Dimitrios Tziafas

Designing new treatment strategies in vital pulp therapy

OBJECTIVES The development of strategies in vital pulp therapy, which aim to maintain vitality and function of the dentine-pulp complex, represents a major focus of attention. Recent progress in understanding the molecular and cellular changes during tooth development and how they are mimicked during dental tissue repair offers the opportunity to now assess whether this knowledge can be exploited to design new treatment strategies in vital pulp therapy. DATA SOURCES AND STUDY SELECTION Current literature on the molecular and cellular basis of tooth development and dental tissue repair has been reviewed in the context of stimulating dentinogenic responses in the tooth together with pertinent published abstracts of relevant conferences and personal communications. Tissue events of direct relevance to clinical application for vital pulp therapy are discussed. CONCLUSIONS The involvement of growth factors and extracellular matrix molecules in signalling and regulating dentinogenic events during tooth development has been identified. During dental tissue repair, many of the processes are mimicked leading to responses of focal deposition of tertiary dentine at injury sites. The nature and specificity of these responses are determined in part by the extent of tissue injury. Traditional clinical strategies are capable of exploiting endogenous signalling molecules in the tissues to develop more effective treatment modalities. Application of exogenous signalling molecules offers opportunities for development of new therapies, although a number of delivery considerations must be addressed before these can be introduced into clinical practice.

The Future Role of a Molecular Approach to Pulp-Dentinal Regeneration

The ultimate goal of a regenerative pulp treatment strategy is to reconstitute normal tissue continuum at the pulp-dentin border, regulating tissue-specific processes of tertiary dentinogenesis. Experimental investigations in mature teeth have shown that a network of extracellular matrix molecules and growth factors signal tertiary dentinogenesis. Application of dentin matrix components or growth factors in deep dentinal cavities stimulated up-regulation of biosynthetic activity of primary odontoblasts (reactionary dentin formation). Pulp-capping studies with a broad spectrum of biological agents, including growth factors and extracellular matrix molecules, showed formation of osteodentin and/or tertiary dentinogenesis (reparative dentin formation). Promising biologically active substances should be subjected to careful evaluation in well-designed preclinical investigations as well as in long-term clinical trials before their introduction in clinical practice.

Differentiation Potential of Dental Papilla, Dental Pulp, and Apical Papilla Progenitor Cells

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.

Effects of recombinant basic fibroblast growth factor, insulin-like growth factor-II and transforming growth factor-β1 on dog dental pulp cells in vivo

The effects of recombinant basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-II and transforming growth factor (TGF)-beta 1 on dental pulp cells were investigated by light and transmission electron microscopy after their implantation for 1 and 3 weeks at central sites of mechanically exposed pulps in dog molar and canine teeth. The implants were Millipore filters that have been soaked with solutions containing 100 or 500 ng/ml of bFGF or IGF-II or 100 ng/ml of TGF-beta 1. Control filters were soaked with dog albumin. No changes in cell organization or matrix synthesis were seen after implantation of control filters. Groups of columnar, polarized cells with numerous mitochondria and Golgi elements or elongated cells unassociated with any matrix deposition were demonstrated after 1 or 3 weeks, respectively, in close proximity to the filters that had been soaked with bFGF solution; at a distance from these implants enhanced formation of an osteotypic matrix was seen beneath the exposure site. No particular response was found in close proximity to the filters that had been soaked with IGF-II solution after 1 or 3 weeks implantation but thick zones of osteodentine were found beneath the exposure site and at adjacent circumferential dentine sites. Numerous elongated, polarized cells with long cytoplasmic extensions invading the filter pores were consistently seen after 1 week in close proximity to the filters that had been soaked with TGF-beta 1 solution. After 3 weeks implantation of these filters, deposition of a tubular matrix surrounding the implants was seen in association with the highly elongated odontoblast-like cells, while enhancement of circumferential dentine formation was also found at adjacent peripheral sites. These experiments demonstrate that TGF-beta 1 when implanted for short term periods at central pulp sites exerted dentine-specific effects, inducing differentiation of odontoblast-like cells and stimulating primary odontoblasts. Implantation of bFGF and IGF-II did not result in reparative dentine formation, but did stimulate osteotypical matrix deposition at a distance from the implants.

