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A facial pore is an empty funnel-shaped structure filled with cornified cylindrical plugs that can be cosmetically bothersome to some patients. In the previous report, the new unipolar radiofrequency (RF) device with a vacuum showed excellent skin tightening and patient satisfaction with improved pores. This study aims to confirm the treatment's efficacy with the new unipolar RF device on facial dilated pores by measuring quantitative sebum production differences and doing a histologic examination of pore size. Twelve patients who visited the dermatologic clinic without other underlying inflammatory facial skin diseases were included, regardless of the patient's age or sex. All patients received five successive treatments at 2-week intervals. We assessed all changes in sebum production levels, melanin index, erythema index before and after treatment, along with overall improvement (reduction of pore size, skin tone, skin texture, and skin laxity). In the five patients who agreed in advance, a biopsy of the pore lesion was taken before and 1 month after the last treatment with hematoxylin and eosin (HE) staining, Masson's trichrome (M-T) staining, and Victoria blue staining. We observed a significant reduction of sebum production and melanin index after using the new unipolar RF device with a vacuum (sebum production, p = 0.011; MI, p = 0.004). In evaluating patient satisfaction for four categories, the patients showed a moderate to the excellent improvement of more than 50% in their condition except for skin tones. The average pore size decreased by 41.7% in the histological examination, from 64.98 to 37.86 μm. Additionally, we observed an overall decreased sebaceous gland in the dermis and the proliferation of dermal collagen fiber. The number of elastic bundles in the D-E junction was increased after treatment. The nonablative unipolar RF devices with a vacuum can improve dilated pores with a dual mechanism (collagen regeneration and reduction of sebum production), with much less pain than other RF devices.

Mitochondrial dysfunction is linked to age-related senescence phenotypes. We report here the pathway increasing nucleoid remodeling and biogenesis in mitochondria during the senescence of foreskin human diploid fibroblasts (fs-HDF) and WI-38 cells. Replicative senescence in fs-HDF cells increased mitochondrial nucleoid remodeling as indicated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and mitochondrial transcription factor A (TFAM) expression in enlarged and fused mitochondria. Mitochondrial nucleoid remodeling was accompanied by mitochondrial biogenesis in old cells, and the expression levels of OXPHOS complex-I, -IV and -V subunits, PGC-1α and NRF1 were greatly increased compared to young cells. Activated protein kinase C zeta (PKCζ) increased mitochondrial activity and expressed phenotypes of delayed senescence in fs-HDF cells, but not in WI-38 cells. The findings were reproduced in the doxorubicin-induced senescence of young fs-HDF and WI-38 cells via the PKCζ-LKB1-AMPK signaling pathway, which was regulated by the p53-p21WAF1 pathway when p16INK4a was silenced. The signaling enhanced PGC-1α-NRF1-TFAM axis in mitochondria, which was demonstrated by Ingenuity Pathway Analysis of young and old fs-HDF cells. Activation of the p53-p21WAF1 pathway and silencing of p16INK4a are responsible for mitochondrial reprogramming in senescent cells, which may be a compensatory mechanism to promote cell survival under senescence stress.

It has been reported that PI3K/AKT pathway is altered in various cancers and AKT isoforms specifically regulate cell growth and metastasis of cancer cells; AKT1, but not AKT2, reduces invasion of cancer cells but maintains cancer growth. We propose here a novel mechanism of the tumor suppresser, TIS21/BTG2, that inhibits both growth and invasion of triple negative breast cancer cells via AKT1 activation by differential regulation of mTORc1 and mTORc2 activity. Transduction of adenovirus carrying TIS21/BTG2 gene and transfection of short interfering RNAs were employed to regulate TIS21/BTG2 gene expression in various cell lines. Treatment of mTOR inhibitors and mTOR kinase assays can evaluate the role of mTORc in the regulation of AKT phosphorylation at S473 residue by TIS21/BTG2 in breast cancer cells. Open data and immunohistochemical analysis were performed to confirm the role of TIS21/BTG2 expression in various human breast cancer tissues. We observed that TIS21/BTG2 inhibited mTORc1 activity by reducing Raptor-mTOR interaction along with upregulation of tsc1 expression, which lead to significant reduction of p70S6K activation as opposed to AKT1S473, but not AKT2, phosphorylation via downregulating PHLPP2 (AKT1-specific phosphatase) in breast cancers. TIS21/BTG2-induced pAKTS473 required Rictor-bound mTOR kinase, indicating activation of mTORc2 by TIS21/BTG2 gene. Additionally, the TIS21/BTG2-induced pAKTS473 could reduce expression of NFAT1 (nuclear factor of activated T cells) and its target genes, which regulate cancer microenvironment. TIS21/BTG2 significantly lost in the infiltrating ductal carcinoma, but it can inhibit cancer growth via the TIS21/BTG2–tsc1/2–mTORc1–p70S6K axis and downregulate cancer progression via the TIS21/BTG2–mTORc2–AKT1–NFAT1–PHLPP2 pathway.

