负载脱落乳牙干细胞外泌体的透明质酸可注射水凝胶的制备及其对小鼠牙周炎抗炎成骨的研究

doi:10.12439/kqhm.1005-4979.2023.05.003

摘要/Abstract

摘要：

目的：制备负载人脱落乳牙干细胞（stem cells from human exfoliated deciduous teeth，SHED）外泌体（exosomes，EXO）的可注射透明质酸（hyaluronic acid，HA）水凝胶，探究其在牙周炎中的抗炎及成骨作用。方法：提取SHED外泌体，以高分子量HA为基质，在其中负载SHED外泌体以合成可注射HA水凝胶（SHED-HA），并测定其降解率。采用CCK-8法分析SHED-HA的生物相容性，应用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）研究SHED-HA的成骨诱导作用及炎症抑制作用。随后，通过micro-CT来分析SHED-HA对小鼠牙槽骨缺损的挽救作用。结果：6% w/v透明质酸水凝胶具有较合适的降解性。CCK-8结果显示，SHED-HA具有较好的生物相容性能。RT-qPCR结果显示，SHED-HA组炎症基因IL-1β、IL-6的表达水平下调，而成骨分化相关基因Runx2、OPN、OCN的表达水平上调。体内实验显示，SHED-HA能减轻小鼠牙槽骨破坏。结论：成功构建负载了SHED外泌体的HA水凝胶SHED-HA，同时证实了其对小鼠牙周炎具有抗炎、成骨的作用。

关键词: 透明质酸, 脱落乳牙干细胞, 外泌体, 成骨分化

Abstract:

Objective: Preparation of injectable hyaluronic acid (HA) hydrogels loaded with exosomes (EXO) of stem cells from human exfoliated deciduous teeth (SHED) to investigate its anti-inflammatory and osteogenic effects in periodontitis. Methods: SHED EXO was extracted and loaded with high molecular weight HA as matrix to synthesize injectable HA hydrogel (SHED-HA) and its degradation rate was determined. The biocompatibility of SHED-HA was analyzed by CCK-8 method, and the osteogenic induction and inflammatory inhibition of SHED-HA were studied by real-time quantitative polymerase chain reaction(RT-qPCR). Subsequently, the effect of SHED-HA on alveolar bone defect in mice was analyzed by micro-CT. Results: SHED-HA had a suitable degradability at the concentration of 6% w/v HA. CCK-8 results showed that SHED-HA had good biocompatibility. RT-qPCR results showed that the expression levels of inflammatory genes IL-1β and IL-6 were down-regulated in the SHED-HA group, while the expression levels of osteogenic differentiation related genes Runx2,OPN and OCN were up-regulated. In vivo experiments showed that SHED-HA reduced alveolar bone destruction in mice. Conclusion: The HA hydrogel SHED-HA of SHED exosome was successfully constructed, and it was found that SHED-HA has anti-inflammatory and osteogenic effects on mouse periodontitis.

Key words: hyaluronic acid, deciduous tooth stem cells, exosome, osteogenic differentiation

中图分类号:

R782

应乔, 俞懿强, 苏俭生. 负载脱落乳牙干细胞外泌体的透明质酸可注射水凝胶的制备及其对小鼠牙周炎抗炎成骨的研究[J]. 《口腔颌面外科杂志》, 2023, 33(5): 292-297.

YING Qiao, YU Yiqiang, SU Jiansheng. Preparation of hyaluronic acid injectable hydrogel with SHED-derived exosomes and its anti-inflammatory and osteogenic effects on periodontitis: An experimental study in the rat[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(5): 292-297.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I5/292

图/表 7

表1 RT-qPCR引物序列

Table 1 Primer sequence of RT-qPCR

基因	正向引物(5′-3′)	反向引物(5′-3′)
IL-6	CTGCAAGAGACTTCCATCCAG	AGTGGTATAGACAGGTCTGTTGG
IL-1β	GAAATGCCACCTTTTGACAGTG	TGGATGCTCTCATCAGGACAG
GAPDH	AGGTCGGTGTGAACGGATTTG	GGGGTCGTTGATGGCAACA
Runx2	TGGTTACTGTCATGGCGGGTA	TCTCAGATCGTTGAACCTTGCTA
OPN	CTCCATTGACTCGAACGACTC	CAGGTCTGCGAAACTTCTTAGAT
OCN	CACTCCTCGCCCTATTGGC	CCCTCCTGCTTGGACACAAAG
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT

图1 不同浓度HA水凝胶随时间降解的质量变化

Figure 1 Degradation quality of HA hydrogels with time in different concentrations

图2 扫描电镜下SHED外泌体呈现出囊泡样结构 A. 放大1 000倍；B. 放大500倍。

Figure 2 The SHED exosomes showed vesicle-like structure under scanning electron microscope

图3 培养SHED-HA表面1、3、7 d的hPDLCs细胞活性 *表示P<0.05，与control组比较。

Figure 3 Cell activity of hPDLCs cultured on SHED-HA surface for 1, 3, 7 d

图4 RAW264.7细胞炎症相关基因的表达水平 A. IL-1β；B. IL-6。*表示P<0.05，与LPS刺激组比较。

Figure 4 Expression levels of inflammation-related genes in RAW264.7 cells

图5 在SHED-HA中培养5 d的hPDLCs成骨分化相关基因的表达水平 A. Runx2；B. OCN；C. OPN。*表示P<0.05，与control组比较。

Figure 5 Expression levels of osteogenic differentiation related genes of hPDLCs cultured in SHED-HA for 5 d

图6 SHED-HA对牙周炎模型小鼠上颌第二磨牙牙槽骨促再生作用 A. micro-CT影像（红色箭头指示骨缺损区域）；B. 各组CEJ-ABC（近中、中点、远中牙槽骨嵴）的距离比较（标准组：自然生长14 d；牙周炎组：牙周炎建模拆线后自然生长14 d；材料组：牙周炎建模拆线后隔天局部给药14 d）。

Figure 6 Effect of SHED-HA on alveolar bone regeneration of maxillary second molar in periodontitis model mice

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