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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., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Introduction: Regenerative endodontic procedures use the differentiation potential of embryonic and adult pulp progenitor cell populations to reconstitute dental structures., Methods: An in-depth search of the literature was accomplished to review biologic knowledge from basic research on tooth morphogenesis and differentiation, root development, dentin-pulp regeneration, pulp revascularization and apexification, experimental and clinical studies on the dentinogenic differentiation potential of progenitor cells in the embryonic dental papilla, dental pulp, and associated mesenchymal tissues of the developing root., Results: Odontogenic potential is determined during early tooth morphogenesis in the odontogenic mesenchyme. Progenitor cells from the odontogenic mesenchyme give rise to primary dentin-forming cells (odontoblasts) in the presence of stage-specific enamel epithelium and/or basement membrane and tertiary dentin-forming cells (odontoblast-like cells) in experimental conditions. The specificity of odontogenic mesenchymal cells to form tertiary dentin might be related to the repertoire of signaling pathways operated by the temporospatial pattern of epithelial-mesenchymal interactions during tooth formation. Dental papilla cells isolated from tooth germs before the onset of odontoblast differentiation have not shown any competence to become odontoblasts in the absence of enamel epithelium. On the other hand, the specificity of progenitor cells in the mesenchymal cell populations of the developing root apex remains to be determined., Conclusions: It seems evident that the dental pulp might be only used as a source of progenitor cells with dentinogenic competence for the regeneration of the dentin-pulp complex. The nature of dental or apical papilla progenitor cells in terms of their specificity for dentin regeneration has to be first characterized., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Some clinical case reports have shown that immature permanent teeth with periradicular periodontitis or abscess can undergo apexogenesis after conservative endodontic treatment. A call for a paradigm shift and new protocol for the clinical management of these cases has been brought to attention. Concomitantly, a new population of mesenchymal stem cells residing in the apical papilla of permanent immature teeth recently has been discovered and was termed stem cells from the apical papilla (SCAP). These stem cells appear to be the source of odontoblasts that are responsible for the formation of root dentin. Conservation of these stem cells when treating immature teeth may allow continuous formation of the root to completion. This article reviews current findings on the isolation and characterization of these stem cells. The potential role of these stem cells in the following respects will be discussed: (1) their contribution in continued root maturation in endodontically treated immature teeth with periradicular periodontitis or abscess and (2) their potential utilization for pulp/dentin regeneration and bioroot engineering.

The difference between stem-cell-mediated bone and dentin regeneration is not yet well-understood. Here we use an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). Elevated expression of basic fibroblast growth factor (bFGF) and matrix metalloproteinase 9 (MMP-9, gelatinase B) was found to be associated with the formation of hematopoietic marrow in BMSSC transplants, but not in the connective tissue of DPSC transplants. The expression of dentin sialoprotein (DSP) specifically marked dentin synthesis in DPSC transplants. Moreover, DPSCs were found to be able to generate reparative dentin-like tissue on the surface of human dentin in vivo. This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and non-mineralized tissues.

Close regulation of odontoblast differentiation and subsequent secretory activity is critical for dentinogenesis during both embryogenesis and tissue repair. Some dental papilla cells achieve commitment and specific competence, allowing them to respond to epithelially derived inductive signals during the process of odontoblast differentiation. Temporo-spatial regulation of odontoblast differentiation is dependent on matrix-mediated interactions involving the basement membrane (BM). Experimental studies have highlighted the possible roles of growth factors in these processes. Regulation of functional activity of odontoblasts allows for both ordered secretion of the primary dentin matrix and maintenance of vitality and down-regulation of secretory activity throughout secondary dentinogenesis. After injury to the mature tooth, the fate of the odontoblast can vary according to the intensity of the injury. Milder injury can result in up-regulation of functional activity leading to focal secretion of a reactionary dentin matrix, while greater injury can lead to odontoblast cell death. Induction of differentiation of a new generation of odontoblast-like cells can then lead to reparative dentinogenesis. Many similarities exist between development and repair, including matrix-mediation of the cellular processes and the apparent involvement of growth factors as signaling molecules despite the absence of epithelium during repair. While some of the molecular mediators appear to be common to these processes, the close regulation of primary dentinogenesis may be less ordered during tertiary dentinogenic responses.