The periorbital wrinkles are easily perceived evidence of aging, so become a major concern for many patients. Various treatments have been attempted to improve periorbital wrinkles, but the need for new treatments that are less invasive and more effective is still high. In this study, we evaluated the safety, clinical and histological effects of intense focused ultrasound using only a 1.5 mm transducer in the management of periorbital wrinkles. Ten adult Korean females were enrolled. The treatment effect and safety profile were evaluated up to 3 months after 1 session of IFUS treatment on the periorbital wrinkles. The mean subjective satisfaction score was 3.2 ± 0.79 (mean ± standard deviation) by 5- point scale. The mean objective clinical improvement score was highest in the fine wrinkle on the crow's feet area and lowest in the deep wrinkles of the infraorbital and crow's feet area. Histometrically, increase of collagen and elastic fiber density was observed in the all layers of dermis. No serious side effects occurred after the treatment. In conclusion, intense focused ultrasound treatment using a 1.5-mm transducer alone can significantly improve periocular wrinkles after a single treatment with a good safety profile.

Mitochondrial dysfunction is linked to age-related senescence phenotypes. We report here the pathway increasing nucleoid remodeling and biogenesis in mitochondria during the senescence of foreskin human diploid fibroblasts (fs-HDF) and WI-38 cells. Replicative senescence in fs-HDF cells increased mitochondrial nucleoid remodeling as indicated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and mitochondrial transcription factor A (TFAM) expression in enlarged and fused mitochondria. Mitochondrial nucleoid remodeling was accompanied by mitochondrial biogenesis in old cells, and the expression levels of OXPHOS complex-I, -IV and -V subunits, PGC-1α and NRF1 were greatly increased compared to young cells. Activated protein kinase C zeta (PKCζ) increased mitochondrial activity and expressed phenotypes of delayed senescence in fs-HDF cells, but not in WI-38 cells. The findings were reproduced in the doxorubicin-induced senescence of young fs-HDF and WI-38 cells via the PKCζ-LKB1-AMPK signaling pathway, which was regulated by the p53-p21WAF1 pathway when p16INK4a was silenced. The signaling enhanced PGC-1α-NRF1-TFAM axis in mitochondria, which was demonstrated by Ingenuity Pathway Analysis of young and old fs-HDF cells. Activation of the p53-p21WAF1 pathway and silencing of p16INK4a are responsible for mitochondrial reprogramming in senescent cells, which may be a compensatory mechanism to promote cell survival under senescence stress.

SummaryBackground Previous studies have shown that radiofrequency (RF) energy is safe and effective for improving skin laxity. Unlike monopolar and bipolar devices, little has been studied with the unipolar hand piece. Objectives We sought to evaluate the safety and efficacy of a novel fractional unipolar RF device on facial tightening. Patients and Methods This was a retrospective, single-center study of 14 subjects with age-related facial laxity who underwent five sessions of fractional unipolar RF at an interval of 2 weeks, and then followed-up for 3 months. Standardized photos were taken at baseline and at 3-months follow-up, and were assessed by two independent dermatologists using a 4-point scale (0=no improvement, 1=mild improvement, 2=moderate improvement, 3=significant improvement). Punch biopsies (2 mm) were performed and a questionnaire was used to evaluate the patient's satisfaction and the incidence of adverse reactions. Results Fourteen subjects with mild to moderate age-related facial laxity were included in the study. The mean age of the subjects was 49.7 years (range 32-80). 35.7% of the subjects showed significant improvement, 50% moderate improvement, and 14.3% slight improvement of facial laxity in their follow-up photos. About 85.7% of the patients replied that they were either greatly satisfied or satisfied with the results at 3-months follow-up. Skin biopsies revealed an increase in collagen in the dermis. None of the subjects experienced any serious adverse events during or after the procedure. Conclusion Our findings suggest that fractional Unipolar RF can be safely performed on the face and is effective in skin tightening. It has a great advantage over other forms of RF by being entirely painless.

Ablative CO2 fractional lasers have recently been introduced for burn scar treatment because of pronounced clinical outcomes with fewer treatment sessions than nonablative fractional laser. This study was conducted to observe clinical as well as histologic changes of burn scars after treatment with CO2 fractional laser. Eleven patients (one female and 10 males, aged 31–59 years) with skin phototypes III to V with burn scars received 10 sessions of fractional CO2 laser treatments (UltraPulse® Encore; Lumenis, Santa Clara, CA, USA) over an average 5-week interval. Two passes were performed using the following parameters: deep FX mode, 12.5–30 mJ, with a density setting of 5–10 %. Clinical evaluations by three blinded dermatologists were obtained at baseline and at 6 months after the final treatment via photographs using the Vancouver scar scale (VSS). Skin biopsies were done on four patients before and after treatment. All patients showed clinical improvement in their scars with significant decrease in VSS. Histologic findings showed the changes in the upper dermis with newly formed dermal papilla. This characteristic upper dermis change was presented as improvement in surface smoothness and skin tension clinically. Postinflammatory hyperpigmentation and itching sensation were the most common adverse effects. Burn scar treatment by fractional CO2 laser is effective by forming new collagen fibers mainly in the upper dermis. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Background: Various modalities have been used to treat acne scars. CO2 fractional laser is an effective and commonly used treatment. CO2 gas injection into the dermis by needle with high pressure can cause fibrotic collagen breakage, producing the effects of subcision. CO2 also stimulates collagen synthesis by increasing neovascularization and releasing oxygen. Objective: This study evaluated the efficacy and the safety of the combined treatment with CO2 gas subcision and CO2 fractional laser for acne scars. Methods and materials: Fourteen patients with acne scars were treated with three sessions of CO2 gas subcision at 2-week intervals and two sessions of fractional laser at 4-week interval. The clinical improvement was assessed using a 4-point scale. For histologic analysis, punch biopsy was performed before and after treatment in 10 patients. Results: All patients experienced clinical improvements. Excellent, marked, moderate, and mild response was achieved in 1 (7%), 8 (57%), 4 (29%), and 1 patient (7...

High-intensity focused ultrasound (HIFU) and radiofrequency (RF) are used for non-invasive skin tightening. Neocollagenesis and neoelastogenesis have been reported to have a mechanism of controlled thermal injury.To compare neocollagenesis and neoelastogenesis in each layer of the dermis after each session of HIFU and monopolar RF.We analyzed the area fraction of collagen and elastic fibers using the Masson's Trichrome and Victoria blue special stains, respectively, before and after 2 months of treatments. Histometric analyses were performed in each layer of the dermis, including the papillary dermis, and upper, mid, and deep reticular dermis.Monopolar RF led to neocollagenesis in the papillary dermis, and upper, mid, and deep reticular dermis, and neoelastogenesis in the papillary dermis, and upper and mid reticular dermis. HIFU led to neocollagenesis in the mid and deep reticular dermis and neoelastogenesis in the deep reticular dermis. Among these treatment methods, HIFU showed the highest level of neocollagenesis and neoelastogenesis in the deep reticular dermis.HIFU affects deep tissues and impacts focal regions. Monopolar RF also affects deep tissues, but impacts diffuse regions. We believe these data provide further insight into effective skin tightening.

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Recently, it has been reported that amniotic epithelial cells (AECs) could differentiate into multiple cell types including neural, epidermal (ectodermal), and pancreatic and hepatic (endodermal) cells. Therefore, AECs are thought to be a good stem cell source for tissue engineering and regenerative medicine. However, there have been no available data on the proliferation of AECs, especially according to their original anatomical sites, i.e., reflected, placental and cord amnions. In this study, we isolated the AECs from these three amnions by explant outgrowth and also performed in situ stratification by culturing each fresh amnion for 2 days in the submerged condition followed by 7 days in the air-liquid interface condition. As a result, the isolation success rates of AEC from reflected, placental, and cord amnions were 13.7, 44.5, and 71.5%, respectively, and in situ stratification success rates were 0, 53.3, and 76.5%, respectively. To our knowledge, this is the first report that AECs from different anatomical sites have different proliferation capacities in vitro.

Intense focused ultrasound (IFUS) is a novel treatment modality for skin laxity. The delivery of thermal energy to the deeper tissue layers effectively tightens the skin but can also cause significant fat atrophy, limiting its use in patients with a lean face.We sought to evaluate the safety and efficacy of modified IFUS on facial rejuvenation.This was a retrospective, single-center study of 28 subjects with age-related facial laxity who underwent 3 sessions of IFUS (UltraskinTwenty-eight subjects with mild-to-moderate age-related facial laxity were included in the study. The mean age of the subjects was 48 (range 29-74) years. About 32.1% of the subjects showed significant improvement and 57.1% showed improvement of facial laxity in their follow-up photographs. All of them (100%) replied that they were either greatly satisfied or satisfied with the results at three-month follow-up. None of the subjects experienced any serious adverse events including fat atrophy after the procedure.Modified IFUS (three sessions, four weeks apart, 200-300 treatment lines per session) can be safely performed with good clinical results.

Various modalities have been used to treat acne scars. COThis study evaluated the efficacy and the safety of the combined treatment with COFourteen patients with acne scars were treated with three sessions of COAll patients experienced clinical improvements. Excellent, marked, moderate, and mild response was achieved in 1 (7%), 8 (57%), 4 (29%), and 1 patient (7%), respectively. Histologic evaluation of the biopsy specimens showed increased dermal collagen with dermal thickening and elastic fiber straightening in the reticular dermis after the treatment.The combination therapy with CO

Obesity increases the risk of many adult diseases, such as atherosclerosis, diabetes, hypertension, ischemic heart disease and breast cancer. Inhibition of adipogenesis is an effective way in the anti-obesity management. Because of main components of Saururus chinensis is flavonoid, it has been showed some improvement by its antioxidant effects on the atherosclerosis, heart disease and diabetic hyperlipidemia. But mechanism of anti-obesity effect of S. chinensis is not clear. We investigated the effects of ethanol extracts of S. chinensis on adipogenesis in the 3T3-L1 pre-adipocyte. The 3T3-L1 cell line is commonly used to study adipogenesis in vitro. In this study, ethanol extracts of S. chinensis significantly decrease the lipid accumulation in the 3T3-L1 cells proved by measuring triglyceride contents and Oil red O staining. The proposed mechanism of inhibition of adipogenesis in the 3T3-L1 cells with ethanol extracts of S. chinensis is down-regulation of transcriptional factors and adipocytespecific genes such CCAAT/enhancer binding protein α (C/EBPα) and Peroxisome proliferator activated receptor γ (PPARγ) in concentration dependent pattern. These results suggest that ethanol extracts of S. chinensis inhibits adipognesis in the 3T3-L1 cells and can be used as a safe and efficient natural substance to manage anti-obesity.

Infraorbital laxity is a common problem that increases with age. Blepharoplasty with lipectomy is a very commonly performed surgical procedure to treat this problem; however, it is invasive and is associated with the potential for re-emergence. Therefore, young patients may prefer a non-surgical procedure rather than to a surgical procedure. Intense-focused ultrasound (IFUS) has emerged as an effective, non-surgical, tissue-tightening procedure.This study assessed the safety and efficacy of IFUS (Ulthera system, Ulthera Inc, Mesa, AZ, U.S.A.) for facial tightening in Asian patients with infraorbital laxity.We studied 15 patients who were treated with an IFUS device applied to both lower eyelids. The primary outcome measure was an objective improvement in a paired comparison of pre-treatment and post treatment (6 months) photographs. A secondary outcome measure was patient satisfaction as measured by a questionnaire.The mean patient age was 50 years (range, 27-69). All patients received one to two treatments with intense-focused ultrasound. All patients in the study experienced both subjective and objective improvement.IFUS can be used as a non-invasive, skin-tightening procedure for infraorbital laxity. No serious, permanent, or delayed side effects were noted up to 6 months post treatment. Thus, this procedure can be effective and safe in the treatment of decreased laxity of the lower eyelids.

Striae distensae (striae alba) is a challenging cosmetic problem for which various treatment modalities have been applied. However, the treatment of striae distensae has not been satisfactory.This study was done to evaluate the effectiveness and the safety of enhanced penetration of platelet rich plasma with ultrasound after plasma fractional radiofrequency for the treatment of striae distensae.Eighteen participants with striae distensae were treated with a Legato system (Alma Lasers, Israel) every two weeks for a total of four sessions. Thereafter, in order to enhance platelet-rich plasma penetration, ultrasound is applied. Clinical photographs were taken before first treatment and two months after the final treatment. Objective and subjective improvement scores were evaluated to demonstrate the efficacy. Abdominal skin biopsies were obtained from three individuals and histological changes were analyzed by light microscopy.During the two months after the last treatment, the average width of the widest striae had decreased from 0.75 to 0.27 mm. In the objective assessment, 71.9% of the participants reported ''good" or "very good'' overall improvement. In the subjective assessment, and 72.2% of the participants reported ''very satisfied" or "extremely satisfied'' with overall improvement. The only reported side effect was post-inflammatory hyperpigmentation (11.1%).The plasma fractional radiofrequency and transepidermal delivery of platelet-rich plasma using ultrasound is useful in the treatment of striae distensae.

Adult stem cells are considered multipotent. Especially, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM-MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency-electromagnetic fields (ELF-EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF-EMFs inhibited the growth of hBM-MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF-L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF-EMFs. We suggest that EMFs can induce neural differentiation in BM-MSCs without any chemicals or differentiation factors.

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.

BACKGROUND Laxity and wrinkles of the aging face are common cosmetic concerns. Intense focused ultrasound (IFUS), a novel treatment modality for skin laxity, produces thermal effects at various depths while sparing overlying epidermis. OBJECTIVE To evaluate the safety and efficacy of IFUS in facial skin tightening. METHODS AND MATERIALS Twenty-two Korean patients with facial laxity were analyzed after a single IFUS treatment. Patient assessments were recorded, and two blinded, experienced clinicians who assessed improvement of nasolabial folds and jaw tightening evaluated photographs of patients and rated skin laxity. Skin biopsies were taken from 11 patients before and 2 months after treatment. RESULTS Objectively, nasolabial folds and jaw lines were improved in all patients. Subjectively, 77% of patients reported much improvement of nasolabial folds, and 73% of patients reported much improvement at the jaw line. Histologic evaluation of skin biopsy samples using hematoxylin and eosin and Victoria blue stains showed greater dermal collagen with thickening of the dermis and straightening of elastic fibers in the reticular dermis after treatment. CONCLUSION IFUS is a safe, effective, noninvasive procedure to tighten the facial skin of Asian patients. Improvement is associated with greater production of dermal collagen and straightening of dermal elastic fibers. The authors have indicated no significant interest with commercial supporters.

The adult hair follicle has well-defined dermal and epithelial populations that display distinct developmental properties. The follicular dermal cells, namely the dermal papilla and dermal sheath, are derived from the same mesenchymal cells as dermal fibroblasts and therefore, we believed that follicular cells could be useful sources of interfollicular keratinocytes and fibroblast for skin wound repair. In this study, we evaluated the relative effect of various mesenchymal-derived cells on wound healing following skin injury. Human dermal cells, including two different follicular dermal cells and skin fibroblasts were cultured in collagen sponges and compared with respect to wound healing. Results indicated that there was no significant difference in wound contraction and angiogenesis among the cell types. Further, dermal sheath cells exhibited relatively poor results compared with other cells in new collagen synthesis. Finally, basement membrane reformation and new collagen synthesis for the dermal papilla cell grafts was superior to those of the dermal sheath cells or fibroblasts.

BACKGROUND Various trials have been conducted on the management of male pattern hair loss (MPHL). A variety of laser and light sources have been used for the treatment of MPHL. OBJECTIVE To understand the effects of a 1,550-nm fractional erbium-glass laser on the hair cycle in an alopecia mouse model and to study the clinical effects of the same laser used as treatment for MPHL. MATERIALS AND METHODS Irradiation was applied to the shaved skin of C3H/HeN mice using various energy and density settings and varied irradiation intervals. In a clinical pilot study involving human subjects, 20 participants were treated over five sessions at 2-week intervals. A fractional photothermolysis laser was used at the energy of 5 mJ and a total density of 300 spots/cm2. RESULTS In the animal study, the hair stimulation effects were dependent upon the energy level, density, and irradiation interval. The anagen conversion of hair and the increase in Wnt 5a, β-catenin signals were observed. In the human pilot study, incremental improvements in hair density and growth rate were observed. CONCLUSIONS This pilot study showed that a 1,550-nm fractional erbium-glass laser might induce hair growth, but more intensive studies are required to clarify the clinical applications of this treatment. The authors have indicated no significant interest with commercial supporters.

Recently hepatic differentiation of mesenchymal stem cells (MSCs) from bone marrow (BM), adipose tissue (AT), umbilical cord blood (UCB), and umbilical cord (UC) has been reported. In this study, the four-step sequential exposure with oncostatin M (OSM) plus trichostatin A (TSA) or OSM plus dimethyl sulfoxide (DMSO) at the final step induced hepatic differentiation of UC-MSCs. As a result, the morphology and protein expression were sequentially changed in the step-dependent manner. And the urea synthesis rates of (OSM plus TSA)- and (OSM plus DMSO)-treated cells on day 21 reached to 23 ± 0.4 and 20 ± 0.5 μg/106 cells/day, respectively. The ammonia concentrations 24 h after culture with 1 mM NH4Cl-supplemented medium dropped to 0.41 ± 0.08 mM (OSM plus TSA) and 0.57 ± 0.05 mM (OSM plus DMSO). Also the ethoxyresorufin O-deethylase (EROD) activities were 3.4-fold (OSM plus TSA) and 2.2-fold (OSM plus DMSO) higher than non-induced controls on day 21. It is thought that TSA and DMSO cause the expression of key genes through epigenetic change. Although sequential exposure with TSA or DMSO induced some liver-specific functions to some extent, the degree of activities are yet lower than those of mature hepatocytes. So it will be necessary to optimize the concentration and exposure time for achieving comparable activities to normal hepatocytes in the future.

There are many different types of periodontal diseases. One such disease causes a defect of alveolar bone that is considered serious. Hence, researchers have examined potential treatments for this type of disease using tissue engineering techniques. Periodontal tissues are exposed to mechanical stress caused by occlusion and mastication, and both the cells and extracellular matrix in these tissues undergo architectural modifications to compensate for the applied stress. Therefore, in this study we analyzed the effect of mechanical tension on the osteogenesis of human dental pulp stem cells (DPSCs). To identify osteogenesis induced by mechanical stress in dental pulp, we examined the effects of tension on DPSCs. We evaluated the effects of mechanical stimuli on the osteogenesis of human dental pulp cells grown on silk scaffolds subjected to 10% strain using a bioreactor. The tension was applied with 0.2 Hz over the course of 5 days and was then continuously applied for 10 more days. We evaluated cell differentiation by RT-PCR, Western blotting and immunohistochemistry. Applying 10% tension to the culture resulted in increases in collagen type I, fibronectin, osteoprotegerin, and bone sialoprotein expression and decreases in a-smooth muscle actin expression. These data suggest that mechanical stimulation promotes osteogenesis in DPSCs.

The purpose of this study was to evaluate the cell compatibility of collagen-hyaluronan (HA) substrate in vitro by assessing ACL cell cultures. Additionally, the use of collagen-HA substrate as a dressing material for partial ACL defects as well as its effect on collagen synthesis and angiogenesis were evaluated in vivo. The initial attachment and proliferation of dog ACL cells on silk matrix covered with collagen-HA substrate (SMCH) was greater than that observed on silk matrix alone. Silk matrix and SMCHs were implanted as dressing materials into partial ACL defects located at the knees of dogs, and they were harvested six weeks after implantation. A histological evaluation of the collagen-HA substrates revealed the presence of monocytes and the absence of giant cells in all cases. MT staining of the SMCH-grafted group showed a higher level of granulation tissue formation consisting of fibroblasts and collagen fibers compared to the silk matrix-grafted group. In addition, CD31 staining revealed that the SMCH-grafted area showed more blood vessel formation than the silk matrix-grafted area. These results suggest that the collagen-HA substrate was cell-compatible in vitro and enhanced collagen synthesis and new blood vessel formation in vivo.

We previously showed that lysophosphatidic acid (LPA) and an adenylyl cyclase inhibitor (ACI) stimulate mitogenic activation of senescent human diploid fibroblasts. Because the modulation of cell proliferation may affect wound healing in aged organisms, we studied the effects of LPA and ACI on in vivo skin wound healing in aged Fisher 344 rats. We found that, in aged rats, wound healing improved in animals treated with LPA and/or ACI (relative to untreated controls), as assessed by histological analysis of reepithelialization and immunostaining for proliferating cell nuclear antigen. The age-dependent activation of mitogenic responses by LPA and ACI was confirmed in other cell types. Taken together, our findings suggest that the activation of mitogenic potential in senescent cells by LPA and/or ACI may translate into enhanced in vivo wound healing and tissue regeneration in aged animals.

The major risk factor of postmenopausal osteoporosis is estrogen deficiency. Hormone replacement therapy is efficacious against osteoporosis, but it induces several significant adverse effects. In this study, therefore, we compared therapeutic potencies of three phytoestrogens: genistein, daidzein, and formononetin. Our result showed that in Saos-2 cells, formononetin and genistein (5 x 10(-7) M) treatment increased alkaline phosphatase activity by 33.0 +/- 5.8% and 21.1 +/- 4.0%. Genistein inhibited osteoclast formation in a dose-dependent manner. In OVX rats, formononetin-treated groups given 1 and 10 mg/kg/day displayed increased trabecular bone areas (TBAs) within the tibia. Genistein- and daidzein-treated groups also displayed increased tibial TBAs. TBAs of the lumbar vertebrae were higher in all treated groups than in the control group. In conclusion, formononetin as well as other isoflavones, such as daidzein and genistein, inhibited bone loss caused by estrogen-deficiency.

In this study, we investigated the effects of a natural mineral extract on damaged skin keratinocytes. We have focused on the possible effects of the natural mineral extract on UV-irradiated normal human skin keratinocytes. Human keratinocytes were UV-irradiated and were treated with 0.1, 1.0, or 10% of natural mineral extract. Study controls included non-UV-irradiated cells, UV-irradiated cells (no extract), and UV-irradiated cells treated with 25 nM ascorbic acid. Cell viability following UV irradiation was significantly higher in the groups treated with the natural mineral extract at doses of 0.1, 1.0, and 10% than in the control group with UV irradiation only. The UV-irradiated cells demonstrated increased secretion of laminin relative to non-irradiated controls. We found that treating these UV-irradiated keratinocytes with natural mineral extract reduced vacuole size and number, and it dose-dependently increased laminin secretion and reduced IL-2 synthesis. Also, MMP-1 and MMP-2 activity were significantly less in cells treated with natural mineral extract than in UV-irradiated controls. The UV irradiation reduced MMP-1 levels to 45±5 ng/mL from 130±5 ng/mL. In addition, MMP-2 production in keratinocytes was significantly reduced by 11±1 ng/mL compared to the UV-irradiation control. In conclusion, the results of this work suggest that natural mineral extract has effects on keratinocytes damaged by UV exposure.

The purpose of this study was to evaluate the biocompatibility of silk and collagen-hyaluronan (HA) in vitro by assessing anterior cruciate ligament (ACL) cell and T-lymphocyte cultures on scaffolds. The use of composite scaffolds as artificial ligaments in ACL reconstruction and their effects on angiogenesis were evaluated in vivo. The silk scaffold was knitted by hand and dry coated with collagen-HA, whereas the composite silk scaffold was made by covering a silk scaffold with a lyophilized collagen-HA substrate. The initial attachment and proliferation of human ACL cells on the composite silk scaffold was superior to the attachment and proliferation observed on the silk scaffold. The immune response was higher in both scaffolds after 72 h (p < 0.05) compared with the control culture condition without scaffolding, as assessed by T-lymphocyte cultures in vitro. There was no significant difference in the immune response in vitro between the silk and composite silk scaffolds. Silk and composite silk scaffolds were implanted as artificial ligaments in ACLs removed from the knees of dogs, and they were harvested 6 weeks after implantation. On gross examination, the onset of an inflammatory tissue reaction, such as synovitis, was seen in both the silk scaffold and the composite silk scaffold groups. An histological evaluation of the artificial ligament implants revealed the presence of monocytes in the silk composite scaffold and the absence of giant cells in all cases. MT staining in the composite silk scaffold-grafted group showed granulation tissue consisting of fibroblasts, lymphocytes, monocytes, and collagen fibers. In addition, CD31 staining revealed the formation of new blood vessels. On the other hand, no reparative tissues, such as blood vessels, collagen, and cells, were observed in the silk scaffold-grafted group. These results suggest that the lyophilized collagen-HA substrate is biocompatible in vitro and enhances new blood vessel and cell migration in vivo.

