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The clinical management of traumatised permanent anterior teeth is complicated by the unreliability of current methods of assessing dental pulp vitality. This study investigated the reliability of laser doppler flowmetry as a method of assessing the pulpal status of traumatised permanent anterior teeth. The study found that although laser doppler flowmetry could record pulpal blood flow, non-pulpal signals were an unavoidable component of the signal obtained. The optimum non-pulpal/pulpal flux signal ratio was obtained when the laser doppler flowmeter probe was supported by a two-stage elastomeric impression jig, perpendicular to the tooth surface, at a distance of between 2-3 mm from the gingival margin. This method resulted in only 10% of the flux signal obtained from a vital dental pulp being of non-pulpal origin. Other recording methods reported in the literature were found to have between 15-45% of the flux signal of non-pulpal origin. Flux signals from vital permanent anterior teeth were found to have two characteristics which distinguished them from flux signals originating from non-vital anterior teeth; a minimum Mean Flux value of 7 perfusion units (P.U.) and a rhythmical variation in the flux signal (termed Slow Wave Vasomotion) with a frequency of between 1-10 cycles a minute, and a mean amplitude of at least 1.6 P.U. Diagnostic criteria based on these variables were used to discriminate between flux signals from 84 vital anterior teeth and 67 non-vital anterior teeth, and were found to have a diagnostic sensitivity and specificity of 1.0. All currently used methods of assessing dental pulp vitality were found to be less reliable (have a lower sensitivity and specificity) than laser doppler flowmetry. The sensitivity and specificity of the majority of the tests remained relatively unchanged whether applied to traumatised or untraumatised teeth. The exception were tests of pulpal sensibility, which showed a large fall in specificity when applied to traumatised incisors as compared with untraumatised incisors. For example, it was found that vital luxated incisors were more likely than not to fail to respond positively to sensibility testing. Although laser doppler flowmetry was found to have a sensitivity and specificity in excess of other pulpal diagnostic tests in current use, it was also found that the pulpal status of teeth was not always determined at the time of injury but could change subsequent to the injury. Both pulpal necrosis and pulpal revascularisation were possible late consequences of dental trauma, occurring months or even years following the injury. Therefore, the reliable diagnosis of pulpal vitality provided by laser doppler flowmetry should only be regarded as an aid to treatment planning the dental care of traumatised incisors. It would appear, however, that the technique of laser doppler flowmetry has potential in increasing current knowledge regarding the pathogenesis of dental pulp disease following traumatic injury.

Direct pulp tissue grafting has been proposed in the past to void the tedious task of laboratory culturing. Migration of cells from dental pulp tissue explantation have shown multi differentiation potential in vitro like DPSC. These cells were named MP-MSC (minced pulp derived mesenchymal stem cells) by our group. Establishing that intact pulp tissue can be a potential source of dental pulp derived stem cells, we further wanted to investigate the possibility of pulp tissue banking. Cord tissue banking has gained popularity and success in the past decade and has shown successful in medicine. Out study demonstrated that cryopreserved pulp tissue has a potential to sustain viable cells and these cells additionally showed that they can maintain the multi potent differentiation in vitro like unfrozen tissue as well as expression of odontogenic marker. Additionally, our data also suggests that the perivascular niche can be maintained intact in the minced pulp tissue as well as frozen minced pulp tissue, suggesting the mesenchymal origin of the cells. Ex vivo tissue engineering showed migration of cells and attachment to dentin surface in presence of scaffold from frozen tissue is similar to unfrozen tissue. Additionally, RT-PCR data from our study shows that frozen pulp tissue derived cells showed differentiation and expression of ALP, OCN, DSPP markers suggesting that they maintained the differentiation potential after cryopreservation.