Glucocorticosteroids are widely used in the treatment of chronic illnesses and have been reported to cause premature obliteration of the pulp space. During the active stages of dentinogenesis, odontoblasts are growth hormone receptor (GHr) positive. The aims of this study were to determine if the glucocorticosteroid, prednisone, affected the rate of dentine deposition and odontoblast expression of GHr in the rat molar. Following subcutaneous injection of 0.05 mg/kg, 1.0 mg/kg or 5.0 mg/kg prednisone for 20 days, immature and mature molars from rats aged 3 and 6 weeks respectively, were examined histologically. Distribution of GHr expression was determined immunohistochemically. No morphological differences were observed in molars from prednisone treated animals. Prednisone did not appear to enhance dentine deposition in immature molars but in mature molars significantly increased dentine deposition on the roof of the pulp chamber at a dosage of 5.0 mg/kg (p < 0.001). In all immature molars, odontoblasts and pulp cells expressed GHr immunoreactivity. In mature molars, odontoblasts and pulpal cells from controls did not show GHr immunoreactivity. However, odontoblasts and pulp cells were GHr immunoreactive in mature molars from animals treated with prednisone.

c-jun and jun-B are nuclear proto-oncogenes induced by growth factors such as bone morphogenetic proteins (BMPs). These gene products enhance the expression of many genes, including osteocalcin and collagen types, indicating that c-jun and jun-B play important roles in the cell differentiation process. It is also known that BMPs affect the differentiation of pulp cells to odontoblast-like cells during reparative dentinogenesis, but little is known about the transcriptional regulation of genes in cells associated with reparative dentinogenesis. In this study, we examined the expression of c-jun and jun-B in pulp cells during reparative dentinogenesis after cavity preparation of rat molars by in situ hybridization. In rat tooth germs, c-jun and jun-B were co-expressed in the odontoblastic lineage. In rat adult molars, c-jun was expressed in the odontoblast layer, but the jun-B expression was absent in all pulp cells. After cavity preparation, we found that c-jun and jun-B were coexpressed in pulp cells underneath cavities. During the early phase of reparative dentinogenesis, levels of c-jun and jun-B greatly increased in pulp cells within and around the reparative dentin matrix formed adjacent to the cavity floor. Fourteen days after cavity preparation, c-jun and jun-B were expressed only in pulp cells lining the irregular surface of the thick reparative dentin. These results suggest that c-jun and jun-B may play important roles both in physiological and in reparative dentinogenesis; in particular, the limited distribution of the jun-B expression suggests a specific role of jun-B only in cells involved with the active formation of the dentin matrix during primary and reparative dentinogenesis.

Pulp tissue responds to dentin injury by laying down reactionary dentin secreted by existing odontoblasts or reparative dentin elaborated by odontoblast-like cells that differentiated from precursor cells in the absence of inner dental epithelium and basement membrane. Furthermore, growth factors or active dentin matrix components are fundamental signals involved in odontoblast differentiation. In vitro, dental pulp cells cultured under various conditions are able to express typical markers of differentiation, but no culture system can re-create pulp response to dentin drilling. This paper reports the behavior of thick slices from human teeth drilled immediately after extraction and cultured from 3 days to 1 month. Results show that the damaged pulp beneath the cavity is able to develop, in vitro, some typical aspects correlated to tissue healing, evidenced by cell proliferation (BrdU-positive cells), neovascularization (positive with antitype-IV collagen antibodies), and the presence of functional (3H proline-positive) cuboidal cells close to the injured area. After 30 days of culture, elongated spindle-shaped cells can be seen aligned along the edges of the relevant dentin walls, whereas sound functional odontoblasts are well-preserved beneath healthy areas. This tissue recovery leads us to believe that such a culture model will be a useful system for testing factors regulating pulp repair.

