Form Meets Function: Fiber Architecture Directs Proliferation and Differentiation in Gingival Keratinocytes

Area of Science:

• Biomaterials Science
• Tissue Engineering
• Cell Biology

Background:

• Oral epithelial regeneration requires precise control over keratinocyte proliferation and differentiation.
• Mechanobiological cues from biomaterial scaffolds significantly influence cell fate.
• The specific roles of scaffold architecture, such as fiber orientation and diameter, in directing gingival keratinocyte behavior are not fully understood.

Purpose of the Study:

• To systematically investigate how electrospun polycaprolactone (PCL) scaffolds with varying fiber orientations (aligned vs. random) and diameters influence human gingival keratinocyte behavior.
• To assess the impact of these scaffold properties on cell morphology, proliferation, differentiation, and the role of basal keratins (KRT5/KRT14).

Main Methods:

• Fabrication of PCL scaffolds with controlled fiber orientations and diameters (600-800 nm, 1.2-1.7 µm, 2.0-2.5 µm).
• Culture of immortalized human gingival keratinocytes on these scaffolds.
• Quantitative analysis of cell and nuclear morphology, proliferation (EdU assays), gene expression (ddPCR) of keratin and differentiation markers, protein expression (IVL, FLG), and effects of KRT5/KRT14 knockdown.

Main Results:

• Aligned, medium-diameter (1.2-1.7 µm) fibers induced elongated cell/nuclear morphology and sustained proliferation.
• Random, smaller-diameter (600-800 nm) fibers promoted rounded cell/nuclear shapes, a transient proliferative burst, and enhanced expression of basal keratins (KRT5/KRT14) and differentiation markers (KRT1, KRT10, IVL, FLG).
• KRT5/KRT14 were essential for keratinocyte viability on random scaffolds but not aligned ones, differentially affecting downstream markers and mechanotransduction pathways (LMNB1, YAP1) based on scaffold type.

Conclusions:

• Fiber orientation and diameter are critical design parameters for controlling keratinocyte proliferation and differentiation on PCL scaffolds.
• Random scaffolds with smaller fibers promote differentiation, while aligned scaffolds support sustained proliferation, suggesting distinct mechanobiological signaling.
• Layered scaffolds combining aligned and random fibers offer a promising strategy for spatially controlled regeneration of oral epithelia.