Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

For patients with large, full-thickness burn wounds, sufficient donor sites for autografting are not available and thus alternate strategies must be used to close these wounds. Cultured epithelial autografts (CEAs) can aid in closing these wounds but are often associated with slow deposition of basement membrane proteins, leading to blistering and graft loss. Rete ridges and dermal papillae present at the dermal-epidermal junction (DEJ) play a key role in epidermal adhesion and skin homeostasis. Promoting the development of an interdigitated DEJ may enhance basement membrane protein deposition and provide enhanced physical interlock of the epidermis and dermis. To develop a dermal template with stable dermal papillae, an electrospun collagen scaffold was seeded with human dermal fibroblasts. Ridged topographies were patterned into the cell-seeded dermal template using laser ablation, creating wide and shallow (ActiveFX) or narrow and deep (DeepFX) wells. Micropatterned or flat (control) dermal templates were combined with CEAs immediately prior to grafting to full-thickness excisional wounds on immunodeficient mice. CEAs grafted in conjunction with ridged templates showed rete ridge formation at two weeks after grafting and led to increased epidermal thickness, proliferation, and stemness compared to templates with a flat DEJ. As this technology is further developed, the dermal papillae-containing dermal templates may be utilized in combination with CEAs to improve adhesion and clinical function.

Objective: Despite the development of a number of treatment modalities, scarring remains common postburn injury. To reduce burn scarring, pressure garment therapy has been widely utilized but is complicated by low patient adherence. To improve adherence, reduced hours of daily garment wear has been proposed. Approach: To examine the efficacy of pressure garment therapy at reduced durations of daily wear, a porcine burn-excise-autograft model was utilized. Grafted burns were treated with pressure garments (20 mmHg) for 8, 16, or 24 h of daily wear with untreated burns serving as controls. Scar area, thickness, biomechanical properties, and tissue structure were assessed over time. Results: All treatment groups reduced scar thickness and contraction versus controls and improved scar pliability and elasticity. Pressure garments worn 24 h per day significantly reduced contraction versus the 8- and 16-h groups and prevented alignment of collagen within the dermis. Innovation: Though pressure garment therapy is prescribed for use 23 h per day, the need for almost continuous use has not been previously examined. Adjustable, low-fatigue pressure garments were developed for this porcine study to examine the role of daily duration of wear without confounding factors such as garment fatigue and patient adherence. Conclusion: For maximum efficacy, pressure garments should be worn 23 to 24 h per day; however, garments worn as little as 8 h per day significantly improve scar outcomes versus no treatment.

Rete ridges play multiple important roles in native skin tissue function, including enhancing skin strength, but they are largely absent from engineered tissue models and skin substitutes. Laser micropatterning of fibroblast-containing dermal templates prior to seeding of keratinocytes was shown to facilitate rete ridge development in engineered skin (ES) both in vitro and in vivo. However, it is unknown whether rete ridge development results exclusively from the microarchitectural features formed by ablative processing or whether laser treatment causes an inflammatory response that contributes to rete ridge formation. In this study, laser-micropatterned and non-laser- treated ES grafts were developed and assessed during culture and for four weeks post grafting onto full-thickness wounds in immunodeficient mice. Decreases in inflammatory cytokine secretion were initially observed in vitro in laser-treated grafts compared to non-treated controls, although cytokine levels were similar in both groups five days after laser treatment. Post grafting, rete ridge-containing ES showed a significant increase in vascularization at week 2, and in collagen deposition and biomechanics at weeks 2 and 4, compared with controls. No differences in inflammatory cytokine expression after grafting were observed between groups. The results suggest that laser micropatterning of ES to create rete ridges improves the mechanical properties of healed skin grafts without increasing inflammation.

The success of cultured epithelial autografts (CEAs) in treating large surface area burns is limited by their fragility and poor adhesion to the wound bed. Rete ridges, interdigitations at the dermal-epidermal junction, are stem cell niches critical to dermal-epidermal adhesion and distribution of mechanical forces within the skin, but are slow to develop in CEA-treated burn wounds. A porcine burn-excise-autograft model was used to investigate the efficacy of an allogeneic dermal template (DT), seeded with fibroblasts and laser micropatterned to have dermal papillae-like topography, in improving outcomes with CEA. Autologous CEAs were applied to full-thickness wounds alone or with DTs that were flat (CEA+Flat) or micropatterned (CEA+Ridged). The use of a DT improved graft handleability and integration at the wound margins, and restored epidermal barrier function two weeks faster than CEAs alone. In CEA+Ridged grafts, increases in keratinocyte proliferation and basement membrane deposition were observed at 2 weeks post-grafting. Rete ridges were only present in CEA+Ridged grafts at week 2, and developed less frequently and shallower in CEA and CEA+Flat grafts over the course of the study. These results suggest that laser micropatterned DTs are suitable for co-transplantation with CEAs and can significantly improve graft adhesion and development.

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 adult 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 follicular-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 adult 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 3D 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.