The bicentenary of the birth of Sir John Tomes.

John Tomes was initially destined for a career in medicine but after being introduced to dental histology, changed careers and gave the first series of lectures on dentistry in Middlesex Hospital. His early academic career resulted in four major papers on the structure of teeth and bone that were read before the Royal Society, establishing his reputation as a leading histologist. He was awarded an FRS at the age of 35. The last 40 years of his life were devoted to establishing dentistry as a regulated profession with the founding of a dental hospital, examinations undertaken by the Royal College of Surgeons, a dental register and regulation by the British Dental Association. He was knighted in 1886. The bicentenary of his birth was recognised with the restoration of his grave.

An immunohistochemical analysis of human Merkel cell density in gingival epithelium from dentate and edentulous subjects.

OBJECTIVE: To investigate whether there is a difference in the density of Merkel cells between the gingiva of dentate and edentate subjects. METHODS: One hundred and two blocks of human mandibular (n=55) and maxillary (n=47) gingiva from 69 white skinned individuals (44 males, 25 females, mean age 70.1 years, range 42--92 years) were analysed. Twenty-six individuals had teeth remaining in at least one jaw (mean age of dentate 64.5 years, edentate 72.1 years), of which 15 were dentate in both jaws. Overall, 24/55 mandibles and 17/47 maxillae were dentate. Five micrometer sections were stained with a monoclonal antibody to cytokeratin 20 (CK 20) using standard immunoperoxidase or immunoalkaline phosphatase methods. Positive cells were counted in 20 consecutive high power fields using the x 40 objective in three sections from each biopsy, and the results analysed for differences related to age, sex, site and presence of teeth. RESULTS: CK 20-positive Merkel cells were present either singly or in clusters in the basal epithelial layers, particularly at the tips of rete ridges. Numbers of gingival Merkel cells were significantly higher (p<0.005, Mann-Whitney) in edentate (cells per field mean, median, standard deviation, respectively: 1.39, 0.64, and 1.85) when compared to dentate (0.67, 0.20, and 1.21) specimens. The differences were not statistically significant for any other variable. CONCLUSION: The data raise the possibility that reduced perception following loss of teeth may be compensated for by an increase in the local Merkel cell population.

The differentiation profile of the epithelium of the human lip.

The aim of this study was to analyse the immunohistochemical differentiation profile of the stratified squamous epithelium of the adult human lip. Full-thickness lower lips taken from 31 cadavers were analysed. Sections were stained with haematoxylin and eosin, periodic acid-Schiff (PAS), cytokeratins (CK), loricrin, involucrin, profilaggrin and filaggrin. The stratified squamous epithelium covering the lip could be divided into: (i) appendage-bearing, orthokeratinised epidermis; (ii) orthokeratinised vermilion which had a more prominent rete pattern than the epidermis; (iii) parakeratinised, PAS-positive intermediate zone; and (iv) non- or parakeratinised labial mucosal epithelium. Epithelial thickness increased gradually from the skin to the mucosal aspect. The CK pattern changed across the intermediate zone, with gradual loss of CK 1 and 10 from the skin, and CK 4, 13 and 19 from the mucosal, aspect. CK 5 and 14 were consistently expressed basally, and variably expressed suprabasally. Apart from labelling Merkel cells, CK 8, 18 and 20 were negative. Involucrin, which was present at all sites, was restricted to the stratum granulosum in skin, but extended into the stratum spinosum, and gradually into parabasal keratinocytes, across the vermilion and mucosa. Loricrin, profilaggrin and filaggrin were present in the stratum granulosum of orthokeratinised sites, but expression was abruptly lost at the junction between the vermilion and the intermediate zone. In conclusion, the phenotype of the stratified squamous epithelium covering the lip changes at, or across, the intermediate zone of the adult vermilion. It is possible that changes in the composition of the stratified squamous epithelium affect the colour of the vermilion.

Detailed morphology and distribution of gap junction protein associated with cells from the intra-articular disc of the rat temporomandibular joint.