This study evaluated the potential toxicity, genotoxicity, and cellular uptake of various sizes and concentrations of chitosan (CS) nanoparticles cultured with normal human dental pulp cells. Normal human dental pulp cells (hDPCs) were derived from human dental pulp tissues and cultured with (50–67) nm and (318–350) nm CS-nanoparticles in concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL as study groups and 0 mg/mL as a control group for time intervals of 16 hours, 24 hours, 3 days, 7 days and 14 days. Attachment efficiency and proliferation rate were assessed by measuring the optical density of crystal violet-stained cells. Cell viability was determined by the activity of mitochondrial dehydrogenase enzymes. Genotoxicity was assessed using the cytokinesis-block micronucleus method and by measuring the fluorescent intensity of phosphorylated H2AX nuclear foci. Cellular uptake was determined by tagging chitosan nanoparticles with FITC stain and then measuring the fluorescence intensity of FITC-tagged chitosan nanoparticles using a spectrophotometer. Statistical analysis was performed using chi-square, one-way ANOVA, and post-hoc Tukey tests. All concentrations of the (50–67) nm group significantly reduced attachment efficiency in comparison with control (P< 0.01) and with (318–350) nm group (p

The development of pulp capping agents has been instrumental in promoting reparative dentin formation and facilitating pulpal repair in response to pulp exposure during extensive caries excavation. Although calcium hydroxide has been used as the gold standard for pulp capping since decades, the development of mineral trioxide aggregate (MTA) and its derivatives, also known as hydraulic calcium-silicate cements (HCSCs) have ushered in a new wave of therapeutic pulp capping agents. Nonetheless, there is limited evidence about the pulpal toxicity and dentin regenerative capacity of these materials. In the current study, the effects of 4 of these HCSCs (PROROOT� MTA, TheraCal LC, EndoSequence BC RRM, Endo-Eze™ MTAFlow) along with the control groups, composite and UltraCal� XS (Dycal)on reparative dentin and dentinal bridge formation and periapical bone loss were evaluated radiographically and histologically in mice. Pulp exposure was induced bilaterally in maxillary first molars of C57/BL6 mice. The maxillae were harvested, fixed, and subjected to μCT scanning, three-dimensional volumetric analysis and Hematoxylin and Eosin staining (H &E) was done to evaluate the reparative potential of the pulp capping materials. Tartrate-Resistant Acid Phosphotase (TRAP) staining was performed, and osteoclasts were quantified. Among the five HCSCs used for pulp capping, only UltraCal� XS (Dycal) failed to induce reparative dentin and dentinal bridge formation, instead inducing periapical bone loss. Although TheraCal LC also induced periapical bone loss, significantly more teeth treated with UltraCal � XS (Dycal) developed periapical radiolucency’s. Histologically, dentinal bridge formation was observed in all HCSCs except UltraCal� XS (Dycal). Histologically, TRAP+ osteoclasts were absent in all the HCSCs. Although some samples of Theracal LC showed a few TRAP+ osteoclasts, significantly more were seen in the UltraCal � XS (Dycal) group. These findings indicate that with the exception of UltraCal� XS (Dycal), all HCSC derivative materials evaluated are comparatively effective to the original PROROOT� MTA in promoting reparative dentin formation and pulpal repair.

BACKGROUND: Thymoquinone (TQ) is one of the most active ingredients of Nigella sativa seed. TQ has a variety of pharmacologic properties including possess antinociceptive, antibacterial, anti-inflammatory, anti-oxidative activities and osteogenic effects on bone cells. Because of these properties, TQ might play an important role in odontogenic effects on human dental pulp cells and be used as a pulp capping material. PURPOSE: This study evaluates the effect of TQ on the attachment efficiency, proliferation, and odontogenic differentiation of Human Dental Pulp Cells (HDPC's). The effect of TQ on odontogenic differentiation was also determined by evaluating alkaline phosphatase (ALP) activity and Dentin Sialoprotein (DSP) expression. METHODS: Human dental pulp cells were cultured in triplicate using growth media with various TQ concentrations: 5 μM, 10μM, 15μM, 30μM, and 0μM as a control group at 7 and 21 days. Crystal violet staining was used to determine cell attachment efficiency and cell proliferation. The proliferation rates were normalized to cell numbers of each group at 16 hours. Cell differentiation was assessed by evaluating ALP activity and DSP expression. The data were normalized on per million cells basis. Univariate analyses including ANOVA and Student’s t-test were conducted. RESULTS: Higher cell attachment efficiency was shown in all TQ groups at 16 hours (P

