This study reports a three-dimensional (3D) bioprinting technique that is capable of producing a full-thickness skin model containing pigmentation. Multiple layers of fibroblast (FB)-containing collagen hydrogel precursor were printed and crosslinked through neutralization using sodium bicarbonate, constituting the dermal layer. Melanocytes (MCs) and keratinocytes (KCs) were sequentially printed on top of the dermal layer to induce skin pigmentation upon subsequent air-liquid interface culture. Histological analysis was performed not only to confirm the formation of distinct skin layers, but also to identify the presence of pigmentation. The bioprinted skin structure showed the dermal and epidermal layers as well as the terminal differentiation of the KC that formed the stratum corneum. Moreover, the MC-containing epidermal layer showed freckle-like pigmentations at the dermal-epidermal junction, without the use of external ultraviolet light or chemical stimuli. The presented method offers the capability of producing engineered ephelides in biomimetic skin, thus rendering 3D bioprinting techniques as productive on-demand options for the creation of skin models available for therapeutic or research use.

Objective Skin ageing is inevitably exposed through its typical features such as wrinkles and sagging. Therefore, skin anti‐ageing is a major issue in cosmetic research to prevent and improve ageing symptoms using effective ingredients. Chondroitin sulphate (CS), a type of glycosaminoglycan, is an important structural component of the extracellular matrix (ECM) and is involved in various biological processes, such as cell proliferation, differentiation and migration. Here, we aimed to investigate the effects of CS on skin regeneration and examine its efficacy as a potential safe and effective skin anti‐ageing ingredient. Methods We investigated the effects of CS on cell proliferation in normal human keratinocytes and fibroblasts. Then, cell migration, ECM synthesis and related signalling pathways were examined in fibroblasts through gene and protein expression analysis. Finally, the effect on skin wound healing and regeneration was validated using a full‐thickness skin wound model and an aged skin model. Results Chondroitin sulphate treatment increased the proliferation of keratinocytes and fibroblasts. It also stimulated the migration and synthesis of ECM components of fibroblasts. Further analysis revealed that CS induced the expression of type I procollagen by activating the extracellular signal‐regulated kinase pathway. Using a full‐thickness skin wound model and an aged skin model, we confirmed that CS treatment promoted skin wound healing and regeneration. Conclusion Together, our results indicated that CS has the potential to facilitate skin regeneration, implying that CS could be clinically applied to improve skin ageing.

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide–dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

Mitochondrial dysfunction can drive cellular senescence, which is accompanied by changes in metabolism and increases in senescence-associated secretory phenotypes. Although pyruvate, a key metabolite for numerous aspects of metabolism, has been used as general supplement in synthetic media, the physiological function of pyruvate underlying its protective role against cellular senescence under normal conditions has remained unknown. Here, we show that extracellular pyruvate prevents senescence in normal human dermal fibroblasts through increasing the generation of oxidized nicotinamide adenine dinucleotide (NAD) during the conversion to lactate. Acetylated peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), vacuolar-type H-ATPaseV0A1 (v-ATPaseV0A1), NF-κB p65 subunit (RelA), and histone H3 accumulate under pyruvate deprivation conditions, resulting in the onset of senescence in normal human dermal fibroblasts through the accumulation of abnormal mitochondria generated by lysosomal inactivation-induced mitophagy defects, and through an increase in senescence-associated secretory phenotypes. Furthermore, pyruvate showed a protective effect against aging phenotypes in skin equivalents, which consist of a dermis and epidermis that act similarly to in vivo skin tissues. Our findings reveal a connection between pyruvate and mitochondrial dysfunction in the progression of senescence that is, to our knowledge, previously unreported. These results suggest that the pyruvate deprivation-induced senescence model can be used to study the connection between metabolism and senescence under normal conditions.