The present study details structural aspects of the cells of the rat intra-articular disc (IAD) of the temporomandibular joint. At the light and electron microscope level, the outline of the cells varied, although a number appeared oval/rounded. Ultrastructurally, the cells contained moderate amounts of the intracellular organelles associated with protein synthesis. Cell processes were not a prominent feature seen using these techniques. However, using confocal microscopy, phalloidin staining revealed the presence of numerous, actin-rich, long, thin, branching processes emanating from the cell membranes. Double staining for connexin 43 revealed that the cell processes showed dense, punctate positive staining for this gap junction protein along their whole length. These observations provide the basis for a transport mechanism for nutritive molecules from the periphery of the IAD (where blood vessels are present) to the central portions of the disc (where blood vessels are absent).

Morphometric appraisal of the skull of Caroline Crachami, the Sicilian "dwarf" 1815?-1824: a contribution to the study of primordial microcephalic dwarfism.

The skeleton of Caroline Crachami is a rare historical example of primordial microcephalic dwarfism (PMD). Studies show the condition to be heterogeneous, with at least three types, for which the assessment criteria rely on descriptive evaluations and/or simple measures with regard to cranial features. Advances in noninvasive imaging allow for a more complete morphometric examination of the skull of Caroline Crachami with the aim of clarifying aspects of the condition. In the present study, the skull of Caroline Crachami was three-dimensionally imaged with computed tomography (CT) and reconstructed in virtual space. Coordinates for a number of measurements were taken to represent interesting anatomies with an emphasis on those measures not easily replicated on the skull itself. Volumes of the endocranial cavity and sella turcica were also computed. These data were compared with normative values taken from the literature and measured from CT images of the Bosma collection. Findings indicate that the general size of the skull is equivalent to that of a 6- to 8-month-old, that the endocranial volume and cranial base angle are commensurate with that of a newborn, and that the sella volume is the same as that for an 8- to 15-month-old. Apart from these traits, the skull was well proportioned and within the range of normal variation for a skull aged between 2-9 years. We conclude that further quantitative analysis on related skulls is warranted in the study of PMD using the methods and techniques described.

Ultrastructure of the human intra-articular disc of the temporomandibular joint.

The ultrastructural appearance of the human intra-articular disc (IAD) was investigated in three discs that had been surgically removed due to disease from three female patients aged 47, 50, and 54 years of age. Regions of the IAD were selected from central areas that appeared to be least affected by disease. Sections were fixed in 2.5 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 7.3 immediately after surgery. The regions examined showed no obvious signs of inflammation. The cells showed moderate amounts of the intracellular organelles associated with protein synthesis and secretion, and possessed considerable amounts of microfilamentous material, thus resembling those described in other mammals. Despite the large number of cells examined, only one cell showed evidence of a chondrocyte-like morphology in that it possessed an incomplete pericellular zone of microfilamentous material separating the cell membrane from the adjacent collagen bundles of the extracellular matrix (ECM). Thus, on morphological grounds, fibrocartilage was virtually non-existent in the specimens examined. The mean collagen fibril diameter was 43.9 nm and the fibril diameter distribution was not unimodal. Although the majority of fibrils had a relatively small diameter, two of the three specimens possessed many fibrils with diameters of over 100 nm, this being consistent with tissue subjected to tension. The mean area of a fibre bundle occupied by collagen (as opposed to the ground substance) was approximately 56 per cent.

The distribution of retinaldehyde dehydrogenase-2 in rat and human orodental tissues.

Retinoic acid is an important signalling molecule in embryological development and continues to be important in the adult animal because it modulates growth and differentiation in many epithelial tissues. The distribution of the enzyme retinaldehyde dehydrogenase-2 (RALDH 2), which is involved in the synthesis of retinoic acid, was studied using immunocytochemical techniques in: (1) the developing orodental region of rats aged between 15 days in utero and 6 months; and (2) in archival human autopsy material consisting of abdominal skin and mucosa from various regions of the mouth. In developing tooth germs, RALDH 2 was absent in the enamel organ and dental papilla, its presence only being noted at the periphery of the dental follicle adjacent to parts of the developing alveolar crypt. In adult teeth, the presence of RALDH 2 was limited to blood vessels in the periodontal ligament. In embryos, the connective tissue beneath the nasal epithelium and the meninges stained strongly positively for RALDH 2, as did the connective tissue beneath nasal epithelium in an adult rat. Both keratinized and non-keratinized human oral epithelia and abdominal skin stained positively for RALDH 2. Staining was present throughout the stratified epithelium, except in the keratinized layer and in the basal layer associated with the dorsal surface of the tongue. In addition, the adnexia as well as the ductal lining of mucous glands stained positively for RALDH 2.

