Non melanoma skin cancer (NMSC) is a serious condition caused by chronic ultraviolet (UV) exposure that leads to DNA damage in skin. UV radiation has the potential to lead to DNA damage, which triggers biochemical pathways within a cell. The result is that the cell either undergoes cell cycle arrest, giving the cell time to repair DNA damage, or apoptosis. Sunscreen is the most commonly used treatment for preventing UV induced skin damage, but it involves a number of undesirable and toxic side effects including damaging the dermis, premature aging of skin and underweight child births. This has led to interest in finding safer alternatives to prevent UV damage without the negative side effects of sunscreen. In particular, bovine milk sphingomyelin (SM) is a compound that has the potential to protect against UV damage without any of the dangerous side effects of sunscreen. Here we present the use of SM for UV protection of human keratinocytes (KRTs) to prevent DNA mutations that result from UV exposure. In particular, analysis of the expression of DNA damage biomarkers p21 and p53 was done to determine the potential of SM to prevent DNA damage associated with UV exposure. Both non-SM treated KRTs and KRTs treated with 0.1% SM media 24 hours prior to UV radiation were fixed and IF-stained at 24 hours following 40 mJ/cm2 of UV exposure. Significant differences in both p21 and p53 were observed between the SM treated and non-SM treated cells at the UV dosage level (via t-test; p

DEVELOPMENT AND CHARACTERIZATION OF PHOSPHOLIPID ENCAPSULATED QUANTUM DOT CONSTRUCTS FOR BIOLOGIC APPLICATIONS The American Cancer Society predicts that 577,190 cancer-related deaths and 1,638,910 newly diagnosed cases of cancer will occur in 2012. As these statistics show, cancer is a prevalent and devastating health issue; determined by the Mayo Clinic to be the second leading cause of death in the United States. Skin cancer is the most common form of cancer in the United States. In 2012 more than 68,000 Americans will be diagnosed with melanoma, 48,000 will be diagnosed with an early form of the disease that has not yet reached the lower levels of the epidermis, and more than 2 million people will be treated for basal cell or squamous cell skin cancer. Early and accurate detection is the most reliable way to ensure a positive outcome and the ultimate survival of the patient. As the most aggressive form of skin cancer, survival of melanoma is especially connected to early detection. Current methods for the initial detection of potential cancerous masses and lesions rely on visual examination, palpitation, and biopsy. Accurate determination of the presence of cancerous cells in a biopsy is especially difficult at the early stages when only a small percentage of cells in the biopsied mass show the morphological traits associated with being cancerous. This circumstance often results in a false negative (FN), delaying the necessary treatment until the cancer has reached a more developed stage. Developing more accurate methods for the detection of cancerous cells within a biopsy would aid in alleviating this problem. An improvement to the conventional method of visually examining biopsied tissues for the presence of cells with abnormal morphologies can be offered by utilizing the model of functionalized quantum dot (QD) constructs. Quantum dots are nano-particles composed of semi-conducting materials that fluoresce at discrete wavelengths when irradiated by a high energy UV source. QD constructs are cadmium-selenium/zinc-sulfide (CdSe/ZnS) quantum dots encapsulated within a bovine derived milk phospholipid micelle. QD constructs provide a potential mechanism for the identification of cancerous cells within a biopsy. Appreciating the scope of the clinical problem and understanding the potential of QDs, the objective of this thesis is to develop a primary model for the solubilization, encapsulation, and primary phospholipid functionalization of two distinct sizes of CdSe/ZnS QDs. The first stage of this thesis optimized the currently utilized protocol for synthesizing cadmium-selenium (CdSe) quantum dots to develop a set of parameters for consistently producing white fluorescing CdSe cores (WFCs) and CdSe/ZnS QDs of 505nm and 555nm (+/- 10nm). The application of synthesis times, temperatures, and quenching methods were employed to achieve this. The second stage developed a phospholipid encapsulation method for the initial functionalization and suspension of the hydrophobic QDs in aqueous media via encapsulation within phospholipid micelles. The final stage of this thesis focused on the successful introduction of the QD constructs into keratinocyte cells. Calcein and Ethidium homodimer-1 stains were applied to determine cell viability, Histochoice was applied as a fixative, and Hoechst staining was employed for cell nuclei identification. Analysis using confocal microscopy suggests successful attachment of QD constructs, in 0.1% w/v keratinocyte media, to the exterior of keratinocyte cell membranes with a 30% average cell survival rate at 24 hours after sample introduction. Future research investigating the interaction of QD constructs with biologic mediums of greater physiological complexity, as well as application of a secondary functionalization, are the next steps on the path toward achieving a viable mechanism for targeting and identifying cancerous cells within a biopsy.

No / Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

no / Canities or senile hair graying, a universally recognized sign of aging, remains unresolved in terms of physiological causes, although a strong genetic contribution is understood (Gunn et al., 2009). As the hair fiber continues to grow long after melanin production ceases, we suggest that melanocytes in the hair follicle may be more sensitive to the impact of chronological aging than are keratinocytes. Moreover, follicular melanocytes also age more markedly than those in the overlying epidermis. The hair follicle provides a unique opportunity to decouple the impact of age on two hair follicular tissue functions: hair formation and hair pigmentation. ... This study provides analysis of race, age, and anatomically matched cultures of adult human epidermal and hair follicle melanocytes (HFMs).

No / Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve < 20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome.

Yes / Background: Melanin is synthesized by melanocytes in the basal layer of the epidermis. When transferred to surrounding keratinocytes it is the key UVR-protective biopolymer responsible for skin pigmentation. Most melanin is observable in the proliferative basal layer of the epidermis, and only sparsely distributed in the stratifying/differentiating epidermis. The latter has been explained, despite formal evidence, to ‘melanin degradation’ in supra-basal layers. Objectives: Our aim was to re-evaluate this currently-accepted basis for melanin distribution in the human skin epidermis, and whether this pattern is altered after a regenerative stimulus. Methods: Normal epidermis of adult human skin, at rest and after tape-stripping, was analysed by a range of (immuno)histochemical and high-resolution microscopy techniques. In vitro models of melanin granule uptake by human keratinocytes were attempted. Results: We propose a wholly different fate for melanin in the human epidermis. Our evidence indicates that the bulk of melanin is inherited only by the non-differentiating daughter cell post mitosis in progenitor keratinocytes, via asymmetric organelle inheritance. Moreover, this preferred pattern of melanin distribution can switch to a symmetric or equal daughter cell inheritance mode under conditions of stress including regeneration. Conclusions: We provide in this preliminary report a plausible and histologically-supportable explanation for how human skin pigmentation is efficiently organized in the epidermis. Steady state epidermis pigmentation may involve much less redox-sensitive melanogenesis than previously thought, and at least some pre-made melanin may be available for re-use. The epidermal-melanin unit may be an excellent example to study organelle distribution via asymmetric or symmetric inheritance in response to micro-environment and tissue demands. / Walgreens Boots Alliance

Yes / Fabrication of extracellular matrix (ECM)-like scaffolds (in terms of structural-functional) is the main challenge in skin tissue engineering. Herein, inspired by macromolecular components of ECM, a novel hybrid scaffold suggested which includes silk/hyaluronan (SF/HA) bio-complex modified by PCP: [polyethylene glycol/chitosan/poly(ɛ-caprolactone)] copolymer containing collagen to differentiate human-adipose-derived stem cells into keratinocytes. In followed by, different weight ratios (wt%) of SF/HA (S1:100/0, S2:80/20, S3:50/50) were applied to study the role of SF/HA in the improvement of physicochemical and biological functions of scaffolds. Notably, the combination of electrospinning-like and freeze-drying methods was also utilized as a new method to create a coherent 3D-network. The results indicated this novel technique was led to ~8% improvement of the scaffold's ductility and ~17% decrease in mean pore diameter, compared to the freeze-drying method. Moreover, the increase of HA (>20wt%) increased porosity to 99%, however, higher tensile strength, modulus, and water absorption% were related to S2 (38.1, 0.32 MPa, 75.3%). More expression of keratinocytes along with growth pattern similar to skin was also observed on S2. This study showed control of HA content creates a microporous-environment with proper modulus and swelling%, although, the role of collagen/PCP as base biocomposite and fabrication technique was undeniable on the inductive signaling of cells. Such a scaffold can mimic skin properties and act as the growth factor through inducing keratinocytes differentiation.

No description available.

No / The ability of a cell to adhere and transmit traction forces to a surface reveals the cytoskeleton integrity of a cell. Shear sensitive liquid crystals were discovered with new function in sensing cell traction force recently. This liquid crystal has been previously shown to be non-toxic, linear viscoelastic and sensitive to localized exerted forces. This paper reports the possibility of extending the application of the proposed liquid crystal based cell force sensor in sensing traction forces of osteoblast-like (MG-63) and human keratinocyte (HaCaT) cell lines exerted to the liquid crystal sensor. Incorporated with cell force measurement software, force distributions of both cell types were represented in force maps. For these lowly contractile cells, chondrocytes expressed regular forces (10 – 90 nN, N = 200) around the circular cell body whereas HaCaT projected forces (0 – 200 nN, N = 200) around the perimeter of poly-hedral shaped body. These forces are associated with the organisation of the focal adhesion expressions and stiffness of the LC substrate. From the results, liquid crystal based cell force sensor system is shown to be feasible in detecting forces of both MG63 and HaCaT.

Yes / Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies. / Malaysia Ministry of Education (Fundamental Research Grant Scheme, FRGS Vot. 1482 and IGSP Vot. 679).