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Objectives and Design: The MELFO (MELanoma FOllow-up) study is an international phase III randomized controlled trial comparing an experimental low-intensity schedule against current national guidelines. Background: Evidence-based guidelines for the follow-up of sentinel node-negative melanoma patients are lacking. Methods: Overall, 388 adult patients diagnosed with sentinel node-negative primary melanoma patients were randomized in cancer centers in the Netherlands and United Kingdom between 2006 and 2016. The conventional schedule group (control: n=196) was reviewed as per current national guidelines. The experimental schedule group (n=192) was reviewed in a reduced-frequency schedule. Quality of life was the primary outcome measurement. Detection rates and survival outcomes were recorded. Patient satisfaction rates and compliance with allocated schedules were compared. Results: At 5 years, both arms expressed high satisfaction with their regimens (>97%). This study found no significant group effect on any patient-reported outcome measure scores between the follow-up protocols. In total, 75/388 (19.4%) patients recurred, with no difference in incidence found between the 2 arms (hazard ratio=0.87, 95% confidence interval: 0.54-1.39, P=0.57). Self-examination was the method of detection for 25 experimental patients and 32 control patients (75.8% vs. 76.2%; P=0.41). This study found no difference in any survival outcomes between the 2 study arms (disease-free survival: hazard ratio=1.00, 95% confidence interval: 0.49-2.07, P=0.99). Conclusions: A reduced-intensity, American Joint Committee on Cancer (AJCC) stage-adjusted follow-up schedule for sentinel node-negative melanoma patients is a safe strategy, and patient self-examination is effective for recurrence detection with no evidence of diagnostic delay. Patients' acceptance is very high.

Purpose Coregistered SPECT/CT can improve accuracy of sentinel node biopsy (SNB) for staging melanoma. This benefit has implications for pathology services and surgical practice with increased diagnostic and surgical workload. The purpose of this study was to investigate the effectiveness of SPECT/CT imaging. Methods SNB data were collected over a 10-year period. Preoperative SLN mapping was performed by using planar lymphoscintigraphy (LSG) for all patients (n = 1522) and after October 2015, patients underwent a second co-registered SPECT/CT scan (n = 559). The patients were stratified according to the imaging protocol. The number of nodes and nodal basins were assessed. The reasons for cancellation also were assessed. Results A total of 95% (1446/1522) of patients underwent a successful SNB procedure. Significantly more sentinel nodes were identified by the SPECT/CT protocol (3 vs. 2; p < 0.0001). More patients were cancelled in the SPECT/CT cohort (9.3% vs. 2.5%; p < 0.0001). Head & neck, lower limb, and AJCC IB primaries were significantly less likely to proceed to SNB. SPECT/CT identified significantly more positive SNBs (20.9% vs. 16.5%; p = 0.038). SPECT/CT imaging was associated with improved disease-free (hazard ratio [HR] = 0.74; 95% confidence interval [CI]: 0.54–1.0); p = 0.048) and disease-specific survival (HR = 0.48; 95% CI: 0.3–0.78; p = 0.003). Patients who did not proceed to SNB had a significantly increased nodal relapse rate (23.5% vs. 6.8%; HR = 3.4; 95% CI: 1.9–6.2; p < 0.0001) compared with those who underwent SNB. Conclusions This large cohort study confirms the increased accuracy of SPECT/CT for identifying SLN metastases, which would appear to have a significant therapeutic benefit, although an increased risk of cancellation of the SNB procedure on the day of surgery.

Extracapsular spread (ECS) is recognized to be a high-risk factor in melanoma patients with macrometastatic (N+) nodal disease; however, ECS risk in sentinel lymph node (SLN) biopsy, micrometastatic stage III disease is ambiguous. The aim of this study was to examine ECS incidence and its prognostic significance. A two-center, retrospective analysis of all patients with micro/macrometastatic lymphadenopathy undergoing nodal surgery from 2008 to 2014 was performed. Patient demographics, tumor characteristics, nodal ECS status, and patient outcomes were collected. Overall, 515 patients with nodal disease were identified (males/females = 277/238); median age was 63 years (range 17–94). There was an increased frequency of ECS disease in N+ disease compared with SLN+ disease (52.4% vs. 16.2%; p

