三种方法建立大鼠种植体周炎模型的比较

doi:10.19723/j.issn.1671-167X.2023.01.004

北京大学学报（医学版） ›› 2023, Vol. 55 ›› Issue (1): 22-29. doi: 10.19723/j.issn.1671-167X.2023.01.004

三种方法建立大鼠种植体周炎模型的比较

孟令玮¹,李雪²,高胜寒¹,李悦¹,曹瑞涛¹,张毅^2,*(),潘韶霞^1,*()

1. 北京大学口腔医学院·口腔医院修复科，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，口腔数字医学北京市重点实验室，国家卫生健康委员会口腔医学计算机应用工程技术研究中心，国家药品监督管理局口腔生物材料重点实验室，北京 100081
2. 军事科学院军事医学研究院辐射医学研究所，北京 100850

收稿日期:2022-10-11 出版日期:2023-02-18 发布日期:2023-01-31
通讯作者: 张毅,潘韶霞 E-mail:zhangyi612@hotmail.com;panshaoxia@vip.163.com
基金资助:
国家重点研发计划(2020YFC2009005);国家重大疾病多学科合作诊疗能力建设项目(PKUSSNMP-202004);北京市自然科学基金(7222228)

Comparison of three methods for establishing rat peri-implantitis model

Ling-wei MENG¹,Xue LI²,Sheng-han GAO¹,Yue LI¹,Rui-tao CAO¹,Yi ZHANG^2,*(),Shao-xia PAN^1,*()

1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digi-tal Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
2. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

Received:2022-10-11 Online:2023-02-18 Published:2023-01-31
Contact: Yi ZHANG,Shao-xia PAN E-mail:zhangyi612@hotmail.com;panshaoxia@vip.163.com
Supported by:
the National Key R & D Program of China(2020YFC2009005);National Program for Multidisciplinary Cooperative Treatment on Major Diseases(PKUSSNMP-202004);Beijing Natural Science Foundation(7222228)

摘要/Abstract

摘要：

目的: 比较传统棉线结扎与种植体周围局部注射牙龈卟啉单胞菌脂多糖(lipopolysaccharide，LPS)诱导法，以及两种方法联合建立大鼠种植体周炎模型的效率和效果，以探索大鼠种植体周炎模型建模的最佳方法。方法: 纳入39只雄性SD大鼠，拔除上颌左侧第一磨牙，拔牙窝愈合4周后植入钛种植体。种植体植入4周骨结合后，将39只大鼠随机分为4组，分别采取棉线结扎(n=12)、种植体周围局部注射LPS(n=12)，以及两种方法联合(n=12)诱导大鼠种植体周炎，同时设无任何处理对照组(n=3)。记录实验组大鼠诱导前及诱导后2周和4周时的探诊深度(probing depth，PD)、探诊出血(bleeding on probing，BOP)和牙龈指数(gingival index，GI)。收集种植体周围牙龈组织，提取RNA，实时荧光定量分析(real time quantitative PCR，RT-qPCR)检测炎症因子白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-alpha，TNF-α)表达。分别于诱导2周和4周时处死实验组动物，收集上颌骨，采用微型CT(micro-CT)观察种植体边缘骨吸收状况。结果: 种植4周后，种植体骨结合良好。3个实验组均可见牙龈红肿，质软，种植体边缘骨吸收。诱导2周和4周时，各实验组PD、GI和BOP均较诱导前增加，但仅BOP在3组间差异有统计学意义(P < 0.05)。炎症诱导2周时，棉线结扎组和联合组在每个位点均可见边缘骨吸收，且联合组每个位点边缘骨吸收程度均大于棉线结扎组，但差异不具有统计学意义(P > 0.05)；局部注射组部分种植体的部分位点可见边缘骨吸收。炎症诱导4周时，各实验组各位点均可见边缘骨吸收，其中棉线结扎组和联合组边缘骨吸收大于局部注射组，差异有统计学意义(P < 0.05)。诱导2周和4周时，实验组TNF-α和IL-1β均较对照组表达增加，差异有统计学意义(P < 0.05)。结论: 相比于种植体周围局部注射LPS，棉线结扎及两种方法联合可以更好且更快地诱导大鼠种植体周炎。

关键词: 种植体周炎, 动物模型, 大鼠, 棉线结扎, 脂多糖局部注射

Abstract:

