五种牙科合金细胞遗传毒性的体外实验和颊粘膜细胞微核试验研究
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摘要
目的：研究五种牙科合金(三种Ni-Cr合金VeraBond, Kera N, TILITF "V",两种Co-Cr合金N.P. Partial,StarLoy C)体外诱发人B淋巴母细胞的细胞毒性和遗传毒性以及Ni-Cr合金修复体对人口腔颊粘膜细胞的遗传损伤。
方法：人B淋巴母细胞为体外试验靶细胞,长春新碱为阳性对照,培养基(IMDM)为阴性对照。五种合金在IMDM中浸泡72小时制备浸出液。采用电感耦合等离子体质谱仪(ICP-MS)检测浸出液中Ni、Cr、Co等主要金属成分离子浓度。分别用CCK-8、中性红摄入(NRU)试验检测五种合金的细胞毒性,用彗星试验和胞质阻断微核试验(CBMN)检测遗传毒性。暴露浓度分别为56.5、112.5、225、450和900μl浸出液/ml,暴露时间为48h。采用微核试验检测68名患者的颊粘膜细胞的遗传损伤(34名戴用Ni-Cr合金全冠修复体患者为实验组,平均冠修复时间为5.32年；34名与实验组年龄、性别匹配志愿者为对照组)。
结果：五种合金浸出液中,VeraBond浸出液中Ni的含量(87.6ug/L)高于KeraN浸出液(57.6ug/L)和TILITE "V"浸出液(70.0ug/L),差异有显著性(P<0.05)。TILITE "V"浸出液中Cr的含量(19.6ug/L)明显低于Kera N浸出液(28.7ug/L)和N.P.Partial浸出液(29.1ug/L)(P<0.05)。StarLoy C浸出液中Mo的含量(6.7ug/L)明显低于Kera N浸出液(15.4ug/L)和VeraBond浸出液(15.9ug/L)(P<0.05)。仅在VeraBond浸出液中检出Be。NRU试验中,除暴露浓度为56.5μl/ml的N.P. Partial浸出液外,其它各组细胞活性均低于阴性对照组(P<0.01or P<0.05)。在相同暴露浓度下,112.5-900μl/ml的VeraBond各组浸出液细胞活性均低于Kera N, TILITF "V", N.P. Partial和StarLoy C(P<0.01)。暴露浓度为450-900μl/ml的Kera N浸出液细胞活性低于N.P. Partial (P<0.01).TILITE"V"浸出液在暴露浓度为225和900μl/ml时,细胞活性低于N.P. Partial (P<0.01). StarLoy C浸出液在暴露浓度为112.5和900μ1/ml时细胞活性低于N.P. Partial (P<0.01).CCK-8试验结果显示,N.P. Partial浸出液在暴露浓度为112.5-900μl/ml时细胞活性明显高于、/eraBond (P<0.01),在暴露浓度为900μl/ml时细胞活性明显高于Kera N (P<0.01). StarLoy C浸出液在暴露浓度为900μl/ml时细胞活性明显高于VeraBond (P<0.01). CBMN试验中,五种合金浸出液在各暴露浓度诱发的人B淋巴母细胞的微核率(MNF)和核分裂指数(NDI)与阴性对照组比较无显著性差异(P>0.05)。彗星试验结果显示,人B淋巴母细胞暴露于五组合金浸出液诱发的%tail DNA(尾部DNA百分比)和Olive尾矩(OTM)与阴性对照组比较,均无明显增加(P>0.05)。人群口腔颊粘膜微核试验调查结果表明,Ni-Cr合金修复体戴入后颊粘膜细胞MNF和微核细胞率(MCF)分别为1.68±2.04‰和1.29±1.43‰,对照组MNF和MCF分别为1.62±1.89‰和1.09±1.16‰,两组之间无显著性差异(P>0.05)。
结论：五种牙科合金用彗星试验和CBMN试验体外实验未能检测出有明显的遗传毒性,与人群调查口腔粘膜微核试验也未检测出使用牙科合金病人的口腔粘膜细胞有明显的遗传损伤的结果相符合,根据我们的实验说明这些牙科合金使用还是基本安全的。但NRU和CCK-8体外试验检测五种牙科合金细胞毒性,发现有明显的差异,而且这些差异与合金中析出的Ni和Be离子浓度有关,建议临床应选用细胞毒性低的牙科合金,以提高合金与周围组织的相容性及减少对周围组织的刺激。
Objective The purposes of present investigation were to detect whether5kinds of dental alloys can induce the cyto-genotoxicity in vitro and to determine whether the increased MNFs of exfoliated buccal cells appear in patients exposed to dental alloys.