Bone morphogenetic proteins (BMP) have been shown to induce reparative dentine formation experimentally but the cells responsible, which respond to BMPs, have not been identified. The BMP signal is probably mediated by interaction of type I and II BMP receptors (R). Here, the RNA of human adult dental pulp and pulp cells in culture was examined by reverse transcription (RT) polymerase chain reaction (PCR) for evidence of mRNA for BMPs. mRNAs for BMP-2, -4, osteogenic protein-1, ActR-1 (activin-like kinase receptor), BMPR-IA, -IB and -II were detected by RT-PCR. The 698-bp PCR fragment for BMPR-IB was used to probe pulp cells for expression of that receptor. Cell expression of BMPR-IB was detected by the hybridization probe. The findings suggest that resident pulp cells may be able to respond to BMPs to initiate tissue formation.

The ability of 2 calcium hydroxide-containing materials to induce initiation of reparative dentinogenesis was tested at sites remote from the dentinogenically-active regions of the pulp periphery. Pieces of the cements, Dycal and Life, were implanted in central parenchymal sites of dog dental pulps for periods of 6, 14 and 42 days, respectively. Similar pieces were placed in peripheral capping sites as controls. The responses were analyzed by light and transmission electron microscopy. Induction of tubular dentin matrix lined with elongated and polarized odontoblast-like cells was only seen at peripheral capping sites. In response to the centrally implanted cements, only atubular hard tissue with lining fibroblast-like cells was deposited.

The effects of Ledermix+calcium hydroxide (Ca(OH)2) or Ca(OH)2 alone on inflamed pulp tissues of dogs were studied. Fifty-nine upper incisor teeth of 10 dogs were used. Class V cavities were prepared and filled with amalgam after placement of decayed dentin particles. After 7 days, the decayed dentin and alloy were removed, and the pulps of the teeth were exposed. Ledermix and Ca(OH)2 mixtures or Ca(OH)2 alone were applied to the cavities. At the end of 7, 30, and 90 days, the teeth were extracted and examined histopathologically. Inflammation was found to be more prevalent in the 7- and 30-day groups that were treated with the Ledermix+Ca(OH)2 combination, whereas fibrosis and necrosis were nearly similar in both groups. In the 90-day groups, no inflammation was seen. No difference between the two 90-day groups with regard to reparative dentin was found.

Exposed dental pulp is known to possess the ability to form a hard-tissue barrier (dentin bridge). The exact mechanisms by which pulp cells differentiate into odontoblasts in this process are unknown. Fibronectin has been demonstrated to play a crucial role in odontoblast differentiation during tooth development. This study tested the hypothesis that fibronectin is involved in the initial stages of replacement odontoblast differentiation and reparative dentin formation. We observed its immunohistochemical localization during dentin bridge formation in human teeth, after pulp was capped with calcium hydroxide [Ca(OH)2]. One day after the capping, precipitation of crystalline structures was observed at the TEM level in association with cell debris at the interface between the superficial necrotic zone and underlying pulp tissue. This layer of dystrophic calcification showed positive reaction for fibronectin, and pulp cells appeared to be closely associated with this layer, seven to ten days post-operatively. At 14 days, an alignment of cells, some of which were elongated and odontoblast-like, was observed adjacent to the fibronectin-positive irregular matrix. Between the cells, corkscrew fiber-like fluorescence was visible. At 28 days, the irregular fibrous matrix was followed by the formation of tubular dentin-like matrix lined with odontoblast-like cells. Therefore, it would seem that fibronectin associated with the initially formed calcified layer might play a mediating role in the differentiation of pulp cells into odontoblasts during reparative dentinogenesis, after pulp was capped with Ca(OH)2.

The purpose of this research was to study the presence and the ultrastructural features of interodontoblastic collagen fibrils and their contribution to the formation of reparative dentin in dog incisors and premolars for a period of 30 days following experimental pulpotomy. On the seventy day after pulp exposure and capping with calcium hydroxide, short cylindrical-shaped cells collected at the coronal end of the vital pulp tissue. Many collagen fibrils were synthesized in the intercellular spaces and in the direction of the long axis of these cells. On the fourteenth day, large bundles of collagen fibrils were observed in the intercellular spaces of young odontoblasts. These bundles spread out in a fan-shaped arrangement. On the thirtieth day, the odontoblasts formed a tubular dentin matrix. Small numbers of twisted collagen bundles crossed the distal junctional complex of the odontoblast cell bodies and entered the dentin matrix.

