Trans-nasal endo-assisted pharyngoplasty: a cadaver study.

A possible new technique of endoscopic pharyngoplasty is described and has been developed in cadavers. The trans-nasal route is used for endoscopic visualisation of the velopharyngeal sphincter. Hynes pharyngoplasty is performed using both trans-oral and trans-nasal routes. This approach allows better visualisation and performance of the Hynes pharyngoplasty at the desired level, "high" in the nasopharynx, without splitting the soft palate.

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.

Keratinocytes suppress TRP-1 expression and reduce cell number of co-cultured melanocytes - implications for grafting of patients with vitiligo.

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well-integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co-cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down-regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase-related protein-1 (TRP-1) negative in confluent well-integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP-1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP-1 expression.

The in vitro percutaneous penetration of chlorpyrifos.

Chlorpyrifos is a widely used organophosphate pesticide. In order to study the pharmacokinetics of the penetration of chlorpyrifos through human skin we measured the percutaneous penetration of chlorpyrifos through human skin using an in vitro flow through apparatus. The chlorpyrifos was applied to the skin as a commercial concentrate or as a reference standard dissolved in ethanol. There was a significant difference (P=0.03) between the rate of penetration from the commercial concentrate (9.0 nmoles cm(-2) h(-1)) and that from the reference standard (4.9 nmoles cm(-2) h(-1)). Each experiment was run for 24 h. The recoveries from experiments where chlorpyrifos was applied to the skin as a commercial concentrate and as a reference standard dissolved in ethanol were, respectively, in total 91 and 87% of the applied dose of which 15 and 10% was recovered from the skin, 56 and 66% was recovered from the surface of the skin and 20 and 11% was recovered from the receptor fluid. There was a significant difference in the recoveries from the skin but there was no significant difference in the recoveries from the surface of the skin. We concluded that the majority of a dermal dose of chlorpyrifos was still present at or in the surface of the skin 24 h after application of a dermal dose. Because chlorpyrifos was recovered from the skin after 24 h, it is possible that the skin could act as a reservoir and release chlorpyrifos over a longer period. We also conclude that the solvent vehicle for chlorpyrifos can affect the rate of penetration of the pesticide.

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).

A longitudinal comparison of the psychological impact on mothers of neonatal and 3 month repair of cleft lip.

PURPOSE: This study investigates whether the timing after birth of babies' cleft repairs influences the psychological status of mothers. METHODS: Mothers of infants born with a cleft lip completed psychological assessments and semistructured interviews at four time points: 2-3 weeks, 3 months and 6 months following the birth. In addition, a preoperative assessment and interview was completed within the first week of birth for those with infants undergoing neonatal repair and within the week before surgery for the 3 month repair group. RESULTS: There were no significant differences between mothers of infants with early (neonatal) and late (3 month) repairs on the emotional measures at any time point or preoperatively. Means of measures for anxiety and depressive symptoms and the Impact of Event Scale were within the normal range. Measures of interaction with the infant, perceived infant difficulty, bonding and parental competence failed to show any impact of timing of operation. Women's emotional status improved significantly over the 6 month period regardless of operation timing. Qualitative analysis of interview data indicated most mothers preferred their infant to receive neonatal repair. CONCLUSIONS: There was no evidence to support the idea that repair neonatally or at 3 months led to differential levels of anxiety or depressive symptoms or differences in attachment to the infant. Nevertheless mothers expressed a preference for and greater satisfaction with neonatal repair. In the absence of definitive evidence of differences in physical outcome, parental preferences should routinely be considered in deciding the timing of this procedure.

The requirement for basement membrane antigens in the production of human epidermal/dermal composites in vitro.

The importance of a dermal element when providing permanent wound cover for skin loss has become evident as the shortcomings of pure epidermal grafts are recognized. We are developing a skin composite formed from sterilized human de-epidermized acellular dermis, keratinocytes and fibroblasts with the ultimate aim of using this composite to cover full-thickness excised burn wounds. These composites can be prepared with or without basement membrane (BM) antigens initially present on the dermis. This study investigates the importance of retaining BM antigens on the dermis to the production and appearance of these composites in vitro. Skin composites prepared from dermis with BM antigens either present or absent initially were studied throughout 3 weeks. Composites with BM antigens present initially were significantly better than those initially lacking BM antigens in: (i) the degree of epithelial cell attachment to the underlying dermis (hemidesmosomes were seen only in the former); (ii) the morphology of the epithelial layer; (iii) the consistent presence of collagen IV and laminin and the increasing expression of tenascin; and (iv) the amount of soluble collagen IV and fibronectin detected in the conditioned media. We conclude that an initial BM antigen template is vital in this skin composite model for the attachment and differentiation of the epithelial layer and for the subsequent remodelling of the BM in vitro.

New forms of skin grafting: from the laboratory to the clinic.

