Trabecular anisotropy and collagen fibre orientation in the mandibular condyle following experimental functional appliance treatment using sheep.

In order to study the modifying effects of functional appliances on the mechanical environment of the temporomandibular joint (TMJ), we characterised the structure of the mandibular condyle subsequent to an experimental functional appliance intervention. Eight, four-month-old, castrated male Merino sheep, were randomly allocated to experimental and control groups (n = 4 in each group). Forward mandibular displacement was induced with an intraoral appliance. The study period was 15 weeks, during which time fluorochromes were administered to all of the animals. Midsagittal sections of the TMJ were selected for analysis and trabecular anisotropy was estimated using bone histomorphometry. Only the experimental group demonstrated that the trabecular bone in the central condylar region was less anisotropic when compared to the subchondral region. Also, the variation in trabecular anisotropy of the central condylar region was found to be smaller in the experimental group. The collagen fibre orientation was analysed under polarised light as the proportion of the dark or bright fibres observed in regions which existed before, and regions which formed during the experiment, as determined by the fluorochrome labels. In the experimental group, more bright collagen fibres were found in the most superior region of the mandibular condyle when compared with the controls. These results suggested that the experimental functional appliances changed the orientation and pattern of the mechanical forces acting on the mandibular condyle, and possibly increased the magnitude of the lateral functional forces applied to the most superior part of the condyle during such treatments.

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial.

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.

Oral cancer: role of the basement membrane in invasion.

Invasive growth of cancer cells is a complex process involving specific interactions between tumour cells and the orderly, integrated complexes of the extracellular matrix. Basement membranes have been proposed as one constituent of extracellular matrix which carries responsibility for regulating invasion and metastasis. Using a chemically induced rat tongue carcinoma model, it has been shown that components of the basement membrane and its overall structure are altered during tumour invasion, and methods have been developed to quantitate some of these differences. Since the basement membrane can be specifically characterized by its fibrous protein network of Type IV collagen and laminin, which is embedded in a heparan sulphate-rich proteoglycan matrix, these components have been targeted. In particular, the current paper presents results in the context of current concepts of early changes in neoplastic invasion of underlying connective tissues. In consequence, further elaboration of the underlying mechanisms of epithelial migration in oral cancer may allow an exploration of the use of alterations in expression of basement membrane components as prognostic indicators.

Therapeutic delivery of calcitonin to inhibit external inflammatory root resorption. I. Diffusion kinetics of calcitonin through the dental root.

Insertion of calcitonin into root canals of monkey teeth has been shown to inhibit external inflammatory root resorption and suppress inflammation. Regulation of this therapeutic event depends upon the rate of arrival (diffusion) of the hormone at sites of resorptive activity. In the present study, the diffusion characteristics of calcitonin through the dental root in an extracted human-tooth model are described, and the role of cementum in the diffusion process is also addressed. Root-canals were endodontically prepared to form a reservoir for [125I]-calcitonin, and macerated to remove organic material from dentinal tubules. In teeth with intact cementum, an initial period of delay (4-5 h) prior to the detection of calcitonin at the external tooth-root surface was followed by a rapid release of the calcitonin during the first 10.5 h (rate peaks at 6 h). Slower, sustained releases of calcitonin through intact cementum were measured for the following 9 days. Removal of cementum, to expose "smear-free" dentine, resulted in an earlier efflux of calcitonin (2 h) at external tooth surfaces and increased amounts of calcitonin release over 9 days. Biphasic delivery of calcitonin by such internal diffusion mechanisms suggests that loss of cementum will enhance therapeutic availability, while prolonged delivery to intact external dental-root surfaces following early intra-canal placement may also be useful for the therapeutic prevention of external inflammatory root resorption.

Therapeutic delivery of calcitonin to inhibit external inflammatory root resorption. II. Influence of calcitonin binding to root mineral.

