G. Björn Stark

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19‐wk‐old human fetuses, Prox1 and vascular endothelial growth factor receptor‐3 (VEGFR‐3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL‐E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR‐3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR‐3, but rarely CD34. In the stroma, spindle‐shaped CD34‐positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR‐3 and CD31. VEGFR‐3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

Spheroid-based engineering of a human vasculature in mice.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

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.

Median nerve compression can be detected by magnetic resonance imaging of the carpal tunnel

OBJECTIVE Clinically symptomatic carpal tunnel syndrome is not necessarily accompanied by impaired nerve conduction values. Surgical decompression, however, may immediately lead to complete and lasting relief of symptoms in these patients. Because minimally invasive techniques have reduced perioperative morbidity and vocational impairment related to operative decompression, the decision to decompress symptomatic patients (despite still unimpaired nerve conduction values) might be subject to discussion in the future. New diagnostic tools may be helpful in deciding which therapeutical options to choose. When the wrist is held either in flexion or in extension, the carpal tunnel pressure increases. To investigate the dynamic changes of the carpal tunnel shape during wrist motion, as well as the variations of space for the median nerve and its signal intensity in T2-weighting, magnetic resonance imaging (MRI) was performed on patients and healthy volunteers alike. Restitution and the persistence of pathological findings were assessed pre- and postoperatively. METHODS MRI (1.0 T) was performed on 20 wrists of patients with clinical symptoms of carpal tunnel syndrome (CTS) and pathological nerve conduction values. Healthy volunteers (20 wrists) were matched according to sex and age. MRI was performed in neutral, 45-degree extension, and 45-degree wrist flexion positions. T2-weighted signal intensity of the median nerve was measured in 18 patients pre- and postoperatively. RESULTS The cross-sectional area of the carpal tunnel in patients with CTS tends to be smaller than that found in nonsymptomatic volunteers. The cross-sectional area of the carpal tunnel decreases during wrist flexion at the pisiform and hamate level. During wrist extension, the cross-sectional area of the carpal tunnel decreases at the level of the pisiform. During extension, it increases at the level of the hamate. The cross-sectional area of the median nerve showed an increase at the pisiform level (P < 0.05), a flattening of the median nerve at the hamate hook level (P < 0.05), and palmar deviation of the flexor retinaculum at the pisiform and hamate hook level (P < 0.001). This was significantly greater in CTS patients than in individuals with normal wrists. Postoperatively, the distal flattening of the median nerve recovered in 94% of the cases reviewed. Although the signal intensity of the median nerve on T2-weighted images decreased by 67%, the motor latency recovered in only 39% of the cases. CONCLUSION The carpal tunnel was smaller in CTS patients than in healthy volunteers. During flexion and extension, the space available for the median nerve narrows. This may lead to potential median nerve compression. MRI is accurate and reliable for diagnosis and postoperative follow-up of carpal tunnel syndrome. In cases with obvious clinical symptoms and yet not measurably impaired median nerve conduction values, it may be helpful in making a decision for surgical decompression.

Complications in abdominoplasty: a risk factor analysis.

INTRODUCTION Abdominoplasty is one of the most popular body-contouring procedures. However, it is associated with a significant number of complications, the most common being seroma and wound-healing problems. In this study, we analysed postoperative complications following abdominoplasty and evaluated the presence of statistically significant correlations between pre-existing risk factors and postoperative complications. METHODS We carried out a retrospective chart review of all patients who underwent abdominoplasty from June 1994 to April 2004 at our institution. Patient demographics, risk factors (smoking, previous abdominal surgery, obesity, and male gender) and complications (minor and major) were noted from the patients medical record. RESULTS In all, 139 patients were included in the study (126 female and 13 male), with a mean age of 42.8 years at the time of surgery. Minor and major complications were seen in 40 (28.8%) and 16 patients (11.5%), respectively. Smoking and previous abdominal surgery were not associated with a significantly increased complication rate. However, patients without previous surgery displayed a significantly increased complication rate (43.2% vs. 22.1% for minor and 25% vs. 5.3% for major complications; p<0.05). Obesity (BMI >30 kg/m(2)) was associated with a significant increase in major complications (20.8% vs. 9.7%; p<0.05). Male gender was predisposed to increased minor and major complications; however, this was not statistically significant. CONCLUSION Despite its popularity, abdominoplasty is still associated with a significant rate of complications. In particular, obese patients and those with no previous history of abdominal surgery seem to be at risk for complications. The role of male gender needs to be evaluated further.

