A comparative light microscopic evaluation of oxytalan fiber staining with a variety of dye substances.

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of response to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin, Taenzer-Unna orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing groups should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.     

A Comparative Light Microscopic Evaluation of Oxytalan Fiber Staining with a Variety of Dye Substances

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of responses to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin Taenzera11 Uma orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing group should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.     

Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.     

Ultrastructural localization of fibrillin-1 and fibrillin-2 in oxytalan fibers in periodontal ligament of Japanese Macaca fuscata monkey

Although oxytalan fibers are a ubiquitous component of the periodontal ligament, their composition and function remain to be fully clarified. The purpose of this study was to investigate the localization of fibrillin-1 and fibrillin-2, large cysteine-rich glycoproteins, in monkey molar periodontal ligament by means of immunohistochemistry and electron microscopic immunogold labeling. Fibers positive for both fibrillin-1 and -2 were observed throughout the periodontal ligament, and their pattern of distribution was basically the same as that of oxytalan fibers. Double immunogold electron microscopy revealed that fibrillin-1 co-localized with fibrillin-2 on the microfibrils of the oxytalan fibers. The results suggest that oxytalan fibers in monkey periodontal ligament are made up of fibrillin-rich microfibrils. These results should contribute to a better understanding of the role of oxytalan fibers in development and tissue remodeling in periodontal ligament.     

Oxytalan fibers in the periodontal ligament of the Caiman and the Alligator (Crocodilia, Reptilia)

Histological examination of the fibrous and cellular connective tissue components of the periodontal ligament in the Caiman and the Alligator reveals the presence of reticular fibrillae, collagenic, elastic, and oxytalan fibers, as well as fibrocytes, osteoblasts, cementoblasts and epithelial rests. The oxytalan fibers differentiated by the peracetic acid aldehyde-fuchsin method are most numerous in the coronal region, radiating from the primary cementum into the periodontal ligament a short distance. Oxytalan fibers in fewer numbers are found interspersed between the oblique and the horizontal principal fiber bundles. Inasmuch as the crocodilian teeth have continuous replacement and thus a relatively short functional life, the oxytalan fibers of the Caiman and the Alligator appear to be proportionally fewer in number than they are in the mammalian periodontal tissues. The presence of the oxytalan fibers and epithelial rests in the Order Crocodilia (Crocodilia) adds to the number of dental structures shared with the Class Mammalia (Mammalia) (mammals) such as a stellate reticulum, a primary and secondary cementum and a periodontal ligament. This furnishes additional histological evidence for evaluation of the phylogenetic position of this group.     

The cytology of submandibular glands of the opossum

Tooth attachment in the majority of the bony fish is by ankylosis or fibrous membrane. However, in one group of the osteichthys, the trigger-fish or balistids, tooth attachment is by means of a periodontium composed of a shallow alveolar socket, a periodontal ligament and acellular cementum. Histologically, the balistid periodontal ligament is composed of a dense fibro-cellular connective tissue possessing an abundance of typical fibrocytes, collagen fiber bundles, and oxytalan fibers. The collagen fiber bundles which resemble the principal fiber bundles of the mammalian periodontal ligament are inserted into the bone of the shallow alveolar sockets and are anchored to the teeth by means of a layer of amorphous acellular cementum that covers the radicular dentin. No cementoblasts were found in functional teeth, and epithelial rests are lacking. The mid-central zone of the balistid periodontal ligament is occupied by small blood vessels.     

Electron microscopic study on the elastic and elastic related fibres in the human fascia transversalis at different ages

An electron microscopic study of the elastic fibre and elastic related fibres of the fascia transversalis of the human inguinal triangle was performed in 20 male patients aged 13 to 81 a with right indirect inguinal hernia submitted to surgical repair. The 3 fibre types comprising the elastic system (oxytalan, elaunin, and elastic fibres) tend to be ordered in a precise manner and sequence among the fibrils, fibres, and collagen fibre bundles, respectively. The present findings show that with aging, there is a decrease in the oxytalan fibres and an increase in the amorphous substance of the elastic fibres. The authors concluded that the decrease in oxytalan fibres as a function of age may be responsible for alteration in the resistance of the transversalis fascia.     

