Mathematical model for promotion of wound closure with ATP release.

To computationally investigate the recent experimental finding such that extracellular ATP release caused by exogeneous mechanical forces promote wound closure, we introduce a mathematical model, the Cellular Potts Model (CPM), which is a popular discretized model on a lattice, where the movement of a "cell" is determined by a Monte Carlo procedure. In the experiment, it was observed that there is mechanosensitive ATP release from the leading cells facing the wound gap and the subsequent extracellular Ca2+ influx. To model these phenomena, the Reaction-Diffusion equations for extracellular ATP and intracellular Ca2+ concentrations are adopted and combined with CPM, where we also add a polarity term because the cell migration is enhanced in the case of ATP release. From the numerical simulations using this hybrid model, we discuss effects of the collective cell migration due to the ATP release and the Ca2+ influx caused by the mechanical forces and the consequent promotion of wound closure.

Role of Skin Stretch on Local Vascular Permeability in Murine and Cell Culture Models.

Excessive mechanical forces, particularly skin stretch, have been implicated in pathological cutaneous scarring. We hypothesize that this reflects, in part, stretch-induced vessel leakage that provokes prolonged wound/scar inflammation. However, this has never been observed directly. Here, a mouse model was used to examine the effect of skin flap stretching on vascular permeability. An in vitro model with pseudocapillaries grown in a stretchable chamber was also used to determine the effect of stretching on endothelial cell morphology and ion channel activity. METHODS: Murine skin flaps were stretched with a biaxial stretching device, after which FITC-conjugated-dextran was injected and imaged with fluorescence stereomicroscopy. Endothelial cells were induced to form pseudocapillary networks in an elastic chamber. The chamber was stretched and differential interference contrast microscopy was used to assess cell morphology. In other experiments, markers for Ca2+ influx and K+ efflux were added before a single stretch was conducted. Histamine served as a positive-control in all experiments. RESULTS: Cyclic stretching (20%) increased the vascular permeability of skin flaps almost as strongly as histamine. Both stimuli also partially disrupted the pseudocapillary networks, induced cell contraction, and created gaps between the cells. Both stimuli caused sustained K+ efflux; stretching had a milder effect on Ca2+ influx. CONCLUSIONS: Excessive cyclical stretching strongly increased the vascular permeability of skin vessels and in vitro pseudocapillaries. This effect associated with increased K+ efflux and some Ca2+ influx. Inhibiting such early stretch-induced signaling events may be an effective strategy for treating and preventing hypertrophic scars and keloids.

A New Model for Specific Visualization of Skin Graft Neoangiogenesis Using Flt1-tdsRed BAC Transgenic Mice.

BACKGROUND: Neovascularization plays a critical role in skin graft survival. Up to date, the lack of specificity to solely track the newly sprouting blood vessels has remained a limiting factor in skin graft transplantation models. Therefore, the authors developed a new model by using Flt1-tdsRed BAC transgenic mice. Flt1 is a vascular endothelial growth factor receptor expressed by sprouting endothelial cells mediating neoangiogenesis. The authors determined whether this model reliably visualizes neovascularization by quantifying tdsRed fluorescence in the graft over 14 days. METHODS: Cross-transplantation of two full-thickness 1 × 1-cm dorsal skin grafts was performed between 6- to 8-week-old male Flt1 mice and KSN/Slc nude mice (n = 5). The percentage of graft area occupied by tdsRed fluorescence in the central and lateral areas of the graft on days 3, 5, 9, and 14 was determined using confocal-laser scanning microscopy. RESULTS: Flt1+ endothelial cells migrating from the transgenic wound bed into the nude graft were first visible in the reticular dermis of the graft center on day 3 (0.5 ± 0.1; p < 0.05). Peak neovascularization was observed on day 9 in the lateral and central parts, increasing by 2- to 4-fold (4.6 ± 0.8 and 4.2 ± 0.9; p < 0.001). Notably, some limited neoangiogenesis was displayed within the Flt grafts on nude mice, particularly in the center. No neovascularization was observed from the wound margins. CONCLUSION: The ability of the Flt1-tdsRed transgenic mouse model to efficiently identify the origin of the skin-graft vasculature and visualize graft neovascularization over time suggests its potential utility for developing techniques that promote graft neovascularization.

Photodynamic Therapy Delays Cutaneous Wound Healing in Mice.