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp (DP) and periodontal ligament (PDL) tissues using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) and compared their capacity to undergo multilineage differentiation. Fluorescence-Activated Cell Sorting (FACS) revealed that 27.3% of DPCs were CD51+/CD140α+, 10.6% were CD271+, and 0.3% were STRO-1+/CD146+; whereas 24% of PDLCs were CD51+/CD140α+, 0.8% were CD271+, and 2.4% were STRO-1+/CD146+. Sorted cell populations were further assessed for their multipotent properties by inducing odonto/osteogenic, chondrogenic, and adipogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into odonto/osteogenic and chondrogenic lineages but with varying degrees. CD271+ DMSCs demonstrated the greatest odonto/osteogenic potential with strong induction of odonto/osteogenic and periodontal markers such as DLX5, RUNX2, BGLAP, DMP1, DSPP, and PLAP-1. Based on these results, the role of CD271 in odontogenic differentiation was further evaluated. CD271 is one of the receptors which Nerve Growth Factor (NGF) exerts its biological effects through. Although NGF is known for its role in the the development and maintenance of the nervous system, it’s capability to promote odontogenic differentiation has made it an interesting candidate for its role as a mineralizing agent in regenerative therapies. We found that exogenous treatment of NGF further increased odonotogenic potential of CD271+ DMSCs from DP with induction of odontogenic markers DLX5, RUNX2, and BGLAP, and increased MAPK, AKT, MTOR, and NF-B signaling pathways in a time dependent manner. Furthermore, knockdown of tropomyosin trkA tyrosine kinase receptor (trkANGFR) and low affinity nerve growth factor receptor (p75NTR/CD271/LNGFR) decreased odontogenic differentiation of CD271+ DMSCs. Our study provides important insights into the use of DMSCs and growth factors for regenerative therapies in dentistry, and improves our understanding of the molecular mechanisms involved in odontogenic differentiation

The aim of this study is to understand the role of immune regulators such as Natural Killer Cells and Monocytes with Dental Pulp Stem Cells (DPSCs). In this study we will demonstrate that DPSCs are very sensitive to Natural Killer cells attack and will undergo cell-mediated lysis. However Monocytes are able to increase the survival of DPSCs and rescue DPSCs from Natural Killer cells cell-mediated lysis. We further analyze the different subpopulation of Monocytes CD16- and CD16+ cell surface receptors to provide an immunotherapeutic tool for tissue regeneration and repair of DPSCs in an event of a stress to a cell.

High mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is released into the extracellular space and promotes inflammation. HMGB1 binds to related cell signaling transduction receptors, including receptor for advanced glycation end products (RAGE), which actively participate in vascular and inflammatory diseases. The aim of this study was to examine whether RAGE and HMGB1 are involved in the pathogenesis of pulpitis and investigate the effect of Prevotella intermedia (P. intermedia) lipopolysaccharide (LPS) on RAGE and HMGB1 expression in odontoblast-like cells (OLC-1). RAGE and HMGB1 expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. Upregulated expression of RAGE was observed in odontoblasts, stromal pulp fibroblasts-like cells, and endothelial-like cell lining human pulpitis tissue. Strong cytoplasmic HMGB1 immunoreactivity was noted in odontoblasts, whereas nuclear HMGB1 immunoreactivity was seen in stromal pulp fibroblasts-like cells in human pulpitis tissue. LPS stimulated OLC-1 cells produced HMGB1 in a dose-dependent manner through RAGE. HMGB1 translocation towards the cytoplasm and secretion from OLC-1 in response to LPS was inhibited by TPCA-1, an inhibitor of NF-κB activation. These findings suggest that RAGE and HMGB1 play an important role in the pulpal immune response to oral bacterial infection.

