Influence of TGF-β1 on the expression of BSP,DSP, TGF-β1 receptor I and Smad proteins during reparative dentinogenesis

Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-β1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TβRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TβRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-β1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-β1.     

Dentin non-collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages

Background information. Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non‐collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. Results. DFCs were isolated from tooth germs of SD (Sprague—Dawley) rats and then co‐cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization‐associated proteins and genes were up‐regulated after induction, whereas the expression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum‐like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. Conclusions. dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.     

Dentin regeneration based on tooth tissue engineering: A review

Missing or damaged teeth due to caries, genetic disorders, oral cancer, or infection may contribute to physical and mental impairment that reduces the quality of life. Despite major progress in dental tissue repair and those replacing missing teeth with prostheses, clinical treatments are not yet entirely satisfactory, as they do not regenerate tissues with natural teeth features. Therefore, much of the focus has centered on tissue engineering (TE) based on dental stem/progenitor cells to create bioengineered dental tissues. Many in vitro and in vivo studies have shown the use of cells in regenerating sections of a tooth or a whole tooth. Tooth tissue engineering (TTE), as a promising method for dental tissue regeneration, can form durable biological substitutes for soft and mineralized dental tissues. The cell-based TE approach, which directly seeds cells and bioactive components onto the biodegradable scaffolds, is currently the most potential method. Three essential components of this strategy are cells, scaffolds, and growth factors (GFs). This study investigates dentin regeneration after an injury such as caries using TE and stem/progenitor cell-based strategies. We begin by discussing about the biological structure of a dentin and dentinogenesis. The engineering of teeth requires knowledge of the processes that underlie the growth of an organ or tissue. Then, the three fundamental requirements for dentin regeneration, namely cell sources, GFs, and scaffolds are covered in the current study, which may ultimately lead to new insights in this field.     

不同年龄组修复性牙本质生成情况探讨

目的观察不同年龄段人的牙齿在受到龋损刺激后,修复性牙本质的生成情况。方法收集5组不同年龄组含有牙本质浅层龋的离体牙,每组各5颗牙齿。制作标本切片,HE染色,显微镜下观察。测量修复性牙本质厚度,作统计学检验。结果5组不同年龄牙齿的修复性牙本质平均厚度分别为:3.36、2.6、2.8、4.2、3.36,统计学检验无明显差异。结论受龋损刺激后,不同年龄牙齿的修复性牙本质生成情况无明显差异。     

Distribution of Small Integrin-binding Ligand, N-linked Glycoproteins (SIBLING) in the Articular Cartilage of the Rat Femoral Head

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis. (J Histochem Cytochem 58:1033–1043, 2010)     

Dentin hardness differences across various mammalian taxa

Differences in dentin microstructure have been used as a tool for dietary reconstruction; however, the extent that diet is associated with this aspect of dental morphology has yet to be empirically tested. We conducted microhardness tests of mammalian dentin sections, hypothesizing that species with adaptations to particularly hard diets would have softer dentin, owing to a higher proportion of soft intertubular dentin. Species adapted to abrasive diets, in contrast, should have harder dentin, resulting from a higher proportion of hypermineralized peritubular dentin. We examined molar dentin hardness in ten mammalian taxa with durophagous diets, abrasive diets, and a comparative “control” group of mechanical generalists. Samples included six primate taxa and four non-primate species representing various dietary regimes. Our results reveal significant variation among taxa in overall hardness, but the data do not distinguish between hard and abrasive diets. Several taxa with generalized (i.e., mechanically diverse) diets resemble each other in exhibiting large variance in hardness measurements and comparably soft dentin. The high variation in these species appears to be either a functional signal supporting the niche variation hypothesis or indicate the absence of sustained unidirectional selective pressure. A possible phylogenetic signal of dentin hardness in the data also holds promise for future systematic investigations.     

预酸蚀乳牙牙本质对自酸蚀粘接系统粘接强度的影响

目的:探讨预酸蚀乳牙牙本质对自酸蚀粘接系统粘接强度的影响。方法:随机选取28颗健康乳磨牙,磨除颊舌面釉质,暴露牙本质粘接面,沿近远中向劈开形成56个样本,随机分为7组(n=8)。直接涂布组(A1组,A2组和A3组)分别涂布AdperTM Easy One(AEO),Xeno-V(XV)和OptiBond All In One(AIO)三种自酸蚀粘接剂,预酸蚀组(B1组,B2组和B3组)在涂布三种自酸蚀粘接剂前先使用35%磷酸酸蚀乳牙牙本质15 s,对照组(C组)使用Prime&Bond NTTM(NT)全酸蚀粘接剂,每个样本用Z350复合树脂堆砌成直径为3 mm的树脂小柱,通过剪切试验测试剪切粘接强度,并通过扫描电子显微镜观察断裂表面形态。结果:B1组,B2组的剪切粘接强度值明显高于A1组,A2组(P<0.001);B3组与A3组的剪切粘接强度值比较无明显差别(P=0.94)。A2组的剪切粘接强度值低于C组(P<0.05);B1组的剪切粘接强度值明显高于C组(P<0.001)。扫描电镜观察结果显示各组试件断裂面形态多为牙本质和复合树脂界面破坏。直接涂布组(A1组,A2组和A3组)断裂多发生在混合层的底部,树脂突较少且低于小管口。B1组和B2组试件断裂面可见多数牙本质小管被树脂突填满,断裂多发生在混合层的中上部。B3组试件断裂面可见牙本质小管空虚,树脂突较少。结论:预酸蚀乳牙牙本质可以提高AEO,XV两种自酸蚀粘接剂的剪切粘接强度。自酸蚀粘接剂处理乳牙牙本质可以达到全酸蚀粘接剂处理的粘接强度,但应用自酸蚀粘接剂前预酸蚀乳牙牙本质可以获得更高的粘接强度。     

