牙种植高分子材料聚甲基丙烯酸甲酯的生物相容性

doi:10.3969/j.issn.2095-4344.2015.47.012

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (47): 7613-7618.doi: 10.3969/j.issn.2095-4344.2015.47.012

• 材料生物相容性 material biocompatibility • 上一篇下一篇

牙种植高分子材料聚甲基丙烯酸甲酯的生物相容性

李小东¹，李新梅¹，孙晓晨¹，孙翔²

1延安市人民医院口腔科，陕西省延安市 716000；2解放军第四军医大学口腔医院修复科，陕西省延安市 710032

收稿日期:2015-10-13 出版日期:2015-11-19 发布日期:2015-11-19
通讯作者: 孙翔，副主任医师，医学博士，解放军第四军医大学口腔医院修复科，陕西省延安市 710032
作者简介:李小东，男，1976年生，2010年解放军第四军医大学口腔医院毕业，硕士，副主任医师。
基金资助:
国家自然基金项目(81371187)；延安市科技计划项目(2011ks-05)

Biocompatibility of polymethylmethacrylate as a polymer material for dental implants

Li Xiao-dong¹, Li Xin-mei¹, Sun Xiao-chen¹, Sun Xiang²

1Department of Stomatology, Yan’an People’s Hospital, Yan’an 716000, Shaanxi Province, China; 2Department of Prosthodontics, School of Stomatology, the Fourth Military Medical University of PLA, Yan’an 710032, Shaanxi Province, China

Received:2015-10-13 Online:2015-11-19 Published:2015-11-19
Contact: Sun Xiang, Associate chief physician, M.D., Department of Prosthodontics, School of Stomatology, the Fourth Military Medical University of PLA, Yan’an 710032, Shaanxi Province, China
About author:Li Xiao-dong, Master, Associate chief physician, Department of Stomatology, Yan’an People’s Hospital, Yan’an 716000, Shaanxi Province, China
Supported by:
the National Natural Science Foundation of China, No. 81371187; Science and Technology Program of Yan’an of China, No. 2011ks-05

摘要/Abstract

摘要：

BACKGROUND: As a kind of dental implant material, the application of titanium has certain restrictions because of its higher probability of postoperative bleeding rate, infection and gingival hyperplasia. Studies have shown that polymethylmethacrylate has been used in artificial joints and artificial bones, but rarely reported to be used as dental implant material.
OBJECTIVE: To explore the biocompatibility indexes such as cytotoxicity, cell adhesion rate, relative cell proliferation rate and post-implantation inflammatory response of human osteoblasts when pure titanium and polymethylmethacrylate are used as dental implant materials, so as to provide certain reference basis for the clinical usage of polymethylmethacrylate as the dental implant material.
METHODS: Human osteoblasts were cultured in vitro. Three groups were divided as follows: control group
(cells cultured normally), pure titanium group (cells cultured with titanium extract) and polymethylmethacrylate group (cells cultured with polymethylmethacrylate extract).
RESULTS AND CONCLUSION: Compared with the control group, the cell adhesion rate was significantly decreased after 2, 4, 8 and 16 hours of culture with pure titanium and polymethylmethacrylate extracts (P < 0.05); the cell adhesion rate in the polymethylmethacrylate group was higher than that in the pure titanium group (P < 0.05). Compared with the control group, the cells were sparse and grew slowly after 2 days of culture with pure titanium and polymethylmethacrylate extracts. Cells in the polymethylmethacrylate group grew faster with fusiform distribution and obvious drawing phenomenon. Compared with the control group, the relative cell proliferation rate was significantly decreased after 2 days of culture with pure titanium and polymethylmethacrylate extracts (P < 0.05); the relative cell proliferation rate of polymethylmethacrylate group was higher than that of the pure titanium group (P < 0.05). The expression of inflammatory factors in rat serum was significantly increased after 7 days of implantation of titanium and polymethylmethacrylate materials (P < 0.05), the expression of inflammatory factors in the polymethylmethacrylate group was less than that in the titanium group (P < 0.05). There was only one rat developing allergic reaction, but no pyrogen reaction and no death in the polymethylmethacrylate group; and three rats presented with allergic reaction, one rat present with pyrogen reaction and no death occurred in the pure titanium group. These results demonstrate that as the dental implant material, polymethylmethacrylate is superior to pure titanium in the cell toxicity, inflammatory response and biocompatibility.
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 生物材料, 口腔生物材料, 聚甲基丙烯酸甲酯, 纯钛, 牙种植材料, 生物相容性, 细胞毒性, 相对增殖率, 炎症因子, 急性毒性, 过敏, 热原反应, 国家自然科学基金

