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Background The primary dentin, secondary dentin, and reactive tertiary dentin are formed by terminal differentiated odontoblasts, whereas atubular reparative tertiary dentin is formed by odontoblast-like cells. Odontoblast-like cells differentiate from pulpal stem cells, which express the neural stem cell markers nestin, S100β, Sox10, and P0. The denticle (pulp stone) is an unique mineralized extracellular matrix that frequently occurs in association with the neurovascular structures in the dental pulp. However, to date, the cellular origin of denticles in human dental pulp is unclear. In addition, the non-collagenous extracellular dentin matrix proteins dentin matrix protein 1 (DMP1), dentin sialoprotein (DSP), and dentin phosphoprotein (DPP) have been well characterized in the dentin matrix, whereas their role in the formation and mineralization of the denticle matrix remains to be clarified. Methods To characterize the formation of denticle, healthy human third molars (n = 59) were completely sectioned and evaluated by HE staining in different layers at 720 µm intervals. From these samples, molars with (n = 5) and without denticles (n = 8) were selected. Using consecutive cryo-sections from a layer containing denticles of different sizes, we examined DMP1, DSP, and DPP in denticle lining cells and tested their co-localizations with the glial stem cell markers nestin, S100β, Sox10, and P0 by quantitative and double staining methods. Results DMP1, DSP and DPP were found in odontoblasts, whereas denticle lining cells were positive only for DMP1 and DSP but not for DPP. Nestin was detected in both odontoblasts and denticle lining cells. S100β, Sox10, and P0 were co-localized with DMP1 and DSP in different subpopulations of denticle lining cells. Conclusions The co-localization of S100β, Sox10, and P0 with DMP1 and DSP in denticle lining cells suggest that denticle lining cells are originated from glial and/or endoneurial mesenchymal stem cells which are involved in biomineralization of denticle matrix by secretion of DMP1 and DSP. Since denticles are atubular compared to primary, secondary, reactionary tertiary dentin and denticle formed by odontoblasts, our results suggest that DPP could be one of the proteins involved in the complex regulation of dentinal tubule formation.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the &alpha, 1- and &beta, 1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the &alpha, 1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both &alpha, 1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α1β1, α2β1) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α1-, β1-, and α2-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α1-, β1-, and α2-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α1-, β1-, and α2-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization.Decalcified free-floating sections of rat molars with PDL were incubated using total (t)-ERK1/2 and phosphorylated (p)-ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p-ERK1/2. To test whether p-ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p-ERK1/2 with calcitonin gene-related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point-counting method.In cervical, midroot, and apical zones of the PDL, t-ERK1/2- and p-ERK1/2-labeled nerve fibers were found in close association with blood vessels. The p-ERK1/2-labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p-ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin.The perivascular distribution of t-ERK1/2 and p-ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p-ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.

The aim of this study was to evaluate the role of host periosteum and recipient bed perforation on the healing of an onlay graft to the mandible based on histologic and immunohistochemical analysis. Each of the 12 adult sheep used in the study received four iliac corticocancellous onlay bone grafts on the lateral surface of the mandible. In experiment 1, the block graft was placed in direct contact with the recipient bed and fixed with micro-screws, and in experiment 2, the recipient cortical bed was perforated before graft placement. The host periosteum around the graft was excised before flap replacement in experiment 3, and in experiment 4, a sheet of silicone membrane was placed between the graft and the recipient bed. The animals were euthanised at 4, 8, 12 and 16 weeks after surgery and the findings were analysed by routine microscopy (haematoxylin and eosin and tartrate-resistant acid phosphatase [TRAP] stains) and immunohistochemistry for proliferation and apoptotic markers (Ki67, caspase-3 and TUNEL stains). After 8 weeks, full graft–host integration was seen in all the experimental groups except experiment 4. After 16 weeks, pronounced graft resorption and volume reduction was observed in experiments 1 and 2, whilst experiment 3 was characterised with extensive connective tissue infiltration and severe resorption of the graft. The number of osteoclasts expressed peaked at 4 weeks in experiments 1 and 2 and at 16 weeks in experiment 3. Immunoreactivity for Ki67 by osteoblasts lining the trabecular bone of the graft’s spongiosa expressed moderate level of Ki67 at 8 weeks, and thereafter declined markedly. The strongest expression of caspase-3 on the bone surface was observed after 16 weeks. Recipient cortical bed perforation offered no advantage over non-perforated bed regarding healing and integration of a bone graft. Excision of the host overlying the periosteum was accompanied with rapid absorption of the grafts and partial or complete replacement with fibrous connective tissue. This study also demonstrated that cell death by apoptosis is a fundamental component of osteoblastic phenotypic differentiation during healing of corticocancellous bone graft.

