Dorsal Skin Of Nude Mice Research Articles

Prophylactic Application of Human Adipose Tissue-Derived Products to Prevent Radiation Disorders.

Radiation therapy is a mainstay treatment for malignancies, but it can induce deterministic adverse effects in surrounding healthy tissues, including atrophy, fibrosis, ischemia, and impaired wound healing. This exploratory study investigated whether prophylactic administration of products containing adipose tissue-derived stem cells immediately after radiotherapy could prevent the development of long-term functional disorders in irradiated tissues. A total irradiation dose of 40 Gy (10 Gy, four times weekly) was delivered to the dorsal skin of nude mice. Subsequently, a prophylactic treatment with vehicle, fat tissue, stromal vascular fraction, or micronized cellular adipose matrix was injected subcutaneously into the irradiated area. Six months after these prophylactic treatments, a cutaneous punch wound was created to evaluate histologic changes and wound healing. Histologic assessments demonstrated dermal thickening, atrophy, and increased collagen deposits in the subcutaneous fatty layer 6 months after radiotherapy. In addition, wound healing was significantly delayed. The prophylactic treatments with three different types of human adipose tissue-derived products significantly prevented radiation-induced histologic changes and accelerated wound healing compared with the vehicle-treated irradiated group. This is the first study to demonstrate potential for prophylactic treatments after radiotherapy, which could prevent the progression of chronic radiation therapy disorders. The results could have a substantial impact on current anticancer radiotherapies; a next-generation radiation therapy may need to be always combined with a stem cell therapy. Such prophylactic treatments have the potential to improve wound healing of irradiated tissue and clinical outcomes of reconstructive surgery required after cancer radiotherapy. The results of this study suggest that it is clinically possible to prevent the development of radiation disorders using stem cell therapies. This study may provide a new concept of prophylactic treatment, which would be a paradigm shift in radiotherapy.

Cell-Free Fat Extract Increases Dermal Thickness by Enhancing Angiogenesis and Extracellular Matrix Production in Nude Mice

Although adipose-derived stem cells (ADSCs) and nanofat exert antiaging effects on skin, they contain cellular components that have certain limitations in clinical practice. Cell-free fat extract (Ceffe) is a fraction purified from nanofat through removal of cellular components and lipid remnants that contains various growth factors. The purpose of this study was to evaluate the effects of Ceffe on cultured human dermal fibroblasts in vitro and on the dermis of nude mice in vivo. In the in vitro study, human dermal fibroblasts were cultured with Ceffe for 72 hours, followed by flow cytometry measurement of cell proliferation and cell cycle. In the in vivo study, different concentrations of Ceffe were injected into the dorsal skin of nude mice for 4 weeks. The thickness of the dermis; proliferation of cells; density of the capillary; and expressions of type I and III collagen (Col-1 and Col-3), matrix metalloproteinase-1, matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-3 were measured through histologic and Western blot analyses. Ceffe significantly increased cell proliferation in cultured dermal fibroblasts. In the mouse skin, Ceffe significantly increased the thickness of the dermis, number of proliferating cells, density of the capillary, and expressions of Col-1 and Col-3. Ceffe increased the dermal thickness of nude mice, possibly by enhancing angiogenesis and extracellular matrix production, and can therefore be used for skin rejuvenation.

Protective Effect of Fat Extract on UVB-Induced Photoaging In Vitro and In Vivo.

Background Nanofat can protect against ultraviolet B- (UVB-) induced damage in nude mice. Fat extract (FE) is a cell-free fraction isolated from nanofat that is enriched with a variety of growth factors. Objective To determine whether FE can protect against UVB-induced photoaging in cultured dermal fibroblasts and in nude mice. Method For the in vitro study, human dermal skin fibroblasts were pretreated with FE 24 h prior to UVB irradiation. Generation of reactive oxygen species (ROS) was analyzed immediately following irradiation, while cell cycle analysis was performed 24 h after UVB irradiation. Senescence-associated β-galactosidase (SA-β-gal) expression, cell proliferation, and expression of glutathione peroxidase 1 (GPX-1), catalase, superoxide dismutase-1 (SOD-1), SOD-2, and collagen type 1 (COL-1) were investigated 72 h after UVB irradiation. For the in vivo study, the dorsal skin of nude mice was irradiated with UVB and mice were then treated with FE for 8 weeks. The thickness of the dermis, capillary density, and apoptotic cells in skin tissue sections were investigated after treatment. The expression of GPX-1, catalase, SOD-2, SOD-1, and COL-1 in the tissue was also measured. Result FE significantly increased cell proliferation and protected cells against UVB-induced cell death and cell cycle arrest. FE reduced ROS and the number of aged cells induced by UVB irradiation. FE promoted the expression of COL-1 and GPX-1 in cultured dermal fibroblasts. FE treatment of UVB-irradiated skin increased dermal thickness and capillary density, decreased the number of apoptotic cells, and promoted the expression of COL-1 and GPX-1. Conclusion FE protects human dermal fibroblasts and the skin of nude mice from UVB-induced photoaging through its antioxidant, antiapoptotic, and proangiogenic activities.

Mechanism of Skin Improvement in Radiation Wounds Following Fat Grafting: The Fate of Adipose-derived Stem Cells and Role of Stromal Vascular Fraction

PURPOSE: Consequences of radiation include thickened, fibrotic and inelastic skin. Fat grafting helps alleviate this damage by decreasing epidermal thickness, increasing vascularization, and decreasing fibrosis. The exact mechanism is not understood and carries many hypotheses including the effects of adipose-derived stem cells (ADSCs) within fat. The first aim of this study was to determine which components of fat cause benefits seen with postradiation fat grafting with the hypothesis that pure ADSC group would have the greatest difference in epidermal thickness. The second aim was to determine the mechanism by which these skin changes are mediated with the hypothesis that human ADSCs can differentiate into epithelial stem cells to regenerate the skin. METHODS: The dorsal skin of nude mice was directly radiated. Four weeks postradiation, injections were performed under the radiated skin with either human lipoaspirate, stromal vascular fraction, or pure ADSCs. The pure ADSCs were confirmed as p63− with flow cytometry before injection. The mice were euthanized at 2 and 4 weeks postinjection. Epidermal thickness was measured to determine treatment effect. Immunohistochemistry was performed using an antibody specific for human epithelial stem cell marker p63 and imaged using confocal microscopy. Nuclei positive for DAPI or p63 were quantified using ImageJ. RESULTS: At 2 weeks, all experimental groups that were injected with human cells (ADSC, SVF, and lipoaspirate) had statistically thinner epidermis compared to the radiation-only control group without statistical differences between experimental groups. At 4 weeks, lipoaspirate and SVF groups remained statistically thinner than control groups with no statistical difference between the two. At 4 weeks, the epidermal thickness of the ADSC group was not statistically different than controls. There was a significant decrease in epidermal thickness from week 2 to week 4 in the lipoaspirate, SVF, and matrigel-only groups. Immunohistochemistry showed the presence of p63+ human cells in all experimental groups and absence in control groups. At 2 weeks, there is a statistically higher percent of p63+ cells in the ADSC and SVF groups compared to all other groups. From week 2 to week 4, there was a significant increase in the percent of p63+ cells present in the lipoaspirate group. At week 4, all experimental groups had a statistically higher percentage of p63+ cells than control groups without statistical differences between the experimental groups. CONCLUSIONS: These findings suggest that improvements seen in radiated skin after fat grafting are due to presence of transferred ADSCs. ADSCs are likely not the only factor necessary to mediate the changes and the other components present within the SVF and lipoaspirate are likely important because these 2 groups maintained significantly thinner epidermis at 4 weeks, whereas the pure ADSC group did not. The ADSCs seem to convert into epithelial stem cells as evidenced by the presence of p63+ human cells within the epidermis of the experimental groups and absence in control groups. The increase in percentage of p63+ cells from week 2 to week 4 suggests that these stem cells are continuing to divide and regenerate the skin.

Therapeutic Effects of Human Adipose-Derived Products on Impaired Wound Healing in Irradiated Tissue.