Methods Human B lymphoblast cells served as target cells in vitro. Vincristine was used as positive control. IMDM was used as negative control. The extracts of five alloys were prepared by being immersed in IMDM culture medium for72hours. The released metal elements of extracts were detected by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The cytotoxic effects induced by five dental alloy extracts on human B lymphoblast cells were detected using neutral red uptake (NRU) and CCK-8assays in vitro. The genotoxicity induced by five alloy extracts were detected using comet and cytokinesis-block micronucleus (CBMN) assays in vitro. The concentrations of five alloy extracts were56.5s112.5、225、450and900μl extracts/ml. The cells were exposed for48h in four assays. Chromosomal damage of exfoliated buccal cells in68subjects were investigated using micronucleus(MN) assay. Thirty four patients exposed to dental alloys for1-13years (mean5.32years) and34controls were matched with patient group according to age and sex.
Results The Ni content (87.6ug/L) in VeraBond (VB) extract was significantly higher than those (57.6ug/L and70.0ug/L) in Kera N (KN) and TILITE"V"(TV) extracts (P<0.05). The Cr content (19.6ug/L) in TV extracts was significantly lower than those (28.7ug/L and29.1ug/L) in KN and N.P. Partial (NP) extracts (P<0.05). The Be content was detectable only in VB extract. The results of NRU assay showed that the cell viability of all groups was significantly lower than that of the negative group (P<0.01or P<0.05) except for the NP alloy extract at the dose of56.5μl extracts/ml. At the doses of112.5-900μl extracts/ml, the cell viability of VB group was significantly lower than that of KN, TV, NP and Starlog C (SC) groups (P<0.01). At the doses of450-900μl extracts/ml, the cell viability of KN group was significantly lower than that of NP group (P<0.01). At the doses of225and900μl extracts/ml, the cell viability of TV group was significantly lower than that of NP group (P<0.01). At the doses of112.5and225μl extracts/ml, the cell viability of SC group was significantly lower than that of NP group (P<0.01). According to the results of CCK-8assay, The cell viability of the NP group was significantly higher than that of the VB group at the doses of112.5-900μl extracts/ml (P<0.01). At the exposure dose of900μl extract/ml, the cell viability of the SC group was significantly higher than that of the VB group (P<0.01). When the exposure dose was900μl extract/ml, the cell viability of NP group was significantly higher than that of KN group (P<0.01). While the DNA damage (%tail DNA) and MNFs induced by VB, KN, TV, NP and SC extracts did not increase significantly when compared with the control (P>0.05) according to the results of comet and CBMN assays. The results of oral buccal cell MN assay revealed that the mean MNF and micronucleated cell frequency (MCF) of oral buccal cells in patient group were1.68±2.04and1.29±1.439‰, respectively.The mean MNF and MCF of oral buccal cells in control group were1.62±1.89and1.09±1.16‰, respectively. There were no significant differences of MNF and MCF between patient group and control group (P>0.05).
Conclusion The extracts of the tested five dental alloys did not significantly induce DNA damage and chromosomal damage on human B lymphoblast cells in comet assay and CBMN assay in vitro. Also the genotoxic effects of buccal cells in68cases with dental alloys were not detectable with micronucleus assay of human buccal cells. However, in CCK-8assay and NRU assay in vitro, there were differences of the cytotoxic effects among5dental alloy extracts to some extent, which may be related to the Ni and Be ions released from the dental alloys. The present investigation suggested that the dental alloys with low cytotoxicity should be used for clinical work so that the biocompatibility could be improved and the stimulant effects could be decreased for around tissues of dental alloys.
