Christoph Andree

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.

Dry, Moist, and Wet Skin Wound Repair

Effects of wet (saline in a vinyl chamber), moist (hydrocolloid dressing), and dry (sterile gauze dressing) environments on wound repair were studied in a porcine partial-thickness wound model. Chambers were exchanged and refilled daily with normal saline containing penicillin G (100 U/ml) and streptomycin (100 µg/ml). Hydrocolloid and gauze dressings were kept in place until biopsy of the wound site. Wounds in wet, moist, and dry environments were completely epithelialized on days 6, 7, and 8, respectively. Thickness of the epidermis in wet, moist, and dry wounds was 204 ± 23, 141 ± 12, and 129 ± 18 (mean ± SEM), respectively. Moist wounds had more subepidermal inflammatory cells than wet wounds. In comparison to dry wounds, the moist or the wet healing environment resulted in less necrosis and faster and better quality of healing in the formation of the newly regenerated epidermis.

Growth factors in the repair of partial thickness porcine skin wounds.

&NA; In 28 porcine partial thickness excisional wounds, the presence of several growth factors was first studied by enzyme‐linked immunoadsorbent assay on wound fluid collected in sealed wound chambers. Basic fibroblast growth factor (bFGF) peaked on day 1 at 31.4 pg/ml; platelet derived growth factor (PDGF)‐AB on day 3 reached 45.2 pg/ml, and transforming growth factor‐beta (TGF‐&bgr;) on day 7 was 726.1 pg/ml. The same chamber system was used in 48 partial thickness excisional wounds for delivery of nanogram doses of bFGF, PDGF‐AB, insulin‐like growth factor (IGF)‐1, epidermal growth factor (EGF), and cholera toxin. PDGF and EGF accelerated healing (1.1 days and 0.3 days, respectively), whereas bFGF and IGF‐1 had no effect. Cholera toxin retarded healing by 1.9 days. Furthermore, in 100 excisional wounds EGF in the concentration range of 10 to 1,000 ng/ml had the same stimulating effect on healing. EGF at 10,000 ng/ml significantly delayed healing. The wound chamber model is useful for detecting of endogenous growth factors as well as for delivering exogenous factors. (Plast. Reconstr. Surg. 100: 657, 1997.)

Morbidity of microsurgical breast reconstruction in patients with comorbid conditions.

Background: Although free tissue-transfer with the deep inferior epigastric perforator (DIEP) flap is one of the best forms of autologous breast reconstruction, surgeons have remained guarded over selecting patients for the procedure in the presence of comorbid conditions. This study has investigated the relevance of these conditions. Methods: A prospective review of all free flap breast reconstructions (n = 624) was performed over a 2-year period at the Department of Plastic Surgery at the Sana Kliniken Düsseldorf. Patients were placed into three groups based on comorbid conditions such as age 65 years or older, active smoking, and body mass index greater than or equal to 30. Flap and donor-site complications were analyzed. Results: Six hundred twenty-four breast reconstructions with DIEP or muscle-sparing transverse rectus abdominis musculocutaneous (TRAM) flaps were performed in 558 patients (66 bilateral reconstructions). There were 36 patients older than 65 years at the time of surgery, 94 active smokers, and 79 patients with a body mass index of greater than or equal to 30. Flap complications such as venous congestion (n = 5), partial flap loss (n = 10), marginal necrosis (n = 15), and total flap loss (n = 5) occurred in 35 cases (5.6 percent). Donor-site complications such as delayed abdominal wound healing (n = 9), seroma (n = 8), abdominal hernia (n = 3), and bulging (n = 11) occurred in 31 cases (5 percent). Conclusions: Despite having significantly higher complications in the form of delayed donor-site wound healing in active smokers and higher total flap loss in obese patients, the overall complication rates compared with other reconstructive procedures are low. Microsurgical reconstruction with DIEP and muscle-sparing TRAM flaps is associated with low complication rates, excellent aesthetic outcome, and high patient satisfaction, even in patients with known risk factors.

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.

Tissue engineering in plastic reconstructive surgery

Tissue engineering (TE) is a new interdisciplinary field of applied research combining engineering and biosciences together with clinical application, mainly in surgical specialities, to develop living substitutes for tissues and organs. Tissue engineering approaches can be categorized into substitutive approaches, where the aim is the ex vivo construction of a living tissue or organ similar to a transplant, vs. histioconductive or histioinductive concepts in vivo. The main successful approaches in developing tissue substitutes to date have been progresses in the understanding of cell–cell interactions, the selection of appropriate matrices (cell–matrix interaction) and chemical signalling (cytokines, growth factors) for stimulation of cell proliferation and migration within a tissue‐engineered construct. So far virtually all mammalian cells can be cultured under specific culture conditions and in tissue specific matrices. Future progress in cell biology may permit the use of pluripotent stem cells for TE. The blueprint for tissue differentiation is the genome: for this it is reasonable to combine tissue engineering with gene therapy. The key to the progress of tissue engineering is an understanding between basic scientists, biochemical engineers, clinicians, and industry. Anat Rec 263:372–378, 2001.

