Use of 0.4-Tesla static magnetic field to promote reparative dentine formation of dental pulp stem cells through activation of p38 MAPK signalling pathway.

AIM: To investigate whether static magnetic fields (SMFs) have a positive effect on the migration and dentinogenesis of dental pulp stem cells (DPSCs) to promote reparative dentine formation. METHODOLOGY: In vitro scratch assays and a traumatic pulp exposure model were performed to evaluate the effect of 0.4-Tesla (T) SMF on DPSC migration. The cytoskeletons of the DPSCs were identified by fluorescence immunostaining and compared with those of a sham-exposed group. Dentinogenic evaluation was performed by analysing the expressions of DMP-1 and DSPP marker genes using a quantitative real-time polymerase chain reaction (qRT-PCR) process. Furthermore, the formation of calcified deposits was examined by staining the dentinogenic DPSCs with Alizarin Red S dye. Finally, the role played by the p38 MAPK signalling pathway in the migration and dentinogenesis of DPSCs under 0.4-T SMF was investigated by incorporating p38 inhibitor (SB203580) into the in vitro DPSC experiments. The Student's t-test and the Kruskal-Wallis test followed by Dunn's post hoc test with a significance level of P < 0.05 were used for statistical analysis. RESULTS: The scratch assay results revealed that the application of 0.4-T SMF enhanced DPSCs migration towards the scratch wound (P < 0.05). The cytoskeletons of the SMF-treated DPSCs were found to be aligned perpendicular to the scratch wound. After 20 days of culture, the SMF-treated group had a greater number of out-grown cells than the sham-exposed group (nonmagnetized control). For the SMF-treated group, the DMP-1 (P < 0.05) and DSPP genes (P < 0.05), analysed by qRT-PCR, exhibited a higher expression. The distribution of calcified nodules was also found to be denser in the SMF-treated group when stained with Alizarin Red S dye (P < 0.05). Given the incorporation of p38 inhibitor SB203580 into the DPSCs, cell migration and dentinogenesis were suppressed. No difference was found between the SMF-treated and sham-exposed cells (P > 0.05). CONCLUSION: 0.4-T SMF enhanced DPSC migration and dentinogenesis through the activation of the p38 MAPK-related pathway.

Dynamics of bromodeoxyuridine label-retaining dental pulp cells during pulpal healing after cavity preparation in mice.

INTRODUCTION: This study aimed at clarifying the dynamics of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) and their relationship to cell proliferation and apoptosis during pulpal healing after cavity preparation in mice. METHODS: A groove-shaped cavity was prepared on the mesial cervical surface of the upper first molars, and the samples were collected at intervals of 12 hours-14 days. The demineralized paraffin sections were processed for immunohistochemistry for BrdU, nestin, and Ki-67 and apoptosis assay using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and in situ hybridization for dentin sialophosphoprotein (Dspp). RESULTS: During hour 12-day 1, odontoblasts and subodontoblastic cells beneath the affected dentin showed degenerative features and TUNEL-positive reactions, and the expressions of nestin and Dspp were lost in the damaged odontoblasts. TUNEL-positive reactions were observed even in the center of the pulp tissue, whereas dense and granular LRCs remained there. On days 2-3, Ki-67-positive cells were significantly increased in number in the center of mesial dental pulp. During days 3-5, granular and few dense LRCs were committed into some (not all) nestin-positive newly differentiated odontoblast-like cells, and these differentiated cells began to express nestin and Dspp. Until day 14, tertiary dentin formation occurred next to the preexisting dentin at the mesial pulp floor in addition to the mesial coronal pulp. CONCLUSIONS: These results suggest that odontoblasts and subodontoblastic cells degenerate after tooth drilling, and, subsequently, dental pulp stem/progenitor cells actively proliferate and differentiate into new odontoblast-like cells.

Nuclear expression of p27(Kip1) is associated with in vivo differentiation of adult human odontoblasts.

