Stretching stimulates fibulin-5 expression and controls microfibril bundles in human periodontal ligament cells

Background and Objective:  The elastic fiber system comprises oxytalan, elaunin and elastic fibers, differing in their relative microfibril and elastin contents. Human periodontal ligaments contain oxytalan fibers (pure microfibrils). Periodontal ligaments are continuously exposed to various functional forces, such as tooth movement and occlusal loading. We have reported that bundles of microfibrils coalesce in response to mechanical strain in cultured periodontal ligament fibroblasts, as assessed in terms of their positivity for fibrillin-1 (the major component of microfibrils). However, the mechanism of microfibril coalescence is unclear. We hypothesized that the fibrillin-1-binding molecule, fibulin-5, contributes to oxytalan fiber formation under mechanical strain.
Material and Methods:  We subjected periodontal ligament fibroblasts to stretching in order to examine the effects of fibulin-5 on the formation of oxytalan fibers in cell/matrix layers. We transfected periodontal ligament cells with small interference RNA for fibulin-5, then examined oxytalan fibers using immunofluorescence and electron microscopy.
Results:  Immunofluorescence showed that fibrillin-1-positive microfibrils coalesced as a result of stretching, compared with cells that were not subjected to stretching. Fibulin-5 colocalized on fibrillin-1-positive microfibrils. Stretching increased fibulin-5 gene expression and protein deposition. Immunofluorescence and immunogold electron microscopy analysis revealed that fibulin-5 suppression inhibited the coalescence of microfibrils under stretching conditions.
Conclusion:  These results suggest that fibulin-5 up-regulated in response to tension strain may control the formation of microfibril bundles in periodontal ligament.     

Expression of fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin-1 and fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin-1 and fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.     

Type VI collagen is associated with microfibrils and oxytalan fibers in the extracellular matrix of periodontium,mesenterium and periosteum

Type VI collagen was immunolocalized in several soft connective tissues at the light and electron microscopic level. Positive labeling was found in all tissues examined, periodontal ligament, gingiva, mesenterium and periosteum. The labeled structures could be divided into 2 categories: microfibrils intermingling with collagen fibrils, and those that formed bundles (oxytalan fibres and elastin-associated microfibrils). Control sections incubated with antibody pre-absorbed to purified type VI collagen, or with non-immune antibody, proved to be negative. Our observations indicate that the structural organization of type VI collagen varies from small microfibrillar structures associated with the collagen and elastin fibre systems to highly ordered parallel arrays of oxytalan bundles.     

Ultrastructural cytochemistry of oxytalan fibers in the periodontal ligament and microfibrils in the aorta with the periodic acid-thiocarbohydrazide-silver proteinate method

Fullmer's oxytalan fibers are special connective tissue fibers in periodontal ligaments and some non-dental sites of certain animal species, and, ultrastructurally appear to resemble microfibrils related to clastogenesis. The present study has ultrastructurally examined the applicability of Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing complex carbohydrates to the study of oxytalan fibers in rat periodontal ligaments and microfibrils of the tunica adventitia in the rat aorta, where microfibrils are often associated with amorphous elastin and are thought to be oxytalan fibers. In the periodontal ligaments, the PA-TCH-SP method weakly to moderately stained collagen fibrils, moderately stained thin fibrils composing the oxytalan fibers, and intensely stained cytoplasmic granules of fibroblasts. In the aortic adventitia, the PA-TCH-SP method moderately stained collagen fibrils. Heavier staining was observed in microfibrils, whereas the amorphous elastin lacked staining. The most intense staining was seen in cytoplasmic granules and glycogen of mural cells. These studies demonstrate that oxytalan fibers in the periodontal ligament of rats and microfibrils in the aorta of rats contain vicinal glycol-containing glycoproteins and the PA-TCH-SP method is a useful tool in ultrastructural studies of oxytalan fibers and microfibrils of rats.     