Purpose of Study: To determine the rate of formation of tertiary dentine in dogs' teeth, in response to five commonly used lining materials. Also, to record the resulting differences in quality of the formed dentine., Materials and Methods: Representatives of five types of materials commonly used in general practice in the UK were placed in deep standardised cavities with a remaining dentine thickness of 400-500 microns, and sealed to the cavo-surface with zinc oxide-eugenol. The daily rate of tertiary dentine formed in response to each material was measured over a period of 119 days using a tetracycline staining method. A concomitant study to show the quality of the formed dentine was undertaken using the same materials and experimental conditions., Results: There was a significant difference between the rates of tertiary dentine formation for all materials except zinc oxide-eugenol and Ledermix paste. For each material there was a increased rate of tertiary dentine formation in the second period (29-49 days) compared to the first period (0-28 days). This is considered to be due, in part, to a lag period when damaged odontoblasts are differentiating from mesenchymal elements. The rate of tertiary dentine formation in response to all the test materials was always greater than that of normal physiological dentine formation in unprepared teeth. Qualitatively, there was difference in the formed tertiary dentine beneath each material. All the materials caused odontoblast destruction, altered ground substance production and alterations in the calcification process., Conclusion: None of the materials routinely used at present can be considered to be 'ideal' as odontoblast destruction, altered ground substance production and altered calcification occur beneath zinc oxide-eugenol preparations, calcium hydroxide preparations as well as in response to corticosteroid-containing materials such as Ledermix paste and cement.

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.

Close association between nerve terminals and preodontoblasts, odontoblasts and predentine was observed during healing after pulpotomy. The nerve terminals frequently contained large numbers of synaptic vesicles. Terminals with many vesicles tended to be fewer in the predentine than in the odontoblastic layer. The distribution of terminals was more dense at the stage before the regenerated odontoblasts became arranged regularly beneath the predentine. It is suggested that these terminals have some efferent role(s), especially during collagen synthesis at the early stage of dentinogenesis. The nerves may release their abundant synaptic vesicles, in addition to serving a sensory role for monitoring the increased sensitivity in the injured areas.

To investigate the effects of a high sucrose diet and xylitol on secondary dentinogenesis and dentinal caries, a part of the sucrose in a high-cariogenic diet was replaced by xylitol. Fifty-four 3-wk-old Wistar rats were labeled with tetracycline and divided into groups. One group received a high sucrose diet (43% sucrose). In two other groups 5% and 20% of the sucrose was replaced by xylitol. A control group received non-cariogenic food. Six weeks later the mandibles were sectioned sagittally. Schiff staining was used to classify the caries, and the areas of dentin formed during the experiment and dentinal caries in first and second molars were measured planimetrically. The high sucrose diet reduced dentin formation of the molars, and this reduction was further increased by xylitol. Caries initiation and dentinal caries progression were significantly reduced by 20% xylitol, whereas only a slight reduction in caries progression was observed with 5% xylitol. Also, a negative correlation between the dentin formation and dentinal caries progression was observed in the high sucrose and 5% xylitol groups. In conclusion, xylitol together with high sucrose reduced dentin formation and dentinal caries progression, and the effect was dose-dependent.

This investigation was designed to study the formation of secondary dentin in permanent teeth of young cats after denervation. In eight animals, cervical dentin was exposed bilaterally in the mandibular canines, 7-10 d after unilateral resection of the inferior alveolar nerve. The observation intervals were 30 d, 90 d and 180 d, after which histological examination of dentin was performed. In order to verify the loss and regeneration of pulpal innervation and an intact blood supply, blood flow responses to electrical stimulation of the tooth and to i.v. injections of substance P (SP) were recorded by laser Doppler flowmetry before and at 7-10 d after denervation, at the end of the experiments, and at predetermined intermediate intervals. SP-induced vasodilation was significantly enhanced at 1 wk and 30 d postoperatively and was normalized to control values at 90 and 180 d. Vasodilation in response to electrical tooth stimulation, which was absent after denervation, reappeared after 90 d in two of four cats. There was no irregular dentin formation under the exposed dentin at any time in denervated or control teeth. Formation of regular secondary dentin appeared to be enhanced on the denervated side at 30 d and 90 d postoperatively, whereas at 180 d there was no difference between sides. The results indicate that intradental nerves influence secondary dentin formation in feline permanent teeth.

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.

