CN109966315A - 一种用于牙齿保护的组合物 - Google Patents
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Abstract
本发明提供一种用于牙齿保护的组合物，能联合窝沟封闭术结合有效地预防和治疗龋病，在实施窝沟封闭术后涂覆本发明的牙齿保护剂能在牙齿表面形成一层富勒烯氟化钙保护层，一方面可以克服封闭剂脱落的问题，另一方面，能更有效地长期保护牙齿。
Description
技术领域
本发明涉及牙齿保护技术领域，具体地涉及一种用于牙齿保护的组合物。
背景技术
龋病是由于牙面菌斑内的致龋微生物和碳水化合物作用产生酸，引起牙体硬组织脱矿和无机物崩解的一种慢性进行性破坏的疾病。根据龋病发生的三联因素理论，龋病的预防主要是增强牙的抗龋能力、控制菌斑特别是抑制致龋菌生长和限制进食碳水化合物或寻找糖的代用品三方面。就防龋药物而论，主要作用是增强牙的抗龋能力（例如氟化物等）以及控制菌斑抑制细菌生长（例如氯己定等）。
目前窝沟封闭是防治龋病有效方法，窝沟封闭指不损伤牙体组织，将窝沟封闭材料涂布于牙冠咬合面、颊舌面的窝沟点隙，当它流入并渗透窝沟后固化变硬，形成一层保护性的屏障，覆盖在窝沟上，能够阻止致龋菌及酸性代谢产物对牙体的侵蚀，以达到预防窝沟龋的方法。窝沟封闭预防窝沟龋的原理是用高分子材料把牙齿的窝沟填平，使牙面变得光滑易清洁，一方面，窝沟封闭后，窝沟内原有的细菌断绝了营养的来源，逐渐死亡；另一方面外面的致龋细菌不能再进入，从而达到预防窝沟龋的目的。
现有窝沟封闭存在的缺点是对人员的操作要求高，容易产生酸蚀不充分，牙面干燥后没有呈现白垩状外观，同时操作过程中唾液或者气枪压缩空气中容易混有水/油，污染了酸蚀后的牙面，致使封闭剂脱落。
发明内容
本发明为了解决上述现有技术存在的问题，提供一种用于牙齿保护的组合物。
本发明实现发明目的采用的技术方案是，
一种用于牙齿保护的组合物，所述组合物为溶解有富勒烯和氟化钙的乙酸乙酯溶液。
优选地，所述富勒烯与氟化钙的质量比为（60-120）：1。
优选地，所述富勒烯与氟化钙的质量比为（80-100）：1。
优选地，所述富勒烯为富勒烯C50、富勒烯C60、富勒烯C70、富勒烯C76、富勒烯C78、富勒烯C84、富勒烯C90和内嵌结构富勒烯中的一种或几种的组合。
优选地，所述富勒烯为富勒烯C60。
本发明的有益效果是，本发明提供一种用于牙齿保护的组合物，能联合窝沟封闭术结合有效地预防和治疗龋病，在实施窝沟封闭术后涂覆本发明的牙齿保护剂能在牙齿表面形成一层富勒烯氟化钙保护层，一方面可以克服封闭剂脱落的问题，另一方面，能更有效地长期保护牙齿。
具体实施方式
实施例1：用于牙齿保护的组合物
一种用于牙齿保护的组合物，将富勒烯C60和氟化钙按照质量比为60:1的比例完全溶解在乙酸乙酯溶剂中，配得富勒烯C60和氟化钙的乙酸乙酯溶液。作为其他实施方式，本发明的富勒烯还可采用富勒烯C50、富勒烯C70、富勒烯C76、富勒烯C78、富勒烯C84、富勒烯C90和内嵌结构富勒烯中的一种或几种的组合。作为其他实施方式，富勒烯C60和氟化钙按照质量比可以在（60-120）:1进行选择。
实施例2：组合物用于牙齿保护的动物试验
1、实验方法