Clinical sequelae of irradiation result in tissue devitalization (e.g., ischemia, fibrosis, and atrophy) where wound healing capacity is impaired. Fat-derived products may work to treat such pathology. Nonlethal irradiation at various doses (5, 10, and 15 Gy) and frequencies (one to three times on sequential days) was delivered to dorsal skin of nude mice, and subsequent gross and microscopic changes were evaluated for up to 4 weeks. Cutaneous punch wounds were then created to compare wound healing in irradiated and nonirradiated states. Wounds were also locally injected with vehicle, cultured adipose-derived stem cells, centrifuged fat tissue, or micronized cellular adipose matrix, and the therapeutic impact was monitored for up to 15 days. Nude mice given total doses greater than 15 Gy spontaneously developed skin ulcers, and radiation damage was dose-dependent; however, a fractionated irradiation protocol was able to reduce the damage. Histologic assessment revealed dose-dependent dermal fibrosis/thickening and subcutaneous atrophy. Dose-dependent (5 to 15 Gy) impairment of wound healing was also evident. At the highest dosage (15 Gy three times), open wounds persisted on day 15. However, wounds injected with cultured adipose-derived stem cells were nearly healed on day 12, and those treated with injection of centrifuged fat or micronized tissue healed faster than untreated controls (p < 0.05). There was no significant differences between treated groups. Tissue devitalization by irradiation was dose-dependent, although fractionated protocols helped to reduce it. Adipose-derived stem cells and other fat-derived products harboring adipose-derived stem cells successfully revitalized irradiated tissues and accelerated wound healing.

Moderate intermittent negative pressure increases invasiveness of MDA-MB-231 triple negative breast cancer cells

BackgroundTo investigate the effect moderate intermittent negative pressure breast reconstructive model exerts on human triple negative breast cancer cell (TNBC) invasion and explore the related mechanism. MethodsThe human TNBC cell line MDA-MB-231 was used. Cells in external volume expansion (EVE) group were exposed to an intermittent −25 mmHg for 12 h; the pressure for non-EVE group was constantly 0 mmHg. In vivo, MDA-MB-231 cell suspensions were injected subcutaneously into dorsal skin of nude mice (n = 27 mice/group). Tumors on mice in EVE group received −25 mmHg suction 3 h/day; while mice in non-EVE group were under normal pressure. Cell invasion assay, ELISA, RT-PCR, western blot analysis and immunohistochemistry were used to evaluate the inflammation, epithelial-mesenchymal transition (EMT) and angiogenesis between the two groups in both vitro and vivo experiments. ResultsMDA-MB-231 cells in the EVE group were more invasive and had higher expressions of IL-8 (30.02 ± 10.44 pg/ml vs. 18.82 ± 9.26 pg/ml, P < 0.05) and TNF-α (20.59 ± 4.72 pg/ml vs. 14.10 ± 3.36 pg/ml, P < 0.05) than the non-EVE group. Grafted MDA-MB-231 tumors in EVE group showed a more obvious epithelial-mesenchymal transition at 2 week and better angiogenesis at 2 and 4 week, respectively. ConclusionModerate intermittent negative pressure induces MDA-MB-231 cells to be more invasive. Future studies should figure out other effects this intervention may bring. Clinical studies should also be conducted to further evaluate its safety and optimize the clinical model.

Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation.

Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.

Promotional Effect of Platelet-Rich Plasma on Hair Follicle Reconstitution in vivo

Platelet-rich plasma (PRP) containing various growth factors has attracted attention in various medical fields. PRP has recently been used during hair transplantation to increase hair density. To investigate the effects of PRP on hair follicle (HF) reconstitution. Freshly isolated epidermal cells and cultured dermal papilla cells (DPCs) were mixed with various concentrations of activated PRP and transferred to a grafting chamber that was implanted onto the dorsal skin of nude mice. The chambers were removed 1week after grafting, and HF formation was monitored for 4weeks. We observed a significant difference (p<.05) in the number of newly formed follicles in the area of reconstituted skin (344±27 with 10% PRP vs 288±35 without PRP). PRP also shortened the time of hair formation significantly; the first hairs were observed in 18±1days using 10% PRP, versus 20±1days without PRP. A considerable effect of PRP on the time of hair formation and the yield of HF reconstitution was observed in this study. Considering the limited evidence available to judge its efficacy, further studies are required to investigate the mechanism of action of PRP.

