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.     

Cementum annulation and age determination in Homo sapiens. II. Estimates and accuracy

The cementum annulation aging technique was evaluated in a sample of 80 clinically extracted premolars (age range 11–70 years). Demineralized thin sections (7μm) stained with hematoxylin were used. The correlation (r) between age and adjusted count (number of annulations added to age of tooth eruption) was 0.78 for the entire sample (N = 73) and 0.86 for a subsample in which teeth with periodontal disease were excluded (N = 55). Standard error of the estimates ranged from 4.7 to 9.7 years depending on sex and health status of the tooth. The technique provided significantly better estimates for females than for males. The overall inaccuracy (mean absolute error) of the technique was 6.0 years, with a bias (mean error) of 0.26 years. Reduced major axis regression of adjusted count on age produced a slope of 0.797 for the entire sample and 0.889 for the nonperiodontal disease subsample. These slopes are consistent with a hypothesis of annual deposition of cementum rings given a decrease in cementogenesis with increasing age.     

垩质年轮法在旧石器时代动物考古学中的应用

在旧石器时代考古遗址研究中,动物的死亡年龄往往能够反映古人类的生存能力及猎物选择趋向;死亡季节则是研究古人类人口数量变化、聚居模式等生存适应问题的重要途径。牙齿垩质切片研究表明,垩质的沉积与树木年轮的生长相似,具有较强的季节规律性和终生生长的特点,可被用于同时推断动物的死亡年龄与季节。垩质年轮法在国外已经有不少较为成功的应用范例,但由于其自身局限及我国旧石器时代动物考古学的发展状况,这一方法至今并未得到较好的应用。本文主要介绍垩质年轮法原理及其在旧石器时代考古学研究中的应用实例,并对该方法在国内遗址相关研究中的应用前景做出展望。     

In-vitro formation of a collagenous matrix upon previously diseased and experimentally treated cemental root surfaces

Summary A diseased and mechanically treated surface of root cementum is known, clinically, to favor periodontal regeneration. The present investigation was undertaken to test whether previously diseased and experimentally treated root surfaces can support the in-vitro formation of a new collagenous matrix. Three teeth extracted for advanced periodontitis were treated first with 5% sodium hypochlorite for 2 h to remove all organic material from the root surface. After the healthy, apical one third of the root was cut off, the roots were scaled with moderate pressure to remove visible calculus. Non-demineralized root discs were cut and placed on a co-culture of periodontal ligament- and alveolar bone-derived cells. After 7 weeks in culture, either one of two matrix types was found along the root surface. The most frequent matrix consisted of clusters of cells layered within densely aggregated collagen fibrils. The other, less frequent matrix consisted of loosely arranged collagen fibrils adjacent to the cemental surface. The findings support the notion that, in vitro, a collagenous matrix is formed in contact to diseased and experimentally treated root surfaces. However, the smooth, non-demineralized and scaled cemental surface does not appear to be a suitable substrate for interdigitation with newly produced collagen fibrils.     

Fate of HERS during tooth root development

Tooth root development begins after the completion of crown formation in mammals. Previous studies have shown that Hertwig's epithelial root sheath (HERS) plays an important role in root development, but the fate of HERS has remained unknown. In order to investigate the morphological fate and analyze the dynamic movement of HERS cells in vivo, we generated K14-Cre;R26R mice. HERS cells are detectable on the surface of the root throughout root formation and do not disappear. Most of the HERS cells are attached to the surface of the cementum, and others separate to become the epithelial rest of Malassez. HERS cells secrete extracellular matrix components onto the surface of the dentin before dental follicle cells penetrate the HERS network to contact dentin. HERS cells also participate in the cementum development and may differentiate into cementocytes. During root development, the HERS is not interrupted, and instead the HERS cells continue to communicate with each other through the network structure. Furthermore, HERS cells interact with cranial neural crest derived mesenchyme to guide root development. Taken together, the network of HERS cells is crucial for tooth root development.     

Attempts to label matrix synthesis of human root cementum in vitro

The present study describes the dynamic process of both acellular extrinsic (AEFC) and acellular/cellular intrinsic fiber cementum (AIFC/CIFC) matrix production on growing human teeth. Selected erupting maxillary and mandibular premolars with roots grown to about 70%–95% of their final length were placed in organ culture immediately following extraction. Twelve teeth for short-time labeling were pulse-incubated for 15 min in medium containing ³H-proline and chased for various times in order to follow the migration and secretion of the tracer. Eight teeth for long-time incubation were labeled continuously for 5 h before being chased for 1–8 days in order to label cementum matrix accumulation. After decalcification in ethylene diaminetetraacetic acid (EDTA), their roots were subdivided into about 20 slices each. Epon-embedded sections were prepared for light- and electron-microsopic as well as autoradiographic examination. During CIFC-formation, cementoblasts revealed high intracytoplasmic silver grain concentrations within the first hour after ³H-proline administration. The release of the tracer occurred between 60 to 120 min after administration. After 2 h, cementoblasts and the cementum matrix appeared to be labeled about equally. After 5 h, most of the labeled proteins appeared to be localized in the cementoid. Silver grains increased in number over the cementum matrix from 5–24 h. Very high intracellular grain concentrations within very large cementoblasts corresponded to regions of rapid cementum formation. Tracer-halos around entrapped cells lend support to a multipolar mode of matrix production during CIFC-initiation. The fate of the tracer during the development of early AEFC-matrix was less clear. However, fibroblasts revealed dense intracytoplasmic grain accumulations within the first hour after ³H-proline administration. Thereafter, the tracer localization was vague. This indistinct grain localization reflected the particular mode of AEFC-matrix production characterized by addition of new fibril segments to pre-existing fibers of a collagenous fringe.     

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.     

Formation of a new fibrous attachment to human dental roots

Summary This study was performed to improve currently employed in vitro models for the study of periodontal regeneration by using a porous filter upon which periodontal ligament cells were grown. Periodontal ligament cells were harvested and 0.3 mm root discs cut from three partially erupted and extracted third molar teeth of one patient. Experimental culturing was performed by seeding periodontal ligament cell suspensions on Puropor-200 filters supported by wire-mesh grids in Grobstein Petri dishes. The following day, an interdental space of 0.1 to 0.3 mm was created by gently placing two dental root discs upon the filter. Cultures were terminated after 42, 56, 112 and 124 days, and processed for light- and electron microscopy. Collagen fibril diameters were measured. Adjacent and often attached to large areas of cementum-lined root discs, a dense fiber fringe developed. This fiber fringe was not found on dentinlined root discs. Although less organized, older cultures demonstrated a similar disc-culture interface, which depended upon the presence or absence of original root cementum. Collagen fibrils of early cultures had a mean diameter of about 42 nm, while in older cultures the diameters ranged from 47 to 68 nm. It is concluded that the fibrous matrix attached to cementum-lined root discs somewhat resembles the initial stages of the formation of dental root cementum in vivo.     

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb^lox(ex3)/+ mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.     

Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M_r 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of β-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.