Biomedical subjects
Publications and source records attributed to C Andree.
The principles and techniques of gene transfer are presented. Common concepts are defined, and the frequently used transfer vectors are described herein. Several gene transfer applications are discussed briefly. Areas of particular interest to the plastic surgeon such as gene transfer to skin and wounds are included.
This report details the transfer of a human epidermal growth factor (hEGF) expression plasmid to porcine partial-thickness wound keratinocytes by particle-mediated DNA transfer (Accell). After gene transfer an external sealed fluid-filled wound chamber was used to protect the wound, provide containment of the exogenous DNA and expressed peptide, and permit sampling of the wound fluid. Analysis of wound fluid for hEGF and total protein, an indicator of reformation of the epithelial barrier, showed that wounds bombarded with the hEGF plasmid exhibited a 190-fold increase in EGF concentration and healed 20% (2.1 days) earlier than the controls. EGF concentrations in wound fluid persisted over the entire 10-day monitored period, decreasing from 200 pg/ml to 25 pg/ml over the first 5 days. Polymerase chain reaction results showed that plasmid DNA was present in the wound for at least 30 days. These findings demonstrate the possible utility of in vivo gene transfer to enhance epidermal repair.
Normal and retrovirally transfected keratinocyte suspensions expressing either the beta-galactosidase gene or the human growth hormone (hGH) gene were transplanted into chamber-enclosed skin full-thickness wounds of Yorkshire pigs. Immunostaining of sequential skin biopsies obtained for 4 weeks after transplantation showed survival of the transplanted keratinocytes as well as expression of beta-galactosidase. Transfected keratinocytes were first seen in the neodermal portions of the wounds, then in the regenerating basal epidermal layer, and finally in the terminally differentiating cells of the stratum spinosum. When keratinocytes transfected with the hGH gene were transplanted into similar wounds, hGH was detected for 10 days in wound fluid. In contrast, hGH was detected in vitro for 47 days. Wounds transplanted with either transfected or normal keratinocytes restored the epithelial barrier function significantly faster than nontransplanted controls (P < 0.05). The study confirms the successful transplantation of keratinocyte suspensions, their reconstitution of the epidermis, and their acceleration of repair. Further, this apparently normal incorporation of genetically engineered transplanted keratinocytes expressing either beta-galactosidase or hGH suggests the possibility of introducing other genes expressing therapeutic proteins into wounds to favorably affect healing. Wound fluid detection of the expressed peptide provided early demonstration of successful transfer of the hGH gene.
Adherence of a biological graft to the wound surface is the most important factor influencing the ultimate success of graft viability. A machine has been developed to test the adherence of biological graft materials to a substrate such as a wound surface. The peeling mode, which yields reproducible quantitative measurements of adherence, is a standard method for testing adhesives. The device is designed to continuously measure the force required to peel the graft from the substrate at a constant rate. This force is a function of the energy of adhesion per unit area of adhered surface. This device has been used to measure the peeling force of (2 x 2 cm) skin grafts which are applied to full-thickness wounds on mice. Results of tests on adherence of autografts on mice show that the peeling force increases significantly with time over the first 9 days of healing. Thus, this device is useful in quantitative comparison of various skin grafting techniques and artificial grafts.
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.
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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.
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.
BACKGROUND: Growth factors play an important role in tissue repair. While the effectiveness of growth factor therapy in animal wound healing models and limited human clinical trials has been demonstrated, the ideal method for their administration to the wound remains unclear. Experimental data suggest that the continuous presence in the early stages of wound repair is beneficial. MATERIALS AND METHODS: We have constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene is fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF can be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 pg/ml to 140 pg/ml on day 7. No EGF was found in the wound at day 14. CONCLUSIONS: A single application of irradiated EGF genetransfected fibroblasts to wounds can thus continuously deliver the transgene in vivo and could be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.