Effect of PRP on the proliferation of dermal papilla cells and hair follicle regeneration in mice

To investigate the effects of platelet-rich plasma (PRP) on the proliferation of dermal papilla cells (DPCs) and hair follicle regeneration. PRP was prepared using the double-spin method and applied to DPCs. The proliferative effect of activated PRP on DPCs was measured using MTT assay. To understand the influence of activated PRP on the hair-inductive capacity of DPCs, freshly isolated epidermal cells and DPCs of passage 4 were resuspended, mixed with various concentrations of a PRP (0%, 5% or 10%) and were then transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting and HF formation was monitored for 4 weeks; the graft site was harvested and processed for histological examination. Activated PRP increased the proliferation benefited the aggregative growth of DPCs. There are significant difference in the yield of hair follicles compared with 10% PRP (344 +/- 27) with 0% PRP (288 +/- 35) in the area of reconstituted skin (P < 0.05). The areas treated with PRP demonstrated an increase in hair follicles density of 19.4%. Ten percent PRP (18 +/- 1) d also can significantly shorten the time of hair formation, compared with 0% PRP (20 +/- 1) d (P < 0.05). There is a considerable effect of PRP on the time of hair formation and the yield of hair follicles reconstitution.

The influence of photoaging on drug and sunscreen absorption via the skin

Ultraviolet (UV) exposure is the predominant cause of skin aging. A systematic evaluation of drug skin permeation via photoaged skin is lacking. The aim of this work was to investigate whether UVA and UVB affect absorption by the skin of drugs and sunscreens, including tetracycline, quercetin, and oxybenzone. The dorsal skin of nude mice was subjected to UVA (24 and 39J/cm2) or UVB (150, 200, and 250mJ/cm2) irradiation. Levels of skin water loss and erythema were evaluated, and a histological examinations of COX-2 and claudin-1 expressions were carried out.

Adipose-Derived Stem Cells Seeded on Acellular Dermal Matrix Grafts Enhance Wound Healing in a Murine Model of a Full-Thickness Defect

The promotion of wound healing using dermal substitutes has become increasingly widespread, but the outcomes of substitute-assisted healing remain functionally deficient. Adipose-derived stem cells (ASCs) have been investigated widely in regenerative medicine and tissue engineering, and they have the potential to enhance wound healing. In this study, we focused on investigating the effects and mechanism of ASCs combined with an acellular dermal matrix (ADM) to treat full-thickness cutaneous wounds in a murine model. The ADM was prepared from the dorsal skin of nude mice by decellularization by treatment with trypsin followed by Triton X-100. The human ASCs were isolated and cultured from abdominal lipoaspirate. We created a rounded, 8-mm, full-thickness cutaneous wound in nude mice and divided the mice into the following 4 groups: silicon sheet cover only, silicon sheet with spreading ASCs, ADM only, and ASCs seeded on ADM. The granulation thickness was evaluated by histology after 7 days. Further comparisons between the ADM only and ASC-seeded ADM groups were undertaken by assessing the reepithelialization ratio and blood vessel density at postoperative days 9 and 14. Statistical analyses were conducted using Student 2-tailed t test. Immunofluorescent histology and ASC labeling were also performed to identify possible mechanisms. The ADM was successfully prepared, and the cytometry analysis and differentiation assay provided the characterization of the human ASCs. A marked improvement in granulation thickness was detected in the ADM-ASC group in comparison with other 3 groups. A significantly increased rate of reepithelialization in the ADM-ASC group (80 ± 6%) compared to the ADM only group (60 ± 7%) was noted on postoperative day 9. The blood vessel density was evidently increased in the ADM-ASC group (7.79 ± 0.40 vessels per field) compared to the ADM only group (5.66 ± 0.23 vessels) on day 14. Cell tracking experiments demonstrated that labeled ASCs were colocalized with staining for VEGF or endothelial cell maker vWF after the transplantation of ADM-ASCs on postoperative day 14. Adipose-derived stem cells seeded on an ADM can enhance wound healing, promote angiogenesis, and contribute to newly formed vasculature, and VEGF-expressing ASCs can be detected after transplantation. This model could be used to improve the other clinical applications of ASCs and to decipher the detailed mechanism by which ASCs interact with wound tissue.