An immunohistological study of cytokeratin 20 in human and mammalian oral epithelium.

Cytokeratin (CK) 20 is a low molecular-weight intermediate filament reportedly expressed only by benign and malignant gastrointestinal epithelium, urothelium and Merkel cells. The main aims here were to map its expression in normal oral mucosa of humans and other mammals, and to determine whether it was expressed by abnormal human oral epithelium. Salivary and odontogenic epithelium were also analysed. An immunoperoxidase method was used on wax-embedded and cryostat sections. In addition, double-labelling experiments were undertaken to determine the association between CK 20 expression and that of CK 8/18 or S100 protein. Normal human oral mucosa from four sites, together with abdominal skin, was studied in autopsy samples from 32 individuals. CK 20-positive, basally situated, round or angular cells, consistent with Merkel cells, were recorded in 24/32 (75.0%) samples of mandibular gingiva, 25/32 (78.1%) samples of hard palate, 7/32 (21.9%) samples of buccal mucosa, 0/32 samples of lateral border of tongue, and 2/32 (6.3%) samples of abdominal skin. Double-labelling showed that all CK 20-positive Merkel cells also expressed CK 8/18 and S100. The only other cells to express CK 20 were human taste buds. There was no expression by dysplastic or invasive oral epithelium from biopsy samples. Colonic mucosa showed luminal-cell positivity in man, marmoset, ferret, rabbit and guinea-pig, but oral mucosa was universally negative in non-human species. It is concluded that in oral mucosa CK 20 is a specific marker of Merkel cells and taste buds, that Merkel cells are more frequently present in keratinized than non-keratinized oral mucosa, that CK 20-positive Merkel cells are also S100-positive, that there may be interspecies variations in CK 20 polypeptide composition and that, by contrast to urothelium, CK 20 has no value in the diagnosis of oral epithelial dysplasia.

Age changes in the cells of the intra-articular disc of the temporomandibular joints of rats and marmosets.

Cells in the intra-articular disc of the temporomandibular joint were studied ultrastructurally at three different ages to investigate any age changes. Rats aged 2, 15.5 months, and 2.5 years, and marmosets aged 21 months, 7 years, and between 10.5 and 14 years were studied. In the first two age groups of the rat and the first of the marmoset, the cells were generally rounded and had moderate amounts of rough endoplasmic reticulum and other organelles associated with protein synthesis and secretion. Many cells had conspicuous amounts of microfilamentous material and cell membranes were closely applied to the collagen fibrils of the extracellular matrix. Occasionally, a narrow, irregular space containing microfilamentous material lay adjacent to the cell membrane. In the 2.5-year-old rats and the two older age groups of marmosets, cells with chondrocyte-like morphology were present. These cells were surrounded by a conspicuous pericellular matrix devoid of collagen fibrils and composed of microfilamentous material embedded in an amorphous ground substance. They resembled cells described in fibrocartilage from other sites, but differed from chondrocytes in hyaline cartilage by lacking a pericellular capsule. Thus, rats and marmosets both show cellular age changes in the intra-articular disc of the mandibular joint, which can be considered as changing from fibrous to fibrocartilaginous with age, a condition similar to that reported in humans.

Crimping of collagen in the intra-articular disc of the temporomandibular joint: a comparative study.