This article reviews current research on skin replacements used in the management of extensive burns and in reconstructive surgery. It describes how progress is being made in developing both biological skin substitutes and artificial materials with the aim of providing skin cover by means other than traditional skin grafting.

Methicillin-resistant Staphylococcus aureus in burns patients--why all the fuss?

Procedures designed to limit spread of methicillin-resistant Staphylococcus aureus (MRSA) in burns units demand time and resources. To assess the significance of MRSA in burns patients we performed a retrospective review of MRSA colonization in in-patients over a 41-month period at the North Trent Sub-regional Burns Unit. Patients were compared with MRSA free controls, matched for age and percentage body surface area (BSA) burn and admitted during the same time period. Length of stay, number of operations and deaths were outcome indicators. All patients managed non-operatively were excluded, leaving 40 MRSA patients and 46 controls. There was no statistical difference between the two groups with regard to number of operations (p= 0.07), duration of admission (p = 0.12) or mortality (p = 0.09). Of the control group, 83% had wound swabs positive for methicillin-sensitive Staphylococcus aureus (MSSA). there was no statistical difference in any outcome variables between this sub-group of controls and MRSA patients. Colonization with S. aureus (both MRSA and MSSA) was associated with larger burns (p<0.05), twice as many operative procedures (p<0.05) and prolonged admissions (p<0.01). Mortality was unaltered by staphylococcal colonization (p = 0.8). Although our study lacks power, we would suggest that methicillin resistance per se is not associated with increased morbidity or mortality in burns patients.

Skin banking in the UK: the need for proper organization.

Skin banking was set up in Sheffield in 1991 to provide a readily available source of allograft material to be used both for research purposes and also as a means of providing immediate wound cover for major burns patients. Once skin was available, however, clinical demand for it both within and outside Sheffield, outstripped the resources to run the bank. Logistical difficulties were encountered in the day to day running of the bank. These revolved around shortage of staff available for harvesting, the relative lack of public awareness of skin donation, shortage of banked skin as the bank became more widely known and lack of space and finance to expand. The decision was made to transfer the now established skin bank to the National Blood Service where it now operates with staff and resources dedicated specifically to this purpose. Experience leads to the suggestion that there is a clinical need for allograft skin in the UK which is not being met at the present time. There is a need for dedicated properly resourced skin banks and for the Department of Health to introduce regulation, monitoring and inspection of skin bank facilities in order to safeguard standards.

Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model.

Composites of human de-epidermised acellular dermis and normal adult human keratinocytes and fibroblasts were examined for the ability of cells to contract these composites. Image analysis of the outline of the composites showed that, in this model, keratinocytes alone or in the presence of fibroblasts caused highly significant contraction (of the order of 25% by day 12). There was no significant contraction of the dermis with fibroblasts alone or in the absence of cells. The presence or absence of basement membrane antigens did not influence the effect of keratinocytes on dermal contraction. Analysis of the conditioned media from these composites showed that the greatest fibronectin production was seen with fibroblasts alone in the presence of basement membrane. Keratinocytes alone produced little fibronectin irrespective of the presence of the basement membrane. If keratinocytes were present with fibroblasts, however, then fibronectin production was significantly reduced both in the presence and absence of the basement membrane, indicating that keratinocytes modify dermal fibroblast extracellular matrix production. This study shows that while keratinocytes and fibroblasts are clearly influencing each other's activity in this human skin composite model, under the circumstances we describe it is the keratinocyte and not the fibroblast which causes contraction of the human de-epidermised acellular dermis.

A comparison of methodologies for the preparation of human epidermal-dermal composites.

The purpose of this study was to compare methodologies for the preparation of human skin composites based on deepidermized acellular dermal matrix, epidermal keratinocytes, and dermal fibroblasts with the aim of preparing a clinically useful skin substitute. Dermal matrices were prepared from normal human skin and we compared methods of sterilization (glycerol treatment, ethylene oxide treatment, and gamma irradiation), methods of removing the epidermis (sodium chloride, phosphate buffered saline, and dispase), and methods of seeding the composites with fibroblasts and keratinocytes. We report protocols for reproducibly preparing composites that share many of the features of normal skin after 7 days culture at an air-liquid interface. Such composites can be based on allodermis pretreated with either glycerol or ethylene oxide (although the latter gave less consistent results than the glycerol treatment). Fibroblast penetration into the dermis could be achieved by culture of cells on the reticular or papillary surface of the dermis. However, we report for the first time that fibroblast entry from the papillary surface only occurred when keratinocytes were also present.

Extracellular matrix proteins induce changes in intracellular calcium and cyclic AMP signalling systems in cultured human keratinocytes.

