Dorothy M. Supp

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 properties 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 demonstrates extensive KC reprogramming toward a folliculogenic gene expression profile, while DP fibroblasts retain features associated with hair-inductive states. 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 follicular structures. Although full maturation into fully developed hair follicles has yet to be achieved in vivo or under prolonged ex vivo conditions, 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.

Scarring remains problematic post-burn injury even with pressure garment therapy, the current standard of care for scar suppression. Unfortunately, there is no consensus regarding the minimum pressure magnitude required for efficacy. Thus, to fill this knowledge gap, the relationship between the magnitude of applied pressure and scar outcomes was assessed using a porcine burn-excise-autograft model. Scars were treated with adjustable, low-fatigue pressure garments fit to 10, 20, or 30 mmHg; control scars were untreated (n = 16/group). Scar contraction, thickness, histological appearance, and biomechanics were assessed, and garment fatigue was measured. At all magnitudes of applied pressure, use of garments significantly reduced scar contraction and thickness, and improved scar pliability and elasticity, versus controls. Utilizing adjustable, low-fatigue garments, pressures of 10, 20, and 30 mmHg were reliably maintained throughout the study, demonstrating the efficacy of pressure garment therapy at pressures as low as 10 mmHg. While 30 mmHg pressure garments significantly reduced contraction compared to 10 or 20 mmHg, these garments required greater adjustment of the garment to reach the target pressure, which in practice could necessitate more frequent garment replacement. The results showed that although greatest improvement was observed at 30 mmHg, pressure as low as 10 mm Hg is effective at reducing post-burn scarring if that pressure is maintained throughout the garment’s use.

Engineered skin (ES) can serve as an advanced therapy for treatment of large full-thickness wounds, but delayed vascularization can cause ischemia, necrosis, and graft failure. To accelerate ES vascularization, this study assessed incorporation of polydopamine (PDA) microparticles loaded with different concentrations of basic fibroblast growth factor (bFGF) into collagen scaffolds, which were subsequently seeded with human fibroblasts to create dermal templates (DTs), and then keratinocytes to create ES. DTs and ES were evaluated in vitro and following grafting to full-thickness wounds in immunodeficient mice. In vitro, metabolic activity of DTs was enhanced with PDA+bFGF, though this increase was not observed following seeding with keratinocytes to generate ES. After grafting, ES with bFGF-loaded PDA microparticles displayed dose-dependent increases in CD31-positive vessel formation vs. PDA-only controls (p < 0.001 at day 7; p < 0.05 at day 14). Interestingly, ES containing PDA+bFGF microparticles exhibited an almost 3-fold increase in water loss through the skin and a less-organized basal keratinocyte layer at day 14 post-grafting vs. controls. This was associated with significantly increased inflammatory cell infiltrate vs. controls at day 7 in vivo (p < 0.001). The results demonstrate that PDA microparticles are a viable method for delivery of growth factors in ES. However, further investigation of bFGF concentrations, and/or investigation of alternative growth factors, will be required to promote vascularization while reducing inflammation and maintaining epidermal health.

Background: Keloids are disfiguring, fibrotic scar-like lesions that are challenging to treat and commonly recur after therapy. A deeper understanding of the mechanisms driving keloid formation is necessary for the development of more effective therapies. Reduced vitamin D receptor (VDR) expression has been observed in keloids, implicating vitamin D signaling in keloid pathology. Vitamin D exhibits anti-proliferative and anti-inflammatory properties, suggesting it could have therapeutic utility in keloid disorder. The current study investigated vitamin D-regulated gene expression in keloid keratinocytes and the effects of inhibiting an enzyme involved in vitamin D metabolism on the phenotype of keloid-derived keratinocytes. Methods: Normal and keloid-derived primary keratinocytes were isolated from normal skin and keloid lesions, respectively, and were cultured in the absence or presence of vitamin D. In some experiments, inhibitors of the vitamin D metabolizing enzyme CYP24A1, ketoconazole or VID400 were added in the absence or presence of vitamin D. Cellular proliferation, migration and gene expression were measured. Results: We observed significant overexpression of CYP24A1 mRNA in keloid versus normal keratinocytes and increased CYP24A1 protein levels in keloids versus normal skin. CYP24A1 encodes 24 hydroxylase and is induced by vitamin D in a feedback loop that regulates vitamin D levels; thus, inhibition of CYP24A1 activity may locally increase active vitamin D levels. Ketoconazole, a non-specific cytochrome P-450 inhibitor, reduced proliferation of keloid and normal keratinocytes, but VID400, a specific CYP24A1 inhibitor, only significantly affected keloid keratinocyte proliferation. Neither inhibitor significantly reduced keratinocyte migration. The two inhibitors had different effects on vitamin D target gene expression in keratinocytes. Specifically, ketoconazole treatment reduced CYP24A1 expression in normal and keloid keratinocytes, whereas VID400 increased CYP24A1 expression. Both inhibitors decreased expression of profibrotic genes, including periostin and hyaluronan synthase 2, in keloid-derived cells. Combined treatment of keloid keratinocytes with vitamin D and ketoconazole or VID400 increased the effects of vitamin D treatment on target genes, although the effects were gene- and cell type-specific. Conclusions: The data suggest that reduction of vitamin D inactivation with CYP24A1 inhibitors may reduce profibrotic gene expression in keloid-derived cells. Therefore, CYP24A1 inhibitors may serve as adjunctive therapies to suppress keloid-associated gene expression changes.