载他克莫司可注射温敏型水凝胶促牙周组织再生的研究

doi:10.3969/j.issn.1005-4979.2020.05.002

《口腔颌面外科杂志》 ›› 2020, Vol. 30 ›› Issue (5): 272-278. doi: 10.3969/j.issn.1005-4979.2020.05.002

载他克莫司可注射温敏型水凝胶促牙周组织再生的研究

陈希¹^,²(), 任春霞²^,⁴, 刘莉莉¹^,², 陈雨蒙¹^,², 郑梦丹¹^,², 崔家森³, 卢金金⁵, 孙宏晨¹^,²^,³()

¹ 吉林大学口腔医学院病理科，吉林长春 130021
² 吉林省牙发育及颌骨重塑与再生重点实验室，吉林长春 130021
³ 中国医科大学附属口腔医院口腔病理科，辽宁沈阳 110001
⁴ 吉林大学口腔医院牙周科，吉林长春 130021
⁵ 吉林大学口腔医院儿童口腔科，吉林长春 130021

收稿日期:2020-03-18 修回日期:2020-04-22 出版日期:2020-10-28 发布日期:2020-10-27
通讯作者: 孙宏晨，教授. E-mail: hcsun@mail.jlu.edu.cn
作者简介:
陈希（1992—），女，河南省新乡市人，硕士研究生. E-mail: chenxixi813717@163.com
基金资助:
国家自然科学基金资助项目(81970903); 国家自然科学基金资助项目(81600843); 国家自然科学基金资助项目(81600823); 吉林省财政厅科技项目(JCSZ2019378-6)

Study on Injectable and Thermosensitive Hydrogel Loaded with Tacrolimus Promoting Periodontal Tissue Regeneration

CHEN Xi¹^,²(), REN Chunxia²^,⁴, LIU Lili¹^,², CHEN Yumeng¹^,², ZHENG Mengdan¹^,², CUI Jiasen³, LU Jinjin⁵, SUN Hongchen¹^,²^,³()

¹ Department of Pathology, School and Hospital of Stomatology, Jilin University, Changchun 130021, Jilin Province
² Key Laboratory of Tooth Development and Jaw Remodeling and Regeneration,Changchun 130021, Jilin Province
³ Department of Oral Pathology, Hospital of Stomatology, China Medical University, Shenyang 110001, Liaoning Province
⁴ Department of Periodontology, School and Hospital of Stomatology, Jilin University, Changchun 130021, Jilin Province
⁵ Department of Pediatric Dentistry, School and Hospital of Stomatology, Jilin University, Changchun 130021, Jilin Province, China

Received:2020-03-18 Revised:2020-04-22 Online:2020-10-28 Published:2020-10-27

摘要/Abstract

摘要：

目的： 制备载FK506（他克莫司，tacrolimus）的可注射温敏型水凝胶，研究其在大鼠牙周炎模型中促牙周组织再生的作用。方法： 以壳聚糖（chitosan，CS）、β-甘油磷酸钠、明胶和FK506为原料合成为载FK506的可注射温敏型水凝胶，扫描电镜下观察其微结构。建立Wistar大鼠牙周炎模型，分为空白组、牙周炎组、牙周炎+水凝胶组、牙周炎+载FK506水凝胶组及牙周炎+FK506组。应用Micro-CT观察牙周骨组织破坏情况、苏木精-伊红染色 (HE染色)法进行组织学分析，并结合免疫组织化学染色检测炎症因子环氧化酶2（cyclooxygenase-2, COX-2）和基质金属蛋白酶9（matrix metalloproteinase-9, MMP-9）的表达情况。进一步使用四甲基偶氮唑盐微量酶反应比色法（MTT法）检查水凝胶载FK506对骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）增殖的影响，评估其生物安全性。结果： 扫描电镜图像显示，冻干水凝胶孔径大小均一；Micro-CT扫描分析结果表明，水凝胶载FK506可以减轻牙周炎导致的牙槽骨破坏；组织学分析表明，在牙周炎+载FK506水凝胶组中几乎没有炎性细胞浸润或牙槽骨破坏，并且在根分叉区及上颌第一和第二磨牙之间可见少量新生骨组织；免疫组织化学染色结果显示，牙周炎+载FK506水凝胶组及牙周炎+FK506组中，COX-2和MMP-9阳性细胞数量较少；MTT结果显示水凝胶载FK506对BMSCs 的增殖活性无明显抑制作用。结论： 可注射温敏型水凝胶缓释FK506可用于抗炎和牙周组织再生，在牙周炎治疗中具有良好的应用前景。

关键词: 水凝胶, FK506, 牙周组织再生, 抗炎

Abstract:

