Three-Dimensional Culture of Keratinocytes and the Formation of Basement Membrane for Canine Footpad Substitute

Abstract

ABSTRACT. A pad equivalent for a dog was prepared as a substitute for the loss of footpad. In addition to the time course of formation on epidermal morphogenesis, we investigated expressions of α 6 integrin subunit as adhesive molecule, and laminin and type IV and VII collagens as extracellular matrices of basement membrane components. Epithelium of the pad equivalent was thick enough to be easily confirmed at 5 days at the air-liquid interface, but many creases appeared on it at 7 days, and it shrank at 10 and 14 days. Keratinocytes were increased in 4 to 5 cell layers at 1 day at the air-liquid interface, differentiating into basal cell layer. Granular and corneal cell layers were confirmed until 5 days, and maintained their shape at least until 14 days. Alpha 6 integrin was expressed at almost the same fluorescent intensity as native pad tissue at 1 day at the dermal-epidermal junction. Laminin and type IV collagen were intermittently expressed at 5 and 10 days, respectively, at the dermal-epidermal junction, and at 14 days the fluorescence showed almost the same intensity as native pad tissue. The expression of type VII collagen was discontinuous at 2 days at the dermal-epidermal junction, but remained as it was at 14 days. The present findings suggested that although the formation of anchoring fibrils in basement membrane was incomplete, the pad equivalent in the dog was reconstructed similar to a native pad by epidermal morphogenesis. KEY WORDS: air-liquid interface, canine, epidermis, footpad, keratinocyte.

Adjuvanting Allergen Extracts for Sublingual Immunotherapy: Calcitriol Downregulates CXCL8 Production in Primary Sublingual Epithelial Cells

“…All sublingual epithelial cells stained positive for this marker. This is in agreement with findings in humans and dogs, which showed that CD49f is a reliable marker to identify epithelial cells (30)(31)(32)(33).…”

Phenotypic Analysis for a Cell Line of Canine Epidermal Keratinocytes

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“…The present study showed that CPEK was positive for cytokeratin 14 and PCNA but negative for cytokeratin 10, demonstrating phenotypic similarity with basal keratinocytes. For additional characterization of CPEK, we also investigated expression of an adhesion molecule, α 6 integrin, which is exclusively expressed on the epidermal-dermal junction of basal keratinocytes [2,11,15]. In basal keratinocytes, flowcytometric analysis could separate two subpopulations into proliferative undifferentiated (α 6 bri ) or postmitotic differentiating (α 6 dim ) keratinocytes based on expression of α 6 integrin [9].…”

Reconstruction of stratum corneum in organotypically cultured canine keratinocyte-derived CPEK cells

“…An organotypic culture of the CPEK cells was performed on the basis of the protocol reported by Yamazoe et al (2007) with modifications. The CPEK cells were suspended in complete CnT-09 medium (0.5×10 6 cells/ml) supplemented with 10 −8 M all trans-retinoic acid, 0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin, 5 μg/ml transferrin, 2 nM 3,3′,5-triiodothyronine, 10 ng/ml recombinant human epidermal growth factor (Invitrogen, Carlsbad, CA), and 5 μg/ml recombinant human insulin (Invitrogen).…”