John H. Lillie

Fine structure of subcultivated stratified squamous epithelium grown on collagen rafts.

Abstract Subcultivated rat lingual epithelial cells when grown on collagen gels at a liquid-gas interface achieve a highly ordered state that closely resembles the parent tissue. Three distinct cell layers are present; basal, spinous, and keratinized. Basal cells are cuboidal in shape and form a complex interface with the underlying collagen fibrils. Spinous cells form a layer 5–10 cells thick and, with the exception of keratohyalin granules, possess an organellar complement identical with native cells, including membrane-coating granules. The keratinized cell layer increases in thickness as a function of time spent in culture. Forty or more plies of terminally differentiated cells are observed following a 30-day culture period. Terminally differentiated cells while retaining pycnotic nuclei and some other organellar debris are principally envelope-enclosed squames filled with tonofilaments. Keratinization is a continuing process which occurs simultaneously across the full expanse of the culture surface. The high degree of tissue organization observed appears to be the result of feeding the cultures from the undersurface.

Bovine corneal endothelium in vitro: Elaboration and organization of a basement membrane

Abstract Bovine corneal endothelial cells can be easily grown in culture using conventional techniques. The cultured cells closely resemble the parent, native endothelium. In culture the endothelium synthesizes and deposits, in a polar fashion, a well organized basement membrane that contains molecules which are characteristic of all basement membranes. Membrane deposition continues for at least a year and, during that time, it begins to acquire the unique, ordered substructure characteristic of the native membrane.

Collagen structure: evidence for a helical organization of the collagen fibril.

The collagen fibrils of human or guinea pig dermis when exposed to the denaturing agents, urea or guanidine—HCl, dissociated into smaller, disparate subunits, probably aggregates of microfibrils. The process of dissociation demonstrates that the fibrils are assembled helically. Initially, diagonal clefts appear on the surface of the fibril. These clefts are surface manifestations of a spirally oriented, internal space. Continued exposure to these denaturants resulted in progressive dissociation of the fibril into helically oriented subunits. It is suggested that water-miscible compounds such as glycols or hydroxypropyl methacrylate, in addition to the urea—guanidinium class of denaturants used in this study, affect the observed fbrillar changes through the disruption of hydrogen bonds between the microfibrils making up the fibril. Such a mode of action may explain why freeze-fractured or “inert embedded” collagen demonstrates helical organization while other, more conventional methods of tissue processing do not. Further support for the proposed mode of action of these dissociative agents was provided by the observation that mature collagen, in which extensive intra- and intermolecular covalent crosslinks are present, is more resistant to dissociation than newly formed collagen.

Fine structure of subcultivated stratified squamous epithelium

Abstract Subcultivated rat lingual epithelium derived from primary expiants remains mitotically active, possesses an organellar complement similar to the parent tissue, and undergoes terminal differentiation. Successful growth of primary cultures requires an incubation temperature below 34 °C and the addition of dimethyl sulfoxide (DMSO) to the medium. The subcultures retain a stable morphological phenotype through a minimum of 15 passages. Cultures are long-lived and may be maintained for one year or more in any passage.

Penetrating Keratoplasty in the Cat: A Clinically Applicable Model

A series of 28 consecutive penetrating keratoplasties were performed on adult cats. Donor corneas (n = 14) were maintained in culture medium for 14--24 hours prior to transplantation. Rotational autografts (n = 7) were used to control for cell loss caused by culture maintenance as well as for the effects of surgery. Additional homografts (n = 7) were transplanted following removal of the corneal endothelium to study the extent of host corneal endothelial cell regeneration. Pre- and post-operative endothelial cell counts of the homografts made from specular micrographs demonstrated an average cell loss of 30% one month following surgery. A similar 30% average cell loss was present in the rotational autografts. Clinically, both homografts and autografts remained clear and were near normal in thickness. Homografts lacking endothelium exhibited persistent, severe edema that correlated with the inability of the host corneal endothelium to resurface the graft. Clinical and morphologic evidence of mild homograft rejection as observed in 15% of the animals that received normal homografts. Corneal endothelial cell loss following penetrating keratoplasty in the cat approximates that observed following the same procedure in the human. Additionally, regenerative capacity of the corneal endothelium in the cat, like that of the human, is limited. These features suggest that this cooperative, hardy animal is an excellent model in which to study many aspects of corneal transplantation that have direct application to the treatment of human corneal disease.

