G. B. Stark

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.

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.

Comparison of Pre-Adipocyte Yield, Growth and Differentiation Characteristics from Excised versus Aspirated Adipose Tissue

Adipose tissue precursor cells (pre-adipocytes) are part of a stromal vascular fraction that can be easily isolated from fat tissue. Adipose tissue can be harvested by 2 methods: aspiration and excision. We analyzed whether the pre-adipocyte yield, growth characteristics and ability to differentiate into mature adipose tissue are influenced by the type of harvesting procedure. Adipose tissue was simultaneously harvested from the abdomen by surgical excision or aspiration according to the Coleman procedure in 10 individuals. This permitted inter- and intra-individual comparisons. Cell viability and yield were determined directly after isolation of pre-adipocytes. The growth kinetics were investigated in culture. Furthermore, pre-adipocytes were cultured under adipogenic conditions to compare their differentiation potential. The number of viable pre-adipocytes was significantly higher after excision of adipose tissue compared to aspiration. The proliferation kinetic was not influenced by the type of harvesting. No differences were observed in the differentiation potential of the pre-adipocytes between both groups. Compared to excision, aspiration of adipose tissue negatively affects the yield of pre-adipocytes. However, growth characteristics and differentiation potential of viable cultured cells are not influenced by the type of surgical harvesting. Due to its reduced donor site morbidity, we conclude that aspiration of adipose tissue is a valid harvesting method for isolation of pre-adipocytes.

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds

Abstract Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds. The technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (KFGS) has been recently developed to overcome common disadvantages of standard cultured epidermal sheet grafts. The combination of this method with glycerolized (nonvital) xenograft overlays in standardized nude mice full-thickness wounds, as compared to KFGS alone or controls with no grafts, showed enhancement of epithelial regeneration in terms of epithelial thickness and diminished wound contraction during the 6-week follow-up. Total scar thickness was increased after the combined KFGS/xenograft technique. The time taken to complete wound reepithelialization was similar in the two groups. Reconstitution of the dermo-epidermal junction zone as shown by electron microscopy and immunohistochemistry was enhanced by the KFGS+xenograft technique, showing structures resembling rete ridges 6 weeks postoperatively. The combined KFGS/xenograft technique is able to transfer proliferative single keratinocytes. The method simplifies the application when compared to conventional epithelial sheet grafting and reduces wound contraction when compared to pure keratinocyte grafting.

Management of hypovascularized wounds not responding to conventional therapy by means of free muscle transplantation.

Background: Chronic ulceration as a complication of arteriosclerotic disease, venous congestion or diabetes mellitus is still a serious clinical problem, resulting in immobilization, extended hospitalization and cost-intensive treatment. Other than standard conservative treatment protocols or early amputation, microsurgical free transfer of well vascularized muscle tissue onto chronic wounds can induce angiogenesis and improve wound healing even in the hypovascularized wound. Patients: From 1993–1999 we treated 12 patients (mean age: 46 years) with vascular ulcers of the lower extremity with free muscle or fasciocutaneous tissue transfer. Results: The average hospitalization was 51.4 days. The perioperative mortality was zero. In one patient with factor V deficiency a partial flap necrosis occurred. Two revisions of the micro anastomoses had to be performed. Two seromas occurred at the donor site. No secondary flap loss was observed. Extremity or stump length preservation was achieved in all cases. Conclu...BACKGROUND Chronic ulceration as a complication of arteriosclerotic disease, venous congestion or diabetes mellitus is still a serious clinical problem, resulting in immobilization, extended hospitalization and cost-intensive treatment. Other than standard conservative treatment protocols or early amputation, microsurgical free transfer of well vascularized muscle tissue onto chronic wounds can induce angiogenesis and improve wound healing even in the hypovascularized wound. PATIENTS From 1993-1999 we treated 12 patients (mean age: 46 years) with vascular ulcers of the lower extremity with free muscle or fasciocutaneous tissue transfer. RESULTS The average hospitalization was 51.4 days. The perioperative mortality was zero. In one patient with factor V deficiency a partial flap necrosis occurred. Two revisions of the micro anastomoses had to be performed. Two seromas occurred at the donor site. No secondary flap loss was observed. Extremity or stump length preservation was achieved in all cases. CONCLUSIONS Optimal postoperative treatment with physiotherapy and orthopaedic shoe support is important. If all these factors are present and if the patient is highly motivated a reintegration into normal life can be achieved.

