Secreted Frizzled-Related Protein 1 Promotes Odontoblastic Differentiation and Reparative Dentin Formation in Dental Pulp Cells.

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.

Evaluation of secondary dentin deposition in lower first molars to indicate a legal age threshold of 14 years using receiver operating characteristic curves.

The age threshold of 14 years has become vital in proving legal violations involving children, particularly in cases of suspected child labour, child pornography and the minimum age of criminal responsibility. In recent years, there has been great interest in the evaluation of age in children and sub-adults using analysis of regressive changes in teeth, with a particular focus on age thresholds that are of medico-legal importance. This research aimed to compare the sensitivity and specificity of stages of root pulp visibility by Olze et al. in a sample of South Indian children aged between 12 and 16 years, with an age threshold of 14 years, using receiver operating characteristic curves and the area under the curve (AUC). Among the four stages of root pulp visibility, stage 2 showed the highest AUC in both female and male cohorts. For stage 2, lower sensitivity and higher specificity values were recorded, indicating the possibility of type II errors (i.e. false negatives). In both sexes, stage 2 had a higher AUC (i.e. 0.696 in females and 0.706 in males, respectively). Based on our findings, it can be concluded that this staging method in lower first molars is of limited value in indicating the legal age threshold of 14 years. Future research should validate the proposed approach in a larger sample and consider how to improve predictions in this area.

Characterization of Odontoblast-like Cell Phenotype and Reparative Dentin Formation In Vivo: A Comprehensive Literature Review.

INTRODUCTION: The primary aim was to explore the criteria used in characterization of reparative cells and mineralized matrices formed after treatment of pulp exposures, and the sequence of relative events. The secondary aim was to evaluate whether the reparative events depend on the experimental model species, age, and therapeutic intervention. METHODS: A literature search of databases using different combinations of the key words was undertaken. Data analysis was based only on studies having histological or histochemical assessment of the pulp tissue responses. The search yielded 86 studies, 47 capping material-based and 39 bioactive application-based experiments, which provided data on morphological or functional characterization of the mineralized matrices and the associated cells. RESULTS: In 64% of capping material-based and 72% of bioactive application-based experiments, a 2-zone mineralized matrix formation (atubular followed by tubular) was detected, whereas characterization of odontoblastic differentiation is provided in only 25.5% and 46.1% of the studies, respectively. In 93.3% of the studies showing odontoblast-like cells, differentiated cells were in association with tubular mineralized matrix formation. Analyses further showed that cell- and matrix-related outcomes do not depend on experimental model species, age, and therapeutic intervention. CONCLUSIONS: The evidence of the reviewed scientific literature is that dental pulp cells secrete a dentin-like matrix of tubular morphology in relation to primitive forms of atubular or osteotypic mineralized matrix. Furthermore, data analysis showed that dental pulp cells express in vivo the odontoblastic phenotype, and secrete matrix in a predentin-like pattern, regardless of the model species, age, and therapeutic intervention used.

Regulation of Reactionary Dentine Formation.

During the treatment of dental caries that has not penetrated the tooth pulp, maintenance of as much unaffected dentine as possible is a major goal during the physical removal of decayed mineral. Damage to dentine leads to release of fossilized factors (transforming growth factor-ß [TGF-ß] and bone morphogenic protein [BMP]) in the dentine that are believed to stimulate odontoblasts to secrete new "tertiary" dentine (reactionary dentine). This is formed on the pulpal surface of existing dentine and rethickens the dentine. We have previously shown that activation of Wnt/ß-catenin signaling is pivotal for tooth repair in exposed pulp injury, and the pathway can be activated by small-molecule GSK-3 antagonists, resulting in enhanced reparative dentine formation. Here, we use a nonexposed pulp injury model to investigate the mechanisms of reactionary dentine formation in vivo, using small molecules to modulate the Wnt/ß-catenin, TGF-ß, and BMP pathways. We found that a local increase of Wnt activation at the injury site enhances reactionary dentine secretion. In addition, inhibition of TGF-ß, BMP, or Wnt pathways does not impede reactionary dentine formation, although inhibition of TGF-ß and/or BMP signaling does result in more disorganized, nontubular reactionary dentine. This suggests that Wnt/ß-catenin signaling plays no major role in the formation of reactionary dentine, but in common with reparative dentine formation, exogenous elevation of Wnt/ß-catenin signaling can enhance tertiary dentine formation. Release of latent TGF-ß or BMPs from dentine is not required for the deposition of mineral to form reactionary dentine but does play a role in its organization.