Very occasional reference is made to the presence of collagen crimps in the fibre bundles of the intra-articular disc (IAD) of the temporomandibular joint (TMJ). As the crimp structure may confer important biomechanical properties on the IAD, the present study was undertaken to determine its distribution in a variety of mammals, including humans. IADs from the rat, rabbit, guinea pig, ferret, sheep, marmoset and human were either sectioned or examined as whole mount preparations with polarized light. Apart from the guinea pig, where fibre bundles in the central region of the disc showed considerable overlap and masking of the crimp structure, the anteroposterior aligned fibres in the central region of all remaining animals showed the conspicuous presence of crimps. The periodicity of the crimp distance varied between about 10 and 30 microm. There was variation of the crimp periodicity between animals and between regions of the same disc. The crimping was associated with undulations along the length of the collagen fibres visible at the light microscope level using routine staining. The possible significance of crimping in association with internal derangement is discussed.

Collagen crimping in the intra-articular disc and articular surfaces of the human temporomandibular joint.

The presence of crimping within soft fibrous connective tissues has a considerable role in determining the biomechanical properties of the tissue. However, there is little or no information on crimping of collagen in the human temporomandibular joint. To remedy this situation, the presence and nature of any crimping was studied in sections of human temporomandibular joints from individuals varying in age from between 4.5-63 years, using polarized light microscopy and differential interference contrast microscopy. The presence of crimping was looked for in collagen within the intra-articular disc and the articular surfaces of the mandibular fossa and mandibular condyle. By polarized light, crimping was seen throughout all three tissues at all ages studied. Quantification from micrographs enlarged to x250 showed that the periodicity of the banding (representing half a complete crimp wave) had a mean varying between about 15-20 microm. Crimping was also directly visualized by differential interference contrast microscopy. The presence of such a fundamental feature needs to be considered when explaining the normal function of the temporomandibular joint.

The ultrastructure of the intra-articular disc of the temporomandibular joint, with special reference to fibrocartilage.

Cells in the intra-articular disc of the temporomandibular joint of the rat, guinea pig, rabbit, ferret, marmoset and sheep were studied at the ultrastructural level. The cells were generally rounded in outline and possessed moderate amounts of roughened endoplasmic reticulum and other organelles associated with protein synthesis and secretion. No intracellular collagen profiles were observed. Many of the cells possessed conspicuous amounts of microfilamentous material. Cell membranes in the rat, guinea pig, rabbit, ferret and sheep were closely applied to the collagen fibrils of the extracellular matrix. Occasionally in these animals, a narrow, irregular space containing microfilamentous material surrounded the cell membrane. Many cells in the marmoset differed from this description in being completely surrounded by an obvious pericellular matrix devoid of collagen fibrils and being comprised of microfilamentous material embedded in an amorphous ground substance. These chondrocyte-like cells in the intra-articular disc of the marmoset differed from chondrocytes in hyaline cartilage by lacking a pericellular capsule.

Interspecies variations in oral epithelial cytokeratin expression.

The aim of this study was to determine the degree to which the epidermis and oral epithelium of species other than man express cytokeratin (CK) intermediate filaments, which are markers of epithelial differentiation. Fixed, wax-embedded samples of skin, buccal mucosa and gingiva from rhesus monkey, marmoset, cow, sheep, pig, ferret, hamster, axolotl and trout were tested for CK expression using a panel of antihuman CK antibodies and an immunoperoxidase procedure. Human skin and oral mucosa were also stained to act as positive control. The results showed that antihuman CK antibodies stained animal tissues, but the patterns of staining were not always identical to the established human CK profile. Of particular interest was the expression of CK18, typically only detected in 'simple' epithelium in man, in bovine, ferret and hamster stratified epithelium from different sites. However, there was evidence of variable anti-CK antibody cross-reactivity, both as a result of intrinsic variations in CK polypeptide structure and as artifacts of fixation. We conclude that some CK are conserved between species, but that biological variables, for example local functional requirements, and technical factors affect the results. These considerations need to be borne in mind in animal studies of epithelial differentiation employing CK immunohistochemistry. Biochemical characterisation is ultimately necessary to determine specific differences between human and animal CK.

Cytokeratin intermediate filaments in oral and odontogenic epithelia.