选择SPF-SD 大鼠80 只，雌雄各半，均于出生后18d 断奶，记录体重，喂以Keyes致龋饲料2000#和5%蔗糖水溶液。19～21d 鼠龄期间给予含有广谱抗生素抑菌（2000g饲料加氨苄西林2g、1000g5%蔗糖水溶液加头孢拉定1g），其他时间的饲料中不加抗生素。鼠龄22d 以无菌棉签取大鼠唾液，涂平板，检测内源性变形链球菌抑制情况，目的在于杀灭大鼠口腔内原有细菌后，再进行变形链球菌国际标准菌株的接种。在鼠龄23～26d 连续4d 于大鼠口腔内接种变形链球菌（1ml/只），在鼠龄24～26d 随机抽取10 只大鼠，标定，乙醚吸入麻醉下，腹腔注射皮罗卡品（0.8mg/100g），待唾液自然流出时，收集唾液（100μl/只），稀释涂平板，37℃厌氧培养，检测细菌是否定植，并记录菌落形成单位，观察细菌生长情况。鼠龄27d 为洗脱期，对大鼠随机分组，编号记录体重。
自鼠龄28d 起用无菌棉签分别蘸取不同溶液（富勒烯乙酸乙酯溶液、氟化钙乙酸乙酯溶液、富勒烯氟化钙乙酸乙酯溶液、乙酸乙酯、蒸馏水对照组）每组依次进行牙合面、颊舌面、口腔黏膜及舌背清理涂擦。剩余药液以钝头注射器注于大鼠口腔保持10s 用于漱口，确保药液作用到位，剂量为1ml/只。整个过程为1min，处理后2h 禁饮食。每天早晚各处理1次，连续5 周，始终由同一操作者完成。每周记录大鼠体重2 次，同时每天观察记录大鼠健康状况。鼠龄62d 时，采集大鼠刺激性唾液（100μl/只），用PBS缓冲液或肉汤培养基倍比稀释（2 个梯度），各梯度分取10μl，立即涂于BHI 血琼脂（记录总菌菌落数）和MS 琼脂（记录变形练球菌菌落数），每个稀释度作2 个平行样本，37℃厌氧培养，记录变形链球菌菌落数和总菌落数（CFU），求两者比值，并以此作为对细菌作用的观察指标。
在鼠龄664 时，乙醚窒息处死大鼠，断头去皮，清洗制备牙颌骨标本，用0.4%紫脲酸铵溶液浸泡12h 染色，洗净干燥，用超薄金刚砂片沿上下领骨磨牙牙合近远中向矢状半切，清洗干燥后，在解剖显微镜下根据Keyes 评分法划分龋齿进展为釉质层（E 级）、牙本质浅层（Ds）、牙本质中层（Dm）及牙本质深层（Dx）4 个级别对大鼠磨牙光滑面和窝沟面龋损进行记分，以此评定不同实验药物抑龋情况（在龋损Keyes 评分过程中应由两名实验者盲法下完成）。本实验数据以大鼠的食物、水消耗、体重、大鼠唾液变形链球菌水平值以及大鼠磨牙窝沟面和光滑面龋损的Keyes评分值作为实验的观察指标，且其均为计量资料。选用Excel2003软件数据库对实验数据进行整理，计量资料统计分析采用均数和标准差表示。实验数据采用SPSS15.0 统计软件包进行单因素方差分析，比较计算各组数据F值。在F值有意义的情况下，再进行多组样本均数两两比较的SNK Dunnett检验和LSD检验。检验水准α=0.05，P＞0.05，表示各组间比较无统计学差异；P＜0.05，表示各组间有比较统计学差异。实验动物应选择容易构建龋病模型的动物，且级别较高的SPF 动物，动物要严格照致龋食谱饲养，接种S.mutans(ATCC25175)后，应连续多次进行细胞形态学和生化鉴定，并检测定植情况记数。对实验分组按照随机原则，编号记录体重。在使用不同实验药物处理大鼠口腔时，均由同一操作者完成，以免造成操作上的误差。大鼠磨牙光滑面和窝沟面龋损Keyes 记分是由两名实验者盲法下完成一人记录评分，另一人核对。实验数据采用SPSS15.0 统计软件包进行统计学分析。
2、实验结果
体重情况：实验过程中对大鼠的食物、水消耗进行记录，各组大鼠间未见明显差异。药物口腔处理期间，大鼠的一般状况良好，口腔黏膜未见异常表现。实验初期，各组大鼠的体重与对照组相比无显著差异；实验期间，各组大鼠的体重均匀增加；实验结束时，各组大鼠的体重也是均匀增加且与蒸馏水对照组相比无显著差异，以上表明该牙齿保护组合物无毒性，动物试验无安全性问题。
