CN116549726A - 一种高亲水性的表面装载纳米铌/igf-1复合涂层的种植体及其制备方法 - Google Patents
一种高亲水性的表面装载纳米铌/igf-1复合涂层的种植体及其制备方法 Download PDFInfo
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Abstract
本发明具体公开了一种高亲水性的表面装载纳米铌/IGF‑1复合涂层的种植体的制备方法,步骤如下:对纯钛表面喷砂处理;将纳米铌溶液逐滴加入涡旋的IGF‑1溶液中,得含水量为75%的溶液,干燥后得纳米铌/IGF‑1复合材料;将上述复合材料溶解于PBS中,滴加到喷砂后的纯钛表面;将上述表面装载纳米铌/IGF‑1复合涂层的种植体在氮气中持续进行碱热反应,即得。本发明制备的纳米铌/IGF‑1种植体表面复合涂层,能够在种植体植入后同时释放促进成骨的纳米铌和IGF‑1,从而在骨缺损部位的近远端均有促成骨成分的持续作用。同时本发明通过亲水改性,加快了种植体早期骨整合,缩短了种植体负重的时间,具有良好的临床前景。
Description
技术领域
本发明涉及口腔种植体,具体涉及一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
背景技术
种植体表面与患者骨组织形成表面有序的结构和稳定的功能连接,是影响种植体长期稳定性及种植效果的关键因素。目前,通过种植体表面改性手段,增强种植体的骨结合和骨整合能力是当前种植体技术优化的核心思路之一。
纳米铌是一种具有优良成骨效果的新兴材料,且具有良好的生物相容性。我们的前期研究发现,纳米铌可以明显增强骨修复过程中早期的成骨分化和后期的成熟矿化。因此,纳米铌有望成为种植体表面涂层改性的重要原料。
骨生长因子在骨组织的愈合和再生的动态过程中起到重要的调节作用。其中,胰岛素样生长因子IGF-1可以促进骨祖细胞的增殖和分化,并刺激骨基质形成和成骨细胞增殖,现已广泛应用于种植修复相关领域的研究。
另外,形成具有良好亲水性的种植体表面可以增加成骨细胞的附着、增殖、迁移,并促进成骨细胞分化从而加速骨愈合。并且亲水表面也可通过免疫调节,在糖尿病、骨质疏松等骨质状况较差的条件下改善骨结合。
种植后的骨修复是一个非常复杂的过程,并受多种因素影响。然而,现有的种植体表面单一涂层改性技术多针对于骨整合过程中的一个因素。因此,针对骨整合过程中的不同因素,对种植体表面进行复合涂层改性有望达到更好的治疗效果。
发明内容
本发明针对上述现有技术的缺点与不足,提出了一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法。
本发明的第二个目的在于提供通过上述方法制备得到的高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
为实现本发明的目的,提供如下的技术方案:
一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,包括以下步骤:
(1)将纯钛浸泡于氢氧化钠溶液中,丙酮和蒸馏水冲洗后,对纯钛表面进行喷砂处理;
(2)将纳米铌颗粒溶解于去离子水中得到纳米铌溶液,并将其逐滴加入涡旋的IGF-1溶液中,得到含水量为75 wt%的纳米铌/IGF-1溶液,清洗干燥后得到纳米铌/IGF-1复合材料;
(3)将步骤(2)得到纳米铌/IGF-1复合材料溶解于PBS中得到纳米铌/IGF-1混合溶液,并将其滴加到步骤(1)中制得的喷砂处理后的纯钛基体表面并使其均匀分布,真空干燥后即得表面装载纳米铌/IGF-1复合涂层的种植体。
(4)将氢氧化钠溶液抽真空后通入氮气,并将步骤(3)得到的表面装载纳米铌/IGF-1复合涂层的种植体用氮气吹干后,在加热环境下浸入氢氧化钠溶液中,持续通入氮气,即得高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
优选的,步骤(1)纯钛在80℃的条件下浸泡于氢氧化钠溶液20 min;所述步骤(1)中纯钛在冷却至室温后,分别在丙酮和蒸馏水中冲洗20~30 min。
优选的,步骤(1)中的喷砂处理是用砂纸打磨抛光后,利用20目的金刚砂均匀喷砂。
优选的,步骤(2)中所述的涡旋为以600~800 rpm的速度磁力搅拌。
优选的,步骤(2)中所述的IGF-1溶液是将IGF-1溶解于去离子水中得到浓度为1~3μg/mL的溶液。