To study provision of this environment, wound healing was examined by light and electron microscopy following pulp amputation and direct capping with hyaluronic acid. Molar pulps of female Sprague-Dawley rats were mechanically exposed and directly capped; the cavities were then restored with glass-ionomer cement. As an experimental control. Calvital (a commercial preparation of calcium hydroxide paste) was used as the direct pulp-capping agent in other rats, and its effect on pulp healing was compared with that of hyaluronic acid. In hyaluronic acid-treated pulps, at 2 days after amputation, wound surfaces were covered with blood and fibrin clots and inflammatory cells such as neutrophils and macrophages. At 1 week, differentiation of fibroblastic and odontoblast-like cells was observed beneath the wound layer; and odontoblast-like cells produced globular calcified nodules along the existing dentine walls. At 2 weeks, a layer of reparative dentine had been formed by odontoblast-like cells over the dentine walls. Between 30 and 60 days, the formation of reparative dentine had extended throughout the pulp chamber. The healing observed after direct capping with hyaluronic acid was, except for the formation of dentine bridges, similar to that seen after Calvital capping. These results suggest that high molecular-weight hyaluronic acid can provide an environment suitable for reparative dentine formation through mesenchymal cell differentiation during healing of the amputated dental pulp.

A quantitative assessment of dentin bridges was conducted on sections prepared from teeth capped with four pulp-capping agents (Bioglass, Life, Demineralized Dentin Matrix, and Teflon) in a micro-swine model. The degree of mineralization of the dentin bridges relative to the adjacent primary dentin was measured using a computer-based image analysis of microradiographs prepared from the sections. The rate of formation of the dentin bridge was measured from fluorescent bands formed in the same sections by a Demeclocycline dentin marker. There were no statistically significant differences in the relative mineral densities of the dentin bridges and the rate of dentin bridge formation under the four pulp-capping agents. These findings support the suggestion that components of the extracellular matrix rather than pulp-capping agents may be important in the formation and mineralization of dentin bridges in repairing dental pulps.

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.

The role of fibronectin during the events initiating the post-developmental histogenesis of dentine was investigated by exposing the pulp to implants of autogenous demineralized dentine or calcium hydroxide-containing cement for short periods. Implants exposed for 3 days were processed for immunoelectron-microscopic analysis of fibronectin adsorption on to their surfaces. The localization of fibronectin in the critical area of interaction was examined by immunofluorescence 6, 14 and 21 days after implantation. Heavy adsorption of fibronectin on to the dentine implants and the crystalline structures that had been deposited on the cement implants was demonstrated. Positive fluorescence was consistently seen around dentine implants. Strongly immunopositive fibroblast-like cells and weakly reactive, differentiating odontoblast-like cells were found in association with the implanted matrix. Uncalcified matrix secreted by the polarized or non-polarized cells was consistently rich in fibronectin. Fibroblast-like cells exhibiting intense immunoreaction only at 14 and 21 days were mainly associated with the crystalline precipitates on the cement surfaces or within the surrounding pulp. The findings indicate that the specific inductive effects of demineralized dentine on pulp cells are initiated by exposure of the pulp to a fibronectin-containing surface; adhesion of pulp cells and synthesis of a fibronectin-rich matrix characterize the development of new dentine. The reparative response to non-specific inductive influences such as calcium hydroxide seems to be mediated by progressive enhancement of fibronectin synthesis in pulp cells.

Dentinogenesis can be initiated secondarily as an intrinsic ability of the dental pulp to repair, or after interaction of pulp cells with specific exogenous inductive factors. In the present article the basic developmental aspects, highlighting the mechanism by which dentinogenesis is initiated during tooth development, are discussed. Furthermore, clinical and experimental observations concerning the events taking place during secondary initiation of dentine formation, as part of exposed or non-exposed pulp tissue repair, or as a result of dentine matrix or other chemical-pulp cell interactions, are reviewed. Discussion includes hypotheses relating to the crucial biological steps leading to expression of odontoblastic-like cell phenotype and secondary initiation of dentine histogenesis.

