Efficacy of topically applied rapamycin-loaded redox-sensitive nanocarriers in a human skin/T cell co-culture model.

Rapamycin, also known as Sirolimus, is a promising anti-proliferative drug, but its therapeutic use for the topical treatment of inflammatory, hyperproliferative skin disorders is limited by insufficient penetration rates due to its high molecular weight (MW of 914.172 g/mol) and high lipophilicity. We have shown that core multi-shell (CMS) nanocarriers sensitive to oxidative environment can improve drug delivery to the skin. In this study, we investigated the mTOR inhibitory activity of these oxidation-sensitive CMS (osCMS) nanocarrier formulations in an inflammatory ex vivo human skin model. In this model, features of inflamed skin were introduced by treating the ex vivo tissue with low-dose serine protease (SP) and lipopolysaccharide (LPS), while phorbol 12-myristate 13-acetate and ionomycin were used to stimulate IL-17A production in the co-cultured SeAx cells. Furthermore, we tried to elucidate the effects of rapamycin on single cell populations isolated from skin (keratinocytes, fibroblast) as well as on SeAx cells. Further, we measured possible effects of the rapamycin formulations on dendritic cell (DC) migration and activation. The inflammatory skin model enabled the assessment of biological readouts at both the tissue and T cell level. All investigated formulations successfully delivered rapamycin across the skin as revealed by reduced IL-17A levels. Nevertheless, only the osCMS formulations reached higher anti-inflammatory effects in the skin compared to the control formulations with a significant downregulation of mTOR activity. These results indicate that osCMS formulations could help to establish rapamycin, or even other drugs with similar physico-chemical properties, in topical anti-inflammatory therapy.

[Current Perceptions of Lipofilling on the Basis of the New Guideline on "Autologous Fat Grafting"]. / Aktuelle Erkenntnisse zur Eigenfett Transplantation anhand der neuen Leitlinie "Autologe Fetttransplantation".

Introduction: Autologous fat transfer has recently become an increasingly popular surgical procedure and comprises harvesting, processing and transplantation of adipose tissue, as well as professional follow-up care. This method, as a surgical procedure, can be utilised for trauma-, disease- or age-related soft tissue volume deficits and soft tissue augmentation. As usage is increasing, but the variables of fat harvest, specific indications and fashion of fat transfer are poorly defined, there is a great demand for development of a guideline in the field of reconstructive and aesthetic surgery. Methods: All relevant points were discussed within the scope of a consensus conference including a nominal group process of all societies involved in the procedure and ratified with a strong consensus (>95%). Literature from the standard medical databases over the last 10 years was retrieved, studied and specific guidelines were concluded. Results: Consensus was achieved among all professionals involved on the following points: 1. definition 2. indication/contraindication, 3. preoperative measures 4. donor sites 5. techniques of processing 6. transplantation 7. follow-up care 8. storage 9. efficacy 10. documentation 11. evaluation of patient safety. Conclusion: Definite indications and professional expertise are paramount for autologous fat tissue transfer. Successful transfers are based on the use of correct methods as well as specific instruments and materials. Autologous adipose tissue transplantation is considered to be a safe procedure in reconstructive and aesthetic surgery, due to the low rate of postoperative complications and sequelae.

The cost of post-burn scarring.

Deep burns lead to scarring and contractures for which there is little or no published data on treatment costs. The purpose of this study was to fill this gap by analysing treatment costs for burn sequelae. To do this, German-DRG for in-patient treatment was collected from the Burn Centre Lower Saxony. DRG-related T95.-coding served as a tool for burn-associated sequelae. Data on scar occurrence, plastic-reconstructive surgery and sick leave were collected by a questionnaire. The findings showed that 44.6% patients reported post-burn scarring and 31% needed surgical intervention. The expected risk for readmission was significantly higher (p=0.0002) with scars compared to without. Significantly higher costs for pressure garments were noted for scarred patients (p=0.04). No differences were found for ointments, silicone dressings or pain medication. Treatment costs for patients with scars were 5.6 times higher compared with no scar assessed by G-DRG. No differences were stated subsuming multiple readmissions for post-burn treatment per individual. Significantly higher costs (p=0.03) were noted for patients with burn sequelae other than scars with regard to individual readmissions. It has been revealed that treatment of scars causes higher costs than for other burn sequelae because of multiple surgical interventions. To reduce post-burn scarring and costs, specialized burn centres provide optimal and state-of-the-art treatment. As well as this, more emphasis should be laid on promoting research for the development of novel anti-scarring therapies.