Experimentally-induced external inflammatory tooth-root resorption can be inhibited by therapeutic doses of calcitonin. Such doses can be delivered by an intrinsically slow diffusion pathway, from a reservoir in endodontically-debrided root canals, via the dentinal tubules. While the kinetics of this journey have been followed in an earlier report, the binding characteristics of calcitonin to the tooth mineral, which will be responsible, in part, for these kinetics, have not been reported before. The current study examines the binding potential of calcitonin to root mineral and addresses the potential role of non-specific binding proteins. A modified Scatchard plot indicated that a simple non-reactive type of ligand binding exists between calcitonin and root mineral, represented by a small number of identical binding sites. This interaction is both strong and reversible. Furthermore, it appears to be time-dependent with more time being required for the residual ligands to interact with the diminishing numbers of free calcitonin-binding sites. While preloaded [125I]-calcitonin could be incompletely (75-91%) displaced from dental-root material by non-radioactive calcitonin, its release was slow over 23 h. Calcitonin was four times as effective as bovine-serum albumin in competing for common "calcitonin binding sites" on macerated dental-root material. Thus, even in the presence of extraneous protein, calcitonin will bind tightly but reversibly to tooth-root material, making it a good candidate for therapeutically protracted delivery to external root surfaces from root canals.

Identification of basal lamina acidic glycoconjugates, particularly heparan sulphate proteoglycans, using a poly-L-lysine-gold probe in induced oral carcinomas.

Acidic glycoconjugates represent the major non-fibrous macromolecular components that form the extracellular and cell-associated matrices of all animal tissues. The constituent molecules are principally structural glycoproteins and proteoglycans. While their protein component is determined by gene pools, it is the polyanionic (acidic) nature of the polysaccharides, determined by their degrees of carboxylation and sulphation, which confers both functional and diagnostic status on these molecules. Sulphated glycoconjugates in the basal laminae have been reported to play a role in tumour invasion and metastasis. In this study, we used cationic colloidal gold together with transmission electron microscopic methods to compare the expression of acidic glyconconjugates in the basal lamina of both normal rat tongue mucosa and experimentally induced oral carcinomas. Results indicated that heparan sulphate rich glycoconjugates were predominant and were mostly confined to the lamina lucida of the basal lamina in normal oral mucosa. Conversely, observation of basal laminae associated with induced carcinomas showed less intense and more widely dispersed gold labelling for heparan sulphate. The observed differences in gold labelling may reflect modified metabolism of sulphated glycoconjugates or result from the action of degradative enzymes in the induced tumours.

Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis.

Cultured chondrocytes respond to insulin-like growth factors (IGFs) by increasing the production of proteoglycans and insulin-like growth factor binding proteins (IGF-BPs). To investigate the biological effects of IGFs and IGF-BPs, isolated bovine articular and ovine growth-plate chondrocytes were cultured at high density in the presence of IGF-1, and its truncated form, des (1-3) IGF-I. Both growth factors stimulated the production of IGF-BPs in articular and growth-plate chondrocyte monolayers. Western ligand blots showed that bovine articular chondrocytes released two forms of IGF-BPs into conditioned medium with molecular weights of 29 and 31 kDa. Ovine growth-plate chondrocytes released four different forms of IGF-BPs of approx. 22, 24; 29-30 and 34 kDa. IGF-I and des (1-3) IGF-I stimulated total proteoglycan synthesis by articular chondrocytes up to 1.5-fold. The truncated analogue was more potent at lower concentrations, particularly in stimulating incorporation of newly synthesized proteoglycans into the cell-layer. The maximal stimulation of proteoglycan synthesis in ovine growth-plate chondrocyte culture was 3-fold with des (1-3) IGF-I, while IGF-I enhanced proteoglycan production by only 2-fold over the concentrations used. Our results suggest that endogenous IGF-BPs in chondrocyte cultures act as a part of a feed-back mechanism which diminishes the bioactivity of IGF-I.

Proteoglycan changes in carcinogen (4NQO)-treated rat tongue mucosa.

