Absence of premature senescence in Werner's syndrome keratinocytes.

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.

Dosing regimen of meropenem for adults with severe burns: a population pharmacokinetic study with Monte Carlo simulations.

OBJECTIVES: To develop a population model to describe the pharmacokinetics (PK) of intravenous meropenem in adult patients with severe burns and investigate potential relationships between dosage regimens and antimicrobial efficacy. PATIENTS AND METHODS: A dose of 1 g every 8 h was administered to adult patients with total body surface area burns of ≥15%. Doses for subsequent courses were determined using results from the initial course and the patient's clinical condition. Five plasma meropenem concentrations were typically measured over the dosage interval on one to four occasions. An open, two-compartment PK model was fitted to the meropenem concentrations using NONMEM and the effect of covariates on meropenem PK was investigated. Monte Carlo simulations investigated dosage regimens to achieve a target T>MIC for ≥40%, ≥60% or ≥80% of the dose interval. RESULTS: Data comprised 113 meropenem concentration measurements from 20 dosage intervals in 12 patients. The parameters were CL (L/h) = 0.196 L/h/kg × [1 - 0.023 × (age - 46)] × [1 - 0.049 × (albumin - 15)], V1 = 0.273 L/kg × [1 - 0.049 × (albumin - 15)], Q = 0.199 L/h/kg and V2 = 0.309 L/kg × [1 - 0.049 × (albumin - 15)]. For a target of ≥80% T>MIC, the breakpoint was 8 mg/L for doses of 1 g every 4 h and 2 g every 8 h given over 3 h, but only 4 mg/L if given over 5 min. CONCLUSIONS: Although 1 g 8 hourly should be effective against Escherichia coli and CoNS, higher doses, ideally with a longer infusion time, would be more appropriate for empirical therapy, mixed infections and bacteria with MIC values ≥4 mg/L.

The in vitro characterization of a gelatin scaffold, prepared by cryogelation and assessed in vivo as a dermal replacement in wound repair.

A sheet gelatin scaffold with attached silicone pseudoepidermal layer for wound repair purposes was produced by a cryogelation technique. The resulting scaffold possessed an interconnected macroporous structure with a pore size distribution of 131 ± 17 µm at one surface decreasing to 30 ± 8 µm at the attached silicone surface. The dynamic storage modulus (G') and mechanical stability were comparable to the clinical gold standard dermal regeneration template, Integra®. The scaffolds were seeded in vitro with human primary dermal fibroblasts. The gelatin based material was not only non-cytotoxic, but over a 28 day culture period also demonstrated advantages in cell migration, proliferation and distribution within the matrix when compared with Integra®. When seeded with human keratinocytes, the neoepidermal layer that formed over the cryogel scaffold appeared to be more advanced and mature when compared with that formed over Integra®. The in vivo application of the gelatin scaffold in a porcine wound healing model showed that the material supports wound healing by allowing host cellular infiltration, biointegration and remodelling. The results of our in vitro and in vivo studies suggest that the gelatin based scaffold produced by a cryogelation technique is a promising material for dermal substitution, wound healing and other potential biomedical applications.

Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

Generation and characterization of multipotent stem cells from established dermal cultures.

Human multipotent skin derived precursor cells (SKPs) are traditionally sourced from dissociated dermal tissues; therefore, donor availability may become limiting. Here we demonstrate that both normal and diseased adult human dermal fibroblasts (DF) pre-cultured in conventional monolayers are capable of forming SKPs (termed m-SKPs). Moreover, we show that these m-SKPs can be passaged and that cryopreservation of original fibroblast monolayer cultures does not reduce m-SKP yield; however, extensive monolayer passaging does. Like SKPs generated from dissociated dermis, these m-SKPs expressed nestin, fibronectin and versican at the protein level. At the transcriptional level, m-SKPs derived from normal adult human DF, expressed neural crest stem cell markers such as p75NTR, embryonic stem cell markers such as Nanog and the mesenchymal stem cell marker Dermo-1. Furthermore, appropriate stimuli induced m-SKPs to differentiate down either mesenchymal or neural lineages resulting in lipid accumulation, calcification and S100ß or ß-III tubulin expression (with multiple processes). m-SKP yield was greater from neonatal foreskin cultures compared to those from adult DF cultures; however, the former showed a greater decrease in m-SKP forming capacity after extensive monolayer passaging. m-SKP yield was greater from adult DF cultures expressing more alpha-smooth muscle actin (αSMA). In turn, elevated αSMA expression correlated with cells originating from specimens isolated from biopsies containing more terminal hair follicles; however, αSMA expression was lost upon m-SKP formation. Others have shown that dissociated human hair follicle dermal papilla (DP) are a highly enriched source of SKPs. However, conversely and unexpectedly, monolayer cultured human hair follicle DP cells failed to form m-SKPs whereas those from the murine vibrissae follicles did. Collectively, these findings reveal the potential for using expanded DF cultures to produce SKPs, the heterogeneity of SKP forming potential of skin from distinct anatomical locations and ages, and question the progenitor status of human hair follicle DP cells.

