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Ultrastructure of Three‐dimensional Bovine Hoof Keratinocyte Cultures
Author(s) -
Nebel U.,
Mülling Ch.,
Budras K. D.
Publication year - 2005
Publication title -
anatomia, histologia, embryologia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 35
eISSN - 1439-0264
pISSN - 0340-2096
DOI - 10.1111/j.1439-0264.2005.00669_84.x
Subject(s) - hoof , ultrastructure , epidermis (zoology) , keratinocyte , biology , stratum spinosum , anatomy , electron microscope , keratin , cytoskeleton , hemidesmosome , microbiology and biotechnology , cell , stratum corneum , cell culture , optics , paleontology , physics , genetics
The aim of this study was to characterize the ultrastructure of three‐dimensional keratinocyte colonies in relation to ex vivo bovine hoof epidermis. Keratinocytes were isolated from the claw and cultured in equal parts of Dulbecco's modified Eagle's medium (Sigma, Taufkirchen) supplemented with 10% fetal bovine serum (Sigma, Taufkirchen) and serum‐free MCDB 153 complete medium (Biochrom, Berlin). Cells were allowed to grow until formation of colonies occurred. Then the colonies were isolated by removing the surrounding cells with a cell scraper every 3 days. The colonies were harvested when the reached diameters of 6–8 mm and were fixed in Karnovsky's solution. They were routinely processed for the embedding in Agar 100. Semithin and ultrathin sections were prepared and examined under the light and transmission electron microscope. These colonies revealed the morphological properties and tissue architecture, which are specific for claw epidermis in vivo . We detected up to 22 layers of keratinocytes and could discover between three stages of differentiation: a basal cell layer, followed by several cell layers corresponding morphologically to the cells of the stratum spinosum and a few cornified superficial cell layers. Throughout all layers cells were connected by desmosomes, which were linked to the cytoskeleton by dense bundles of filaments. In addition we observed the formation of desmosomes. The cells of the upper un‐cornified layers generated an cornified envelope. In the following layers cell death and subsequent cornification accompanied by the characteristic alterations of the nuclei occurred. The cells at the superficial layers were filled by electron dense, solid horn masses. Additionally we detected intercellular cementing substance in the intercellular space between the upper layers. Our electron microscopical characterization of the colonies provides evidence that the bovine hoof keratinocytes in vitro undergo basically a similar pattern of proliferation and differentiation like in the claw in vivo . These findings provided a basis for further studies like the long‐term culture and differentiation of bovine hoof keratinocytes in perfusion chambers.