Objective: To investigate the effect of an injectable and thermosensitive hydrogel loaded with FK506 (tacrolimus) on periodontal tissue regeneration in rat periodontitis model. Methods: Chitosan(CS), β-glycerophosphate, gelatin and FK506 were used as raw materials to synthesize injectable and thermosensitive hydrogel loaded with FK506. The microstructure was observed under scanning electron microscope (SEM). At the same time, the periodontitis model was established on Wistar rats. These Wistar rats were randomly divided into five groups, i.e. the blank control group, periodontitis without treatment group, periodontitis treated with hydrogels group, periodontitis treated with hydrogels loaded with FK506 group, and periodontitis treated with FK506 group. The damage of the periodontal bone tissue was observed by Micro-CT. The histological analysis was performed by HE staining, and the expression of cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) were detected by immunohistochemical staining. The effect on bone marrow mesenchymal stem cells (BMSCs) proliferation was examined by MTT method. Results: SEM images showed that the lyophilized hydrogel possessed a uniform pore size. Micro-CT analysis showed that hydrogel loaded with FK506 can reduce alveolar bone damage caused by periodontitis. Histological analysis showed that there was almost no inflammatory cells or alveolar bone destruction in the periodontitis treated with hydrogels loaded with FK506 group, and a small amount of new bone was seen in furcation area and between the first maxillary and second maxillary molar regions. Besides, immunohistochemical staining results showed that COX-2 and MMP-9 positive cells were less in the periodontitis treated with hydrogels group and periodontitis treated with hydrogels loaded with FK506 group. The results of MTT showed that the hydrogels loaded with FK506 had no obvious inhibitory effect on the proliferation of BMSCs. Conclusion: Injectable and thermosensitive hydrogel sustained-release FK506 can be used for anti-inflammatory, periodontal tissue regeneration and has great potential in the treatment of periodontitis.

Key words: hydrogel, FK506, periodontal tissue regeneration, anti-inflammation

中图分类号:

R781.4

陈希, 任春霞, 刘莉莉, 陈雨蒙, 郑梦丹, 崔家森, 卢金金, 孙宏晨. 载他克莫司可注射温敏型水凝胶促牙周组织再生的研究[J]. 《口腔颌面外科杂志》, 2020, 30(5): 272-278.

CHEN Xi, REN Chunxia, LIU Lili, CHEN Yumeng, ZHENG Mengdan, CUI Jiasen, LU Jinjin, SUN Hongchen. Study on Injectable and Thermosensitive Hydrogel Loaded with Tacrolimus Promoting Periodontal Tissue Regeneration[J]. 《Journal of Oral and Maxillofacial Surgery》, 2020, 30(5): 272-278.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2020/V30/I5/272

图/表 6

图1 Micro-CT扫描结果显示牙槽骨再生情况注：A. 空白组Micro-CT三维重建图片；B. 空白组Micro-CT矢状面图片；C. 牙周炎组Micro-CT三维重建图片；D. 牙周炎组Micro-CT矢状面图片；E. 牙周炎+水凝胶组Micro-CT三维重建图片；F. 牙周炎+水凝胶组Micro-CT矢状面图片； G. 牙周炎+载FK506水凝胶组Micro-CT三维重建图片；H. 牙周炎+载FK506水凝胶组Micro-CT矢状面图片； I. 牙周炎+FK506组Micro-CT三维重建图片；J. 牙周炎+FK506组Micro-CT矢状面图片。

Figure 1 Micro-CT images showing regeneration of the alveolar bone

图2 各组CEJ至ABC的距离比较注：与空白组比较，*表示 P<0.05，**表示 P<0.01。

Figure 2 Quantitative comparison on the distance from CEJ to ABC

图3 各组Micro-CT 相关参数比较注：A. BMD；B. TMD；C. BV/TV；D. Tb.Th；E. Tb.Sp；F. Tb.N。与空白组比较，* P<0.05，** P<0.01，*** P<0.001；与牙周炎组比较，#P<0.05，###P<0.001。

Figure 3 Comparison of Micro-CT related parameters of each group

图4 牙周组织HE染色结果注: A. 空白组牙周组织HE染色结果（×40）；B. 空白组结合上皮处（×100）；C. 空白组牙槽骨和牙骨质（×100）；D. 牙周炎组牙周组织HE染色结果（×40）；E. 牙周炎组结合上皮处（×100）；F. 牙周炎组牙槽骨和牙骨质（×100）； G. 牙周炎+水凝胶组牙周组织HE染色结果（×40）；H. 牙周炎+水凝胶组结合上皮处（×100）；I. 牙周炎+水凝胶组牙槽骨和牙骨质（×100）；J. 牙周炎+载FK506水凝胶组牙周组织HE染色结果（×40）；K. 牙周炎+载FK506水凝胶组结合上皮处（×100）；L. 牙周炎+载FK506水凝胶组牙槽骨和牙骨质（×100）；M. 牙周炎+FK506组牙周组织HE染色结果（×40）；N：牙周炎+FK506组结合上皮处（×100）；O. 牙周炎+FK506组牙槽骨和牙骨质（×100）。

Figure 4 HE staining of the periodontium

图5 牙周组织免疫组织化学染色结果（X400）注：A~E. 各组牙周组织中COX-2的免疫组织化学染色结果；F~J. 各组牙周组织中MMP-9的免疫组织化学染色结果。

Figure 5 Immunohistochemical staining results of the periodontal tissue（X400）

图6 MTT法检测载FK506水凝胶对BMSCs增殖活性的影响

Figure 6 The effect of FK506-containing hydrogel on the proliferation of BMSCs by MTT method

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