The Burden of Narcolepsy in Adults: A Population Sampling Study Using Personal Media.

OBJECTIVE: To obtain insight in the spectrum of narcolepsy symptoms and associated burden in a large cohort of patients. METHODS: We used the Narcolepsy Monitor, a mobile app, to easily rate the presence and burden of 20 narcolepsy symptoms. Baseline measures were obtained and analyzed from 746 users aged between 18 and 75 years with a reported diagnosis of narcolepsy. RESULTS: Median age was 33.0 years (IQR 25.0-43.0), median Ullanlinna Narcolepsy Scale 19 (IQR 14.0-26.0), 78% reported using narcolepsy pharmacotherapy. Excessive daytime sleepiness (97.2%) and lack of energy were most often present (95.0%) and most often caused a high burden (79.7% and 76.1% respectively). Cognitive symptoms (concentration 93.0%, memory 91.4%) and psychiatric symptoms (mood 76.8%, anxiety/panic 76.4%) were relatively often reported to be present and burdensome. Conversely, sleep paralysis and cataplexy were least often reported as highly bothersome. Females experienced a higher burden for anxiety/panic, memory, and lack of energy. CONCLUSIONS: This study supports the notion of an elaborate narcolepsy symptom spectrum. Each symptom's contribution to the experienced burden varied, but lesser-known symptoms did significantly add to this as well. This emphasizes the need to not only focus treatment on the classical core symptoms of narcolepsy.

On-the-road driving performance of patients with central disorders of hypersomnolence.

INTRODUCTION: Excessive Daytime Sleepiness is a core symptom of narcolepsy and idiopathic hypersomnia, which impairs driving performance. Adequate treatment improves daytime alertness, but it is unclear whether driving performance completely normalizes. This study compares driving performance of patients with narcolepsy and idiopathic hypersomnia receiving treatment to that of healthy controls. METHODS: Patients diagnosed with narcolepsy type 1 (NT1, n = 33), narcolepsy type 2 (NT2, n = 7), or idiopathic hypersomnia (IH, n = 6) performed a standardized one-hour on-the-road driving test, measuring standard deviation of lateral position (SDLP). RESULTS: Results showed that mean SDLP in patients did not differ significantly from controls, but the 95%CI of the mean difference (+1.02 cm) was wide (-0.72 to +2.76 cm). Analysis of subgroups, however, showed that mean SDLP in NT1 patients was significantly increased by 1.90 cm as compared to controls, indicating impairment. Moreover, four NT1 patients requested to stop the test prematurely due to self-reported somnolence, and two NT1 patients were stopped by the driving instructor for similar complaints. CONCLUSION: Driving performance of NT1 patients may still be impaired, despite receiving treatment. No conclusions can be drawn for NT2 and IH patients due to the low sample sizes of these subgroups. In clinical practice, determination of fitness to drive for these patients should be based on an individual assessment in which also coping strategies are taken into account.

Suppression of myocardial glucose metabolism in FDG PET/CT: impact of dose variation in heparin bolus pre-administration.

INTRODUCTION: Adequate suppression of physiologic myocardial glucose uptake is important to ensure the interpretability and diagnostic reliability of [18F]fluorodeoxyglucose (FDG) PET/CT studies performed in the context of cardiac inflammation and infection. This study describes our experience with 4 preparatory protocols used in our institution. METHODS: FDG PET/CT scans were performed according to 4 preparatory protocols (716 scans total), i.e. 6-h fast (group 1), low-carbohydrate diet plus 12-h fast (group 2), low-carbohydrate diet plus 12-h fast plus intravenous heparin pre-administration (15 IU/kg) (group 3), and low-carbohydrate diet plus 12-h fast plus intravenous heparin pre-administration (50 IU/kg) (group 4). Consecutive scans were retrospectively included from time frames during which the particular protocol was used. FDG uptake in normal myocardium was scored on a scale ranging from 0 (uptake less than that in the left ventricular blood pool) to 4 (diffuse uptake greater than that in the liver). Complete suppression was defined as uptake less than or equal to the blood pool (scores 0-1). RESULTS: Complete suppression was accomplished in 27% in group 1, 68% in group 2, 69% in group 3 and 81% in group 4. Complete suppression was significantly lower in group 1 compared with all other groups (P < 0.0001 for all comparisons) and significantly higher in group 4 compared with group 2 (P = 0.005) and group 3 (P = 0.007). Groups 2 and 3 did not differ significantly (P = 0.92). CONCLUSION: A total of 50 IU/kg single-dose heparin administration before FDG PET/CT in addition to a low-carbohydrate diet and prolonged fast significantly outperformed protocols with no or lower dose (15 IU/kg) heparin in completely suppressing myocardial glucose metabolism.