The purpose of this study was to investigate whether extracellular matrix proteins which influence human keratinocyte behaviour are capable of altering intracellular signalling systems in these cells. The effects of extracellular matrix proteins on two major signal transduction pathways, intracellular calcium and cyclic adenosine monophosphate (cyclic AMP), were investigated. The extracellular matrix proteins examined were the basement membrane preparation matrigel, collagens type I and IV, vitronectin and its active tripeptide component Arg-Gly-Asp (RGD). Acute additions of matrigel, vitronectin and RGD caused rapid transient increases in intracellular calcium and, together with collagen type I, also caused sustained elevations in basal calcium when cells were grown on these substrates. Cyclic AMP production was unaffected by acute exposure to these extracellular matrix proteins. Culture of cells on matrigel, collagen type I or IV, however, significantly reduced basal cyclic AMP accumulation and increased the response of the cells to the receptor-independent agonist forskolin. It is concluded that in vitro some extracellular matrix proteins can initiate both acute and sustained changes in intracellular signalling in human keratinocytes.

Oxidative damage to protein and alterations to antioxidant levels in human cutaneous thermal injury.

Evidence that oxygen free radicals may be contributory to further tissue damage in the events following cutaneous thermal injury supports a role for interventional therapy using antioxidants. However, previous work has relied almost entirely on animal-based models with little clinical information available. Also, methods used to support an oxidative role in thermal injury have relied almost exclusively upon the use of lipid peroxidation studies. Further work substantiating a contributory role of free radicals is therefore required using additional methodology before considering antioxidant therapy aimed at retarding tissue damage. We investigated general oxidative damage to protein in burn blister fluid by quantifying the protein carbonyl levels from 11 patients admitted with superficial or partial thickness burns. Total antioxidant capacity was also assessed, together with measurement of protein and the antioxidants uric acid and bilirubin. Data were compared with values obtained for serum in healthy volunteers. Following thermal injury, burn blister fluid protein carbonyl level was increased by almost 50 per cent (P = 0.005) compared with normal serum. Antioxidant scavenging capacity, protein and bilirubin were all significantly reduced, but uric acid unaltered compared with control values. The present data support a role for oxidative damage in cutaneous thermal injury.

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Effect of glycerol on intracellular virus survival: implications for the clinical use of glycerol-preserved cadaver skin.

Glycerol has long been used for the preservation of skin allografts. The antimicrobial activity of glycerol has not been fully documented. This paper reports the results of an investigation of a model studying the effect of glycerol on the inactivation of intracellular viruses. Two viruses--herpes simplex type I (HSV-1) and poliovirus--were cultured within human dermal fibroblasts. These intracellular viruses were incubated with 50 per cent, 85 per cent and 98 per cent glycerol at 4 degrees C and 20 degrees C for 4 weeks. Each week, the cultures in glycerol and controls in fibroblast maintenance medium were assayed for virus infectivity by examining the ability of harvested viruses to infect further fibroblasts. At 4 degrees C, 85 per cent glycerol could not fully inactivate intracellular HSV-I or poliovirus even after 4 weeks; 98 per cent glycerol inactivated intracellular HSV-I (after 3 weeks) but could not fully inactivate intracellular poliovirus after 4 weeks. At 20 degrees C, 85 per cent glycerol inactivated intracellular HSV-I (within 1 week) but could not fully inactivate intracellular poliovirus after 4 weeks; 98 per cent glycerol inactivated intracellular HSV-I (within 1 week) and inactivated intracellular poliovirus (after 2 weeks). It is suggested that, on the basis of this study, glycerol can reduce intracellular virus infectivity but that its effects are very dependent on concentration, time and temperature such that we would recommend that allograft skin be exposed to 98 per cent glycerol for a minimum of at least 4 weeks at a minimum temperature of 20 degrees C before clinical use.

A simple human dermal model for assessment of in vitro attachment efficiency of stored cultured epithelial autografts.

This study investigated the effect of short-term storage on the viability and in vitro attachment efficiency of cultured epithelial autograft sheets. Four storage protocols were investigated: overnight at 37 degrees C in keratinocyte culture medium, overnight at 4 degrees C in phosphate-buffered saline solution, overnight at -80 degrees C in cryopreservation medium (containing 10% dimethyl sulphoxide), and 1 week at -80 degrees C in cryopreservation medium. Viability was assessed before and after storage by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. All the storage conditions significantly reduced viability compared with fresh sheets, and no significant decrease was seen when the sheets stored under the different protocols were compared with each other. The best viability obtained was 60% of that of the fresh sheets. The in vitro viability of these stored sheets was then compared with that of the fresh sheets by culturing them on deepidermized acellular allodermis and assessing the composites formed by light microscopy and the MTT assay. The fresh sheets attached and formed a histologically demonstrable composite with the dermal substrate, whereas none of the stored sheets formed demonstrable composites. The MTT assay demonstrated that composites formed with the stored sheets had less than 10% viability compared with composites formed with fresh sheets. It is concluded that under the conditions of storage examined, the viability of cultured epithelial autograft sheets was significantly reduced, but up to 60% of viability could be retained in some cases. However, the subsequent in vitro attachment and proliferation of such preserved sheets on allogeneic deepidermized dermis was poor compared with that of fresh cultured epithelial autograft sheets.