The purpose of this study was to undertake preliminary analyses of the extracellular proteoglycans in carcinogen [4-nitroquinoline N-oxide (4NQO)]-treated rat tongue mucosa. Experimental rats were exposed to twice-weekly applications of 4NQO in propylene glycol for six months, after which the animals were killed. Control and 4NQO-treated tissues were subjected to sequential aqueous extractions of proteoglycans under associative and dissociative conditions, followed by alkaline cleavage of protein-glycosaminoglycan linkages to yield a glycosaminoglycan residue. Tissues subjected to 4NQO applications contained smaller proportions of proteoglycans which were readily soluble under associative and dissociative conditions. Proportionately more proteoglycan remained strongly associated with other intercellular tissue components, being released only by alkaline cleavage. These biochemical alterations in preinvasive 4NQO-treated epithelium and connective tissues, together with an observed associated change in water retention by the connective tissue, occurred prior to actual neoplastic invasion and suggest differences in macromolecular conformation and orderliness. We hypothesize that these changes are related to the phenomenon of neoplastic epithelial invasion.

Distribution of basal lamina type IV collagen and laminin in normal rat tongue mucosa and experimental oral carcinoma: ultrastructural immunolocalization and immunogold quantitation.

The relationship of basal lamina, a form of specialised extracellular matrix which separates epithelial cells and other cell types from adjacent stroma, to the behaviour of malignant neoplasms of epithelial origin is not well understood. However, it is widely acknowledged that the properties of local invasion and metastasis of carcinomas are linked to extracellular matrix (including basal lamina) changes. In the present study, the distribution of the major basal lamina components, type IV collagen and laminin, in normal rat tongue mucosa and experimentally induced oral carcinomas was investigated using post-embedding immunogold techniques and electron microscopy. The expression of these components was also quantitatively analysed using morphometry and immunocytochemistry. Results indicated that type IV collagen and laminin were confined to the lamina densa of normal oral epithelial basal lamina, and that both components were also detected in the lamina densa of basal lamina associated with carcinomas, and in the extracellular matrix of tumours. Furthermore, laminin was detected within stromal fibroblasts in normal tissues and experimental carcinomas. Quantitative analysis indicated that expression of laminin was significantly increased in carcinomas. In contrast, type IV collagen expression was significantly decreased. The quantitative changes observed in the two basal lamina constituents may be related to the process of tumour invasion, reflecting altered metabolic activities of tumour and stromal cells. These observations may be of use in understanding the architectural characteristics of oral mucosa basal lamina and in assessing the malignant potential of epithelial dysplasias or "premalignant" lesions.

Laminin and type IV collagen in experimental rat oral carcinomas.

The pattern of staining and the distribution of laminin and type IV collagen in normal rat tongue mucosa and induced tongue carcinomas were investigated by immunohistochemical techniques. Both normal and neoplastic epithelial basement membrane revealed positive staining for laminin and type IV collagen. However, compared with normal tissue, carcinomas exhibited areas of increased density and thickness for laminin. Focal tumour basement membrane discontinuities were observed in some specimens stained for type IV collagen.

Ultrastructural features of normal epithelium and 4-nitroquinoline 1-oxide-induced carcinomas of the rat tongue.

An electron microscopical examination of normal rat lingual mucosa and 4-nitroquinoline 1-oxide (4NQO)-induced tongue carcinomas was undertaken. In normal rat tongue, the epithelium of papillae and interpapillary regions exhibited two distinct keratohyalin granule types and essentially similar ultrastructural cellular features in the different epithelial compartments. The interface between epithelium and connective tissue showed a continuous basal lamina. Compared with normal rat tongue epithelium, 4NQO-induced oral carcinomas revealed cellular and nuclear pleomorphism, atypical tonofilament aggregates, increased and swollen mitochondria, dilated intercellular spaces, local discontinuities and thickening of the basal lamina.

Cell responses to Hydron by a new in-vitro method.

