We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.

Human melanocytes in monolayer culture are extremely dependent on a wide range of soluble signals for their proliferation and melanogenesis. The advent of three-dimensional models of reconstructed skin allows one to ask questions of how these cells are regulated within a setting which more closely approximates normal skin. The purpose of this study was to investigate to what extent melanocytes within a reconstructed skin model are sensitive to regulation by dermal fibroblasts, basement membrane (BM) proteins and the addition of alpha-melanocyte-stimulating hormone (alpha-MSH). Sterilized acellular de-epidermized dermis (prepared to retain BM proteins or deliberately denuded of BM by enzymatic treatment) from skin type I or II was reconstituted with fibroblasts, melanocytes and keratinocytes. In all but one case (9/10), cell donors were skin type I or II. The presence of BM antigens was found to be necessary for positional orientation of the melanocytes; in the absence of BM, melanocytes moved into the upper keratinocyte layer pigmenting spontaneously. Addition of fibroblasts suppressed the extent of spontaneous pigmentation of melanocytes within this model. Neither alpha-MSH nor cholera toxin induced pigmentation in this model despite the fact that melanocytes clearly had the ability to synthesize pigment.

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.

Background Evidence-based guidelines for follow-up treatment of American Joint Committee on Cancer (AJCC) stages 1B to 2C melanoma patients are lacking. The MELanoma FOllow-up study is an international phase 3 randomized trial, and the 3-year interim data were recently reported from the Netherlands. The study was undertaken concurrently with a British cohort for comparison and validation of the Dutch study. Methods The study enrolled and stratified 207 patients by AJCC stage. The conventional schedule group (CSG; n = 103) cohort was reviewed as per UK guidelines. The experimental schedule group (ESG; n = 104) cohort was reviewed in a reduced-frequency nurse-led, consultant-supervised clinic. Quality of life (QoL) was measured at baseline (T1), a 1 year (T2), and at 3 years (T3) using the State-Trait Anxiety Inventory, the Cancer Worry Scale, the Impact-of-Event Scale, and the Mental and Physical Component scales (PCS/MCS) of the RAND-36. Results Of the 207 QoL questionnaires, 170 (82.1%) were completed at T3. Both cohorts expressed high satisfaction (> 93%) with their regimens. At T3, no significant group effect was found on any patient-reported outcome measures scores, indicating no QoL difference between the follow-up protocols. Recurrence had developed in 33 patients Conventional follow-up (CFU), 16 [15.5%]; Experimental follow-up (EFU), 17 [16.3%]. Self-examination was the method of detection for 12 ESG patients (70.6%) and 11 CSG patients (68.8%). The melanoma-specific survival was identical. Conclusion The UK 3-year data were consistent with the previous Dutch report. The reduced follow-up strategy was shown to be safe, with significant resource usage benefits for national cancer services. Patient anxiety levels were not increased by a less-intensive follow-up regimen, and acceptance was high. The study data indicate that patient self-examination is very effective for recurrence detection.