背景：牙釉质发育不全是一种影响牙釉质的遗传性疾病。在其表型中，低钙化AI的特征是矿物质缺乏，导致组织磨损，因此,牙齿敏感性过量的氟化物摄入（通过饮用水，氟化物补充剂，牙膏,或通过摄入杀虫剂或杀虫剂等产品）可能导致称为氟斑牙的疾病，这表现为污渍和牙齿变色影响其结构。我们最近的研究表明，哥伦比亚本地植物的提取物，Ilexguayusa和Pipermarginatum,将磷酸盐和正磷酸盐等矿物离子沉积到牙釉质结构中；然而，目前尚不清楚这些提取物是否会对牙髓产生毒性作用。
目的：评估暴露于从I.guayusa和P.marginatum分离的提取物对人牙髓干细胞（hDPSC）的细胞毒性作用，因此，其临床使用的安全性。
方法：拉曼光谱，荧光显微镜,采用流式细胞术技术。对于拉曼光谱，将hDPSC接种到纳米生物芯片上，旨在提供表面增强拉曼光谱（SERS效应），这将它们的拉曼信号增强了几个数量级。经过八天的培养，加入不同浓度(10、50和100ppm)的I.guayusa和P.marginatum提取物。在施用提取物后的0、12和24小时进行拉曼测量。使用OLIMPUSfv1000显微镜进行荧光显微镜检查，使用采用BDFACSCantoTMII流式细胞仪的试剂盒进行活死测定，数据分析是使用FlowJo程序确定的。
结果:拉曼光谱结果显示与活细胞一致的光谱。使用荧光显微镜和流式细胞术技术证实了这些发现，证实高细胞活力。
结论：分析的提取物表现出低细胞毒性，表明它们可以安全地应用在搪瓷上用于再矿化目的。使用用于SERS效果的纳米生物芯片改善了细胞活力评估。
BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp.
OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use.
METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program.
RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability.
CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.
PDF(Pubmed)