3. Exploring the Differential Effects of Titanium Dioxide (TiO2) Nanoparticles on Keratinocyte Functions and Structural Integrity in Tissue Engineered Skin Models

PURPOSE: Our team has produced bioprinted tissue engineered skin constructs that developed an ordered differentiated epidermis.1 Different substances’ effects on cell functionality can be tested on these constructs. TiO2nanoparticles are ubiquitous components in products i.e., colorants, electronics, plastics, and hence a significant component of environmental pollution. When directly exposed to multiple cell types, adverse effects were reported.1Since skin is the main protective barrier against environmental pollution, we explored rutile TiO2’s impact on keratinocytes’ and dermal fibroblasts’ ability to self-assemble and form functional skin equivalents. METHODS: Human keratinocytes and dermal fibroblasts (IRB#20022283) were treated for 24 hours with 0.4 and 0.8 mg/mL TiO2 nanoparticles. Fluorescence-activated cell sorting was conducted on the cells. Cells containing nanoparticles were separated from those without nanoparticles. Skin equivalents were constructed from keratinocytes and fibroblasts, where both cell types or one cell type contained nanoparticles. They were compared to the control produced from cells sorted without particles. TiO2’s effect on functionality was determined from collagen contraction and colony formation efficiency assays for sorted fibroblasts and keratinocytes, respectively. The impact on cell differentiation was determined from qRT-PCR using mRNA levels of filaggrin, involucrin, corneodesmosin, keratin 10, and mucin 15, all normalized to housekeeping gene GAPDH. RESULTS: Sorting demonstrated nanoparticle uptake in 86% of fibroblasts and 46% of keratinocytes, which produced excessive mucin-type secretion. Functional assays indicated no change in collagen contraction. Colony forming efficiency was reduced by 63% relative to the control. Histology indicated constructs using keratinocytes containing TiO2 (0.4 mg/mL) exhibited 50% reduction in stratum corneum and epidermal thickness relative to the control and contained large TiO2 nanoparticle aggregates. No effects in dermis’ structure and no particulate aggregates were observed when fibroblasts were exposed to TiO2. qRT-PCR indicated significant mRNA upregulation of differentiation markers filaggrin, involucrin, corneodesmosin, keratin 10, and mucin 15. Keratinocyte differentiation’s early onset is a factor in their reduced proliferation. Differentiation was postulated to trigger nanoparticle secretion from keratinocytes and encapsulation in a mucin-like substance, resulting in large aggregates. As skin equivalents developed, aggregates continuously moved into the stratum corneum, from which we observed elimination. Increasing TiO2 concentration to 0.8 mg/mL for keratinocytes resulted in larger aggregates, damage to the epidermis, and no stratum corneum formation. CONCLUSION: TiO2 exposure affects keratinocytes in skin equivalents, inhibiting proliferation, causing early differentiation, and resulting in a thinner epidermis and stratum corneum. Particle agglomeration was followed by encapsulation and secretion from the stratum corneum, showing the epidermal layer’s capacity in protecting skin from environmental pollutants. We need to further understand TiO2 nanoparticle interactions in biological systems and applications in dermatological contexts. REFERENCE:1Li J, Fu S, Lu KW, Christie O, Gozelski MT, Cottone MC, Cottone P, Kianian S, Feng KC, Simon M, Rafailovich M, Dagum AB, Singh G. Engineering functional skin constructs: A quantitative comparison of three-dimensional bioprinting with traditional methods. Exp Dermatol. 2022 Apr;31(4):516-527. doi: 10.1111/exd.14488.