优选的, 步骤(3)中所述的纳米铌/IGF-1混合溶液的浓度为10 μg/mL。
优选的,步骤(3)中所述的滴加到种植体表面的纳米铌/IGF-1混合溶液的体积为200~300 μL。
优选的,步骤(3)中所述的真空干燥条件是在室温下干燥1~2 h。
优选的,步骤(4)中将步骤(3)中得到的表面装载纳米铌/IGF-1复合涂层的种植体与浓度10 M的氢氧化钠溶液在50~100 ℃的氮气环境下持续反应时间为4~6 h。
与现有技术相比,本发明具有以下优点及有益效果:
本发明构建了一个纳米铌/IGF-1的复合涂层,纳米铌具有优良成骨性能,种植体植入后可在种植体周围释放有效成分,起到在骨缺损区域近端的促进成骨分化和促矿化功能。IGF-1是在成骨过程中发挥重要生理功能的细胞因子,可以促进成骨细胞的增殖黏附与分化,并促进胶原的产生。种植体植入后,IGF-1可以响应体内环境的趋化等作用,从而能实现在骨缺损区域远端的促成骨功能。同时,本专利通过反沉淀法制备了纳米铌/IGF-1的复合材料,一定程度上可以起到两种有效成分的缓释效果,更有利于种植体的骨结合。
本发明首先对种植体进行了喷砂预处理,从而在种植体表面形成了良好的拓扑结构,随后在种植体表面形成复合涂层,最后再使用氢氧化钠处理,使其每层微结构上都充满致密的-OH,并持续通入氮气保护亲水的-OH层面,从而最终获得稳定的亲水表面。 亲水表面能有效增强种植体与骨组织的亲和力,增加成骨细胞的附着和增殖,并可以通过免疫调节促进骨愈合,提高种植成功率,具有良好的临床前景。
附图说明
下面结合附图对本发明技术方案做进一步的详细说明,但不构成对本发明的任何限制。
图1为高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的扫描电镜图。
图2为高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的表面亲水性结果。
图3为高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体对骨髓间充质干细胞的ALP活性影响的结果图。
图4为高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的对骨髓间充质干细胞细胞外基质矿化程度影响的结果图。
实施方式
下面将结合说明书附图和具体实施例进一步说明本发明的内容,但不应理解为对本发明的限制。不背离本发明精神和实质的情况下,对本发明方法、步骤、条件所作的修改或替换,均属于本发明的范围。若无特别说明,实施例中所用的实验方法均为本领域技术人员所熟知的常规方法和技术,试剂或材料如无特殊说明均为通过商业途径得到。本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
实施例1:高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备
(1)采用商业医用TA2纯钛加工成Φ 3.3 mm*6 mm的牙科柱状种植体基体(同时加工直径为20 mm,厚度为1mm的种植体试样用于体外成骨性能检测)用浓度30wt%的氢氧化钠溶液在80℃环境下浸泡20 min,冷却至室温后分别用丙酮和蒸馏水超声冲洗20~30 min,然后再用砂纸打磨抛光后,最后用20目金刚砂进行均匀喷砂处理。
(2)将直径为40~60 nm的纳米铌颗粒溶解于去离子水中得到纳米铌溶液,并将其以0.5~0.75秒/滴的速度逐滴加入浓度为1~3 μg/mL的IGF-1去离子水溶液中,并以600~800rpm的速度磁力搅拌1~2 h,直至得到含水量为75 wt%的纳米铌/IGF-1溶液,清洗干燥后得到纳米铌/IGF-1复合材料。
(3)将步骤(2)中得到纳米铌/IGF-1复合材料溶解于PBS中得到浓度为5 μg/mL的纳米铌/IGF-1混合溶液,并将200~300 μL的混合溶液滴加到步骤(1)中制得的喷砂处理后的纯钛基体表面并使其均匀分布,室温下真空干燥1~2 h后,即得表面装载纳米铌/IGF-1复合涂层的种植体。
(4)将浓度为10 M的氢氧化钠溶液抽真空后通入氮气,并将步骤(3)得到的表面装载纳米铌/IGF-1复合涂层的种植体用氮气吹干后,浸入氢氧化钠溶液中,并在50~100 ℃的氮气环境下持续反应4~6 h,即得高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
