Stabilization of Lipid Lamellar Bilayer Structure of Stratum Corneum Modulated by Poly (2-methacryloyloxyethyl phosphorylcholine) in Relation to Skin Hydration and Skin Protection

BACKGROUND@#One crucial factor in skin tissue engineering is to understand the hydration and barrier property of skin. We investigated the skin hydration and stabilization strategy of inter-lamellar structure of stratum corneum (SC) using poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC). @*METHODS@#The unique hydration and stabilization potency of PMPC on the barrier function of the SC examined using freshly excised hairless mouse skin as a model membrane and the relationship between the stabilization of the lipid lamellar bilayer (LLB) and its enhanced water holding capacity was established. @*RESULTS@#Differential scanning calorimeter based on the phase-transition temperature of lipid domain of SC demonstrated that PMPC stabilized the LLB. The ratio of the heat of lipid phase transition (△H) of SC exposed to water and PMPC for 24 h was 1.51. X-ray crystallography showed the presence of well- organized lipids in intercellular membranes exhibiting short and long periodicity of lamellar phases. The peak at 4.4 nm attributed to the long periodicity phase (LPP) was missing in water-treated SC, where, the presence of 4.2- 4.4 nm peak in PMPC treated SC indicated that PMPC stabilized LPP. Transmission electron microscopy study demonstrated that the LLB structure became more rigid and orderly in PMPC treated SC. @*CONCLUSION@#The unique ion paired structure of PMPC enhances the barrier function of the SC by stabilizing LLB structure and hydration by inducing weakly bound water. The unique hydration state and stabilization effect from extended water exposure could provide a valuable information to prepare reliable artificial skin matrix and skin tissue.

Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements

BACKGROUND: Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. METHODS: Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPGΔNLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. RESULTS: CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPGΔNLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22 nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20 nm, n = 563). CONCLUSION: Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

Effect of Palmitoyl-Pentapeptide (Pal-KTTKS) on Wound Contractile Process in Relation with Connective Tissue Growth Factor and α-Smooth Muscle Actin Expression

To evaluate whether Palmitoyl-pentapeptide (Pal-KTTKS), a lipidated subfragment of type 1 pro-collagen (residues 212–216), plays a role in fibroblast contractility, the effect of Pal-KTTKS on the expression of pro-fibrotic mediators in hypertropic scarring were investigated in relation with trans-differentiation of fibroblast to myofibroblast, an icon of scar formation. α-SMA was visualized by immunofluorescence confocal microscopy with a Cy-3-conjugated monoclonal antibody. The extent of α-SMA-positive fibroblasts was determined in collagen lattices and in cell culture study. Pal-KTTKS (0–0.5 µM) induced CTGF and α-SMA protein levels were determined by western blot analysis and fibroblast contractility was assessed in three-dimensional collagen lattice contraction assay. In confocal analysis, fibroblasts were observed as elongated and spindle shapes while myofibroblast observed as squamous, enlarged cells with pronounced stress fibers. Without Pal-KTTKS treatment, three quarters of the fibroblasts differentiates into the myofibroblast; α-SMA-positive stress fibers per field decreased twofold with 0.1 µM Pal-KTTKS treatment (75 ± 7.1 vs 38.6 ± 16.1%, n = 3, p<0.05). The inhibitory effect was not significant in 0.5 µM Pal-KTTKS treatment. Stress fiber level and collagen contractility correlates with α-SMA expression level. In conclusion, Pal-KTTKS (0.1 µM) reduces α-SMA expression and trans-differentiation of fibroblasts to myofibroblast. The degree of reduction is dose-dependent. An abundance of myofibroblast and fibrotic scarring is correlated with excessive levels of α-SMA and collagen contractility. Delicate balance between the wound healing properties and pro-fibrotic abilities of pentapeptide KTTKS should be considered for selecting therapeutic dose for scar prevention.

Inhibition of fibrotic contraction by C-phycocyanin through modulation of connective tissue growth factor and α-smooth muscle actin expression.