Differentiation of odontoblasts is important for dentin formation in tooth germs and mature teeth. Although previous reports have indicated that there may be a kind of inductive agent that could induce mesenchymal cells in dental pulps to differentiate into odontoblasts, and secrete dentin matrix, the primary inductive factor of odontoblasts has not been found. Bone morphogenetic protein (BMP), which induces the formation of cartilage and bone when implanted in muscle tissue, is found in dentin matrix. The relationship between the differentiation of odontoblasts and BMP was observed by means of immunohistochemical staining with monoclonal antibody (MAb) against BMP in dental pulp tissue and cell culture; [3H]thymidine incorporation; and measurement of alkaline phosphatase activity. The conclusions are: (1) BMP exists in odontoblasts, ameloblasts, and dentin matrix (the positive reaction in ameloblasts appeared earlier and remained stronger); (2) BMP promotes incorporation of [3H]thymidine and increases the activity of alkaline phosphatase in cultured dental pulp cells; (3) BMP-induced dental pulp cells in dental pulp tissue cultures differentiate from mesenchymal to odontoblast-like cells; and (4) BMP induces formation of osteodentin and tubular dentin when used as a dental capping agent of dogs' teeth. Bone morphogenetic protein plays an important role in differentiation of odontoblasts and might be one of the inductive agents of odontoblasts. Further investigations of BMP as a biologic dental capping agent are warranted.

Autogenous dentinal matrix was exposed to the pulp cells of adult dogs in order to determine whether the mature pulp cells possess the ability to differentiate into odontoblast-like cells as a direct response to known inductive influences. The pulps of molars, premolars, and canines of three dogs (2 to 4 yr old) were mechanically exposed through buccal class V cavities. Pieces of demineralized or native dentin and predentin were implanted in the pulp sites for periods of 2 to 6 wk. The reactions were analyzed by light microscopy. Induction of dentin formation was observed only after native dentin implantation; either as early response to exposure of predentinal surfaces or around mineralized dentin after 3 postoperative wk. Encapsulation by fibrous connective tissue or matrix degradation was seen around demineralized dentin implants. A characteristic enhancement of circumferential pulpal dentin deposition around the implantation site was demonstrated after native dentin exposure to light and scanning electron microscopic examination. These data indicate that specific inductive influences given by the native but not the acid-conditioned dentin, when it is exposed to the pulp environment of adult teeth, are able to direct differentiation of odontoblast-like cells and to enhance the biosynthetic activity of primary odontoblasts.

An extra oral approach was made to 26 pairs of Wistar rat incisors and a small bur used to expose the pulps which were then dressed with either calcium hydroxide paste (Pulpdent) zinc oxide-eugenol cement (Kalzinol) or a triamocinolone acetonide/tetracycline containing cement (Ledermix). Animals were sacrificed at 7 days, incisors removed, a window cut to the pulp opposite the site of exposure, soft tissues removed with 7% NaOCl for 30 minutes before teeth were dehydrated in graded concentrations of acetone and gold coated for examination under SEM. Calcium hydroxide produced rapid complete repair with a remarkably regular formation of calcospherites, except for a zone of relative inhibition perforated by nutrient canals at the periphery of each lesion. Both zinc oxide-eugenol and triamcinolone acetonide cements inhibited both bridging of the defect created by the exposure and dentine formation in the surrounding pulpal wall. Data was grouped according to the degree of hard tissue repair. The results were statistically significant (Chi-square p < or = 0.001). This method demonstrated qualitative and quantitative differences in the repair process resulting from chemical variations in the dressings applied.

The effects of demineralized dentin and bone matrix on dental ectomesenchymal cells were evaluated after observation periods of two or three weeks. Autogenous dentin and bone matrix, obtained from the crowns of primary molars or maxillary cortical bone, respectively, were demineralized with 3% acetic acid and implanted into pulpal or papilla sites of erupting dog teeth: first molars, fourth premolars, and canines. Dentin histogenesis associated with odontoblastic arrangement was demonstrated in relation to all dentin implants in pulpal sites. Deposition of osteodentin, followed in some areas by tubular predentin formation, was observed in contact with bone implants in pulpal sites. In papilla sites, the dentin implantation exhibited bone-like matrix formation, while bone implants were encapsulated by connective tissue. The interactions of pulp cells with demineralized dentin matrix constitute a model for experimental induction of secondary dentinogenesis and odontoblast-like cell differentiation.