Nitric oxide (NO) mediates different cellular functions by activating soluble guanylate cyclase (sGC) that converts guanosine-5'-triphosphate (GTP) to cyclic guanosine-3',5'-monophosphate (cGMP). Membrane-bound GCs produce cGMP in response to natriuretic peptides in osteoblasts, but neither the NO-target enzyme sGC, nor the phosphorylation sites of NOS III, nor their regulation by extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt/protein kinase B (Akt/PKB) in osteoclasts have been established.Rat molars with periodontium were perfusion- and post-fixed, decalcified, and frozen-sectioned. Free-floating sections were stained using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and tartrate-resistant acid phosphatase (TRAP) histochemical techniques and immunoreacted with antisera against NO-synthase (NOS) I-III, NOS III phoshorylated at Thr495, NOS III phoshorylated at Serine1177 (Ser1177), ERK1/2, phosphorylated ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2/beta1), and cGMP.NADPH-d staining and immunostaining of NOS I-III, NOS III phosphorylated at Ser1177, ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (alpha2 and beta1-subunits), and cGMP were detected in osteoclasts. Immunohistochemical reaction products for NOS III phosphorylated at threonine495 (Thr495) and phosphorylated ERK1/2 could not be identified in osteoclasts. Comparison of TRAP activity and immunostaining for sGC beta1-subunit revealed that sGC beta1-subunit is only expressed in a sub-population of osteoclasts.NO is likely to be generated by NOS I and NOS III in osteoclasts. The inconstant expression of NOS II in some osteoclasts may be explained with inducible expression of NOS II upon physiological cell activation. Localization of the sGC alpha2- and beta1-subunits and cGMP in osteoclasts is compatible with an involvement of NO-sGC signaling in the homeostasis of osteoclasts. The phosphorylation site of NOS III at Ser1177 and phosphorylated Akt/PKB are involved in regulation of NO production by NOS III in osteoclasts under basal conditions.

It is known that total endothelial nitric oxide synthase (eNOS) is localized in peripheral nerve fibers, but the existence of the phosphorylation site/s of eNOS in peripheral nerve fibers is unknown. In the perfusion-fixed and decalcified sections of rat palates, eNOS and eNOS phosphorylated at Ser(116) were identified in the nerve fibers of the glandulae palatinae. The localization of eNOS phosphorylated at Ser(1177) and Thr(495) in nerve fibers was undetectable. It is concluded that eNOS is phosphorylated at Ser(116) in nerve fibers of the glandulae palatinae under basal conditions. The basal Ser(1177) and Thr(495) phosphorylation of eNOS are missing in nerve fibers of the glandulae palatinae.

Knowledge of the histochemical substrates of cellular and neurovascular connections in the gingiva is essential in order to understand the initial mechanisms of inflammation in the periodontium. Since the localization of the neuroendocrine peptide galanin in the gingiva is still unclear, we used immunohistochemical, in situ hybridization and immunoblot techniques to assess the localization of galanin in the gingiva of rat molars. Galanin-immunoreactive nerve fibers were located around blood vessels in the lamina propria, beneath the epithelium, in the epithelial-proprial junction and in the basal layer of the epithelium. Galanin was highly expressed in the suprabasal keratinocytes of the gingival epithelium. The localization of galanin in gingival nerve fibers and the expression of galanin in keratinocytes of the gingival epithelium indicate that galanin may be a possible regulator of different cellular functions in the gingiva.