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies.

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

Is Pulp Inflammation a Prerequisite for Pulp Healing and Regeneration?

The importance of inflammation has been underestimated in pulpal healing, and in the past, it has been considered only as an undesirable effect. Associated with moderate inflammation, necrosis includes pyroptosis, apoptosis, and nemosis. There are now evidences that inflammation is a prerequisite for pulp healing, with series of events ahead of regeneration. Immunocompetent cells are recruited in the apical part. They slide along the root and migrate toward the crown. Due to the high alkalinity of the capping agent, pulp cells display mild inflammation, proliferate, and increase in number and size and initiate mineralization. Pulp fibroblasts become odontoblast-like cells producing type I collagen, alkaline phosphatase, and SPARC/osteonectin. Molecules of the SIBLING family, matrix metalloproteinases, and vascular and nerve mediators are also implicated in the formation of a reparative dentinal bridge, osteo/orthodentin closing the pulp exposure. Beneath a calciotraumatic line, a thin layer identified as reactionary dentin underlines the periphery of the pulp chamber. Inflammatory and/or noninflammatory processes contribute to produce a reparative dentinal bridge closing the pulp exposure, with minute canaliculi and large tunnel defects. Depending on the form and severity of the inflammatory and noninflammatory processes, and according to the capping agent, pulp reactions are induced specifically.

Minimally invasive clinical approach in indirect pulp therapy and healing of deep carious lesions.

Indirect pulp treatment is a conservative vital pulp procedure performed in deep carious lesion approximating the pulp, but without signs or symptoms of pulp degeneration. Removing the carious biomass along with sealing the residual caries from extrinsic substrate and oral bacteria makes residual caries after the first excavation less active. This allows time for pulpo dentinal complex to form tertiary dentine so that at the second excavation, there is less likelihood of pulpal exposure. It has also been suggested that by changing the cavity environment from an active lesion into a more slowly progressing lesion, will be accompanied by more regular tubular tertiary dentin formation. The success of this approach has been demonstrated by various randomized controlled studies comparing conventional treatment of such lesions with stepwise excavation. These results are echoed at clinical, radiographic, macroscopic, microscopic and ultrastructural level during follow up visits. This study reviews promising concepts and rationale of minimally invasive indirect pulp therapy technique where conventional wisdom of caries removal is challenged

Is hard tissue formation in the dental pulp after the death of the primary odontoblasts a regenerative or a reparative process?

OBJECTIVES: Conceptually, two types of tertiary dentine may be produced in response to caries and environmental irritations: "reactionary dentine" that is secreted by existing primary odontoblasts and "reparative dentine", formed after the death of the odontoblasts by proliferation and differentiation of progenitor cells into odontoblast-like cells. Because histologic evidence for tubular dentine generated by newly differentiated odontoblast-like cells is lacking in human teeth, the present study examined pulpal cellular changes associated with caries/restorations, in the presence or absence of pulpal exposures. METHODS: Ninety-six extracted human teeth were histologically processed and serial sectioned for light microscopy: 65 contained untreated enamel/dentine caries; 20 were heavily restored and 11 had carious exposures managed by direct pulp-capping. RESULTS: Sparsely distributed, irregularly arranged dentinal tubules were identified from the tertiary dentine formed in teeth with unexposed medium/deep caries and in restored teeth; those tubules were continuous with the tubules of secondary dentine; in some cases, tubules were absent. The palisade odontoblast layer was reduced to a single layer of flattened cells. In direct pulp-capping of pulp exposures, the defects were repaired by the deposition of an amorphous dystrophic calcified tissue that resembled pulp stones more than dentine, sometimes entrapping pulpal remnants. This atubular hard tissue was lined by fibroblasts and collagen fibrils. CONCLUSIONS: Histological evidence from the present study indicates that reparative dentinogenesis cannot be considered as a regenerative process since the so-formed hard tissue lacks tubular features characteristic of genuine dentine. Rather, this process represents a repair response that produces calcified scar tissues by pulpal fibroblasts. CLINICAL SIGNIFICANCE: Formation of hard tissue in the dental pulp after the death of the primary odontoblasts has often been regarded by clinicians as regeneration of dentine. If the objective of the clinical procedures involved is to induce healing, reduce dentine hypersensitivity, or minimise future bacteria exposure, such procedures may be regarded as clinical success. However, current clinical treatment procedures are not adept at regenerating physiological dentne because the tissues formed in the dental pulp are more likely the result of repair responses via the formation of calcified scar tissues.

