Reparative Dentin Formation Research Articles

Ability of stem and progenitor cells in the dental pulp to form hard tissue

Summary Dental pulp has an important ability to form mineralized hard tissue in response to a variety of external stimuli. The formation of mineralized tissue within the pulp cavity has been widely examined in both clinical and animal studies. Despite these studies focusing on the phenomena of reparative dentin and dentin bridge formation, the mechanisms of their induction remain unknown. Recently, several morphological studies revealed that the source of cells for hard tissue formation is the dental pulp itself, even after pulp injury. This finding indicates that the dental pulp tissue contains undifferentiated cells participating in dentin and pulp regeneration. Additionally, stem and progenitor cells isolated from the dental pulp were found to differentiate into odontoblasts as well as osteoblasts. This review presents current evidences for the multipotent ability of dental pulp cells and their usefulness in tissue engineering applications as a cell resource.

Disruption of Tgfbr2 in Odontoblasts Leads to Aberrant Pulp Calcification

Transforming growth factor β (TGF-β) signaling has been implicated in dentin formation and repair; however, the molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-β signaling in dentin formation, we analyzed odontoblast-specific Tgfbr2 conditional knockout mice. The mutant mice had aberrant teeth with thin dysplastic dentin and pulpal obliteration, similar to teeth from human patients with dentinogenesis imperfecta type II and dentin dysplasia. In mutant, the odontoblasts lost their cellular polarity, and matrix secretion was disrupted after mantle dentin formation. As a consequence, the amount of predentin decreased significantly, and an ectopic fibrous matrix was formed below the odontoblast layer. This matrix gradually calcified and obliterated the pulp chamber with increasing age. Immunohistochemistry revealed decreased expression of alkaline phosphatase in mutant odontoblasts. In mutant dentin, Dsp expression was reduced, but Dmp1 expression increased significantly. Collagen type I, biglycan, and Dsp were expressed in the ectopic matrix. These results suggest that loss of responsiveness to TGF-β in odontoblasts results in impaired matrix formation and pulpal obliteration. Our study indicates that TGF-β signaling plays an important role in dentin formation and pulp protection. Furthermore, our findings may provide new insight into possible mechanisms underlying human hereditary dentin disorders and reparative dentin formation.

Possible mechanisms of lack of dentin bridge formation in response to calcium hydroxide in primary teeth

Introduction: The usage of Calcium hydroxide (CaOH 2 ) has wide applications due to the property of osteo-inductive, protective, and antibacterial actions. However, it is not used in primary teeth, as it fails to form reparative dentin and the exact mechanism has not been explained. The hypothesis: The authors propose an explanation that lack of dentin bridge formation in response to (CaOH 2 ) in primary teeth could be multifactorial: inability of the deciduous stem cells to generate complete dentin-pulp-like tissue; the absence of calcium-magnesium-dependent adenosine triphosphatase (Ca-Mg ATPase) in the odontoblasts; the pre-existing predilection of deciduous dentine pulp to form odontoclasts; the solubility of (CaOH 2 ). Evaluation of the hypothesis: The hypothesis discusses the innate traits of the deciduous stem cells that lack the ability to form the dentin bridge, the absence of Ca-Mg ATPase enzyme and increased solubility of (CaOH 2 ) together fail to stimulate the odontoblasts. Alternatively, pre-existing progenitor cells with proclivity to change into odontoclasts may cause internal resorption and hamper formation of reparative dentin.

Odontogenic effects of a fast-setting calcium-silicate cement containing zirconium oxide

A fast-setting calcium-silicate cement (Endocem) was introduced in the field of dentistry for use in vital pulp therapy. Similar to mineral trioxide aggregate (MTA), it contains bismuth oxide to provide radiopacity. Recently, another product, EndocemZr, which contains zirconium oxide (ZrO2) as a radiopacifier, was developed by the same company. In this study, the biological/odontogenic effects of EndocemZr were investigated in human primary dental pulp cells (hpDPCs) in vitro and on capped rat teeth in vivo. The biocompatibility of EndocemZr was similar to that of ProRoot and Endocem on the basis of cell viability tests and cell morphological analysis. The mineralization nodule formation, expression of odontogenic-related markers, and reparative dentin formation of EndocemZr group was similar to those of other material groups. Our results suggest that EndocemZr has the potential to be used as an effective material for vital pulp therapy, similar to ProRoot and Endocem.

A review on vital pulp therapy in primary teeth.

