Determination of two different types of cellular cementogenesis in rat molars: a histological and immunohistochemical study.

To elucidate the attachment mechanism of dentin and cellular cementum, developing and developed cellular cementum of rat molars was examined by light microscopy. Routine histological staining, immunohistochemical staining for bone sialoprotein (BSP) and osteopontin (OPN), and digestion tests with trypsin were conducted. Two different types of cellular cementogenesis were established, one on the mesial (type I cementogenesis) and one on the distal sides (type II cementogenesis) of the examined roots. In the type I cementogenesis a thin initial cementum layer, which was fibril-poor, hematoxylin-stained, and immunopositive for BSP and OPN, appeared on the mineralized dentin. With cellular cementogenesis, the layer became the cemento-dentinal junction. The cementum mineralization did not precede the dentin mineralization. After trypsin treatment the cemento-dentinal junction lost immunoreactivity for BSP and OPN and the cementum was detached from the dentin. In the type II cementogenesis the cellular cementum formed directly on the predentin without the initial cementum layer and the cementum mineralization preceded the dentin mineralization. Cemental and predentinal fibrils appeared to intermingle, as the cemento-dentinal junction was indiscernible by any staining. Trypsin treatment did not cause cementum detachment. The findings of the present study suggest that: (1) The type I cementogenesis requires the intervening initial cementum to bind cementum and dentin and to induce the cementum mineralization. (2) In the type II cementogenesis the cemento-dentinal attachment depends on fibril intermingling and the cementum mineralization advances apically and very rapidly, probably producing mineralization foci. (3) The formation of the initial cementum depends on the speed of the cementogenesis in the apical direction.     

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.     

Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.This study was supported by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, University of Berne, Berne, Switzerland.     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

Ultrastructural immunolocalization of dentin matrix protein 1 on Sharpey's fibers in monkey tooth cementum

Despite the importance of dentin matrix protein 1 (DMP1) in the formation of mineralized tissue, including dentinogenesis and osteogenesis, its precise role in cementogenesis remains to be clarified fully. The purpose of our study was to demonstrate the ultrastructural immunolocalization of DMP1 in monkey molar tooth cementum. Japanese Macaca fuscata monkeys were fixed by perfusion. The upper molar teeth and accompanying periodontium then were dissected and demineralized with EDTA. Cryosections were obtained, incubated in anti-DMP1 polyclonal antibody, and processed by immunoperoxidase and immunogold labeling. Intense immunoperoxidase staining for DMP1 was observed in acellular extrinsic fiber cementum, particularly in Sharpey's fibers. Cementocyte lacunae with canaliculi showed DMP1 staining in the apical region of the tooth root. Electron immunomicroscopy revealed the close proximity of DMP1 to collagen fibrils in Sharpey's fibers at the mineralization front. Intense immunogold labeling was localized on the walls of the cementocyte lacunae in cellular cementum. These results should contribute to better understanding of the role of DMP1, not only in Sharpey's fiber biomineralization, but also in the maintenance of the cementocyte lacunar space in cementum.     

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.     

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.     

Sulfated hyaluronan derivatives reduce the proliferation rate of primary rat calvarial osteoblasts

Glycosaminoglycans (GAG) and proteoglycans, which are components of the extracellular bone matrix, are also localized in and at the membrane of osteoblasts and in the pericellular matrix. Due to their interaction with several growth factors, water and cations these molecules play an important role in regulating proliferation and differentiation of osteoblasts and bone development. The aim of this study was to assess in vitro the effects of two chemically sulfated hyaluronan (HyaS) derivatives on the proliferation of rat calvarial osteoblasts and to compare with those of native hyaluronan (Hya) and natural sulfated GAG such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS) and heparan sulfate (HS). Moderately and highly sulfated HyaS derivatives caused a time-dependent reduction of osteoblast proliferation. The anti-proliferative effect of HyaS was accompanied by a cell cycle arrest in the G1 phase, but was not associated with cell death. Whereas non-sulfated high molecular weight (HMW)- and low molecular weight (LMW)-Hya as well as C4S, C6S, DS and HS showed no effect on the cell proliferation.     

The hydration of proteoglycans of bovine cornea

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a disaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin

N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation.