S. Elizabeth James

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.

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes

We have used the T-Lymphocyte cloning technique as a method of monitoring the human population for somatic cell mutant frequency. We present a statistical analysis of the experimental factors which may influence the observed mutant frequency. We have obtained consistently high plating efficiencies of T-cells from the mononuclear cell fraction from donor blood samples (mean of 56%, based on 123 observations from 70 individuals). Nevertheless, an inverse correlation of mutant frequency with plating efficiency was observed, and some experimental factors (serum and interleukin-2 batch, and worker) may have a significant effect on the observed mutant frequency. We discuss the difficulties that these possible effects present in establishment of a reference database and design of long-term studies. No significant effect of donor sex on mutant frequency was observed, but age (1.3% increase per year for normal adults) and smoking (56% increase over normal non-smokers) both significantly increased the mutant frequency. We discuss the utility of the assay for the monitoring of populations for heritable DNA damage, and we compare the results to those obtained with lymphocytes using other endpoints, e.g. chromosome aberrations, micronuclei and sister-chromatid exchange.

Comparative Human Cellular Radiosensitivity: II. The Survival Following Gamma-irradiation of Unstimulated (G0) T-lymphocytes, T-lymphocyte Lines, Lymphoblastoid Cell Lines and Fibroblasts from Normal Donors, from Ataxia-telangiectasia Patients and from Ataxia-telangiectasia Heterozygotes

We have measured clonal survival following gamma-irradiation of unstimulated (G0) T-lymphocytes from 35 donors, of 11 T-lymphocyte cell lines, of six lymphoblastoid cell lines, and of nine primary fibroblast strains for which we have G0 T-lymphocyte material from the same donor. Amongst the G0 lymphocytes we have results from nine normal donors, from eight cord bloods, from seven ataxia-telangiectasia (A-T) patients and from nine A-T heterozygotes. Although there is some variation between samples, G0 T-lymphocytes from normal donors appear to be slightly more radioresistant than T-lymphocyte lines, with a more shouldered survival curve. From our limited sample, lymphoblastoid cell lines appear to be slightly more radiosensitive than T-lymphocytes. The overall radiosensitivity of primary fibroblasts appears to be broadly similar to that of G0 T-lymphocytes. In nine instances, five A-Ts and four A-T heterozygotes, both G0 T-lymphocytes and primary fibroblasts from the same donor were tested. In five cases there was closely similar radiosensitivity in the two cell types, but in four cases there was some discrepancy. Further work, especially with normal donors, will be required in order to establish how reliably radiosensitivity in other cell types can be predicted from that of G0 T-lymphocytes. In all cell types the hypersensitivity of A-T cells was confirmed. Furthermore, the marginally greater sensitivity of A-T heterozygotes, when compared as a group with normals, was confirmed with G0 T-lymphocytes. Our results also suggest a slightly increased radiosensitivity in G0 T-lymphocytes from some, but not all, cord blood samples.

The potential for eye bank limbal rings to generate cultured corneal epithelial allografts.

Purpose. Patients with severe limbal deficiencies are unable to maintain a stable corneal surface. If sheets of cultured allogeneic corneal epithelium could be prepared from eye banked corneal limbal rings, which are normally discarded after keratoplasty, the sheets may be beneficial for grafting onto patients with limbal stem cell deficiencies. Method. Biopsies of limbal tissue (2–3 mm2) removed from organ-cultured corneal limbal rings or from fresh whole globes were either trypsinized or set up as explants to assess their potential for corneal epithelial cell production. Results. Several biopsies were taken from each of 21 organ-cultured limbal rings and 10 fresh cadaveric globes. Cultures were generated from every cadaveric eye (10/10), although not all biopsies from the same eye gave rise to cultures. Confluent sheets of cultured cells were also produced successfully from limbal rings that had been in organ culture for up to 25 days, but the success rate from limbal ring material was variable (14/21). An analysis of parameters associated with each limbal ring was carried out in an attempt to identify the reasons for the different efficiencies of epithelial production. No obvious single parameter correlation was detected, although there was a trend to poorer efficiency with increased donor age. Conclusions. Confluent sheets of cultured corneal epithelial cells, suitable for grafting, can be produced from limbal tissue taken from eye bank organ-cultured corneas, although it takes longer, on average, to reach confluence (17–21 days) than an equivalent sample from a fresh eye (9–12 days).

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. (WOUND REP REG 2003;11:132–138)

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 S100β or β-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.

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.

Werner's syndrome T lymphocytes display a normal in vitro life-span

Werners syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

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.

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 patients 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.