Immunohistochemical localization of elastin, fibrillins and microfibril-associated glycoprotein-1 in the developing periodontal ligament of the rat molar

Sugawara Y, Sawada T, Inoue S, Shibayama K, Yanagisawa T. Immunohistochemical localization of elastin, fibrillins and microfibril‐associated glycoprotein‐1 in the developing periodontal ligament of the rat molar. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2008.01196.x. © 2009 John Wiley & Sons A/S Background and Objective: Elastic system fibers are a major component of the periodontal ligament, but little information is available about their detailed composition or the mechanism of elastogenesis in the developing periodontal ligament. The purpose of this study was to investigate immunolocalization of elastin, fibrillins and microfibril‐associated glycoprotein‐1 (MAGP‐1) in the developing periodontal ligament of the rat molar. Material and Methods: Frozen sections of demineralized as well as non‐demineralized periodontal ligament of Wistar rats of various ages from 19 days to 7 weeks were incubated with anti‐elastin, anti‐fibrillin‐1 and ‐2 and anti‐MAGP‐1 antibodies followed by peroxidase‐conjugated secondary antibodies. After incubation with diaminobenzidine solution, immunoreaction products were observed with a light microscope. Results: In the developing periodontal ligament of 19‐day‐old rats, fibers immunopositive to elastin were not present, but fibers positively stained for fibrillin‐2 and MAGP‐1 were widely distributed throughout the ligament. The latter fibers were arranged in the apico‐occlusal direction along with blood vessels. In 3‐week‐old rats, fibers stained for elastin were observed for the first time in the apical region of the ligament. The number and distribution pattern of these elastin‐positive fibers was basically the same as those in rats aged 5 and 7 weeks. In contrast, fibrillin‐2‐ and MAGP‐1‐positive fibers were more extensively distributed in the ligament, and their pattern of distribution was comparable to that of reported oxytalan fibers. Fibrillin‐1 was, however, not detected either in demineralized sections or in non‐demineralized sections, indicating its absence in periodontal ligament. Conclusion: Elastin expressed in the periodontal ligament assembled into elaunin fibers in the vicinity of blood vessels. Both fibrillin‐2 and MAGP‐1 are structural components not only of the elastin‐associated microfibrils but also of elastin‐free microfibrils, with possible roles in elastogenesis and in periodontal ligament homeostasis.     

Intracellular interaction of EMILIN-1 with fibrillin-1 in human periodontal ligament cells

The elastic system fibers comprise oxytalan, elaunin and elastic fibers, differing in their relative microfibril and elastin contents. Human periodontal ligaments (PDLs) contain oxytalan fibers (pure microfibrils), which are composed mainly of fibrillin-1, the major component of microfibrils. We recently demonstrated that EMILIN-1, located at the interface between elastin and microfibrils, controls the amount of fibrillin-1 assembly in PDL fibroblast cell/matrix layers [8], although the mechanism involved was unclear. We subjected cultured PDL fibroblasts to immunofluorescence and immunoprecipitation assays in order to examine the intracellular interaction of EMILIN-1 with fibrillin-1. Immunofluorescence showed that EMILIN-1 was colocalized with fibrillin-1, both inside and outside the cells. Additionally, EMILIN-1 formed a complex with fibrillin-1 in the intracellular fraction. These results suggest that EMILIN-1 may form complexes with fibrillin-1 in cellular vesicles, thus contributing effectively to the initial assembly of pericellular fibrillin-1 during the process of oxytalan fiber formation.     

Light and ultrastructural relationship between oxytalan fibers in the periodontal ligament of the guinea pig.

The interfaces and the relationships between collagen and oxytalan fibers were observed under light and electron microscopy. Guinea pig periodontal ligament was prepared for light and electron microscopy with perfusion using Peter's buffered formalin for light microscopy and GTA-S-collidine and OSO4 for electron microscopic studies. The tissue for light microscopy was stained with a modified Gomori's aldehyde fuchsin technique, in which pre-oxidization with potassium monopersulfate was carried out before staining so as to demonstrate the oxytalan fibers. EM tissues were routinely stained with lead citrate and uranyl acetate. Two different structural relationships were observed. First, the subcomponents of the collagen and oxytalan fiber types interweave with each other; and, second, some of these two-fiber subcomponents appear attached to each other. These relationships and the known orientation of oxytalan fibers as seen in the periodontal ligament provide insight as to the function of oxytalan fibers. The oxytalan fibers may provide increased structural integrity and increased distribution of forces over a wider area of the periodontal ligament. Because of their close relationship to blood and lymph vessels in the periodontal ligament, they may also help to stabilize these elements by the same structural relationships to collagen fibers.     

