Histological and Histochemical Methods for the Evaluation of Tissue Engineered Skin Substitutes

A thorough evaluation and characterization of advanced therapy medicinal products (ATMPs), including tissue-engineered skin substitutes (TESS), is an essential requirement of the European and National Medicines Agencies. Although clear regulatory criteria have been established for different parameters such as stability, sterility or viability, the histological characterization of bioengineered tissues remains inadequately defined. Histological analyses are crucial for assessing both the in vitro features and in vivo behavior of TESS. This chapter describes the essential histological methods used for assessing TESS, including hematoxylin and eosin staining, and several methods focused on the analysis of specific components of the skin cells and extracellular matrix, such as Picrosirius Red for collagen fibers, Fontana Masson Picrosirius for melanin and collagen, Verhoeff for elastic fibers, Alcian Blue for proteoglycans, and Periodic acid-Schiff for glycoproteins. Relevant immunohistochemical methods for the identification of some specific markers are also included in this chapter. These methods provide a comprehensive practical approach to assessing both in vitro and in vivo histological features and evolution of TESS.