Les brûlures profondes entraînent des cicatrices et des contractures pour lesquels il n'existe pas de données publiées dés coûts de traitement. Le but de cette étude était de combler cette lacune en analysant les coûts de traitement des séquelles de brûlures. Nous avons recueillies les données sur les séquelles de brûlure du Centre de Brûlés de Basse-Saxe en utilisant un questionnaire. Toutes les informations sur les cicatrices, la chirurgie plastique reconstructive et les congés de maladie ont été recueillies. Les résultats ont montré que 44.6% des patients avaient des cicatrices et 31% ont eu besoin d'une intervention chirurgicale. Le risque de réadmission était significativement plus élevé (p = 0,0002) parmi les patients avec des cicatrices. Pour ces patients les coûts étaient considérablement plus élevés pour les vêtements de compression (p = 0,04) mais, en ce qui concerne les pommades, les pansements siliconés ou les médicaments contre la douleur aucune différence n'a été trouvée. Les coûts de traitement pour les patients porteurs de cicatrices étaient 5,6 fois plus élevés par rapport aux patients sans aucune cicatrice. Les coûts plus élevés (p = 0,03) ont été observés chez les patients avec des séquelles de brûlures autre que cicatrices dues aux réadmissions individuelles. Nous avons noté aussi que le traitement des cicatrices entraîne des coûts plus élevés par rapport aux autres séquelles à cause des interventions chirurgicales multiples. Pour réduire les cicatrices post-brûlures, et donc les coûts, les centres spécialisés fournissent un meilleur traitement. De plus, l'accent devrait être mis sur la recherche pour le développement de nouvelles thérapeutiques anti-cicatrices.

[Impact of Martian and Lunar dust simulants on cellular inflammation in human skin wounds ex vivo]. / Einfluss von Mars- und Mondstaubanaloga auf die Wundheilung humaner Haut im ex-vivo Modell.

BACKGROUND: In 2006 NASA published its plans to build a manned lunar station in order to undertake missions to Mars in the future. Thus research projects have been conducted on the influence of Lunar and Martian dust on human health. The present study investigated the effect of Lunar and Martian dust simulants (LDS, MDS) in comparison with earth dust (ED) on viability, migration and inflammatory reaction during wound closure in an ex vivo human skin wound model. MATERIALS AND METHODS: 6 mm full-thickness skin explants, with a central 3 mm epidermal wound were cultured with LDS, MDS or ED for 4 and 8 days and compared to wound closure without dust exposure. Tissue and conditioned medium were submitted to histological, immunohistochemical (Ki67, Caspase-3) and biochemical analyses (hydroxyproline assay, zymography, IL-6, TNF-α and TGF-ß ELISA). RESULTS: All dusts increased proinflammatory markers with significant increases in MDS-treated samples (IL-6: p<0.05; MMP-9: p<0.005) and reduced MMP-2 (p<0.05) compared to no dust controls over time. No significant differences were found regarding wound closure, proliferation, apoptosis and tissue degradation. CONCLUSION: Highly oxidative Martian dust may cause increased cutaneous inflammation. As is currently advocated for wounds contaminated with earth dust, surgical wound debridement should be performed to ensure uncompromised wound healing.