The effects of C-phycocyanin (C-pc), a phycobiliprotein, on the expression of pro-fibrotic mediators in hyper-tropic scarring such as connective tissue growth factor (CTGF) and α-smooth muscle actins (α-SMA) were investigated in relation to trans-differentiation of fibroblast to myo-fibroblast, an icon of scar formation. C-pc was isolated from Spirulina Platensis extract using sonication method and C-pc concentration was determined by Bennet and Bogorad equation. α-SMA and CTGF levels in wounded primary human dermal fibroblasts were determined by western blot analysis and immuno-fluorescence confocal microscope was employed. Fibroblast contractility was examined by three-dimensional collagen lattice contraction assay. There was an elevation of α-SMA (121%) and CTGF (143%) levels in wound cells as compared with non-wound cells. The does-response profiles of down regulation demonstrated that the maximum inhibitions of α-SMA by 63% (p<0.05) and CTGF by 50% (p<0.1) were achieved by C-pc (6 nM) treated cells. In confocal assay, non-wound fibroblasts exhibited basal level of α-SMA staining, while wounded cells without C-pc treatment showed strong up-regulation of α-SMA by 147% (p<0.05). C-pc (6 nM) inhibited α-SMA expression by 70% (p<0.05) and reduced collagen contraction by 29% (p<0.05). C-pc seemed to lessen the over expression of CTGF, α-SMA, subsequently alleviating the fibrotic contracture. This study suggests the potential application of C-pc to regulation of the expression of pro-fibrotic mediators in scarring process and its potential usage as an efficient means for anti-fibrosis therapy.

Inhibition of Fibrotic Contraction by C-Phycocyanin through Modulation of Connective Tissue Growth Factor and α-Smooth Muscle Actin Expression

The effects of C-phycocyanin (C-pc), a phycobiliprotein, on the expression of pro-fibrotic mediators in hyper-tropic scarring such as connective tissue growth factor (CTGF) and α-smooth muscle actins (α-SMA) were investigated in relation to trans-differentiation of fibroblast to myo-fibroblast, an icon of scar formation. C-pc was isolated from Spirulina Platensis extract using sonication method and C-pc concentration was determined by Bennet and Bogorad equation. α-SMA and CTGF levels in wounded primary human dermal fibroblasts were determined by western blot analysis and immuno-fluorescence confocal microscope was employed. Fibroblast contractility was examined by three-dimensional collagen lattice contraction assay. There was an elevation of α-SMA (121%) and CTGF (143%) levels in wound cells as compared with non-wound cells. The does-response profiles of down regulation demonstrated that the maximum inhibitions of α-SMA by 63% (p<0.05) and CTGF by 50% (p<0.1) were achieved by C-pc (6 nM) treated cells. In confocal assay, non-wound fibroblasts exhibited basal level of α-SMA staining, while wounded cells without C-pc treatment showed strong up-regulation of α-SMA by 147% (p<0.05). C-pc (6 nM) inhibited α-SMA expression by 70% (p<0.05) and reduced collagen contraction by 29% (p<0.05). C-pc seemed to lessen the over expression of CTGF, α-SMA, subsequently alleviating the fibrotic contracture. This study suggests the potential application of C-pc to regulation of the expression of pro-fibrotic mediators in scarring process and its potential usage as an efficient means for anti-fibrosis therapy.

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T m values of 85.03 +/- 0.54degrees C for stx1 and 87.47 +/- 0.35degrees C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/microL), and quantifiable (R 2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.

Optimum scratch assay condition to evaluate connective tissue growth factor expression for anti-scar therapy.

