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Biomedical subjects

G B Stark

Publications and source records attributed to G B Stark.

At least 37 records · Page 2Linked to original sources
PubMed2001

Fibrin glue as matrix for cultured autologous urothelial cells in urethral reconstruction.

In the present study, we have established a technique to create an artificial urethra in a rat animal model by transplantation of in vitro-expanded urothelial cells onto an in vivo-prefabricated tube formation using tissue engineering methods. Urothelial cells from isogenic rats were harvested for culture. A silicon catheter was used to induce a connective tissue capsule-tube formation underneath the abdominal skin. Two weeks later, the cultivated urothelial cells were seeded onto the lumen of this tube using fibrin glue as delivery matrix. The histomorphological and immunohistochemical studies revealed a viable multilayered urothelium, lining the inner surface of the prior formed connective tissue tube-formation 4 weeks after grafting the cells. We have shown that cultured and in vitro-expanded urothelial cells can be successfully reimplanted onto a prefabricated tube-like structure using fibrin glue as a delivery matrix and native cell expansion vehicle. The results suggest that the creation of an artificial urethra may be achieved in vivo using tissue engineering methods, showing potential for urethral reconstruction and providing autologous urothelium for reconstructive surgery in the genitourinary tract.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

[Cell transplantation in surgery--reality and prospects for tissue engineering].

Traditionally surgical repair of tissue defects and loss or failure of function has relied on mechanical means, medical (drug) treatment, autologous and allogenic transplantation, and alloplastic/synthetic devices. Tissue engineering represents 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. The understanding of cell-cell interactions and chemical signalling (growth factors) and the selection of appropriate matrices (cell-matrix interaction) is the key for success. Gene therapy represents the logical combination with tissue engineering on the molecular biology level. Application of cultivated skin and cartilage has already become reality, engineering of vascularized, more complex organs remains a challenge for this century.

Cells, Cultured↗
PubMed2000

The contralateral bilobed trapezius myocutaneous flap for closure of large defects of the dorsal neck permitting primary donor site closure.

BACKGROUND: Various reliable local flaps can be used to cover soft tissue defects of the posterior neck. Among others, the (either vertical or transverse) trapezius myocutaneous flap is a reliable option in this context. However, for defects with a diameter larger than 15 cm, these flaps require skin grafting of the donor site. METHODS: To prevent this additional morbidity we describe a contralaterally based bilobed trapezius musculocutaneous flap successfully applied in two elderly patients with large tumors of the posterior neck in palliative situations. RESULTS: This novel approach makes use of a standard vertical skin paddle to cover the neck, together with a transverse cervical flap in a bilobed design to primarily close the paravertebral donor site. CONCLUSIONS: The bilobed technique allows the primary closure of massive soft tissue neck wounds. Preservation of the accessory nerve to the superior fibers of the trapezius muscle permitted normal arm elevation in our patients.

Accessory Nerve↗
PubMed2000

[Tissue engineering with mesenchymal stem cells for cartilage and bone regeneration].

Tissue engineering offers the possibility to fabricate living substitutes for tissues and organs by combining histogenic cells and biocompatible carrier materials. Pluripotent mesenchymal stem cells are isolated and subcultured ex vivo and then their histogenic differentiation is induced by external factors. The fabrication of bone and cartilage constructs, their combinations and gene therapeutic approaches are demonstrated. Advantages and disadvantages of these methods are described by in vitro and in vitro testing. The proof of histotypical function after implantation in vivo is essential. The use of autologous cells and tissue engineering methods offers the possibility to overcome the disadvantages of classical tissue reconstruction--donor site morbidity of autologous grafts, immunogenicity of allogenic grafts and loosening of alloplastic implants. Furthermore, tissue engineering widens the spectrum of surgical indications in bone and cartilage reconstruction.

Bone Regeneration↗
PubMed2000

Plasminogen activation in venous leg ulcers.

BACKGROUND: Venous leg ulceration results from chronic venous insufficiency of the lower extremities. We recently showed that matrix metalloproteinase (MMP) -2 plays a major part in the pathogenesis of venous leg ulcers. In vitro activation of recombinant MMP-2 is controlled by the activity of the urokinase-type plasminogen activator (uPA), which acts as a fibrin-independent plasminogen activator. The activity of MMP-2 is potentiated by binding of uPA to the uPA receptor (uPAR). OBJECTIVES: We aimed to clarify the role of plasminogen activation in venous leg ulcers. METHODS: The expression of uPA, uPAR, the tissue-type plasminogen activator, and plasminogen activator inhibitor (PAI) -1 and PAI-2 was investigated using reverse transcription followed by polymerase chain reaction and Western blotting. RESULTS: These provided direct evidence of elevated expression of uPA and uPAR at the mRNA and protein levels in venous leg ulcers, in comparison with healthy skin. By immunohistochemistry, elevated expression of uPA and uPAR was detected. Fibrin zymography showed significantly elevated endogenous uPA activity in venous leg ulcers in comparison with healthy controls. CONCLUSIONS: Our findings indicate venous leg ulcers to be characterized by elevated plasminogen activation, suggesting that this enzyme cascade plays a crucial part in maintaining proteolytic activity in venous leg ulcers.

