CN214485171U - 一种具有生物活性表面的牙种植体 - Google Patents
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Abstract
本实用新型提供一种具有生物活性表面的牙种植体，包括种植体本体和覆盖在种植体本体外表面上的缓释层，缓释层具体为负载有EPO的壳聚糖纳米微球复合层。负载有EPO的壳聚糖纳米微球附着于牙种植体本体表面，在牙种植体本体表面形成含有EPO的壳聚糖纳米微球缓释层结构。本实用新型所得牙种植体上的EPO可持续释放15d，具有持久、高效的促进血管内皮细胞生长，促进血管化的作用，进一步能够支持多种细胞(如成骨细胞、软骨细胞)的粘附、存活、生长与分化，具有广阔的应用前景。
Description
技术领域
本实用新型涉及牙种植体技术领域，具体而言，尤其涉及一种具有含EPO生物活性表面的牙种植体。
背景技术
为了提高钛和钛合金的生物活性，缩短种植周期，加快骨愈合和提高骨与种植体结合力，种植材料的表面改性技术逐渐发展并日趋成熟。种植体表面改性后，表面物理和化学特性随之发生改变。然而如何使种植体早期负重甚至即刻负重，从而提高患者生活水平及修复质量，广大学者对种植体表面研究仍然在不断探索。为了缩短种植体负重时间，如何构建一种能够早期固位较好的新型种植体，使即刻负重成为可能，是先进研究的焦点。
Tresguerres IF等进行动物实验在兔股骨植入螺纹种植体，在种植体植入局部添 加重组人生长素冻干粉末，局部使用生长素的一组皮质骨区骨一种植体结合率高于对照组30个百分点，差别明显。该项研究首次证实生长素局部使用对骨整合的促进作用，并且证明，局部使用生长素可促进种植初期骨结合。Brama等在种植体表面涂布骨形成蛋白并且观察其在动物实验中的骨结合，接着这种技术被应用在在种植体表面涂布人重建骨形成蛋白-2或者TGF-β1，这种技术能早期提高种植体周围骨形成。但这些细胞因子涂层存在种植早期快速释放降解，单一细胞因子作用局限以及在种植体表面涂布不均匀并且涂布浓度低等诸多不足。
因此，如何实现牙种植体表面的药物缓释十分重要。采用离子凝胶法制备的壳聚糖-三聚磷酸钠纳米微球被证明是一种理想的可溶性纳米载体，其上负载有EPO后可明显延长EPO的药物作用浓度，起到缓释效果。
实用新型内容
根据上述提出的技术问题，本实用新型提出一种牙种植体，其表面设置有壳聚糖-EPO纳米微球复合层，促红细胞生成素(EPO)均匀涂布于种植体表面，在种植体植入早期，缓释分泌EPO促进早期血管化和骨形成，从而获得理想、具生物相容性的生物材料以及最符合口腔生理要求的种植体表面。
本实用新型采用的技术手段如下：
一种具有生物活性表面的牙种植体，包括种植体本体和缓释层；种植体本体表面设有螺纹，种植体本体的两端分别为冠口端和植入端，种植体本体靠近植入端一侧的表面设有自攻槽，缓释层覆盖于种植体本体外表面，螺纹、冠口端、植入端、自攻槽的外表面均覆盖缓释层；缓释层为包载EPO的壳聚糖纳米微球复合层。
进一步地，种植体本体呈倒圆锥体，植入端远离冠口端的一端为圆弧面，冠口端横截面直径大于植入端横截面直径。
进一步地，自攻槽个数为2～4个，自攻槽均匀设置在种植体本体外表面。
进一步地，缓释层壳聚糖纳米微球的粒径为150～350nm，包载EPO的包封率为72％～80％。
较现有技术相比，本实用新型具有以下优点：
本实用新型的牙种植体，在种植本体外表面涂覆有包载EPO的壳聚糖纳米微球复合缓释层，可有效促进种植体周围骨再生和提高骨结合的效果。
本申请牙种植体植入体内后，随着壳聚糖纳米微球涂层表面的缓慢溶解，EPO被逐步释放于周围组织中，可以持续10～15天，具有缓释作用，可持久、高效的促进血管内皮细胞生长，促进血管化，进一步能够支持多种细胞(如成骨细胞、软骨细胞)的粘附、存活、生长与分化，具有广阔的应用前景。
附图说明