Maintaining deciduous teeth in function until their natural exfoliation is absolutely necessary. Vital pulp therapy (VPT) is a way of saving deciduous teeth. The most important factors in success of VPT are the early diagnosis of pulp and periradicular status, preservation of the pulp vitality and proper vascularization of the pulp. Development of new biomaterials with suitable biocompatibility and seal has changed the attitudes towards preserving the reversible pulp in cariously exposed teeth. Before exposure and irreversible involvement of the pulp, indirect pulp capping (IPC) is the treatment of choice, but after the spread of inflammation within the pulp chamber and establishment of irreversible pulpitis, removal of inflamed pulp tissue is recommended. In this review, new concepts in preservation of the healthy pulp tissue in deciduous teeth and induction of the reparative dentin formation with new biomaterials instead of devitalization and the consequent destruction of vital tissues are discussed.

Chronic inflammation and angiogenic signaling axis impairs differentiation of dental-pulp stem cells.

Dental-pulp tissue is often exposed to inflammatory injury. Sequested growth factors or angiogenic signaling proteins that are released following inflammatory injury play a pivotal role in the formation of reparative dentin. While limited or moderate angiogenesis may be helpful for dental pulp maintenance, the induction of significant level of angiogenesis is probably highly detrimental. Hitherto, several studies have addressed the effects of proinflammatory stimuli on the survival and differentiation of dental-pulp stem cells (DPSC), in vitro. However, the mechanisms communal to the inflammatory and angiogenic signaling involved in DPSC survival and differentiation remain unknown. Our studies observed that short-term exposure to TNF-α (6 and 12 hours [hrs]) induced apoptosis with an upregulation of VEGF expression and NF-κB signaling. However, long-term (chronic) exposure (14 days) to TNF-α resulted in an increased proliferation with a concomitant shortening of the telomere length. Interestingly, DPSC pretreated with Nemo binding domain (NBD) peptide (a cell permeable NF-κB inhibitor) significantly ameliorated TNF-α- and/or VEGF-induced proliferation and the shortening of telomere length. NBD peptide pretreatment significantly improved TNF-α-induced downregulation of proteins essential for differentiation, such as bone morphogenic proteins (BMP)-1 & 2, BMP receptor isoforms-1&2, trasnforming growth factor (TGF), osteoactivin and osteocalcin. Additionally, inhibition of NF-κB signaling markedly increased the mineralization potential, a process abrogated by chronic exposure to TNF-α. Thus, our studies demonstrated that chronic inflammation mediates telomere shortening via NF-κB signaling in human DPSC. Resultant chromosomal instability leads to an emergence of increased proliferation of DPSC, while negatively regulating the differentiation of DPSC, in vitro.

Preliminary study of light-cured hydrogel for endodontic drug delivery vehicle.

Direct pulp capping is the treatment of an exposed vital pulp with a dental material to facilitate the formation of reparative dentin and maintenance of vital pulp. A bioengineered drug delivery vehicle has the potential to increase the success rate of pulp capping. The aim of this study was to develop an injectable and light-curing drug delivery vehicle for endodontic treatment including direct pulp capping. Polyethylene glycol-maleate-citrate (PEGMC) hydrogel was synthesized as a drug delivery vehicle that is composed of PEGMC (45% w/v), acrylic acid (AA) (5% w/v), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (0.1% w/v), and deionized water. The association between prehydrogel-solution volume and visible light-curing was examined. The cytotoxicity of the hydrogel was tested using L929 cells in a cell culture system. Ca(2+) release from the hydrogel was determined using calcium hydroxide as the incorporated medicine. The results showed that the light-curing time for hydrogel is comparable to composite resin. The hydrogel had cell toxicity similar to adhesive systems. Moreover, controlled Ca(2+) release was obtained from the calcium hydroxide incorporated hydrogel. The data suggest that hydrogel should be explored further as a promising drug delivery vehicle for vital pulp therapy and regenerative endodontics.

Pulp Stem Cells: Implication in Reparative Dentin Formation

Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

A short-term treatment with tumor necrosis factor-alpha enhances stem cell phenotype of human dental pulp cells