Aged↗
PubMed2000

Cultured human keratinocytes on type I collagen membranes to reconstitute the epidermis.

The development of new techniques and modifications to overcome some of the disadvantages in cultured keratinocyte grafting has been motivated by several well-known drawbacks in the use of cultured epithelial autografts such as long culture periods, lack of adherence, difficulty in handling, lack of dermal substrates, and high costs. Two recent insights have influenced further research. On the one hand, it has been shown that the use of undifferentiated proliferative cells in fibrin glue suspensions is effective in epithelial reconstitution. On the other hand, the enzymatic release of cells from the culture surfaces is a critical step leading to at least temporary destruction of anchoring structures of the cultured cells. In this study, we tried to combine these two aspects in an attempt to modify common modalities of keratinocyte transplantation. To avoid dispase dissolving of the cultured cells, keratinocytes were seeded onto bovine collagen type I membranes without feeder layers and under serum-free culture conditions. Subconfluent monolayers of cultured human keratinocytes were transplanted as an upside-down graft on collagen membranes (keratinocyte collagen membrane grafts [KCMG], n = 12) after 3 days of culture or as membrane grafts alone (n = 12) onto standard nude mice full-thickness wounds. Fully differentiated epidermis was found at 21 days after grafting KCMG with persistence of human keratinocytes. This study demonstrates that upside-down grafts of undifferentiated monolayers of keratinocytes on non-cross-linked bovine type I collagen membranes do lead to an early reconstitution of multilayered squamous epithelium with enhanced wound healing compared to the control group. The upside down KCMG grafting technique is able to transfer actively proliferative keratinocytes and simplifies the application compared to conventional epithelial sheet grafting.

Animals↗
PubMed2000

[Tissue engineering: possibilities and perspectives].

Successful tissue engineering requires intensive co-oporation between clinicians, biologists (cell culture, gene therapy), chemical engineers (biomaterials) and industrial partners. In case of wound healing tissue engineered constructs have already been applied successfully in burns and chronic wounds. In order to improve carrier and matrix function biomaterials still have to be optimized. The potential of such constructs might even be enhanced by gene therapeutical methods. The complex mammalian organism has to be considered as the gold standard and the model for perfect tissue engineering. The problem of vascularization of complex organs yet has to be solved. In general it seems to be more promising to substitute deficient components in vivo and to rely on modulating influences within the host organism rather than to create complex organs ex vivo.

Animals↗
PubMed2000

[Gene therapy perspectives in modulation of wound healing].

A variety of reasons can afflict wound healing. Current research is focussed on the acceleration of wound healing by stimulating molecular processes. Gene therapy may offer completely new ways to treat chronic wounds. Possible advantages of gene therapeutic modulation of wound healing might be a long term efficiency, systemic or local regulation of gene expression and low side-effects. Current goals comprise the improvement of transfection efficiency and specificity. In vivo applications are therefore focussed on optimized inducible or even cell-type specific promotors, as well as on improved local application techniques. Studies from our laboratory demonstrate the possibility to combine modern cell culture techniques with different types of gene transfer. This enables the simultaneous grafting of manipulated cells to the wound with the continuous delivery of specific proteins of interest. Experimentally, this lead to accelerated closure of partial and full thickness animal wounds. Clinically, gene therapy for the treatment of chronic wounds seems to be a realistic goal within the next years and might be applicable for a variety of novel indications.

Animals↗
PubMed2000

[Treatment of chronic wounds with cultured autologous keratinocytes as suspension in fibrin glue].

Cultivated keratinocytes have been used for treatment of chronic wounds. Our group developed a new application form, using a suspension of subconfluently cultivated keratinocytes in fibrin glue (keratinocyte-fibrin-glue-suspension = KFGS), which has successfully been used in burn patients. Altogether 8 patients (average: 57 yrs.) with complex chronic wounds of different origin were treated with KFGS. All wounds, which had been existing from 4 months to 14 years, showed good reepithelization. Up to now there has been observed stable wound closure for 4 years after grafting. This study demonstrates the wound healing potency of a keratinocyte-fibrin-glue-suspension also for chronic wounds. Fibrin glue seems to be both an ideal application vehicle as a biological matrix for the cultivated keratinocytes. The transplantation of cultivated autologous keratinocytes as suspension in fibrin glue is a promising way in the treatment of chronic wounds.

Adult↗
PubMed1999

[Quality of life after breast reduction-plasty].

We have investigated the improvement in the quality of life in 384 breast reduction patients within a seven-year postoperative follow-up. Through the use of a questionnaire (recall of n = 223 patients), we sought to establish the patient's most compelling reason for seeking this surgical procedure. In addition, the effect of the operation on the patient's quality of life was investigated. Preoperatively, the main complaint was pain in the shoulder girdle in 77% of the patients, followed by 67% patients suffering from lower and thoracic back pain symptoms, whereas 51% defined mastodynia as their primary complaint. 95% of the patients noted a remarkable improvement in the quality of their professional and social lifes. 94% were able to specify this improvement as an increase in the feeling of self-esteem, 53% showed up with amelioration of selfconfidence. 95% of the patients felt that the breast reduction had a positive influence on their sexual behaviour and personal relationships. Our study reveals that breast reduction surgery offers not only an aesthetic improvement but also the relief of preoperative pain and discomfort, leading to important improvements in the patient's quality of life.