INTRODUCTION: Odontoblasts are terminally differentiated cells of ectomesenchymal origin that produce the dentin. Differentiated odontoblasts cannot be identified yet by a single phenotypic marker protein; therefore, a combination of markers is currently used. Up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27(Kip1) protein was shown in many adult mouse tissues, but no information is available on the expression of p27(Kip1) in mammalian dental pulp. METHODS: Healthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27(Kip1) and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections. RESULTS: p27(Kip1) showed strong nuclear expression in many differentiated human molar odontoblasts at the odontoblastic layer. Most cells of the cell-rich zone displayed low levels of p27(Kip1) despite the fact that preodontoblasts localized in the cell-rich zone of the subodontoblastic layer have been identified as quiescent cells. The nuclear expression of p27(Kip1) in stromal cells of the dental pulp was variable, indicating that subpopulations of these cells were in distinct states of differentiation. Odontoblasts generating reparative dentin showed comparable nuclear expression of p27(Kip1) in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status. CONCLUSIONS: Our findings show that increased expression of nuclear p27(Kip1) occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27(Kip1) can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo.

Dental pulp stem cells, niches, and notch signaling in tooth injury.

Stem cells guarantee tissue repair and regeneration throughout life. The decision between cell self-renewal and differentiation is influenced by a specialized microenvironment called the 'stem cell niche'. In the tooth, stem cell niches are formed at specific anatomic locations of the dental pulp. The microenvironment of these niches regulates how dental pulp stem cell populations participate in tissue maintenance, repair, and regeneration. Signaling molecules such as Notch proteins are important regulators of stem cell function, with various capacities to induce proliferation or differentiation. Dental injuries often lead to odontoblast apoptosis, which triggers activation of dental pulp stem cells followed by their proliferation, migration, and differentiation into odontoblast-like cells, which elaborate a reparative dentin. Better knowledge of the regulation of dental pulp stem cells within their niches in pathological conditions will aid in the development of novel treatments for dental tissue repair and regeneration.

Immunohistochemical study of small integrin-binding ligand, N-linked glycoproteins in reactionary dentin of rat molars at different ages.

Small integrin-binding ligand, N-linked glycoproteins (SIBLING) are believed to play key roles in the process of biomineralization. Reactionary dentin (RD), formed by odontoblasts in response to external stimuli, differs morphologically from primary dentin (PD). To test our hypothesis that the microscopic changes reflect variations in molecular mechanisms involved in formation of the two forms of dentin, and to characterize RD further, we compared the distributions of four SIBLING proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP)] in naturally occurring RD with those in PD. Molars of rats aged 12, 18, 24 and 36 wk were analyzed using immunohistochemistry with antibodies against BSP, OPN, DMP-1, and dentin sialoprotein (a fragment of DSPP). Differences in the distribution of the four SIBLING proteins were evident. Bone sialoprotein, not seen in PD, was consistently observed in RD. Osteopontin, almost absent from PD, was clearly observed in RD. The expression levels of DMP-1 and DSP in RD were lower than in PD. Elevated expression of BSP and OPN, along with a marked decrease of dentin sialoprotein and DMP-1 in RD, suggests a difference in the mechanism of formation of the two forms of dentin.

Pulp-dentin biology in restorative dentistry. Part 7: The exposed pulp.

Exposure of the dental pulp, through a caries lesion, accidentally during routine cavity preparation, or as a result of tooth fracture, is a clinical reality that requires optimal treatment. The potential for healing by formation of a dentinal bridge is good, provided that the pulp is not inflamed. Calcium hydroxide has a long history of inducing dentinal bridge formation to promote successful healing. Resin composites are emerging as alternative materials for pulp capping, but healing is slower, and relatively little clinical experience is available for analysis. The prognosis for healing is poor after exposure of an inflamed pulp. Pulpotomy should be considered to create a wound in an uninflamed location. If an exposure is expected through a caries lesion, stepwise excavation of the carious dentin should be considered to allow healing of the dentin and pulp prior to the final excavation of the carious dentin.

Hierarchy of variables correlated to odontoblast-like cell numbers following pulp capping.