方法：人B淋巴母细胞为体外试验靶细胞,长春新碱为阳性对照,培养基(IMDM)为阴性对照。五种合金在IMDM中浸泡72小时制备浸出液。采用电感耦合等离子体质谱仪(ICP-MS)检测浸出液中Ni、Cr、Co等主要金属成分离子浓度。分别用CCK-8、中性红摄入(NRU)试验检测五种合金的细胞毒性,用彗星试验和胞质阻断微核试验(CBMN)检测遗传毒性。暴露浓度分别为56.5、112.5、225、450和900μl浸出液/ml,暴露时间为48h。采用微核试验检测68名患者的颊粘膜细胞的遗传损伤(34名戴用Ni-Cr合金全冠修复体患者为实验组,平均冠修复时间为5.32年；34名与实验组年龄、性别匹配志愿者为对照组)。
结果：五种合金浸出液中,VeraBond浸出液中Ni的含量(87.6ug/L)高于KeraN浸出液(57.6ug/L)和TILITE "V"浸出液(70.0ug/L),差异有显著性(P<0.05)。TILITE "V"浸出液中Cr的含量(19.6ug/L)明显低于Kera N浸出液(28.7ug/L)和N.P.Partial浸出液(29.1ug/L)(P<0.05)。StarLoy C浸出液中Mo的含量(6.7ug/L)明显低于Kera N浸出液(15.4ug/L)和VeraBond浸出液(15.9ug/L)(P<0.05)。仅在VeraBond浸出液中检出Be。NRU试验中,除暴露浓度为56.5μl/ml的N.P. Partial浸出液外,其它各组细胞活性均低于阴性对照组(P<0.01or P<0.05)。在相同暴露浓度下,112.5-900μl/ml的VeraBond各组浸出液细胞活性均低于Kera N, TILITF "V", N.P. Partial和StarLoy C(P<0.01)。暴露浓度为450-900μl/ml的Kera N浸出液细胞活性低于N.P. Partial (P<0.01).TILITE"V"浸出液在暴露浓度为225和900μl/ml时,细胞活性低于N.P. Partial (P<0.01). StarLoy C浸出液在暴露浓度为112.5和900μ1/ml时细胞活性低于N.P. Partial (P<0.01).CCK-8试验结果显示,N.P. Partial浸出液在暴露浓度为112.5-900μl/ml时细胞活性明显高于、/eraBond (P<0.01),在暴露浓度为900μl/ml时细胞活性明显高于Kera N (P<0.01). StarLoy C浸出液在暴露浓度为900μl/ml时细胞活性明显高于VeraBond (P<0.01). CBMN试验中,五种合金浸出液在各暴露浓度诱发的人B淋巴母细胞的微核率(MNF)和核分裂指数(NDI)与阴性对照组比较无显著性差异(P>0.05)。彗星试验结果显示,人B淋巴母细胞暴露于五组合金浸出液诱发的%tail DNA(尾部DNA百分比)和Olive尾矩(OTM)与阴性对照组比较,均无明显增加(P>0.05)。人群口腔颊粘膜微核试验调查结果表明,Ni-Cr合金修复体戴入后颊粘膜细胞MNF和微核细胞率(MCF)分别为1.68±2.04‰和1.29±1.43‰,对照组MNF和MCF分别为1.62±1.89‰和1.09±1.16‰,两组之间无显著性差异(P>0.05)。
结论：五种牙科合金用彗星试验和CBMN试验体外实验未能检测出有明显的遗传毒性,与人群调查口腔粘膜微核试验也未检测出使用牙科合金病人的口腔粘膜细胞有明显的遗传损伤的结果相符合,根据我们的实验说明这些牙科合金使用还是基本安全的。但NRU和CCK-8体外试验检测五种牙科合金细胞毒性,发现有明显的差异,而且这些差异与合金中析出的Ni和Be离子浓度有关,建议临床应选用细胞毒性低的牙科合金,以提高合金与周围组织的相容性及减少对周围组织的刺激。
Objective The purposes of present investigation were to detect whether5kinds of dental alloys can induce the cyto-genotoxicity in vitro and to determine whether the increased MNFs of exfoliated buccal cells appear in patients exposed to dental alloys.