Abstract:

Key words: Tissue Engineering, Biocompatible Materials, Titanium

李小东，李新梅，孙晓晨，孙翔. 牙种植高分子材料聚甲基丙烯酸甲酯的生物相容性[J]. 中国组织工程研究, 2015, 19(47): 7613-7618.

Li Xiao-dong, Li Xin-mei, Sun Xiao-chen, Sun Xiang. Biocompatibility of polymethylmethacrylate as a polymer material for dental implants[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(47): 7613-7618.

图/表（结果） 5

2.1 体外细胞实验结果 2.1.1 两种材料浸提液培养不同时程后细胞贴壁率实验结果显示，与空白对照组相比，加入纯钛提取液或聚甲基丙烯酸甲酯浸提液分别培养hFOB1.19细胞2，4，8，16 h后，细胞贴壁率均明显降低(P < 0.05)，其中，聚甲基丙烯酸甲酯组的细胞贴壁率明显高于纯钛组(P < 0.05)。见表1。

2.1.2 两种材料浸提液培养2 d后细胞的生长情况对比与空白对照组相比，加入纯钛浸提液和聚甲基丙烯酸甲酯提取液培养hFOB1.19细胞2 d后，细胞较稀疏，生长缓慢，其中，聚甲基丙烯酸甲酯组与纯钛组相比，细胞生长较快，形态饱满，呈梭形，拉丝现象不明显。见图1。

2.1.3 MTT法检测两种材料浸提液培养2 d后细胞的相对增殖率以及材料毒性等级 MTT实验结果显示，与空白对照组相比，加入纯钛或聚甲基丙烯酸甲酯提取液培养细胞 2 d后，细胞相对增殖率明显降低(P < 0.05)，其中，聚甲基丙烯酸甲酯组的细胞相对增殖率明显高于纯钛组(P < 0.05)。纯钛组和聚甲基丙烯酸甲酯组的材料毒性等级均合格，见表2。

2.2 动物体内实验结果

2.2.1 实验动物数量分析纳入大鼠30只均进入结果分析，无实验外的死亡和感染，无脱失值。

2.2.2 大鼠血清中炎性因子的表达情况 ELISA结果显示，与空白对照组相比，Wistar大鼠上颌种植纯钛体或聚甲基丙烯酸甲酯体7 d后，血清中炎性细胞因子肿瘤坏死因子a和白细胞介素1β表达明显增加(P < 0.05)，其中，种植聚甲基丙烯酸甲酯材料组的炎性因子较纯钛组少(P < 0.05)，见表3。

2.2.3 两种材料种植后，对大鼠全身毒性、过敏和热原反应的影响种植聚甲基丙烯酸甲酯材料组仅有1只大鼠发生过敏反应，无热原反应和死亡；种植纯钛材料组的大鼠有3只发生过敏反应，1只发生热原反应，无死亡。见表4。

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引言

材料方法

1.1 设计随机对照动物实验。

1.2 时间及地点：于2014年7月至2015年7月在延安市人民医院中心实验室完成。

1.3 材料

实验动物：健康8周龄Wistar大鼠30只，购于南京君科生物工程有限公司，体质量200-300 g，许可证号：SYXK (苏)2014-0014；动物于SPF级环境中饲养，温度18-26 ℃；相对湿度40%-70%；给予pH 2.5-2.8的酸化水，动物自由摄食。

牙种植材料：聚甲基丙烯酸甲酯牙种植材料购自上海众荣新材料科技有限公司，型号：MB-1PW，由日本东亚公司合成。纯钛牙种植材料：购自北京莱顿生物材料有限公司，型号：YY0304-1998。

