Fibroblasts accelerate culturing of mucosal substitutes

Reconstruction of large mucosal defects of the floor of the mouth is typically performed with keratinizing skin. Drawbacks include donor site defects and hair bearing of the flaps. Cultured mucosal substitutes (CMSs) have been developed for clinical use to replace keratinizing skin. Acellular dermis is often used as a dermal carrier for autologous cells, because it reduces wound contraction and is easier for the surgeon to handle than, for example, collagen gels. A major problem of CMSs using acellular dermis is variation in epidermal quality. To improve the quality of the CMSs, human fibroblasts were incorporated into the acellular dermis and seeded with human keratinocytes. To study the role of the fibroblasts in epidermal morphology and basement membrane formation, CMSs were stained for differentiation markers beta1 integrin, cytokeratin 10, and involucrin after 1 and 2 weeks in culture. Basement membrane formation was analyzed using laminin 5 and collagen IV and VII staining; proliferation was analyzed using Ki-67 staining. The epidermises of fibroblast-containing CMSs matured faster into a well-organized epithelium than did those that did not contain CMSs. A 52.7% increase in basal cells, a 53.5% increase in mitosis index, and a 78.0% increase in keratinocyte cell layers were observed. Addition of fibroblasts reduced culturing time and enhanced proliferation, maturation, and quality of the epidermis.