STRUCTURAL BIOLOGY OF THE FIBRES OF THE COLLAGENOUS AND ELASTIC SYSTEMS

The different types of fibres of the collagenous and elastic systems can be demonstrated specifically in tissue sections by comparing the typical ultrastructural picture of each of the fibre types with studies using selective staining techniques for light microscopy. A practicalmodus operandi, which includes the recommended staining procedures and interpretation of the results, is presented. Micrographs and tables are provided to summarize the differential procedures. Reticulin fibres display a distinct argyrophilia when studied by means of silver impregnation techniques, and show up as a thin meshwork of weakly birefringent, greenish fibres when examined with the aid of the Picrosirius-polarization method. In addition, electron-microscopic studies showed that reticulin fibres are composed of a small number of thin collagen fibrils, contrasting with the very many thicker fibrils that could be localized ultrastructurally to the sites where non-argyrophilic, coarse collagen fibres had been characterized by the histochemical methods used. The three different fibre types of the elastic system belong to a continuous series: oxytalan—elaunin—elastic (all of the fibre types comprising collections of microfibrils with, in the given sequence, increasing amounts of elastin). The three distinct types of elastic system fibres have different staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic elastic fibre consists of two morphologically different components: a centrally located solid cylinder of amorphous and homogeneous elastin surrounded by tubular microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed amorphous material (elastin) is intermingled among the microfibrils.     

Structure and Development of the Notochord ‘Elastica Externa’ and Nearby Components of the Elastic Fibre System of Agnathans

Between 15 days and 3 months in age, the ‘elastica externa’ of the notochord sheath of larval lampreys develops from patches of moderately dense and amorphous material into a thick, continuous and electron-dense layer. In both lampreys and hagfish, this layer stains strongly with Verhoeff's elastic stain and aldehyde fuchsin and is penetrated by collagen fibrils on both its outer and inner boundaries. Peroxidase labelling using an antibody raised against human elastin specifically labels both the notochord ‘elastica externa’ and the elastic fibre system of lampreys. The diameters of the microfibrils (10–13 nm) of the oxytalan, elaunin and elastic fibres of lampreys and hagfish are the same as those of higher vertebrates. The connective tissue immediately dorsal and ventral to the notochord of lampreys contains mainly oxytalan fibres in very young ammocoetes, a combination of oxytalan, elaunin and elastic fibres in older ammocoetes, and predominantly elastic fibres in adult lampreys. While the region of the endomeninx at the base of the spinal cord contains almost exclusively oxytalan fibres in young ammocoetes, it also possesses numerous elastic fibres in adult lampreys. These findings indicate that, as in higher vertebrates, the elastic fibres of lampreys develop from oxytalan fibres via elaunin fibres.     

A micromechanically-based, three-dimensional interface finite element for the modelling of the periodontal ligament

Some ideas are presented for the implementation of an interface finite element capable to model in 3-dimensions several mechanical features of the periodontal ligament. Such an element is based on a simple 2-cable micromechanical model, able to reproduce the periodontal ligament stiffness and strength under any loading condition, including the pure torsion of a tooth. A single cable represents a sufficiently populated sample of collagen fibres, each with an initially crimped geometry; a single collagen fibre can provide a mechanical response, in tension, only when it is completely uncoiled. The macroscopic interface behaviour is obtained by statistical integrations over the uncoiled length of each collagen fibre, up to the fibre failure. Such a model can reproduce the periodontal ligament anisotropy due to the variable fibre orientation along the tooth root, its different behaviour in tension/compression/shear, its different behaviour for extrusive/intrusive loading, and so forth. Some numerical examples illustrate the potentialities of this interface element, quite simple in essence but rather complete from an engineering viewpoint.     

Loose front teeth: radiological and histological correlation with grooming function in the impala Aepyceros melampus

Casual observations have revealed that the anterior dentition of impala and other antelope is loosely embedded, with the tips of the teeth movable over a distance of 1middot;5 to 2mm. The comb-like anterior dentition of impala Aepyceros melampus is utilized extensively for grooming purposes, and it was hypothesized that the looseness of the teeth might be related to the grooming function. A sample of 12 impala mandibles was obtained from Pilanesberg National Park. Boputhatswana. Six of the incisor canine (IC) complexes were examined macroscopically, radiographically and histologically, while the remaining six were used to determine the alveolar depth relative to total root length, The findings were: (1) wide periodontal ligament spaces, most prominent in the apical region; (2) a loose, highly vascular periodontal ligament; (3) well-developed trans-septal periodontal ligament fibres; and (4) relatively shallow alveoli, with only approximately two-thirds of the roots included within the alveoli. In no case could looseness be ascribed to pathological changes in the periodontal ligament, cementum or alveolar bone. These features suggest that the looseness of the teeth is associated with a see-saw action of the teeth about a fulcrum below the alveolar bone crest. with the maintenance of the closed resting position of the teeth being facilitated by the well-developed trans-septal fibres. It is suggested that the minimal interdental space maintained by this arrangement during grooming assists in the efficient removal of parasites from the pelage by impala.     