Improved safety of autologous breast reconstruction surgery by stabilisation of microsurgical vessel anastomoses using fibrin sealant in 349 free DIEP or fascia-muscle-sparing (fms)-TRAM flaps: A two-centre study

BACKGROUND We routinely perform free DIEP flap and fascia-muscle-sparing (fms) TRAM flap procedures using fibrin sealant to stabilise anastomosed vessels, thus avoiding some of the difficulties associated with microsurgical anastomoses. METHODS Women undergoing elective, autologous breast reconstruction with free DIEP flaps or fms-TRAM flaps between June 2004 and June 2007 in two Interdisciplinary Breast Centres were included in a retrospective chart review. RESULTS A total of 349 breast reconstructions were performed in 325 women. Of these, 201 (57.6%) were free DIEP flap procedures and 148 (42.4%) were fms-TRAM flap procedures. Average hospital stay was 9.8 days. Complete flap loss was seen in 3 cases (0.9%). Low rates of post-operative complications were observed. CONCLUSIONS Microsurgical breast reconstruction using free DIEP flaps and fms-TRAM flaps, with fibrin sealant for stabilisation of microvascular anastomoses, provides good post-operative outcome featuring a low incidence of flap loss or other common post-operative complications.

Effect of Ultrasonic Assisted Lipectomy (UAL) on Breast Tissue: Histological Findings

Abstract. As the use of ultrasound-assisted liposuction (UAL) increases, the technique grows more popular in breast surgery, especially in reduction mammaplasty and treatment of gynecomastia. The aim of our study was to investigate the effect of UAL on breast tissue using histological examinations, and analyze the effect of this technique on a cellular level.Biopsies from 10 patients undergoing ultrasonically assisted lipectomy prior to classic reduction mammaplasty were taken from the treated areas of the breast. Biopsies were fixed in formalin and embedded in paraffin. Sections were stained with hematoxilin-eosin, and analyzed for defective adipocytes, and the effects of UAL on breast tissue. Untreated breast tissue and breast tissue that had been treated only with conventional aspiration lipectomy served as controls. Sections were analyzed using light microscopy. Compared to the breast tissue treated only with conventional lipectomy, a stronger destruction of the cellular structure of adipocytes could be detected. The destruction was visible even in areas more distant from the aspiration channel. In contrast, the breast tissue was mostly intact, no signs of ultrasonic-induced cellular destruction were visible. The glandular structure was kept intact. Beside the direct mechanical destruction by the probe and the canula, no further alterations of the cellular integrity of the glandular parts were visible.In conclusion our results indicates that UAL is also a safe technique for use in breast surgery. Besides easy handling and improved modelling, the destructive effect of the ultrasound does not include the glandular breast tissue.

Treatment of Chronic, Nonhealing Abdominal Wound in a Liquid Environment

A 66-year-old woman with an abdominal wound caused by infected synthetic mesh had failed to heal in spite of many surgical attempts to close the wound. A sealed transparent vinyl chamber was glued to the periphery of the wound and antibiotics in high concentrations (up to 2,500 times the minimum inhibitory concentration) were delivered through the chamber. The wound fluid in the chamber was used for analysis of microbial activity, concentration of residual antibiotics, and growth factor activity. After 10 weeks of treatment, the wound was closed and has not recurred in 24 months.

Orthogonal polarization spectral imaging: a tool for assessing burn depths?

The estimation of burn depths is still a challenge, and even experienced surgeons often fail. In search of an objective method for differentiation between deep- and partial-thickness burns, we investigated the use of orthogonal polarization spectral (OPS) imaging to visualize the microcirculation in burn wounds.Twenty-seven burned patients were included in the study, 81 burn areas were investigated at day 1 and 4 post burn. The final therapy was compared with the therapy that would have been carried out as a consequence of the OPS imaging result. The patients were treated conservatively with dressings or surgically with necrectomy and split skin grafts.A comparison of the estimation of the burn depths of OPS imaging with the final therapy showed a correlation of 76.5%, about 5% less than the clinical assessment. It has proved a useful tool in particular for the prognosis as to whether the burn will heal spontaneously within 14 days or not.The presented OPS-Imaging device provides additional qualitative and quantitative information about the perfusion of the skin and therefore facilitates decisions about the follow on therapy. It is not an alternative to an experienced burn surgeon but provides important additional information.