A wide variety of stimuli can trigger activation of the transcription factor CREB (cAMP-responsive element binding protein), pointing toward a central role for CREB in the integration of various signaling inputs. No data are available on the expression and phosphorylation of CREB in mammalian teeth. Using immunohistochemical analysis of free-floating sections, we show here that CREB was strongly expressed and phosphorylated at Ser-133 within the nucleus of a subpopulation of adult human molar odontoblasts. Many dental pulp stromal cells and periodontal ligament fibroblasts expressed CREB and showed phosphorylation of CREB at Ser-133. In addition, cementoblasts displayed nuclear expression and phosphorylation of CREB at Ser-133. The epithelial rests of Malassez revealed strong nuclear expression of CREB, but phosphorylation at Ser-133 was variable. Our results provide the first evidence that the constitutively phosphorylated transcription factor CREB is involved in the biomineralization process of adult human molar odontoblasts and cementoblasts.

The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation-dependent sensitization of nociceptors, and the inflammatory mediator bradykinin (BK) led to a reduced threshold in the nociceptor terminals, activating intracellular signaling by phosphorylating receptors and ion channels. The effects of BK on the non-transcriptional modulation of the ERK1/2 in the peripheral nociceptor terminals, including in nerve endings of the dentin-pulp complex, are unknown. The time-dependent effects of BK (10−7 M) on the ERK1/2 phosphorylation in nerve terminals of the dentin-pulp complex were investigated by quantitative and double immunolabeling with organ bath experiments. In nerve terminals, total and p-ERK1/2 were detected. In comparison with the controls, the numbers of p-ERK1/2-positive nerve endings increased after 1 and 3 min and decreased after 10 min of BK treatment. Analysis of the data indicates that BK induces phosphorylation-mediated local activation of ERK1/2 in nerve terminals modulating nociception in the dentin-pulp complex.

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10−7 M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10−6 M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.

Large clinical studies provided evidence of a lower incidence of a reduced cardiac morbidity in diabetic patients treated with angiotensin type 1 (AT1) receptor blockers. The AT1 receptor is coupled to the Gq class of G-proteins, which stimulate proteinkinase C (PKC) via activation of phospholipase Cβ. To study the role of the Gq protein Gα11 and its signaling through PKC in diabetic heart disease, we induced diabetes in wildtype and Gα11 knockout mice using streptozotocin.After eight weeks of stable hyperglycemia, cardiac morphology and function were assessed by echocardiography, myocardial expression and translocation of PKC isoforms by immunohistochemistry and immunoblot after tissue fractionation.Wildtype mice (n=8) but not Gα11 knockout animals (n=8) developed ventricular hypertrophy upon induction of hyperglycemia. PKC isoforms α, βII, δ, e, ζ and θ were all detected in myocardium of wildtype animals. Compared to normoglycemic control animals, PKC isoforms α and ζ showed increased expression levels in diabetic wildtype mice. In addition, PKC ζ was phosphorylated at Thr410/403 and showed strong translocation to nuclear membranes in cardiomyocytes of diabetic but not of control animals. Hearts from normoglycemic Gα11-knockout mice showed lower expression levels of PKC α and δ compared to wildtype animals. Upon induction of hyperglycemia in knockouts, expression of PKC δ was only slightly increased to the level found in non-diabetic wildtypes. Expression, phosphorylation and translocation of PKC isoforms, however, remained unaffected by the induction of diabetes in Gα11-deficient mice.We conclude that Gα11 is central to the induction of myocardial hypertrophy in type 1 diabetes. Activation of PKC α and ζ appear to be important pathways in hypertrophic signaling via Gα11. The inhibition of this pathway may in part explain the strong therapeutic benefit of AT1 receptor blockade in diabetic patients.

By the formation of cyclic guanosine 3′,5′-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC α2-subunit/β1-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC α2, and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10−5 M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10−4 M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.