The Four Pillars for Successful Regenerative Therapy in Endodontics: Stem Cells, Biomaterials, Growth Factors, and Their Synergistic Interactions.

Endodontics has made significant progress in regenerative approaches in recent years, thanks to advances in biologically based procedures or regenerative endodontic therapy (RET). In recent years, our profession has witnessed a clear conceptual shift in this therapy. RET was initially based on a blood clot induced by apical bleeding without harvesting the patient's cells or cell-free RET. Later, the RET encompassed the three principles of tissue engineering, stromal/stem cells, scaffolds, and growth factors, aiming for the regeneration of a functional dentin pulp complex. The regenerated dental pulp will recover the protective mechanisms including innate immunity, tertiary dentin formation, and pain sensitivity. This comprehensive review covers the basic knowledge and practical information for translational applications of stem cell-based RET and tissue engineering procedures for the regeneration of dental pulp. It will also provide overall information on the emerging technologies in biological and synthetic matrices, biomaterials, and signaling molecules, recent advances in stem cell therapy, and updated experimental results. This review brings useful and timely clinical evidence for practitioners to understand the challenges faced for a successful cell-based RET and the importance of preserving or reestablishing tooth vitality. The clinical translation of these current bioengineering approaches will undoubtedly be beneficial to the future practice of endodontics.

Histologic response and tenascin and fibronectin expression after pulp capping in pig primary teeth with mineral trioxide aggregate or calcium hydroxide.

This study evaluated the histological response and the expression of tenascin (TN) and fibronectin (FN) after pulp capping with mineral trioxide aggregate (MTA) or calcium hydroxide (CH). Class V cavities and pulp exposure were performed in 40 primary pig teeth. The pulps were capped with either MTA or CH, and the cavities were sealed with resin-modified glass ionomer cement. CH was used as a control. Seven and 70 days posttreatment, the animals were sacrificed and teeth were prepared for histological evaluation. TN and FN were detected by immunostaining. A severe inflammatory response was observed after 7 days in the CH group (p<0.043), while in the MTA group, a mild response was observed. Similar reparative dentin deposition was observed after 70 days for both groups (p<0.005). The expression of FN and TN was similar for both groups in the two periods evaluated. TN and FN were expressed during pulp reparative events, independently of the capping material.

Adhesive resin induces apoptosis and cell-cycle arrest of pulp cells.

The application of an adhesive resin near or directly over the pulp was shown to induce pulp inflammation and lack of dentin regeneration. We hypothesize that the absence of dentin bridging is due to adhesive-resin-induced apoptosis of cells responsible for pulp healing and dentin regeneration. Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), or macrophages (RAW 264.7) were exposed to SingleBond polymerized for 0-40 seconds. Annexin V and propidium iodide assays demonstrated that SingleBond induced apoptosis of MDPC-23, OD-21, and macrophages. The proportion of apoptotic cells was dependent on the degree of adhesive resin polymerization. Adhesive-resin-induced death of pulp cells was associated with activation of the pro-apoptotic cysteine protease Caspase-3. Interestingly, most cells exposed to adhesive resin that did not undergo apoptosis showed cell-cycle arrest. We conclude that an adhesive resin induces apoptosis and cell-cycle arrest of cells involved in the regeneration of the dentin-pulp complex in vitro.

MAPK signaling is required for LPS-induced VEGF in pulp stem cells.

Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR- 4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC zeta) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis.

Angiogenic signaling triggered by cariogenic bacteria in pulp cells.

The inflammation observed in the dental pulp of teeth with deep caries lesions is characterized by a significant increase in blood vessel density. It is known that lipoteichoic acid (LTA) from Gram-positive cariogenic bacteria induces expression of vascular endothelial growth factor (VEGF) in dental pulp cells. The hypothesis underlying this study was that LTA induces VEGF expression in dental pulp cells through TLR2 and PI3k/Akt signaling. Odontoblast-like cells (MDPC-23) and undifferentiated pulp cells (OD-21) were exposed to LTA from Streptococcus sanguis, and the role of TLR2, PI3K/Akt, and IKK signaling in LTA-induced VEGF expression was evaluated. These studies demonstrated that TLR2 signaling through the PI3K-Akt pathway is necessary for LTA-induced VEGF expression in pulp cells. In contrast, inhibition of IKK signaling did not prevent VEGF up-regulation in response to LTA. Understanding signaling pathways triggered by cariogenic bacteria may reveal novel therapeutic targets for the clinical management of pulpitis.

Effects of VEGF and FGF2 on the revascularization of severed human dental pulps.

The long-term outcome of replanted avulsed permanent teeth is frequently compromised by lack of revascularization, resulting in pulp necrosis. The purpose of this study was to evaluate the effects of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) on the revascularization of severed human dental pulps. Tooth slices were prepared from non-carious human molars and treated with 0-50 ng/mL rhVEGF(165) or rhFGF-2 for 7 days in vitro. Both angiogenic factors enhanced pulp microvessel density compared with untreated controls (p < 0.05). Tooth slices were also treated with 0 or 50 ng/mL rhVEGF(165) for one hour prior to implantation into the subcutaneous space of immunodeficient mice. Treatment with rhVEGF(165) increased pulp microvessel density in vivo (p < 0.05). These results demonstrate that rhVEGF(165) enhanced neovascularization of severed human dental pulps and suggest that topical application of an angiogenic factor prior to replantation might be beneficial for the treatment of avulsed teeth.

