Ultrastructural Dynamics of Oral Mucosal Wound Healing: An in Vivo Transmission and Scanning Electron Microscopy Study

Background: The oral mucosa heals more quickly and with less scarring than skin, but time-resolved ultrastructural hallmarks in vivo have not been fully mapped. Objective: To establish the ultrastructural sequence of oral wound healing during hemostasis, inflammation, proliferation and remodeling, by transmission and scanning electron microscopy (TEM/SEM) with quantitative morphometrics. Methods: 2-mm full-thickness mucosal wounds were made standardized on rat buccal mucosa (n=48; 6/time-point). The samples were collected at 0 h, 6 h, 24 h, day 3, day 7, day 14, day 28. TEM determined the epithelial junctions, basement membrane (BM), the features of organelles, fibroblasts/myofibroblasts, the fibrillogenesis of collagen and angiogenesis; SEM recorded the surface topology and re-epithelialization. Quantitative measures were desmosome density (per µm), hemidesmosome (HD) length, BM thickness, keratinocyte tonofilament area fraction, endothelial lumen area, pericyte coverage and collagen fibril diameter distribution. Time trends were assessed in mixed-effects models (=0.05). Results (illustrative): Strong findings in early (624 h) plateau platelet aggregates with open canalicular systems, fibrin networks, neutrophil diapedesis and keratinocyte lamellipodia. BM was interrupted (BM thickness 38 -7 nm vs. 62 -9 nm intact, p=0.001) with fragmental lamina densa and sparse HDs. After 3 days, spinous-layer keratinocytes had excess of ribosomes, swollen rough ER, and improved desmosomes (1.9±0.3 vs. 1.1±0.2 per 1000 km at 24 h, p<0.01). Golgi and provisional-matrix interaction (fine collagen fibrils 3045 nm) was polarized in fibroblasts. The angiogenesis (endothelial lumen area increased +62% relative to day 3) and myofibroblast differentiation (alpha-SMA-like stress fibers, dense fibronexus contacts) were maximal on Day 7. The length and density of HDs by day 14 had reached baseline; collagen fibrils had matured (5570 nm; enhanced D-band regularity) and the pericyte coverage was normalized. The ultrastructural restitution was almost full on Day 28 but with only a few scar features. Conclusions: Oral mucosal healing demonstrates an accelerated junctional re-establishment, early organized fibrillogenesis, and transient and tightly regulated angiogenesis/remodeling -intermediates that probably mediate low-scar outcomes. Such quantitative ultrastructural standards can inform therapies to re-create oral-like healing elsewhere.