Evaluation of drug and sunscreen permeation via skin irradiated with UVA and UVB: Comparisons of normal skin and chronologically aged skin

Ultraviolet (UV) exposure is the predominant cause of skin aging. A systematic evaluation of drug skin permeation via photoaged skin is lacking. The aim of this work was to investigate whether UVA and UVB affect absorption by the skin of drugs and sunscreens, including tetracycline, quercetin, and oxybenzone. The dorsal skin of nude mice was subjected to UVA (24 and 39 J/cm(2)) or UVB (150, 200, and 250 mJ/cm(2)) irradiation. Levels of skin water loss, erythema, and sebum were evaluated, and histological examinations of COX-2 and claudin-1 expressions were carried out. Permeation of the permeants into and through the skin was determined in vitro using a Franz cell. In vivo skin uptake was also evaluated. Senescent skin (24 weeks old) was used for comparison. Wrinkling and scaling were significant signs of skin treated with UVA and UVB, respectively. The level of claudin-1, an indicator of tight junctions (TJs), was reduced by UVA and UVB irradiation. UVA enhanced tetracycline and quercetin skin deposition by 11- and 2-fold, respectively. A similar enhancement was shown for flux profiles. Surprisingly, a lower UVA dose revealed greater enhancement compared to the higher dose. The skin deposition and flux of tetracycline both decreased with UVB exposure. UVB also significantly reduced quercetin flux. The skin absorption behavior of chronologically aged skin approximated that of the UVA group, with photoaged skin showing higher enhancement. UV generally exhibited a negligible effect on modulating oxybenzone permeation. Skin disruption produced by UV does not necessarily result in enhanced skin absorption. It depends on the UV wavelength, irradiated energy, and physicochemical properties of the permeant. To the best of our knowledge, this is the first report establishing drug permeation profiles for UV-irradiated skin.

Comparisons of mouse models for hair follicle reconstitution

Three methods are currently available to reconstitute hair follicles in mice. Direct comparisons have yet to be made to determine which method is most efficient. In this study, mouse epithelial cells (MECs) and mouse dermal cells (MDCs) were grafted onto the dorsal skin of nude mice using the chamber, flap or patch assays. Comparisons were made based on gross, scanning electron microscopic and histological observations. MDCs alone induced hair follicle reconstitution with the production of hairs yielding false-positive results caused by contamination by hair follicle remnants. Neither primary MECs nor cultured MDCs alone formed hair follicles but did result in hair follicle formation when mixed together. Frozen MECs or MDCs resulted in decreased hair follicle-inductive activity but could still regenerate hairs. The hair patch assay was the quickest model (20±3 days) to determine whether cell mixtures would reconstitute hair follicles that produce hairs; however, the hair follicles were randomly orientated and often associated with foreign body granulomas. The flap assay took the longest time (29±2 days) to produce follicles and hairs to develop with a clinically natural appearance, but an epidermal sheet was needed. The chamber assay was the most labour-intensive and cell number-dependent procedure but follicles developed in a dense, clinically normal manner.

Recapitulation of Fibromatosis Nodule by Multipotential Stem Cells in Immunodeficient Mice

