纳米蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料生物安全性的临床前评价
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摘要
目的:
本研究通过将纳米级蒙脱土(OMMT)粉体在聚合物(PMMA)基材中均匀分散,获得新型的纳米蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料,并对蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料进行生物安全性的临床前评价。为义齿复合材料的研究和开发提供理论依据和参考。
方法:
根据ISO7405-1997“牙科学——用于牙科的医疗器械生物相容性临床前评价——牙科材料实验方法”国际标准,对纳米蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料进行了细胞毒性试验、溶血试验、急性经口全身毒性试验、口腔粘膜刺激试验、皮肤致敏试验和骨髓细胞微核试验。
1、细胞毒性试验细胞毒性试验是一类在离体状态下模拟生物体生长环境,检测材料和器械接触机体后生物学反应的体外试验,它是生物学体系评价中最重要的检测指标之一,一般列为首选和必选项目。本试验是采用Verocell(非洲绿猴肾细胞,CCL81)体外培养的四唑盐比色实验和材料浸提液培养细胞的形态学观察法。
2、溶血试验本研究采用材料直接以及采用经不同浸提条件下制得的材料浸提液两种方式,在体外模拟生物体的温度与稀释兔血接触,相互作用一定时间后,测定其光吸收度,从而分别判断材料的溶血性能,其目的是研究生物材料不同接触方式和浸提条件对溶血作用的影响,以试图提高体外方法检测生物材料生物安全性的可靠性和科学性。材料样品的生理盐水浸提液加稀释兔血0.2ml,离心5min,吸取上清液移入比色皿内,测定各管吸光度。
3、急性经口全身毒性试验用材料样品的生理盐水浸提液以1500mg/kg的剂量给昆明小鼠灌胃,灌胃后一周内观察小鼠一般状态及毒性表现和死亡数。
4、口腔粘膜刺激试验将材料圆片用眼科针线缝合固定在健康仓鼠颊囊两侧,缝合固定后观察两周,观察动物全身反应,接触局部的粘膜反应,两周处死动物,局部粘膜作组织切片,HE染色,观察粘膜的组织细胞反应。
5、皮肤致敏试验选白色雄性健康豚鼠,分别用阳性对照为10%二硝基氯苯,阴性对照为生理盐水,材料样品的生理盐水浸提液,完全弗氏佐剂(FCA)及完全弗氏佐剂和受试物的乳化剂进行诱导接触,激发接触,在24h,48h观察激发接触结果。
6、骨髓细胞微核试验选用健康昆明种小白鼠设高、中、低三个剂量组,分别按15.0g/kg、7.5g/kg、3.75g/kg的比例给予材料混悬液灌胃;阳性对照为40mg/kg的环磷酰胺,用腹腔注射;阴性对照为生理盐水灌胃。第一次染毒后24小时再次染毒,末次染毒后6h处死动物,制取胸骨骨髓推片,固定,染色,观察计数红细胞,每只小鼠计数1000个PCE,观察微核的数。
结果:
体内外的生物学试验结果表明:该材料无明显细胞毒性、溶血性,无急性全身毒性、致敏性、口腔粘膜刺激性和至突变性。
1.细胞毒性试验纳米基托树脂、普通基托树脂与阴性对照在试验中的吸光度值无显著性差异(P＞0.05);毒性均为1级;倒置相差显微镜观察细胞形态未见改变。
2.溶血试验材料样品的溶血率小于5%,参照ISO/TR7405-1984(E)规定溶血试验合格。
3.急性经口全身毒性试验一周观察期内,动物未见中毒症状和不良反应,亦无死亡,LD50＞15000mg/kg,根据急性毒性分级标准,受试物样品混悬液属实际无毒。
4.口腔粘膜刺激试验实验观察:通过两周的观察发现,动物活动、饮食正常:局部粘膜无异常反应,如充血、出血、糜烂、溃疡等,与阴性对照无显著性差异。
组织切片:两种材料组织切片均显示上皮连续性完好,细胞层次清楚,排列均匀。
皮下组织疏松,腺体形态规则,细胞排列整齐,结缔组织中无炎细胞浸润,与阴性对照组的组织切片无显著性差异。
5.皮肤致敏试验
激发接触后注射受试物样品的各点及阴性对照的各点皮肤无不良的变化;阳性对照的各点激发后24h皮肤水肿显著,红斑中度或重度,后期糜烂,节痂。根据致敏率分级标准,该材料属弱致敏物。
6.骨髓细胞微核试验
本试验各组骨髓细胞组成PCE/NCE在正常范围内,未见明显抑制。雌雄小鼠微核未见显著性差异(P＞0.05),故将数据合并统计,得出受试物样品各剂量组与阴性对照无显著性差异(P＞0.05),阴性对照与阳性对照有显著性差异(P＜0.01)。
结论:
纳米蒙脱土/聚甲基丙烯酸甲酯纳米义齿基托复合材料经初步筛选实验证明具有良好的生物安全性。该新型口腔材料经细胞毒性实验,溶血实验,急性经口毒性试验、皮肤致敏反应试验、口腔黏膜刺激试验、和骨髓细胞微核试验等综合分析显示,该材料无明显细胞毒性、溶血性,无急性全身毒性、致敏性、口腔黏膜刺激性和至突变性,其可靠的生物学安全性为之尽快进入临床应用阶段提供了有利依据。
Objective:
To evaluate the biological safety of the nanometer OMMT/(P)MMA denture composite material before clinical application,but also improve the mechanical performance of the artificial teeth.
Methods:
According to the standard of ISO 7405-1997(E),cytotoxicity test,haemolysis test,acute systemic toxicity test(by oral),oral mucous membrane irritating test, sensitization test and micronucleus assay were investigated on the nanometer OMMT(P)MMA denture composite material