实施例2:高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备
(1)采用商业医用TA2纯钛加工成Φ 3.3 mm*6 mm的牙科柱状种植体基体(同时加工直径为20 mm,厚度为1mm的种植体试样用于体外成骨性能检测)用浓度30 wt%的氢氧化钠溶液在80℃环境下浸泡20 min,冷却至室温后分别用丙酮和蒸馏水超声冲洗20~30 min,然后再用砂纸打磨抛光后,最后用20目金刚砂进行均匀喷砂处理。
(2)将直径为40~60 nm的纳米铌颗粒溶解于去离子水中得到纳米铌溶液,并将其以0.5~0.75秒/滴的速度逐滴加入浓度为1~3 μg/mL的IGF-1去离子水溶液中,并以600~800rpm的速度磁力搅拌1~2 h,直至得到含水量为75 wt%的纳米铌/IGF-1溶液,清洗干燥后得到纳米铌/IGF-1复合材料。
(3)将步骤(2)中得到纳米铌/IGF-1复合材料溶解于PBS中得到浓度为10 μg/mL的纳米铌/IGF-1混合溶液,并将200~300 μL的混合溶液滴加到步骤(1)中制得的喷砂处理后的纯钛基体表面并使其均匀分布,室温下真空干燥1~2 h后,即得表面装载纳米铌/IGF-1复合涂层的种植体。
(4)将浓度为10 M的氢氧化钠溶液抽真空后通入氮气,并将步骤(3)得到的表面装载纳米铌/IGF-1复合涂层的种植体用氮气吹干后,浸入氢氧化钠溶液中,并在50~100 ℃的氮气环境下持续反应4~6 h,即得高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
对照例1:
(1)采用商业医用TA2纯钛加工成Φ 3.3 mm*6 mm的牙科柱状种植体基体(同时加工出直径为20 mm,厚度为1mm的种植体试样用于体外成骨性能检测)用浓度30wt%的氢氧化钠溶液在80℃环境下浸泡20 min,冷却至室温后分别用丙酮和蒸馏水超声冲洗20~30min,然后再用砂纸打磨抛光后,最后用20目金刚砂进行均匀喷砂处理。
以实施例2制备的高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体,并结合相关附图对本发明进行进一步说明。通过扫描电镜(SEM)对实施例2制得的种植体进行表面形貌检测,结果如图1所示。可以发现,高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体形成具有多孔结构的复合涂层结构,且在孔隙中可见IGF-1分布。
实施例4:高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的亲水性测试
采用静态水接触角测试仪测试材料表面润湿性。通过注射器将4 μL超纯水垂直慢速悬滴到实施例2和对照例1的种植体试样表面,使用机器自带成像系统拍摄液滴照片并分析接触角大小。结果如图2所示。可以发现,相对于对照例1中纯钛组的种植体表面较大的接触角,而实施例2中高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体表面的接触角显著减小,具有更好的亲水性能。
实施例5:高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的促成骨性能测试
通过对实施例1~2和对照例1中牙科种植体试样作用下大鼠骨髓间充质干细胞的的碱性磷酸酶活性进行检测,探究高亲水性的表面装载纳米铌/IGF-1复合涂层种植体的早期促成骨分化效果,具体步骤如下:
大鼠骨髓间充质干细胞rBMMSC在α-MEM培养基中37 ℃、5% CO2条件下培养。每3 d换1次培养基,待细胞融合达80 %左右时,用胰蛋白酶消化传代,选用第3~4代细胞进行试验;将实施例1~2和对照例1中的种植体试样置入6孔板中,然后在试样表面滴加2 mLrBMMSC细胞悬液,放入培养箱中,期间每3天更换一次细胞培养液。rBMMSCs的碱性磷酸酶活性用ALP活性检测试剂盒测定;培养至7天和14天后,用PBS清洗试样两次,将细胞更换至新的6孔培养板,每孔滴加0.5 mL的细胞裂解液,将孔板置于冰块上裂解40 min。将裂解细胞洗脱下来,4℃下离心(8000 rpm,10 min),并取上清液,向上清液中加入磷酸对硝基苯酯,并于37℃下培养30 min。加入1M NaOH溶液终止反应,在荧光显微镜下观察ALP的染色情况。结果如图3所示。
