Three-Dimensional Skin Tissue Printing with Human Skin Cell Lines and Mouse Skin-Derived Epidermal and Dermal Cells.

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

Effects of Melandrium firmum Rohrbach on RANKL­induced osteoclast differentiation and OVX rats.

Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate­resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor­κB ligand­induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c­Fos were measured using western blotting and reverse transcription­PCR. The expression levels of osteoclast­related genes were also examined. To further investigate the anti­osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro­computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast­related genes by downregulating transcription factors, such as NFATc1 and c­Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis­like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.

Effect of Cone of Pinus densiflora on DNCB-Induced Allergic Contact Dermatitis-Like Skin Lesion in Balb/c Mice.

Cone of Pinus densiflora (CP), or Korean red pinecone, is a cluster of Pinus densiflora fruit. CP has also been verified in several studies to have anti-oxidation, anti-fungal, anti-bacterial, and anti-melanogenic effects. However, anti-inflammatory effects have not yet been confirmed in the inflammatory responses of pinecones to allergic contact dermatitis. The purpose of this study is to prove the anti-inflammatory effect of CP on allergic contact dermatitis (ACD) in vitro and in vivo. CP inhibited the expression of TSLP, TARC, MCP-1, TNF-α, and IL-6 in TNF-α/IFN-γ-stimulated HaCaT cells and MCP-1, GM-CSF, TNF-α, IL-6, and IL-8 in PMACI (phorbol-12-myristate-13-acetate plus A23187)-stimulated HMC-1 cells. CP inhibited the phosphorylation of mitogen-activated protein kinase (MAPKs), as well as the translocation of NF-κB on TNF-α/IFN-γ stimulated in HaCaT cells. In vivo, CP decreased major symptoms of ACD, levels of IL-6 in skin lesion, thickening of the epidermis and dermis, infiltration of eosinophils and mast cells, and the infiltration of CD4+ T cells and CD8+ T cells. This result suggests that CP represents a potential alternative medicine to ACD for diseases such as chronic skin inflammation.

Anti-inflammatory effects of Fritillaria thunbergii Miquel extracts in LPS-stimulated murine macrophage RAW 264.7 cells.

The aim of the present study was to demonstrate that Fritillaria thunbergii Miquel extract exerts anti-inflammatory and antioxidant effects on lipopolysaccharide-stimulated RAW 264.7 cells. To confirm the inhibitory effect of ethyl acetate fraction of FTM (EAFM) on inflammation, the expression of nitric oxide (NO) and inflammatory cytokines was assessed by performing ELISA. Expression of intracellular mRNA and protein was confirmed by reverse transcription PCR and western blotting. In addition, the anti-inflammatory and anti-oxidant mechanisms of NF-κB, MAPK and heme oxygenase-1 (HO-1) were also investigated. EAFM significantly inhibited the expression of inflammatory factors including NO, IL-6 and TNF-α at non-toxic concentrations. EAFM also inhibited the mRNA and protein expression of inducible nitric oxide synthase in a concentration-dependent manner, but did not alter the expression of cyclooxygenase-2. Pre-treatment with EAFM inhibited the nuclear translocation of NF-κB, and suppressed the phosphorylation of ERK and JNK. In addition, EAFM induced 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity and an increase in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. The results indicated that EAFM inhibited the expression of pro-inflammatory cytokines by inhibiting ERK/JNK phosphorylation and NF-κB translocation. EAFM also exerted antioxidant effects via Nrf2/HO-1 stimulation. Collectively, the results of the present study indicated that EAFM may be a valuable alternative for the treatment of a variety of inflammatory diseases.

Solanum nigrum Linne improves DNCB­induced atopic dermatitis­like skin disease in BALB/c mice.

The present study aimed to investigate the effects of Solanum nigrum Linne (SNL) in a model of 1­chloro­2,4­dinitrobenzene (DNCB)­induced atopic dermatitis (AD) and in TNF­α/IFN­Î³­stimulated HaCaT cells. AD is a chronic inflammatory skin disease and is characterized by erythema, edema, increased pruritus and eczema. Steroids are most commonly used for anti­inflammatory therapy; however, their long­term use is limited due to side­effects, such as osteoporosis, brittle skin, muscle weaknesses and diabetes. Therefore, patients with AD require alternative treatment strategies. In previous studies, SNL has been reported to be effective against oxidants and cancer. However, to the best of our knowledge, the effects of SNL on AD have not yet been investigated. The present study examined the effects of SNL ethanol extract on a model of DNCB induced AD and on TNF­α/IFN­Î³­stimulated HaCaT cells. The skin tissue was sectioned to measure the thicknesses of the epidermis and dermis, as well as the numbers of eosinophils, mast cells and CD8 infiltration by H&E, toluidine blue, Masson's trichrome and IHC staining. ELISA was performed using serum to measure IgE levels. The present study also examined the expression of various inflammatory cytokines, MAPK and NF­κB in TNF­α/IFN­Î³­stimulated HaCaT cells. SNL significantly reduced the levels of cytokines released from HaCaT cells stimulated with TNF­α/IFN­Î³. SNL also significantly reduced the levels of p­p38 at 30 min and significantly reduced the activation of NF­κB in a time course experiment. In addition, SNL significantly reduced the level of serum IgE and dermal thickness and the infiltration of mast cells and CD8 in the BALB/c mouse model of DNCB­induced AD. The results of the current study suggest that SNL exerts a suppressive effect on pro­inflammatory cytokines in vitro and in vivo through the regulation of the immune system.