1.Cytotoxicity test:
The proliferations of the Vero cells were investigated using atetrazolium-based colorimetric assay(MTT assay).Eluates from two tested materials were added into 96-well culture plates.The samples were tested with ultraviolet spectrophotometer in 492 nm wavelength at 2,4,7 days after the adding of the extracts. Morphology of these cells was also observed with phase-contrast microscope.
2.Haemolysis test:
Added the dilute blood of rabbit 0.2ml into the normal saline maceration extract of the sample-material,centrifugated for five minutes,took supematant and moved it into color comparator,assayed every cylinder's optical densit(O.D.).
3.Acute systemic toxicity test by oral(limit experiment):
Irrigated the KunMing mice's stomach with the normal saline maceration extract of the sample-material at the dose of 15000mg/kg,observed the little mice's general condition,venenous manifest and the number of the dead in a week after they were irrigated.
4.Oral mucous membrane irritating test:
Sutured the materials round pieces with ophthalmology bullet and line on bilateral buccal bursa of healthy Golden hamsters,In two weeks observed the general reaction,oral mucosa reaction in contacted site,killed the animals after two weeks and did pathological examination of local oral mucosa with hematoxylin and eosin(HE)stain and observed the tissue cells changes of them
5.Skin sensitization test:
Selected white healthy male guinea pig,induced to contact them with the positive control of 10%dinitrocholorobenzne(DNCB),the negative control of normal saline maceration extract of the sample-material,Freund complete adjuvant(FCA),the emulsion of the tested material and FCA,stimulated to contact and observed the result of stimulation in 24 and 48 hours.
6.Micronucleus assay:
Selected healthy KunMing white mice and divided into high,middle,low dose groups,irrigated stomachs with suspended liquid on dose of 15.0g/kg,7.5g/kg,3.75g/kg individually.The positive control was injected into abdomens with Cyelophosphamide on 40 mg/kg.The negative control was irrigating stomachs with normal saline.Contaminated the mice and contaminated again after 24 hours.After 6 hours killed the animals,made sternum marrow slicings,fixed,stained,observed red cell count,every mouse was counted 1000 polychromatic erythrocytes(PCE),observed the number of micronucleus.
Results:
The intracorporal and extracorporal biology experimental results indicated:there was almost no obvious cytotoxicity,no heamolysis,no acute systemic toxicity,no sensitivity,no irritant reaction on oral mucous membrane and no potential mutation..