The presence of open dentinal tubules affects the biological properties of dental pulp cells <Emphasis Type="Italic">Ex Vivo</Emphasis>

To investigate the effects of open dentinal tubules on the morphological and functional characteristics of dental pulp cells. Morphological changes in human dental pulp cells that were seeded onto dentin discs with open dentinal tubules were investigated on days 1, 2, 4, and 10 of culture using scanning electron microscopy and fluorescence microscopy. Samples collected on days 1, 3, 6, 8, and 10 of culture were evaluated for cell proliferation rate and alkaline phosphatase activity. Cultured human dental pulp cells developed a columnar or polygonal morphology and monopolar cytoplasmic processes that extended into the dentinal tubules. The cells formed a multilayer and secreted an extracellular matrix onto the cell surface. Scanning electron microscopy and fluorescence microscopy revealed polarized organization of odontoblasts. Cells seeded onto dentin discs proliferated minimally but showed high levels of ALP activity. Dental pulp cells seeded onto treated dentin discs develop an odontoblastlike phenotype, which may be a potential alternative for use in experimental research on dentinogenesis.     

骨涎蛋白在大鼠磨牙第三期牙本质形成过程中的表达

目的:观察大鼠牙髓修复第三期牙本质形成过程中骨涎蛋白(bone sialoprotein,BSP)的表达变化及意义。方法:选取6周龄雄性Wistar大鼠10只,建立实验大鼠模型,利用免疫组化法检测大鼠第三期牙本质中骨涎蛋白的表达情况。结果:盖髓2周后,在盖髓处下方有第三期牙本质形成。与原发性牙本质(PD)相比,第三期牙本质小管数目少且形态不规则。BSP在原发性牙本质中没有表达,但在盖髓下方和髓角下第三期牙本质中都有表达。结论:BSP可能通过参与羟基磷灰石(HA)的形成以及调节新分化的成牙本质细胞向新形成的牙本质基质的粘附来参与早期的第三期牙本质的生成。     

Generation and characterization of DSPP‐Cerulean/DMP1‐Cherry reporter mice

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3‐GFP, pOBCol3.6‐GFP, and DMP1‐GFP), similar to the endogenous proteins, are also expressed by bone‐forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP‐Cerulean/DMP1‐Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24‐258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1‐Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP‐Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.     

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract

Summary In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.     

In vitro behaviour of human osteoblasts on dentin and bone

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.     

Near tubule and intertubular bovine dentin mapped at the 250 nm level

In this study, simultaneous diffraction and fluorescence mapping with a (250 nm)², 10.1 keV synchrotron X-ray beam investigated the spatial distribution of carbonated apatite (cAp) mineral and elemental Ca (and other cations including Zn) around dentin tubules. In 1 μm thick sections of near-pulp root dentin, where peritubular dentin (PTD) is newly forming, high concentrations of Zn, relative to those in intertubular dentin (ITD), were observed adjacent to and surrounding the tubule lumens. Some but not all tubules exhibited hypercalcified collars (high Ca signal relative to the surrounding ITD), and, when present, the zone of high Ca did not extend around the tubule. Diffraction rings from cAp 00.2 and 11.2 + 21.1 + 30.0 reflections were observed, and cAp was the only crystal phase detected. Profiles of Ca, Zn and cAp diffracted intensities showed the same transitions from solid to tubule lumen, indicating the same cAp content and organization in ITD far from the tubules and adjacent to them. Further, the matching Ca and diffraction profiles demonstrated that all of the Ca is in cAp or that any noncrystalline Ca was uniformly distributed throughout the dentin. Variation of 00.2 and 11.2 + 21.1 + 30.0 diffracted intensity was consistent with the expected biaxial crystallographic texture. Extension of X-ray mapping from near 1 μm resolution to the 250 nm level, performed here for dentin and its tubules, will provide new understanding of other mineralized tissues.     

Whole‐field macro‐ and micro‐deformation characteristic of unbound water‐loss in dentin hard tissue

High‐resolution deformation measurements in a functionally graded hard tissue such as human dentin are essential to understand the unbound water‐loss mediated changes and their role in its mechanical integrity. Yet a whole‐field, 3‐dimensional (3D) measurement and characterization of fully hydrated dentin in both macro‐ and micro‐scales remain to be a challenge. This study was conducted in 2 stages. In stage‐1, a stereo‐digital image correlation approach was utilized to determine the water‐loss and load‐induced 3D deformations of teeth in a sagittal section over consecutively acquired frames, from a fully hydrated state to nonhydrated conditions for a period up to 2 hours. The macroscale analysis revealed concentrated residual deformations at the dentin‐enamel‐junction and the apical regions of root in the direction perpendicular to the dentinal tubules. Significant difference in the localized deformation characteristics was observed between the inner and outer aspects of the root dentin. During quasi‐static loadings, further increase in the residual deformation was observed in the dentin. In stage‐2, dentin microstructural variations induced by dynamic water‐loss were assessed with environmental scanning electron microscopy and atomic force microscopy (AFM), showing that the dynamic water‐loss induced distention of dentinal tubules with concave tubular edges, and concurrent contraction of intertubular dentin with convex profile. The findings from the current macro‐ and micro‐scale analysis provided insight on the free‐water‐loss induced regional deformations and ultrastructural changes in human dentin.      

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.     

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion.We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors.The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.