An isolated hand tumour as primary manifestation of sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic disorder with unknown etiology, characterized by non-caseating granulomas in numerous organs and tissues. In 90% of patients lung and lymph nodes are involved. The incidence of sarcoidal granulomas in the upper extremities is low. Here we present the case of a primary hand manifestation of sarcoidosis without clinical systemic involvement. OBJECTIVES: A young woman presented with a painful swelling in her right hand. There were no signs of inflammation. Normal perfusion, mobility and sensibility were found. Magnetic resonance imaging (MRI) revealed a tumour infiltrating the muscles and flexor tendons of the third digit around the metacarpal bone and with pathological signal enhancement after administration of contrast medium. RESULTS: Intraoperatively, nodular masses and fat tissue were seen. Histological examination after radical tumour resection showed sarcoidal granulomas. Postoperative staging diagnostics with computed tomography (CT) demonstrated multiple thoracic lymph node swellings in the mediastinum and bilateral hill. Follow-up after one year we saw normal scars in the palmar hand. There was no sign of local recurrence. The pulmological care is still going on. CONCLUSIONS: Sarcoidosis is a rare, often asymptomatic disease. Patients present with dyspnoe and cough caused by the inflammation of the lung. The first clinical manifestation of sarcoidosis as a tumor in the palmar hand is unusual. Extrapulmonary systemic or progressive sarcoidosis is regarded as an indication for therapy with glucocorticosteroids. This case demonstrates that surgical excision enabled complete local cure without necessity of systemic and/or local treatment with steroids.

Epithelial regeneration from bioengineered skin explants in culture.

BACKGROUND: Artificial skin substitutes are beneficial in the treatment of chronic wounds although their performance relative to authentic human skin is unclear. OBJECTIVES: We compared the rate of outgrowth and morphology of neoepidermis from a bioengineered skin construct (Apligraf) with normal adult human skin explants on de-epidermized human dermal growth substrate with or without intact epidermal basement membrane zone. METHODS: Epithelial outgrowth of air-exposed cultures in serum-supplemented keratinocyte medium was quantified by fluorescence imaging, morphology by light microscopy, biomarkers of keratinocyte activation, proliferation and migration by immunohistochemical analysis, and gelatinases by zymography. RESULTS: Resurfacing from bioengineereed skin explants started earlier than from normal skin but subsequently, from day 3 to day 9, the rate of epidermalization from bioengineered skin was only 40% (206 +/- 23 microm day(-1), mean +/- SEM) of that of authentic skin (521 +/- 17 microm day(-1), P < 0.001). At culture termination at day 11, normal human skin had formed a multilayered and well-structured neoepidermis covering 41.0 +/- 1.2 mm2 of the dermal substrate while bioengineered skin produced a thinner, less organized epithelium covering 20.4 +/- 3.0 mm2. At this later stage, a higher expression of beta-defensin-2, keratin 16, Ki67 and matrix metalloproteinase (MMP)-9 was found in neoepidermis formed from authentic skin than from bioengineered skin. Activated MMP-2 was elevated in bioengineered skin-derived neoepidermis. Minor epithelial outgrowth was noted with either skin type on the dermal substrate devoid of basement membrane zone. CONCLUSIONS: Cultured normal skin explants produced a more uniform and expansive in vivo-like neoepidermis than bioengineered skin explants.

Effect of a matrix metalloproteinase activity and TNF-alpha converting enzyme inhibitor on intra-abdominal adhesions.

BACKGROUND: Formation of intra-abdominal adhesions depends, in part, on the activity of serine proteinases. Matrix metalloproteinases (MMP) are required for epithelialization of skin wounds but their involvement in mesothelialization of peritoneal wounds and in adhesion pathogenesis is not known. Early tumor necrosis factor-alpha (TNF-alpha) levels have been proposed to reflect propensity to adhesion formation. OBJECTIVE: The impact of MMP activity and secreted TNF-alpha on peritoneal adhesion formation and healing was investigated through systemic administration of the synthetic broad-spectrum MMP and TNF-alpha-converting enzyme (TACE) inhibitor GM 6001. METHODS: Female Sprague-Dawley rats of 4-6 weeks of age were injected subcutaneously daily with GM 6001 100 mg/kg (n = 12) or vehicle (n = 10) starting two days before surgery. In each rat, two standardized peritoneal wounds, 20 mm x 5 mm, were made. One peritoneal wound was sutured whereas the contralateral wound healed by secondary intention. Adhesion formation and peritoneal healing, cell proliferation, and hydroxyproline concentrations were evaluated on postoperative day 7. RESULTS: Total serum TNF-alpha levels increased in vehicle-treated rats (p = 0.019) while GM 6001 treatment effectively prevented the rise in the postoperative phase (p < 0.001). No significant differences were observed in the extent of adhesion formation (p = 0.67) between control (65.0%) and GM 6001-treated (61.5%) animals, or peritoneal wound healing or cell proliferation. Hydroxyproline levels increased in the wounds (p = 0.014) but were not different between the two groups (p = 0.14). CONCLUSIONS: Lack of a striking effect of the MMP and TACE antagonist GM 6001 on postoperative adhesions suggests that MMP activity and TNF-alpha might not be major adhesiogenic factors.