In this work, a novel type of composite scaffold was designed, which has the suitability of both high biocompatibility and strong mechanical properties, for use in bioartificial dermis applications. The reinforced scaffold consisted of a lyophilized collagen sponge formed around a cross-linked collagen meshwork with an average thread diameter of approximately 55 μm. Fibroblasts were cultured in the reinforced collagen sponge for 7 days, during which time the pores in the sponge became filled with cells that secreted extracellular matrix (ECM) to form a bioartificial dermis. Results of ultimate tensile strength (UTS) measurements and compression tests indicated that the bioartificial dermis formed around the reinforced collagen sponge showed about ten times the strength of the bioartificial dermis formed around a typical collagen sponge (1.5 ± 0.05 vs. 0.15 ± 0.05 and 2.5 ± 0.1 vs. 0.2 ± 0.08 MPa, respectively). As a result, reinforced collagen mesh improved mechanical properties and this technique will be possible to make stronger scaffolds, not only for artificial skin applications but also various artificial tissues, such as synthetic cartilage, bone, and blood vessels.

In this study, we have evaluated the effects of mechanical tension on the proliferation and extracellular matrix (ECM) production of human dental pulp stem cells (DPSCs) using a flexwell system that imposed cyclic mechanical tension at 0.03 Hz with 0, 5, and 8% strains. In the early stage (4 days), DPSCs at 5 and 8% strains had a similar proliferation, which was higher than the control. However, in the late stage (10 days), DPSCs at 8% strain had a higher proliferation than the control and 5% strains. This result clearly demonstrated that DPSC proliferation under tension varied with culture time. In addition, mechanical tension was shown to increase the amount of lactate dehydrogenase (LDH) released during culture. RT-PCR analysis was used to show that mechanical tension also increased collagen and osteopontin expression and decreased α-smooth muscle actin (α-SMA) expression. Furthermore, FACS analysis showed that CD105 expression did not change in all groups but CD 90 expression decreased at 8% strain. In conclusion, our results suggest that an appropriate level of mechanical tension can serve as a potent positive modulator of proliferation, differentiation and ECM production in DPSCs.

This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

Herein, we reconstructed a rabbit corneal epithelium on a lyophilized amniotic membrane (LAM) using a modified version of two Teflon rings (the Ahn’s supporter). We compared the corneal epithelial cells we had differentiated in vitro using air-liquid interface (6 days, 12 days) and submerged (6 days, 12 days) cultures and followed a six-day tilting dynamic air-liquid interface culture with a six-day tilting submerged culture. We characterized the reconstructed corneal epithelium using digital photography, histological imaging, and transmission electron microscopy. The reconstructed corneal epithelium created under air-liquid interface culture exhibited a healthier basal corneal epithelial layer than that created under submerged culture. The reconstructed corneal epithelium on the LAM that was produced using the tilting dymanic culture exhibited a healthy basal layer. We therefore proposed that tilting submerged culture not only supplied nutrients from the medium to the corneal epithelial cells on the LAM, but it also removed the horny layer in the upper part of the reconstructed corneal epithelium, presumably by mimicking the effects of blinking. This study demonstrated that corneal epithelium reconstruction on a LAM using a tilting submerged culture after a tilting air-liquid interface culture may be useful not only for allogeneic or autologous transplantation, but also for in vitro toxicological test kits.

Background:Striae distensae (SD) are dermal scars characterized by linear atrophic depressions. Fractional photothermolysis (FP) is an effective modality for the treatment of cutaneous scars. Objective:To evaluate the safety and efficacy of FP for the treatment of SD in Asian skin. Methods:Six female volunteers aged between 20 and 35 years with chronic striae albae phase SD on both buttocks were enrolled. SD lesions on the right buttock were irradiated with a 1550-nm FP laser and patients were followed every 4 weeks for a total of 8 weeks. Outcome assessments included serial photographs, erythema index and melanin index tracking, skin elasticity, histologic examination, and the patients’ subjective satisfaction scores. Results:FP treatment was associated with a substantial improvement in the appearance of SD at 8 weeks post-treatment. The erythema index and melanin index of the treated SD lesions tended to converge with those of the control skin at 4-week follow-up. Skin elasticity was found to be partially normalized after FP. In addition, there was a significant increase in epidermal thickness, collagen, and elastic fiber deposition after FP as demonstrated by histologic examination. Adverse effects of FP included mild and transient pain and hyperpigmentation. Conclusion:FP can reduce SD by stimulating new collagen and elastic fiber synthesis. In addition, repetitive treatments might constitute an effective approach to improving the treatment of SD.

