The evolution of human periodontal tissues with ageing.

In this research, the structural modifications with ageing of clinically healthy periodontal tissues were analyzed by means of polarization microscopy and morphometrical methods for light microscopy. The new findings may be summarized as follows. The periodontal ligament was found to be widened in the cervical and apical regions. The thickening of cementum with ageing was shown to be accompanied by a modification in the shape of Sharpey's fibres, which in the elderlies were wavy instead of straight as in the control. Lamellar bone, forming an osteone, was found to substitute in part for cementum in one tooth. These results are interpreted as indicating that: (1) late active eruption occurs in man, causing the observed modification in the thickness of periodontal ligament and cementum in the apical region and in the direction of Sharpey's fibres within cementum; (2) cementum may undergo renewal during lifetime and in this case bone may be deposited in contact with dentin.     

The mosasaur tooth attachment apparatus as paradigm for the evolution of the gnathostome periodontium

SUMMARY Vertebrate teeth are attached to jaws by a variety of mechanisms, including acrodont, pleurodont, and thecodont modes of attachment. Recent studies have suggested that various modes of attachment exist within each subcategory. Especially squamates feature a broad diversity of modes of attachment. Here we have investigated tooth attachment tissues in the late cretaceous mosasaur Clidastes and compared mosasaur tooth attachment with modes of attachment found in other extant reptiles. Using histologic analysis of ultrathin ground sections, four distinct mineralized tissues that anchor mosasaur teeth to the jaw were identified: (i) an acellular cementum layer at the interface between root and cellular cementum, (ii) a massive cone consisting of trabecular cellular cementum, (iii) the mineralized periodontal ligament containing mineralized Sharpey's fibers, and (iv) the interdental ridges connecting adjacent teeth. The complex, multilayered attachment apparatus in mosasaurs was compared with attachment tissues in extant reptiles, including Iguana and Caiman . Based on our comparative analysis we postulate the presence of a quadruple-layer tissue architecture underlying reptilian tooth attachment, comprised of acellular cementum, cellular cementum, mineralized periodontal ligament, and interdental ridge (alveolar bone). We propose that the mineralization status of the periodontal ligament is a dynamic feature in vertebrate evolution subject to functional adaptation.     

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.     

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.     

Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.     

Immunohistochemical detection of matrix metalloproteinase-1 in the periodontal ligament of equine cheek teeth

The hypsodont equine cheek tooth erupts continuously throughout life. The collagen fibers of the periodontal ligament (PDL) have to remodel constantly to allow the tooth to move in an occlusal direction. Remodeling of the collagen fiber bundles needs to be well-coordinated in order to maintain functional tooth support. The aim of this study was to examine the role of matrix metalloproteinase-1 (MMP-1) in the collagen remodeling of the equine PDL under physiological conditions. Specimens containing the PDL interposed between the dental cementum and the alveolar bone were taken from nine Warmblood horses at three designated horizontal levels: subgingival, middle, and apical. The expression of MMP-1 was detected immunohistochemically. MMP-1 was found to be present in the specimens of all horses. Immunopositive fibroblasts/fibrocytes were accumulated within individual single collagen fascicles. Our results suggest that MMP-1 induced collagen degradation plays a central role in the physiological remodeling of the equine PDL. The distribution of MMP-1 positive fascicles indicates well-directed remodeling which occurs as an asynchronous process, so that only single collagen fascicles are remodeled at the same time. Due to this remodeling of one fascicle at a time, the overall anchorage of the tooth is preserved at all times.     

The molecular organization of myosin in stress fibers of cultured cells

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

大鼠咬合创伤牙周膜中MCP-1、ICAM-1的表达变化

目的:研究咬合创伤大鼠牙周组织中MCP-1、ICAM-1的表达情况。方法:12周龄雄性SD大鼠24只,随机分为4组(1个正常对照组和3个实验组),每组6只。正常对照组不作任何处理,实验组通过在大鼠左上颌第一磨牙颌面粘接树脂并内置不锈钢丝形成高出颌面0.6-0.8 mm的树脂层以建立同侧下颌咬合创伤实验动物模型,分别于建模后3、5、7 d处死各组大鼠,分离大鼠下颌组织,运用HE、Masson染色观察咬合创伤牙周组织形态变化,同时用免疫组织化学染色法检测MCP-1和ICAM-1的表达变化。结果:HE染色显示,正常组牙周膜纤维排列整齐,牙骨质表面较为平整,牙槽骨结构致密。实验组牙周膜纤维排列紊乱,牙周膜血管水肿充血、间隙改变,牙槽骨和牙骨质表面不平整,出现骨吸收。Masson染色显示,正常组牙周组织未见异常表现;实验组牙周膜纤维排列紊乱,可见水解断裂,局部有血流障碍和血管破裂。免疫组织化学显示,各实验组MCP-1和ICAM-1的表达变化均较正常对照组增多,差异有显著性(P0.05)。其中7 d组表达水平最高,与其他2组相比有统计学意义(P0.05)。结论:咬合创伤可引起大鼠牙周组织形态变化,MCP-1、ICAM-1的表达随时间呈现递增的趋势。     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Dilated capillaries, disorganized collagen fibers and differential gene expression in periodontal ligaments of hypomorphic fibrillin-1 mice