Frequently asked questions in direct pulp capping of permanent teeth.

UNLABELLED: Direct pulp capping is a proven method of preserving tooth vitality of a mature permanent tooth in cases of pulp exposures. The indications for this treatment, treatment modalities and materials are discussed in this paper. CLINICAL RELEVANCE: This paper answers many of the frequently asked questions by general practitioners, dental students and specialists about direct pulp capping.

Regenerative endodontics: regeneration or repair?

Recent advances in biotechnology and translational research have made it possible to provide treatment modalities that protect the vital pulp, allow manipulation of reactionary and reparative dentinogenesis, and, more recently, permit revascularization of an infected root canal space. These approaches are referred to as regenerative procedures. The method currently used to determine the origin of the tissue secreted during the repair/regeneration process is largely based on the identification of cellular markers (usually proteins) left by cells that were responsible for this tissue production. The presence of these proteins in conjunction with other indicators of cellular behavior (especially biomineralization) and analysis of the structure of the newly generated tissue allow conclusions to be made of how it was formed. Thus far, it has not been possible to truly establish the biological mechanism controlling tertiary dentinogenesis. This article considers current therapeutic techniques to treat the dentin-pulp complex and contextualize them in terms of reparative and regenerative processes. Although it may be considered a semantic argument rather than a biological one, the definitions of regeneration and repair are explored to clarify our position in this era of regenerative endodontics.

Histologic examinations of teeth treated with 2 scaffolds: a pilot animal investigation.

INTRODUCTION: A growing body of evidence is building a case for the possibility of tissue regeneration within the root canal of necrotic teeth, allowing for continued root development. However, it remains unknown what type of tissue is produced after regenerative endodontics. The purpose of this study was to use blood clots and platelet-rich plasma (PRP) as scaffolds in regenerative endodontics under ideal conditions in a ferret model to examine the tissues generated within the root canals. METHODS: The pulps of 21 canine teeth from 7 young ferrets were extirpated using broaches without filing the canal walls. Bleeding was stimulated from the periapical tissues, and a blood clot was induced in the canal space to the level of the cementoenamel junction in 12 teeth. PRP was prepared and placed in the canals to the level of the cementoenamel junction in 9 teeth. The coronal access was sealed with mineral trioxide aggregate. Seven canines were not operated on and served as controls. Three months later, block sections including each canine and its surrounding tissues were removed for histologic evaluation. The tissues found in the canals of experimental teeth were compared with those in the control teeth. RESULTS: Almost all of the experimental teeth showed the presence of intracanal bonelike tissue. No evidence of dentinal wall thickening or apical narrowing was noted in the experimental teeth. CONCLUSIONS: In this experimental model, the use of either PRP or blood clots during regenerative endodontics leads to the formation of intracanal bonelike tissue without continual root maturation.

Dentina / Dentin

Si bien la dentina y la pulpa tienen marcadas diferencias en su composición y estructura, ambas están tan íntimamente ligadas por su origen embriológico, que cualquier cosa que afecte a la dentina lo hará sobre la pulpa y viceversa. El ejemplo más claro en ese sentido está dado por el líquido intersticial. Este líquido, semejante al plasma pero con menos proteínas, constituye una continuidad entre ambos tejidos y sus efectos hidrodinámicos son muy importantes, tanto en los estados fisiológicos como en los patológicos

Analysis of the contribution of nonresident progenitor cells and hematopoietic cells to reparative dentinogenesis using parabiosis model in mice.