Development of oxytalan fibers in the rat molar periodontal ligament

Although oxytalan fibers are known to be a ubiquitous component of the periodontal ligament, little information has been available concerning their organization in the developing periodontal ligament. In the present study, growth and distribution of oxytalan fibers were examined in the developing periodontal ligament of rat molars aged 11, 14, 19, 21 and 28 days. A quantitative analysis of the fibers was made and the spatial relationship between the fibers and blood vessels was studied by means of a three-dimensional reconstruction of serial sections. At the beginning of root formation, oxytalan fibers appeared at first as dot-like structures around the root sheath as well as in areas very close to blood vessels. These structures were resolved in the electron microscope to be made up of 12-nm-wide microfibrils in the vicinity of the surface of the cells of the root sheath. In the process of development, these dot-like structures elongated into entities with helical appearances. As the development further proceeded, longer oxytalan fibers were produced in the apico-occlusal direction along with blood vessels. Quantitative analysis showed that an increase in oxytalan fibers coincided with an increase in the density of the vascular network in the developing periodontal ligament. Based on the results of the present study, the role of oxytalan fibers in the developing periodontal ligament may be in the maintenance of the integrity of the vascular system as previously suggested.     

Light and ultrastructural relationship between oxytalan fibers in the periodontal ligament of the guinea pig

Abstract The interfaces and the relationships between collagen and oxytalan fibers were observed d light and electron microscopy. Guinea pig periodontal ligament was prepared for light and electron microscopy with perfusion using Peter's buffered formalin for light microscopy and GTA-S-collidine and O_SO₄ for electron microscopic studies. The tissue for light microscopy was stained with a modified Gomori's aldehyde fuchsin technique, in which pre-oxidization with potassium monopersulfate was carried out before staining so as to demonstrate the oxytalan fibers. EM tissues were routinely stained with lead citrate and uranyl acetate. Two different structural relationships were observed. First, the subcomponents of the collagen and oxytalan fiber types interweave with each other; and, second, some of these two-fiber subcomponents appear attached to each other. These relationships and the known orientation of oxytalan fibers as seen in the periodontal ligament provide insight as to the function of oxytalan fibers. The oxytalan fibers may provide increased structural integrity and increased distribution of forces over a wider area of the periodontal ligament. Because of their close relationship to blood and lymph vessels in the periodontal ligament, they may also help to stabilize these elements by the same structural relationships to collagen fibers.     

Ultrastructural evidence of nerve and oxytalan fibre associations in the endoneurium of human periodontal ligament

Human periodontal ligament from premolars contained numerous fibres of the oxytalan meshwork system affiliated with unmyelinated endoneurium and myelinated nerves in the endoneurium. These fibres consisted of bundles of microfibrils and were present within 0.5 μ of Schwann cells and their unmyelinated axons. This ultrastructural association has not been previously demonstrated.     

Oxytalan-vascular relationships observed in histologic examination of the periodontal ligaments of man and mouse

Buccolingual and mesiodistal sections through the periodontal ligaments of human premolars and mouse molars revealed similar oxytalan systems. The fibres formed a dense three-dimensional meshwork extending from the dentine-cementum junction to the peripheral vessels of the periodontal ligament. Apical to the alveolar crest, the meshwork exhibited a predominantly occluso-apical orientation with a laterally intercommunicating system of fine branching fibrils. Oxytalan fibres displayed two types of vascular association. One arrangement was a generalized relationship with individual periodontal vessels of all types. The second type of association involved the formation of oxytalan-vascular structures in which the oxytalan meshwork enclosed groups of vessels, as well as the individual vessels within each group, to form complex units. It is postulated that the oxytalan-vascular association may form part of a periodontal receptor mechanism which mediates vascular control.     

The oxytalan fiber system in the mandibular periodontal ligament of the lathyritic mouse.

Experimental lathyrism was produced in young albino mice with a diet containing 50% sweet pea seed (Lathyrus odoratus). After 7 days on the lathyritic diet, sections of themandibular molar and incisor periodontal ligaments, when oxidized and treated with aldehyde fuchsin, demonstrated enhanced staining of the oxytalan fibers and numerous vessels. At this time aldehyde fuchsin or orcein also revealed marked pathological changes in the periodntal ligament of all molars. When athyrism was prolonged for 12 weeks, both the molar and incisor oxytalan systems were still readily identifiable although the molar periodontal ligament continued to be serverely affected by lathyrism. The oxytalan fibers retained their characteristic tooth-vascular association in all of the lathyritic mice. Oxytalan fibers of the lathyritic and control animals showed similar reactions to enzyme digestion with beta-glucuronidase, elastase, and pepsin. However, gingival elastic fibers reacted in a different way from oxytalan fibers with beta-glucuronidase and elastase treatment. These findings indicate that in the lathyritic mouse the oxytalan fiber system of functioning teeth possesses a high degree of permanence and is metabolically distinct from collagen and elastic fibers.     