Musculoskeletal fibromatosis remains a disease of unknown etiology. Surgical excision is the standard of care, but the recurrence rate remains high. Superficial fibromatosis typically presents as subcutaneous nodules caused by rapid myofibroblast proliferation followed by slow involution to dense acellular fibrosis. In this study, we demonstrate that fibromatosis stem cells (FSCs) can be isolated from palmar nodules but not from cord or normal palm tissues. We found that FSCs express surface markers such as CD29, CD44, CD73, CD90, CD105, and CD166 but do not express CD34, CD45, or CD133. We also found that FSCs are capable of expanding up to 20 passages, that these cells include myofibroblasts, osteoblasts, adipocytes, chondrocytes, hepatocytes, and neural cells, and that these cells possess multipotentiality to develop into the three germ layer cells. When implanted beneath the dorsal skin of nude mice, FSCs recapitulated human fibromatosis nodules. Two weeks after implantation, the cells expressed immunodiagnostic markers for myofibroblasts such as α-smooth muscle actin and type III collagen. Two months after implantation, there were fewer myofibroblasts and type I collagen became evident. Treatment with the antifibrogenic compound Trichostatin A (TSA) inhibited the proliferation and differentiation of FSCs in vitro. Treatment with TSA before or after implantation blocked formation of fibromatosis nodules. These results suggest that FSCs are the cellular origin of fibromatosis and that these cells may provide a promising model for developing new therapeutic interventions.

Abstract 681: Knockdown of connective tissue growth factor and treatment with temozolomide inhibited invasion, angiogenesis, and tumorigenesis of human glioblastomas

Abstract Connective tissue growth factor (CTGF) is a putative proto-oncogene and plays a crucial role in endothelial cell migration and tumor angiogenesis. CTGF is highly upregulated in proliferating endothelial cells and glioblastoma cells. In this investigation, we examined whether knockdown of CTGF at the mRNA level and treatment with temozolomide (TMZ) could inhibit cell invasion, angiogenesis, and growth of human glioblastoma SNB-19 and T98G cells in vivo. The cells were stably transfected with a plasmid vector carrying the human CTGF siRNA cDNA and then treated with 10 μM TMZ for 48 h. Semiquantitative PCR, Western blotting, and immunohistochemical staining demonstrated 80% downregulation of CTGF both at mRNA and protein levels after stable transfection. Matrigel invasion, cell migration from spheroids, and cell proliferation studies demonstrated significant inhibition of cell invasion, migration, and proliferation, respectively, of both cell lines after downregulation of CTGF and treatment with TMZ. In vitro and in vivo angiogenesis assays demonstrated inhibition of network formation of endothelial cells and neovascularization under the dorsal skin of nude mice, respectively, after knockdown of CTGF and treatment with TMZ. Both subcutaneous and intracerebral tumorigenesis in nude mice was markedly reduced in CTGF downregulated cells after TMZ treatment. Mechanistic studies demonstrated significant reduction of PCNA, VEGF, c-Myc, CDK2, CDK4, and cyclin D1 and upregulation of the cell cycle inhibitors p21Waf1 and p27Kip1. Flow cytometric analysis showed cell cycle arrest at G2/M phase in both cell lines after CTGF knockdown and TMZ treatment. Taken together, our study indicates that the CTGF knockdown and TMZ treatment effectively prevents cell invasion, migration, angiogenesis, and growth of glioblastomas in vivo. Therefore, CTGF knockdown during TMZ treatment offers a novel and potential therapeutic strategy for controlling the growth of glioblastomas. This investigation was supported in part by the R01 grants (CA-91460 and NS-57811) from the National Institutes of Health (Bethesda, MD). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 681.

Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model

BackgroundIt was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy.ResultsA mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 μl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo.ConclusionAs a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma

The limitless invasive and proliferative capacities of tumor cells are associated with telomerase and expression of its catalytic component, human telomerase reverse transcriptase (hTERT). IFN-gamma modulates several cellular activities, including signaling pathways and cell cycle, through transcriptional regulation. Using a recombinant plasmid with hTERT siRNA cDNA, we downregulated hTERT during IFN-gamma treatment in human glioblastoma SNB-19 and LN-18 cell lines and examined whether such a combination could inhibit angiogenesis and tumor growth in nude mice. In vitro angiogenesis assay was done using coculture of tumor cells with human microvascular endothelial cells. In vivo angiogenesis assay was done using diffusion chambers under the dorsal skin of nude mice. In vivo imaging of intracerebral tumorigenesis and longitudinal solid tumor development studies were conducted in nude mice. In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-gamma. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression. Our study showed that the combination of hTERT siRNA and IFN-gamma effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-gamma is a promising therapeutic strategy for controlling the growth of human glioblastoma.