IntroductionDuring normal pulp tissue healing, inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) or interleukins, act in the initial 48 hours (inflammatory phase) and play important roles not only as chemo-attractants of inflammatory cells and stem/progenitor cells but also in inducing a cascade of reactions toward tissue regeneration or reparative dentin formation or both. Previous reports have shown that inflammatory cytokines regulate the differentiation capacity of dental pulp stem/progenitor cells (DPCs), but none has interrogated the impact of these cytokines on the stem cell phenotype of stem/progenitor cells. This study investigated the effects of a short-term treatment with TNF-α on the stem cell phenotype and differentiation ability of human DPCs.MethodsAn in vivo mouse model of pulp exposure was performed for analysis of expression of the mesenchymal stem cell marker CD146 in DPCs during the initial stage of inflammatory response. For in vitro studies, human DPCs were isolated and incubated with TNF-α for 2 days and passaged to eliminate TNF-α completely. Analysis of stem cell phenotype was performed by quantification of cells positive for mesenchymal stem cell markers SSEA-4 (stage-specific embryonic antigen 4) and CD146 by flow cytometry as well as by quantitative analysis of telomerase activity and mRNA levels of OCT-4 and NANOG. Cell migration, colony-forming ability, and differentiation toward odontogenesis and adipogenesis were also investigated.ResultsThe pulp exposure model revealed a strong staining for CD146 during the initial inflammatory response, at 2 days after pulp exposure. In vitro experiments demonstrated that a short-term (2-day) treatment of TNF-α increased by twofold the percentage of SSEA-4+ cells. Accordingly, STRO-1, CD146, and SSEA-4 protein levels as well as OCT-4 and NANOG mRNA levels were also significantly upregulated upon TNF-α treatment. A short-term TNF-α treatment also enhanced DPC function, including the ability to form cell colonies, to migrate, and to differentiate into odontogenic and adipogenic lineages.ConclusionsA short-term treatment with TNF-α enhanced the stem cell phenotype, migration, and differentiation ability of DPCs.

Odontogenic Effect of a Fast-setting Pozzolan-based Pulp Capping Material

IntroductionMineral trioxide aggregate (MTA) is widely used as a pulp capping material. Recently, a MTA-derived fast-setting pozzolan cement (Endocem; Maruchi, Wonju, Korea) was introduced in the endodontic field. Our aim in this study was to investigate the odontogenic effects of this cement in vitro and in vivo. MethodsHuman dental pulp cells (hDPCs) were cultured, and the effects of Endocem and a previously marketed MTA (ProRoot; Dentsply, Tulsa, OK) on biocompatibility were evaluated by assessing cell morphology and performing a cell viability test. Chemical composition of each material was analyzed by energy-dispersive X-ray spectroscopic analysis. Odontoblastic differentiation was analyzed by alkaline phosphatase activity and alizarin red S staining. The expression of odontogenic-related markers, namely dentin sialophosphoprotein, dentin matrix protein 1, and osteonectin, was evaluated by real-time polymerase chain reaction, Western blotting, and immunofluorescence analysis. Pinpoint pulp exposures were made on rat teeth and then capped with ProRoot or Endocem. After 4 weeks, reparative tertiary dentin formation and inflammatory responses were investigated histologically. ResultsThe biocompatibility of Endocem was similar to that of ProRoot. Energy-dispersive X-ray spectroscopic analysis showed that ProRoot and Endocem contained similar elemental constituents such as calcium, oxygen, and silicon. Alkaline phosphatase activity and mineralized nodule formation increased in ProRoot- and Endocem-treated cells compared with medium only–treated cells in the control group (P < .05). The expression of odontogenic-related markers was significantly higher in the ProRoot- and Endocem-treated groups than the control group (P < .05), but there was no significant difference in the expression of these markers between the 2 experimental groups (P > .05). Four weeks after the pulp capping procedure, continuous tertiary dentin had formed directly underneath the capping materials and the pulp exposure area in all samples in the 2 treated groups. Furthermore, most specimens either had no inflammation or minor pulpal inflammation. ConclusionsOur results indicate that ProRoot and Endocem have similar biocompatibility and odontogenic effects. Therefore, Endocem is as effective a pulp capping material as ProRoot.

β-Catenin Enhances Odontoblastic Differentiation of Dental Pulp Cells through Activation of Runx2

An intense stimulus can cause death of odontoblasts and initiate odontoblastic differentiation of stem/progenitor cell populations of dental pulp cells (DPCs), which is followed by reparative dentin formation. However, the mechanism of odontoblastic differentiation during reparative dentin formation remains unclear. This study was to determine the role of β-catenin, a key player in tooth development, in reparative dentin formation, especially in odontoblastic differentiation. We found that β-catenin was expressed in odontoblast-like cells and DPCs beneath the perforation site during reparative dentin formation after direct pulp capping. The expression of β-catenin was also significantly upregulated during odontoblastic differentiation of in vitro cultured DPCs. The expression pattern of runt-related transcription factor 2 (Runx2) was similar to that of β-catenin. Immunofluorescence staining indicated that Runx2 was also expressed in β-catenin–positive odontoblast-like cells and DPCs during reparative dentin formation. Knockdown of β-catenin disrupted odontoblastic differentiation, which was accompanied by a reduction in β-catenin binding to the Runx2 promoter and diminished expression of Runx2. In contrast, lithium chloride (LiCl) induced accumulation of β-catenin produced the opposite effect to that caused by β-catenin knockdown. In conclusion, it was reported in this study for the first time that β-catenin can enhance the odontoblastic differentiation of DPCs through activation of Runx2, which might be the mechanism involved in odontoblastic differentiation during reparative dentin formation.