A review of tissue-engineered skin bioconstructs available for skin reconstruction.

Situations where normal autografts cannot be used to replace damaged skin often lead to a greater risk of mortality, prolonged hospital stay and increased expenditure for the National Health Service. There is a substantial need for tissue-engineered skin bioconstructs and research is active in this field. Significant progress has been made over the years in the development and clinical use of bioengineered components of the various skin layers. Off-the-shelf availability of such constructs, or production of sufficient quantities of biological materials to aid rapid wound closure, are often the only means to help patients with major skin loss. The aim of this review is to describe those materials already commercially available for clinical use as well as to give a short insight to those under development. It seeks to provide skin scientists/tissue engineers with the information required to not only develop in vitro models of skin, but to move closer to achieving the ultimate goal of an off-the-shelf, complete full-thickness skin replacement.

Use of a novel porcine collagen paste as a dermal substitute in full-thickness wounds.

A commercially available porcine collagen sheet material has been found previously to be useful as an implant for reconstructive surgery. However, its use as a dermal substitute has been hindered by slow cell penetration and vascularization. A novel paste formulation of this material was investigated for its potential role as a dermal substitute in full-thickness wounds. A porcine punch biopsy model was initially used to assess the integration of a wide range of material formulations. Selected formulations were then assessed further in a larger wound-chamber model. Paste formulations were compared with those of sheet and another commercially available dermal regeneration template. The porcine collagen paste became integrated into full-thickness wounds without rejection and without excessive inflammation. It was detected in wounds up to day 27 postimplantation. Porcine collagen paste was readily infiltrated by host cells by day 2 and supported migrating keratinocytes on its surface. Staining for endothelial cells indicated neovasculature formation as early as day 4 and functional newly formed microvessels were noted at day 7. This was comparable with neovascularization of an alternative and clinically proven dermal regeneration template and was significantly superior to the sheet material formulation at the same time points. Our findings suggest that porcine collagen paste may be suitable as an alternative to current dermal substitutes in full-thickness wounds.

Effect of artificial dermal substitute, cultured keratinocytes and split thickness skin graft on wound contraction.

In this study, the effect of different wound treatments on contraction was evaluated in an established porcine model. In two separately conducted experiments full thickness wounds treated with artificial dermal substitute, split thickness skin graft (STSG), meshed STSG applied in combination with cultured keratinocytes or meshed STSG alone were compared with untreated wounds. The surface area of all wounds was quantified at regular time intervals. After 20 days wounds from some groups were subjected to histological analysis to establish the degree of epithelialization. Wounds treated with STSG contracted more than with artificial dermal substitute until day 21. From day 21 to day 35 wounds treated with STSG showed the least contraction. Wounds sprayed with cultured keratinocytes demonstrated a slower rate of contraction than those with meshed STSG alone after 20 days. The untreated control wounds showed a greater rate of contraction and had almost closed by day 20. This study demonstrates that there is a significant difference in contraction between wounds treated with artificial dermal substitute and control wounds and between wounds treated with STSG with cultured keratinocytes and meshed STSG alone. STSG with cultured keratinocytes, unmeshed STSG, and artificial dermal substitute all reduced wound contraction significantly.

Survival of cultured allogeneic limbal epithelial cells following corneal repair.

In this study a technique for determining donor cell fate following corneal grafting was evaluated. Patients treated for limbal deficiency with allogeneic cultured corneal epithelial cells were studied to determine the fate of the grafted cells. The technique was evaluated initially through the use of donor eyes and then applied to the clinical analysis of 7 patients who had received a cultured corneal epithelial allograft. Cells removed from the cornea and any retrieved tissue were analyzed via polymerase chain reaction (PCR) genotyping to determine the origin of the cells populating the patients' healed cornea. A mixture of genotypes was detected in a cornea retrieved from a patient following a fully penetrating keratoplasty who had received a mixture of allogeneic tissue. Donor cells were no longer detected on the corneal surface of all 7 cases beyond 28 weeks postgraft. At these later time points, only patient genotype could be detected. These results demonstrate that PCR genotyping can be used to determine the origin of cells populating the surface of the cornea following the grafting of cultured allogeneic cells and demonstrates that transplanted cultured limbal epithelial cells do not persist on the surface of the host cornea for more than 28 weeks.

Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction.

PURPOSE: To investigate the outcome of a new technique of ex vivo expanded stem cell allograft for limbal stem cell deficiency (LSCD), and to characterize the ocular surface genotype after surgery. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Ten eyes of 10 patients with profound LSCD arising from ectodermal dysplasia (3 eyes), Stevens-Johnson syndrome (3 eyes), chemical injury (2 eyes), thermal injury (1 eye), and rosacea blepharoconjunctivitis (1 eye). INTERVENTION: Allogeneic corneal limbal stem cells were cultured on plastic and transplanted to the recipient eye after removal of conjunctival pannus. Amniotic membrane was applied in a bandage capacity. The procedure was combined with other reconstructive surgery in 2 cases. Nine patients received systemic cyclosporin A immunosuppression, and the DNA genotype was investigated with surface impression cytology. MAIN OUTCOME MEASURES: Parameters of LSCD, including vascularization, conjunctivalization, inflammation, epithelial defect, photophobia, and pain. RESULTS: The mean follow-up period was 28 months (range, 12-50). Seven of 10 eyes (70%) had improved parameters of LSCD at final follow-up and were considered successes. Four (40%) had improved visual acuity, including 3 having had further procedures for visual rehabilitation. Three patients failed to improve-1 with a thermal burn and lid deformity, 1 with Stevens-Johnson syndrome and severe dry eye, and 1 with ectodermal dysplasia who developed an epithelial defect at 26 months. DNA analysis of the first 7 cases showed no ex vivo donor stem cell DNA present beyond 9 months. CONCLUSIONS: Ex vivo expanded stem cell allograft is a useful technique for restoring the ocular surface in profound LSCD. The absence of donor DNA beyond 9 months suggests that ongoing immunosuppression may be unnecessary and raises questions regarding the origin of the host corneal epithelium.

A comparison of keratinocyte cell sprays with and without fibrin glue.

Fibrin glue is an excellent template for cellular migration and has been shown to be an effective delivery system for cultured autologous keratinocytes. We have investigated whether fibrin glue has any benefit on the percentage of epithelial cover when cultured autologous keratinocytes are sprayed onto a freshly debrided wound bed. Three pigs were used for this study. This provided a total of 18 full thickness, vertically orientated wounds, each 4cm in diameter and isolated in PTFE chambers to prevent re-epithelialisation from the wound margins. Eight wounds were sprayed with cultured autologous keratinocytes suspended in 2ml culture medium and eight wounds were sprayed with cultured autologous keratinocytes suspended in 1ml of the fibrin/aprotinin component of Tisseel fibrin glue (Baxter) mixed with 1ml of culture medium. In the latter group the thrombin component of the fibrin glue kit was applied to the wound bed immediately prior to grafting. The remaining two wounds were used as controls and sprayed with either culture medium or fibrin glue without cells. Epithelial cover was calculated in whole-wound biopsies at 3 weeks using image analysis, histology and immunohistochemistry. The cell suspension in fibrin glue appeared to spread more evenly over the wound surface, with no pooling in the inferior aspect of the wound. However, mean epithelial area at 3 weeks in the fibrin group was 1.6cm(2) per wound compared with 1.8cm(2) for the non-fibrin group, as measured by image analysis of digital photographs. There was no statistically significant difference between the two groups (P=0.802). This surprising result was confirmed by histological analysis of the wound biopsies, with a good correlation between histological and image analysis data (R=0.967). There was no observable difference in the quality of the epithelium on histological and immunohistological analysis of either group.

Upward migration of cultured autologous keratinocytes in Integra artificial skin: a preliminary report.

The combination of cultured autologous keratinocytes with the dermal regeneration template Integra could offer increased possibilities for reconstructive surgery and wound healing. A single-step application of cells, centrifuged deep into an Integra-like matrix at the silicone-matrix junction, has been described but might prove technically complex for clinical use. We have investigated the possibility of simplifying this procedure by applying cultured cells directly to the underside of the Integra or directly to the wound bed immediately prior to grafting. The objective was to see whether cells would migrate through the matrix in an upward direction. We tested the principle of this concept using a pig wound healing model. Integra was seeded directly with cultured cells and grafted onto fresh full-thickness wounds, or unseeded Integra was applied to freshly excised wound beds that had just been seeded with the same number of cells. Biopsies were taken at 3, 7, 11, and 14 days. Histological sections showed that the cells moved through the Integra to give a confluent surface epithelium. Direct seeding onto the Integra was the most efficient method. Transduction of cultured autologous keratinocytes in vitro with a MFGlacZnls retrovirus confirmed that the epidermis was derived from the cultured autologous keratinocytes.