1.Cytotoxicity test:
There was no significant difference between the nanometer OMMT/(P)MMA denture composite material and the common PMMA denture resin material (P＞0.05);The toxicity grades.of the two kinds of material are 1,The cells growing in the extract showed normal morphology.
2.Haemolysis test:
The Haemolysis rate of the sample-material was lower than 5%,consulted ISO/TR 7405-1984(E)the result of haemolysis experiment was up to ISO standard.
3.Acute systemic toxicity test by oral:
In a week there were no toxicity symptoms on the animals and there was no death. LD50 was more than 15000mg/kg.According to the standard of acute toxicity grade, the suspended liquid tested was no toxicity actually.
4.Oral mucous membrane irritating test:
Experiment observation:The animals' exercise and diet was normal with two week's observation.There was no abnormal reaction on local mucous membrane such as hyperemia,haemorrhage,erosion,ulcer and so on..There was no significant difference with the negative control.
Slicings of tissue:
The tissue slicings of the two kinds of materials showed:the epitheliums were complete,cells stratifications were clear,the permutations were well-distributed. Hypodermises were Rarefaction,the forms of glands were regular,there was no inflammation cell infiltration in connective tissue,all these had no significant difference with tissue slicings of the negative control group.
5.Skin sensitization test:
There was no patho-change on the skin of tested sample-material and the negative contrast,but the skin of postive contrast showed obvious oedema and middle or serious erythema in 24 hours after stimulated,later showed erosion and scab.
6.Micronucleus assay:
The sternum marrow cells formation(PCE/NCE)of this experiment was normal and no obvious inhibition was found.There was no significant difference between female and male little mice's micronucleus(P＞0.05),so amalgamated and counted the date,founded that there was no significant difference between every dose group of tested sample-material and the negative contrast(P＞0.05),there was significant difference between the postive control and the negative control(P＜0.01)
Conclusions:
The nanometer OMMT/(P)MMA denture composite material was proved to have good biological safety by preliminary selected tests.
本研究通过将纳米级蒙脱土(OMMT)粉体在聚合物(PMMA)基材中均匀分散,获得新型的纳米蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料,并对蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料进行生物安全性的临床前评价。为义齿复合材料的研究和开发提供理论依据和参考。
方法:
根据ISO7405-1997“牙科学——用于牙科的医疗器械生物相容性临床前评价——牙科材料实验方法”国际标准,对纳米蒙脱土/聚甲基丙烯酸甲酯义齿基托复合材料进行了细胞毒性试验、溶血试验、急性经口全身毒性试验、口腔粘膜刺激试验、皮肤致敏试验和骨髓细胞微核试验。