Objective: To compare the efficiency and effect of establishing rat peri-implantitis model by traditional cotton thread ligation and local injection of Porphyromonas gingivalis lipopolysaccharide (LPS) around the implant, as well as the combination of the two methods. Methods: Left side maxillary first molars of 39 male SD rats were extracted, and titanium implants were implanted after four weeks of healing. After 4 weeks of implant osseointegration, 39 rats were randomly divided into 4 groups. Cotton thread ligation (n=12), local injection of LPS around the implant (n=12), and the two methods combined (n=12) were used to induce peri-implantitis, the rest 3 rats were untreated as control group. All procedures were conducted under 5% isoflurane inhalation anesthesia. The rats were sacrificed 2 weeks and 4 weeks after induction through carbon dioxide asphyxiation method. The maxilla of the rats in the test groups were collected and marginal bone loss was observed by micro-CT. The gingival tissues around the implants were collected for further real time quantitative PCR (RT-qPCR) analysis, specifically the expression of tumor necrosis factor-alpha (TNF-α) as well as interleukin-1β (IL-1β). The probing depth (PD), bleeding on probing (BOP) and gingival index (GI) of each rat in the experimental group were recorded before induction of inflammation and before death. Results: After 4 weeks of implantation, the osseointegration of implants were confirmed. All the three test groups showed red and swollen gums, obvious marginal bone loss around implants. After 2 weeks and 4 weeks of inflammation induction, PD, GI and BOP of the three test groups increased compared with those before induction, but only BOP was statistically significant among the three test groups (P < 0.05). At the end of 2 weeks of inflammation induction, marginal bone loss was observed at each site in the cotton thread ligation group and the combined group. At each site, the bone resorption in the combined group was greater than that in the cotton thread ligation group, but the difference was not statistically significant (P > 0.05), bone resorption was observed at some sites of some implants in LPS local injection group. At the end of 4 weeks of inflammation induction, marginal bone loss was observed at all sites in each group. The marginal bone loss in the cotton thread ligation group and the combined group was greater than that in the LPS local injection group, and the difference was statistically significant (P < 0.05). At the end of 2 weeks and 4 weeks of induction, the expression of TNF-α and IL-1β in the test groups were higher than those in the control group (P < 0.05). Conclusion: Compared with local injection of LPS around the implant, cotton thread ligature and the two methods combined can induce peri-implantitis in rats better and faster.

Key words: Peri-implantitis, Animal models, Rats, Cotton thread ligation, Lipopolysaccharide local injection

中图分类号:

R781.42

孟令玮,李雪,高胜寒,李悦,曹瑞涛,张毅,潘韶霞. 三种方法建立大鼠种植体周炎模型的比较[J]. 北京大学学报（医学版）, 2023, 55(1): 22-29.

Ling-wei MENG,Xue LI,Sheng-han GAO,Yue LI,Rui-tao CAO,Yi ZHANG,Shao-xia PAN. Comparison of three methods for establishing rat peri-implantitis model[J]. Journal of Peking University (Health Sciences), 2023, 55(1): 22-29.

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参考文献 21

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Gene	Primer sequences
TNF-α	Forward: 5′-CTTCTCATTCCTGCTCGTG-3′
	Reverse: 5′-TTTGGGAACTTCTCCTCCT-3′
IL-1β	Forward: 5′-TTCATCTTTGAAGAAGAGCCC-3′
	Reverse: 5′-CTGTCTAATGGGAACATCACAC-3′
GAPDH	Forward: 5′-AACTCCCATTCTTCCACCT-3′
	Reverse: 5′-TTGTCATACCAGGAAATGAGC-3′

Time point	Group	Items	PD/mm, ${\bar x}$±s				GI	BOP+/%
Time point	Group	Items	Medial	Distal	Palatal	Buccal	GI	BOP+/%
2 weeks	LPS	Baseline	0.75±0.31	0.42±0.15	0.92±0.45	0.50±0.18	0.67±0.21	0
		2 weeks	1.50±0.48	0.83±0.28	1.58±0.52	1.08±0.44	1.33±0.42	20.8
		P	0.220	0.220	0.359	0.246	0.240	0.025
	Ligation	Baseline	2.58±0.33	1.75±0.36	1.67±0.42	2.08±0.37	0.33±0.21	0
		2 weeks	3.00±0.26	2.83±0.31	2.67±0.21	3.00±0.26	2.00±0.00	50.0
		P	0.341	0.045	0.060	0.072	0.002	< 0.001
	LPS+Ligation	Baseline	2.50±0.34	2.25±0.40	1.75±0.44	2.17±0.30	0.17±0.17	0
		2 weeks	2.91±0.20	2.75±0.25	2.83±0.21	2.92±0.27	2.00±0.00	83.3
		P	0.318	0.317	0.052	0.097	0.002	< 0.001
4 weeks	LPS	Baseline	1.67±0.48	0.75±0.48	1.08±0.47	1.00±0.43	0.67±0.21	0
		4 weeks	2.17±0.38	1.33±0.42	2.00±0.48	1.67±0.44	1.83±0.17	58.3
		P	0.432	0.382	0.205	0.304	0.009	< 0.001
	Ligation	Baseline	1.75±0.36	1.58±0.37	2.08±0.45	2.00±0.37	0.50±0.22	0
		4 weeks	2.83±0.28	2.91±0.20	2.92±0.20	3.00±0.13	2.17±0.17	91.7
		P	0.039	0.011	0.125	0.027	0.002	< 0.001
	LPS+Ligation	Baseline	2.17±0.31	2.00±0.45	1.67±0.42	2.17±0.40	0.17±0.17	0
		4 weeks	2.92±0.20	3.08±0.27	3.17±0.17	3.17±0.28	2.33±0.21	95.8
		P	0.068	0.065	0.008	0.068	0.002	< 0.001

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三种方法建立大鼠种植体周炎模型的比较

Comparison of three methods for establishing rat peri-implantitis model

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