Use of a collagen/elastin-membrane for the tissue engineering of dermis.

In an experimental model in rats, xenogeneic membranes consisting of processed native collagen and elastin were grafted to serve as a template for the formation of a neo-dermis, while in vitro-cultured autogeneic keratinocytes were applied on top of this to restore an epidermis. The process of tissue reconstruction and the digestion of the grafted membrane components were analysed by histological and immunohistochemical methods as well as electron microscopy. Approximately 3 weeks post grafting the membranes were completely vascularised and colonized by different types of cells. After 6 weeks, the collagenous fibres of the graft were mostly replaced by newly formed collagenous texture, whereas elastic membrane components were still present even after 20 weeks. In a second step, in vitro-cultured keratinocytes were applied onto the partially integrated membranes, resulting in an epithelial coverage of approximately 47% of the grafted area after 8-11 days. As early as on day 6 post application, a multilayered, partially differentiated epithelium, together with lymphocytes and Langerhans' cells, could be observed. After 10 days the formation of a basement membrane including anchoring fibrils appeared to be complete. This three-dimensional matrix structure offers a promising scaffold for a tissue engineering strategy to restore skin structure and function. Further experimental studies are in progress to test its applicability to human skin replacement.

Treatment of skin defects using suspensions of in vitro cultured keratinocytes.

The in vitro cultivation of keratinocytes and their application in the form of confluent sheets to cover various kinds of skin defects involves a number of problematical steps which could be improved by using single cell suspensions instead. Therefore we developed a method to apply keratinocytes suspended as single cells in a fibrin gel. By testing the feasibility of this method in different experimental animal models we found that it facilitates cultivation as well as application of the cells, moreover, this method allows a much more flexible use of the cells, i.e. it is easier to consider the clinical condition of the patient than by the conventional method.

Synthesis and application of new microcarriers for animal cell culture. Part I: Design of polystyrene based microcarriers.

In this work (Part I), surface modified styrene polymers as new microcarrier material for animal cell culture were extensively investigated. The first synthesis steps--carried out by chloromethylation, sulphonation and nitration of the polystyrene matrix--resulted in precursors with a defined surface layer thickness. The obtained hydrophobic bulk phase showed a limited absorption of hydrophilic media components compared to polysaccharides matrices like dextran. By varying reaction conditions for microcarrier synthesis and/or by using similar styrene type polymer matrices like polyvinyltoluene, the specific density (1.028-1.05 g/cm3) of the microcarrier matrix was adjusted without problems. Chemical varying of the microcarrier surface by reaction of the precursors with different amines, saccharides or proteins led to new microcarriers with optimal conditions for cell adhesion and cell growth. All biological investigations were carried out with a BHK 21 (c-13) cell line. Detailed results will be discussed and summarized in Part II of this work.

Synthesis and application of new microcarriers for animal cell culture. Part II: Application of polystyrene microcarriers.

In this work (Part II) the application of new polystyrene based microcarriers in cell culture technology is demonstrated. Carriers with a variety of surface modifications were tested as a growth support for cell line BHK 21. The growth behavior of the cells and cell to surface attachment were compared to Cytodex 3 (Pharmacia), which was used as a reference carrier. To select carriers with growth supporting surfaces, broad screening in petri dish experiments was carried out. Candidates with the highest growth rates were investigated in spinner flash experiments in further detail. Polystyrene carrier with a surface modification like triethylamine, maltamine or N-methylglucosamine were able to support growth as good or better as the reference carrier Cytodex 3. Economies of ingredients and ease in laboratory handling could make amine-modified polystyrenes a competitive alternative to currently commercially available microcarrier types.