基于单细胞测序技术解析人炎性牙槽窝微环境下肉芽组织的转归

Deciphering the fate of granulation tissue in the human inflammatory alveolar microenvironment using single-cell RNA sequencing

目的探究人牙槽窝肉芽组织的细胞组成与异质性并构建单细胞图谱,解析肉芽组织在炎性微环境下通过自然转归的潜在结局。方法纳入2022年9月至2023年8月就诊于第四军医大学口腔医院颌面外科,因中、重度牙周炎行牙拔除术并拟行延期位点保存术或自体牙移植术的患者12例,收集其牙槽窝肉芽组织,分别用HE染色和单细胞转录组测序技术(ScRNA-seq)观察不同类型肉芽组织细胞构成、组织形态学变化,获得特定微环境下的炎性牙槽窝肉芽组织细胞序列差异,构建出人牙槽窝不同类型肉芽组织的单细胞图谱,并探索炎性肉芽组织转归过程中细胞类型分布及关键基因变化的时空规律。结果HE染色显示,牙槽窝炎性肉芽组织中含有大量炎性细胞浸润,保留炎性肉芽组织待自然转归3周后,其组织学形态与修复性肉芽组织愈合形态基本一致。ScRNA-seq共捕获肉芽组织细胞20448个,基因表达定量检测分析炎性肉芽组织、自然转归性肉芽组织及修复性肉芽组织基因总数分别为33835、36058、34719个。肉芽组织在单细胞水平被注释为血管内皮细胞、间充质干细胞、成纤维细胞等10个细胞亚群。炎性肉芽组织与自然转归性肉芽组织在细胞组成占比存在差异。拟时序分析展示了肉芽组织中免疫反应与血管新生参与组织转归愈合的两种主要结局。参与炎症调控和免疫反应的基因胰岛素样生长因子结合蛋白5(Igbp5)、锌指蛋白(Zfp36)和热休克蛋白(Hspa1b),以及参与细胞外基质分泌及血管、纤维形成的相关基因组织金属蛋白酶抑制因子3(Timp3)、骨膜蛋白(Postn)和G蛋白信号调节因子5(Rgs5)在两种肉芽组织中的表达存在显著差异。结论炎性肉芽组织在细胞组成占比、基因表达、生物学功能等方面与自然转归性肉芽组织存在异质性,不刮除牙槽窝炎性肉芽组织待其自然转归后在组织学及单细胞水平展现出与修复性肉芽组织相似的细胞结构组成,并通过免疫反应及组织重塑调节炎症反应及愈合过程。

Objective To investigate the cellular composition and heterogeneity of granulation tissue in human alveolar sockets and construct single-cell transcriptomic maps to elucidate the potential outcomes of natural resolution in the inflammatory microenvironment.Methods Granulation tissues from 12 alveolar sockets undergoing tooth extraction due to periodontitis and scheduled for delayed site preservation or autologous tooth transplantation were collected in the Department of Oral and Maxillofacial Surgery,School of Stomatology,The Fourth Military Medical University from September 2022 to August 2023.This study used HE staining and single-cell RNA sequencing(scRNA-seq)to observe the cellular composition and morphological changes of different types of granulation tissues.This approach enabled us to identify cellular sequence variations in the inflammatory dental alveolar granulation tissue within specific microenvironments,construct single-cell atlases for different types of human dental alveolar granulation tissues,and explore the spatiotemporal patterns of cell type distribution and key gene changes during the resolution process of inflammatory granulation tissue.Results HE staining revealed extensive infiltration of inflammatory cells in the dental alveolar inflammatory granulation tissue.After allowing the inflammatory granulation tissue to naturally resolve for three weeks,its histological morphology was essentially consistent with that of reparative granulation tissue.ScRNA-seq captured a total of 20448 cells from granulation tissues,and the gene expression quantification analysis revealed total gene counts of 33835 for inflammatory granulation tissue,36058 for naturally resolved granulation tissue,and 34719 for reparative granulation tissue.At the single-cell level,granulation tissue was annotated into ten cell subgroups,including vascular endothelial cells,mesenchymal stem cells,and fibroblasts.Differences were observed in the proportion of cell compositions between inflammatory and naturally resolved granulation tissues.Pseudo-temporal analysis illustrated two main outcomes in tissue resolution and healing,involving immune responses and angiogenesis.Among these,genes associated with inflammation regulation and immune response,such as Igbp5,Zfp36,and Hspa1b,as well as genes involved in extracellular matrix secretion and the formation of vessels and fibers such as Timp3,Postn,and Rgs5,showed significant differences in expression between the two types of granulation tissues.Conclusions Inflammatory granulation tissue exhibits heterogeneity in cell composition,gene expression,and biological functions compared to naturally resolved granulation tissue.When the inflammatory granulation tissue in the alveolar socket is left undisturbed to undergo natural resolution,it displays a cellular composition similar to that of reparative granulation tissue at both the histological and single-cell levels.Moreover,it modulates the inflammatory response and the healing process through immune reactions and tissue remodeling.