The Oxytalan Fiber System in the Mandibular Periodontal Ligament of the Lathyritic Mouse

Abstract Experimental lathyrism was produced in young albino mice with a diet containing 50 % sweet pea seed (Lathyrus odoratus). After 7 days on the lathyritic diet, sections of the mandibular molar and incisor periodontal ligaments, when oxidized and treated with aldehyde fuchsin, demonstrated enhanced staining of the oxytalan fibers and numerous vessels. At this time aldehyde fuchsin or orcein also revealed marked pathological changes in the periodontal ligament of all molars. When lathyrism was prolonged for 12 weeks, both the molar and incisor oxytalan systems were still readily identifiable although the molar periodontal ligament continued to he severely affected by lathyrism. The oxytalan fibers retained their characteristic tooth-vascular association in all of the lathyritic mice. Oxytalan fibers of the lathyritic and control animals showed similar reactions to enzyme digestion with beta-glucuronidase, elastase, and pepsin. However, gingival elastic fibers reacted in a different way from oxytalan fillers with beta-glucuronidase and elastase treatment. These findings indicate that in the lathyritic mouse the oxytalan fiber system of functioning teeth possesses a high degree of permanence and is metabolically distinct from collagen and elastic fibers.     

Reconstitution of the human oxytalan system during orthodontic tooth movement.

Orthodontic tooth movement in man has revealed that the oxytalan fiber system possesses a high order of maintenance. Oxytalan fibers did not merely increase in number during orthodontic movement. On the contrary, the oxytalan fiber system underwent reconstruction and adaptation to extensive metabolic and anatomic changes within the periodontium. With the use of light orthodontic forces, the oxytalan fiber system was constantly remodeled on both the tension and compression sides and maintained a characteristic cementum-vascular relationship even when teeth were moved a significant distance through the alveolar bone. In contrast, heavier forces caused localized destruction of the oxytalan system in regions of excessive pressure and tension. Reconstitution of the oxytalan system provided evidence against the concept that oxytalan fibers are stretched by orthodontic movement and subsequently contribute to relapse by elastic rebound. In man the oxytalan fiber system of the periodontal ligament is arranged as a three-dimensional fiber meshwork and exhibits a complex geometry like other fiber systems in the connective tissues. Knowledge of the interaction between elastic and collagen fibrillar assemblies has been used to derive some speculative concepts of oxytalan-collagen interaction. These concepts have been put forward with the intention of stimulating further interest in the oxytalan fiber meshwork. The present investigation emphasizes that the use of the light microscope to examine and record static images of complex biologic changes can provide new knowledge of the structure and function of human connective tissues.     

Stretching modulates oxytalan fibers in human periodontal ligament cells

Background and Objective:  Oxytalan fibers, as well as collagen fibers, are the structural components of periodontal ligaments. Periodontal ligaments are continuously exposed to various functional forces. However, the behavior of oxytalan fibers under mechanical strain has not been investigated. We hypothesized that strain would alter the amount and appearance of oxytalan fibers in terms of positivity for their major components, fibrillin-1 and fibrillin-2.
Material and Methods:  We subjected periodontal ligament fibroblasts to stretching strain to examine the effects on their formation of oxytalan fibers in cell/matrix layers.
Results:  Stretching increased the levels of fibrillin-1 and fibrillin-2 by 25% relative to the control, but did not affect the gene expression level of either type of fibrillin. Immunofluorescence and immunogold electron microscopy analysis revealed that bundles of oxytalan fibers became thicker under stretching conditions.
Conclusion:  These results suggest that tension strain functionally regulates microfibril assembly in periodontal ligament fibroblasts and thus may contribute to the homeostasis of oxytalan fibers in periodontal ligaments.     

Electron-microscopic affiliations of oxytalan fibres,nerves and the microvascular bed in the mouse periodontal ligament

Throughout the ligament, oxytalan fibres were contiguous to myelinated nerves, unmyelinated exposed axons and free nerve endings. In the cervical and apical regions, accumulations of vessel-related simple and complex mechanoreceptor units were associated with collagen fibrils and fibres of the oxytalan system. The various receptors and nerve endings penetrated to the abluminal surface of the endothelial wall in the different categories of vessels constituting the microvascular bed. Periodontal receptors with oxytalan fibres were also present in the septal wall of dividing vessels and related to endothelial protrusions into the lumen of microvessels. Similarities existed between periodontal mechanoreceptors and baroreceptors. Anatomically, the oxytalan-fibre meshwork provided coupling between the various mechanoreceptor units in the microvascular bed. This periodontal model has morphological characteristics which support the hypothesis that the oxytalan-fibre meshwork forms part of a proprioceptor system for the regulation of vascular flow.     