为了更清楚地说明本实用新型实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图做以简单地介绍，显而易见地，下面描述中的附图是本实用新型的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1中，(a)为牙种植体结构示意图，(b)为局部剖面示意图。
图2为实施例2所得牙种植体的EPO释药率。
图3为碱性磷酸酶活性检测结果。
图4为细胞骨钙素分泌检测结果。
具体实施方式
以下通过实施例对本实用新型技术方案作详细说明，实施例所涉及的设备与原料如无特别说明，均可通过常规途径获得。
实施例1
一种具有生物活性表面的牙种植体，包括种植体本体6和缓释层5，种植体本体6外表面设置有螺纹，种植体本体6的两端分别为冠口端1和植入端2，种植体本体6呈倒圆锥体，植入端2远离冠口端1的一端为圆弧面4，冠口端1横截面直径大于植入端2横截面直径。
植入端2的外表面上设置有自攻槽3，自攻槽3个数为两个，均布在植入端2的外表面；自攻槽3的深度自植入端2远离冠口端1的一端向植入端2靠近冠口端1的方向逐渐变浅。
缓释层5覆盖于种植体本体6外表面，螺纹、冠口端1、植入端2、自攻槽3的外表面均覆盖缓释层5；缓释层5为包载EPO的壳聚糖纳米微球复合层；壳聚糖纳米微球为水溶性的羟丙基壳聚糖(HCS)纳米微球，粒径为200～350nm；EPO包封率为75％左右。
实施例2
一种具有含EPO生物活性表面的牙种植体的制备方法，将种植体本体浸泡于包载EPO的壳聚糖纳米微球溶液中，使种植体本体表面形成缓释层，种植体本体的螺纹、冠口端、植入端、自攻槽的外表面均覆盖缓释层。
本实用新型优选的一种制备方法如下：
(1)水溶性羟丙基壳聚糖纳米微球的制取
取2g壳聚糖(Chitosan,CS)于三口烧瓶中，加入15ml质量分数为20％NaOH溶液和24ml异丙醇，于室温下搅拌1h，加入1ml催化剂四甲基氢氧化铵，缓慢滴加15ml环氧丙烷，于60℃温度水浴下震荡搅拌混匀5h，反应结束后冷却到室温。用HCl调节pH值为7，再加入丙酮水溶液中搅拌，抽滤，最后用无水乙醇洗涤数次，60℃真空干燥。
(2)水溶性羟丙基壳聚糖-EPO纳米微球制备
采用离子凝胶法，0.2gHCS和0.2gEPO溶解于50ml双蒸水中，搅拌后静置1h使其充分溶解，0.45微米滤纸过滤，用少量醋酸调节pH值为6；将0.1g三聚磷酸钠(TPP)溶解于50ml双蒸水中，以0.22微米滤纸过滤；取20mlHCS溶液，以20滴/分速度滴加TPP溶液20ml，磁力持续搅拌30min下，HCS终浓度2.0g/l，TPP终浓度1.0g/l；纳米微球粒直径为150～350nm；4℃低温保存。EPO在纳米微球中包封率75％左右，EPO从纳米微微球中持续释放时间可达10d以上。
(3)种植体本体表面的前处理
用去离子水清洗待处理种植体本体，干燥后进行喷砂处理；喷砂砂材为氧化铝，喷砂压力为0.5Mpa，时间为70s，喷嘴与种植体本体间距为8cm，喷砂角度为20°；清洗喷砂后的种植体本体，干燥后，在70℃下酸蚀30min；所用酸蚀药水按体积分数计含35％的硫酸、25％的盐酸和余量的水；酸蚀后将种植体本体置于60℃、5mol/L NaOH溶液孵育48h，调整pH值7.0；去离子水清洗后，先用纯丙酮浸泡10分钟，再用无水乙醇浸泡10分钟；将种植体本体放入150mM、pH为7的氯化钠电解液中保存。
前处理后的种植体本体表面用激光共聚焦显微镜检测，粗糙度参数RoughnessParameter(微米)如下：
Sa,(算术平均偏差arithmetic mean deviation of the surface)：1.3+0.02
Sq,(均方根偏差root-mean-square deviation of the surface)：1.6+0.03
St,(最大峰谷高度maximum peak-to-valley height of the surface)：8.1+0.2
Sk,(振幅分布偏斜amplitude distribution skew)：4.9+1.8
(4)具有水溶性羟丙基壳聚糖-EPO纳米微球表面的牙种植体制备