Daytime sleep state misperception in a tertiary sleep centre population.

STUDY OBJECTIVES: Sleep state misperception is common in various sleep disorders, especially in chronic insomnia with a prevalence ranging between 9-50%. Most prior studies used nocturnal polysomnography (PSG) for the identification of sleep state misperception during nighttime. Our objective was to assess sleep state misperception during daytime in people with sleep disorders with excessive daytime sleepiness (EDS). METHODS: In this prospective observational study, we assessed the occurrence of, and factors influencing sleep state misperception in consecutive patients undergoing a routine multiple sleep latency test (MSLT) in a tertiary sleep-wake centre included between 2014 and 2017. Mixed models were applied to assess the influence of patients' clinical data on sleep state perception. RESULTS: People with narcolepsy type 1 (NT1, n = 33) and type 2 (NT2, n = 14), idiopathic hypersomnia (IH, n = 56), obstructive sleep apnea (OSA, n = 31) and insufficient sleep syndrome (ISS, n = 31) were included. The prevalence of both classical and reverse sleep state misperception did not differ between the sleep disorders (mean 25%, range 8-37%) after correction for sleep stage, sleep onset latency and age. Longer sleep onset latency and reaching only non-rapid eye movement (REM) sleep stage 1 were significant predictors for classical sleep state misperception. CONCLUSIONS: Sleep state misperception is common in people with NT1 and NT2, IH, OSA, and ISS. Classical sleep state misperception is more frequent in patients with longer sleep onset latencies who only reach non-REM sleep stage 1 during a nap.

H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported. T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls. HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected. These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

Chronotypes and circadian timing in migraine.

Background It has been suggested that migraine attacks strike according to circadian patterns and that this might be related to individual chronotype. Here we evaluated and correlated individual chronotypes, stability of the circadian rhythm, and circadian attack timing in a large and well-characterised migraine population. Methods In 2875 migraine patients and 200 non-headache controls we assessed differences in: (i) distribution of chronotypes (Münich Chronotype Questionnaire); (ii) the circadian rhythm's amplitude and stability (Circadian Type Inventory); and (iii) circadian timing of migraine attacks. Data were analysed using multinomial and linear regression models adjusted for age, gender, sleep quality and depression. Results Migraineurs more often showed an early chronotype compared with controls (48.9% versus 38.6%; adjusted odds ratio [OR] = 2.42; 95% confidence interval [CI] = 1.58-3.69; p < 0.001); as well as a late chronotypes (37.7% versus 38.1%; adjusted OR = 1.69; 95% CI = 1.10-2.61; p = 0.016). Migraineurs, particularly those with high attack frequency, were more tired after changes in circadian rhythm (i.e. more languid; p < 0.001) and coped less well with being active at unusual hours (i.e. more rigid; p < 0.001) than controls. Of 2389 migraineurs, 961 (40.2%) reported early morning attack onset. Conclusion Migraine patients are less prone to be of a normal chronotype than controls. They are more languid and more rigid when changes in circadian rhythm occur. Most migraine attacks begin in the early morning. These data suggest that chronobiological mechanisms play a role in migraine pathophysiology.

Experimental validation of the AVIVET trap, a tool to quantitatively monitor the dynamics of Dermanyssus gallinae populations in laying hens.

