CN105935317A - 一种表面载药缓释颌面种植经皮基台及其制备方法 - Google Patents

一种表面载药缓释颌面种植经皮基台及其制备方法 Download PDF

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CN105935317A
CN105935317A CN201610207011.6A CN201610207011A CN105935317A CN 105935317 A CN105935317 A CN 105935317A CN 201610207011 A CN201610207011 A CN 201610207011A CN 105935317 A CN105935317 A CN 105935317A
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魏洪波
赵铱民
李德华
王嘉
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Fourth Military Medical University FMMU
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Abstract

本发明公开了一种表面载药缓释颌面种植经皮基台及其制备方法,属于颌面种植材料技术领域。首先在基台表面制备纳米管涂层,另外制备介孔二氧化硅纳米颗粒(MSN),并在MSN三维孔道内加载结缔组织生长因子(CTGF);然后,将加载有CTGF的MSN装载入基台表面的纳米管管道中,建立复合缓释涂层,长期缓释CTGF,有望有效的促进经皮部位软组织与经皮种植基台之间的生物学结合的形成,从而能抵御细菌和外界刺激对种植体的侵袭,降低经皮种植体的失败率。本发明能够有效促进经皮部位软组织与经皮种植基台之间的生物学结合的形成,从而能抵御细菌和外界刺激对种植体的侵袭,降低经皮种植体的失败率。