Induction of reparative dentin formation on exposed dental pulp by dentin phosphophoryn/collagen composite.

The ultimate goal of vital pulp therapy is to regenerate rapidly dentin possessing an excellent quality using a biocompatible, bioactive agent. Dentin phosphophoryn (DPP), the most abundant noncollagenous polyanionic protein in dentin, cross-linked to atelocollagen fibrils was applied to direct pulp capping in rats. After 1, 2, and 3 weeks, the teeth applied were examined on the induction of reparative dentin formation and the response of pulp tissue, compared to calcium hydroxide-based agent conventionally used. The reparative dentin formation induced by DPP/collagen composite was more rapid than by calcium hydroxide. In the morphometrical analysis, the formation rate of reparative dentin by DPP/collagen composite was approximately the same as that by calcium hydroxide at 3 weeks. Nevertheless, the compactness of reparative dentin formed by DPP/collagen composite was much superior to what resulted from calcium hydroxide. Also, DPP/collagen composite showed high covering ability of exposed pulp. Moreover, DPP/collagen composite led only to slight pulp inflammation at the beginning whereas calcium hydroxide formed necrotic layer adjacent to the material and induced severe inflammation in pulp tissue at 1 week. The present study demonstrates a potential for DPP/collagen composite as a rapid biocompatible inducer for the formation of reparative dentin of excellent quality in rats.

A histological comparison of a new pulp capping material and mineral trioxide aggregate in rat molars.

Recent investigations have attempted to improve regenerative endodontics with the help of stem cell therapy. In vitro studies have shown the ability of different agents to stimulate the differentiation of dental pulp stem cells (DPSC) into odontoblast-like cells. A combination of dexamethasone, β-glycerophosphate and Vitamin D has been proven to induce a successful differentiation. The aim of this animal study was to evaluate the effect of this combination, named odontoblastic differentiating material (ODM), on pulp tissue when used as a capping material. Sixty maxillary right and left molars of 30 Sprague-dawley rats were selected for this study. The teeth were exposed under sterile condition. Half of the teeth were capped with mineral trioxide aggregate (MTA) and the other half with ODM. All cavities were restored with glass ionomer. The rats were sacrificed at post-operative intervals of 2 weeks and 2 months. Samples were histologically evaluated for the degree of inflammation and reparative dentin formation. Finally the data was analyzed with Mann-Whitney and Chi-Square tests. Reparative dentin formed in all groups within both time periods and there was no statistically significant difference between the groups in the mentioned time periods. The MTA group, however, showed a statistically significant reduction in inflammation at both time intervals (P<0.05). Compared to MTA, ODM samples showed a greater amount of inflammation in the pulp tissue. ODM, as a pulp capping material, can induce dentinal bridge formation.

Porous chitosan bilayer membrane containing TGF-β1 loaded microspheres for pulp capping and reparative dentin formation in a dog model

ObjectivesThe objectives of this study were to develop a chitosan bilayer membrane containing microspheres with sustained TGF-β1 release to enhance odontoblast-like cell function in vitro, and to investigate pulp-capping in a dog model to promote reparative dentin formation in vivo for the first time. MethodsA chitosan bilayer membrane was synthesized consisting of a dense film on one side and a macroporous sponge on the other side. The rationale was to use the dense film to block the perforated pulp from bacterial invasion, and the porous sponge to be loaded with microspheres containing TGF-β1 (MS-TGF) for sustained release. Pulp capping in 48 teeth of six beagle dogs was performed to test four groups: Control with no pulp capping material, commercial Dycal, chitosan membrane without MS-TGF, and chitosan membrane with MS-TGF. The dog teeth were harvested for histological analysis at two time points (10 and 60d). ResultsThe spongy side of the membrane had macropores with a mean size of 151μm. The porosity of the membrane was 83%. Chitosan microspheres containing TGF-β1 showed sustained release, gradually releasing 42% of the TGF-β1 in 7d. The proliferation of odontoblast-like cells on membrane with MS-TGF was much greater than that without TGF (p<0.05). At 10d, H&E staining revealed mild to moderate pulp inflammation in all four groups, with no dentin bridge formation. At 60d, pulp inflammation disappeared, but there was no reparative dentin bridge in the group with no pulp-capping material. Chitosan membranes with MS-TGF generated reparative dentin with a thickness of (142±29)μm, 3–6 times thicker than that with Dycal or chitosan bilayer membrane without TGF (p<0.05). SignificanceA novel chitosan bilayer-microsphere construct containing TGF-β1 for pulp-capping generated 3–6 times more reparative dentin than the controls in a dog model for the first time. The chitosan bilayer-microsphere construct with growth factor delivery may be useful for a wide range of dental and regenerative medicine applications.

