Vasoactive intestinal polypeptide-like immunoreactivity in the bovine heart: high degree of coexistence with neuropeptide Y-like immunoreactivity

Summary Recent studies have accomplished the establishment of a collagenous fiber-fringe matrix upon dental root surfaces in vitro. The present study was undertaken to follow the development of such a matrix in vitro and to test the possible effects of root surface treatments upon this matrix. Periodontal ligament cells, 0.1 to 0.2-mm thick dental root discs, and alveolar bone cells were derived after extraction from four partially erupted third molars and the accompanying interradicular bony septa of 1 male patient. Autologous serum was obtained by venipuncture. Cultures were initiated by delivering a 1-ml suspension of 50000 tritiated thymidine-labeled periodontal ligament cells and 50000 alveolar bone cells onto each of 42 culture sets. The following day, demineralized or non-demineralized root discs treated with autologous serum, fibronectin or complete medium were placed in pairs, separated by a 0.1–1.0 mm gap, upon the initial cell layer. Representative cultures were terminated after 2, 3, 4, 5 and 6 weeks, and processed for light- and electron microscopy, morphometric analysis and autoradiography. An outstanding feature of the early cultures (2, 3 and 4 weeks) was a patchwise, random distribution of matrix making a precise developmental study impossible, although collagen fibrils were produced within the first 2 weeks. Some 3-week cultures already demonstrated a mature fiber-fringe characterized electron-microscopically as oriented, densely packed collagen fibrils closely abutting the cementum-lined root discs. The treatments (including autologous serum) used in this study had no appreciable morphologic or morphometric effect upon the fiber-fringe formed. Because none of the cultures in the present or past studies have demonstrated a true cementoid matrix, this model may not be suitable for the in-vitro study of cementum formation.     

Establishment of acellular extrinsic fiber cementum on human teeth

Summary The present study describes for the first time the development of early acellular extrinsic fiber cementum (AEFC) until its establishment on human teeth. Precisely selected premolars with roots developed to 50%–100% of their final length were prefixed in Karnovsky's fixative and most of them were decalcified in EDTA. Their roots were subdivided into about 10 blocks each, cut from the mesial and distal root surfaces. Following osmication, these blocks were embedded in Epon and sectioned for light-and transmission electron microscopy. Some blocks were cut non-demineralized. From semithin stained sections, the density of the collagenous fiber fringe protruding from the root surface was measured by using the Videoplan-system. After initiation of this fiber fringe and its attachment to the dentinal root surface followed by mineralization, the fringe gradually increased in length and subsequently became mineralized. Fringe elongation and the advancement of the mineralization front appeared to progress proportionally. Thus, in all stages of AEFC development, a short fiber fringe covered the mineralized AEFC. Its density remained constant, irrespective of AEFC thickness. The latter gradually increased and reached an early maximum of 15–20 m in the cervical region. At this stage, the AEFC fringe appeared to fuse with the future dentogingival or other collagen fibers of the tooth supporting apparatus. Mineralization of the fringe commenced with isolated, spherical or globular centers, which later fused with the mineralization front and became incorporated in AEFC.Abbreviations AEFC acellular extrinsic fiber cementum - CIFC cellular intrinsic fiber cementum - CMSC cellular mixed stratified cementum - CEJ cemento-enamel junction - CM centers of mineralization - D dentin - DCJ dentino-cemental junction - EDTA ethylene diaminetetraacetic acid - FF fiber fringe - GL glycogen storage granules - MF mineralization front - PL periodontal ligament - PLF periodontal ligament fibers     

Initiation of acellular extrinsic fiber cementum on human teeth

Summary The development of acellular extrinsic fiber cementum (AEFC) has never before been studied in human teeth. We have therefore examined the initiation of AEFC in the form of a collagenous fiber fringe and its attachment to the underlying dentinal matrix, in precisely selected, erupting human premolars with roots developed to 50%–60% of their final length. Freshly extracted teeth were prefixed in Karnovsky's fixative, decalcified in EDTA and subdivided into about 10 blocks each, cut from the mesial and distal root surfaces, vertical to and along the root axis. The blocks were postfixed in osmium tetroxide, embedded in Epon and cut for light- and electron-microscopic investigation. Starting at the advancing edge of the root, within a region extending about 1 mm coronal to this edge, fibroblast-like cells were seen closely covering the external root surface. Along the first 100 m from the root edge, these cells extended cytoplasmic processes and contacted the dentinal collagen fibrils. Between these cells and the dentinal matrix, new collagen fibrils and very short collagen fibers gradually developed. Within the second 100 m from the root edge, this resulted in the formation of a cell-fiber fringe network. Newly formed fibers of the fringe were directly attached to the non-mineralized matrix containing dentinal collagen fibrils and could be distinguished from the latter by differences in fibril orientation. During the process of dentin mineralization, the transitional zone between the fiber-fringe base and the dentinal matrix, i.e., the future dentino-cemental junction, also mineralized. It is suggested that this fiber fringe is the base of AEFC, which later increases in thickness by fiber extension and subsequent mineralization.Abbreviations AEFC acellular extrinsic fiber cementum - AIFC acellular intrinsic fiber cementum - CIFC cellular intrinsic fiber cementum - CMSC cellular mixed stratified cementum - ARE advancing root edge - CP cytoplasmic process - D dentin - DCJ dentinocemental junction - E enamel - EBL external basal lamina - EC epithelial cell - EDTA ethylene diaminetetraacetic acid - ERM epithelial rests of Malassez - FF fiber fringe - HRS Hertwig's epithelial root sheath - IBL internal basal lamina - MD mineralized dentin - NMD non-mineralized dentin - OB odontoblast - PD predentin - PL periodontal ligament     

Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum

We examined the ultrastructural localization of collagens Type I, V, VI and of procollagen Type III in decalcified and prefixed specimens of the periodontal ligament and cementum, by immunoelectron microscopy using ultra-thin cryostat sections. Immunostaining for collagen Type I was pronounced on the major cross-striated fibrils entering cementum and in cementum proper, whereas staining for procollagen Type III was almost exclusively observed on the major fibrils in the periodontal ligament situated more remote from cementum. Reactivity for collagen Type V was limited to aggregated, unbanded filamentous material of about 12 nm diameter that was found mainly in larger spaces between bundles of cross-striated collagen fibrils and occasionally on single microfibrils that apparently originated from the ends of the major collagen fibrils, which may support the concept of this collagen as a component of core fibrils. Collagen Type VI was present as microfilaments appearing to interconnect single cross-striated fibrils. In the densely packed fibril bundles of the periodontal ligament, no collagen type VI was detected. Neither Type V or Type VI collagen was observed in cementum.     

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.     

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.     

Bone-like nodules formed in vitro by rat periodontal ligament cells

The periodontal ligament has been shown to possess the ability to regenerate both new cementum and alveolar bone as well as a self-regenerative capacity; however, the source of cementoblasts and osteoblasts is not still clear. We investigated the development of bone-like tissue in vitro by periodontal ligament cells, in order to determine whether the periodontal ligament contains osteoprogenitor cells. Periodontal ligament cells were obtained from periodontal ligament tissue attached to the maxillary incisors of 6-week-old WKA rats by means of the explant technique. Cells at passage #3 were cultured for long term in α-minimum essential medium containing 10% fetal bovine serum, antibiotics, and 50 μg/ml ascorbic acid, and were then examined using phase-contrast microscopy, histochemistry, transmission electron microscopy, X-ray microanalysis, and electron diffraction. Nodules were formed in the cultures, and when 10 mM Na-β-glycerophosphate was added, these nodules became mineralized. The mineralized nodules were identified as bone-like elements in view of the presence of osteoblast-like and osteocyte-like cells, collagenous matrix, a mineral composed of hydroxyapatite, and intense alkaline phosphatase activity. The results show that the periodontal ligament contains osteoprogenitor cells, which differentiate into osteoblasts and produce bone-like tissue.     

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.     

Oxytalan fibre organization in marsupial mandibular periodontal tissues

The arrangement and distribution of oxytalan fibers in Australian marsupials has not previously been reported. Periodontal tissues of wombat, wallaby, possum, and marsupial mouse were examined to ascertain oxytalan fibre organization. Despite adaptation of the marsupial masticatory apparatus to different diets the oxytalan fibre organization in the periodontal ligament shows a basic pattern which corresponds with that reported in other animals. The oxytalan system forms a continuous meshwork of fine, branching fibres which completely invests each tooth root and connects adjacent teeth. Thick ribbon-like apico-occlusally orientated oxytalan fibres, thought to form by the coalescence of thinner fibres, are restricted to the periodontal ligament. The oxytalan fibres are embedded in cementum and attached to blood vessels in the periodontal ligament. Oxytalan fibres do not insert into alveolar bone. Histological evidence indicates functional remodelling of the oxytalan fibre system in continuously erupting teeth.     

Directing the differentiation of human dental follicle cells into cementoblasts and/or osteoblasts by a combination of HERS and pulp cells

It is known that the dental follicle (DF) consists of progenitor cells that give rise to the cementum, periodontal ligament, and alveolar bone; but little information is available about the regulation of DF cell differentiation into either cementogenic or osteogenic cell lineages for the regeneration of diseased periodontal tissue. Here, we investigated the roles of DF, Hertwig’s epithelial root sheath (HERS), and pulp cells in the cementum and during alveolar bone formation. We cultured these cells; transplanted them alone or in combination into immunocompromised mice; and observed their effects at 6 and 12 weeks. Histological and immunohistochemical results revealed that DF cells formed cementum-like tissues with immunoreactivity to cementum-derived attached protein, bone sialoprotein, type I collagen, and alkaline phosphatase. In addition, HERS cells played a role in the induction and maturation of cementum-like tissues formed by DF cells. In contrast, implants of DF cells in the presence of pulp cells led to the formation of bone-like tissues. Interestingly, in the presence of both HERS and pulp cells, DF cells formed both cementum-like and bone-like tissues. We demonstrated that while HERS cells are able to induce DF cell differentiation into cementoblasts and promote cementum formation, pulp cells could direct DF cell differentiation into osteoblasts and enhance alveolar bone formation. These results suggest that the combined use of DF, HERS, and pulp cells could direct DF cell differentiation into cementoblasts and/or osteoblasts in vivo, thus providing a novel strategy for the successful repair and regeneration of diseased periodontal tissue.     