A method for rapid evaluation of photoaging by measuring fluorescence intensity of green fluorescent protein due to elastin promoter activity

Summary Background Photoaging increases the deep furrows of the facial skin. This can be seen histologically as solar elastosis in the upper layer of the dermis. In sunlight, ultraviolet B (UVB) light plays a pivotal role in forming the wrinkles, because it enhances elastin gene expression and matrix metalloproteases (MMPs) at the transcriptional level in dermal fibroblasts. Sunscreens are believed to inhibit UVB-induced wrinkle formation in humans, but few methods have accurately assessed the protective effects of sunscreens in vivo to date. A major reason is that it takes a long time to cause the deep facial lines. Objective To develop a rapid and simple in vivo evaluation system, to assess the inhibitory effects of sunscreens against photoaging, a new experimental method was studied. Methods The promoter region of the human elastin gene was cloned into a green fluorescent protein (GFP) vector. The plasmid DNA was transfected into 3T3 cells and stable transfectants (3T3/Eln-GFP) were established. These cells were injected into the dorsal skin of nude mice. After UVB irradiation, the GFP fluorescence intensity was measured by fluorescence emission spectroscopy (FEMS) on the dorsal skin of the mouse. Results UVB irradiation of 3T3/Eln-GFP cells increased both the fluorescence intensity and the expression of GFP. Increased GFP fluorescence intensity could be measured from the body surface with FEMS at the injected site of the cells after UVB irradiation in vivo . Conclusion A rapid and simple evaluation system for human elastin promoter activity by UVB was established, and we propose it to be an useful method for objective assessment of sunscreens designed to prevent wrinkle formation.

Hair follicle reformation induced by dermal papilla cells from human scalp skin

To investigate the possibility of hair follicle reformation induced by dermal papilla cells in vivo and in vitro. Dermal papilla cells, dermal sheath cells obtained from human scalp skin by enzyme digestion were mixed with collagen to form mesenchymal cell-populated collagen gels. Superior and inferior epithelial cells and bulb matrical cells were then cultured on these gels by organotypic culture to recombine bilayer artificial skins. Dermal papilla cells and outer root sheath keratinocytes were mingled together and transplanted under subcutaneous tissue of the dorsal skin of nude mice. The results of histologic examination was observed with HE stain. These recombinants by organotypic culture all reformed bilayer structure like nature skin. Hair follicle-like structure reformation was found in dermal sheath cell-populated collagen gel when combined with superior or inferior epithelial cells. Dermal papilla cells also induced superior and inferior epithelial cells to form hair follicle on nude mice. Low passage dermal papilla cells mixed with hair follicle epithelial cells reformed many typical hair follicle structures and produced hair fibres after transplantation on nude mice. The dermal part of hair follicle, such as dermal papilla cells and dermal sheath cells, has the ability to induce hair follicle formation by interaction with the epithelial cells of hair follicle.

Neocartilage from human mesenchymal stem cells in alginate: Implied timing of transplantation

Previous reports have demonstrated the suitability of alginate microencapsulation for chondrogenesis of human mesenchymal stem cells (MSCs) in vitro. This study examined the MSCs-alginate constructs that were transplanted beneath the dorsal skin of nude mice for 8 weeks after a variety of in vitro culture periods. The in vitro culture had great effects on gross morphology and histological characteristics of transplants. The integrity of alginate of transplants increased as the in vitro culture period increased. Transplants were characterized by an opaque and yellowish color, fair burnish, a firm to elastic texture, but without any evidence of calcification spots. Histological findings agreed with the clinical determination of hyaline cartilage, characterized by isolated cells with basophilic ground substance positive in Safranin-O staining and collagen type II immunohistochemistry. Transplants with exposure to TGF-beta1 for more than 2 weeks before transplantation, lost burnish, were flexible in texture, and had an increased formation of calcification spots. Accordingly, 1-week exposure to TGF-beta1 in vitro before transplantation is appropriate for neocartilage formation of human MSCs in alginate. These findings suggested that regeneration using cell therapy or tissue engineering should assist in ascertaining the optimal timing of transplantation.