To support and enhance thein vitro growth and activity of mesenchymal stem cells (MSCs), the cell culture medium may be supplemented with various proteins and factors to mimic the physiological environment in which the cells optimally proliferate and differentiate. In this study, the effects of mechanical factors on cellular metabolic responses were investigated experimentally using a bioreactor. The effects of various chemical factors, such as growth factors, cytokines, and hormones, were also investigated. Based on previous reports demonstrating the important roles of mechanical factors in the growth and activity of MSCs, we sought to evaluate the effects of mechanical stimuli on the proliferation of bone marrow-derived MSCs using a cell training bioreactor that imposed cyclic mechanical stretch, with parameters of 240 min/day, 0.03 Hz, and 5–15% strain. The application of cyclic stretch (5–15% strain) to the MSCs enhanced their proliferation during the early stage (3 days), but not the late stage (14 days), of batch culture. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from the MSCs during culture. Appropriate levels of mechanical stretch (5–10% strain) increased collagen synthesis, but did not alter MSC surface antigen expression. It is thought that the appropriate level of mechanical stretch was able to serve as a potent positive modulator of MSC proliferation during the initial stages of culture.

Mesenchymal stem cells (MSCs) from bone marrow appear to be an attractive tool for use in tissue engineering and cell-based therapies due to their multipotent capacity. The majority of studies on MSCs have been restricted to the roles of growth factors, cytokines, and hormones. Based on previous reports demonstrating the important roles of amino acids, we sought to evaluate the effect of essential amino acids (EAs) and nonessential amino acids (NEAs) on the proliferation and differentiation of MSCs. The results showed that the EA/NEA compositions during culture could significantly modulate MSC proliferation and differentiation and, especially, that EAs served as a potent positive modulator in the proliferation of MSCs without causing a deficit in the differentiation capacity of the cells. These results will be very useful in the production of MSC-based cell therapy products for use in the field of tissue engineering and regenerative medicine.

WD40-repeat proteins have four to eight repeating units flanked by Gly-His (GH) and Trp-Asp (WD) at both termini and folds into a beta-propeller. A polypeptide deduced from a Clonorchis sinensis cDNA clone analyzed to have seven WD40-repeats and predicted to form a beta-propeller (CsWD1). The CsWD1 protein was expressed stage-specifically in the metacercariae and localized in the tegumental syncytium. The CsWD1 protein is suggested to serve as a platform for interacting partner proteins in the tegumental syncytium of C. sinensis metacercariae.

The aim of this study was to estimate the mechanical properties and evaluate the biocompatibility of silk and PGA scaffolds as an artificial ligament to an ACL reconstruction. The scaffold for the artificial ligament was braided / knitted silk or PGA thread. The mechanical properties, cell growth, and subcutaneous tissue reactions were determined for both types of scaffolds. The breaking load of the PGA scaffold was double that of the sericin removed silk scaffold (SRSS). However, the initial attachment and growth of human ACL cells on the SRSS was superior to the PGA scaffold. In addition, the immune response was significantly higher on the PGA scaffold after 72 h (p

In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.

Hair follicle is a small but very complex and dynamic miniorgan of the human body. It is easy to isolate and culture mesenchymal cells but not epithelial cells of hair follicle. It is necessary for intact and healthy outer root sheath (ORS) cells to be isolated and cultured. In this study we developed an appropriate isolation method to yield 6.4±0.75×104 cells/hair follicle, which is about 9-fold comparing to our previous data. This yield was achieved by modifications such as different kinds of enzyme uses, fragmentation, and mechanical stimuli. Especially we detected that the different kinds of isolation enzyme could affect proliferation of ORS cells during primary culture. In addition, bovine pituitary extract (BPE) was needed for ORS cells to proliferate and to form colonies under serum-free, feeder layer-free culture condition, but type I collagen as a substratum did not have any positive effect. Moreover, ORS cells under BPE-added condition contained stem/progenitor cells expressing β1-integrin. CK19, and CD34. These results can provide useful cell culture information, not only in the study of hair biology but also in the field of tissue engineering and cell therapy for the treatment of alopecia.

BACKGROUND Various lasers have recently been reported as effective treatment modalities for striae distensae, but pigmentary alterations are a major concern to the darker skin type. The Thermage (Therma Cool TC; Thermage Inc, Hayward, CA) is a radiofrequency device for the lifting of face and neck, and there is no report of using it for striae distensae. OBJECTIVE The purpose was to evaluate the effectiveness of the Thermage in combination with pulsed dye laser. MATERIALS AND METHODS Thirty-seven patients with abdominal striae distensae were treated with the Thermage and 585-nm pulsed dye laser in the first session at baseline. An additional two sessions of pulsed dye laser were performed at Weeks 4 and 8. Thermage was used at fluences of 53 to 97 J/cm2 and pulsed dye laser at fluences of 3.0 J/cm2 with 10-mm spot. Skin biopsies were taken of nine patients. RESULTS In the subjective assessment, 89.2% of the patients showed “good and very good” to overall improvement, and 59.4% were graded as “good and very good” in elasticity. All of the nine specimens showed an increase in the amount of collagen fibers, and increased elastic fibers were found in six specimens. CONCLUSION The Thermage and pulsed dye laser appear to be an effective treatment for striae distansae.

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.