Molecular and functional heterogeneity and identification of fibroblasts

Degree type
Master of Science in Oral Biology (MSOB)
Graduate group
Discipline
Dentistry
Subject
fibroblast, dentistry, regeneration, inflammation, periodontitis, periodontology, immunology
Funder
Grant number
Copyright date
2024-06-05
Distributor
Author
Ko, Annette
Sahingur, Sinem Esra
Chang, Yu-Cheng
Contributor
Abstract

Introduction: Oral wounds heal with exceptional regenerative capacity, which contrasts skin wound repair. Paired related homeobox 1 (Prrx1) is a marker for mesenchymal progenitors in bone, and recent studies have demonstrated presence and functional significance of Prrx1+ fibroblasts in dermis. Our previous findings suggest pro-healing role of Prrx1+ oral fibroblasts in mouse model, thus we sought to examine equivalent Prrx1+ cells using human gingival tissue biopsies. Here, we hypothesize that human Prrx1+ fibroblasts reside in different oral gingival tissues and that they exhibit enhanced regenerative potential for wound healing. Methods: Human gingival samples were collected from the anterior rugae, healed sites (distal wedge, edentulous crest), and tooth-associated sites (marginal gingiva from crown lengthening procedure or osseous surgery) in accordance to approved IRB protocol. The tissues were processed in paraffin-embedded block and sectioned at 5um. Immunofluorescence staining was performed with antibodies against Prrx1 and vimentin to detect Prrx1+ oral fibroblasts in various oral anatomic sites. Image analysis was carried out by counting spindle-shaped vimentin+ and Prrx1+ cells in lamina propria, normalized by area (mm2). To examine the regenerative potential of Prrx1+ cells in oral tissues, human anterior rugae samples were collected and processed for single-cell RNA sequencing (scRNA-seq). Transcriptomic analysis was performed after combining with the public scRNA-seq data (GEO: GSE164241, GSE152042). Results: Prrx1+ fibroblasts were present throughout all types of oral soft tissues. Anterior palate tissue had 72% fibroblasts expressing Prrx1, which was significantly more than those found in distal wedge, crestal tissue and marginal gingivae (39%, 30%, 26%, respectively; P<0.05) in the reticular layer of lamina propria and submucosa. ScRNA-seq analysis of the combined data demonstrated upregulation of several WNT-associated genes in Prx1+ fibroblasts, such as SFRP1 and SLPI, which had previously been reported to be involved in stem cell function and regeneration. The transcriptomic analysis further revealed that Prx1+ fibroblasts exhibit progenitor phenotype with projected differentiation trajectory toward chemotactic fibroblasts and expedited wound healing potential via immunomodulation in innate immune response. Further characterization of these inflammatory fibroblasts showed high expression of intercellular adhesion molecule-1 (ICAM-1), suggesting its potential usage as a surface marker for identification. Conclusion: Our results demonstrate that human Prrx1+ oral fibroblasts reside in the deep layer of the lamina propria, particularly in the anterior palate at higher frequency compared to other anatomic sites. These fibroblasts may exhibit improved regenerative potential via modulating WNT-associated pathway and rapid resolution of inflammation. The findings provide new insight into the enhanced healing potential of oral fibroblast subpopulations that may contribute to developing surgical techniques targeted to improve unpredictable soft tissue augmentation.

Advisor
Ko, Kang
Date of degree
2024-06-28
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