An in-vitro biotoxicity test system, suitable for the assessment of endodontic filling materials, has been developed and used to test cell responses to Hydron, AH26 and Tubliseal. A robust, well-characterized and stable cell line (L-cells) which was grown as uniform cultures on Millipore filters, has been used as indicator cells. As they approached confluence they were exposed to test substances for 24 h and biosynthetic activities were measured. The test system is a modification of that described by Wennberg et al. (1979). By inverting the cultures on organ-culture rafts, cells were separated from the test material, which was placed on top of the Millipore filters. Freshly mixed polymerizing Hydron and prepolymerized Hydron were tested. The cell responses were compared with those of cultures exposed to freshly mixed AH26 and Tubliseal. Polymerizing and prepolymerized Hydron depressed both cell division, assessed by 3H-thymidine incorporation, and the synthesis and secretion of matrix material as measured by precipitable 35S-sulphate. Prepolymerized Hydron decreased cell functions by 59% and 56% of live cell controls, respectively, while the freshly mixed polymerizing Hydron inhibited biosynthesis by 89% and 94%, respectively. The data for polymerizing Hydron were compared with results for other root-filling materials and showed similar values to those for Tubliseal (92% and 95%), but greater inhibition of biosynthesis than for AH26 (53% and 50%). The AH26 values were similar to those obtained from cultures exposed to the prepolymerized Hydron. Recovery of biosynthetic capacity by these cultures after removal of all endodontic material was also assessed. Partial biosynthetic recovery of cell cultures was observed 24 h after removal of preopolymerized Hydron.(ABSTRACT TRUNCATED AT 250 WORDS)

Reimplantation of growth plate chondrocytes into growth plate defects in sheep.

Defects in growth plates due to trauma, infection, or genetic causes can result in bone formation across the defect, bridging the epiphysis and metaphysis, resulting in growth arrest and limb deformation. We have investigated the capacity of implanted chondrocyte cultures to prevent this process. Sheep growth plate chondrocytes were isolated, and after culture at high density produced easily manipulated cartilaginous discs. The tissue was implanted into growth plate defects produced in lambs and the response was assessed histologically. Following implantation, cultures continued to proliferate and maintain a cartilage-like matrix. After 8 to 12 weeks, hypertrophic maturation chondrocyte columnation, and associated endochondral calcification were observed. Culture implantation was always associated with local immune inflammatory reaction, which continued throughout the course of investigation. Cellular survival was variable and resulted in the presence of viable implants as well as residual cartilage matrix devoid of chondrocytes; however, implanted chondrocyte discs always prevented bone bridge formation. These findings encourage the expectation that cultured chondrocytes may provide a useful replacement for the inert interpositional materials currently used in the treatment of growth arrest. The potential of this technique for growth plate replacement, however, requires a more predictable rate of implant survival. The likely reasons for implant loss are discussed.

Growth-plate chondrocyte cultures for reimplantation into growth-plate defects in sheep. Characterization of cultures.

Damage to epiphyseal growth plates due to fracture, trauma, or infection can lead to invasion of bone across the cartilage and localized arrest of long-bone growth. The implantation of a viable de novo cartilage plug into such defects may provide the appropriate cartilage presence necessary to inhibit the initial formation of bony bridges across the epiphysis and so maintain the growth potential. De novo cartilage plugs were prepared from ovine growth plates by culturing isolated epiphyseal chondrocytes from fetal lambs. After 14 days of culture, these de novo cartilage discs were composed of chondroitin sulfate, a small amount (5%) of dermatan sulfate, and cartilage-specific collagen. The cellular morphology and the histochemistry resembled resting zones of normal growth-plate cartilage. Those de novo cartilage discs, which had been embedded in gelled Type I collagen, retained their morphology and could be easily manipulated. On the other hand, Type II collagen and a polyuronic acid gauze (Surgicel) were not satisfactory substrates to facilitate subsequent transplantation into growth-plate defects. The use of 5-carboxyfluorescein diacetate succinimidyl ester (CSFE) throughout the cultures of epiphyseal chondrocytes or prolonged incorporation of [3H]-thymidine appeared to label the cells with useful markers for following their fate subsequent to implantation in vivo.

The effects of hyaluronic acid on macrophage Fc receptor binding and phagocytosis are independent of the mode of depolymerization.

In order to determine whether exposure of hyaluronic acid to oxygen radicals caused an alteration in its properties, independent of the change in molecular weight induced, we examined its effect upon macrophage Fc receptor binding. High molecular weight hyaluronic acid (Healon-Pharmacia) caused a dose dependent inhibition of binding between the concentrations of 0.2-1 mg/ml. At a concentration of 0.3 mg/ml both oxygen radical depolymerized and enzymatically degraded hyaluronic acid caused an inhibition of Fc receptor binding at molecular weights of 1 x 10(6), 1.5 x 10(6) and 2 x 10(6). Oxygen radical degraded hyaluronic acid caused a stimulation of Fc receptor binding at molecular weights of 2 x 10(5) and 3.5 x 10(5), and enzyme degraded hyaluronic acid causes stimulation at a molecular weight of 2.5 x 10(6). Thus this "biological property" of hyaluronic acid is dependent upon molecular weight solely and not upon the mode of depolymerization.