Background Perioperative complications following inguinal lymphadenectomy, including seroma formation, are frequent. We have employed a 2-layer negative pressure wound therapy (2-LNPWT) as a method to reduce seroma rate and perioperative complications. We present the outcome of our initial experience with 2-LNPWT and compare the outcomes of its use with traditional closed suction drains (CSDs). Materials and methods A non-randomised retrospective case–control series was analysed. Surgeons performing inguinal lymphadenectomy for metastatic cutaneous melanoma utilised either the 2-LNPWT therapy or traditional CSDs according to their practice preference. Results The study included 111 patients. The cohorts were well matched for gender, disease burden, body mass index and comorbidities. The 2-LNPWT technique was associated with significantly better postoperative outcomes than CSD, in terms of incidence of seroma formation (26.9% vs 49.4%; p p = 0.005) and return to theatre rate (0% vs 15.3%; p = 0.03). The overall seroma rate was 44.1%. The only significant association with seroma initiation was the type of drainage system used (2-LNPWT 31.2% vs CSD 58.3%; p Conclusion This retrospective non-randomised case control study has demonstrated the safe use of a novel application of negative pressure wound therapy that significantly reduced the incidence of seroma formation and postoperative complication rate for inguinal lymphadenectomy for melanoma.

Purpose Coregistered SPECT/CT can improve accuracy of sentinel node biopsy (SNB) for staging melanoma. This benefit has implications for pathology services and surgical practice with increased diagnostic and surgical workload. The purpose of this study was to investigate the effectiveness of SPECT/CT imaging. Methods SNB data were collected over a 10-year period. Preoperative SLN mapping was performed by using planar lymphoscintigraphy (LSG) for all patients (n = 1522) and after October 2015, patients underwent a second co-registered SPECT/CT scan (n = 559). The patients were stratified according to the imaging protocol. The number of nodes and nodal basins were assessed. The reasons for cancellation also were assessed. Results A total of 95% (1446/1522) of patients underwent a successful SNB procedure. Significantly more sentinel nodes were identified by the SPECT/CT protocol (3 vs. 2; p < 0.0001). More patients were cancelled in the SPECT/CT cohort (9.3% vs. 2.5%; p < 0.0001). Head & neck, lower limb, and AJCC IB primaries were significantly less likely to proceed to SNB. SPECT/CT identified significantly more positive SNBs (20.9% vs. 16.5%; p = 0.038). SPECT/CT imaging was associated with improved disease-free (hazard ratio [HR] = 0.74; 95% confidence interval [CI]: 0.54–1.0); p = 0.048) and disease-specific survival (HR = 0.48; 95% CI: 0.3–0.78; p = 0.003). Patients who did not proceed to SNB had a significantly increased nodal relapse rate (23.5% vs. 6.8%; HR = 3.4; 95% CI: 1.9–6.2; p < 0.0001) compared with those who underwent SNB. Conclusions This large cohort study confirms the increased accuracy of SPECT/CT for identifying SLN metastases, which would appear to have a significant therapeutic benefit, although an increased risk of cancellation of the SNB procedure on the day of surgery.

For invasive primary cutaneous melanoma, wider excision is advocated to reduce local recurrence risk and improve patient outcomes. Excision detail is controversial, especially in intermediate- and high-risk primary melanoma (AJCC pT2-pT4). Guidance varies from sizes 1 to 3 cm (translating into large defects of 2-6 cm). The aim of this study was to determine the reconstructive and resource burden of wider excision margins (EMs).Data analysis from our prospective database (2008-2017) included 1184 patients (563F:621 M) with cutaneous melanoma (pT1b-pT4b). Procedure tariff data were sourced from our financial services department.Two hundred twenty-nine patients had a narrower EM (1 cm) and 995 (80.7%) had a wider EM (2-3 cm). Reconstructive requirement significantly increased with a wider EM collectively (11.3% vs 29.3%, odds ratio (OR) = 3.2; p 0.0001), in the extremities (15.2% vs 42.0%; p 0.0001), and in the head and neck (HN) (23.5 % vs 64.7%; p 0.0001). Reconstruction significantly increased hospitalisation rates (26.6% vs 63.0%, OR = 4.7; p 0.0001) collectively, in the HN (26.8 % vs 53.9%), and in the upper (18.9 % vs 42.3%) and lower extremities (34.8% vs 77.3%). Narrower EMs significantly reduced hospitalisation rates in the upper and lower extremities (7.1% vs 28.5%; p = 0.004, 37.9% vs 58.5%; p = 0.005, respectively). Overall procedure cost significantly increased by £180 (mean, p 0.0001) and £346 (median, p = 0.0004) per patient when reconstruction was required.Our data suggest substantial impact of wider EM on patients, which more than doubled in the functionally and cosmetically sensitive extremities and the HN region. Reconstructions add significant financial and healthcare service burden. Without randomised controlled trial (RCT) evidence demonstrating increased efficacy of wider EM, narrower EM is advocated whilst awaiting future planned RCT results specifically investigating on this.