Description

一种表面载药缓释颌面种植经皮基台及其制备方法
技术领域
本发明属于颌面种植材料技术领域,具体涉及一种表面载药缓释颌面种植经皮基台及其制备方法。
背景技术
口腔颅颌面种植技术是提高病人生活质量的一种高科技技术。口腔颅颌面种植技术,是处置口腔颅颌面缺损修复的技术。经过外科手术将和人体有生物相容性的种植体植入人体口腔缺牙部位的颌骨内或颅面缺损部位的骨组织内,作为野生牙根或颅面假体的固位体,中止口腔牙颌及颅面缺损修复。
目前,大量的颌面缺损患者需要采用赝复体来恢复容貌和生理功能。随着种植技术的发展,颌面种植体固位已成为赝复体的首选固位技术。现在国内外颌面种植经皮基台表面均采用光滑处理,颌面部软组织无法像骨组织那样与经皮种植基台形成紧密的生物学结合,结合部位易遭受细菌和外界刺激的侵袭,导致颌面经皮种植体失败率非常高。文献显示:44.4%有放疗史的患者和8.3%无放射史的患者遭遇了颌面经皮种植体的失败,需要拔出失败的种植体。
发明内容
为了克服上述现有技术的缺陷,本发明的目的在于提供一种表面载药缓释颌面种植经皮基台及其制备方法,能够有效促进经皮部位软组织与经皮种植基台之间的生物学结合的形成,从而能抵御细菌和外界刺激对种植体的侵袭,降低经皮种植体的失败率。
本发明是通过以下技术方案来实现:
一种表面载药缓释颌面种植经皮基台的制备方法,包括以下步骤:
1)制备二氧化钛纳米管涂层
采用阳极氧化法在基台表面制备二氧化钛纳米管涂层,然后经超声清洗、 真空干燥及射线消毒处理,备用;
2)制备介孔二氧化硅纳米颗粒
将无水乙醇和质量分数为25%的氨水按照(50~150):(3~5)的体积比均匀混合,得混合液,向混合液中加入正硅酸乙酯,搅拌反应24h后,清洗,然后在-80℃下预冻24h后再进行冷冻干燥,制得介孔二氧化硅纳米颗粒;
3)在介孔二氧化硅纳米颗粒中加载CTGF
采用浸泡法,将步骤2)制得的介孔二氧化硅纳米颗粒浸泡入CTGF溶液中,浸泡时间为24h;
4)将加载CTGF的介孔二氧化硅纳米颗粒装载入基台表面的二氧化钛纳米管管道中
首先,用PBS将步骤3)制得的加载CTGF的介孔二氧化硅纳米颗粒进行分装,配制成1~3μg/μL的母液;然后,将该1~3μg/μL的母液稀释成1~10ng/μL的工作液;再次,在消毒好的基台表面滴加工作液,使工作液铺匀基台表面;最后,将滴加有工作液的基台置于无菌冻干机中,温度设置为室温,真空干燥1~2h;
5)重复步骤4),直至试样加载到所需的量,制备得到表面载药缓释颌面种植经皮基台。
步骤1)中,所述基台是由纯钛加工而成,并经抛光、清洗及真空干燥处理;清洗是依次用丙酮、无水乙醇及去离子水进行超声清洗。
步骤2)中,向混合液中加入的正硅酸乙酯的量是按照每3~5mL的氨水加入100~300μL正硅酸乙酯。
步骤2)中,清洗是采用去离子水清洗三遍。
步骤3)中,CTGF溶液的浓度为1~3μg/mL。
步骤4)中,在基台表面滴加5-50μL的工作液。
步骤1)所述的采用阳极氧化法在基台表面制备二氧化钛纳米管涂层,具 体操作为:配制1~2mol/L H3PO4以及0.3%HF的混合电解液,置入电磁搅拌仪中持续搅拌,取干燥后的基台放入上述混合电解液中,与阳极相连,铂为阴极,在电压为10~25V的条件下持续搅拌1~3h,在试样表面生成二氧化钛纳米管涂层。
本发明还公开了采用上述公开的方法制得的表面载药缓释颌面种植经皮基台。
与现有技术相比,本发明具有以下有益的技术效果:
本发明公开的表面载药缓释颌面种植经皮基台的制备方法,首先在基台表面制备纳米管涂层,另外制备介孔二氧化硅纳米颗粒(MSN),并在MSN三维孔道内加载结缔组织生长因子(CTGF);然后,将加载有CTGF的MSN装载入基台表面的纳米管管道中,建立复合缓释涂层,长期缓释CTGF,有望有效的促进经皮部位软组织与经皮种植基台之间的生物学结合的形成,从而能抵御细菌和外界刺激对种植体的侵袭,降低经皮种植体的失败率。本发明能够有效促进经皮部位软组织与经皮种植基台之间的生物学结合的形成,从而能抵御细菌和外界刺激对种植体的侵袭,降低经皮种植体的失败率。
经本发明方法制得的表面载药缓释颌面种植经皮基台,实现了促进软组织与颌面种植体经皮基台生物学结合的目的。涂层体外缓释实验证实CTGF可缓释数十天,细胞学实验证实成纤维细胞的早期粘附、增殖均得到促进。动物实验证实此类经皮基台在1年的观察期内,种植体周围炎的发生率得到有效降低。
