Rebecca A. Dawson

Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use.

The aim of this study was to identify a sterilization technique for the preparation of human allodermis which could be used as a dermal component in wound healing and as the dermal base for production of dermal–epidermal composites for one‐stage grafting in patients. We report that it is possible to produce dermal–epidermal composites which perform well in vitro and in vivo using a standard ethylene oxide sterilization methodology. Prevention of ethylene oxide‐induced damage to the dermis was achieved using gentle dehydration of the skin prior to ethylene oxide sterilization. The issue of whether viable fibroblasts are required for composite production was examined in comparative studies using glycerol vs. ethylene oxide sterilized dermis. Where good collagen IV retention was achieved following preparation of acellular de‐epidermized dermis there was no advantage to having fibroblasts present in vitro or in vivo; however, where collagen IV retention was poor or where keratinocytes were initially expanded in culture then there was a significant advantage to introducing fibroblasts to the composites during their preparative 10‐day period in vitro. The requirement for fibroblasts became less evident when composites were grafted on to nude mice. In conclusion, we report a protocol for the successful sterilization of human allodermis to achieve an acellular dermis with good retention of collagen IV. This acellular dermis would be appropriate for clinical use as a dermal replacement material. It can also be used for the production of dermal–epidermal composites using autologous keratinocytes (with or without fibroblasts).

Attachment of human keratinocytes to plasma co-polymers of acrylic acid/octa-1,7-diene and allyl amine/octa-1,7-diene

Plasma co-polymers (PCPs) of acrylic acid/octa-1,7-diene and allyl amine/octa-1,7-diene have been prepared and characterised using X-ray photoelectron spectroscopy (XPS). The use of a hydrocarbon diluent in the monomer feed allowed the deposition of films with controlled concentrations of carboxylic acid and nitrogen-containing functional groups. Human keratinocytes were cultured on these PCP surfaces, tissue culture poly(styrene) (TCPS) and collagen I. The level of keratinocyte attachment over 24 h was measured. PCP surfaces containing low concentrations of carboxylic acid groups (2.3%) were found to promote keratinocyte attachment. The performance of these PCPs was similar to collagen I, a well established substratum for attachment. Nitrogen-containing PCP surfaces were found to promote attachment at higher functional group concentrations, although the attachment did not attain the level achieved on the acid functionalised PCP surfaces.

A new autologous keratinocyte dressing treatment for non‐healing diabetic neuropathic foot ulcers

Aims   To evaluate the use of a new cell‐tailored carrier surface (TranCell) for delivery of autologous keratinocytes to promote wound healing in patients with chronic neuropathic foot ulcers.

Clinical experience using cultured epithelial autografts leads to an alternative methodology for transferring skin cells from the laboratory to the patient

We report a 10-year audit using cultured epithelial autografts (CEAs) for patients with extensive burns. Clinical take using CEAs averaged only 45% (as has been reported by others) but over half of all cells cultured for these patients had to be discarded owing to difficulties of timing the production of CEA sheets to the needs of the patients. CEAs could not be used until they had reached confluence and formed an integrated sheet, which took, on average, 12 days. However, once formed, they needed to be used within 2-3 days or they lost the ability to attach to wound beds. In response to this we developed a simpler carrier dressing methodology for transferring cultured subconfluent keratinocytes from the laboratory to the wound bed. This methodology offers an increase in speed of delivery but its major contribution is the greater flexibility in timing the transfer of cells from the laboratory to the changing needs of the patients.

Characterization of an in vitro model of human melanoma invasion based on reconstructed human skin

The purpose of this study was to compare the invasive properties of normal human cutaneous melanocytes and of a cutaneous melanoma cell line (HBL) in a three‐dimensional model of reconstructed human skin. Specifically, we asked to what extent the pigmentary and invasive behaviour of both cells is influenced by their interaction with adjacent skin cells (keratinocytes and fibroblasts) and the basement membrane (BM). In the presence of a BM, normal human melanocytes within this model remained within the basal layer of keratinocytes and did not pigment spontaneously. When the BM was removed, melanocytes were found suprabasally and pigmented extensively. No significant invasion of melanocytes into the dermis was detected in the presence or absence of the BM. HBL melanoma cells showed no significant ability to invade into the dermis in the absence of other cells, irrespective of the presence or absence of the BM. However, when added to keratinocytes and fibroblasts, HBL cells showed a capacity to invade into the dermis, both in the presence and absence of the BM. Associated with HBL invasion into the dermis, we noted significant keratinocyte entry into the dermis. On their own, keratinocytes entered the dermis in the absence of the BM but showed no significant penetration into the dermis when the BM was present. In summary, this model demonstrates clear differences between melanocytes and a melanoma cell line with respect to their invasive properties. It also allows demonstration of interactions between cells, and between cells and the BM. The study also provides evidence for a synergistic interaction between this melanoma cell line and keratinocytes in penetrating the BM.