Adolescent↗
PubMed1999

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.

Adult↗
PubMed1999

Prefabrication of bilaminar-epithelialized composite flap with tissue expander and cultured keratinocytes.

This study investigated the feasibility of prefabrication of a bilaminar-epithelialized flap by using a tissue expander and cultured keratinocytes, for reconstruction of perforate defects in the oral cavity and upper aerodigestive tract. In each of six rats, a 10-ml volume expander was implanted under the inferior epigastric flap and a thin silicon catheter was introduced into periexpander space. Seven days after implantation, 10 x 10(6) cultured keratinocytes, isolated from inbred donor rats, were suspended in fibrin glue and injected into the periexpander space through the catheter (n = 4 of 6). The expansion was started immediately after cell inoculation and lasted at least 3 weeks at the speed of 2 to 3 ml every 5 to 7 days. At the end of expansion, the periexpander space was opened and the capsule around the tissue expander was found to be covered completely with a neoepithelium. Thus, a bilaminar-epithelialized flap based on femoral vessels was elevated and successfully transferred to cover the excisional perforate defect in the oral cavity with the neoepithelial side as inner lining. All flaps treated with 10 x 10(6) cultured keratinocytes survived with complete wound healing during a 1-week follow-up (n = 4 of 6). Both macroscopic and histologic findings demonstrated that a bilaminar-epithelialized composite flap can be fabricated by using a tissue expander and keratinocyte-fibrin glue suspension.

Animals↗
PubMed1999

The rectus abdominis free flap as an emergency procedure in extensive upper extremity soft-tissue defects.

Stable wound coverage after extensive soft-tissue loss of the upper extremity remains a difficult problem in the management of large defects of the upper limb. To prevent further tissue loss owing to infection or inadequate cover when important structures such as vessels, tendons, nerves, joints, and bones are exposed, various free flaps have been introduced into the therapeutic armamentarium of acute plastic surgical management options. Emergency or delayed early reconstruction has been proposed to prevent chronic infection and further tissue loss. We report a series of 12 emergency and delayed early reconstructions of the forearm, wrist, carpus, metacarpus, and hand using the free rectus abdominis muscle flap with split-skin coverage, demonstrating the versatility of this flap within this special context. Emergency free rectus muscle flap transfer is safe, technically easy, and expandable.

Abdomen↗
PubMed1999

[Free microsurgical flap-plasty in reconstructive therapy of diabetic foot ulcer].

The diabetic foot ulcer is a significant clinical problem often resulting in frustranous conservative treatment or early amputation. In certain cases transfer of well vascularized tissue can improve wound healing and lead to a length-spearing therapy. In this study the concept of microsurgical tissue transfer in the treatment of diabetic foot ulcers is introduced. Following a radical debridement and, if necessary a atypical length-spearing amputation the wound is covered by a free transplanted muscle flap followed by a split-thickness skin graft. In six patients treated with this procedure the extremity could be saved. The perioperative mortality was 0%, average hospitalization was 47.6 days. One flap was lost, two vascular revision were necessary. Several necrectomies and skin grafts were performed and one donor side seroma was drained. One hernia was observed after free rectus abdominus transfer. Compared to conservative treatment or amputation this concept leads to a length-spearing therapy and can increase success rates of rehabilitation and quality of life.

Amputation, Surgical↗
PubMed1999

[Keratinocyte transplantation and tissue engineering. New approaches in treatment of chronic wounds].

Cultured keratinocytes have been used for the treatment of extensive burns since disease lethality is reduced. Consequently, the treatment of chronic wounds with keratinocytes may be promising. Cell culture technology allows to expand keratinocytes up to 6000-fold in vitro after taking a single biopsy from patient. Today the transplantation of these in vitro cultured keratinocytes in different modifications is an established clinical treatment regimen for therapy of extensive wounds. For example, keratinocyte-fibrin-glue-suspensions, mainly consisting of proliferative epidermal basal cells, were used for the treatment of burns in experimental and clinical settings to bypass the disadvantages of conventional sheet grafts. Other approaches in tissue engineering for wound healing aim at the (epi-)dermal repair by the combination of allodermis and biomaterials, i.e. collagen-sponges and microspheres. Due to most recent efforts in keratinocyte culture techniques, developments in tissue engineering, research for novel biomaterials and gene therapy, therapy of chronic wounds may prove to be more efficient. Furthermore, from the socio-economical point of view, overall costs for treatment of chronic wounds could be reduced.

Cell Division↗
PubMed1999

[Continuous delivery of epidermal growth factor to wounds in vivo by genetically modified fibroblasts transfected with a novel chimeric construct].

OBJECTIVE: This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS: It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS: Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION: A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.

Amino Acid Sequence↗