BACKGROUND: Cutaneous wound healing is a complex, dynamic physiological process. Traditional methods of promoting wound healing are not always effective. Consequently, alternative modalities, such as photodynamic therapy (PDT), are needed. We examined the effectiveness and underlying mechanisms of PDT in a murine model of acute wound healing. METHODS: Two excisional wounds were produced, one on each side of the midline, in C57bL/6J mice. Methyl 5-aminolevulinate hydrochloride (MAL) was applied to the right-side wound. After 1 to 3 hours of incubation, the wound was irradiated with red light. The left-side wound was not treated with MAL or red light. On Day 14, the wounds were excised and subjected to histological and immunohistochemical analysis. RESULTS: During the first week, no difference was seen between the two sides. However, at week 2, PDT-treated wounds exhibited delayed re-epithelialization. On Day 14, hematoxylin and eosin (HE) staining showed a continuous epithelial lining in untreated wounds. In contrast, PDT-treated wounds partially lacked epithelium in the wound bed. Masson's Trichrome (MTC) staining showed a thicker dermis and more collagen fibers and inflammatory cells in PDT-treated wounds than in untreated wounds. Immunohistochemical analyses showed significantly fewer CD31+ blood vessels and greater collagen III density in PDT-treated wounds than in untreated wounds. However, treated and untreated wounds did not differ in collagen I density. CONCLUSIONS: PDT delayed acute wound healing in a murine model of secondary intention wound healing.

Noncontact Phased-Array Ultrasound Facilitates Acute Wound Healing in Mice.

BACKGROUND: The authors developed a noncontact low-frequency ultrasound device that delivers high-intensity mechanical force based on phased-array technology. It may aid wound healing because it is likely to be associated with lower risks of infection and heat-induced pain compared with conventional ultrasound methods. The authors hypothesized that the microdeformation it induces accelerates wound epithelialization. Its effects on key wound-healing processes (angiogenesis, collagen accumulation, and angiogenesis-related gene transcription) were also examined. METHODS: Immediately after wounding, bilateral acute wounds in C57BL/6J mice were noncontact low-frequency ultrasound- and sham-stimulated for 1 hour/day for 3 consecutive days (10 Hz/90.6 Pa). Wound closure (epithelialization) was recorded every 2 days as the percentage change in wound area relative to baseline. Wound tissue was procured on days 2, 5, 7, and 14 (five to six per time point) and subjected to histopathology with hematoxylin and eosin and Masson trichrome staining, CD31 immunohistochemistry, and quantitative polymerase-chain reaction analysis. RESULTS: Compared to sham-treated wounds, ultrasound/phased-array-treated wounds exhibited significantly accelerated epithelialization (65 ± 27 percent versus 30 ± 33 percent closure), angiogenesis (4.6 ± 1.7 percent versus 2.2 ± 1.0 percent CD31 area), and collagen deposition (44 ± 14 percent versus 28 ± 13 percent collagen density) on days 5, 2, and 5, respectively (all p < 0.05). The expression of Notch ligand delta-like 1 protein (Dll1) and Notch1, which participate in angiogenesis, was transiently enhanced by treatment on days 2 and 5, respectively. CONCLUSIONS: The authors' noncontact low-frequency ultrasound phased-array device improved the wound-healing rate. It was associated with increased early neovascularization that was followed by high levels of collagen-matrix production and epithelialization. The device may expand the mechanotherapeutic proangiogenesis field, thereby helping stimulate a revolution in infected wound care.

Hyperforin/HP-ß-Cyclodextrin Enhances Mechanosensitive Ca2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing.

Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca2+ signaling in keratinocytes by hyperforin treatment. However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl-ß-cyclodextrin- (HP-ß-CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP-ß-CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca2+ increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca2+ waves via TRPC6. This process might facilitate wound closure, because the Ca2+ response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca2+ signaling. We also applied hyperforin/HP-ß-CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP-ß-CD treatment for 24 h improved the stretch-induced Ca2+ responses and oscillations which failed in atopic skin.

Mechanosensitive ATP release from hemichannels and Ca²âº influx through TRPC6 accelerate wound closure in keratinocytes.