Methods Human B lymphoblast cells served as target cells in vitro. Vincristine was used as positive control. IMDM was used as negative control. The extracts of five alloys were prepared by being immersed in IMDM culture medium for72hours. The released metal elements of extracts were detected by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The cytotoxic effects induced by five dental alloy extracts on human B lymphoblast cells were detected using neutral red uptake (NRU) and CCK-8assays in vitro. The genotoxicity induced by five alloy extracts were detected using comet and cytokinesis-block micronucleus (CBMN) assays in vitro. The concentrations of five alloy extracts were56.5s112.5、225、450and900μl extracts/ml. The cells were exposed for48h in four assays. Chromosomal damage of exfoliated buccal cells in68subjects were investigated using micronucleus(MN) assay. Thirty four patients exposed to dental alloys for1-13years (mean5.32years) and34controls were matched with patient group according to age and sex.
Results The Ni content (87.6ug/L) in VeraBond (VB) extract was significantly higher than those (57.6ug/L and70.0ug/L) in Kera N (KN) and TILITE"V"(TV) extracts (P<0.05). The Cr content (19.6ug/L) in TV extracts was significantly lower than those (28.7ug/L and29.1ug/L) in KN and N.P. Partial (NP) extracts (P<0.05). The Be content was detectable only in VB extract. The results of NRU assay showed that the cell viability of all groups was significantly lower than that of the negative group (P<0.01or P<0.05) except for the NP alloy extract at the dose of56.5μl extracts/ml. At the doses of112.5-900μl extracts/ml, the cell viability of VB group was significantly lower than that of KN, TV, NP and Starlog C (SC) groups (P<0.01). At the doses of450-900μl extracts/ml, the cell viability of KN group was significantly lower than that of NP group (P<0.01). At the doses of225and900μl extracts/ml, the cell viability of TV group was significantly lower than that of NP group (P<0.01). At the doses of112.5and225μl extracts/ml, the cell viability of SC group was significantly lower than that of NP group (P<0.01). According to the results of CCK-8assay, The cell viability of the NP group was significantly higher than that of the VB group at the doses of112.5-900μl extracts/ml (P<0.01). At the exposure dose of900μl extract/ml, the cell viability of the SC group was significantly higher than that of the VB group (P<0.01). When the exposure dose was900μl extract/ml, the cell viability of NP group was significantly higher than that of KN group (P<0.01). While the DNA damage (%tail DNA) and MNFs induced by VB, KN, TV, NP and SC extracts did not increase significantly when compared with the control (P>0.05) according to the results of comet and CBMN assays. The results of oral buccal cell MN assay revealed that the mean MNF and micronucleated cell frequency (MCF) of oral buccal cells in patient group were1.68±2.04and1.29±1.439‰, respectively.The mean MNF and MCF of oral buccal cells in control group were1.62±1.89and1.09±1.16‰, respectively. There were no significant differences of MNF and MCF between patient group and control group (P>0.05).
Conclusion The extracts of the tested five dental alloys did not significantly induce DNA damage and chromosomal damage on human B lymphoblast cells in comet assay and CBMN assay in vitro. Also the genotoxic effects of buccal cells in68cases with dental alloys were not detectable with micronucleus assay of human buccal cells. However, in CCK-8assay and NRU assay in vitro, there were differences of the cytotoxic effects among5dental alloy extracts to some extent, which may be related to the Ni and Be ions released from the dental alloys. The present investigation suggested that the dental alloys with low cytotoxicity should be used for clinical work so that the biocompatibility could be improved and the stimulant effects could be decreased for around tissues of dental alloys.
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