INTRODUCTION: The aim of this study was to analyze the contribution of nonresident progenitor/stem cells and hematopoietic cells to reparative dentinogenesis. METHODS: Parabiosis was established between C57BL/6-TgN(ACTbEGFP)10sb/J transgenic mice (GFP+) and C57BL/6 wild-type mice (GFP-) to ensure blood cross-circulation between animals. Reparative dentinogenesis was stimulated by pulp exposures and capping on the first maxillary molar in the GFP- mice. Histologic sections of injured molars from GFP- mice were analyzed by epifluorescence microscopy to examine the contributions of GFP+ cells (nonresident progenitor cells and hematopoietic cells originating from GFP+ mice) to reparative dentinogenesis. RESULTS: GFP+ cells were detected in close association with reparative dentin formed at the site of pulp exposure in the maxillary first molars of the GFP- mice. CONCLUSIONS: The present study suggests the participation of the nonresident progenitor cells and hematopoietic cells in reparative dentinogenesis.

On the repair of the dentine barrier.

The overall aim of this thesis was to study some aspects of the repair of the dentine barrier, especially in conjunction with dental pulp capping. Understanding the events leading to the healing of the dentine and pulp, and hence successfully preserving the vitality and functions of the tooth, would lead to a scientific basis for a less invasive treatment of pulp exposures than performing root canal treatments. The surfaces of the body have physiological barrier functions aimed at protecting the body from external noxious agents. In the tooth, the odontoblasts, which line the outermost part of the pulp and are responsible for the formation of dentine, play a central role in the barrier function and thus in the defence mechanisms of the tooth. The micro-organisms in the caries lesion can reach the pulp via the dentinal tubules. However, the barrier function helps to prevent microbial invasion and thereby avoid deleterious inflammation and subsequent necrosis of the pulp. Dentine repair is an important part of the barrier function. There are however doubts as to whether the repair also leads to restitution of the function and the ability to withstand bacterial influx over the longer term. Pulp capping is a treatment method used when the pulp has been exposed in order to stimulate healing of the pulp and dentine. The evidence for repair of the dentine after pulp capping in humans has been studied by means of a systematic review. The focus of the literature search was studies performed in humans where hard tissue formation had been studied with the aid of a microscope. We concluded, based on the limited evidence available, that calcium hydroxide based materials but not bonding agents promote formation of a hard tissue bridge. Scientific evidence was lacking as to whether MTA was better than calcium hydroxide based materials in this regard. A gel (Emdogain Gel) containing amelogenin, known to be involved in dentinogenesis, was evaluated with regard to formation of hard tissue in a clinical study. A greater amount of hard tissue was formed after application of the gel compared to the control. Characterization of the tissue concluded it to be dentine, based on its content of type 1 collagen and dentine sialoprotein, although it was not formed as a continuous bridge covering the pulp wound. Beneath a deep caries lesion an important part of the barrier function is the odontoblasts' response to bacteria with the formation of new dentine. A cell model with odontoblasts was used to study the effects of clinical isolates from a deep carious lesion on their viability and production of type 1 collagen, the major component of the dentine in the early stages of its formation. There were bacteria that negatively affected the viability of the odontoblast-like cells and different bacteria varied in their effects on type 1 collagen production, suggesting that some bacteria may have a direct influence on the odontoblasts' ability to form dentine. In summary; Emdogain Gel initiated dentine formation, though not in a form that could constitute a barrier and there are indications that bacteria may differentially affect the odontoblasts' ability to repair the dentine barrier.

Dentin regeneration using deciduous pulp stem/progenitor cells.