Gene technology and tissue engineering

The interest in gene therapy to treat human diseases has increased with the advances in recombinant DNA technology and the improved physical, chemical and biological methods of delivering genes to mammalian cells. Areas of therapeutic interest for gene therapy relevant for tissue engineering are, for example, in the treatment of wounds, skin diseases, nerve, bone, and muscle diseases. The transfer of a gene into a cell can lead to the addition or modification of a function and may be an attractive alternative to the pharmacological use of proteins. The complementation of defective functions could also be an effective treatment for inherited skin diseases with a gene defect. The two major challenges facing gene technology in tissue engineering are the problem of identifying appropriate genes that are effective in tissue repair, and the reliable expression of the therapeutic gene at clinically beneficial levels. This review discusses principles and methods of delivering genes encoding growth factors into cells, together with their respective advantages and disadvantages.

Experiences with rigid internal fixation using a low volume titanium implant system in metacarpal and phalangeal fractures

Abstract Our experience using the low volume titanium mini-fragment system ”Profile Titan” is presented retrospectively. The system contains self-tapping miniscrews with countersinking heads and very small adaptable plates with rounded edges. The study consists of 53 patients who underwent osteosynthesis, arthrodesis, or corrective osteotomy of the hand skeleton from 1 January 1994 to 31 March 1995. Functional results were evaluated and standard postoperative X-rays were taken at 1, 2, and 4 weeks after injury. Fifty-three patients with 56 fractures were treated. Results show that 78% of patients regained complete recovery of active range of motion. Fractures with and without negative prognostic factors were compared. There were three major postoperative complications not related to the type of implant. Compared to the AO mini system this system has several advantages.

Naturally occurring growth factors in porcine wounds treated with autologous keratinocytes in a liquid environment

Abstract A step toward an improved understanding of the complex mechanisms of growth factor interactions may lie in the detection of endogenous growth factors during normal wound healing. The findings of this study on standardized full thickness wounds in swine, provide direct evidence that growth factors were present in the wound fluid in the picogram range (highest concentrations ranging from 1273 pg/ml for transforming growth factor-beta (TGFβ) to 85.6 pg/ml for platelet-derived growth factor-AB (PDGF-AB ) during healing. The presence of transplanted autologous keratinocyte suspensions and cultured epithelial sheet graft had no significant effect upon the observed growth factor levels, although transplanted keratinocyte cell suspensions (KCS) and cultured epidermal autografts (CEA) did accelerate healing in comparison to control wounds in our model (KCS treated wounds healed in 13.2±0.9 days, CEA in 13.7 days±0.8 and control wounds in 14.7 days±0.3). The variable occurrence of growth factors during normal wound healing may suggest possible mechanisms of growth factor interaction which could have an impact on the future design of their therapeutic use.

Management therapierefraktärer hypovaskularisierter Wunden durch freie Muskelverpflanzung