SHORT-TERM DENTINOGENIC RESPONSE OF DOG DENTAL PULP TISSUE AFTER ITS INDUCTION BY DEMINERALIZED OR NATIVE DENTINE, OR PREDENTINE

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

Experimental Formation of Dentin-like Structure in the Root Canal Implant Model Using Cryopreserved Swine Dental Pulp Progenitor Cells

OBJECTIVES The purpose of the present study was to present histological and immunohistochemical evidence showing the regenerative capacity of swine dental pulp stem cells (S-DPSCs) seeded on organic or synthetic scaffolds and implanted as hybrid root implants in the jaw bone of minipigs. METHODS Immature permanent incisor teeth and unerupted premolars at the early root-forming stage were extracted from three 7-month-old minipigs, and mesenchymal stem/progenitor cells were isolated from dental pulp. Cells were cryopreserved in liquid nitrogen. A year later, new permanent incisor and premolar teeth were extracted; pulp tissue was removed; and pieces of root canals of the extracted teeth, containing collagen or Poly(lactic-co-glycolic acid) scaffolds seeded with the autologous cryopreserved DPSCs, were implanted into the fresh post-extraction socket of the mini pig jaw. The resulting constructs were harvested after 6 and 10 weeks and evaluated by histological and immunohistochemical analyses. RESULTS Six weeks postoperatively, the central canal space of the root implants showed degrading scaffold material. New extracellular matrix had been deposited in a polar predentin-like pattern on the canal dentinal walls by cuboidal nonpolarized cells. Ten weeks postoperatively, newly formed organic matrix had been consistently deposited on the canal walls. The presence of a continuous layer of polarized cells showing typical columnar morphology adjacent to the newly deposited organic matrix was evident. CONCLUSIONS The interactions of S-DPSCs with the dentin matrix of roots implanted in the jawbone of minipigs constitute a model to study in vivo organization and differentiation potential of DPSCs.

Experimental bacterial anachoresis in dog dental pulps capped with calcium hydroxide

The pulps of 36 permanent dog teeth were mechanically exposed and capped with Dycal, calcium hydroxide powder mixed with saline, or Teflon. At 2, 14, and 28 days postoperatively, nine teeth treated with the materials were extracted (treated control teeth): A suspension of streptococci was then injected intravenously. Twenty-four h later the dogs were killed and both the 27 treated teeth (experimental group) and 6 unoperated control teeth were removed in tissue blocks. Tissue sections were examined for the presence of bacteria, hard tissue formation, inflammatory cell response and necrosis. Bacteria were not observed in the unoperated and treated control teeth or in three of four teeth capped with Teflon for 29 days. In all the remaining specimens colonies of gram-positive cocci were found.

IMMUNOLOCALIZATION OF FIBRONECTIN DURING THE EARLY RESPONSE OF DOG DENTAL PULP TO DEMINERALIZED DENTINE OR CALCIUM HYDROXIDE-CONTAINING CEMENT

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.

Dentinogenic responses after direct pulp capping of miniature swine teeth with Biodentine.

INTRODUCTION The aim of this study was to evaluate pulpal responses after experimental direct pulp capping of mechanically exposed teeth with a new calcium silicate-based dentin replacement material. METHODS Thirty-four anterior and posterior teeth of 3 miniature swine were used. Class V or I cavities were prepared on the buccal or occlusal surfaces, respectively. Pulpal exposures were further performed using a round carbide bur 0.8 mm in diameter. Exposures were treated with white MTA Angelus (Angelus, Londrina, PR, Brazil) or Biodentine (Septodont, Saint Maur des Fosses, France), and the cavities were further restored with Biodentine. The pulpal tissue responses were histologically assessed at postoperative periods of 3 and 8 weeks. Data were statistically analyzed using the Kruskal Wallis and the Mann-Whitney U tests. RESULTS Inflammatory infiltration or pulp tissue necrosis was not found in any of the specimens. All teeth showed mineralized matrix formation in the form of a complete hard tissue bridge composed of osteodentin or osteodentin followed by a discontinuous or continuous reparative dentin zone. A significantly higher thickness of the hard tissue bridge was found in the group of teeth treated with Biodentine at both 3 and 8 weeks. A number of teeth, which were under root development at the onset of the experimental procedures, exhibited ectopic pulp calcification. CONCLUSIONS The application of both calcium silicate-based materials in direct contact with the mechanically exposed pulp of healthy miniature swine teeth led to pulp repair with complete hard tissue bridge formation. The thickness of hard tissue bridges was significantly higher after pulp capping with Biodentine.

Inductive effect of native dentin on the dentinogenic potential of adult dog teeth

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.