The elastic fibre system in the veins of the human lower oesophagus

The elastic, elaunin and oxytalan fibres in the tunica mucosa and tela submucosa of veins of the human lower oesophagus were studied in 30 necropsy and biopsy specimens using appropriate histological and ultrastructural methodologies. Elaunin fibres predominate in the veins endowed with muscle cells. Scattered oxytalan and elastic fibres were also observed, the latter being more numerous at the vein periphery. No noteworthy fibre arrangement was encountered in veins lacking muscle cells. Those fibres disposed around the veins were considered to belong to the connective tissue of the tunica mucosa and tela submucosa in which they are embedded. The veins of the lower oesophagus seem to be of low elasticity, which may be related to a blockage mechanism described for the gastro-oesophageal blood stream in cases of portal hypertension.     

Changes in the Distribution of Periodontal Nerve Fibers during Dentition Transition in the Cat

The periodontal ligament has a rich sensory nerve supply which originates from the trigeminal ganglion and trigeminal mesencephalic nucleus. Although various types of mechanoreceptors have been reported in the periodontal ligament, the Ruffini ending is an essential one. It is unknown whether the distribution of periodontal nerve fibers in deciduous teeth is identical to that in permanent teeth or not. Moreover, morphological changes in the distribution of periodontal nerve fibers during resorption of deciduous teeth and eruption of successional permanent teeth in diphyodont animals have not been reported in detail. Therefore, in this study, we examined changes in the distribution of periodontal nerve fibers in the cat during changes in dentition (i.e., deciduous, mixed and permanent dentition) by immunohistochemistry of protein gene product 9.5. During deciduous dentition, periodontal nerve fibers were concentrated at the apical portion, and sparsely distributed in the periodontal ligament of deciduous molars. During mixed dentition, the periodontal nerve fibers of deciduous molars showed degenerative profiles during resorption. In permanent dentition, the periodontal nerve fibers of permanent premolars, the successors of deciduous molars, increased in number. Similar to permanent premolars, the periodontal nerve fibers of permanent molars, having no predecessors, increased in number, and were densely present in the apical portion. The present results indicate that the distribution of periodontal nerve fibers in deciduous dentition is almost identical to that in permanent dentition although the number of periodontal nerve fibers in deciduous dentition was low. The sparse distribution of periodontal nerve fibers in deciduous dentition agrees with clinical evidence that children are less sensitive to tooth stimulation than adults.     

Geometrical changes of knee ligaments and patellar tendon during passive flexion

Patterns of fibre elongation and orientation for the cruciate and collateral ligaments of the human knee joint and for the patellar tendon have not yet been established in three-dimensions. These patterns are essential for understanding thoroughly the contribution of these soft tissues to joint function and of value in surgical treatments for a more conscious assessment of the knee status. Measurements from 10 normal cadaver knees are here reported using an accurate surgical navigation system and consistent anatomical references, over a large flexion arc, and according to current recommended conventions. The contours of relevant sub-bundles were digitised over the corresponding origins and insertions on the bones. Representative fibres were calculated as the straight line segments joining the centroids of these attachment areas. The most isometric fibre was also taken as that whose attachment points were at the minimum change in length over the flexion arc. Changes in length and orientation of these fibres were reported versus the flexion angle. A good general repeatability of intra- and inter-specimens was found. Isometric fibres were found in the locations reported in the literature. During knee flexion, ligament sub-bundles slacken in the anterior cruciate ligament, and in the medial and lateral collateral ligaments, whereas they tighten in the posterior cruciate ligament. In each cruciate ligament the two compounding sub-bundles have different extents for the change in fibre length, and also bend differently from each other on both tibial planes. In the collateral ligaments and patellar tendon all fibres bend posteriorly. Patellar tendon underwent complex changes in length and orientation, on both the tibial sagittal and frontal planes. For the first time thorough and consistent patterns of geometrical changes are provided for the main knee ligaments and tendons after careful fibre mapping.     