Reparative dentin formation is essential for maintaining the integrity of dentin structure during disease or trauma. In this study, we investigated stem/progenitor cell-based tissue engineering for dentin regeneration in a large animal model. Porcine deciduous pulp stem/progenitor cells (PDPSCs) were mixed with a beta-tricalcium phosphate (ß-TCP) scaffold for dentin regeneration. Different concentrations of PDPSCs were tested to determine the optimal density for dentin regeneration. Aliquots of 5×10(5) PDPSCs in 1 mL resulted in the highest number of cells attached to the scaffold and the greatest alkaline phosphatase activity. We labeled PDPSCs with green fluorescent protein (GFP) and used the optimal cell numbers mixed with ß-TCP to repair pulp chamber roof defects in the premolars of swine. Four weeks after transplantation, GFP-positive PDPSCs were observed in PDPSC-embedded scaffold constructs. At 16 weeks after transplantation, the PDPSCs mixed with ß-TCP significantly regenerated the dentin-like structures and nearly completely restored the pulp chamber roof defects. This study demonstrated that the PDPSC/scaffold construct was useful in direct pulp-capping and provides pre-clinical evidence for stem/progenitor cell-based dentin regeneration.

Hypoxia promotes mineralization of human dental pulp cells.

INTRODUCTION: Dental pulp can be exposed to hypoxic conditions in case of trauma or inflammation. Dental pulp cells (DPCs) have mineralization potential, which plays a key role in pulp repair and reparative dentinogenesis process. Little information is available about DPC mineralization in hypoxic condition. The purpose of this study was to assess the influence of hypoxia on DPC mineralization to pave the way for a better understanding of dental pulp regeneration and reparative dentin formation. METHODS: Human DPCs were obtained by using tissue explant technique in vitro and cultured in normoxia (20% O(2)) or hypoxia (5% O(2)). Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell mineralization was assessed by von Kossa staining and alizarin red S staining. Important mineral genes such as osteocalcin (OCN), dentin matrix acidic phosphoprotein-1 (DMP-1), bone sialoprotein (BSP), and dentin sialophosphoprotein (DSPP) were determined by real-time polymerase chain reaction. RESULTS: Cell viability of DPCs increased more in hypoxia than in normoxia from day 3 to day 5. Von Kossa staining and alizarin red S staining showed DPCs in hypoxia had higher mineralization activity than in normoxia. Expression of mRNAs for OCN, DMP-1, BSP, and DSPP was greater in hypoxia than in normoxia. CONCLUSIONS: These results imply that hypoxia promotes DPC mineralization.

Pulp healing and regeneration: more questions than answers.

Differences between pulp repair and regeneration guide different strategic options. After mild carious dentin lesions, odontoblasts and Hoehl's cells are implicated in the formation of reactionary dentin. Reparative dentin formation and/or pulp regeneration after partial degradation is under the control of pulp progenitors. A series of questions arise from recent researches on tissue engineering. In this series of questions, we compare the therapeutic potential of pluripotent embryonic and adult stem cells, both being used in cell-based dental therapies. Crucial questions arise on the origin of stem cells and the localization of niches of progenitors in adult teeth. Circulating progenitor cells may also be candidate for promoting pulp regeneration. Then, we focus on strategies allowing efficient progenitors recruitment. Along this line, we compare the potential of embryonic stem cells versus adult stem cells. Re-programming adult pulp cells to become induced pluripotent stem cells constitute another option. Genes, transcription factors and growth factors may be used to stimulate the differentiation cascade. Extracellular matrix molecules or some bioactive specific domains after enzymatic cleavage may also contribute to the formation of an artificial pulp and ultimately to its mineralization.

Pulpal progenitors and dentin repair.

Mesenchymal stem cells are present in the dental pulp. They have been shown to contribute to dentin-like tissue formation in vitro and to participate in bone repair after a mandibular lesion. However, their capacity to contribute efficiently to reparative dentin formation after pulp lesion has never been explored. After pulp exposure, we have identified proliferative cells within 3 zones. In the crown, zone I is near the cavity, and zone II corresponds to the isthmus between the mesial and central pulp. In the root, zone III, near the apex, at a distance from the inflammatory site, contains mitotic stromal cells which may represent a source of progenitor cells. Stem-cell-based strategies are promising treatments for tissue injury in dentistry. Our experiments focused on (1) location of stem cells induced to leave their quiescent state early after pulp injury and (2) implantation of pulp progenitors, a substitute for classic endodontic treatments, paving the way for pulp stem-cell-based therapies.