The periodontal ligaments (PDLs) are soft connective tissue between the cementum covering the tooth root surface and alveolar bone. PDLs are composed of collagen and elastic system fibers, blood vessels, nerves, and various types of cells. Elastic system fibers are generally formed by elastin and microfibrils, but PDLs are mainly composed of the latter. Compared with the well-known function of collagen fibers to support teeth, little is known about the role of elastic system fibers in PDLs. To clarify their role, we examined PDLs of mice underexpressing fibrillin-1 (mgR mice), which is one of the major microfibrillar proteins. The PDLs of homozygous mgR mice showed one-quarter of the elastic system fibers of wild-type (WT) mice. A close association between the elastic system fibers and the capillaries was noted in WT, homozygous and heterozygous mgR mice. Interestingly, capillaries in PDLs of homozygous mice were dilated or enlarged compared with those of WT mice. A comparable level of type I collagen, which is the major collagen in PDLs, was expressed in PDL-cells of mice with three genotypes. However, multi-oriented collagen fiber bundles with a thinner appearance were noted in homozygous mice, whereas well-organized collagen fiber bundles were seen in WT mice. Moreover, there was a marked decrease in periostin expression, which is known to regulate the fibrillogenesis and crosslinking of collagen. These observations suggest that the microfibrillar protein, fibrillin-1, is indispensable for normal tissue architecture and gene expression of PDLs.     

Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.     

OPN deficiency suppresses appearance of odontoclastic cells and resorption of the tooth root induced by experimental force application

Osteopontin (OPN) is a major non-collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN-deficient and wild-type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro-CT section and the level of tartrate resistant acid phosphatase (TRAP)-positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force-induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP-positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN-deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application.     

Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues

Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.     

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.     

Initial formation of cellular intrinsic fiber cementum in developing human teeth

Summary The present study describes the formative process of the initiation of cellular intrinsic fiber cementum (CIFC) in still growing human teeth. From 29 premolars and molars with incomplete roots developed to 60–90% of their final length, 8 premolars (with roots formed to three quarters of their final length) were selected for electron-microscopic investigation. All teeth were clinically intact and prefixed in Karnovsky's fixative immediately after extraction. Most of them were decalcified in ethylene diaminetetraacetic acid (EDTA), and the apical part of the roots was divided axially into mesial and distal portions that were subdivided in about 5 slices each. Following osmication and embedding in Epon, these blocks were cut for light- and electron-microscopic examination. In addition, 5 teeth with incomplete roots were freed from organic material and processed for scanning electron microscopy. It was found that CIFC-initiation commenced very close to the advancing root edge and resulted in a rapid cementum thickening. Thereafter, appositional growth continued on the already established cementum surface. Large, basophilic and rough endoplasmic reticulum-rich cementoblasts, some of which became cementocytes, were responsible for both fast and slow CIFC-formation. The CIFC-matrix was free of Sharpey's fibers and composed of more or less organized intrinsic collagen fibrils, in part fibril bundles, that ran roughly parallel to the root surface. Initially, the cementum fibrils intermingled with those of the dentinal collagen fibrils, which were not yet mineralized. This boundary subsequently underwent calcification. The development of collagen fibril bundles and their extracellular arrangement were associated with cytoplasmic processes probably involved in fibril formation and fibril assembly. Many cementoblasts contained intracytoplasmic, membrane-bounded collagen fibrils, which probably were related to fibril formation rather than degradation.     

Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections

This study was designed to detect tissue non-specific alkaline phosphatase (TNSALP) by Azo-dye staining, calcium by glyoxal bis (2-hydroxyanil) (GBHA) staining, bone sialoprotein (BSP) and osteopontin (OPN) by immunoperoxidase staining in developing rat molars, and also to discuss the mineralization process during acellular cementogenesis. To restrain a reduction in histochemical and immunohistochemical reactions, fresh-frozen undemineralized sections were prepared. Where the epithelial sheath was intact, TNSALP reaction was observed in the dental follicle, but not in the epithelial sheath. With the onset of dentin mineralization, the BSP- and OPN-immunoreactive, initial cementum layer appeared. At this point, cementoblasts had shown intense TNSALP reaction and GBHA reactive particles (=calcium-GBHA complex) appeared on the root surface. With further development, the reaction of TNSALP and GBHA became weak on the root surface. Previous studies have shown that the initial cementum is fibril-poor and that matrix vesicles and calciferous spherules appear on the root surface only during the initial cementogenesis. The findings mentioned above suggest that: during the initial cementogenesis, cementoblasts release matrix vesicles which result in calciferous spherules, corresponding to the GBHA reactive particles. The calciferous spherules trigger the mineralization of the initial cementum. After principal fiber attachment, mineralization advances along collagen fibrils without matrix vesicles.