The generation of reducing ends by exposure of hyaluronic acid to oxygen derived free radicals.

Enzymatic depolymerization of hyaluronic acid (HA) is accompanied by the release of reducing ends irrespective of the linkage cleaved. In this investigation we have examined HA exposed to oxygen-derived free radicals (oxy radicals) in order to determine whether reducing ends are released upon depolymerization. Reducing ends were detected by the assay of Park and Johnson, which is a non-specific but sensitive assay for reducing ends, but only to a minimal degree by the Reissig modification of the Morgan Elson reaction, which will detect N-acetylglucosamine at the reducing end. Exposure of a sample of HA, pre-exposed to streptomyces hyaluronidase, to an oxy radical flux did not result in a decrease in Morgan Elson reactivity thus indicating that post cleavage modification of the reducing end by further reaction with oxy radicals does not account for the lack of Morgan Elson reactivity. In addition reducing ends were also detected by reaction with radiolabelled cyanide to the degree predicted by molecular weight. These findings were interpreted as being consistent with the hypothesis that oxy radical induced depolymerization of HA occurs by preferential cleavage of the glucuronidic linkage thus leaving D-glucuronic acid at the reducing end.

Depolymerisation products of hyaluronic acid after exposure to oxygen-derived free radicals.

Preparative chromatographic fractions of human umbilical cord hyaluronic acid (HA) of a molecular weight of 10(6) were subjected to graded oxygen-derived free radical (oxy radical) fluxes produced by: (a) the autoxidation of ferrous ions; (b) the action of xanthine oxidase (XO) on hypoxanthine (HX); and (c) by peripheral blood polymorphonuclear leucocytes that had been stimulated by phorbol myristate acetate (PMA). Analysis by gel chromatography of the products obtained with each of the oxy radical generating systems showed polydispersity in size. The smallest molecules detected had a molecular weight of 10(4). This limiting size was not reduced further by exposure to a second oxy radical flux. The relative proportions of large, medium, and small degradation products were established for various levels of oxy radical flux. Consistently a relatively rapid transition from large to small material was seen on Sepharose 2B chromatography, suggesting an ordered element to the breakdown process. Although the decrease in molecular weight after oxy radical exposure was confirmed by analytical ultracentrifugation, this procedure showed that those samples of lowest viscosity did not have the lowest sedimentation values, possibly reflecting oxy radical-induced repolymerisation. If the size and possibly the conformational characteristics of HA are altered, oxy radical exposure might be expected to alter its biological properties.

Surgicel: its fate following implantation.

Surgicel, a local haemostatic gauze, is claimed to consist of oxidised regenerated cellulose. It is a polyanion, the functional unit of which is termed polyanhydroglucuronic acid. The ability of tissues to absorb Surgicel and its inherent haemostatic properties have been extensively investigated. This study was undertaken a) to determine the time required for absorption of Surgicel from implantation sites in the chest wall muscles of rats, and b) to establish mechanisms for its removal. Data derived from sequential uronic acid assays, histochemistry using the stain alcian blue, and transmission electron microscopy of implanted Surgicel were interpreted to reveal that Surgicel consists of at least two active components. These are a soluble uronic acid component which is lost after 6 h, and a fibrous component which persists. The latter material resembles Surgicel in the electron microscope and is still evident at the implantation site at 48 h post-implantation. Moreover, Surgicel can be characterized in vitro into at least two components according to its solubility under dissociative salt conditions (4M guanidinium chloride). A residual fibrous material could then be hydrolysed with 0.3N sodium hydroxide. We postulate that the absorption of the former salt soluble uronate in vivo is by early degradation and/or systemic clearance, whilst removal of the fibrous material requires phagocytosis.