将前处理好的酸蚀刻加喷砂表面种植体本体置于水溶性羟丙基壳聚糖-EPO纳米微球溶液中浸泡30min，种植体本体表面吸附纳米微球达到饱和状态，调整pH值为7.0，钴60辐射灭菌下消毒24h，得到具有含EPO生物活性表面的牙种植体。
实施例3
将实施例2制备所得的牙种植体进行药物缓释试验测试，具体试验步骤如下：
(1)将处理好的水溶性羟丙基壳聚糖-EPO纳米微球表面的牙种植体置于20ml磷酸缓冲液中(pH＝7.2)，37℃水浴；
(2)每隔一段时间取出0.5ml的上清液，用考马斯亮蓝蛋白测定试剂盒测定上清液中游离的EPO含量，同时用等量磷酸缓冲液保持体积恒定；
(3)每次试验重复3次，取3次测定的平均值。
结果如图1所示，200h后仍展现良好的EPO缓释效果。
实施例4
制备不同表面实验组及对照组通过碱性磷酸酶和骨钙素检测进行骨形成能力测试，具体试验步骤如下：
对照组1(Plastic)：24孔细胞培养板(直径15mm)；
对照组2(Plastic+EPO)：水溶性羟丙基壳聚糖-EPO纳米微球处理孔细胞培养板；
实验组1(SLA)：表面酸蚀和喷砂处理钛盘，钛盘直径15mm高1mm；
实验组2(SLA+EPO)：水溶性羟丙基壳聚糖-EPO纳米微球处理酸蚀和喷砂处理钛盘，钛盘直径15mm高1mm。
(1)碱性磷酸酶检测
取第3代人牙槽骨成骨细胞以9300cells/cm2的细胞浓度接种于含有实验组和对照组24孔细胞培养板中，按实验分组干预；于37℃、体积分数5％CO2细胞培养箱内培养，每48h更换培养液，7天后移除培养液收获细胞；将细胞离心，洗涤沉淀物，加入200μL细胞裂解液(含0.2％Triton X-100的PBS溶液)，4℃下超声裂解细胞；用蛋白质检测BCA试剂盒测定细胞总蛋白含量。
碱性磷酸酶活性测定是利用将无色的磷酸对硝基苯酯转换为着色的对硝基苯酚，实验严格根据碱性磷酸酶测定试剂盒实验流程完成。颜色变化通过分光光度计在405nm时测得，量化细胞释放的酶，并用同一个标准曲线进行比较。碱性磷酸酶水平被标准化处理为相对总蛋白含量，结果如图2所示，SLA+EPO生成的蛋白总量最高。
(2)细胞骨钙素分泌检测
取第3代人牙槽骨成骨细胞以9300cells/cm2的细胞浓度接种于含有实验组和对照组24孔细胞培养板中，按实验分组干预；于37℃、体积分数5％CO2细胞培养箱内培养，每48h更换培养液，8天后移去培养液备用；培养液中骨钙素分泌量通过商用骨钙素检测试剂盒完成，实验采用酶联免疫测定法测得，测量操作流程严格按照试剂盒说明书执行。该测量常用的灵敏度(0.4μg/L)，结果如图3所示，SLA+EPO展现出良好的骨形成能力。
最后应说明的是：以上各实施例仅用以说明本实用新型的技术方案，而非对其限制；尽管参照前述各实施例对本实用新型进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本实用新型各实施例技术方案的范围。
Claims (4)
1.一种具有生物活性表面的牙种植体，其特征在于，所述牙种植体包括种植体本体(6)和缓释层(5)；所述种植体本体(6)表面设有螺纹，所述种植体本体(6)的两端分别为冠口端(1)和植入端(2)，所述种植体本体(6)靠近植入端(2)一侧的表面设有自攻槽(3)，所述缓释层(5)覆盖于种植体本体(6)外表面，所述螺纹、冠口端(1)、植入端(2)、自攻槽(3)的外表面均覆盖缓释层(5)；所述缓释层(5)为包载EPO的壳聚糖纳米微球复合层。
2.根据权利要求1所述的牙种植体，其特征在于，所述种植体本体(6)呈倒圆锥体，所述植入端(2)远离冠口端(1)的一端为圆弧面(4)，所述冠口端(1)横截面直径大于所述植入端(2)横截面直径。
3.根据权利要求1所述的牙种植体，其特征在于，所述自攻槽(3)个数为2～4个，所述自攻槽(3)均匀设置在种植体本体(6)外表面。
4.根据权利要求1所述的牙种植体，其特征在于，所述缓释层壳聚糖纳米微球的粒径为150～350nm，包载EPO的包封率为72％～80％。
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