Recent advances in microfluidic cell cultures enable the construction of in vitro human skin models that can be used for drug toxicity testing, disease study. However, current in vitro skin model have limitations to emulate real human skin due to the simplicity of model. In this paper, we describe the development of ‘skin-on-a-chip’ to mimic the structures and functional responses of the human skin. The proposed model consists of 3 layers, on which epidermal, dermal and endothelial components originated from human, were cultured. The microfluidic device was designed for co-culture of human skin cells and each layer was separated by using porous membranes to allow interlayer communication. Skin inflammation and edema were induced by applying tumor necrosis factor alpha on dermal layer to demonstrate the functionality of the system. The expression levels of proinflammatory cytokines were analyzed to illustrate the feasibility. In addition, we evaluated the efficacy of therapeutic drug testing model using our skin chip. The function of skin barrier was evaluated by staining tight junctions and measuring a permeability of endothelium. Our results suggest that the skin-on-a-chip model can potentially be used for constructing in vitro skin disease models or for testing the toxicity of cosmetics or drugs.

Infantile hemangioma (IH) is the most common tumor of infants and young children.^1,2 Although small IH lesions typically do not cause problems, they often require treatment because of the site of occurrence and associated complications such as abnormal eye development or vision problems, airway obstruction, and bowel obstruction.^3,4 Historically, steroids have been used as the primary treatment for IH.^5,6 Steroids have been shown to be antiangiogenic in a number of in vitro settings and also have shown good therapeutic effects clinically. However, the use of steroids may lead to various complications including gastroesophageal reflux and growth disorders, although these complications are associated with long-term use and high dose.⁷ A type of anticancer drug or immunomodulator, interferon alfa, may be used for severe IH in cases where patients did not respond to steroids.^8,9 However, interferon alfa also has several possible adverse effects, including fever, muscle pain, systemic myalgia, and in severe cases, liver damage, blood toxic effects, thyroid hormonal abnormality, and neurological and neurodevelopmental toxic effects.⁸ Because of concerns about these adverse effects, many guardians of pediatric patients prefer to wait rather than accept treatment.

The number of patients requiring flat bone transplantation continues to increase worldwide. Cell transplantation has been successfully applied clinically; however, it causes another defect site and the time requirements to harvest cells and expand them are considerable. In this study, KLD12/KLD12-SP (KLD12+KLD12-substance P [SP]) was designed to mimic endogenous tissue-healing processes. The structures of KLD12, KLD12-SP, and KLD12/KLD12-SP were observed by transmission electron microscopy and circular dichroism spectra. KLD12/KLD12-SP nanofibers (5-10 nm) were created under physiological conditions by formation of a β-sheet structure. The ability of mesenchymal stem cells (MSCs) to recruit KLD12/KLD12-SP was observed by using an in vivo fluorescence imaging system. Labeled human bone marrow stromal cells supplied via an intravenous injection were recruited to the scaffold containing KLD12/KLD12-SP. Polylactic acid/beta-tricalcium phosphate (PLA/β-TCP) scaffolds filled with KLD12/KLD12-SP were applied to repair calvarial defects. The composite constructs (groups: defect, PLA/β-TCP, PLA/β-TCP/KLD12, and PLA/β-TCP/KLD12/KLD12-SP) were implanted into rat defect sites. Bone tissue regeneration was evaluated by observing gross morphology by hematoxylin and eosin and Masson's trichrome staining at 12 and 24 weeks after surgery. Gross morphology showed that the defect site was filled with new tissue that was integrated with host tissue in the KLD12/KLD12-SP group. In addition, from the staining data, cells were recruited to the defect site and lacunae structures formed in the KLD12/KLD12-SP group. From these results, the PLA/β-TCP+KLD12/KLD12-SP composite construct was considered for enhancement of bone tissue regeneration without cell transplantation.

Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen. In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used. Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast-like cells. To discover the mechanism of type I pro-collagen expression in hADSCs in F-CM, we observed that phospho-smad 2/3 levels were increased in the TGF-β/Smad signaling pathway. For in-vivo analysis, we injected various cell types into balb/c nude mouse skin carrying a 10-mm punch wound, and observed a significantly positive wound healing effect in this full-thickness excision model with F-CM-treated hADSCs rather than with untreated hADSCs or the PBS injected group. We differentiated F-CM-treated hADSCs into fibroblast-like cells and demonstrated their efficiency in wound healing in a skin wound model.

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