Engineering spatially organized hair peg-like structures in human skin constructs using keratinocyte-dermal papilla fibroblast spheroids and micropatterned dermal scaffolds.

Tissue-engineered skin substitutes (TESS) represent a key therapeutic option for extensive cutaneous injuries when autologous grafting is limited; however, current constructs lack skin appendages, particularly hair follicles, resulting in incomplete functional and aesthetic restoration. A central barrier to engineering follicular structures within TESS is the rapid loss of hair-inductive features in cultured human dermal papilla (DP) fibroblasts and the difficulty of recreating epithelial-mesenchymal interactions required for early folliculogenesis. Here, we report a bioengineering platform that integrates self-organizing composite spheroids composed of human epidermal keratinocytes (KCs) and DP fibroblasts with laser-micropatterned collagen scaffolds to generate spatially defined hair peg-like structures within human TESS. Under optimized three-dimensional culture conditions, mixed KC-DP spheroids reproducibly undergo polarization, budding, and elongation to form hair peg-like structures that exhibit compartmentalized organization and expression of key follicle-associated markers. Transcriptomic analysis reveals activation of follicle-associated transcriptional programs in KCs, while DP fibroblasts retain DP-associated features under these conditions. When implanted into predefined microwells within collagen-based dermal scaffolds, these spheroids continue coordinated morphogenesis alongside epidermal stratification and dermal matrix formation, yielding engineered skin constructs containing spatially integrated hair peg-like structures. Although maturation into fully developed hair follicles remains to be achieved, this study establishes a scalable, clinically relevant platform for integrating early-stage follicular units into TESS. Collectively, these findings provide a foundation for future efforts toward appendage-inclusive skin regeneration using human cells. STATEMENT OF SIGNIFICANCE: Engineering human hair follicles in skin substitutes remains challenging because dermal papilla cells rapidly lose hair-inductive function during laboratory expansion, and existing organoid models are difficult to scale or integrate into engineered skin. Here, we present a laser-micropatterned collagen scaffold that spatially incorporates early-stage follicular units formed from human keratinocytes and dermal papilla cells. Through systematic optimization of cell state, spheroid configuration, culture conditions, and scaffold integration, mixed keratinocyte-dermal papilla cell spheroids reproducibly undergo polarization, budding, and elongation in three-dimensional culture and retain hair peg-like organization after incorporation into micropatterned dermal scaffolds. This work advances hair follicle engineering toward clinically relevant skin constructs for studying human hair biology and regenerative skin therapies.