OBJECTIVES: Following tooth pulp exposure, pulpal repair is accomplished by dentine bridge secretion by odontoblast-like cells. However, little information is available about the hierarchy of variables, which influence odontoblast-like cell numbers. The purpose of this study was to examine correlations between pulp capping events and odontoblast-like cell numbers. METHODS: Two hundred and fifty standardised pulp exposed cavities were prepared in non-human primate teeth according to ISO usage guidelines. Exposed pulps were capped with Calcium hydroxide [Ca(OH)(2)], and multi-step and self-etching primer composite resins. Teeth were collected from 3 to 60-days to observe pulp reactions. Statistical analysis was evaluated using analysis of variance. RESULTS: The hierarchy of variables correlated to odontoblast-like cells were the dentine bridge area (P = 0.0001), time since pulp exposure (P = 0.0001), odontoblast numbers opposite the exposure site (P = 0.0002), and pulp capping materials (P = 0.0313). Other pulp capping variables were found to be less likely to be correlated with odontoblast-like cell numbers. CONCLUSIONS: The area of dentine bridge formation is directly related to the numbers of odontoblast-like cells, cell activity is time dependent, and the cell numbers are much lower than original odontoblast cells. The time-lag between the appearance of odontoblast-like cells at the site of pulp exposure, and the limited numbers of these cells, explain why pulpal repair is difficult to achieve successfully following pulp exposure.

Immunohistochemistry of bone sialoprotein and osteopontin during reparative dentinogenesis in vivo.

OBJECTIVE: To investigate the stage-specific and tissue expression of bone sialoprotein (BSP) and osteopontin (OPN) during reparative dentinogenesis in vivo. METHODS: Direct pulp-capping with Ca(OH)2 developed a model used for the investigation of reparative dentinogenesis in the exposed dental pulp. In this model, standardized class V cavities were prepared close to the gingival margin on the buccal surface of each tooth. Animals were sacrificed 3, 7, and 10 days post-operation. Immunohistochemical staining determined the tissue-specific expression of BSP and OPN during the process. RESULTS: Odontoblast-like cells and reparative dentin reacted positively with BSP and OPN. The expression of BSP reached a peak at day 7, then gradually decreased, while the expression of OPN showed no significant difference between day 7 and day 10. CONCLUSION: BSP and OPN may play different roles in reparative dentinogenesis. BSP may serve as a nucleator of hydroxyapatite crystal formation.

Stimulation of the dentine-pulp complex of rat incisor teeth by transforming growth factor-beta isoforms 1-3 in vitro.

Expression of transforming growth factor-beta (TGF-beta) isoforms by odontoblasts leads to their sequestration within dentine matrix. TGF-beta1 and -beta3 stimulate matrix secretion and also initiate odontoblast cytodifferentiation in vitro and in vivo. Using a recently established organ-culture model, the aim here was to examine the effects of TGF-beta isoforms on the response of the dentine-pulp complex during culture. Agarose beads were soaked in isoforms 1-3, and placed on the odontoblast area of slices of 28-day-old rat incisor tooth. The slices were maintained in Trowel-type cultures for 7 days. Both TGF-beta1 and -beta3 stimulated a local increase in predentine secretion at the site of the bead application when compared to control cultures. Mitogenic effects on the cells of the subodontoblast layer were also seen and occasionally small foci of newly differentiated odontoblast-like cells could be observed a little distant from the application site of TGF-beta3. TGF-beta2 had a minimal effect on the cultured tissues. These results demonstrate that TGF-beta1 and -beta3 can stimulate secretion of extracellular matrix by odontoblasts, are mitogenic to pulp cells, and that TGF-beta3 may have inductive effects on pulpal cells. Such activities might be important during reparative processes in the dentine-pulp complex after tissue injury.

In vitro culture of human dental pulp cells: some aspects of cells emerging early from the explant.

Cells emerging from a dental pulp explant were studied in order to elucidate the origin of precursor cells implicated in the formation of reparative dentin in vivo. Such cells observed at the very early stage of culture (days 5-10) were different from fibroblast-like cells obtained after one subculture. These early cells were round or elongated, with thin spinous processes, highly mobile, and contained numerous lipid vesicles. Incorporation of 1-[14C]palmitic acid did not show an increased incorporation of radiolabeled total lipids or triglycerides into these early cells compared to cells after four subcultures or control skin fibroblasts, suggesting that the lipids in vesicles were not synthesized by the cells. Since these cells also show a high level of fluid phase uptake via macropinocytosis, it is suggested that these lipids originate from macropinocytosis. Between days 10 and 20, these cells spontaneously start conversion into cells that have a fibroblast-like morphology, are less mobile, and lack lipid vesicles. Their morphology, movement, and macropinocytosis suggest that these cells, which migrate from the pulp explants, are mesenchymal cells related to mononuclear phagocytes/histiocytes.

Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components.

The effects of dentine extracellular matrix components on dental mesenchymal cells were studied by light and transmission electron microscopy after their implantation at central sites of mechanically exposed pulps in dog molar teeth. The implants were Millipore filters that had been soaked with solutions containing 30 or 300 micrograms/ml of an EDTA-soluble fraction of rabbit incisor dentine. Control filters were soaked with dog albumin or phosphate buffered saline. Columnar, polarized cells were consistently seen after 8 days in close proximity to the filters coated with both concentrations of dentine matrix components. Characteristic features of these polarized cells included widened cisternae of the rough endoplasmic reticulum, a rich microfilamentous network in the long cytoplasmic extensions invading the filter pores and numerous cytoplasmic bodies. These cells also showed evidence of functional as well as cytological differentiation. Polarized processing of secretory granules could be observed after 8 days' implantation, and also the presence of matrix vesicles and deposition of a fine, collagenous matrix into the filters apically to the distal end of the cytoplasmic processes. After 24 days' implantation, secretion of a tubular matrix could be consistently seen in association with the odontoblast-like cells. No changes in cell organization or matrix synthesis were seen after implantation of control filters. These studies demonstrate that bioactive components present in the EDTA-soluble dentine matrix fraction are able to directly induce cell polarization and apical secretion of tubular matrix when implanted in contact with dental pulp cells at sites remote from the odontoblast layer.

Induction of reparative dentin formation in dogs by bovine bone morphogenetic protein bound to ceramic dentin.

Bone morphogenetic protein (BMP) refers to a group of bone-inducing growth factors used to enhance experimental osseous repair. Previous studies have indicated that BMP promotes the proliferation and differentiation of fibroblasts from human dental pulp both in vitro and in vivo. Furthermore, when BMP is used as a pulp capping agent, it has ability to induce the formation of both osteodentin and tubular dentin. In this study, we sought to explore whether or not BMP, complexed to ceramic dentin (BMP/CD), would constitute a more effective pulp capping agent than BMP alone. Experimental pulp exposures were created in dogs, and BMP/CD was placed on the surface of the exposed dental pulp. The inductive effect of such stimulation on reparative dentin formation was observed morphologically. Results showed that two weeks after exposure, some osteodentinal matrix was formed in the space surrounding the implanted BMP/CD particles while the remainder of the space was occupied by proliferating fibroblasts. At 4 weeks postoperatively, the dential bridge was complete. When ceramic dentin without BMP was used, there was minimal bone-like matrix formed even at 4 weeks postoperatively. These results suggest that the BMP/CD complex is effective as a stimulant of reparative dentin formation and shows potential as a therapeutically useful pulp-capping agent in a clinical setting.

Characterization of cellular responses involved in reparative dentinogenesis in rat molars.

During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programmed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

Odontoblast processes in dentin revealed by fluorescent Di-I.

There has been controversy about the length and structure of the odontoblast process within dentin since the earliest histologic studies of teeth. Our objective was to use the fluorescent carbocyanine dye Di-I combined with a new gelatin embedment procedure and confocal microscopy to determine the structure and extent of odontoblast processes in developing and mature rat teeth, injured rat molars, reparative dentin, and adult monkey teeth. We found that odontoblast processes do not extend into outer dentin or to the dentin-enamel junction except during early stages of development. Those in innervated regions of crown are long and straight, whereas those in roots are extensively branched and shorter. Cavity injury to crown dentin caused odontoblast fragments to be aspirated into outer dentin. In reparative dentin the odontoblast processes were branched and similar to those in roots. We used photoconversion and electron microscopy to show that Di-I fills the entire odontoblast after gelatin embedment, including the cytoplasm. This is a different type of carbocyanine staining from any previously reported, and it also stains other cells in adjacent hard tissues such as bone and cementum. The Di-I-gelatin method is a new way to use carbocyanine dyes. It has enabled us to solve a long-standing controversy about the histology of teeth, and it should be useful for many other studies of cell structure.

Induction of reparative dentinogenesis in vivo: a synthesis of experimental observations.