Cutaneous wound healing is accelerated by exogenous mechanical forces and is impaired in TRPC6-knockout mice. Therefore, we designed experiments to determine how mechanical force and TRPC6 channels contribute to wound healing using HaCaT keratinocytes. HaCaT cells were pretreated with hyperforin, a major component of a traditional herbal medicine for wound healing and also a TRPC6 activator, and cultured in an elastic chamber. At 3 h after scratching the confluent cell layer, the ATP release and intracellular Ca(2+) increases in response to stretching (20%) were live-imaged. ATP release was observed only in cells at the frontier facing the scar. The diffusion of released ATP caused intercellular Ca(2+) waves that propagated towards the rear cells in a P2Y-receptor-dependent manner. The Ca(2+) response and wound healing were inhibited by ATP diphosphohydrolase apyrase, the P2Y antagonist suramin, the hemichannel blocker CBX and the TRPC6 inhibitor diC8-PIP2. Finally, the hemichannel-permeable dye calcein was taken up only by ATP-releasing cells. These results suggest that stretch-accelerated wound closure is due to the ATP release through mechanosensitive hemichannels from the foremost cells and the subsequent Ca(2+) waves mediated by P2Y and TRPC6 activation.

Facile synthesis of highly water-soluble fullerenes more than half-covered by hydroxyl groups.

Using a novel hydrogen peroxide heating method, we synthesized milky white, water-soluble polyhydroxylated fullerenes (fullerenols) with 36-40 hydroxyl groups (estimated average) along with 8-9 secondary bound water molecules. The fullerenols exhibited high water solubility up to 58.9 mg/mL in a neutral (pH = 7) condition. Dynamic light scattering analysis showed a high dispersion property, to give a narrow particle size distribution within 0.7-2.0 nm.

Water-soluble C60 fullerene prevents degeneration of articular cartilage in osteoarthritis via down-regulation of chondrocyte catabolic activity and inhibition of cartilage degeneration during disease development.

OBJECTIVE: Studies have shown the roles of oxidative stress in the pathogenesis of osteoarthritis (OA) and induction of chondrocyte senescence during OA progression. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against catabolic stress-induced degeneration of articular cartilage in OA, both in vitro and in vivo. METHODS: In the presence or absence of C60 (100 microM), human chondrocytes were incubated with interleukin-1beta (10 ng/ml) or H2O2 (100 microM), and chondrocyte activity was analyzed. An animal model of OA was produced in rabbits by resection of the medial meniscus and medial collateral ligament. Rabbits were divided into 5 subgroups: sham operation or treatment with C60 at 0.1 microM, 1 microM, 10 microM, or 40 microM. The left knee joint was injected intraarticularly with water-soluble C60 (2 ml), while, as a control, the right knee joint received 50% polyethylene glycol (2 ml), once weekly for 4 weeks or 8 weeks. Knee bone and cartilage tissue were prepared for histologic analysis. In addition, in the OA rabbit model, the effect of C60 (10 microM) on degeneration of articular cartilage was compared with that of sodium hyaluronate (HA) (5 mg/ml). RESULTS: C60 (100 microM) inhibited the catabolic stress-induced production of matrix-degrading enzymes (matrix metalloproteinases 1, 3, and 13), down-regulation of matrix production, and apoptosis and premature senescence in human chondrocytes in vitro. In rabbits with OA, treatment with water-soluble C60 significantly reduced articular cartilage degeneration, whereas control knee joints showed progression of cartilage degeneration with time. This inhibitory effect was dose dependent, and was superior to that of HA. Combined treatment with C60 and HA yielded a significant reduction in cartilage degeneration compared with either treatment alone. CONCLUSION: The results indicate that C60 fullerene is a potential therapeutic agent for the protection of articular cartilage against progression of OA.

Antioxidant activity of supramolecular water-soluble fullerenes evaluated by beta-carotene bleaching assay.

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C(60), and gamma-cyclodextrin (CD)-bi-capped C(60), based on comparable beta-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k(obs)) as well as the persistent absorbency of beta-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of beta-carotene in any system.

Preclinical studies on safety of fullerene upon acute oral administration and evaluation for no mutagenesis.

Fullerenes characterized as an antioxidant are believed to reduce various reactive chemical species, such as free radicals, and their characteristic features have been disclosed to furnish many useful medical technologies. Despite the numerous applications for the biological efficacy of fullerenes, less is known about the toxicity of fullerenes in mammals. Hence, the protocol was designed to determine the acute oral median lethal dose and evaluate the acute toxicity of fullerenes when administrated as a single dose to Sprague-Dawley rats. In an acute toxicity test, fullerenes were administered once orally to a single group of male and female at a dose level of 2000 mg/kg. No deaths were observed and the body weights in both sexes of 2000 mg/kg group increased in a similar pattern to the control group. Genotoxicity of fullerenes was also assessed in a bacterial reverse mutation assay (Ames test) and the chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells. Although structural chromosomal aberrations were induced at up to 5000 microg/mL, there was no significant increase in the frequency of chromosomal aberrations at any dose level regardless of presence of S9. Fullerenes did not cause genetic damage in Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and Escherichia coli WP2uvrA/pKM101. These results indicate that fullerenes are not of high toxicological significance.

