The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we ...studied postnatal incisor development in K320E-Twinkle
mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-Twinkle
mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.
The binding of nitric oxide (NO) to heme in the β1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1β1 and α2β1 isoforms of the enzyme, leading to the increased production ...of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and β1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and β1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the β1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.
The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum ...and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.
While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated ...protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³) results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise.
Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers.
7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques.
While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle.
Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and concomitantly pAMPKThr¹⁷² levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser²⁸⁴³ can mechanistically impact Ca²⁺handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.
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 α1- and β1-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 α1- and β1-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 α1- and β1-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.
Orthodontic tooth movement to therapeutically align malpositioned teeth is supposed to impact blood flow in the surrounding tissues. Here, we evaluated actual vascularization in the tension area of ...the periodontal ligament during experimental tooth movement in rats (
= 8) with magnetic resonance imaging (MRI). We inserted an elastic band between the left upper first and the second rat molar; the right side was not treated and served as control. After four days of tooth movement, we recorded T1-weighted morphologic and dynamic-contrast-enhanced MRI sequences with an animal-specific 7 Tesla MRI to assess of local vascularization. Furthermore, we quantified osteoclasts and monocytes in the periodontal ligament, which are crucial for orthodontic tooth movement, root resorptions as undesirable side effects, as well as the extent of tooth movement using paraffine histology and micro-CT analysis. Data were tested for normal distribution with Shapiro-Wilk tests followed by either a two-tailed paired
-test or a Wilcoxon matched-pairs signed rank test. Significant orthodontic tooth movement was induced within the four days of treatment, as evidenced by increased osteoclast and monocyte activity in the periodontal ligament as well as by µCT analysis. Contrast enhancement was increased at the orthodontically-treated side distally of the moved upper first left molar, indicating increased vascularization at the tension side of the periodontal ligament. Accordingly, we detected reduced time-to-peak and washout rates. Our study using MRI to directly assess local vascularization thus seems to confirm the hypothesis that perfusion is enhanced in tension zones of the periodontal ligament during orthodontic tooth movement.
Various local and systemic factors compromise oral wound healing and may lead to wound dehiscence, inflammation, or ulcers. Currently, there is a lack of topical therapeutical options. Thus, this ...study aimed to investigate the effect of Aloe vera (AV) and Rheum palmatum root (RPR) on oral wound healing capacity in vitro. The effect of AV and RPR on human primary fibroblast viability and migration was studied by measuring metabolic activity and gap closure in a scratch assay. Furthermore, cell cycle distribution and cytoskeletal features were analyzed. Antimicrobial activity against the oral pathogen Porphyromonas gingivalis was evaluated by broth microdilution assay. AV and RPR increased fibroblast migration after single agent treatment. Synergistic effects of the plant extract combination were observed regarding cellular migration which were confirmed by calculation of the phenomenological combination index (pCI), whereas the cell cycle distribution was not influenced. Furthermore, the combination of AV and RPR showed synergistic antibacterial effects as determined by the fractional inhibitory concentration index. This study demonstrated that the combination of AV and RPR can promote the migration of human primary fibroblasts in vitro and exert antimicrobial efficacy against P. gingivalis, suggesting these compounds for the topical treatment of wound healing disorders.
VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. ...However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.
The activity of endothelial nitric oxide synthase (eNOS) in endothelial cells increased with the phosphorylation of the enzyme at Ser1177 and decreased at Thr495. The regulation of the ...phosphorylation sites of eNOS at Ser1177 and Thr495 in blood vessels of the healthy and inflamed human dental pulp is unknown. To investigate this, healthy and carious human third molars were immersion-fixed and decalcified. The localization of eNOS, Ser1177, and Thr495 in healthy and inflamed blood vessels was examined in consecutive cryo-sections using quantitative immunohistochemical methods. We found that the staining intensity of Ser1177 in healthy blood vessels decreased in inflamed blood vessels, whereas the weak staining intensity of Thr495 in healthy blood vessels strongly increased in inflamed blood vessels. In blood vessels of the healthy pulp, eNOS is active with phosphorylation of the enzyme at Ser1177. The phosphorylation of eNOS at Thr495 in inflamed blood vessels leads to a decrease in eNOS activity, contributing to eNOS uncoupling and giving evidence for a decrease in NO and an increase in O2− production. Since the formation of the tertiary dentin matrix depends on intact pulp circulation, eNOS uncoupling and phosphorylation of eNOS at Thr495 in the inflamed pulp blood vessels should be considered during caries therapy.
Abstract Introduction Odontoblasts are terminally differentiated cells of ectomesenchymal origin that produce the dentin. Differentiated odontoblasts cannot be identified yet by a single phenotypic ...marker protein; therefore, a combination of markers is currently used. Up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 has been associated with exit from the cell cycle and terminal differentiation of mammalian cells. Immunoreactivity for p27Kip1 protein was shown in many adult mouse tissues, but no information is available on the expression of p27Kip1 in mammalian dental pulp. Methods Healthy and carious adult human molars with reparative dentin formation were decalcified, cryoprotected, frozen embedded, and frozen sectioned. The expression of p27Kip1 and nestin in cells of adult human dental pulp was analyzed by immunohistochemistry using free floating sections. Results p27Kip1 showed strong nuclear expression in many differentiated human molar odontoblasts at the odontoblastic layer. Most cells of the cell-rich zone displayed low levels of p27Kip1 despite the fact that preodontoblasts localized in the cell-rich zone of the subodontoblastic layer have been identified as quiescent cells. The nuclear expression of p27Kip1 in stromal cells of the dental pulp was variable, indicating that subpopulations of these cells were in distinct states of differentiation. Odontoblasts generating reparative dentin showed comparable nuclear expression of p27Kip1 in comparison with odontoblasts synthesizing primary/secondary dentin. This result indicates that odontoblasts synthesizing primary/secondary or reparative dentin exhibit a similar differentiation status. Conclusions Our findings show that increased expression of nuclear p27Kip1 occurred during differentiation from preodontoblasts to odontoblasts in adult healthy and carious molars. p27Kip1 can be used as a novel nuclear marker protein for differentiated human odontoblasts in vivo.
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