2制备)在EDC/NHS溶液中活化6h,用去离子水洗涤两次后,将支架和微球浸入2.4mM的多肽G-1溶液中6h,最后用去离子水轻轻洗掉支架表面未粘接的微球,真空干燥后,得到粘结了微球的多肽接枝明胶微球支架/明胶微球系统。
2、杂乱肽G-Ctrl粘接的微球支架:步骤同上,区别是杂乱肽G-Ctrl替换多肽G-1。
制备流程图见图2。
试验例1:微观变化
1、样本:明胶微球支架、多肽G-1和杂乱肽G-Ctrl粘接的微球支架。
2、观察方法:将微球支架放在体视镜下拍照,放大4倍观察。
3、结果:见图3
结果显示:经过多肽G-1和杂乱肽G-ctrl接枝的明胶微球支架其表面含有一层黄色的明胶微球,表明多肽G-1和杂乱肽G-ctrl接枝的微球均成功接枝。
试验例2:在细菌培养基中的响应性释放
1、实验菌株:
试验菌株革兰氏阳性菌金黄色葡萄球菌(S.aureus,CMCC26003)采购于上海保藏生物技术中心。
2、实验方法:
将6g蛋白胨置于锥形瓶中,加入200mL蒸馏水,超声至完全溶解后,制得液体培养基(即对照组培养基),压胶塞,高压灭菌(表压0.7kg/cm2,121℃,30min),取出放凉,备用。
用无菌接种环蘸取革兰氏阳性菌金黄色葡萄球菌菌液于液体培养基中37℃培养24h,得到金黄色葡萄球菌培养基,备用。
分别量取5ml对照组培养基或金黄色葡萄球菌培养基到无菌透明玻璃瓶中。
将多肽G-1接枝的明胶微球分别放入到装有对照组培养基或金黄色葡萄球菌培养基的玻璃瓶中,将杂乱肽G-Ctrl接枝的明胶微球放入到装有金黄色葡萄球菌培养基的玻璃瓶中两小时后观察拍照。
3、实验结果:见图4(多肽接枝的明胶微球支架/明胶微球系统在细菌培养基中的响应性释放)
多肽G-1的表现:多肽G-1接枝的明胶微球支架在对照组培养基中无法释放,溶液澄清;而在金黄色葡萄球菌培养基中释放出其表面接枝的微球,溶液变得浑浊;
杂乱肽G-Ctrl接枝的明胶微球也无法在金黄色葡萄球菌菌液中释放,溶液澄清。
4、结果分析:
金葡菌培养基中含有细菌分泌的明胶酶能够特异性切割多肽G-1,从而实现支架表面的微球响应性释放;杂乱肽G-ctrl不含有明胶酶切割位点,故无法实现微球的释放。
试验例3:对兔骨髓炎模型治疗8周后的X光图
1、实验动物:
新西兰大白兔2~3kg,雌雄比随机,由四川大学华西医院科技园提供,生产许可证号:SCXK(川)2015-030,使用许可证号:SYXK(川)2018-113)。
2、实验方法及分组:
细菌培养:将试验例2中接种的金黄色葡萄球菌菌株用接种环接种一部分到细菌培养基中,于37℃恒温培养16-18h,经测量OD值测得菌液浓度为1OD=6.0×108CFU/mL,PBS洗涤重悬后将菌液制备为1×108CFU/mL备用。
胫骨骨髓炎模型制作:所有动物适应性饲养14d,用3%戊巴比妥钠麻醉,在其右侧胫骨以金葡菌联合鱼肝油酸钠渗入骨窗的方法建立慢性骨髓炎模型。将兔右下肢手术部位去毛,取仰卧位,四肢固定于手术台上,常规消毒、铺巾,在右侧胫骨近端内侧纵行切口,逐层分离皮肤、皮下及筋膜,骨膜剥离器剥离骨膜,暴露胫骨前端上内侧。采用4mm直径的克氏针钻孔,在该处钻1个骨洞,直达髓腔,制成一个骨窗,刮除其内松质骨,修整骨洞边缘。向骨髓腔内依次注入鱼肝油酸钠(质量分数5%,0.1mL)、金葡菌(1×108CFU/mL,0.1mL)的金葡菌液后用骨蜡封闭骨窗,生理盐水冲洗伤口,逐层缝合组织和皮肤。
治疗:造模6周后,对骨髓炎建模处进行清创处理,对于不同的治疗,对照组使用万古霉素溶液反复清洗感染部位,微球支架G-1组用万古霉素溶液清洗后植入微球支架。最后,用骨蜡封住骨缺损,然后缝合伤口。在治疗后的第8周处死兔子收集胫骨并用X-Ray进行成像。
2、实验结果:见图5(多肽G-1接枝的明胶微球支架/明胶微球系统对兔骨髓炎模型治疗8周后的X光图)
从图5的X光图像可以看出:在兔胫骨骨髓炎模型中,对照组兔由于没有骨修复支架的植入,发生了骨折,出现了大量死骨;而实验组由于植入了多肽G-1接枝的明胶微球支架后炎症消失,骨缺损逐渐愈合。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种用于明胶酶酶切响应水解的多肽G-1,其氨基酸序列如下:Arg-Gly-Asp-Gly-Pro-Leu-Gly-Met-Arg-Gly-Asp。
2.一种含多肽G-1的骨缺损修复材料,包括以下成分:明胶微球支架、明胶微球和多肽G-1。
3.根据权利要求2所述的骨缺损修复材料,其特征在于,骨缺损修复材料包括以下配比的原料:1g明胶微球支架、150-300mg明胶微球和2-3mM多肽G-1。
4.根据权利要求3所述的骨缺损修复材料,其特征在于,所述步骤2)中的明胶微球,由以下方法制备:
1)将明胶与水混合,纳米羟基磷灰石与水混合,然后将明胶水溶液加入到纳米羟基磷灰石浆料中,制备得到8~15%明胶/纳米羟基磷灰石溶液;
2)将步骤1)得到的浆料,缓慢加入到40~50℃的油中,搅拌,然后边搅拌边冰浴冷却,再加入丙酮溶解,过滤,干燥,得到明胶微球。
5.根据权利要求3所述的骨缺损修复材料,其特征在于,所述步骤2)中的明胶微球支架,其由以下方法制备:将明胶微球加入到香草醛乙醇溶液中交联,然后丙酮清洗,过滤,干燥,再加入去离子水润湿后,干燥,得到明胶微球支架。
6.根据权利要求5所述的骨缺损修复材料,其特征在于,所述香草醛乙醇溶液中香草醛和乙醇的重量体积比为1:8~12。
7.根据权利要求5所述的骨缺损修复材料,其特征在于,明胶微球与香草醛的质量比为1:0.8~1.2。
8.一种含多肽G-1的骨缺损修复材料的制备方法,该方法包括以下步骤:
按照配比称取各成分,然后将明胶微球和明胶微球支架在EDC/NHS中活化,再浸入多肽G-1中,用去离子水清洗,干燥,即得。
9.根据权利要求8所述的制备方法,其特征在于,所述EDC/NHS溶液中EDC和NHS的比例为1:0.8~1.2。
10.含权利要求2-7任一项所述的多肽G-1的骨缺损修复材料在用于制备治疗骨髓炎的材料中的应用。
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