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.

Extracapsular spread (ECS) is recognized to be a high-risk factor in melanoma patients with macrometastatic (N+) nodal disease; however, ECS risk in sentinel lymph node (SLN) biopsy, micrometastatic stage III disease is ambiguous.The aim of this study was to examine ECS incidence and its prognostic significance.A two-center, retrospective analysis of all patients with micro/macrometastatic lymphadenopathy undergoing nodal surgery from 2008 to 2014 was performed. Patient demographics, tumor characteristics, nodal ECS status, and patient outcomes were collected.Overall, 515 patients with nodal disease were identified (males/females = 277/238); median age was 63 years (range 17-94). There was an increased frequency of ECS disease in N+ disease compared with SLN+ disease (52.4% vs. 16.2%; p 0.0001). The absolute disease-specific survival (DSS) difference for SLN+ patients was approximately 30% at 10 years (66.2% vs. 37.2%; p 0.0001), and the prognosis of SLN+/ECS+ patients was identical to N+/ECS- patients. Multivariate analysis demonstrated that ECS status was an independent prognostic indicator for DSS (hazard ratio 2.47, 95% confidence interval 1.87-3.26; p 0.0001) in patients with SLN+ disease. There were significant differences in nodal burden according to ECS status between the SLN+ and N+ subgroups suggestive of differing biology in ECS+ tumors.We found that ECS is a significant DSS, progression-free survival, and overall survival indicator in SLN+ and N+ disease. We demonstrated that ECS upstages stage III disease, similar to ulceration in primary melanoma (stage I/II disease). A simplified staging system substituting ECS for N stage accurately stages patients according to prognosis.

Background Appropriate reconstruction of the posterolateral mandible remains controversial. Both osseous and soft tissues are vital components for an overall successful outcome and are often combined in complex defects. Their respective effect on oromandibular function in the reconstruction of different degrees of mandibular defects has been less evaluated. This study aimed to compare patient-perceived oromandibular function in osseous and soft tissue-only reconstructions following posterolateral mandibular defects, defined as limited or extended. Patients and Methods A 10-year retrospective review of consecutive patients undergoing mandibular reconstructions of the posterolateral mandible were identified. Limited defects were defined as reaching from the ipsilateral parasymphysis to anterior of the coronoid (sparing insertion of muscles of mastication). Extended defects were defined as reaching from the ipsilateral parasymphysis to posterior of the coronoid (sacrificing the muscle insertions). Functional outcomes were assessed using the University of Washington Quality of Life questionnaire, version 4. Results A total of 163 patients were identified, of which 41 patients had the particular posterolateral mandibular resections sought after. In 23 limited resections, there was no difference in functional outcome between osseous and soft tissue-only reconstructions. In 18 patients undergoing extended resections, osseous reconstructions demonstrated significantly better outcomes (p = 0.011). There were no significant differences in patient demographics between the groups. Conclusion Our study highlights the interest of soft tissue-only reconstructions of the posterolateral mandible. Limited resections seem not to benefit from complex osseous reconstruction for adequate function. Conversely, there is a noteworthy positive impact on functional outcomes in extended posterolateral mandibulectomies reconstructed with osseous tissue, compared with soft tissue only. Although a larger study is needed to identify a stronger relationship, these preliminary results could aid reconstructive decisions, particularly when considering patient morbidity.