EDTA--and/or guanidine HCl--insoluble dentinal matrix, or demineralized dentin which had been treated with plasma fibronectin, or pieces of Millipore filters coated with a recombinant fibronectin-like engineered polymer, incorporating many RGD sequences, were implanted into central parenchymal sites of young dog molars, via mechanical pulp exposures. Furthermore demineralized dentin and Millipore filters coated with plasma fibronectin were placed into the central pulp of old animals. Histological analysis of buffered formalin-fixed tissues showed that: 1. The dentinogenic activity was retained in the EDTA--and/or guanidine-insoluble dentin matrix. 2. Implantation of Millípore filters supplemented with the recombinant polymer did not induce any odontoblast-like cell differentiation, indicating that the interactions of pulp cells with the exogenous fibronectin are not RGD-dependent. 3. Acid-insoluble dentin matrix or plasma fibronectin (both separately inducing dentinogenesis in dental pulp of young animals) did not show any dentinogenic activity when exposed in pulp sites of old animals. Acid-insoluble dentin matrix and plasma fibronectin also failed to induce dentinogenic activity in the young pulpal tissues, when both factors were combined before to their implantation. Synthesizing the present data with previous relevant information it could be suggested that in the mechanism initiating reparative dentinogenesis, growth factors (endogenous or artificially implanted) and fibronectin are involved and this mechanism seems to be more complex than the simple immobilization of pulp cells onto an adhesion substratum.

Short-term dentinogenic response of dog dental pulp tissue after its induction by demineralized or native dentine, or predentine.

The events initiating the expression of odontoblastic potential by pulpal ectomesenchymal cells were investigated by exposing the pulp to demineralized, native and unmineralized autogenous dentine. The pulp responses to implants were histologically evaluated 3, 7 and 10 days postoperatively, while the surface structure of the newly mineralized matrices was examined 12 and 28 days after implantation. Differentiation of odontoblast-like cells in close proximity to the implanted matrix was consistently demonstrated after exposure to predentine. Scattered columnal cells undergoing polarization, characterized ultrastructurally by the orientation of their rough endoplasmic reticulum, were also found in direct contact with the demineralized dentine. However, in response to demineralized implants, groups of differentiated odontoblast-like cells were clearly seen only in association with a zone of matrix secreted in a polar, predentine-like pattern, indicating an asynchronous inductive influence of this type of implant on pulp cells. Further, the response of pulp cells to native dentine was characterized by the elaboration of a two-layered matrix (a fibrous and a polarly deposited matrix) before initiation of secondary dentinogenesis. Scanning electron microscopy of the newly deposited matrices revealed differences between the indirect matrix synthesis, observed in short-term response to implants of demineralized or native dentine, and the specific, dentinogenic function of the odontoblast-like cells. These observations indicate that the dentine-induced dentinogenesis is initiated by two mechanisms--direct induction of odontoblast-like cells as well as indirect matrix synthesis, which further controls cell polarization. Immobilization of the cells on implanted matrix seems to be the critical requirement for direct expression of the odontoblastic phenotype.

Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation, and differentiation.

The findings from the recent literature on pulpal cell responses to the application of calcium hydroxide to exposed pulps are described. The effect of calcium hydroxide on healthy and inflamed pulp is discussed. The effect of incorporation of calcium hydroxide in various pulp-capping agents is presented. The initial effect of calcium hydroxide applied to exposed pulp is the development of a superficial three-layer necrosis. The beneficial effect of calcium hydroxide is regarded as the result of the chemical injury caused by the hydroxyl ions, limited by a zone of firm necrosis against the vital tissue, and the toleration of calcium ions by the tissue. The firm necrosis causes slight irritation and stimulates the pulp to defense and repair. The observed sequence of tissue reactions is that which is expected when connective tissue is wounded. It starts with vascular and inflammatory cell migration and proliferation, to control and elimination of the irritating agent. This is followed by the repair process, including migration and proliferation of mesenchymal and endothelial pulp cells and formation of collagen. When the pulp is protected from irritation, odontoblasts differentiate, and the tissue formed assumes the appearance of dentin, i.e., the function of the pulp is normalized. The mineralization of the collagen starts with dystrophic calcification of both the zone of firm necrosis and the degenerated cells in the adjacent tissue, leading to deposition of mineral in the newly-formed collagen. The presence of calcium ions stimulates precipitation of calcium carbonate in the wound area and thereby contributes to the initiation of mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)