EphB–EphrinB Interaction Controls Odontogenic/Osteogenic Differentiation with Calcium Hydroxide

IntroductionCalcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide–related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide. MethodsPrimary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed. ResultsCalcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1–positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1–EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide–induced mineralization from primary pulp cells. ConclusionsEphrinB1–EphB2 interaction contributes to calcium hydroxide–induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide–induced odontogenic/osteogenic differentiation.

Nuclear Expression of p27Kip1 Is Associated with In Vivo Differentiation of Adult Human Odontoblasts

IntroductionOdontoblasts 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 p27Kip1 has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27Kip1 protein was shown in many adult mouse tissues, but no information is available on the expression of p27Kip1 in mammalian dental pulp. MethodsHealthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27Kip1 and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections. Resultsp27Kip1 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 p27Kip1 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 p27Kip1 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 p27Kip1 in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status. ConclusionsOur findings show that increased expression of nuclear p27Kip1 occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27Kip1 can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo.

Repair processes of experimental root fractures in rat molars examined by histopathological techniques and 3D micro-CT imaging

AimsThe purpose of this study was to investigate the repair processes of experimental intra-alveolar root fractures of immature rat molars with histopathological techniques and 3-dimensional micro-computed X-ray tomography (3D micro-CT) imaging. MethodsTwelve left upper first molars of 4-week-old rats were dislocated toward the palate with modified Hoe's pliers at a load of 900 g for 20 s, and repositioned. After 1 or 2 weeks, the maxillae were dissected and scanned with micro CT for 3D micro-CT imaging. After standard histological preparation, buccolingual serial sections of the maxillae were stained with hematoxylin and eosin or Masson–Goldner stain and observed by light microscopy. Subsequently, we examined the identified root fracture lesions by 3D micro-CT imaging and histopathological sections. ResultsWe identified 6 intra-alveolar vertical incomplete fracture lines in the mesial roots. Histopathological findings showed that newly differentiated odontoblast-like cells had formed immature reparative dentin on the pulpal side of the fracture line at 1 postoperative week, and deposited reparative dentin with a tubular structure at 2 weeks. While surface resorption and granulation-tissue formation occurred on the periodontal side of the fracture line, the narrow fracture line remained unchanged, and the surface resorption lacunae were partly covered with cementum at postoperative 2 weeks. ConclusionsRoot-fracture investigation requires a combination of histopathological observation and 3D-CT imaging. Intra-alveolar fractured root repair accompanied by tubular reparative dentin formation at the pulpal side, and the deposition of cementum at the periodontal side, may occur when the fracture line is too narrow for the formation of granulation tissue.

MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

Effect of Statins with α-Tricalcium Phosphate on Proliferation, Differentiation, and Mineralization of Human Dental Pulp Cells

IntroductionThe aim of this study was to investigate the combined effect of statin and α-tricalcium phosphate (α-TCP) on odontoblastic differentiation of human dental pulp cells and to compare them with mineral trioxide aggregate (MTA). MethodsExperimental cements were prepared with TCP containing simvastatin and atorvastatin. Cell proliferation, cell adherence on a dentin disc, alkaline phosphatase (ALP) activity, expression of osteogenic/odontoblastic markers, and mineralization of the human dental pulp cells on experimental cement and MTA were assessed. ResultsThe cell growth and ALP activity of TCP containing simvastatin–treated cells was greater than MTA-treated cells. The mineralization and messenger RNA expression of markers (ie, dentin sialophosphoprotein, dentin matrix protein 1, bone morphogenetic protein 2, ALP, and osteonectin) of TCP containing simvastatin– and TCP containing atorvastatin–treated cells were comparable with MTA-treated cells. The enhanced cell proliferation and similar level of ALP of TCP-treated cells compared with the control indicate that α-TCP is an effective osteoconductive material. The differentiation effect observed in TCP containing simvastatin– and TCP containing atorvastatin–treated cells is attributed to the effect of statin. ConclusionsThe results suggest that α-TCP can be used for local delivery of statin as a pulp capping material to accelerate reparative dentin formation.

Synthetic octacalcium phosphate-enhanced reparative dentine formation via induction of odontoblast differentiation

Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP.