Double-Unit Superomedio-Central (DUS) Pedicle Inverted-T Reduction Mammaplasty in Gigantomastia: A 7-year Single-Center Retrospective Study.

INTRODUCTION: Reduction mammaplasty in patients with gigantomastia is challenging. The Double-Unit technique with a Superomedio-Central pedicle and inverted-T incision is the standard technique for reduction mammaplasty in our clinic. The aim of this study was to review our approach in cases with gigantomastia in comparison with the current literature. PATIENTS AND METHODS: From 01/2011 to 12/2017, we performed 831 reduction mammaplasties in 630 patients. The Double-Unit  Superomedio-Central  (DUS) pedicle and inverted-T incision was implemented as a standard procedure for gigantomastia. Patient demographics and the outcome parameters complication rate, patient satisfaction with the aesthetic result, nipple sensibility, and surgical revision rate were obtained and retrospectively analyzed. RESULTS: In 37 patients, 55 reduction mammaplasties were performed with more than 1000 g per breast. Mean resection weight was 1311 g on right side and 1289 g on left side. Mean age was 52.5 years, mean body mass index was 32.8 kg/m2, mean sternal-notch-to-nipple distance was 38.3 cm. A free NAC graft was necessary in four breasts. Overall complication rate was 14.5%; secondary surgical revision rate was 12.7%. 91% of the patients were "very satisfied" and "satisfied" with the aesthetic result. Nipple sensibility was rated "high" and "medium" in 83%. CONCLUSION: The Double-Unit technique with a Superomedio-Central pedicle and inverted-T incision is very effective to achieve volume reduction and aesthetically pleasing reproducible results with a low complication rate in cases with gigantomastia. LEVEL OF EVIDENCE: Level of Evidence This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

[Significance of Thoracic Surgery for Treatment of Pleural Empyema in Childhood and Adolescence]. / Stellenwert der Thoraxchirurgie im Gesamtkonzept der Behandlung von Pleuraempyemen bei Kindern.

BACKGROUND: There still is controversy about surgical treatment of pleural empyema in children. PATIENTS AND METHODS: Retrospective analysis of treatment strategy, focussing on indication for surgery and outcome of children treated in 2 centres for pleural complications due to primary pneumonia from January 1(st) 2008 to December 31(st) 2012. RESULTS were compared to studies published within the last 10 years. RESULTS: 1 451 children with pneumonia were treated during the 5 year period. 187 (average age 6.1 years, sex: 86/101 f/m) developed a pleural effusion. THERAPY: pleural punction in 22 children, chest tube in 78 and operation in 37 children. In 9 cases microorganisms were identified. 34 children were operated for empyema stage II, only 3 for stage III. 3 children were operated due to septicaemia not responding to antibiotics. Time from admission to operation (including referring hospital):14.5 days. Time from operation to discharge: 12,5 days. All children but one were operated by thoracoscopy. COMPLICATIONS: 1 bronchopleural fistula, 1 delayed healing of the wound. All children survived and fully recovered mean (observation period 28 months postoperatively). SUMMARY: In experienced hands thoracic surgery yields excellent results for children suffering from pleural empyema stage II and III. Recent randomised prospective trials comparing fibrinolysis with VATS do not convince regarding the treatment protocols of their surgical arms. Fibrinolysis is nevertheless a valuable treatment in early stage II empyema, especially if thoracic surgical experience is not available. However, the further advanced the empyema presents, the sooner surgical experience should be gathered.

Impact of headache disorders in Italy and the public-health and policy implications: a population-based study within the Eurolight Project.

BACKGROUND: Migraine, tension-type headache (TTH) and medication-overuse headache (MOH) are disabling lifelong illnesses. The Eurolight project, a partnership activity within the Global Campaign against Headache, assessed the impact of headache disorders in ten countries in Europe using a structured questionnaire coupled with various sampling methods. Here we present the findings from the Italian population. METHODS: Questionnaires were distributed to a stratified sample (N = 3500) of the adult (18-65 years) inhabitants of Pavia province (1.05 % of the general population), randomly selected in cooperation with the local health service. Questions included demographic and diagnostic enquries, and assessment of various aspects of impact and health-care utilisation. RESULTS: Altogether 500 questionnaires were returned of which 487 were adequately completed for analysis (58 % female, 42 % male). Among these, gender-adjusted lifetime prevalence of headache was 82.5 %, higher in females than in males (91.2 % vs 72.4 %; p < 0.0001). Gender-adjusted 1-year prevalence was 74.2 % (females 87.7 %, males 61.1 %; p < 0.0001). The most prevalent headache type was migraine (gender-adjusted 1-year prevalence 42.9 %; females 54.6 %, males 32.5 %; p < 0.0001), followed by TTH (28.6 %; no gender-related difference); all causes of headache on ≥15 days/month were reported by 7.0 % of participants (females 10.6 %, males 2.0 %; p = 0.0002), of whom 2.1 %,, all female (p = 0.0064) concomitantly overused acute medications (therefore probable MOH). Only 16.6 % of responders reporting headache had received a diagnosis from a doctor, and very few (2.4 %) were taking preventative medications. Headache had negative impacts on different aspects of life: education, career and earnings, family and social life. Each person with headache had lost, on average, 2.3 days from paid work and 2.4 days from household work, and missed social occasions on 1.2 days, in the preceding 3 months. An increasing gradient for impact was observed from episodic to chronic forms of headache. CONCLUSIONS: Our study reveals that in Italy, as in other countries, migraine, TTH and MOH are highly prevalent and are associated with significant personal impact. These findings have important implications for health policy in Italy.

Secondary solution for breast reconstruction following total DIEP flap loss: A single-center experience after 3270 DIEP flaps.

BACKGROUND: Total deep inferior epigastric perforator (DIEP) flap failure is a significant concern in autologous breast reconstruction. Literature on secondary reconstruction options following total flap failure is limited. This study outlines the outcomes of patients who underwent reconstruction post-DIEP flap failure at our institution. METHODS: We conducted a retrospective analysis of patients receiving autologous breast reconstruction between 2004 and 2021. We aimed to identify causes of total DIEP flap failure, outcomes of revision surgery, and outcomes of secondary breast reconstruction procedures. RESULTS: From 2004 to 2021, 3456 free flaps for breast reconstruction were performed, with 3270 being DIEP flaps for 2756 patients. DIEP flap failure was observed in 40 cases (1.22%). Bilateral reconstructions had a higher failure rate (2.31%) than unilateral (0.72%). The primary cause was intraoperative complications during flap harvest (18 cases), followed by insufficient arterial perfusion (seven cases). Other causes included postoperative hematoma (seven cases), venous congestion (six cases), and late-onset fat necrosis (two cases). Post-failure, five patients received a second free flap with three cases of repeated flap failure. Twenty patients received implant-based reconstruction with two cases of reconstruction failure, while seven patients received a pedicled latissimus dorsi (LD) flap reconstructions with no cases of reconstruction failure. Eight patients declined further reconstruction. CONCLUSION: A second free flap post-DIEP failure was associated with a high risk of reconstruction failure, suggesting the need for careful patient selection. Implant-based and pedicled LD flap seem to be reliable secondary reconstruction options.

The cost of headache disorders in Europe: the Eurolight project.

BACKGROUND AND PURPOSE: Headache disorders are very common, but their monetary costs in Europe are unknown. We performed the first comprehensive estimation of how economic resources are lost to headache in Europe. METHODS: From November 2008 to August 2009, a cross-sectional survey was conducted in eight countries representing 55% of the adult EU population. Participation rates varied between 11% and 59%. In total, 8412 questionnaires contributed to this analysis. Using bottom-up methodology, we estimated direct (medications, outpatient health care, hospitalization and investigations) and indirect (work absenteeism and reduced productivity at work) annual per-person costs. Prevalence data, simultaneously collected and, for migraine, also derived from a systematic review, were used to impute national costs. RESULTS: Mean per-person annual costs were €1222 for migraine (95% CI 1055-1389; indirect costs 93%), €303 for tension-type headache (TTH, 95% CI 230-376; indirect costs 92%), €3561 for medication-overuse headache (MOH, 95% CI 2487-4635; indirect costs 92%), and €253 for other headaches (95% CI 99-407; indirect costs 82%). In the EU, the total annual cost of headache amongst adults aged 18-65 years was calculated, according to our prevalence estimates, at €173 billion, apportioned to migraine (€111 billion; 64%), TTH (€21 billion; 12%), MOH (€37 billion; 21%) and other headaches (€3 billion; 2%). Using the 15% systematic review prevalence of migraine, calculated costs were somewhat lower (migraine €50 billion, all headache €112 billion annually). CONCLUSIONS: Headache disorders are prominent health-related drivers of immense economic losses for the EU. This has immediate implications for healthcare policy. Health care for headache can be both improved and cost saving.

The Eurolight project: the impact of primary headache disorders in Europe. Description of methods.

The Eurolight project is the first at European Union level to assess the impact of headache disorders, and also the first of its scale performed by collaboration between professional and lay organizations and individuals. Here are reported the methods developed for it. The project took the form of surveys, by structured questionnaire, conducted in ten countries of Europe which together represented 60% of the adult population of the European Union. In Lithuania, the survey was population-based. Elsewhere, truly population-based studies were impractical for reasons of cost, and various compromises were developed. Closest to being population-based were the surveys in Germany, Luxembourg, the Netherlands, Italy and Spain. In Austria, France and UK, samples were taken from health-care settings. In addition in the Netherlands, Spain and Ireland, samples were drawn from members of national headache patient organizations and their relatives. Independent double data-entry was performed prior to analysis. Returned questionnaires from 9,269 respondents showed a moderate female bias (58%); of respondents from patients' organizations (n = 992), 61% were female. Mean age of all respondents was 44 years; samples from patients' organizations were slightly older (mean 47 years). The different sampling methods worked with differing degrees of effectiveness, as evidenced by the responder-rates, which varied from 10.8 to 90.7%. In the more population-based surveys, responder-rates varied from 11.3 to 58.8%. We conclude that the methodology, although with differences born of necessity in the ten countries, was sound overall, and will provide robust data on the public ill-health that results from headache in Europe.

Epidermal growth factor (EGF) transfection of human bone marrow stromal cells in bone tissue engineering.

A novel therapeutic approach for the treatment of bone defects is gene therapy assisted bone tissue engineering using bone marrow stromal cells (hBMSC). The aim of this study was to investigate the influence of human epidermal growth factor (hEGF) on proliferation and alkaline phosphatase (AP) activity of primary hBMSC in vitro. hBMSC cultures were achieved by explantation culture of bone chips. Following exposure to 0-10 ng recombinant hEGF (rhEGF)/ml cell numbers were determined by automated cell counting and cell bound AP activity was measured spectrophotometrically. hBMSC were transfected with hEGF plasmids and the proliferative effect was studied by cocultivation of transfected and untreated cells using porous cell culture inserts. The persistence of hEGF expression even after cell transfer was studied by the generation of possibly osteogenic constructs introducing transfected hBMSC in fibrin glue and bovine cancellous bone. The maximum increase in proliferation (156 +/- 7%) and AP activity (220 +/- 34%) was detected after exposition to 10 ng rhEGF/ml. In the separation chamber assay transfected cells produced hEGF concentrations up to 3.6 ng/ml, which induced a mean proliferation increase of 93% which could be significantly inhibited by a neutralizing hEGF antibody. Further, EGFsecretion of transfected hBMSC in 3D-culture was verified. Recombinant and transgenic hEGF stimulate proliferation of primary hBMSC in vitro. Lipotransfection of hBMSC with hEGF plasmids allows the transient and site directed delivery of biologically active transgenic hEGF. The introduction of mitogenic, angiogenic and chemoattractive factors in gene therapy assisted bone tissue engineering is discussed by the example of EGF.

Effects of maleimide-polyethylene glycol-modified human hemoglobin (MP4) on tissue necrosis in SKH1-hr hairless mice.

OBJECTIVE: Tissue hypoxia after blood loss, replantation and flap reperfusion remains a challenging task in surgery. Normovolemic hemodilution improves hemorheologic properties without increasing oxygen carrying capacity. Red blood cell transfusion is the current standard of treatment with its attendant risks. The aim of this study was to investigate the potential of the chemically modified hemoglobin, MP4, to reduce skin flap necrosis and its effect on selected blood markers and kidneys. MATERIALS AND METHODS: Tissue ischemia was induced in the ear of hairless mice (n=26). Hemodilution was performed by replacing one third of blood volume with the similar amount of MP4, dextran, or blood. The extent of non-perfused tissue was assessed by intravital fluorescent microscopy. RESULTS: Of all groups, MP4 showed the smallest area of no perfusion (in percentage of the ear +/- SEM: 16.3% +/- 2.4), the control group the largest (22.4% +/- 3.5). Leukocytes showed a significant increase in the MP4 and dextran group (from 8.7 to 13.6 respectively 15.4*109/l). On histology no changes of the kidneys could be observed. CONCLUSION: MP4 causes an increase of leukocytes, improves the oxygen supply of the tissue and shows no evidence of renal impairment.

Expanding the delivery of clinical preventive services through community collaboration: the SPARC model.

BACKGROUND: Population-based rates of adult vaccinations and cancer screenings are low, with less than 40% of older adults up to date with routinely recommended prevention services. Delivery rates are lower still in poor and minority communities. CONTEXT: During the past 10 years, Sickness Prevention Achieved through Regional Collaboration (SPARC), a New England-based nonprofit agency, has developed a promising model for increasing community-wide delivery of prevention services. However, the SPARC model has not been tested in communities elsewhere. In 2006, the Centers for Disease Control and Prevention facilitated a partnership between SPARC and the Aging Services division of the Atlanta Regional Commission to evaluate the program's replicability. METHODS: SPARC coalitions involving local public health agencies, hospitals, social service organizations, and advocacy groups were established in two counties of the region, with the Atlanta Regional Commission providing regional coordination. Using the SPARC model, the coalitions planned, marketed, and implemented community-based activities to deliver adult screenings and vaccinations. CONSEQUENCES: During a 3-week pilot phase, SPARC clinics were held in central Atlanta at three senior housing facilities, a local fire station, and a charter middle school, delivering 353 prevention services to 314 residents. In Fayette County, 634 people received influenza vaccinations on Election Day at SPARC clinics located near 10 polling places. INTERPRETATION: The SPARC model provides a practical framework for improving the community-wide delivery of disease prevention services. The model can galvanize local health services providers to develop successful locally tailored interventions, and the approach is applicable in communities outside of SPARC's home region.

Some resonances between Eastern thought and Integral Biomathics in the framework of the WLIMES formalism for modeling living systems.

Forty-two years ago, Capra published "The Tao of Physics" (Capra, 1975). In this book (page 17) he writes: "The exploration of the atomic and subatomic world in the twentieth century has …. necessitated a radical revision of many of our basic concepts" and that, unlike 'classical' physics, the sub-atomic and quantum "modern physics" shows resonances with Eastern thoughts and "leads us to a view of the world which is very similar to the views held by mystics of all ages and traditions." This article stresses an analogous situation in biology with respect to a new theoretical approach for studying living systems, Integral Biomathics (IB), which also exhibits some resonances with Eastern thought. Stepping on earlier research in cybernetics1 and theoretical biology,2 IB has been developed since 2011 by over 100 scientists from a number of disciplines who have been exploring a substantial set of theoretical frameworks. From that effort, the need for a robust core model utilizing advanced mathematics and computation adequate for understanding the behavior of organisms as dynamic wholes was identified. At this end, the authors of this article have proposed WLIMES (Ehresmann and Simeonov, 2012), a formal theory for modeling living systems integrating both the Memory Evolutive Systems (Ehresmann and Vanbremeersch, 2007) and the Wandering Logic Intelligence (Simeonov, 2002b). Its principles will be recalled here with respect to their resonances to Eastern thought.

Cost of disorders of the brain in Luxembourg.

Brain disorders (psychiatric, neurological and neurosurgical diseases) are leading causes of disease and disability. According to WHO data they cause 35% of the burden of all diseases in Europe. The present study aims to estimate the cost of defined brain disorders and adds all selected disorders to arrive at the total cost for Luxembourg. A model combining published economic and epidemiological data retrieved from the OECD (Organization for Economic Co-operation and Development) and Eurostat databases on brain disorders in Europe (EU member countries, Iceland, Norway and Switzerland) was used. We transformed and converted data for a defined period into the same currency (Euro 2004) and adjusted country specific economic data for purchasing power and relative size of economy and imputed data where no local data were available. There are an estimated 123000 people in Luxembourg currently living with a brain disorder. The total annual cost of brain disorders is estimated at Euro 500 million in 2004 or an average of Euro 1100 per inhabitant. Mental disorders constitute 62% of the total cost (excluding dementia), followed by neurological diseases (excluding dementia) 22%, neurosurgical diseases excluding herniated discs 2.2%. Direct medical expenditures (outpatient care, hospitalization, drugs) have a share of 32%, direct non-medical costs (social services, informal care, adaptation, transportation) 18% and indirect costs (sick leave, early retirement and premature death) 51%.

Myosonographic study of abdominal wall dynamics to assess donor site morbidity after microsurgical breast reconstruction with a DIEP or an ms-2 TRAM flap.

BACKGROUND: Currently, autologous breast reconstruction with a free tissue transfer from the lower abdomen is considered to be a safe method that provides a stable long-term solution. The DIEP-flap and the ms-2-TRAM-flap reconstructions have helped reduce donor site morbidity. In order to assess the potential differences between these techniques, we carried out myosonographic evaluations that assessed the muscle dynamics pre- and post-operatively. In addition to investigating the properties of the rectus abdominis muscle post-operatively, this prospective study also allowed us to analyse the muscle preoperatively and to investigate the prospects for harvesting a DIEP-flap as opposed to a TRAM-flap. MATERIALS AND METHODS: Sixty patients underwent breast reconstruction with 71 (11 bilateral) free abdominal wall flaps (DIEP-: n = 48; ms-2-TRAM-flap: n = 23). Myosonographic examinations were performed preoperatively and at 3 and 6 months post-operatively. The thickness of the muscle at relaxation and maximum contraction and the difference between the muscle thickness measured at the two states were measured. A general-linear-model (GLM) was used for statistical analysis. The main variable was the surgical method, and the co-variables included BMI and patient age. The decision on whether to harvest a DIEP- or ms-2-TRAM-flap was made intra-operatively and based on the dominant perforator. RESULTS: It shows that the patients who underwent breast reconstruction with a DIEP-flap had significantly better muscle function (p < 0.05) in the follow-up. In addition, the analysis revealed that better muscle function before surgery made it more likely that a patient would undergo a DIEP-flap-reconstruction successfully. Patient age also had a highly significant effect on muscle recovery (p < 0.0005). CONCLUSIONS: This prospective study used a dynamic ultrasound evaluation of the abdominal wall and showed that the DIEP-flap significantly reduces donor site morbidity compared to the ms-2-TRAM-flap. The study also showed that good preoperative muscle function might increase the probability of surgeons performing a DIEP-flap reconstruction.

Conciliating neuroscience and phenomenology via category theory.

The paper discusses how neural and mental processes correlate for developing cognitive abilities like memory or spatial representation and allowing the emergence of higher cognitive processes up to embodied cognition, consciousness and creativity. It is done via the presentation of MENS (for Memory Evolutive Neural System), a mathematical methodology, based on category theory, which encompasses the neural and mental systems and analyzes their dynamics in the process of 'becoming'. Using the categorical notion of a colimit, it describes the generation of mental objects through the iterative binding of distributed synchronous assemblies of neurons, and presents a new rationale of spatial representation in the hippocampus (Gómez-Ramirez and Sanz, 2011). An important result is that the degeneracy of the neural code (Edelman, 1989) is the property allowing for the formation of mental objects and cognitive processes of increasing complexity order, with multiple neuronal realizabilities; it is essential "to explain certain empirical phenomena like productivity and systematicity of thought and thinking (Aydede 2010)". Rather than restricting the discourse to linguistics or philosophy of mind, the formal methods used in MENS lead to precise notions of Compositionality, Productivity and Systematicity, which overcome the dichotomic debate of classicism vs. connectionism and their multiple facets. It also allows developing the naturalized phenomenology approach asked for by Varela (1996) which "seeks articulations by mutual constraints between phenomena present in experience and the correlative field of phenomena established by the cognitive sciences", while avoiding their pitfalls.

[The DIEP Flap as Method of Choice in Breast Reconstruction - Results and Protocol for Succesful Reconstruction]. / Der DIEP Lappen als Standard in der autologen Brustrekonstruktion - Ergebnisse und Algorithmus zur erfolgreichen Rekonstruktion.

BACKGROUND: Autologous breast reconstruction is becoming increasingly important, especially in light of the increased rates of prophylactic mastectomies with BRCA mutations. Regarding the indications and complications between free TRAM and free DIEP flaps for autologous breast reconstruction the current data is not clear. MATERIALS AND METHODS: All patients who received an autologous breast reconstruction between January 2010 and January 2014 using free DIEP or free MS-2-TRAM flaps were included in the study. The choice of flap between DIEP and MS-2-TRAM was performed by a standardised algorithm with preoperative CT angiography, intraoperative evaluation of the flap perfusion and Doppler detection. The analysis took into account partial flap necrosis <20% and >20%), complete flap loss, flap necrosis and surgical revision of the anastomosis. RESULTS: The study considered 362 women who received a total of 419 free flaps for breast reconstruction. 66 patients received a bilateral reconstruction (52 double DIEPS, 9 DIEP and MS2 TRAM and 5 double MS2 TRAMs). The total flap loss was 0.24%. Partial necrosis <20% occurred in 0.72% (DIEP 0.82%, MS2 TRAM 0%), partial necrosis >20% in 0.72% (DIEP 0.69% MS2 TRAM 0.98%). One DIEP flap was lost (0.24%). After implementing our protocol the rate of MS-2-Tram flaps could be reduced to 10-15% of all autologous breast reconstruction procedures as compared to the years before. CONCLUSION: Through the application of the presented algorithm for breast reconstruction with free DIEP flap, such surgery is a safe treatment option if it is done at high frequency in a microsurgical centre. Flap loss and complications are not increased compared to MS2 TRAM flaps. The decision for the selection of the appropriate flap for breast reconstruction should be based on the presented algorithm in order to achieve secure and reproducible results.

Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice.

This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2-cm wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, and immunohistochemical microscopy. "Take" was determined separately by a direct immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, there was little difference between the two graft types, with the CEG having occasional blister formation. By light microscopy, the CEG had a dissociation of dermis from epidermis until day 42, which was never apparent with the CG. By day 42, the CG had increased dermoepidermal interdigitations similar to rete ridges, with a mature epithelium. Neither of these findings were seen with the CEG. Immunofluorescent and immunohistochemical microscopy for type IV collagen and laminin, as well as electron microscopy, showed similar retardation of basement membrane formation with the CEG. Using this model, the composite graft had significant advantages over the epidermal sheet graft in the closure of full-thickness wounds.

Cultured epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full-thickness wounds.

Research efforts to modify cultured autologous skin transplants for large full-thickness burn wounds and in chronic ulcers have shifted from multilayered differentiated grafts ("sheet" grafts) toward smaller units of basal undifferentiated single cell suspensions in a transport medium and subconfluently covered static carriers. It has been shown that wounds transplanted with single cell suspensions reconstitute the epidermis. However, this technique requires the detachment of the keratinocytes from the culture flasks by enzymatic digestion-digestion that might alter the anchoring proteins of the cells. A new approach might be to circumvent the enzymatic digestion to harvest the keratinocytes. This study reports a technique to culture epidermal cells on spherical microcarriers as a suspension culture and transport vehicle. The spherical microcarrier consists of a 100-microm-diameter collagen-coated dextran carrier (Cytodex 3 Pharmacia) and has been used previously for enzyme production commercially. With this new approach, we seeded the human keratinocytes in a spinner-like system onto microspheres and transplanted these micrografts onto full-thickness wounds on the back of nude mice. After 14 days, we showed a reconstituted epithelium that was multilayered and keratinized compared to control wounds. We believe that this is the first step of a new approach to increase the cell yield for seeding without altering the anchoring proteins by enzymatic steps, leading to a superior transplantation method for keratinocytes.

Plasmid gene delivery to human keratinocytes through a fibrin-mediated transfection system.

We have developed a matrix-mediated transfection system to deliver plasmids to human keratinocytes. The matrix is a soluble, self-hardening fibrin matrix (Tissucol), Baxter) that has been used clinically. Recently it has been shown that full thickness burn wounds can be successfully treated with a keratinocyte fibrin glue suspension. Further, it has been demonstrated that hEGF transfected cells accelerate wound healing. In this study, we inoculated the matrix with the hEGF expression plasmid and resuspended the matrix with either cultured or noncultured human keratinocytes. We obtained successful transfection rates of these cells (up to a 100-fold increase compared to controls containing no EGF expression plasmid) in vitro. After transplantation to full thickness wounds on athymic mice we were able to show a 180-fold increase in EGF concentration compared to controls, which persisted over the entire 7-day monitored period, decreasing from 180 to 20 pg/mL at day seven. This unique approach indicates the possible utility to combine a matrix for cell transplantation with a transfection system to release therapeutic proteins in vitro and in vivo.

Human recombinant EGF protein delivered by a biodegradable cell transplantation system.

We have previously shown a new approach to expand cultured human keratinocytes and reconstitute the epidermis in full-thickness wounds using a new microsperical transport system. This was a new approach to increase the cell yield for seeding without altering the anchoring proteins by enzymatic steps. That time we used Cytodex 3 which failed to be degraded and induced an inflammatory reaction in a t-cell-deficient organism. Therefore, we have investigated another microcarrier consisting of PLGA, which is a well-known carrier material for cell culture and transplantation. After coating the PLGA carrier with gelatine the seeding time of viable cells reached 4 h and the cell gain after 7 days of spinner culture was 16-fold. At 14 days after transplantation, we could detect a new stratified epithelium in our full-thickness wound healing model. Because cytokines play a major role in wound healing, we loaded this carrier material with different concentrations of rhEGF, showing a dose dependent release of the protein in vitro and in vivo. This result might lead to a different approach in the treatment of wounds.

Single-cell suspensions of cultured human keratinocytes in fibrin-glue reconstitute the epidermis.

To overcome common disadvantages of standard cultured epidermal sheet grafts (CEG) we have developed a new technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (Keratinocyte-fibrin-glue suspension-KFGS). In an athymic mouse model with reproducible standardized full thickness wounds this new technique was compared directly to CEG. Reepithelialization was similar in both groups, but reconstitution of the dermo-epidermal junction zone, as shown by electron microscopy and immunohistochemistry was significantly enhanced by the fibrin-glue suspension technique. The new KFGS technique is earlier available than sheet grafts, is able to transfer actively proliferative single keratinocytes, and simplifies the application.