Sentinel lymph node biopsy (SLNB) is an important staging tool for the management of melanoma. A multicentre study was done to validate previous findings that the timing of lymphoscintigraphy influences the accuracy of SLNB and patient outcomes, particularly survival.Data were reviewed on patients undergoing SLNB for melanoma at three centres in the UK and Sweden, examining the effect of timing of SLNB after nuclear medicine scanning. Kaplan-Meier survival analysis was used to assess overall (OS), disease-specific (DSS) and progression-free (PFS) survival, stratified by timing of lymphoscintigraphy. Independent risk factors for survival were identified by Cox multivariable regression analysis.A total of 2270 patients were identified. Median follow-up was 56 months. Univariable analysis showed a 4·2 per cent absolute and 35·5 per cent relative benefit in DSS (hazard ratio 1·36, 95 per cent c.i. 1·05 to 1·74; P = 0·018) for 863 patients whose SLNB was performed up to 12 h after lymphoscintigraphy compared with 1407 patients who had surgery after more than 12 h. There were similar OS and PFS benefits (P = 0·036 and P = 0·022 respectively). Multivariable analysis identified timing of lymphoscintigraphy as an independent predictor of OS (P = 0·017) and DSS (P = 0·030). There was an excess of nodal recurrences as first site of recurrence in the group with delayed surgery (4·5 versus 2·5 per cent; P = 0·008).Delaying SLNB beyond 12 h after lymphoscintigraphy withLa biopsia de ganglio centinela (sentinel lymph node biopsy, SLNB) es una técnica importante para la estadificación y tratamiento del melanoma. Se realizó un estudio multicéntrico para validar hallazgos previos según los cuales el momento de la linfogammagrafía (lymphoscintigraphy, LS) influye en la precisión de la SLNB y en los resultados de los pacientes, especialmente en la supervivencia. MÉTODOS: Se revisaron los datos de los pacientes a los que se realizó una SLNB por melanoma en 3 centros en el Reino Unido y Suecia, con especial atención al efecto del período entre la inyección el material radioactivo y la SLNB. Se realizó un análisis de supervivencia mediante el método de Kaplan-Meier para la supervivencia específica de la enfermedad (disease-specific survival, DSS), supervivencia global (overall survival, OS) y supervivencia libre de progresión (progression-free survival, PFS), todas ellas estratificadas por el momento de la LS. Los factores de riesgo independientes para la supervivencia se determinaron mediante un análisis de regresión multivariable de Cox.Se incluyeron 2.270 pacientes. La mediana de seguimiento fue de 49 meses. El análisis univariado mostró un beneficio absoluto del 4,2% y relativo del 35,5% (cociente de riesgos instantáneos, hazard ratio, HR: 1,36 (i.c. del 95% 1,05-1,74, P = 0.02)) en la DDS para los pacientes a los que la SLNB se realizó 12 horas después de la LS (n = 863) en comparación con aquellos realizados 12 horas (n = 1407). Se detectaron beneficios similares para la OS y la PFS (P = 0,04 y P = 0,02, respectivamente). El análisis multivariable identificó el tiempo entre la LS y la SLNB como un factor independiente de OS (P = 0,017) y DSS (P = 0,03). Hubo un aumento en las recidivas ganglionares como primer sitio de recidiva en el grupo de 12 horas (2,5% versus 4,5%; P = 0,008). CONCLUSIÓN: Estos datos validan nuestra investigación previa y tienen implicaciones significativas para las unidades de melanoma, en el sentido de que retrasar la SLNB más allá de las 12 horas después de realizar la LS con nanocoloides marcados con Tc99 tiene un impacto negativo significativo en la supervivencia de los pacientes y debe evitarse. Se presenta la hipótesis de que la causa subyacente es la migración temporal del trazador que determina una SLNB incorrecta. .

Objectives: Sentinel lymph node biopsy (SLNB) is standard of care for staging regional LN in AJCC stage IB-IIC melanoma; using dual localization with radiolabelled colloid and blue dye. Combining these gives optimal accuracy; drawbacks include cumulative radiation exposure for healthcare workers, coordination between disciplines and anaphylaxis. An alternative tracer agent is indocyanine green (ICG); an optical enhancer that fluoresces in the near infrared range. This prospective cohort study assesses the feasibility of using ICG as a tracer agent to detect SLN in cutaneous melanoma. Methods: Primary melanoma patients diagnosed with pT1b-pT4b tumours undergoing SLNB were recruited over a 6-month period at a tertiary referral centre. All underwent standard preoperative lymphoscintigraphy (LSG) using 20-40MBq of Tc99radiolabelled nanocolloid plus intraoperative Patent Blue dye (PBD). ICG was administered as a third tracer agent intraoperatively. Results: 62 patients (33M/29F) were recruited; median age was 61 years. Median melanoma Breslow thickness was 1.6mm. 144 specimens containing 135 SLN were excised. Concordance rate for all 3 tracer agents was 88.1%(119/135 LN); that for radioisotope/PBD was 88.2%(95%CI:82.2,93.7). There were no discordance pairs between radioisotope/PBD compared to radioisotope/PBD/ICG. Radioisotope/ICG significantly increased the sensitivity of detecting SLN to 98.5%(95%CI:94.8,99.8); p