通过对实施例1~2和对照例1中种植体试样作用下大鼠骨髓间充质干细胞细胞外基质的矿化情况进行检测,探究高亲水性的表面装载纳米铌/IGF-1复合涂层种植体的促矿化效果,具体步骤如下:
大鼠骨髓间充质干细胞rBMMSC在α-MEM培养基中37 ℃、5% CO2条件下培养。每3 d换1次培养基,待细胞融合达80 %左右时,用胰蛋白酶消化传代,选用第3~4代细胞进行试验;将实施例1~2和对照例1中的种植体试样置入6孔板中,然后在试样表面滴加2 mLrBMMSC细胞悬液,放入培养箱中,期间每3天更换一次细胞培养液;培养至7和14天后,用PBS清洗样品三次,然后用75%的酒精固定1 h,固定完后用PBS漂洗三次,接着用40 mM茜素红在室温下将细胞染色10 min;用去离子水反复漂洗样品至不再脱色,在荧光显微镜明场模式下观察试样表面ECM的矿化情况。结果如图4所示。
可以发现,与对照组1中的纯钛牙科种植体相比,实施例1~2中的高亲水性的表面装载纳米铌/IGF-1复合涂层种植体作用下骨髓间充质干细胞的早期成骨分化水平和细胞外基质矿化程度均明显升高,并且10 μg/mL的纳米铌/IGF-1组作用下的成骨分化和矿化程度优于5 μg/mL的纳米铌/IGF-1组。这证实了本发明高亲水性的表面装载纳米铌/IGF-1复合涂层种植体的促成骨效果优良,并且优选实施例2中由10 μg/mL的纳米铌/IGF-1混合溶液制得的高亲水性的表面装载纳米铌/IGF-1复合涂层种植体材料。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,步骤如下:
(1)将纯钛浸泡于氢氧化钠溶液中,丙酮和蒸馏水冲洗后,对纯钛表面进行喷砂处理;
(2)将纳米铌颗粒溶解于去离子水中得到纳米铌溶液,并将其逐滴加入涡旋的IGF-1溶液中,得到含水量为75 wt%的纳米铌/IGF-1溶液,清洗干燥后得到纳米铌/IGF-1复合材料;
(3)将步骤(2)得到纳米铌/IGF-1复合材料溶解于PBS中得到纳米铌/IGF-1混合溶液,并将其滴加到步骤(1)中制得的喷砂处理后的纯钛基体表面并使其均匀分布,真空干燥后得到表面装载纳米铌/IGF-1复合涂层的种植体;
(4)将氢氧化钠溶液抽真空后通入氮气,并将步骤(3)得到的表面装载纳米铌/IGF-1复合涂层的种植体用氮气吹干后,在加热环境下浸入氢氧化钠溶液中,持续通入氮气,即得高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体。
2.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(1)纯钛在80℃的条件下浸泡于氢氧化钠溶液20 min;所述步骤(1)中纯钛在冷却至室温后,分别在丙酮和蒸馏水中冲洗20~30 min。
3.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(1)中的喷砂处理是用砂纸打磨抛光后,利用20目的金刚砂均匀喷砂。
4.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(2)中涡旋为以600~800 rpm/min的速度磁力搅拌。
5.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(2)中的IGF-1溶液是将IGF-1溶解于去离子水中得到浓度为1~3 μg/mL的溶液。
6.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(3)中得到的纳米铌/IGF-1混合溶液的浓度为10 μg/mL。
7.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(3)中滴加到种植体表面的纳米铌/IGF-1混合溶液的体积为200~300 μL。
8.根据权利要求1所述的一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(3)中真空干燥的条件是在室温下干燥1~2 h。
9.根据权利要求1一种高亲水性的表面装载纳米铌/IGF-1复合涂层的种植体的制备方法,其特征在于,所述步骤(4)中将步骤(3)中得到的表面装载纳米铌/IGF-1复合涂层的种植体与浓度10 M的氢氧化钠溶液在50~100 ℃的氮气环境下持续反应时间为4~6 h。
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