Dermanyssus gallinae (D.gallinae) infestation causes economic losses due to impaired health and production of hens and costs of parasite control across the world. Moreover, infestations are associated with reduced welfare of hens and may cause itching in humans. To effectively implement control methods it is crucially important to have high quality information about the D.gallinae populations in poultry houses in space and time. At present no validated tool is available to quantitatively monitor the dynamics of all four stages of D.gallinae (i.e., eggs, larvae, nymphs, and adults) in poultry houses.This article describes the experimental validation of the AVIVET trap, a device to quantitatively monitor dynamics of D.gallinae infestations. We used the device to study D.gallinae in fully equipped cages with two white specific pathogen free Leghorn laying hens experimentally exposed to three different infestation levels of D.gallinae (low to high).The AVIVET trap was successfully able to detect D.gallinae at high (5,000 D.gallinae), medium (2,500 D.gallinae), and low (50 D.gallinae) level of D.gallinae infestation. The linear equation Y = 10∧10∧(0.47 + 1.21X) with Y = log10 (Total number of D.gallinae nymphs and adults) in the cage and X = log10 (Total number of D.gallinae nymphs and adults) in the AVIVET trap explained 93.8% of the variation.The weight of D.gallinae in the AVIVET trap also appears to be a reliable parameter for quantifying D.gallinae infestation in a poultry house. The weight of D.gallinae in the AVIVET trap correlates 99.6% (P < 0.000) to the counted number of all stages of D.gallinae in the trap (i.e., eggs, larvae, nymphs, and adults) indicating that the trap is highly specific.From this experiment it can be concluded that the AVIVET trap is promising as quantitative tool for monitoring D.gallinae dynamics in a poultry house.

[Kleine Levin Syndrome: more than just periodic hypersomnia]. / Kleine-Levin-syndroom: meer dan periodieke hypersomnie.

BACKGROUND: Kleine Levin Syndrome (KLS) is a rare disease with periodic hypersomnia as its main feature. Hyperphagia and hypersexuality are also described as classical symptoms, although quite recently it has become clear that the full triad is absent in the majority of patients. CASE DESCRIPTION: A 14-year-old boy developed KLS after a period of flu-like symptoms. Over the course of three years he suffered from seven one-week episodes of extreme hypersomnia (sleeping 18 hours a day), depersonalisation, apathy, anxiety, paranoia, confusion, hallucinations and uninhibited sexual behaviour. He ate little. Ancillary investigations did not reveal any abnormalities. In between these episodes he had no symptoms. CONCLUSION: From this case description and a summary of the symptoms of twelve other patients with KLS, it appears that neuropsychiatric symptoms are much more prominent than hyperphagia and hypersexuality. It is important that the typical KLS phenotype be reappraised, so that the condition can be recognised early and patients managed appropriately.

Daily shedding dynamics of E. coli O157 in an Australian grass-fed beef herd.

This study aimed to describe the diurnal shedding dynamics of Escherichia coli O157 in cattle managed on pasture. The purpose was to identify the value of a single measurement for predicting the shedding status on subsequent days. Over a 14-day period, 24 beef cows with known E. coli O157 shedding status were sampled twice daily or daily (21 sampling points) and E. coli O157 was enumerated from faeces. No association between shedding status of individual animals within a 7-h period was identified (odds ratio 1·5, P = 0·08). Short-interval sampling demonstrated substantial diurnal volatility in shedding of E. coli O157 that is not evident in studies based on long-interval (>7 days) sampling. The findings contribute to and support previous findings on the question why it has been difficult to achieve progress in understanding the epidemiology of E. coli O157 infection in cattle.

The Role of the Suprachiasmatic Nucleus in Cardiac Autonomic Control during Sleep.

BACKGROUND: The suprachiasmatic nucleus (SCN) may play an important role in central autonomic control, since its projections connect to (para)sympathetic relay stations in the brainstem and spinal cord. The cardiac autonomic modifications during nighttime may therefore not only result from direct effects of the sleep-related changes in the central autonomic network, but also from endogenous circadian factors as directed by the SCN. To explore the influence of the SCN on autonomic fluctuations during nighttime, we studied heart rate and its variability (HRV) in a clinical model of SCN damage. METHODS: Fifteen patients in follow-up after surgical treatment for nonfunctioning pituitary macroadenoma (NFMA) compressing the optic chiasm (8 females, 26-65 years old) and fifteen age-matched healthy controls (5 females, 30-63 years) underwent overnight ambulatory polysomnography. Eleven patients had hypopituitarism and received adequate replacement therapy. HRV was calculated for each 30-second epoch and corrected for sleep stage, arousals, and gender using mixed effect regression models. RESULTS: Compared to controls, patients spent more time awake after sleep onset and in NREM1-sleep, and less in REM-sleep. Heart rate, low (LF) and high frequency (HF) power components and the LF/HF ratio across sleep stages were not significantly different between groups. CONCLUSIONS: These findings suggest that the SCN does not play a dominant role in cardiac autonomic control during sleep.

Restless legs syndrome in migraine patients: prevalence and severity.

