This study presents a multilayer in vitro human skin platform to quantitatively relate predicted spatial time–temperature history with measured tissue injury response. This information is needed to elucidate high-temperature, short-duration burn injury kinetics and enables determination of relevant input parameters for computational models to facilitate treatment planning. Multilayer in vitro skin platforms were constructed using human dermal keratinocytes and fibroblasts embedded in collagen I hydrogels. After three seconds of contact with a 50–100 °C burn tip, ablation, cell death, apoptosis, and HSP70 expression were spatially measured using immunofluorescence confocal microscopy. Finite element modeling was performed using the measured thermal characteristics of skin platforms to determine the temperature distribution within platforms over time. The process coefficients for the Arrhenius thermal injury model describing tissue ablation and cell death were determined such that the predictions calculated from the time–temperature histories fit the experimental burn results. The activation energy for thermal collagen ablation and cell death was found to be significantly lower for short-duration, high-temperature burns than those found for long-duration, low-temperature burns. Analysis of results suggests that different injury mechanisms dominate at higher temperatures, necessitating burn research in the temperature ranges of interest and demonstrating the practicality of the proposed skin platform for this purpose.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Background: Mucopolysaccharidosis type IIIB (MPSIIIB, Sanfilippo syndrome type B) is a rare lysosomal storage disease caused by deficiency of alpha-N-acetylglucosaminidase (NAGLU) enzyme, leading to progressive accumulation of heparan sulfate and severe neurological decline. MPSIIIB’s significant genetic heterogeneity presents a major barrier to developing broadly effective treatments and suggests a need for personalized therapeutic strategies. Methods: We established a personalized drug repurposing platform using high-content imaging with lysotracker dye as an indirect functional readout of lysosomal dysfunction to screen compounds that correct lysosomal defects in patient-derived fibroblasts. We screened 2807 compounds on cells from an MPSIIIB patient with a homozygous NAGLU p.Arg297Ter mutation. Hits that reduced lysosomal accumulation by at least 25% with minimal cytotoxicity were validated and subsequently tested for efficacy in fibroblasts from a second patient with a different NAGLU genotype. Results: The primary screen yielded 72 hits (2.6% hit rate), with 10 confirmed in dose–response assays. Notably, four clinically approved drugs—baclofen, dextrose, epalrestat and moxifloxacin—reduced lysosomal accumulation in the index patient’s cells. However, none of these four drugs were effective in the second patient’s cells, demonstrating a profound patient-specific effect. Only one non-clinical compound, 6-chlorothymol, showed a trend toward activity in both cell lines. Conclusions: Our study demonstrates a feasible framework for conducting rapid, N-of-1 drug repurposing screens for rare diseases. While we identified four promising candidates for the index patient, the lack of efficacy in a second patient cell line underscores that genetic heterogeneity may preclude a “one-size-fits-all” approach for MPSIIIB. These findings support the integration of individualized drug screening as a potential precision-medicine strategy, offering a potential path toward patient-specific therapies for rare diseases rather than traditional drug development.

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