Use of peracetic acid to sterilize human donor skin for production of acellular dermal matrices for clinical use

We previously reported methods for sterilizng human skin for clinical use. In a comparison of γ‐irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity. The aim of this study was to evaluate peracetic acid as an alternative sterilizing agent. Skin sterilized with peracetic acid was compared with skin sterilized using glycerol alone or glycerol with ethylene oxide. The effect of subsequently storing peracetic acid sterilized skin in glycerol or propylene glycol was also examined. Acellular dermal matrices were produced after removal of the epidermis and cells in the dermis, processed for histological and ultrastructural analysis, and the biological function was evaluated by reconstitution with keratinocytes and fibroblasts. Results showed that sterilized acellular matrices retained the integrity of dermal structure and major components of the basement membrane. There were no overall significant differences in the ability of these matrices to form reconstructed skin, but peracetic acid alone gave a lower histologic score than when combined with glycerol or propylene glycol. We conclude that peracetic acid sterilization followed by preservation in glycerol or propylene glycol offers a convenient alternative protocol for processing of human skin. It is suggested that this sterile acellular dermis may be suitable for clinical use.

Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes.

Melanoma invasion is a complex multi stage process involving changes to the cell/extracellular matrix (ECM) and cell/cell interactions. We have previously shown using an in vitro model of reconstructed human skin (consisting of human dermis with a basement membrane [BM] and populated with human skin cells) that some melanoma cells (HBL cell line) invade more actively in the presence of adjacent normal skin cells. The aim of the present study was to further investigate the relationship between melanoma cells, skin cells and ECM proteins during melanoma cell invasion through reconstructed skin, extending this to a study of three melanoma cell lines. We also examined whether such cell/cell induced invasion is due to increased expression and activation of matrix-metalloproteinase-2 (MMP-2) and MMP-9, or due to increases in general protease activity for keratinocytes, fibroblasts or melanoma lines. Addition of skin cells dramatically altered the invasive behaviour of the three metastatic melanoma cell lines (HBL, C8161 and A375SM) used; they increased the invasive ability of HBLs which were unable to invade on their own; they potentiated the invasion of C8161 cells which were invasive in their own right, but reduced the invasion of A375-SM cells which were aggressive invaders in the absence of skin cells. Latent forms of MMP-2, and MMP-9, were clearly expressed by the normal skin cells whereas all three melanoma lines weakly expressed these proteases. Fibroblast and keratinocyte MMPs were activated specifically by culture on type I collagen and on dermis which retained an intact basement membrane. These findings demonstrate that while there is an active communication between melanoma cells and adjacent skin cells, the invasive process is dictated by the melanoma cells and not the skin cells. However, activation of skin cell derived MMPs may play an important role in facilitating invasion by particular melanoma phenotypes.

Calmodulin antagonists of improved potency and specificity for use in the study of calmodulin biochemistry

Syntheses are described for a range of N-(omega-aminoalkyl)-5-iodo- and -5-cyanonaphthalene-1-sulphonamides. The selective activity of these compounds as inhibitors for calmodulin-dependent phosphodiesterase (EC 3.1.4.17) is compared with their activity for the calmodulin-independent but calcium-dependent enzymes protein kinase C and transglutaminase (EC 2.3.2.13). The results show a drastic improvement in the selectivity of effect for the 5-iodo-compounds compared with the widely-used drug, W7, N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide.

Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents

The crucial role of oxygen during the complex process of wound healing has been extensively described. In chronic or nonhealing wounds, much evidence has been reported indicating that a lack of oxygen is a major contributing factor. Although still controversial, the therapeutic application of hyperbaric oxygen (HBO) therapy can aid the healing of chronic wounds. However, how HBO affects reepithelization, involving processes such as keratinocyte proliferation and differentiation, remains unclear. We therefore used a three‐dimensional human skin–equivalent (HSE) model to investigate the effects of daily 90‐minute HBO treatments on the reconstruction of an epidermis. Epidermal markers of proliferation, differentiation, and basement membrane components associated with a developing epidermis, including p63, collagen type IV, and cytokeratins 6, 10, and 14, were evaluated. Morphometric analysis of hematoxylin and eosin‐stained cross sections revealed that HBO treatments significantly accelerated cornification of the stratum corneum compared with controls. Protein expression as determined by immunohistochemical analysis confirmed the accelerated epidermal maturation. In addition, early keratinocyte migration was enhanced by HBO. Thus, HBO treatments stimulate epidermal reconstruction in an HSE. These results further support the importance of oxygen during the process of wound healing and the potential role of HBO therapy in cutaneous wound healing.

Keratinocyte-driven contraction of reconstructed human skin.

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose‐6‐phosphate to be ineffective and ascorbic acid‐2‐phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.