To evaluate a potential anti-scar therapy, we first need to have a reliable in vitro wound model to understand dermal fibroblast response upon cell injury and how cytokine levels are changed upon different wound heal phases. An in vitro wound model with different scratch assay conditions on primary human foreskin fibroblast monolayer cultures was prepared and cytokine levels and growth properties were evaluated with the aim of determining optimum injury conditions and observation time. Morphological characteristics of differently scratched fibroblasts from 0 to 36 h post injury (1 line, 2 lines and 3 lines) were investigated. The expression of connective tissue growth factor, CTGF, which is a key mediator in hyper-tropic scarring, and relative intensity of CTGF as a function of time were determined by western blot and gelatin Zymography. After injury (1 line), CTGF level was increased more than 2-fold within 1 h and continuously increased up to 3-fold at 6 h and was leveled down to reach normal value at 36 h, at which cell migration was complete. In more serious injury (2 lines), higher expression of CTGF was observed. The down regulation of CTGF expression after CTGF siRNA/lipofectamine transfection in control, 1 line and 2 lines scratch conditions were 40%, 75% and 55%, respectively. As a model anti-CTGF based therapy, CTGF siRNA with different ratios of linear polyethyleneimine (PEI) complexes (1:1, 1:5, 1:10, 1:20 and 1:30) were prepared and down-regulation efficacy of CTGF was evaluated with our optimized scratch assay, which is 1 line injury at 6 h post injury observation time. As the cationic linear PEI ratio increased, the down regulation efficacy was increased from 20% (1:20) to 55% (1:30). As CTGF level was increased to the highest at 6 h and leveled down afterwards, CTGF level at 6 h could provide the most sensitive response upon CTGF siRNA transfection. The scratch assay in the present study can be employed as a useful experimental tool to differentiate between anti-scar therapies for their down regulation efficacy of CTGF.

Gravimetric analysis and differential scanning calorimetric studies on glycerin-induced skin hydration.

A thermal gravimetric analysis (TGA) and a differential scanning calorimetry (DSC) were carried out to characterize the water property and an alteration of lipid phase transition of stratum corneum (SC) by glycerin. In addition, the relationship between steady state skin permeation rate and skin hydration in various concentrations of glycerin was investigated. Water vapor absorption-desorption was studied in the hairless mouse stratum corneum. Dry SC samples were exposed to different conc. of glycerin (0-50%) followed by exposure to dry air and the change in weight property was monitored over time by use of TGA. In DSC study, significant decrease in DeltaH of the lipid transition in 10% glycerin and water treated sample: the heat of lipid transition of normal, water, 10% glycerin treated SC were 6.058, 4.412 and 4.316 mJ/mg, respectively. In 10% glycerin treated SCs, the Tc of water shifts around 129 degrees C, corresponding to the weakly bound secondary water. In 40% glycerin treated SC, the Tc of water shifts to 144 degrees C corresponding to strongly bound primary water. There was a good correlation between the hydration property of the skin and the steady state skin flux with the correlation coefficient (r2=0.94). As the hydration increased, the steady state flux increased. As glycerin concentration increased, hydration property decreased. High diffusivity induced by the hydration effect of glycerin and water could be the major contributing factor for the enhanced skin permeation of nicotinic acid (NA).

Enhancing dermal matrix regeneration and biomechanical properties of 2nd degree-burn wounds by EGF-impregnated collagen sponge dressing.

To better define the relationship between dermal regeneration and wound contraction and scar formation, the effects of epidermal growth factor (EGF) loaded in collagen sponge matrix on the fibroblast cell proliferation rate and the dermal mechanical strength were investigated. Collagen sponges with acid-soluble fraction of pig skin were prepared and incorporated with EGF at 0, 4, and 8 microg/1.7 cm2. Dermal fibroblasts were cultured to 80% confluence using DMEM, treated with the samples submerged, and the cell viability was estimated using MTT assay. A deep, 2nd degree- burn of diameter 1cm was prepared on the rabbit ear and the tested dressings were applied twice during the 15-day, post burn period. The processes of re-epithelialization and dermal regeneration were investigated until the complete wound closure day and histological analysis was performed with H-E staining. EGF increased the fibroblast cell proliferation rate. The histology showed well developed, weave-like collagen bundles and fibroblasts in EGF-treated wounds while open wounds showed irregular collagen bundles and impaired fibroblast growth. The breaking strength (944.1 +/- 35.6 vs. 411.5 +/- 57.0 Fmax, gmm(-2)) and skin resilience (11.3 +/- 1.4 vs. 6.5 +/- 0.6 mJ/mm2) were significantly increased with EGF-treated wounds as compared with open wounds, suggesting that EGF enhanced the dermal matrix formation and improved the wound mechanical strength. In conclusion, EGF-improved dermal matrix formation is related with a lower wound contraction rate. The impaired dermal regeneration observed in the open wounds could contribute to the formation of wound contraction and scar tissue development. An extraneous supply of EGF in the collagen dressing on deep, 2nd degree-burns enhanced the dermal matrix formation.