1、细胞毒性试验细胞毒性试验是一类在离体状态下模拟生物体生长环境,检测材料和器械接触机体后生物学反应的体外试验,它是生物学体系评价中最重要的检测指标之一,一般列为首选和必选项目。本试验是采用Verocell(非洲绿猴肾细胞,CCL81)体外培养的四唑盐比色实验和材料浸提液培养细胞的形态学观察法。
2、溶血试验本研究采用材料直接以及采用经不同浸提条件下制得的材料浸提液两种方式,在体外模拟生物体的温度与稀释兔血接触,相互作用一定时间后,测定其光吸收度,从而分别判断材料的溶血性能,其目的是研究生物材料不同接触方式和浸提条件对溶血作用的影响,以试图提高体外方法检测生物材料生物安全性的可靠性和科学性。材料样品的生理盐水浸提液加稀释兔血0.2ml,离心5min,吸取上清液移入比色皿内,测定各管吸光度。
3、急性经口全身毒性试验用材料样品的生理盐水浸提液以1500mg/kg的剂量给昆明小鼠灌胃,灌胃后一周内观察小鼠一般状态及毒性表现和死亡数。
4、口腔粘膜刺激试验将材料圆片用眼科针线缝合固定在健康仓鼠颊囊两侧,缝合固定后观察两周,观察动物全身反应,接触局部的粘膜反应,两周处死动物,局部粘膜作组织切片,HE染色,观察粘膜的组织细胞反应。
5、皮肤致敏试验选白色雄性健康豚鼠,分别用阳性对照为10%二硝基氯苯,阴性对照为生理盐水,材料样品的生理盐水浸提液,完全弗氏佐剂(FCA)及完全弗氏佐剂和受试物的乳化剂进行诱导接触,激发接触,在24h,48h观察激发接触结果。
6、骨髓细胞微核试验选用健康昆明种小白鼠设高、中、低三个剂量组,分别按15.0g/kg、7.5g/kg、3.75g/kg的比例给予材料混悬液灌胃;阳性对照为40mg/kg的环磷酰胺,用腹腔注射;阴性对照为生理盐水灌胃。第一次染毒后24小时再次染毒,末次染毒后6h处死动物,制取胸骨骨髓推片,固定,染色,观察计数红细胞,每只小鼠计数1000个PCE,观察微核的数。
结果:
体内外的生物学试验结果表明:该材料无明显细胞毒性、溶血性,无急性全身毒性、致敏性、口腔粘膜刺激性和至突变性。
1.细胞毒性试验纳米基托树脂、普通基托树脂与阴性对照在试验中的吸光度值无显著性差异(P＞0.05);毒性均为1级;倒置相差显微镜观察细胞形态未见改变。
2.溶血试验材料样品的溶血率小于5%,参照ISO/TR7405-1984(E)规定溶血试验合格。
3.急性经口全身毒性试验一周观察期内,动物未见中毒症状和不良反应,亦无死亡,LD50＞15000mg/kg,根据急性毒性分级标准,受试物样品混悬液属实际无毒。
4.口腔粘膜刺激试验实验观察:通过两周的观察发现,动物活动、饮食正常:局部粘膜无异常反应,如充血、出血、糜烂、溃疡等,与阴性对照无显著性差异。
组织切片:两种材料组织切片均显示上皮连续性完好,细胞层次清楚,排列均匀。
皮下组织疏松,腺体形态规则,细胞排列整齐,结缔组织中无炎细胞浸润,与阴性对照组的组织切片无显著性差异。
5.皮肤致敏试验
激发接触后注射受试物样品的各点及阴性对照的各点皮肤无不良的变化;阳性对照的各点激发后24h皮肤水肿显著,红斑中度或重度,后期糜烂,节痂。根据致敏率分级标准,该材料属弱致敏物。
6.骨髓细胞微核试验
本试验各组骨髓细胞组成PCE/NCE在正常范围内,未见明显抑制。雌雄小鼠微核未见显著性差异(P＞0.05),故将数据合并统计,得出受试物样品各剂量组与阴性对照无显著性差异(P＞0.05),阴性对照与阳性对照有显著性差异(P＜0.01)。
结论:
纳米蒙脱土/聚甲基丙烯酸甲酯纳米义齿基托复合材料经初步筛选实验证明具有良好的生物安全性。该新型口腔材料经细胞毒性实验,溶血实验,急性经口毒性试验、皮肤致敏反应试验、口腔黏膜刺激试验、和骨髓细胞微核试验等综合分析显示,该材料无明显细胞毒性、溶血性,无急性全身毒性、致敏性、口腔黏膜刺激性和至突变性,其可靠的生物学安全性为之尽快进入临床应用阶段提供了有利依据。
Objective:
To evaluate the biological safety of the nanometer OMMT/(P)MMA denture composite material before clinical application,but also improve the mechanical performance of the artificial teeth.
Methods:
According to the standard of ISO 7405-1997(E),cytotoxicity test,haemolysis test,acute systemic toxicity test(by oral),oral mucous membrane irritating test, sensitization test and micronucleus assay were investigated on the nanometer OMMT(P)MMA denture composite material
1.Cytotoxicity test:
The proliferations of the Vero cells were investigated using atetrazolium-based colorimetric assay(MTT assay).Eluates from two tested materials were added into 96-well culture plates.The samples were tested with ultraviolet spectrophotometer in 492 nm wavelength at 2,4,7 days after the adding of the extracts. Morphology of these cells was also observed with phase-contrast microscope.
