High-resolution optical coherence tomography as a non-destructive monitoring tool for the engineering of skin equivalents.

BACKGROUND: Three dimensional skin equivalents are widely used in dermatopharmacological and toxicological studies and as autologous transplants in wound healing. In pharmacology, there is tremendous need for monitoring the response of engineered skin equivalents to external treatment. Transplantation of skin equivalents for wound healing requires careful verification of their quality prior to transplantation. Optical coherence tomography (OCT) is a non-contact, non-destructive imaging technique for living tissues offering the potential to fulfill these needs. This work presents an analysis of OCT for high-resolution monitoring of skin equivalents at different stages during the culture process. METHODS: We developed a high-resolution OCT imaging setup based on a commercially available OCT system. A broadband femtosecond laser light source replaces the original superluminescence diode. Tomograms of living skin equivalents were recorded with an axial resolution of 3 mum and correlated with histology and immunofluorescence images. Comparison with standard low-resolution OCT is presented to emphasize the advantages of high-resolution OCT for this application. RESULTS: OCT is particularly able to distinguish between different layers of skin equivalents including stratum corneum, epidermal and dermal layer as well as the basement membrane zone. The high-resolution OCT scans correlate closely with two key benchmarks, histology and immunofluorescence imaging. CONCLUSIONS: This study clearly demonstrates the benefits of high-resolution OCT for identifying living tissue structure and morphology. Compared with the current gold standard histology, OCT offers non-destructive tissue imaging, enabling high-resolution evaluation of living tissue morphology and structure as it evolves.

An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts.

To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air-liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi-layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell-adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three-dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.