The water-soluble fullerene derivative "Radical Sponge" exerts cytoprotective action against UVA irradiation but not visible-light-catalyzed cytotoxicity in human skin keratinocytes.

Fullerene was entrapped in polyvinylpyrrolidone of 60-80 kDa at a molar ratio range of 0.42-0.67:1, resulting in a water-soluble derivative with a mean particle diameter of about 688 nm, named "Radical Sponge" because of its ROS-scavenging ability as previously demonstrated, and examined in the present study for its photo-biological actions toward human skin keratinocytes HaCaT. The keratinocytes were repeatedly irradiated with a visible light of wavelengths of 400-2000 nm (approximately 19,800 lux) in the presence or absence of Radical Sponge of 25-75 microM and did not exhibit any photo-cytotoxicity due to coexistent Radical Sponge as compared with the sham-irradiation control. Radical Sponge exerted a more marked cytoprotection at doses of 10-40 microM against UVA irradiation of 30 J/cm(2) when it was pre-irradiationally administered and rinsed out immediately before the irradiation, than when administered only during or after the irradiation, indicating the preventive rather than therapeutic or ray-sheltering effect of Radical Sponge on UVA injuries. Cytoprotection by Radical Sponge against UVA was achieved at the advisable range doses of 10-40 microM in contrast to no effect of polyvinylpyrrolidone alone; its dose-dependency was advantageous over that of VC-IP, a tetra-alkyl-esterized provitamin C, which became less cytoprotective above 20 microM. Thus, Radical Sponge is expected as an anti-UVA-preventive agent without visible-light-catalyzed cytotoxicity toward human skin keratinocytes.

Antioxidant effects of water-soluble fullerene derivatives against ultraviolet ray or peroxylipid through their action of scavenging the reactive oxygen species in human skin keratinocytes.

Chemically generated hydroxyl radicals were scavenged by PEG-modified fullerene, hydroxy-fullerene and isostearate-mixed fullerene as efficiently as ascorbic acid (Asc) or its 2-O-phosphorylated derivative (A2P) as shown by the DMPO-spin trap/ESR method. Enzymatically generated superoxide anion radicals were also scavenged by PEG-modified or PVP-entrapped fullerene similarly as done by Asc or A2P. Some reactive oxygen species (ROS) such as hydroperoxides and hydrogen peroxides were generated preferably in the nuclei of UVB-irradiated human skin keratinocytes HaCaT, and repressed by PVP- or gamma-cyclodextrin-fullerene. In contrast, the cytoplasm in the keratinocytes accumulated the ROS that were generated by the peroxylipid model compound t-BuOOH, and underwent the ROS repression by PVP-fullerene more markedly than by A2P. Thus several fullerene derivatives, especially PVP-fullerene, were shown to diminish the ROS amounts in terms of the molecular and cellular levels against either UVB or t-BuOOH, suggesting the expectation for development as rejuvenation cosmetics.

Amination of ethers using chloramine-T hydrate and a copper(I) catalyst.

Amination of C-H bonds activated by ether oxygen atoms is facile with chloramine-T as nitrene source and copper(I) chloride in acetonitrile as catalyst. For cyclic ethers the hemiaminal products are generally stable and can be isolated pure. For acyclic ethers, the hemiaminal products, as expected, fragment with elimination of alcohol to yield imines. When activation of benzylic positions is remote through a conjugated system, stable benzylamine derivatives are isolated. Mechanistic studies are consistent with concerted insertion of an electrophilic nitrenoid into the C-H bond in the rate-determining step, though in an asynchronous manner with a more activated substrate.

Catalytic Diastereoselective Imidation of Diaryl Sulfides Bearing a Chiral Oxazolinyl Moiety with Chloramine T Trihydrate.

Up to 99 % de is obtained in the imidation of diaryl sulfides bearing a chiral oxazolinyl moiety at the ortho position. The corresponding N-p-tosylsulfimides are produced upon use of chloramine T trihydrate in the presence of Cu(OTf)2 as catalyst [Eq. (1)]. OTf=trifluoromethanesulfonate; Ts=toluenesulfonyl.