变形链球菌的变化：实验中分别在细菌复苏后，接种前及接种后都对细菌进行菌落的形态，革兰染色和生化鉴定，连续鉴定3 次，结果表明接种细菌均为变形链球菌（S.mutans ATCC25175），并在大鼠口腔成功定植。在鼠龄24～26d 连续3 天大鼠的唾液采集样本中变形链球菌（S.mutansATCC25175）水平的变化显示：随着接种时间的延长，大鼠唾液中变形链球菌水平富勒烯乙酸乙酯溶液组基本持平，氟化钙乙酸乙酯溶液组有一定的增加，而富勒烯氟化钙乙酸乙酯溶液组的变形链球菌水平则降低相当明显，乙酸乙酯溶剂组和蒸馏水组的变形链球菌水平均有相当程度的增加。大鼠唾液中变形链球菌水平是以测试时与成功定植时比值表示。由此可以看出：富勒烯和氟化钙的组合产生了协同的抑菌效果。
大鼠磨牙龋损的Keyes 评分：Keyes 于1958 年创建一种龋齿记分系统，以单个牙面龋损范围和以四种方式记录探诊深度即（累及釉质E、浅层牙本质DS、中层牙本质DM、深层牙本质DX）来评估大鼠磨牙的龋损，其中E 级记分反映龋损累及广度，DS、DM、DX 级记分反映龋损累及牙本质深度，两者共同反映了龋损严重程度。龋损广度单位记录结合其深度单位，浅层单位记录包含深层单位如浅层DS 级单位总数将包含中层DM 和深层DX 单位数，因为中层DM 和深DX 层包含的牙面单位数必定经过浅层过程，所有权重分配不使用半个单位计数，同时实验结果统计需计算龋齿降低率。经广为应用，该系统能客观、有效的评估大鼠龋病模型的龋损信息，其科学性己被广为认可。
光滑面龋Keyes 记分：在E 级龋齿记分中，与乙酸乙酯溶剂组和蒸馏水组相比，富勒烯乙酸乙酯溶液组和氟化钙乙酸乙酯溶液组龋齿Keyes 记分均有一定的降低，且有明显的统计学意义（P=0.000），都显示了对釉质龋有一定的防治效果，而富勒烯氟化钙乙酸乙酯溶液组的降低程度最大，显示出对光滑面深度龋损有更好的预防效果。
窝沟龋Keyes 记分：从鼠磨牙牙合面上观察，各组大鼠磨牙均出现不同程度的龋损。在E 级龋齿记分中，与乙酸乙酯溶剂组和蒸馏水组相比，富勒烯乙酸乙酯溶液组和氟化钙乙酸乙酯溶液组龋齿Keyes 记分均有一定的降低，且有明显的统计学意义（P=0.000），都显示了对釉质龋有一定的防治效果，而富勒烯氟化钙乙酸乙酯溶液组的降低程度最大，显示出对窝沟龋损有更好的预防效果；在Ds、Dm 级龋齿记分中，富勒烯氟化钙乙酸乙酯溶液组同样显示出协同增效的有益效果。
最后应说明的是：以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此尽管本说明书参照上述的各个实施例对本发明已进行了详细的说明，但是本领域的技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围中。
Claims (5)
1.一种用于牙齿保护的组合物，所述组合物为溶解有富勒烯和氟化钙的乙酸乙酯溶液。
2.根据权利要求1所述的用于牙齿保护的组合物，其特征在于：所述富勒烯与氟化钙的质量比为（60-120）：1。
3.根据权利要求2所述的用于牙齿保护的组合物，其特征在于：所述富勒烯与氟化钙的质量比为（80-100）：1。
4.根据权利要求1至3任一项所述的用于牙齿保护的组合物，其特征在于：所述富勒烯为富勒烯C50、富勒烯C60、富勒烯C70、富勒烯C76、富勒烯C78、富勒烯C84、富勒烯C90和内嵌结构富勒烯中的一种或几种的组合。
5.根据权利要求4所述的用于牙齿保护的组合物，其特征在于：所述富勒烯为富勒烯C60。
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