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。     

Extracellular matrix-mediated tissue remodeling following axial movement of teeth.

Tooth eruption is a multifactorial process involving movement of existing tissues and formation of new tissues coordinated by a complex set of genetic events. We have used the model of the unopposed rodent molar to study morphological and genetic mechanisms involved in axial movement of teeth. Following extraction of opposing upper molars, lower molars supererupted by 0.13 mm. Labeled tissue sections revealed significant amounts of new bone and cementum apposition at the root apex of the unopposed side following supereruption for 12 days. Newly apposited cementum and alveolar bone layers were approximately 3-fold thicker in the experimental vs the control group, whereas periodontal ligament width was maintained. Tartrate-resistant acid phosphatase staining indicated bone resorption at the mesial alveolar walls of unopposed molars and provided in tandem with new bone formation at the distal alveolar walls an explanation for the distal drift of molars in this model. Microarray analysis and semiquantitative RT-PCR demonstrated a significant increase in collagen I, integrin beta5, and SPARC gene expression as revealed by comparison between the unopposed molar group and the control group. Immunohistochemical verification revealed increased levels of integrin beta5 and SPARC labeling in the periodontal ligament of the unopposed molar. Together our findings suggest that posteruptive axial movement of teeth was accomplished by significant formation of new root cementum and alveolar bone at the root apex in tandem with upregulation of collagen I, integrin beta5, and SPARC gene expression.     

The characteristics of epithelial cell rests of Malassez during tooth eruption of development mice

It has been demonstrated that Hertwig’s epithelial root sheath (HERS) has an important role in root development, closely related to development of cementum epithelial rests of Malassez (ERM) as the residuum after HERS fragment, and is the exclusive epithelial structure in the mature periodontal ligament. Some studies reported that ERM may play a role in maintaining a stable environment of periodontal, and likely to be involved in regeneration of periodontal tissue, especially of cementum. However, the function of the ERM is not well understood. In this study, we observed the morphology and biological characteristics of ERM of the maxillary 1st molar with surrounding periodontal tissues of BALB/c mice during the period of tooth cusp erupted out of the gingiva to occlusion stability. Immunohistochemistry revealed ERM predominately located at the cervical and root furcation regions of the periodontal ligament. The number of ERM cells at the cervical and root furcation regions of the post-built occlusion stage decreased compared to pre-built occlusion stage and occlusion building stage. Transmission electron microscopy analysis showed that epithelial cell nuclei with typical features of apoptosis were observed at the post-built occlusion stage, and consistent with positive bodies labeled by TUNEL(terminal deoxy-transferase (TdT)-mediated dUTP-biotin nick end labeling), while proliferating cell nuclear antigen positive bodies mainly located at occlusion built stage. It suggests that ERM may regulate in alveolar bone remodeling in association with the periodontal ligament during tooth erupting to occlusion stability and may play important roles potentially in regeneration and homeostasis of the periodontal tissues.     

Cementum is key to periodontal tissue regeneration: A review on apatite microstructures for creation of novel cementum-based dental implants

The cementum is the outermost layer of hard tissue covering the dentin within the root portion of the teeth. It is the only hard tissue with a specialized structure and function that forms a part of both the teeth and periodontal tissue. As such, cementum is believed to be critical for periodontal tissue regeneration. In this review, we discuss the function and histological structure of the cementum to promote crystal engineering with a biochemical approach in cementum regenerative medicine. We review the microstructure of enamel and bone while discussing the mechanism underlying apatite crystal formation to infer the morphology of cementum apatite crystals and their complex structure with collagen fibers. Finally, the limitations of the current dental implant treatments in clinical practice are explored from the perspective of periodontal tissue regeneration. We anticipate the possibility of advancing periodontal tissue regenerative medicine via cementum regeneration using a combination of material science and biochemical methods.     

Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine

Periodontal ligament (PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support the teeth in situ and preserve tissue homeostasis. Recent studies have revealed the existence of stem cells in human dental tissues including periodontal ligament that play an important role, not only in the maintenance of the periodontium but also in promoting periodontal regeneration. In this study, human periodontal ligament cells (hPDLCs) were isolated by outgrowth and enzymatic dissociation methods. Expression of surface markers on PDLCs as human mesenchymal stem cells (MSCs) was identified by flow cytometry. In addition, proliferation and differentiation capacity of cultured cells to osteoblasts, adipocytes were evaluated. As a result, we successfully cultured cells from the human periodontal ligament tissues. PDLCs express mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 and do not express CD34, CD45, and HLA-DR. PDLCs also possess the multipotential to differentiate into various types of cells, such as osteoblast and adipocytes, in vitro. Therefore, these cells have high potential to serve as materials for tissue engineering, especially dental tissue engineering.     

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.     

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.     

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.