(56) Investigating oral fibroblast heterogeneity and its wound healing implications in humans

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 preliminary 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 from were collected from the anterior palate, 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, we carried out transcriptomic analysis of Prrx1+ fibroblasts using public single-cell RNA data (GEO: GSE164241).

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. When single-cell RNA sequencing of oral gingival tissues from published dataset (Williams et al., 2021) was examined, we found that Prrx1-high fibroblasts had upregulated expression of several WNT-associated genes such as SFRP1 and SLPI, which had previously been reported to be involved in stem cell function and regeneration.

Conclusions: 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 cells may exhibit improved regenerative potential via modulating WNT-associated pathway. The findings are clinically significant because utilizing the enhanced healing potential of oral fibroblast subpopulations may provide benefit for unpredictable soft tissue augmentation procedures, such as papilla regeneration.

Lead Author's Contributions to the Research Project. : The Lead Author was responsible for the data analysis and the composition of the abstract.