With the development of monoclonal antibodies, the intermediate filaments of the cytoskeleton have attracted a great deal of interest in the last twenty years. Because they are highly antigenic, they could be easily identified using immunohistochemical methods and their specificity for one type of cell offered possibilities in the field of diagnostic pathology. The intermediate filaments most specific for epithelial cells are the cytokeratins (CK). After CK were classified, and certain "rules" regarding CK defined, research proceeded a pace to investigate epithelia in a vast array of anatomical and pathological situations. However, much of the immunohistochemical data that was generated during the 1980's was difficult to digest and some difficult to interpret. Reasons for this include identification of CK by molecular weight rather than CK number, or confusion as to which CK were being labelled by a particular antibody clone known to detect more than one CK. The aim of this article is therefore to present a digest of current knowledge of the CK present within the epithelium of the oral cavity (including the odontogenic epithelium) and its glandular component, and to highlight the significance of the CK phenotype in our understanding of pathological change.

Caroline Crachami, the Sicilian dwarf (1815-1824): was she really nine years old at death?

Caroline Crachami (C.C.) considered to have had the Seckel syndrome was one of the most extreme cases of dwarfism ever recorded. Reputedly born in Sicily in 1815, she attracted much attention when exhibited in England before her death on 3 June 1824. Although she is said to have been 9 years old at death, published descriptions give her a dental age varying from 2 to 7 years. Examination of her skull in the Royal College of Surgeons of England demonstrated a more or less erupted complete deciduous dentition, with no erupted permanent teeth. Radiographs showed agenesis of several permanent teeth. It was concluded that the dental age of C.C. was 3 years (+/- 6 months). Perikymata were evident in the surface enamel encircling the crown of the partially exposed maxillary left first permanent molar. Their distribution and spacing were normal, with no evidence of developmental retardation. The distance between the periradicular bands in the root near the cement-enamel junction was also normal. We conclude that the dental age of C.C. was similar to her chronological age and that at death she was about 3 and not 9 years old. The reason that she was said to be nearly 9 when exhibited in England we believe was related to financial considerations, as people would be generally less impressed with a dwarf only 3 years old. The new age we give C.C. has implications on the diagnosis of her medical condition.

The structure of bovine periodontal ligament with special reference to the epithelial cell rests.

The aim of this study was to determine the structure of the bovine periodontal ligament, with special reference to epithelial cell rests (ECR) and their cytokeratin content. Periodontal ligament was obtained from bovine molar teeth and studied at both the light microscopic and electron microscopic levels. Cytokeratin content was determined using immunohistochemistry against a number of cytokeratin antibodies and specificity tested against bovine and human oral mucosa. Collagen fibril diameters and the area of a fiber bundle occupied by collagen were determined using a digital planimeter with a digitizing tablet. The majority of periodontal fibroblasts possessed considerable quantities of roughened endoplasmic reticulum, indicating rapid synthesis and secretion of collagen, but no intracellular collagen profiles were present. Endothelial cells showed Weibel-Palade bodies. Collagen fibril diameters showed a unimodal distribution with a mean collagen fibril diameter of 55.3 nm. The mean percentage area of the extracellular matrix occupied by collagen was 42%. Structurally, ECR were unusual in exhibiting large numbers of microvilli and conspicuous amounts of cytokeratin filaments. Bovine ECR showed a positive reaction to the pancytokeratin MNF116 (which reacts with the cytokeratins 5, 6, 8, 17, and probably 19), to PCK-26 (which reacts with the type II cytokeratins 1, 5, 6, and 8) and to cytokeratin 13. There was no reaction for cytokeratins 1, 4, 10, 11 and 18. Structurally, bovine periodontal ligament showed features common to other species. However, ECR in terms of both structure and cytokeratin content showed features indicative of important species differences which may have relevance when considering the etiology of radicular cysts.

The distribution of cellular retinoic acid-binding protein I (CRABPI) and cellular retinol-binding protein I (CRBPI) during molar tooth development and eruption in the rat.