The release of zinc ions from and cytocompatibility of two zinc oxide dressings.

OBJECTIVE: These in vitro studies examined the release of zinc ions from and the response of human dermal fibroblasts to two zinc oxide-medicated dressings: one with zinc oxide in an ointment base and one using polyvinylpyrrolidone (PVP), a hydrophilic polymer for the binding of zinc oxide particles. METHOD: Zinc release from the dressings in buffered-saline (pH 7.4) was studied through a high-pore-density membrane (pore size, 0.40 microm) in a two-compartment model at 37 degrees C for three hours. Cytocompatibility of the dressings and 500 micromol/l of zinc ions was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay after exposure to monolayers of confluent normal human dermal fibroblasts to the dressing extracts for four hours. RESULTS: The zinc release rate from PVP-bound zinc oxide was more than two-fold higher than from zinc oxide in the ointment. Extract of the zinc oxide ointment, containing 150 micromol/l solubilised zinc, elicited a cytotoxic reaction, while the zinc oxide-PVP extract, containing 410 micromol/l solubilised zinc, and 500 micromol/l zinc chloride were non-cytotoxic to the fibroblasts. CONCLUSION: Zinc release in a simulated wound milieu appears to be inhibited when zinc oxide is incorporated in a lipophilic vehicle. It is hypothesised that the ointment vehicle induced cytotoxicity rather then the solubilised zinc oxide. DECLARATION OF INTEREST: None.

Topical synthetic inhibitor of matrix metalloproteinases delays epidermal regeneration of human wounds.

Matrix metalloproteinases (MMPs) degrade extracellular proteins during epithelialization of wounds. To evaluate the biological significance of MMPs in epidermal healing, the synthetic broad-spectrum MMP inhibitor GM 6001 (also called Galardin and Ilomastat) was applied topically to standardized human wounds. GM 6001 (10 microg/microl) or vehicle alone was applied every second day onto 4 de-roofed 6 mm suction blister wounds on the volar forearm of healthy male volunteers for 12 days. GM 6001 delayed healing by 2-4 days as assessed macroscopically and microscopically. In situ hybridization or immunohistochemistry showed that MMP-1 (interstitial collagenase) was present in and MMP-2 (gelatinase A) close to laterally migrating keratinocytes whereas MMP-9 (gelatinase B) was seen during maturation of new epidermis. MMP-1 was undetectable in blister roofs (normal epidermis) and found in low levels in normal skin. Total MMP-1 activities increased about 100-fold in wounds, independent of treatment, compared to normal skin as analyzed by specific ELISA-based activity assay. By gelatin zymography, MMP-2, but not MMP-9, was detected in blister roofs and wound healing was associated with increased active MMP-2 and latent MMP-9 levels. GM 6001 prevented activation of MMP-2 and increased latent MMP-9 levels. GM 6001 delayed re-appearance of laminin-5, the synthesis of which correlated with epidermal regeneration. Restoration of stratum corneum, measured indirectly by transepidermal water loss, was also impaired (P<0.05) in the GM 6001 group. In conclusion, pharmacological MMP inhibition delayed epidermal regeneration in vivo, suggesting that MMPs are required to restore epidermis after epidermal ablation in humans.