Tropoelastin expression by periodontal fibroblasts

Elastic system fibers are load-bearing proteins found in periodontal tissue. There are three types--oxytalan, elaunin, and elastic fibers--which differ in their relative microfibril and elastin contents. Oxytalan fibers are known to be distributed in the periodontal ligaments and gingiva, whereas elaunin and elastic fibers are present only in the gingiva. We examined gene expression and accumulation of tropoelastin in the cell-matrix layers of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF) in vitro. HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 8 wks. Northern blotting and RT-PCR analyses showed that only HGF expressed mRNA encoding tropoelastin. Western blotting analysis demonstrated 77-kDa protropoelastin and 68-kDa tropoelastin only in the cell-matrix layer of HGF cultured for 8 wks. These results suggest that the different tropoelastin expression patterns reflect the difference between HGF and HPLF phenotypes.     

Oxytalan connective tissue fibers: a review

Oxytalan connective tissue fibers are a separate and distinct fiber type. Although current histochemical methods cannot distinguish pre-elastic from oxytalan fibers, the two fiber types are readily distinguished by electron microscopy. Oxytalan fibers are found in periodontal membranes of all teeth of man, monkeys, rats, guinea pigs and mice. Increased numbers and size of oxytalan fibers are observed in periodontal membranes of teeth subjected to increased stress, such as those used for bridge abutments. Edwards (1968) observed increased size and number of oxytalan fibers in periodontal membranes of dog incisors subjected to orthodontic forces. Some oxytalan fibers serve to support the blood and lymphatic vessels leading to the teeth. Oxytalan fibers appear to have a protein component and a stainable component digestible with beta-glucuronidase after peracetic acid digestion. Oxytalan fibers develop in repair tissues of the periodontal membrane. Although oxytalan fibers probably develop in relation to tumors developed from dental tissues, electron microscopy must be employed to distinguish oxytalan from developing elastic tissues inasmuch as histochemical methods are inadequate.     

Gene expression and accumulation of fibrillin-1, fibrillin-2, and tropoelastin in cultured periodontal fibroblasts

The elastic system fibers consist of three types--oxytalan, elaunin, and elastic fibers--differing in their relative microfibril and elastin contents. All three types are found in human gingiva, but human periodontal ligaments contain only elastin-free fibers. We examined cultured human gingival fibroblasts (HGF) and cultured human periodontal ligament fibroblasts (HPLF) to determine the gene expression of fibrillin-1 and fibrillin-2 (the major components of microfibrils) and of tropoelastin. In addition, we assessed the degree of accumulation of these proteins in the extracellular matrix. Northern blot analysis revealed that the level of expression of fibrillin-1 and fibrillin-2 was higher in HGF than in HPLF. However, examination of matrix samples from HGF and HPLF cell layers showed that there was no difference in fibrillin-1 accumulation, although fibrillin-2 accumulated to a much greater extent in the HGF-derived matrix. Tropoelastin was expressed only in and around HGF. These results show a correlation between gene expression and the accumulation of tropoelastin and fibrillin-2 in HGF.     

Oxytalan connective tissue fibers: A review

Abstract. Oxytalan connective tissue fibers are a separate and distinct fiber type. Although current histochemical methods cannot distinguish pre-elastic from oxytalan fibers, the two fiber types are readily distinguished by electron microscopy. Oxytalan fibers are found in periodontal membranes of all teeth of man, monkeys, rats, guinea pigs and mice. Increased numbers and size of oxytalan fibers are observed in periodontal membranes of teeth subjected to increased stress, such as those used for bridge abutments. Edwards (1968) observed increased size and number of oxytalan fibers in periodontal membranes of dog incisors subjected to orthodontic forces. Some oxytalan fibers serve to support the blood and lymphatic vessels leading to the teeth. Oxytalan fibers appear to have a protein component and a stainable component digestible with β-glucuronidase after peracetic acid digestion. Oxytalan fibers develop in repair tissues of the periodontal membrane. Although oxytalan fibers probably develop in relation to tumors developed from dental tissues, electron microscopy must be employed to distinguish oxytalan from developing elastic tissues inasmuch as histochemical methods are inadequate.