BACKGROUND AND PURPOSE: Our aim was to study not only the prevalence but more importantly the severity and the correlation between sleep quality and restless legs syndrome (RLS) in a large population of well-defined migraine patients as poor sleep presumably triggers migraine attacks. METHODS: In a large cross-sectional and observational study, data on migraine and RLS were collected from 2385 migraine patients (according to the International Classification of Headache Disorders ICHD-IIIb) and 332 non-headache controls. RLS severity (International RLS Study Group severity scale) and sleep quality (Pittsburgh Sleep Quality Index) were assessed. Risk factors for RLS and RLS severity were calculated using multivariable-adjusted regression models. RESULTS: Restless legs syndrome prevalence in migraine was higher than in controls (16.9% vs. 8.7%; multivariable-adjusted odds ratio 1.83; 95% confidence interval 1.18-2.86; P = 0.008) and more severe (adjusted severity score 14.5 ± 0.5 vs. 12.0 ± 1.1; P = 0.036). Poor sleepers were overrepresented amongst migraineurs (50.1% vs. 25.6%; P < 0.001). Poorer sleep quality was independently associated with RLS occurrence (odds ratio 1.08; P < 0.001) and RLS severity (P < 0.001) in migraine patients. CONCLUSION: Restless legs syndrome is not only twice as prevalent but also more severe in migraine patients, and associated with decreased sleep quality.

Neurology and psychiatry: waking up to opportunities of sleep. : State of the art and clinical/research priorities for the next decade.

In recent years, evidence has emerged for a bidirectional relationship between sleep and neurological and psychiatric disorders. First, sleep-wake disorders (SWDs) are very common and may be the first/main manifestation of underlying neurological and psychiatric disorders. Secondly, SWDs may represent an independent risk factor for neuropsychiatric morbidities. Thirdly, sleep-wake function (SWF) may influence the course and outcome of neurological and psychiatric disorders. This review summarizes the most important research and clinical findings in the fields of neuropsychiatric sleep and circadian research and medicine, and discusses the promise they bear for the next decade. The findings herein summarize discussions conducted in a workshop with 26 European experts in these fields, and formulate specific future priorities for clinical practice and translational research. More generally, the conclusion emerging from this workshop is the recognition of a tremendous opportunity offered by our knowledge of SWF and SWDs that has unfortunately not yet entered as an important key factor in clinical practice, particularly in Europe. Strengthening pre-graduate and postgraduate teaching, creating academic multidisciplinary sleep-wake centres and simplifying diagnostic approaches of SWDs coupled with targeted treatment strategies yield enormous clinical benefits for these diseases.

Synchronization of E. coli O157 shedding in a grass-fed beef herd: a longitudinal study.

This study aims to describe in detail the temporal dynamics of E. coli O157 shedding and risk factors for shedding in a grass-fed beef herd. During a 9-month period, 23 beef cows were sampled twice a week (58 sampling points) and E. coli O157 was enumerated from faecal samples. Isolates were screened by PCR for presence of rfbE, stx 1 and stx 2 . The prevalence per sampling day ranged from 0% to 57%. This study demonstrates that many members of the herd were concurrently shedding E. coli O157. Occurrence of rainfall (P < 0·01), feeding silage (P < 0·01) and lactating (P < 0·01) were found to be predictors of shedding. Moving cattle to a new paddock had a negative effect on shedding. This approach, based on short-interval sampling, confirms the known variability of shedding within a herd and highlights that high shedding events are rare.

Alterations in diurnal rhythmicity in patients treated for nonfunctioning pituitary macroadenoma: a controlled study and literature review.