附图说明
图1为本发明制得的表面载药缓释颌面种植经皮基台表面促进了成纤维细胞在钛表面的增殖生长曲线图;
图2为本发明制得的表面载药缓释颌面种植经皮基台表面促进了成纤维细胞在钛表面的细胞粘附结果图;
图3为评价动物实验软组织评分结果图;
图4为本发明方法流程图。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
参见图4,本发明公开的表面载药缓释颌面种植经皮基台的制备方法,包括以下步骤:
(1)制备二氧化钛纳米管涂层
①选用纯钛加工成基台基体,抛光,依次用丙酮、无水乙醇及去离子水超声清洗,真空干燥。
②采用阳极氧化的方法在基台表面制备二氧化钛纳米管涂层,超声清洗,真空干燥,射线消毒。
阳极氧化方法:配制1~2mol/L H3PO4以及0.3%HF的混合电解液,置入电磁搅拌仪中持续搅拌。取干燥后的基台放入上述混合电解液中,与阳极相连,铂为阴极,在10~25V电压、持续搅拌的条件下进行1~3h的阳极氧化处理,在试样表面生成二氧化钛纳米管。试样制备完成后依次用丙酮、无水乙醇及去离子水进行15min超声清洗,干燥后备用。制备好的试样称为二氧化钛纳米管试样。
(2)制备MSN:取50~150mL无水乙醇,向其中加入3~5mL的质量分数为25%的氨水,均匀混合。用移液器吸取100~300μL正硅酸乙酯,对溶液进行剧烈搅拌,反应24h。去离子水清洗3遍。-80℃预冻24h后冷冻干燥。
(3)在MSN三维孔道中加载CTGF:使用浸泡法将MSN浸泡入高浓度的CTGF溶液(1~3μg/Ml)中,浸泡时间为24小时。
(4)使用简易冻干法将加载有CTGF的MSN装载入基台表面的纳米管管道中。具体方法是:
①用PBS将步骤3)制得的加载CTGF的介孔二氧化硅纳米颗粒进行分装, 配制成1~3μg/μl的母液;
②将该1~3μg/μl的母液稀释成1~10ng/μl的工作液;
③在无菌超净台上,在消毒好基台表面用移液管滴加5~50微升工作液,尽量铺匀整个表面。(上述过程需快速完成。)
④然后将试样放入无菌冻干机中,将温度设定为室温,抽真空,干燥1-2h。
⑤重复上述加载步骤,直到试样加载到需要的量。
实验方法:
1、细胞增殖使用MTT法
对成纤维细胞在三种不同表面(光滑表面,二氧化钛纳米管表面,纳米管载药复合涂层表面(与本专利所述基台表面处理一样)细胞增殖能力的测试选取了MTT法。所有试样采用丙酮2h,无水乙醇1h,双蒸水2h依次超声清洗。在细胞培养前采用Co60照射消毒试样。
(1)将MTT粉用PBS溶解配置5mg/ml溶液,过滤备用。
(2)成纤维细胞铺满瓶底约80%时,于室温条件用0.25%胰蛋白酶溶消化、离心、稀释后,将细胞接种于放有试样的48孔板中的材料表面,细胞的接种浓度常规为1500/孔,加入DMEM细胞培养液继续培养。
(3)将培养1,3,5天后的细胞培养孔里加入MTT溶液50μl/孔,4h后终止培养,去除上清液,加DMSO 500μl/孔,振荡10min。
(4)用酶联免疫仪在490nm处测吸光度OD值。
2、细胞粘附使用DAPI染色荧光显微镜下计数法
通过计算单位面积中粘附的细胞个数,即细胞的密度来反映细胞的粘附数量。
(1)细胞接种及培养
(i)制备细胞悬液:成纤维细胞铺满瓶底约80%时,于室温条件用0.25%胰蛋白酶消化离心稀释后,制备成50000/ml悬液。
(ii)将细胞接种于安放有试样的48孔板中的材料表面,细胞的接种浓度常规为3500/孔。
(iii)加入DMEM细胞培养液在37℃下继续培养2,24小时。
(2)细胞固定:
(i)配置细胞固定液:取多聚甲醛40g,加入1000mlPBS中加热至60℃搅拌至透明。用NaOH调PH至7.3,加PBS定容至1000ml。
(ii)将达到培养时间的孔板中的培养液去除,用PBS将未粘附的细胞冲洗干净,冲洗3次。
(iii)在孔板中加入固定液200ul,固定15分钟。PBS冲洗3次
(3)DAPI染色:
(i)室温避光下滴加200ulDAPI溶液。
(ii)37℃避光孵育5分钟,PBS中洗3次
(iii)用50%甘油PBS把试样固定在盖玻片上。
(4)荧光显微镜观察拍照。在10倍物镜10倍目镜下在每个试样表面随机选取5个区域,拍摄荧光照片。