Distribution and organization of the elastic system fibres in healthy human gingiva

Summary The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.     

Ultrastructural cytochemistry of oxytalan fibres in monkey periodontal ligaments with the high iron diamine method

Summary Monkey periodontal ligaments have been examined at the ultrastructural level to demonstrate the nature of reactive sites in oxytalan fibres. The high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate methods specific for sulphate groups, with and without prior oxidation with monopersulphate, were used. Oxytalan fibres were composed of bundles of microfibrils with a diameter of 11.5 ± 1.7 nm (mean ±s.d.,n = 50). In cross section the microfibrils were found to have a denser periphery, giving them a tubular appearance. The oxytalan microfibrils of non-oxidized specimens showed little reactivity with either HID method, except that the extracellular matrix material in close association with collagen fibrils stained weakly; in oxidized specimens, both HID methods strongly stained oxytalan microfibrils and weakly stained the extracellular matrix material. Such reactivity of oxytalan microfibrils was not altered by digestion with testicular hyaluronidase or chondroitinase ABC, performed prior to or after persulphate oxidation. Further, the sequential thiosulphation and HID method for the demonstration of disulphide and sulphhydryl groups stained oxytalan fibres moderately. These results indicate that the oxidative generation of sulphate groups in oxytalan fibres may occur from either disulphide or sulphhydryl groups, or both, rather than the result of unmasking of sulphated glycosaminoglycans.     

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells.     

5-Hydroxytryptamine immunoreactivity is detectable in sympathetic nerve fibres in rat oral tissues

Summary The aim of this investigation was to examine if 5-hydroxytryptamine (5-HT) is detectable not only in mast cells but also in sympathetic nerve fibres in oral sites of the rat, including the periodontal ligament, pulp, palatal mucosa, and vestibular sulcus. Antibodies against 5-HT and tyrosine hydroxylase were used. Maxillae from rats were dissected free, fixed, decalcified, cut transversally, and processed for immunohistochemistry. Nerve fibres showing 5-HT-like immunoreactivity were regularly observed in the walls of the arteries and arterioles in the vestibular sulcus and the periodontal ligament. However, 5-HT-like immunoreactivity was not seen in the walls of the vessels of the palatal mucosa. Interestingly, 5-HT-like immunoreactivity coexisted with tyrosine hydroxylase-like immunoreactivity in the innervation of the periodontal ligament and the vestibular sulcus. Thus, the present study gives morphological correlate for the occurrence of effects of 5-HT derived not only from mast cells but also from sympathetic nerve fibres in oral tissues. The source of 5-HT in the nerve fibres as well as the functional implications of the observations remain to be determined.     

Ligament fibre recruitment at the human ankle joint complex in passive flexion

Knowledge of ligament fibre recruitment at the human ankle joint complex is a fundamental prerequisite for analysing mobility and stability. Previous experimental and modelling studies have shown that ankle motion must be guided by fibres within the calcaneofibular and tibiocalcaneal ligaments, which remain approximately isometric during passive flexion. The purpose of this study was to identify these fibres.

Three below-knee amputated specimens were analysed during passive flexion with combined radiostereometry for bone pose estimation and 3D digitisation for ligament attachment area identification. A procedure based on singular value decomposition enabled matching bone pose with digitised data and therefore reconstructing position in space of ligament attachment areas in each joint position. Eleven ordered fibres, connecting corresponding points on origin and insertion curves, were modelled for each of the following ligaments: posterior talofibular, calcaneofibular, anterior talofibular, posterior tibiotalar, tibiocalcaneal, and anterior tibiotalar.

The measured changes in length for the ligament fibres revealed patterns of tightening and slackening. The most anterior fibre of the calcaneofibular and the medio-anterior fibre of the tibiocalcaneal ligament exhibited the most isometric behaviour, as well as the most posterior fibre of the anterior talofibular ligament. Fibres within the calcaneofibular ligament remain parallel in the transverse plane, while those within the tibiocalcaneal ligament become almost parallel in joint neutral position. For both these ligaments, fibres maintain their relative inclination in the sagittal plane throughout the passive flexion range.

The observed significant change in both shape and orientation of the ankle ligaments suggest that this knowledge is fundamental for future mechanical analysis of their response to external forces.