The distribution of cellular retinoic acid-binding protein (CRABPI) and cellular retinol binding protein (CRBPI) was studied in a series of prenatal and early postnatal rats, covering the main stages of development and eruption of the molar teeth. CRABPI positive cells were found in the mesenchymal cells of the dental follicle from the cap stage and in the dental papilla from the early bell stage. In the dental papilla, CRABPI positive cells were situated adjacent to the enamel organ in the cervical loop region and in the subodontoblastic region. Newly formed odontoblasts were CRABPI positive for a short period of time. The enamel organ was CRBPI and CRABPI negative, except for the presence of CRABPI positive cells in the internal enamel epithelium over the tip of cusps and in parts of the stratum intermedium. During root formation, CRABPI positive cells were found in the developing periodontal ligament, in the dental papilla adjacent to the epithelial root sheath and in the subodontoblastic zone. During crown formation, CRBPI positive cells were mainly localized to the mesenchymal cells of the dental papilla during the cap stage of crown development. The periosteum of the developing mandible contained CRABPI positive cells while some osteoclasts appeared to show a weak but positive reaction to CRBPI. The findings were considered in terms of the possible significance of retinoid-binding proteins during tooth and bone development.

The effect of rate of eruption on periodontal ligament glycosylaminoglycan content and enamel formation in the rat incisor.

The rate of eruption of rat mandibular incisors was either increased by cutting one tooth out of occlusion or eliminated by means of pinning. The effects of such changes in eruption rate on the sulphated glycosylaminoglycan content of the periodontal ligaments was analysed. The length of the enamel secretory zone and the composition of the developing enamel matrix protein was also compared. Sulphated glycosylaminoglycan content of the periodontal ligament increased fourfold (P < 0.001) during accelerated eruption but decreased to a corresponding extent (P < 0.001) in the absence of eruption, when compared with controls. The length of the enamel secretory zone was also significantly reduced in the immobilised teeth, although the protein content was similar compared with controls. The results demonstrate the differential response to varied eruption rates of the periodontal ligament and enamel, particularly in respect of the extracellular matrix. The data are consistent with the view that the ground substance of the periodontal ligament plays a role in the generation of the eruptive force.

The distribution of cellular retinoic acid-binding protein I during odontogenesis in the rat incisor.

Retinoids are important molecules in various aspects of embryological development. Here the distribution of cellular retinoic acid-binding protein I (CRABPI) was studied in the continuously growing incisor of adult rats using an affinity-purified rabbit polyclonal antibody. CRABPI was present throughout the presecretory and secretory ameloblast layer. The protein disappeared from that layer during its maturation phase. The adjacent dental mesenchyme of the developing pulp stained positively for CRABPI, especially in the layer immediately beneath the fully differentiated odontoblasts. Little CRABPI was present in the odontoblast layer itself. The distribution of CRABPI, both in the undifferentiated basal region of the incisor tooth and associated with the cells during hard-tissue formation, suggests a role for this molecule during differentiation and hard-tissue genesis.

Cellular retinoic acid binding protein in the periodontal ligament.

As retinoic acid is an important signaling molecule during embryological development. Since periodontal tissues are thought to have fetal-like properties, the present study was to determine the presence and distribution of cellular retinoic acid-binding protein I(CRABPI) in the periodontal tissues of the rat. Following demineralization, wax sections of the molar teeth of four adult male rats were cut in a plane parallel to the molar tooth row. Five representative sections from each jaw were then processed and stained to reveal the presence of CRABP. This protein was identified by immunochemistry utilizing an affinity-purified rabbit polyclonal antibody. The IgG antibody was used at a dilution of 1 microgram ml-1 and immunoreactivity detected with a kit according to protocol. Immunoreactivity was detected with diaminobenzidine and sections counterstained with hematoxylin. Results showed that virtually all the cells of periodontal ligament, the vast majority representing fibroblasts, contained CRABP. Fibroblasts in the gingiva above the level of the alveolar crest also stained positively for CRABP. The periodontal tissues of all three molar teeth were similarly affected. Fibroblasts in other regions of the oral cavity, such as the dermis of the skin of the cheek or the lamina propria of the buccal mucosa or of the diastema region, exhibited little or no staining for CRABP. Because of its properties as a signaling molecule and its ability to interact with other regulatory molecules, retinoic acid and cellular retinoic acid-binding protein may play an important role in the biology of the periodontal tissues.