(5)计算每块区域单位面积中的细胞密度。
(6)统计学分析使用SPSS12.0软件进行数据分析,ANOVA和配对t检验联用进行组间比较,检验水准P取α=0.05,n=5。
图1、图2显示的是本专利即表面载药缓释颌面种植经皮基台表面促进了成纤维细胞在钛表面的增殖、粘附结果。
经皮种植体的远期成功建立在形成一个稳定的软组织经皮生物学密封的基础上。感染,上皮下行以及机械应力是导致经皮种植体的成功率减低的主要问题。当种植体经皮部位形成软组织种植体界面的生物学封闭后,会阻碍上皮向下生长以及细菌的侵袭,并可以增加抵抗机械应力的能力。
皮肤是由表皮和真皮组成。真皮在生物密封的形成中起着更重要的作用。 表皮的封闭更多的应该是建立在真皮与种植体形成牢固结合的基础上表皮迁移到种植体表面。成纤维细胞产生ECM和各种结缔组织的重要组成部分,如糖蛋白和胶原蛋白等。在种植体植入早期,成纤维细胞在种植体表面的快速粘附和增殖可以促进经皮部位永久性生物学密封的形成和防止植入部位的早期污染。
本实验研究5天时间内成纤维细胞在复合涂层表面,纳米管表面和光滑钛表面的生理反应,不同实验结果均显示纳米CCN2复合涂层表面促进了成纤维细胞的在材料表面的粘附、增殖;在0.25、0.5、1、2、4和24小时,早期粘附到纳米CCN2复合涂层表面的细胞数量均高于其它两种材料表面。细胞粘附的质量是决定生物材料表面的体外生物相容性的主要因素,并可能决定在体内的生物相容性。
经皮种植体失败的主要原因上皮下行和细菌感染。目前大多数研究的目的是防止细菌感染,而不是修复周围的皮肤与种植体的结合。本发明提出的研究思路是在种植体表面建立含有结缔组织生长因子的纳米缓释涂层,促进手术部位周围的结缔组织生长,形成稳定的经皮封闭,这不仅防止上皮下行,也提供了一道可以永久抵御细菌侵袭的屏障。
被选为药物缓释涂层药物载体的二氧化钛纳米管,已被证明有良好的生物相容性。层药物可以有很多种选择,CCN2是一种很好的模型药物,本研究选用的模型药物是Peprotech公司分子量11.2kDa的人重组CCN2片段,其能发挥CCN2所有的生理功能。我们的研究结果表明,复合涂层中CCN2的增强了改善二氧化钛纳米管表面上的成纤维细胞的生理功能。
成纤维细胞在种植体表面上快速的早期粘附和增殖将促进形成一经皮区域的液体的封闭,阻挡细菌的入侵和防止种植体表面的早期污染,这对形成一个经皮区永久的生物密封是非常重要的。
图3为经皮种植体周围软组织反应评价,显示了:促进了经皮封闭形成的 同时,阻挡了细菌的入侵,降低了种植体周围炎的发生。
实验方法:实验动物选用SD大鼠,种植体和基台选用适合动物尺寸大小的试样。实验选用21只雌性三个月大的SD大鼠,平均体重290g。手术器械采用常规高压灭菌。常规动物麻醉。脱毛剂除去双侧股毛发,消毒常规消毒铺巾,在两侧股骨髁下外侧分层切开,暴露股骨。大鼠的双侧股骨植入种植体,每侧一枚,分别随机植入光滑对照组,纳米管组和复合涂层组中任意一个不同的样品。术后7天每天每只动物注射20万单位青霉素以防感染,碘伏清理伤口。
植入后观察种植体周围软组织愈合情况并记录。植入4周后,根据Holgers,K.M等提出的种植体周围组织反应的评价标准对置入的经皮种植体周围软组织反应反应情况进行评价。评价标准如下表1:
表1经皮种植体周围的评价标准
实验观察过程中,所有动物都处于健康状态,无动物死亡。术后几天内,尽管注射了青霉素和用碘伏清洁,但所有实验动物的伤口处都有不同程度的感染。
光滑对照组和TiO2纳米组比复合涂层组严重。在植入4周内试样都保持稳固,无试样因为感染而脱落。感染的发生可能与伤口愈合初期的炎症反应和脱毛剂对皮肤的烧伤有关。植入2周后,几乎所有复合涂层组种植体周围的感染现象都基本消失,不再有渗出液,伤口周围变得清洁和干燥,种植体与皮肤 紧密贴附。而其它组实验,炎症程度也有好转,但仍有部分实验有少量渗出物和红肿。
4周后,种植体周围软组织反应的情况如图3所示。复合涂层组种植体周围软组织反应评分都集中在2分以下,即红肿、轻微红肿或无反应。而另外两组对照组种植体周围多集中在2分或2分以上,即红肿、肉芽组织增生及感染。
经皮种植体周围软组织反应评价结果显示出植入4周后,复合涂层种植体周围软组织反应要明显小于对照组,这说明采用复合涂层处理后,在促进了经皮封闭形成的同时,阻挡了细菌的入侵,降低了种植体周围炎的发生。

