John R. Gibbins

Epithelial Migration in Organ Culture. A Morphological and Time Lapse Cinematographic Analysis of Migrating Stratified Squamous Epithelium

Summary The migration of stratified squamous epithelium in organ cultures of rat palatal explants has been studied using scanning and transmission electron microscopy. The scanning microscope revealed plate‐like folds at the margins, and microvilli on the bodies of cells. These structures were most highly developed on those cells nearest the leading edge of the sheet of cells and are interpreted as an index of cells that are migrating. The cells at the leading edge have broad flat pseudopodia in direct contact with the collagen bundles. A time lapse cinemicrographic study showed that the net forward movement of cells (nuclei) remote from the leading edge was at least as great as that at the leading edge immediately in front of them and the distance they travelled was greater than that of the leading edge. In transmission electron micrographs of these migrating epithelial cells from in vivo wounds, profiles that could correspond to the microvilli and plate‐like folds could be found on the surface of the migrating cells. The results of this study suggest a simple model for the particular type of movement that occurs in stratified squamous epithelium in healing wounds where a mass of cells is produced that can both migrate into the wound and undergo stratification and cornification. A tracked vehicle shedding a broken track is used as an analogy of the model proposed.

Adipogenic healing in adult mice by implantation of hollow devices in muscle

In mammals, wound healing is thought to result in the formation of scar tissue, with the exception of bony healing after fractures. Here we describe a previously unknown pattern of wound healing in which adipose rather than scar tissue is formed. Adipogenesis is normally confined to the embryo, although there are several experimental models for adipogenesis with highly specific dietary, cytokine, matrix, sex, or age requirements. The adipogenic healing described in this work provides a simple and reproducible experimental mouse model for adipogenesis without these limitations. Mice received intramuscular implants of nylon mesh material. Fibrinous material impregnated implants and within 4 weeks was replaced with highly vascular granulation tissue, typical of wound healing. Also consistent with wound healing was a reduction in vascularity of the newly formed tissue over time (P < 0.05). Lipoblasts were prevalent in granulation tissue, reaching a maximum in week 2 (P < 0.001) but falling to very low levels by week 9. These cells matured to adipocytes, with intermediate forms being seen. The identity of lipoblasts and adipocytes was confirmed by Oil Red O staining and electron microscopy. Control experiments confirmed that adipogenesis was independent of the materials used as well as of the sex and age of the animals. Rather, adipogenesis appeared to be due to replacement of fibrinous material in a space created within muscle. It is possible that adipogenic healing represents an adaptation for limiting the formation of restrictive scar tissues within muscle, and that this is the basis for the formation of traumatic lipomas in humans. Also, muscle tissue is replaced by adipose cells, seemingly derived from pluripotential satellite cells, in several degenerative muscle conditions, suggesting a role for adipogenic healing in these conditions. Anat Rec 267:28–36, 2002.

Differential expression of transforming growth factors-β1, -β2, -β3 and the type I, II, III receptors in the lining epithelia of inflamed gingiva

Aims: To investigate the distribution of transforming growth factor‐&bgr; isoforms in chronically inflamed periodontal tissues. Methods: The present study determined, by immunohistochemistry, the expression patterns of TGF‐&bgr;s and their receptors in the lining epithelia of inflamed gingiva. Frozen sections were obtained from 22 human gingival biopsies. Results: TGF‐&bgr;1 was not detected in gingival epithelial cells in examined sections. Detection of TGF‐&bgr;2 indicated a progressive reduction of staining from the external oral epithelium through to gingival sulcus and the gingival attachment or pocket epithelium. TGF‐&bgr;3 showed intense staining in all domains of both minimally inflamed gingiva and advanced periodontitis tissues. TGF‐&bgr; RI was visualised as focal staining of the spinous layer in the external oral epithelium of both periodontitis lesions and minimally inflamed tissues. TGF‐&bgr; RII was present throughout the strata, but with progressive reduction in intensity from the oral epithelium to gingival attachment or pocket epithelium respectively while, conversely, TGF‐&bgr; RIII showed an increase in diffuse staining intensity from external oral epithelium to pocket epithelium. Conclusions: A distinct expression profile was observed within different individuals for TGF‐&bgr;s and the corresponding receptors. These findings provide a basis for evaluation of the role of these growth factors in the pathogenesis of periodontitis.

MICROVASCULAR ANGIOPATHY IN ADVANCED PERIODONTAL DISEASE

Previous studies have shown a perivascular hyaline thickening affecting restricted regions of the microcirculation in gingivitis and moderate periodontitis and in the pulpal vessels in chronic pulpitis. In the present study of the lesion of advanced periodontitis, immunostaining for type IV collagen and laminin demonstrated widespread deposition of basement membrane material, with manifest involvement of the venous network. Some vessels were associated with an increased deposition of both basement membrane proteins, while others showed preferential deposition of either laminin or type IV collagen. Immunostaining also revealed an extensive trabecular network of type IV collagen throughout the affected gingival tissue that was not related to recognizable vessels but was co‐extensive with less intense staining for laminin. This network was not associated with viable endothelial cells demonstrable by staining with the endothelial marker Ulex agglutinin (UEA‐1). The results indicate extensive vascular pathology in advanced periodontitis that could explain the attenuation of the inflammatory reaction and the restricted ability to develop reparative granulation tissue in this disease.