牙种植高分子材料生物相容性实验用主要试剂及材料：
试剂及材料	来源
聚甲基丙烯酸甲酯牙种植材料	上海众荣新材料科技有限公司
纯钛牙种植材料	北京莱顿生物材料有限公司
MTT试剂盒	上海歌凡生物科技有限公司
ELISA试剂盒	武汉博士德生物工程有限公司
hFOB1.19细胞，DMEM培养基	上海基免生物科技有限公司

1.4 实验方法

1.4.1 分组及处理 ①实验在细胞水平观察分为3组，每组10只，分别为空白对照组(正常DMEM培养基)、纯钛组(纯钛浸提液培养)、聚甲基丙烯酸甲酯(聚甲基丙烯酸甲酯浸提液培养)。②实验根据种植材料不同分组为3组，每组10只(与上述分组动物为同一批)，空白对照组(未种植组)；种植聚甲基丙烯酸甲酯组(聚甲基丙烯酸甲酯组)；种植纯钛组(纯钛组)。

1.4.2 体外细胞实验

细胞计数法检测细胞贴壁率：将2种种植材料用PBS冲洗3次，放入紫外操作台中照射3 h；置于35 mm培养皿中，加入2 mL DMEM培养基，于体积分数5%CO₂、37 ℃

的培养箱中培养48 h，获得浸提液；hFOB1.19细胞以 2×10⁸ L^-¹的细胞浓度铺于35 mm皿中，加入获取的浸提液(每皿加入2 mL)，分别于培养2，4，8，16 h后，去除培养基，用PBS冲洗3次，用0.25%胰酶消化后进行细胞计数。细胞贴壁率=(贴壁细胞数/接种细胞数)×100%^[6]。

细胞培养与形态学观察：将两种材料用PBS冲洗3次，放入紫外操作台中照射6 h；置于35 mm培养皿中，加入 2 mL培养基DMEM，于体积分数5%CO₂、37 ℃的培养箱中培养48 h，获得浸提液；hFOB1.19细胞以2×10⁵ L^-1的细胞浓度种于35 mm小皿中，分别用正常DMEM培养基、DMEM+纯钛提取液、DMEM+聚甲基丙烯酸甲酯提取液各2 mL培养细胞48 h后观察细胞形态学变化^[7]。

MTT法检测细胞毒性：将两种种植材料用PBS冲洗3次，放入紫外操作台中照射3 h；置于35 mm培养皿中，加入2 mL DMEM培养基，于体积分数5%CO₂、37 ℃的培养箱中培养48 h，获得浸提液；hFOB1.19细胞以3 500 L^-¹的浓度铺于96孔板上，加入获取的浸提液(每孔加入200 μL)培养48 h后，在96孔板中加入MTT稀释溶液各20 μL，继续放入培养箱中分别培养2 d后终止，在酶标仪紫外线450 nm处读取各孔的吸光度。计算细胞的相对增殖率RGR=(实验组吸光度/对照组吸光度)×100%^[8]。

ELISA检测：种植材料7 d后，观察大鼠血清中炎症因子的变化情况：选Wistar大鼠右上颌，种植不同材料7 d后，大鼠下腔静脉取血，静置1 h后，离心取上清，将不同浓度的标准品各0.1 mL依次加入一排7孔中，加入稀释的待检测样品0.1 mL，置于37 ℃孵育90 min，甩去酶标板中的液体。将准备好的抗体工作液按每孔0.1 mL依次加入，置于37 ℃孵育60 min，用PBS洗涤3次，每次浸泡约1 min；将准备好的ABC工作液按每孔0.1 mL依次加入，置于37 ℃孵育30 min，用PBS洗涤5次，每次浸泡约1 min；按每孔90 μL依次加入TMB显色液，置于37℃避光反应15- 20 min；按每孔100 μL依次加入TMB终止液，此时蓝色立刻转为黄色，用酶标仪在紫外线450 nm处测定吸光度^[9]。

合成材料毒性等级评分：
合成材料毒性等级	毒性分级
100%及以上	0(合格)
75%-99%	1(合格)
50%-74%	2(待定)
25%-49%	3(不合格)
小于24%	4(不合格)

1.4.3 不良反应评估两种材料种植7 d后，观察大鼠全身毒性、过敏、热原反应的观察指标变化，将大鼠用戊巴比妥钠麻醉，进行上颌牙种植实验，观察期为7 d^[10]。全身毒性：体温升高至41 ℃以上，呼吸急促，打喷嚏，流鼻涕，有时下痢，全身发抖，四肢抽搐、尿便失禁；过敏反应：呼吸急促、爬伏、软瘫；热原反应：体温迅速升高。

1.5 主要观察指标各组细胞贴壁率、细胞相对增殖率、血清中炎性因子表达。

1.6 统计学分析采用SPSS 17.0统计软件进行数据分析，故计量数据以x(_)±s表示，由于数据服从正态分布，组间数据差异的比较采用单因素方差分析和t 检验，P < 0.05表示差异有显著性意义。

讨论

聚甲基丙烯酸甲酯作为牙种植体的分子材料具有较为优越的生物相容性，将取代传统的钛合金牙种植材料。对于牙缺失的患者，种植牙是一个提高生活质量的选择。种植牙是将金属种植体(相当于牙根)植入牙龈下的牙槽骨中，使其能与周围的骨形成类似真牙牙根与牙槽骨结合的稳定形式^[11-13]。然后医生在种植体上面作烤瓷冠修复，以求完美的软组织美学效果^[14]。目前种植牙的材料临床多用钛合金种植体，它有良好的生物相容性。但是近年来的研究发现，钛作为牙种植材料，虽应用广泛，但术后的出血率、发生感染和牙龈增生的概率较高^[15]，因而，使用高分子材料替代钛合金成了研究的热点^[16-17]。聚甲基丙烯酸甲酯是一种合成高分子材料，透明性极好，强度高，耐光老化，是一种质轻而坚韧，在常温下具有优良的拉伸强度，弯曲强度，拉伸强度的材料，现已经广泛应用于灯具、照明器材、医用玻璃等领域^[18-20]；聚甲基丙烯酸甲酯作为人体代谢过程中必不可少的中间代谢产物，主要参与生物体内的尿素循环，用于生物体内瓜氨酸、精氨酸、脯氨酸、多胺的生物合成^[21-24]。

研究主要通过探究纯钛和聚甲基丙烯酸甲酯高分子材料作为牙种植材料对于人成骨细胞的细胞毒性、细胞贴壁率、相对增殖率以及种植后所引起的炎性反应等生物相容性指标的比较^[25-27]。

实验结果显示，纯钛和聚甲基丙烯酸甲酯提取液培养细胞不同时程后，细胞贴壁率均明显较空白对照组降低，其中，聚甲基丙烯酸甲酯组的细胞贴壁率高于纯钛组；纯钛和聚甲基丙烯酸甲酯提取液培养细胞2 d后，细胞较空白对照组稀疏，生长速度缓慢，其中，聚甲基丙烯酸甲酯组细胞生长较快，呈梭形，拉丝现象不明显；纯钛和聚甲基丙烯酸甲酯提取液培养细胞2 d后，细胞相对增殖率与空白对照组相比，均明显降低，其中，聚甲基丙烯酸甲酯组的细胞增殖率高于纯钛组；种植纯钛和聚甲基丙烯酸甲酯材料7天后，大鼠血清中炎性因子明显增加，其中，种植聚甲基丙烯酸甲酯组的炎性因子较纯钛组少；聚甲基丙烯酸甲酯组仅有1只大鼠发生过敏反应，无热原反应和死亡，而纯钛组有3只大鼠发生过敏反应，1只发生热原反应，无死亡。

以上实验结果说明，聚甲基丙烯酸甲酯作为牙种植材料，在细胞毒性、种植后引发的炎症反应以及生物相容性方面均优于纯钛，实验创新点主要在于将种植材料毒性、生物相容性等方面从细胞学角度和动物角度两方面入手分析，这也为临床上牙种植材料的创新和应用提供了科学依据。关于聚甲基丙烯酸甲酯高分子材料在人体牙种植中的具体应用将是作者下一步的研究重点。
中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

牙种植高分子材料聚甲基丙烯酸甲酯的生物相容性

Biocompatibility of polymethylmethacrylate as a polymer material for dental implants

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