Kaempferol is the major flavonol in green tea and exhibits many biomedically useful properties such as antioxidative, cytoprotective and anti-apoptotic activities. To elucidate its effects on the skin, we investigated the transcriptional profiles of kaempferol-treated HaCaT cells using cDNA microarray analysis and identified 147 transcripts that exhibited significant changes in expression. Of these, 18 were up-regulated and 129 were down-regulated. These transcripts were then classified into 12 categories according to their functional roles: cell adhesion/cytoskeleton, cell cycle, redox homeostasis, immune/defense responses, metabolism, protein biosynthesis/modification, intracellular transport, RNA processing, DNA modification/ replication, regulation of transcription, signal transduction and transport. We then analyzed the promoter sequences of differentially-regulated genes and identified over-represented regulatory sites and candidate transcription factors (TFs) for gene regulation by kaempferol. These included c-REL, SAP-1, Ahr-ARNT, Nrf-2, Elk-1, SPI-B, NF-κB and p65. In addition, we validated the microarray results and promoter analyses using conventional methods such as real-time PCR and ELISA-based transcription factor assay. Our microarray analysis has provided useful information for determining the genetic regulatory network affected by kaempferol, and this approach will be useful for elucidating gene-phytochemical interactions.

Demands for safe depigmentation compounds are constantly increasing in the pharmaceutical and cosmetic industry, since the numerous relevant compounds reported to date have shown undesirable side effects or low anti-melanogenic effects. In this study, we reported three novel inhibitors of tyrosinase, which is the key enzyme in melanogenesis, identified using docking-based high throughput virtual screening of an in-house natural compound library followed by mushroom tyrosinase inhibition assay. Of the three compounds, gallacetophenone showed high anti-melanogenic effect in both human epidermal melanocytes and a 3D human skin model, MelanoDerm. The inhibitory effect of gallacetophenone on tyrosinase was elucidated by computational molecular modeling at the atomic level. Binding of gallacetophenone to the active site of tyrosinase was found to be stabilized by hydrophobic interactions with His367, Ile368, and Val377; hydrogen bonding with Ser380 and a water molecule bridging the copper ions. Thus, our results strongly suggested gallacetophenone as an anti-melanogenic ingredient that inhibits tyrosinase.

The outer epidermal skin is a primary barrier that protects the body from extrinsic factors, such as ultraviolet (UV) radiation, chemicals and pollutants. The complete epithelialization of a wound by keratinocytes is essential for restoring the barrier function of the skin. However, age-related alterations predispose the elderly to impaired wound healing. Therefore, wound-healing efficacy could be also considered as a potent function of an anti-aging reagent. Here, we examine the epidermal wound-healing efficacy of the fourth-generation retinoid, seletinoid G, using HaCaT keratinocytes and skin tissues. We found that seletinoid G promoted the proliferation and migration of keratinocytes in scratch assays and time-lapse imaging. It also increased the gene expression levels of several keratinocyte proliferation-regulating factors. In human skin equivalents, seletinoid G accelerated epidermal wound closure, as assessed using optical coherence tomography (OCT) imaging. Moreover, second harmonic generation (SHG) imaging revealed that seletinoid G recovered the reduced dermal collagen deposition seen in ultraviolet B (UVB)-irradiated human skin equivalents. Taken together, these results indicate that seletinoid G protects the skin barrier by accelerating wound healing in the epidermis and by repairing collagen deficiency in the dermis. Thus, seletinoid G could be a potent anti-aging agent for protecting the skin barrier.

Nowadays, laser therapy is a common method for treating various dermatological troubles such as acne and wrinkles because of its efficient and immediate skin enhancement. Although laser treatment has become a routine procedure in medical and cosmetic fields, the prevention of side-effects, such as hyperpigmentation, redness and burning, still remains a critical issue that needs to be addressed. In order to reduce the side-effects while attaining efficient therapeutic outcomes, it is essential to understand the light-skin interaction through evaluation of physiological changes before and after laser therapy. In this study, we introduce a quantitative tissue monitoring method based on optical coherence tomography (OCT) for the evaluation of tissue regeneration after laser irradiation. To create a skin injury model, we applied a fractional CO2 laser on a customized engineered skin model, which is analogous to human skin in terms of its basic biological function and morphology. The irradiated region in the skin was then imaged by a high-speed OCT system, and its morphologic changes were analyzed by automatic segmentation software. Volumetric OCT images in the laser treated area clearly visualized the wound healing progress at different time points and provided comprehensive information which cannot be acquired through conventional monitoring methods. The results showed that the laser wound in engineered skins was mostly recovered from within 1~2 days with a fast recovery time in the vertical direction. However, the entire recovery period varied widely depending on laser doses and skin type. Our results also indicated that OCT-guided laser therapy would be a very promising protocol for optimizing laser treatment for skin therapy.