J. P. Ouhayoun

A comparative biochemical and immunological analysis of cytokeratin patterns in the oral epithelium of the miniature pig and man

In man, cytokeratin constitutes a family of 19 polypeptides that show different but distinct distribution patterns in the various epithelia. Changes in these patterns may occur during epithelial development and differentiation. The cytokeratin patterns in the oral mucosa of the miniature pig, an animal used in studies of wound healing, were investigated. Surgical biopsies were obtained from the gingiva, hard palate and alveolar mucosa of both man and pig. The cytokeratins were analysed by immunofluorescence, two-dimensional gel electrophoresis and by immunoblotting. Nine monoclonal antibodies were used to identify the different cytokeratin polypeptides in cryostat sections. Two-dimensional gel electrophoresis showed that pig oral mucosa contains at least 10 different polypeptides, five of the acidic type I and five of the basic type II cytokeratins. These were different from the human cytokeratin polypeptides and accordingly were designated P1-P10, according to their molecular weight and isoelectric mobility. Their molecular weight varied between 48 and 69 kdalton and the pHi varied between 5 and 7.3. Immunoblotting showed the monoclonal antibody Ks 13.1 (anticytokeratins Nos 13 and 14) to cross-react with the pig polypeptides P10 and P8. Immunolocalization showed that all the antibodies cross-reacted with the pig tissue except Ks 19.1 (anticytokeratin No. 19). It was possible to differentiate between pig alveolar mucosa, which expressed only P3, P4, P5, P8 and P10, and the gingival and hard palatal mucosae, which expressed all 10 polypeptides except P5. This distinction was made by antibody 6B10 (anticytokeratin No. 4), which reacted only with alveolar mucosa; antibody Ks 13.1, which strongly reacted with uncornified mucosa but weakly with cornified mucosa (gingiva and palate); and any of RKSE60, Kk 8.60 or EE21.6 (anticytokeratin No. 10, anticytokeratins Nos 10 and 11 and anticytokeratins Nos 1, 2, 10 and 11, respectively), which reacted strongly with cornified mucosa but weakly, if at all, with uncornified mucosa. These findings provide a baseline for studies on epithelial differentiation in the miniature pig such as in wound healing.

Histological evaluation of natural coral skeleton as a grafting material in miniature swine mandible

Natural coral skeleton (NCS) has recently been proposed as a bone graft substitute that enhances bone formation. The present paper describes the effects of implanting NCS in bone cavities prepared in the mandibles of miniature pig, and compares these with the effects of two alloplastic materials; a tricalcium phosphate (TCP) and a porous hydroxyapatite (PHA). On 11 pigs, 5 × 5 mm windows were created through alveolar bone of the four mandibular incisors. Three cavities were filled with the various materials and the fourth was left unfilled. The animals were slaughtered at 0, 1, 2, 4, 12, 26 and 52 weeks post-operatively and the tissues were examined histologically. Healing completed at 26 weeks for NCS and TCP, and at 52 weeks for PHA. NCS granules provided surface for cell attachment and deposition of a distinguishable organic matrix two weeks post-operatively. This matrix developed to bone after four weeks. The granules gradually resorbed and were replaced by bone at 52 weeks. The excellent properties of NCS, biocompatibility, porosity and osteogenic effect make us suggest that it might be a suitable replacement for bone grafting.

Ultrastructural study of the effects of coral skeleton on cultured human gingival fibroblasts in three-dimensional collagen lattices

Natural coral skeleton has recently been introduced as a bone graft substitute which enhances bone formation in man and animals. The effects of NCS on cultured human cells has not previously been investigated. In the present study we report these effects as studied by light microscopy, transmission and scanning electron microscopy in three-dimensional culture. The results showed that natural coral skeleton does not inhibit the normal function of fibroblasts in contracting collagen lattices. After 8 weeks, the cells maintained a healthy ultrastructural morphology. At the collagen/coral interface, the cells were well-spread and attached to the surface by numerous adhesion plaques. Evidence for biosynthetic activity was also observed; the cells showed numerous ribosomes, mitochondria and prominent rough endoplasmic reticulum. Extracellularly, a perigranular dense matrix, appearing as nodules in the SEM, was deposited on the coral surface. This matrix was made of highly organized fibrils lacking periodicity, and a ground substance. The present study shows that coral was well-tolerated by human gingival fibroblasts, and that it provided a surface for cell spreading, attachment and deposition of the special extracellular matrix.