Vascular co-localization of proteolytic enzymes and proteinase inhibitors in advanced periodontitis

Sections of tissue biopsies obtained from advanced, destructive periodontitis were compared with minimally inflamed periodontal tissues in relation to the distribution of type I (interstitial) collagenase. Immunohistochemistry using a highly specific antiserum showed weak staining of occasional vessels in minimally inflamed specimens but widespread reactivity, localized to the vasculature, in advanced disease. In situ hybridization confirmed the vascular source of type I collagenase. Minimally inflamed tissues did not react with antibody to urokinase‐type plasminogen activator, a potential activator of pro‐collagenase, but there was a consistent strong reaction in advanced disease. Antibody to tissue inhibitor of metalloproteinase (TIMP)‐1 did not react with minimally inflamed tissues, but gave intense, widespread vascular staining in advanced disease, whereas antibody to TIMP‐2 produced localized connective tissue staining. These results indicate that upregulation of proteinases and inhibitors related to the vasculature is an integral component of destructive periodontitis.

Migration of stratified squamous epithelium after injury in vivo and in organ culture

&NA; As a basis for detailed study of the mechanisms of epithelial migration following injury, an in vitro organ culture technique is described and the migration of the epithelial cells is compared in vivo and in vitro. Judged by morphological criteria, the behaviour of the epithelial cells during the first 48 hr. in vitro and in vivo is similar. After 1 wk. the similarity in the appearance and behaviour of the cells decreases.

Analysis of the amplification refractory mutation allele-specific polymerase chain reaction system for sensitive and specific detection of p53 mutations in DNA

The sensitivity of the amplification refractory mutation allele‐specific polymerase chain reaction system (ARMAS‐PCR) to detect known p53 mutations was determined using DNA extracted from two human tumour cell lines collected by cytobrush, as a model for its use in exfoliative cytology. Using DNA extracted from SW480 and CEM cell lines diluted with normal human fibroblasts, a nested ARMAS‐PCR was more sensitive than a non‐nested version and could detect one mutated cell amongst 100 000 normal cells. When compared with PCR–single stranded conformational polymorphism, nested ARMAS‐PCR was 10 000 times more sensitive for detecting mutant p53 in extracted DNA. Primer design proved to be influential on the sensitivity and specificity of the assay; increased specificity was achieved by the use of deliberate mismatches upstream from the 3′ end of mutation‐specific primers. ARMAS‐PCR was confirmed to be specific for the mutation that each primer was designed to detect. Nested ARMAS‐PCR offered a rapid and sensitive method of analysis of cells with predetermined p53 mutations and has the potential to be applied to the study of the molecular progression of cancer, including diagnosis and detection of residual disease. It could also be extended to the in situ detection of aberrant cells. Copyright

Multiple levels of post-transcriptional regulation of collagenase (matrix metalloproteinase 1) in an epithelial cell line.

Multiple levels of regulation of collagenase (matrix metalloproteinase 1; MMP‐1), have been demonstrated in a clonal rat epithelial cell line (A5P/B10). Secreted enzyme could not be demonstrated in culture medium from A5P/B10 cells but, using antibodies specific for collagenase, the enzyme was detected within the cytoplasm and on the surface of the cells. A probe for rat collagenase could not detect a signal for mRNA in the cytoplasm while nuclear run‐on data demonstrated that the gene for collagenase was being transcribed. Incubating the cells with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) significantly increased cytoplasmic mRNA levels and slightly increased the intensity of staining in permeabilized cells, but collagenase activity was still not detected in the conditioned medium. This indicated that the protein was being synthesized by the TPA‐treated cells but was not being secreted into the medium. These data suggest that the secretion of collagenase may be regulated both following transcription and after the completion of translation and it is suggested that multiple levels of control may be operating to determine the rate of collagenase release and hence, the rate of collagen turnover.

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.

Non-coexpression of collagenase and transin in malignant epithelial cells

Increased synthesis and secretion of the matrix metalloproteinase has been associated with the acquisition of the invasive properties of malignant cells. In a rat malignant anaplastic cell line (T952/F7) sporadic expression of mRNA for collagenase-3 against a background of high transin-l mRNA has been shown. Sporadic induction of either metalloproteinase was not found in the benign precursor cells (A5P/B10) of T952/F7 cells, but a coordinate mRNA induction profile for these two genes could be induced by the addition of 12-O-tetradecanoylphorbol-13-acetate. In a different uncloned neoplastic epithelial cell line (BCl) fluctuations in the levels of collagenase and transin appeared coordinately, but coordinate expression was not a feature in cloned lines derived from BCl. The loss of coordinate regulation of the two matrix metalloproteinases may relate to the variability in metastatic potential seen in clonal cell lines.