Evidence for Autoregulation of Cell Division and Cell Transit in Keratinocytes Grown on Collagen at an Air-Liquid Interface

Oral and epidermal rat keratinocytes when cultured on a matrix of type I collagen fibrils at the interface between the gaseous and liquid phases of a culture form a highly ordered stratified squamous epithelium. Autoradiographic studies of cells labeled by tritiated thymidine indicate that the keratinocytes are capable of autoregulating cell division. Early confluent cultures exhibit 51% of basal cells labeled, a percentage that decreases to 18% when a full differentiated stratified squamous epithelium is formed. Such a decrease in labeling occurs in cultures where the mitotically active basal cells have unimpeded access to culture medium supplied from below and when no cell type other than the keratinocyte is present in the culture. Additionally, the transit of keratinocytes from the basal cell layer through other viable cell strata to the layer of terminally differentiated cells can be followed by tracking cells labeled with tritiated thymidine. In cultures of oral keratinocytes, cells move from the basal cell layer to the cornified layer at a maximum rate of 7 days, while virtually all labeled cells (91%) are localized in the terminally differentiated cell layer 14 days following labeling. Keratinocyte cultures grown in culture at an air-liquid interface exhibit tissue organization that closely resembles the native, parent tissue. Such cultures can be useful in studying the effects of pharmacologic mediators of cell division and cell transit.

Magnetic resonance imaging of the normal craniovertebral junction

Sagittal magnetic resonance images of the normal cran-iovertebral junction In 25 patients were examined for visualization of bony, synovial, and ligamentous structures. The excellent delineation of soft tissue by magnetic resonance imaging enabled recognition of the joint space between the dens and anterior arch of C1 in 14 out of 25 patients. High-signal-intensity tissue was noted immediately superior to the dens in all patients; an anatomic specimen confirmed the flbrofatty nature of this tissue. The medullary space of the dens had lower signal intensity than did the marrow in the body of C2 in more than one half of the cases. Additional thin-section images suggested that this was a partial-volume artifact. Understanding of the normal appearances of structures in this region is necessary to assess correctly the presence or absence of disease.

The Culture of Oral Epithelium

Publisher Summary The carefully regulated pattern of stratified squamous epithelial differentiation within the oral cavity has intrigued several generations of investigators, many of whom attempted to remove the mucosa from the body and place it in the “controlled environment” of cell and organ culture to gain insight into factors controlling both epithelial differentiation and the modeling of the epithelial-connective tissue interface. The oral mucosa is also clinically important. Organ culture involves the removal of a small (1–2 mm 3 ) tissue fragment or embryologic organ rudiment from an animal and placing it on a support at an interface between a nutrient medium and a humidified gaseous environment. This is the procedure that most investigators have used when studying oral mucosa in vitro . The objective when using the technique of organ culture with adult tissues is the maintenance of a differentiated state, especially when one or more cell types are present or where the interaction of two tissues is necessary.

Electron microscopy of cytoplasmic crystalloids in rat parotid glands.

Summary-Crystalloid-containing bodies that may be lysosomes were observed by electron microscopy in acinar and striated duct cells of normal fasted rat parotid glands. The crystalloids varied in number from animal to animal and from lobule to lobule. They consisted of either parallel linear densities with a 5Snm periodicity, or of alternating major dense and minor light lines, with a 15 nm distance between successive major lines. it was concluded that the presence of a few such crystalloids in parotid glands of treated rats is not a cytopathic effect and they are significant only when present in large numbers. lNTRODUfflON Cytoplasmic crystalloids, with and without limiting membranes, occur normally in a variety of glands, such as thyroid (Nunez, Gould and Holt, 1970; Elvin, 1971; Neve and Wollman, 1973) and exocrine pancreas (Forsmann and Metz, 1976), in many different organisms. Moreover, crystalloids can be induced to form in these and other organs when animals are subjected experimentally to the appropriate physiological, pharmacological or nutritional stress. In the course of an ultrastructural study of rat parotid gland, we have found crystalloids in the glands in normal fasted animals.

Effect of Age and Keratoplasty on the Postnatal Development of Feline Corneal Endothelium

Unilateral autologous rotational penetrating keratoplasties were performed on 10 4-week-old kittens. Three eyes were lost to glaucoma secondary to anterior synechiae formation, four eyes were examined histologically 2 weeks to 1 month postoperatively, and the three remaining eyes were examined serially for 9 months by slit lamp observation, specular microscopy, and pachometry. Contralateral unoperated control eyes demonstrated a rapid decline in endothelial cell density during the first 8 weeks of life (6000 cells/mm2 to 3800 cells/mm2), a change which correlated with the growth of the cornea to its adult size. Thereafter, the endothelial density declined more gradually (2800 cells/mm2 at 6 months). Transplants exhibited a 50% endothelial cell loss 2 weeks postoperatively that related to the keratoplasty procedure, and the endothelial cell density subsequently declined gradually with age. Corneal thickness comparisons of unoperated and postkeratoplasty eyes suggests that the endothelium of the infant kitten has less functional reserve than the adult endothelium. Corneal endothelial cell counts made from infant and adult dogs, rabbits, and humans indicate that the high postnatal endothelial cell density observed in the cat also occurs in other mammalian species.