Abstract: Traumatic dental injuries are very common in the first and second decades of life. Trauma, if severe, could result in irreversible changes such as root resorption and/or partial or complete pulp obliteration by hard tissue. Dental pulp calcific metamorphosis is the rapid calcification of the pulp soft tissue in response to trauma. It is characterised by the deposition of dentin-like and bone-like tissues inside the dental pulp. Total avulsion of teeth represents almost 16% of dental traumatic injuries where the replantation of teeth back into their socket is the first treatment of choice. Inflammatory cells and osteoclasts were reported in teeth replantation cases, highlighting the role of inflammation in this type of calcification. Accordingly, the hypothesis of the current research was that dental pulp calcification and inflammation are closely integrated mechanisms. The immuno-histochemical localisation of a calcification molecule, dentin matrix protein (DMP-1), in pulp inflammation was performed. This was followed by determining its possible role in mediating inflammation by testing its induction of interleukin-6 (IL-6) and IL-8 expression in pulp fibroblasts. This proinflammatory effect is enhanced using lipopolysaccharide (LPS). The inhibitor of p38 mitogen activated protein kinase (p38MAPK) (SB-203580) blocked this effect. Osteopontin (OPN) and osteocalcin (OCN) were also examined for their expression in pulp inflammation using western blot and immuno-histochemistry respectively. Vascular endothelial growth factor (VEGF) increased in response to OPN stimulation of pulp cells. DMP-1 induced the expression of OPN, OCN, alkaline phosphatase (AP) and VEGF. p38MAPK inhibitor decreased DMP-1- induced OPN, AP and VEGF to their normal expression. Recombinant human DMP-1 showed marked calcification of ferret dental pulp chambers and DMP-1 was localised at 2 and 6 weeks following the replantation of ferret premolars. In vivo blocking of the inflammatory effect of DMP-1 using p38MAPK inhibitor significantly decreased calcification inside the dental pulp at 2 and 6 weeks post replantation. It is concluded that the DMP-1 is involved in the development of calcific metamorphosis partly through its pro inflammatory effect. DMP-1 also promoted pulp cells proliferation, pro-inflammation, and angiogenesis. Our data demonstrate a novel therapeutic strategy by which dental pulp inflammation and calcification are prevented at the same time.

Human teeth are very complex structures that are susceptible to many different pathologies due to poor dental health. Currently, there are many restorative methods to reestablish some of the function that teeth have, but the materials used in these methods all have drawbacks and cannot fully mimic the native teeth. Tissue engineering research groups have begun to explore regenerating bone or dental tissue using mesenchymal stem cells derived from the bone marrow. However, our group focuses on regenerating dental tissues using multipotent stem cells from dental pulp. Dental pulp stem cells (DPSCs) have shown similarities to bone marrow stem cells in in that they can differentiate into many cell types. Also, stem cells in general have shown that differentiation can be induced with microenvironmental factors such as growth factors and substrate properties. If enough is known about the cues that cells receive that induces differentiation, tissues could be engineered using the constructs and growth conditions necessary. To determine the effect of substrate stiffness on human DPSCs, cells were placed on polyacrylamide gels of varying stiffness and in varying growth factor conditions. The cells were then observed with light and confocal microscopy, and the amount of alkaline phosphatase (ALP) activity was measured. These tests gave an indication of growth and differentiation. It was seen that the growth patterns were different on the gels than they were on a glass control, but there was little difference between the two gels. Also, the growth factors did not appear to have a significant contribution to differentiation. Much work has been done to determine the effects of mechanical compression muscleoskeletal tissues, such as cartilage and bone. Dental tissues are also subject to loading throughout the day. Therefore, it was hypothesized that if dental pulp stem cells are compressed with pressure similar to that seen physiologically, it will induce differentiation to a bone or tooth-like lineage. To determine the effects of static compression on dental pulp stem cells, a custom compressive device was fabricated. The device was tested for usability and it was deemed acceptable for use. ALP assays were performed similar to the previous studies. Preliminary results showed that that after only 1 day of culture time, the compression did not have much of an effect on dental pulp stem cells, while it did have an effect on osteoblasts. More work is to be done to determine the effects of compressive forces on dental pulp stem cells.

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.

The purpose of this study is to evaluate the clinical and histological responses of human pulps to two medicaments acting synergisticly in a vital pulpotomy procedure. The drugs to be used are metacresol acetate (Cresatin) and calcium hydroxide (Pulpdent). Both drugs have been used independently in pulpotomy treatment and their effects have been well documented. Calcium hydroxide has long been known to stimulate the healing of the pulp, and to effect the formation of a reparative dentin bridge over the exposed area. The reported rates of success of this treatment has not always been favorable. This is not due to the drug, but rather diagnosis, technique or bacterial invasion ... [TRUNCATED]

The purpose of this study was to evaluate and compare the effectiveness of a number of widely used endodontic medicaments for the emergency pulpotomy procedure. The medicaments used were: camphorated parachlorophenol, Cresatin, eugenol, and formocresol. Physiologic saline was used as a control. In some cases, the patient's saliva was used to determine the response of the pulp to contamination during the operative procedure. [TRUNCATED]