Engineered adipose tissue supplied by functional microvessels.

A volume-persistent culture of adipose tissue under in vivo conditions can be achieved only by early vascularization after cell transplantation. Cotransplantation of autologous preadipocytes with endothelial cells may enable the early formation of a capillary network. Investigations were performed in vivo in a specially adapted chorioallantoic membrane (CAM) model. Fertilized White Leghorn eggs were incubated and opened on day 3 of incubation and human dermal microvascular endothelial cell (HDMVEC) spheroids and preadipocytes were transferred in a fibrin matrix to the CAM. On day 7 after incubation the composites were explanted and immunohistologically investigated. Numerous vessels consisting of HDMVECs could be detected and the lumena of these vessels were perfused by chick erythrocytes. These results show the formation of a capillary network consisting of transplanted HDMVECs. The microcirculation of chick erythrocytes in vessels consisting of human endothelial cells proves the continuity of a newly formed capillary system to the host vessel system. The experiments demonstrate the first patent connection of tissue-engineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or transient transfection. The cotransplantation of endothelial cell spheroids with angiogenic mesenchymal cells may lead to the engineering of complex three-dimensional implants.

In vivo engineering of a human vasculature for bone tissue engineering applications

The neovascularization of three‐dimensional voluminous tissues, such as bone, represents an important challenge in tissue engineering applications. The formation of a preformed vascular plexus could maintain cell viability and promote vascularization after transplantation. We have developed a three‐dimensional spheroidal coculture system consisting of human primary endothelial cells and human primary osteoblasts (hOBs) to improve angiogenesis in bone tissue engineering applications. In this study, we investigated the survival and vascularization of the engineered implants in vivo. Endothelial cell spheroids were cocultured with hOBs in fibrin and seeded into scaffolds consisting of processed bovine cancellous bone (PBCB). The cell‐seeded scaffolds were evaluated for their angiogenic potential in two different in vivo assays: the chick embryo chorioallantoic membrane (CAM) model and the severe combined immunodeficiency disorder (SCID) mouse model. In both assays, the development of a complex three‐dimensional network of perfused human neovessels could be detected. After subcutaneous implantation into immunodeficient mice, the newly formed human vasculature was stabilized by the recruitment of murine smooth muscle α‐actin‐positive mural cells and anastomoses with the mouse vasculature. We conclude that this endothelial cell spheroid system can be used to create a network of functional perfused blood vessels in vivo. The finding that this process takes place with high efficacy in the presence of co‐implanted primary osteoblasts and in an osteoconductive environment provided by the PBCB scaffold, suggests that this system may be suitable for improving vascularization in bone tissue engineering.

Development and Characterization of a Spheroidal Coculture Model of Endothelial Cells and Fibroblasts for Improving Angiogenesis in Tissue Engineering

Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications.

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.

Cultured keratinocytes in fibrin with decellularised dermis close porcine full-thickness wounds in a single step

The purpose of this study was to assess the feasibility of combined keratinocyte and dermal scaffold transplantation performed in a single step for treatment of full-thickness wounds. Cultured autologous keratinocytes were suspended in fibrin and grafted together with decellularised human dermis (Alloderm) in a porcine animal model, involving 10 animals over 4 weeks. Wound healing was evaluated by planimetry. Histology included morphological analysis as well as immunohistochemistry at regular intervals (1, 2 and 4 weeks). The results showed both successful histo-integration of the in vivo composite grafts and reduced wound contraction, compared with the control group (plain epithelial grafts). Histologically a neo-epithelium originated from the grafted cells on top of the decellularised dermis, as well as a reconstituted basement membrane. After 4 weeks cellular ingrowth into the dermal matrix could be observed. The successful combination of a keratinocyte-fibrin suspension and acellular dermis applied in a single step onto full-thickness wounds resulted in closure.