Introduction: Sentinel lymph node biopsy (SLNB) in cutaneous melanoma (CM) is performed to identify patient at risk of regional and distant relapse. We hypothesized that timing of lymphoscintigraphy may influence the accuracy of SLNB and patient outcomes. Methods: We reviewed prospective data on patients undergoing SLNB for CM at a large university cancer-center between 2008-2015, examining patient and tumor demographics and time between lymphoscintigraphy (LS) and SLNB. Kaplan-Meier survival analysis assessed disease-specific (DSS) and overall-survival (OS), stratified by timing of LS. Cox multivariate regression analysis assessed independent risk factors for survival. Results: We identified 1015 patients. Median follow-up was 45 months (IQR 26-68 months). Univariate analysis showed a 6.8% absolute DSS (HR 1.6 [1.03-2.48], p= 0.04) benefit and a 10.7% absolute OS (HR 1.64 [1.13-2.38], p=0.01) benefit for patients whose SLNB was performed < 12 hrs of LS (n= 363) compared to those performed >12 hours (n=652). Multivariate analysis identified timing of LS as an independent predictor of OS (p=0.007) and DSS (p=0.016) when competing with age, sex, Breslow thickness (BT) and SLN status. No difference in nodal relapse rates (5.2% v 4.6%; p=0.67) was seen. Both groups were matched for age, sex, BT and SLN status. Conclusion: These data have significant implications for SLNB services, suggesting delaying SLNB >12 hours after LS using a Tc99-labelled nanocolloid has a significant negative survival impact for patients and should be avoided. We hypothesise that temporal tracer migration is the underlying cause and advocate further trials investigating alternative, 'stable' tracer-agents.

The rising incidence of primary head and neck (H&N) cancers in the elderly presents a dilemma regarding the appropriateness of complex surgery in this assumed frail age group. With limited data on surgical morbidity, survival, and patient quality of life (QOL), this analysis aimed to broaden the understanding of safety and effectiveness of microsurgical treatment in very elderly H&N cancer patients. A prospective database analysis was used to evaluate surgical outcomes (morbidity, survival, and QOL) in all patients aged 80 years and older undergoing microsurgical reconstruction for cutaneous and intra-oral H&N cancers between 2004 and 2014. Outcomes were assessed for their association with surgical, tumour, and patient variables. Comorbidities were categorized by the ACE27 index and postoperative morbidity by the Clavien–Dindo scoring system. QOL was analyzed using the UW-QOLv4. Of 720 microsurgical reconstructions, 96 patients were identified. Median survival was 25 months. The ACE27 index was the only variable significantly associated with survival with a 5-year survival of 59.2 % in the least comorbid group versus 19.7 % in the most comorbid group (p 0.015). ACE-27 showed influence on socioemotional QoL scores. Physical QOL scores were influenced by tumour and operative factors. Patients were found to value physical QOL over socioemotional. Microsurgical reconstructions are well tolerated in the very elderly patients and should be considered predominantly based on comorbidity. Tumour stage, flap type, and cancer site should still form part of the preoperative counseling due to their implication on postoperative physical function.

This report describes a good example of the rare fourth branch of the marginal mandibular nerve. This case emphasizes the need for respecting the variation in the marginal mandibular nerve when carrying out surgery.

Summary Background Complete excision of high-risk extensive non-melanoma skin cancers in the head and neck is paramount to achieving loco-regional control. However, achieving clear margins still remains a significant challenge. Mohs' micrographic surgery (MMS) provides the most accurate method of intraoperative mapping and histological assessment of tumour margins. We have developed a technique combining MMS with reconstruction as a single-stage procedure performed under general anaesthetic. We present our experience and results. Materials and methods Following regional skin cancer multidisciplinary team (MDT) discussion, patients considered appropriate for management as a single-stage combined procedure were referred for assessment. At surgery, a two-team approach was employed consisting of an MMS resection team and a reconstructive team, allowing simultaneous resection and elevation of any free tissue required for reconstruction. Outcome data were retrieved from a prospectively collated MMS database. Results Twenty-six cases were performed between January 2010 and January 2013. Fifty-eight percent of cases were basal cell carcinomas. Clear margins were achieved in 50% of cases following the first Mohs' layer. Free tissue reconstruction was required in 13 cases. Mean anaesthetic time was 445 min. Loco-regional control was achieved in 96% of patients, at a mean follow-up period of 29 months (range 11–50 months). Conclusions This study shows that the combined single-stage MMS and reconstruction surgical model is safe, results in a low recurrence rate and improved patient care. It is a model that can be replicated in other tertiary skin cancer units.

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.

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.