Claims (8)

1.一种表面载药缓释颌面种植经皮基台的制备方法,其特征在于,包括以下步骤:
1)制备二氧化钛纳米管涂层
采用阳极氧化法在基台表面制备二氧化钛纳米管涂层,然后经超声清洗、真空干燥及射线消毒处理,备用;
2)制备介孔二氧化硅纳米颗粒
将无水乙醇和质量分数为25%的氨水按照(50~150):(3~5)的体积比均匀混合,得混合液,向混合液中加入正硅酸乙酯,搅拌反应24h后,清洗,然后在-80℃下预冻24h后再进行冷冻干燥,制得介孔二氧化硅纳米颗粒;
3)在介孔二氧化硅纳米颗粒中加载CTGF
采用浸泡法,将步骤2)制得的介孔二氧化硅纳米颗粒浸泡入CTGF溶液中,浸泡时间为24h;
4)将加载CTGF的介孔二氧化硅纳米颗粒装载入基台表面的二氧化钛纳米管管道中
首先,用PBS将步骤3)制得的加载CTGF的介孔二氧化硅纳米颗粒进行分装,配制成1~3μg/μL的母液;然后,将该1~3μg/μL的母液稀释成1~10ng/μL的工作液;再次,在消毒好的基台表面滴加工作液,使工作液铺匀基台表面;最后,将滴加有工作液的基台置于无菌冻干机中,温度设置为室温,真空干燥1~2h;
5)重复步骤4),直至试样加载到所需的量,制备得到表面载药缓释颌面种植经皮基台。
2.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤1)中,所述基台是由纯钛加工而成,并经抛光、清洗及真空干燥处理;清洗是依次用丙酮、无水乙醇及去离子水进行超声清洗。
3.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤2)中,向混合液中加入的正硅酸乙酯的量是按照每3~5mL的氨水加入100~300μL正硅酸乙酯。
4.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤2)中,清洗是采用去离子水清洗三遍。
5.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤3)中,CTGF溶液的浓度为1~3μg/mL。
6.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤4)中,在基台表面滴加5-50μL的工作液。
7.根据权利要求1所述的表面载药缓释颌面种植经皮基台的制备方法,其特征在于,步骤1)所述的采用阳极氧化法在基台表面制备二氧化钛纳米管涂层,具体操作为:配制1~2mol/L H3PO4以及0.3%HF的混合电解液,置入电磁搅拌仪中持续搅拌,取干燥后的基台放入上述混合电解液中,与阳极相连,铂为阴极,在电压为10~25V的条件下持续搅拌1~3h,在试样表面生成二氧化钛纳米管涂层。
8.采用权利要求1~7中任意一项所述的方法制得的表面载药缓释颌面种植经皮基台。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236981A (zh) * 2017-06-07 2017-10-10 福建师范大学 介孔二氧化硅/二氧化钛纳米管阵列复合材料的制备方法
CN108175527A (zh) * 2018-01-26 2018-06-19 徐文洲 具备可见光机能化功能的种植体基台穿龈结构及制作方法
CN117065089A (zh) * 2023-07-06 2023-11-17 首都医科大学附属北京口腔医院 一种载药TNTs表面SF/MSN复合药物控释涂层

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103480369A (zh) * 2012-06-13 2014-01-01 中国石油天然气股份有限公司 一种铂纳米复合催化剂及其制备和应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103480369A (zh) * 2012-06-13 2014-01-01 中国石油天然气股份有限公司 一种铂纳米复合催化剂及其制备和应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HONGBO WEI, ET AL.: "Increased fibroblast functionality on CNN2-loaded titania nanotubes", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 *
刘湘宁: "纯钛纳米管表面和纳米胶束生物学特性的研究", 《中国博士学位论文全文数据库工程科技I缉》 *
方欣闪: "介孔二氧化硅微球的制备", 《长春理工大学学报(自然科学版)》 *
王文谦: "基于介孔二氧化硅纳米颗粒的可控释放体系", 《化学进展》 *
甘琪: "介孔氧化硅基纳米输送体系的构建及其在骨修复材料中的应用", 《中国博士学位论文全文数据库》 *
郝晓红: "基于介孔二氧化硅的多功能纳米药物输送体系研究进展", 《生物化学与生物物理进展》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236981A (zh) * 2017-06-07 2017-10-10 福建师范大学 介孔二氧化硅/二氧化钛纳米管阵列复合材料的制备方法
CN107236981B (zh) * 2017-06-07 2019-03-01 福建师范大学 介孔二氧化硅/二氧化钛纳米管阵列复合材料的制备方法
CN108175527A (zh) * 2018-01-26 2018-06-19 徐文洲 具备可见光机能化功能的种植体基台穿龈结构及制作方法
CN108175527B (zh) * 2018-01-26 2020-04-17 徐文洲 具备可见光机能化功能的种植体基台穿龈结构及制作方法
CN117065089A (zh) * 2023-07-06 2023-11-17 首都医科大学附属北京口腔医院 一种载药TNTs表面SF/MSN复合药物控释涂层
CN117065089B (zh) * 2023-07-06 2024-03-05 首都医科大学附属北京口腔医院 一种载药TNTs表面SF/MSN复合药物控释涂层

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