Phospholipid polymer, 2-methacryloyloxyethyl phosphorylcholine and its skin barrier function.

The effect of poly[2-methacryloyloxyethyl phosphorylcholine] (pMPC) on the skin permeation property was investigated by performing in vitro skin permeation study of a model drug, nicotinic acid (NA). Effect of pMPC polymer in donor solution on skin permeation rates was evaluated using side-by-side diffusion cells. Also, the structural alterations in the stratum corneum (SC), inter-lamellar bilayer (ILB) and dermis layers in pMPC-treated and -untreated skin sections were investigated with transmission electron microscopy (TEM). The permeation profile of NA without pMPC in donor solution showed biphasic mode: initial 1st phase and 2nd hydration phase. The sudden, more than 10-fold increase in flux from the initial steady state (43.5 microg/cm2/hr) to the 2nd hydration phase (457.3 microg/cm2/h) suggests the disruption of skin barrier function due to extensive hydration. The permeation profile of NA with 3% pMPC in the donor solution showed monophasic pattern: the steady state flux (10.9 microg/cm2/h) without abrupt increase of the flux. The degree of NA permeation rate decreased in a concentration-dependent manner of pMPC. TEM of skin equilibrated with water or 2% pMPC for 12 h showed that corneocytes are still cohesive and epidermis is tightly bound to dermis in 2% pMPC-treated skin, while wider separation between corneocytes and focal dilations in inter-cellular spaces were observed in water-treated skin. This result suggests that pMPC could protect the barrier property of the stratum corneum by preventing the disruption of ILB structure caused by extensive skin hydration during skin permeation study.

Effect of topically applied silver sulfadiazine on fibroblast cell proliferation and biomechanical properties of the wound.

The effect of silver sulfadiazine (SSD) on the proliferation of human dermal fibroblast (HDF) was studied to determine the impact of the drug on the wound healing process and dermal mechanical strength. Human dermal fibroblasts were cultured to 80% confluency using DMEM with 10% FBS and viability of the cell was estimated using neutral red assay. In addition, the 2nd degree burn wound was prepared on the anterior part of rabbit ear skin and dressings containing SSD were applied for 96 h. Presence of inflammatory cells and degree of re-epithelialization were investigated in the wound. After 15 day of the induction of burn wounds, the treated area was excised and dermal mechanical strength was quantitatively measured with a constant speed tensiometer. SSD was found to be highly cyto-toxic in cultured HDF cells. The topical application of SSD (2%) could control the infection as evidenced by the lack of accumulation of inflammatory cells in histological evaluation. Therefore, these observations suggested that the impairment of dermal regeneration and decreased mechanical strength of dermal tissue was resulted from the cyto-toxic effect of SSD on dermal cells. Since the decreased mechanical strength may lead to reduction in resilience, toughness and maximum extension of the tissue, the identification of optimum dose for SSD that limits infection while minimizes the cyto-toxic effect may be clinically relevant.

The Establishment of a Wound Model Induced by Ultrapulse CO2 Laser / 대한피부과학회지

BACKGROUND: Wound model is important for the development of dressing materials used in dermatologic practice. Most of the wound healing models are not standardized, individualized ones for each special situation. OBJECTIVES: The purpose of our study is to establish a standardized wound model for evaluating the efficiency of wound healing of dressing material and the effect of epidermal growth factor(EGF) on artificially induced wounds. METHODS: Using ultrapulse CO2 laser and rats, we calibrates the most effective level of energy, frequency, and pulse duration for a wound model of upper dermis. Then, we evaluated the effect of various dressing materials such as vaseline gauze, collagen sponge, collagen sponge containing EGF and hydrogel on wound healing. RESULTS: We obtained relatively even and uniformed wound models of upper dermal level with the setting of ultrapulse CO2 laser; continuous mode, 800watt, frequency of 100Hz, and pulse duration of 0.3ms. Wounds applied with collagen sponge containing EGF showed relatively accelerated wound healing, but wounds with hydrogel or collagen sponge only showed delayed healing of wounds CONCLUSION: Ultrapulse CO2 laser could make a wound model of uniform depth level and could be used as a tool for making standardized wound models.