Dermal Templates Support Epidermal Regeneration and Maturation Regardless of Cell Donor Age

To provide an optimal wound bed for epidermal regeneration, a viable dermis is needed. As the dermis is destroyed in full-thickness burns, dermal templates (DTs) are used to create a healthy dermis for grafting and other procedures. Neonatal foreskin has exclusively been used as the source of dermal fibroblasts in commercial DTs, as the tissue is readily available and because these cells are assumed to be more proliferative and capable of superior wound healing compared with adult fibroblasts. The goal of this study was to assess the function of adult fibroblasts compared with neonatal fibroblasts for DT construction and epidermal regeneration. Primary fibroblasts were isolated from neonatal or adult surgical discard tissue (n = 4 each). Expression of collagen type 1 A1 (COL1A1) and matrix metalloprotease 1 (MMP1), MMP3, and MMP9 was assessed for each cell strain, and proliferation was quantified in two-dimensional (2D) cultures and 3D DTs. Subsequently, DTs were constructed from each cell strain by inoculating fibroblasts onto electrospun collagen scaffolds. DT contraction, extracellular matrix remodeling, and cell viability were assessed over 7 days in culture, and the ability of the DTs to promote epidermal regeneration was assessed using primary adult keratinocytes. No differences in gene expression were observed in neonatal versus adult fibroblasts in 2D culture. Neonatal fibroblasts were significantly more proliferative at day 7 when cultured in 2D; however, fibroblast proliferation was independent of donor age in 3D culture. Neonatal DTs contracted significantly more than adult DTs (68.8% ± 6.2% vs. 91.7% ± 4.2% original wound area, respectively). Upon seeding with keratinocytes, a robust, stratified epidermis formed in all DT groups, with no statistically significant differences in dermal or epidermal thickness, basal keratinocyte proliferation, epidermal barrier function, or basement membrane deposition. Analysis of gene expression revealed modest differences in the expression of MMP1, COL1A1, and ACTA2 in neonatal versus adult engineered skin in vitro, which were not associated with any discernable histological differences. These results indicate that the fabrication of DTs with adult fibroblasts can promote epidermal regeneration equivalent to that of neonatal fibroblasts but with less in vitro contraction, which may enable the treatment of larger wound areas.