Background: Chronic ulceration as a complication of arteriosclerotic disease, venous congestion or diabetes mellitus is still a serious clinical problem, resulting in immobilization, extended hospitalization and cost-intensive treatment. Other than standard conservative treatment protocols or early amputation, microsurgical free transfer of well vascularized muscle tissue onto chronic wounds can induce angiogenesis and improve wound healing even in the hypovascularized wound. Patients: From 1993–1999 we treated 12 patients (mean age: 46 years) with vascular ulcers of the lower extremity with free muscle or fasciocutaneous tissue transfer. Results: The average hospitalization was 51.4 days. The perioperative mortality was zero. In one patient with factor V deficiency a partial flap necrosis occurred. Two revisions of the micro anastomoses had to be performed. Two seromas occurred at the donor site. No secondary flap loss was observed. Extremity or stump length preservation was achieved in all cases. Conclu...BACKGROUND Chronic ulceration as a complication of arteriosclerotic disease, venous congestion or diabetes mellitus is still a serious clinical problem, resulting in immobilization, extended hospitalization and cost-intensive treatment. Other than standard conservative treatment protocols or early amputation, microsurgical free transfer of well vascularized muscle tissue onto chronic wounds can induce angiogenesis and improve wound healing even in the hypovascularized wound. PATIENTS From 1993-1999 we treated 12 patients (mean age: 46 years) with vascular ulcers of the lower extremity with free muscle or fasciocutaneous tissue transfer. RESULTS The average hospitalization was 51.4 days. The perioperative mortality was zero. In one patient with factor V deficiency a partial flap necrosis occurred. Two revisions of the micro anastomoses had to be performed. Two seromas occurred at the donor site. No secondary flap loss was observed. Extremity or stump length preservation was achieved in all cases. CONCLUSIONS Optimal postoperative treatment with physiotherapy and orthopaedic shoe support is important. If all these factors are present and if the patient is highly motivated a reintegration into normal life can be achieved.

Tissue engineering: a biotechnologic challenge and chance for the future of operative medicine

Tissue engineering (TE) has been defined as “the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes that restore, maintain, or improve tissue function” (Skalak & Fox 1988). There are prognoses that the economic volume of TE will soon exceed that of gene therapy, and that it may be one of the developments in biotechnology with the most impact. TE offers bright prospects especially for surgeons, and clinically practicing surgeons may play a major role to ensure the success of TE. Notably tissue engineering may offer more functional solutions for chronic diseases and degenerative ailments than conventional treatments. These chronic deficiencies constitute an increasing social and economic burden, especially in western societies with aging populations. Two relatively simple tissue types – epithelium (epidermis and oral mucosa) and cartilage – have already been introduced into routine clinical practice (BioSeedS/BioSeedC). They share the peculiarity that they do not need an intrinsic vascular system for parenchymal cell survival and function. Other clinically relevant tissue defects currently under investigation for this kind of tissue substitution include nervous tissue, cornea, liver, endocrine pancreas, kidney tubules, urothelium and bladder, adipose, cardiovascular and musculoskeletal tissues (bone, cartilage, muscle, tendons, ligaments), abdominal wall and many more. For practical application, tissue engineering approaches can be categorized into more substitutive approaches, where the aim is the ex vivo construction of a living tissue or organ similar to a transplant, versus histeoconductive or histeo inductive concepts. With regard to this aspect a frequently used synonym is regenerative medicine. The latter approach attempts to optimize the structure and composition of biogenic or (semi)synthetic matrix materials, boost or select living cells for optimal proliferation in vivo, and to use highly specific growth factors or gene therapy transfecting for such cytokines. For the successful development of products applicable in TE a close co-operation between material sciences, biology (cell biology, molecular biology, genetics), medicine (clinical sciences, embryology, anatomy, physiology) and industry is a prerequisite. Only a comprehensive understanding of the components involved (biomaterials, cells, cytokines, genes) can lead to successful manipulation of reparative processes. With increasing experimental success in the development of TE products, the importance of a complex medical technology (including minimally invasive surgery) to transfer them into clinical application becomes increasingly important. Materials used for TE applications must be biocompatible, non-toxic (including their breakdown products), non-carcinogenic, sterilizable and surgically handable. They should have an intrinsic mechanical stability as requested for the tissue type to be substituted for. In most cases they should be resorbable in the order in which they are replaced in vivo by the newly produced matrix. They should also be tissue conductive (guide migrating and proliferating cells) and possibly tissue inductive (induce specifically the proliferation of cells). To permit the survival of living cells they must be permeable for nutrients, cytokines and waste products. Ideally they may contain binding sites for cells (e.g. fibronectins). Currently used Min Invas Ther & Allied Technol 2002: 11(3) 85–86