2.Haemolysis test:
Added the dilute blood of rabbit 0.2ml into the normal saline maceration extract of the sample-material,centrifugated for five minutes,took supematant and moved it into color comparator,assayed every cylinder's optical densit(O.D.).
3.Acute systemic toxicity test by oral(limit experiment):
Irrigated the KunMing mice's stomach with the normal saline maceration extract of the sample-material at the dose of 15000mg/kg,observed the little mice's general condition,venenous manifest and the number of the dead in a week after they were irrigated.
4.Oral mucous membrane irritating test:
Sutured the materials round pieces with ophthalmology bullet and line on bilateral buccal bursa of healthy Golden hamsters,In two weeks observed the general reaction,oral mucosa reaction in contacted site,killed the animals after two weeks and did pathological examination of local oral mucosa with hematoxylin and eosin(HE)stain and observed the tissue cells changes of them
5.Skin sensitization test:
Selected white healthy male guinea pig,induced to contact them with the positive control of 10%dinitrocholorobenzne(DNCB),the negative control of normal saline maceration extract of the sample-material,Freund complete adjuvant(FCA),the emulsion of the tested material and FCA,stimulated to contact and observed the result of stimulation in 24 and 48 hours.
6.Micronucleus assay:
Selected healthy KunMing white mice and divided into high,middle,low dose groups,irrigated stomachs with suspended liquid on dose of 15.0g/kg,7.5g/kg,3.75g/kg individually.The positive control was injected into abdomens with Cyelophosphamide on 40 mg/kg.The negative control was irrigating stomachs with normal saline.Contaminated the mice and contaminated again after 24 hours.After 6 hours killed the animals,made sternum marrow slicings,fixed,stained,observed red cell count,every mouse was counted 1000 polychromatic erythrocytes(PCE),observed the number of micronucleus.
Results:
The intracorporal and extracorporal biology experimental results indicated:there was almost no obvious cytotoxicity,no heamolysis,no acute systemic toxicity,no sensitivity,no irritant reaction on oral mucous membrane and no potential mutation..
1.Cytotoxicity test:
There was no significant difference between the nanometer OMMT/(P)MMA denture composite material and the common PMMA denture resin material (P＞0.05);The toxicity grades.of the two kinds of material are 1,The cells growing in the extract showed normal morphology.
2.Haemolysis test:
The Haemolysis rate of the sample-material was lower than 5%,consulted ISO/TR 7405-1984(E)the result of haemolysis experiment was up to ISO standard.
3.Acute systemic toxicity test by oral:
In a week there were no toxicity symptoms on the animals and there was no death. LD50 was more than 15000mg/kg.According to the standard of acute toxicity grade, the suspended liquid tested was no toxicity actually.
4.Oral mucous membrane irritating test:
Experiment observation:The animals' exercise and diet was normal with two week's observation.There was no abnormal reaction on local mucous membrane such as hyperemia,haemorrhage,erosion,ulcer and so on..There was no significant difference with the negative control.
Slicings of tissue:
The tissue slicings of the two kinds of materials showed:the epitheliums were complete,cells stratifications were clear,the permutations were well-distributed. Hypodermises were Rarefaction,the forms of glands were regular,there was no inflammation cell infiltration in connective tissue,all these had no significant difference with tissue slicings of the negative control group.
5.Skin sensitization test:
There was no patho-change on the skin of tested sample-material and the negative contrast,but the skin of postive contrast showed obvious oedema and middle or serious erythema in 24 hours after stimulated,later showed erosion and scab.
6.Micronucleus assay:
The sternum marrow cells formation(PCE/NCE)of this experiment was normal and no obvious inhibition was found.There was no significant difference between female and male little mice's micronucleus(P＞0.05),so amalgamated and counted the date,founded that there was no significant difference between every dose group of tested sample-material and the negative contrast(P＞0.05),there was significant difference between the postive control and the negative control(P＜0.01)
Conclusions:
The nanometer OMMT/(P)MMA denture composite material was proved to have good biological safety by preliminary selected tests.
引文
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