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Objective: The present study aims to investigate whether reparative dentinogenesis could be guided at central pulpal sites or at a distance from the amputated pulp of miniature pig teeth, by using set calcium silicate-based carriers containing human recombinant bioactive molecules., Design: Pulp exposures were performed in 72 permanent teeth of 4 healthy miniature swine. The teeth were capped with pre-manufactured implants of set calcium silicate-based material containing BMP-7, TGFβ1 or WnT-1, for 3 weeks. Conical-shaped intrapulpal implants were exposed in the central pulp core, while disc-shaped extrapulpal implants were placed at a distance from the amputated pulp. Implants without bioactive molecules were used as controls. Thickness and forms of new matrix mineralized deposition were assessed histologically at post-operative periods of 3 weeks by light microscopy., Results: Intrapulpal applications: Calcified structures composed of osteodentine were found in contact with the BMP-7 implants. An inhomogeneous calcified tissue matrix was found around the WnT-1 carriers. A two-zone calcified structure composed of osteodentine and a thicker tubular matrix zone was seen at the TGFβ1 carrier-pulp interface. Extrapulpal applications: The space between WnT-1 implants and pulp periphery had been invaded by soft tissue with traces of calcified foci. Thick calcified structures composed of osteodentine were found surrounding pulp exposure sites in response to application of BMP-7. Spindle-shaped cells associated with atubular calcified matrix or elongated polarized cells associated with tubular dentine-like matrix were found along the cut dentinal walls of the TGFβ1 group., Conclusion: The present experiments indicated that set calcium silicate could be used as carrier for biologically active molecules. TGFβ1 was shown to be an effective bioactive molecule in guiding tertiary dentine formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fluoride has been used for decades, either systemically or topically, to prevent dental caries. The purpose of this study was to clarify the effects of low concentrations of fluoride on proliferation, differentiation and extracellular-matrix synthesis in normal human dental pulp cells (DP-1 and DP-2) in vitro. The effects were compared with those on a human osteoblastic osteosarcoma cell line, TE-85. Fluoride at micromolar concentrations significantly and dose-dependently stimulated [3H]thymidine incorporation into DNA in DP-1, DP-2 and TE-85 cells, with optimal effects around 50 microM, by 127 +/- 7%, 124 +/- 0.6% and 152 +/- 13.4%, respectively. To assess the potential influence of fluoride on cell differentiation, the effects of mitogenic concentrations on alkaline phosphatase activity were measured. Fluoride significantly increased the enzyme's activity in DP-1 and TE-85 by 177 +/- 12% and 144 +/- 12.3%. To evaluate the effect on extracellular-matrix synthesis, the synthesis of type I collagen was indirectly determined by an assay of procollagen type I c-peptide production. Fluoride significantly increased that production by 150 +/- 8.7% in TE-85, but not in either DP-1 or DP-2. These observations suggest that fluoride, if used at low concentrations, could be a useful therapeutic agent where increased regeneration of dentine is desired, such as after pulp amputation, by stimulating the proliferation and differentiation of the dental pulp cells.

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 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.

Recombinant human osteogenic protein-1 (OP-1) when applied to freshly cut dentine stimulated significantly more reparative dentine than calcium hydroxide paste in permanent monkey teeth. The response to OP-1 was dependent upon the concentration applied to the tooth as a cavity liner as well as the thickness of the residual dentine. The response to calcium hydroxide was similarly dependent upon the thickness of the residual dentine. These data suggest that dental pulps contain cells, including perhaps mature odontoblasts, responsive to OP-1. Therefore OP-1 may be useful in the therapeutic induction of reparative dentine formation.

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 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.

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.

Recombinant human bone morphogenetic protein (BMP)-2, BMP-4 and transforming growth factor (TGF)-beta 1 combined with collagen matrix as a carrier were examined for their effects on pulp regeneration and dentine formation. Seventy days after implantation of 2 micrograms of BMP-2, mineralized osteodentine-like tissue containing embedded osteodentinocytes was seen in the cavity. Unmineralized fibrous tissue and pulp-like loose connective tissue were also found in the same cavity. In teeth implanted with 660 ng of BMP-2 only unmineralized fibrous and pulp tissues were seen. In teeth with 220 ng of BMP-2 or collagen alone, pulp tissue was seen. It is therefore likely that the cavity fills with pulp tissue and that spindle-shaped cells elaborate extracellular matrix that mineralizes to be osteodentine in a dose-dependent manner. Similar osteodentine was seen in teeth implanted with 4 micrograms of BMP-4 and collagen. No distinct tubular dentine was formed, unlike an earlier experiment in which BMP-2 or -4 was implanted with enriched, inactivated dentine matrix. These findings suggest that both BMP-2 and -4 induce osteodentine formation if combined with collagen matrix; some other matrix component present in inactivated dentine matrix might be essential for further differentiation into odontoblasts. In teeth implanted with TGF-beta 1, the carrier collagen remained in the cavity and little pulp tissue proliferation was seen, suggesting a possible inhibitory effect of TGF-beta 1 in pulp regeneration. It is likely that the response to growth and differentiation factors is dependent on the state of differentiation of pulp cells.