OBJECTIVE: Patients treated for nonfunctioning pituitary macroadenomas (NFMAs) have fatigue and alterations in sleep characteristics and sleep-wake rhythmicity frequently. As NFMAs often compress the optic chiasm, these complaints might be related to dysfunction of the adjacent suprachiasmatic nucleus (SCN). We aimed to explore whether indirect indices of SCN functioning are altered in the long term after surgery for NFMAs. METHODS: We studied 17 NFMA patients in long-term remission after transsphenoidal surgery, receiving adequate and stable hormone replacement for hypopituitarism, and 17 control subjects matched for age, gender, and BMI. Indirect indices of SCN function were assessed from 24-h ambulatory recordings of skin and core body temperatures, blood pressure, and salivary melatonin levels. Altered melatonin secretion was defined as an absence of evening rise, considerable irregularity, or daytime values >3 pg/ml. We additionally studied eight patients treated for craniopharyngioma. RESULTS: Distal-proximal skin temperature gradient did not differ between NFMAs and control subjects, but proximal skin temperature was decreased during daytime (P=0.006). Core body temperature and non-dipping of blood pressure did not differ, whereas melatonin secretion was often altered in NFMAs (OR 5.3, 95% CI 0.9-30.6). One or more abnormal parameters (≥2.0 SDS of control subjects) were observed during nighttime in 12 NFMA patients and during daytime in seven NFMA patients. Similar patterns were observed in craniopharyngioma patients. CONCLUSION: Heterogeneous patterns of altered diurnal rhythmicity in skin temperature and melatonin secretion parameters were observed in the majority of patients treated for NFMAs. On a group level, both NFMA and craniopharyngioma patients showed a lower daytime proximal skin temperature than control subjects, but other group averages were not significantly different. The observations suggest altered function of central (or peripheral) clock machinery, possibly by disturbed entrainment or damage of the hypothalamic SCN by the suprasellar macroadenoma or its treatment.

Carotid body tumors are not associated with an increased risk for sleep-disordered breathing.

PURPOSE: Tumors in the carotid bodies may interfere with their function as peripheral chemoreceptors. An altered control of ventilation may predispose to sleep-disordered breathing. This study aimed to assess whether patients with unilateral or bilateral carotid body tumors (uCBT or bCBT, respectively) or bilateral CBT resection (bCBR) display sleep-disordered breathing and to evaluate the global contribution of the peripheral chemoreceptor to the hypercapnic ventilatory response. METHODS: Eight uCBT, eight bCBT, and nine bCBR patients and matched controls underwent polysomnography. The peripheral chemoreflex drive was assessed using euoxic and hyperoxic CO2 rebreathing tests. Daytime sleepiness and fatigue were assessed with the Epworth Sleepiness Scale and the Multidimensional Fatigue Index. RESULTS: All patient groups reported significant fatigue-related complaints, but no differences in excessive daytime sleepiness (EDS) were found. The apnea/hypopnea index (AHI) did not differ significantly between patient groups and controls. Only in bCBT patients, a trend towards a higher AHI was observed, but this did not reach significance (p=0.06). No differences in the peripheral chemoreflex drive were found between patients and controls. CONCLUSIONS: Patients with (resection of) CBTs have more complaints of fatigue but are not at risk for EDS. The presence or resection of CBTs is neither associated with an altered peripheral chemoreflex drive nor with sleep-disordered breathing.

Prevalence of livestock-associated MRSA on Dutch broiler farms and in people living and/or working on these farms.

This study aimed to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) on 50 Dutch broiler farms. Of 145 persons living and/or working on these farms, eight tested positive for MRSA (5.5%). Investigation of 250 pooled throat samples of broilers and 755 dust samples resulted in four farms where MRSA-positive samples were present (8.0%). All isolates belonged to the CC398 complex. Living and/or working on a MRSA-positive farm was a risk for MRSA carriage; 66.7% of people on positive farms were MRSA positive vs. 1.5% on negative farms (P<0.0001). Due to the low number of positive farms and persons, and high similarity in farm management, it was impossible to draw statistically valid conclusions on other risk factors. For broiler farming, both farm and human MRSA prevalence seem much lower than for pig or veal farming. However, MRSA carriage in people living and/or working on broiler farms is higher compared to the general human population in The Netherlands (5.5% vs. <0.1%). As broiler husbandry systems are not unique to The Netherlands, this might imply that people in contact with live broilers are at risk for MRSA carriage worldwide.

Disturbed subjective sleep characteristics in adult patients with long-standing type 1 diabetes mellitus.

AIMS/HYPOTHESIS: Decreased sleep duration and/or impaired sleep quality negatively influence glucoregulation. The aim of this study was to assess subjective sleep characteristics in patients with type 1 diabetes, to relate sleep characteristics to long-term glycaemic control and to assess possible risk factors for impaired sleep. METHODS: We studied 99 adult patients with type 1 diabetes (55 men, 44 women, duration of diabetes 26.9 ± 1.2 years) and 99 age-, sex- and BMI-matched non-diabetic controls. Subjective sleep characteristics were assessed by validated questionnaires, i.e. Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale and the Berlin Questionnaire. Glucoregulation was assessed by HbA(1c) values. Clinical variables were obtained from medical charts. Depression was assessed by the Hospital Anxiety and Depression Scale (HADS). Peripheral polyneuropathy was assessed by neurological examination and quantitative sensory testing. RESULTS: Of the patients with type 1 diabetes, 35% had subjective poor sleep quality compared with 20% of the control participants (p = 0.021). A higher proportion of the patients with type 1 diabetes were at increased risk for obstructive sleep apnoea (OSA) (17.2% vs 5.1%, p = 0.012). There was no significant association between individual sleep characteristics and HbA(1c) values. On logistic regression analysis, the HADS depression score, presence of peripheral polyneuropathy, habitual snoring and other sleep disturbances (e.g. hypoglycaemia) were independently associated with poor sleep quality. CONCLUSIONS/INTERPRETATION: Adult patients with long-standing type 1 diabetes mellitus have disturbed subjective sleep quality and a higher risk for OSA compared with control participants. Subjective sleep disturbances are part of the complex syndrome of long-standing type 1 diabetes.

Patients previously treated for nonfunctioning pituitary macroadenomas have disturbed sleep characteristics, circadian movement rhythm, and subjective sleep quality.

CONTEXT AND OBJECTIVE: Fatigue and excessive sleepiness have been reported after treatment of nonfunctioning pituitary macroadenomas (NFMA). Because these complaints may be caused by disturbed nocturnal sleep, we evaluated objective sleep characteristics in patients treated for NFMA. DESIGN: We conducted a controlled cross-sectional study. SUBJECTS AND METHODS: We studied 17 patients (8 women; mean age, 54 yr) in remission of NFMA during long-term follow-up (8 yr; range, 1-18 yr) after surgery (n = 17) and additional radiotherapy (n = 5) without comorbidity except for hypopituitarism and 17 controls matched for age, gender, and body mass index. Sleep was assessed by nocturnal polysomnography, sleep and diurnal movement patterns by actigraphy, and quality of life and subjective sleep characteristics by questionnaires. RESULTS: Compared to controls, patients had reduced sleep efficiency, less rapid eye movement sleep, more N1 sleep, and more awakenings in the absence of excessive apnea or periodic limb movements. Actigraphy revealed a longer sleep duration and profound disturbances in diurnal movement patterns, with more awakenings at night and less activity during the day. Patients scored higher on fatigue and reported impaired quality of life. CONCLUSION: Patients previously treated for NFMA suffer from decreased subjective sleep quality, disturbed distribution of sleep stages, and disturbed circadian movement rhythm. These observations indicate that altered sleep characteristics may be a factor contributing to impaired quality of life and increased fatigue in patients treated for NFMA.

Design and in vivo evaluation of a molecularly defined acellular skin construct: reduction of early contraction and increase in early blood vessel formation.

Skin substitutes are of great benefit in the treatment of patients with full thickness wounds, but there is a need for improvement with respect to wound closure with minimal contraction, early vascularisation, and elastin formation. In this study we designed and developed an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes. The epidermal layer was prepared using type I collagen, heparin and fibroblast growth factor 7 (FGF7), while the porous dermal layer was prepared using type I collagen, solubilised elastin, dermatan sulfate, heparin, fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). The construct was biochemically and morphologically characterised and evaluated in vivo using a rat full thickness wound model. The results were compared with the commercial skin substitute IntegraDRT and untreated wounds. The double-layered construct was prepared according to the design specifications. The epidermal layer was about 40 µm thick, containing 9% heparin and 0.2 µg FGF7 mg per layer, localised at the periphery. The dermal layer was 2.5 mm thick, had rounded pores and contained 10% dermatan sulfate+heparin, and 0.7 µg FGF2+VEGF mg per layer. The double-layered skin construct was implanted in a skin defect and on day 7, 14, 28 and 112 the (remaining) wound area was photographed, excised and (immuno) histologically evaluated. The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison with IntegraDRT and/or the untreated wound. On day 14 the double-layered skin construct also had the fewest myofibroblasts present. On day 112 the double-layered skin construct contained more elastic fibres than IntegraDRT and the untreated wound. Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly any were found in IntegraDRT. The results provide new opportunities for the application of acellular skin constructs in the treatment of surgical wounds.

A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials.

Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.