Prostaglandin E production and viability of cells cultured in contact with freshly mixed endodontic materials.

AIM: To determine whether commonly used endodontic sealers could either induce or increase the release of prostaglandin E2 (PGE2) when in contact with cell types found in the periapical tissues. METHODOLOGY: Freshly mixed samples of Roth 801 sealer, Sealapex and ProRoot mineral trioxide aggregate (MTA) were placed in contact with cultured macrophages and fibroblasts for 24 h. The supernatant from the cultures was assayed for PGE2 using enzyme-linked immunosorbent assay. Cell viability counts were made. As a positive control, similar cultures were also exposed to lipopolysaccharide and the supernatant analysed for PGE2. Data were compared by anova. RESULTS: The three materials examined in these experiments did not stimulate increased PGE2 release from either of the cell lines. In control cultures, lipopolysaccharide increased PGE2 release from macrophages but not from fibroblasts. Viability counts revealed that, whilst Roth 801 sealer caused some cell death in both fibroblasts and macrophages, Sealapex led to cell death only in the macrophage cultures. ProRoot MTA did not lead to statistically significant cell death in either culture. CONCLUSIONS: Under 24-h culture conditions, the three freshly mixed test materials did not increase directly either production or release of PGE2 from either macrophages or gingival fibroblasts. Roth 801 decreased cell viability counts for both fibroblasts and macrophages. Sealapex decreases macrophage viability. ProRoot MTA did not affect viability in either cell line.

Effect of ProRoot MTA mixed with chlorhexidine on apoptosis and cell cycle of fibroblasts and macrophages in vitro.

AIM: To compare the percentage of apoptotic cells and the cell cycle profile of fibroblasts and macrophages exposed to either ProRoot mineral trioxide aggregate (MTA) mixed with chlorhexidine (CHX), or exposed to ProRoot MTA mixed with sterile water. METHODOLOGY: Mouse gingival fibroblasts or mouse macrophages were seeded in six-well plates and allowed to attach overnight. Freshly mixed or set (allowed to dry for 24 h) specimens of tooth-coloured (white) ProRoot MTA were prepared with 0.12% CHX gluconate (MTA/CHX) or with sterile water (MTA/H2O). The cells were exposed for 24 h to the MTA specimens, which were placed over permeable membrane inserts to avoid direct contact with the cells. Untreated cells served as controls. Propidium iodide staining followed by flow cytometry was used to evaluate the effects of ProRoot MTA on cell apoptosis and cell cycle. Statistical analyses were performed by one-way anova followed by post-hoc tests with the use of the SigmaStat 2.0 software, and significance was determined at P < or = 0.05. RESULTS: MTA specimens containing CHX induced apoptosis of macrophages and fibroblasts (P < 0.05). In contrast, no change in the proportion of apoptotic cells was observed when sterile water was used to prepare the specimens (P > 0.05). Cell cycle analysis showed that exposure to MTA/CHX decreased the percentage of fibroblasts and macrophages in S phase (DNA synthesis) as compared with exposure to MTA/H2O (P < 0.05). CONCLUSION: This in vitro study demonstrated that the substitution of CHX for sterile water in MTA increases its cytotoxicity. This suggests that the potentially beneficial antimicrobial effect of CHX may be accompanied by an increase in the cytotoxicity of the resulting MTA-based material.

Asociación de paladar fisurado y sindrome de Klippel-Feil / Cleft palate and Klippel-Feil syndrome: a case report

El síndrome de Klippel-Fiel, Distrofia Brevicollis Congénita, Sinostósis Congénito Cervical o Fusión de las Vértebras Cervicales, consiste en la fusión de, al menos, dos de las siete vértebras del cuello. Se caracteriza por la presencia de una tríada clásica compuesta por cuello corto, baja inserción de la línea del cabello y limitación de los movimientos del cuello. Pero además se puede acompañar de una serie de condiciones tales como: escoliosis, tortícolis, deformidad de Sprengel, malformaciones cardiovasculares, renales, auditivas y paladar fisurado entre otros. La aparición del paladar fisurado en pacientes con el síndrome de Klippel-Feil se presenta entre el 5 y el 10 de los casos. Como posibles causas primarias de la falta de fusión del paladar se han reportado las anomalías de la columna cervical superior y alteraciones en la base del cráneo, defectos que impiden la fusión de las dos apófisis horizontales de los maxilares. Con este artículo se reporta el caso de una niña de una niña de 10 años que presenta Síndrome de Klippel-Feil asociado con la deformidad de Sprengel y Paladar fisurado.

Kippel-Feil syndrome is a condition characterized by shortness of the neck resulting from reduction in the number of vertebrae or the fusion of multiple hemivertebrae into one osseous mass. It is characterized by a classic triad: short neck, low hair insertion line and limited neck movements. There are some associated conditions that could be presents like scoliosis, wryneck, Sprengel deformation, cardiovascular, kidney, hearing troubles and fissured palate. Kippel-Feil syndrome had been reported as an important cause of about 5 to 10 of fissured palate because skull basal lesions and spine cervical upper alterations could be the primary troubles for the tow part of the palate bone union. This article presents a case of Klippel-Feil syndrome, Sprengel deformity and clef palate associated in a ten years old girl.