Mesenchymal Wnt/ß-catenin signaling limits tooth number.

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/ß-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/ß-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/ß-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/ß-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/ß-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/ß-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

Concise Review: Cellular and Molecular Mechanisms Regulation of Tooth Initiation.

Development of teeth depends on the reciprocal interactions between the surface epithelium (ectoderm) and the underlying neural crest-derived mesenchyme. These interactions are facilitated by the conserved signaling pathways, which build a complex network of signals and transcription factors. Tooth development starts at specific and predetermined loci in the oral ectoderm and is described as a morphologically distinct thickening of oral ectoderm, named dental lamina. Cells within the dental lamina invaginate into the underlying mesenchyme, generating placodes that mark the onset of individual tooth development. In the following stages of development, the tooth epithelium buds and folds transitioning through various shapes, including bud, cap, and bell shapes, which also identify the specific stages of tooth development. Although much of the molecular regulation of tooth development has been unraveled, the regulation of the initial stages of tooth development, as well as the cellular mechanisms that govern tooth development remain largely unknown. This review provides a systematic overview of the current knowledge on the molecular and cellular mechanisms that guide initial stages of tooth development and outlines the challenges which temper the progress. Stem Cells 2019;37:26-32.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

The association between clinical and laboratory findings of bullous pemphigoid and dipeptidyl peptidase-4 inhibitors in the elderly: a retrospective study.

AIM: To evaluate the association between the use of dipeptidyl peptidase-4 inhibitors (DPP4I) and clinical and laboratory findings of bullous pemphigoid (BP) in patients treated at the European Reference Network - Skin Reference Centre in Croatia. METHODS: This retrospective study enrolled 82 patients treated for BP at the Department of Dermatovenereology, University Hospital Center Zagreb from January 2015 to December 2019. Clinical features of BP, presence of comorbidities, and laboratory findings of anti-BP antibodies and eosinophilia were analyzed in three groups of BP patients: 1) diabetes mellitus (DM) type II patients treated with DPP4I, 2) DM type II patients not treated with DPP4I, and 3) non-DM type II patients. RESULTS: The average age and anti-BP180 titer were similar in all three groups. DPP4I group had a slightly lower eosinophil level in both peripheral blood (4.89%) and biopsy specimens (87.5%), but the difference was not significant. The prevalence of inflammatory BP in DPP4I group was 76.5%. DPP4I group had significantly higher percentage of patients with chronic renal failure and dementia (52.9% and 11.8%, respectively) compared with non-DPP4I DM (14.3% and 0%, respectively) and non-DM type II patients (15.7% and 0%, respectively). CONCLUSION: BP patients treated with DPP4I and those not treated with DPP4Is did not significantly differ in laboratory findings. However, DPP4I treatment was associated with an inflammatory subtype of BP and a higher prevalence of dementia and chronic renal failure. These findings warrant further research into the association of BP and DM with dementia and chronic renal failure.

Omega-3 Versus Omega-6 Polyunsaturated Fatty Acids in the Prevention and Treatment of Inflammatory Skin Diseases.

Omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated fatty acids (PUFAs) are nowadays desirable components of oils with special dietary and functional properties. Their therapeutic and health-promoting effects have already been established in various chronic inflammatory and autoimmune diseases through various mechanisms, including modifications in cell membrane lipid composition, gene expression, cellular metabolism, and signal transduction. The application of ω-3 and ω-6 PUFAs in most common skin diseases has been examined in numerous studies, but their results and conclusions were mostly opposing and inconclusive. It seems that combined ω-6, gamma-linolenic acid (GLA), and ω-3 long-chain PUFAs supplementation exhibits the highest potential in diminishing inflammatory processes, which could be beneficial for the management of inflammatory skin diseases, such as atopic dermatitis, psoriasis, and acne. Due to significant population and individually-based genetic variations that impact PUFAs metabolism and associated metabolites, gene expression, and subsequent inflammatory responses, at this point, we could not recommend strict dietary and supplementation strategies for disease prevention and treatment that will be appropriate for all. Well-balanced nutrition and additional anti-inflammatory PUFA-based supplementation should be encouraged in a targeted manner for individuals in need to provide better management of skin diseases but, most importantly, to maintain and improve overall skin health.

Altered Levels of Sphingosine, Sphinganine and Their Ceramides in Atopic Dermatitis Are Related to Skin Barrier Function, Disease Severity and Local Cytokine Milieu.

Dysfunctional skin barrier plays a key role in the pathophysiology of atopic dermatitis (AD), a common inflammatory skin disease. Altered composition of ceramides is regarded as a major cause of skin barrier dysfunction, however it is not clear whether these changes are intrinsic or initiated by inflammation and aberrant immune response in AD. This study investigated the levels of free sphingoid bases (SBs) sphingosine and sphinganine and their ceramides and glucosylceramide in the stratum corneum (SC) and related them to skin barrier function, disease severity and local cytokine milieu. Ceramides were measured in healthy skin, and lesional and non-lesional skin of AD patients by a novel method based on deacylation of ceramides which were subsequently determined as corresponding sphingoid bases by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytokine levels were determined by multiplex immunoassay. Atopic skin showed increased levels of most investigated markers, predominantly in lesional skin. The largest difference in respect to healthy skin was found for glucosylceramide with respective median values of 0.23 (IQR 0.18-0.61), 0.56 (IQR 0.32-0.76) and 19.32 (IQR 7.86-27.62) pmol/g protein for healthy, non-lesional and lesional skin. The levels of investigated ceramide markers were correlated with disease severity (scoring atopic dermatitis, SCORAD) and skin barrier function (trans-epidermal water loss, TEWL) and furthermore with cytokines involved in innate, Th-1, and Th-2 immune response. Interestingly, the strongest association with SCORAD was found for sphinganine/sphingosine ratio (r = -0.69, p < 0.001; non-lesional skin), emphasizing the importance of SBs in AD. The highest correlation with TEWL was found for glucosylceramide (r2 = 0.60, p < 0.001), which was investigated for the first time in AD. Findings that the changes in SBs and ceramide levels were predominant in lesional skin and their association with disease severity and cytokine levels suggest an immune-system driven effect. a novel analysis method demonstrates a robust and simple approach that might facilitate wider use of lipid biomarkers in the clinics e.g., to monitor (immune) therapy or dissect disease endotypes.

Suppression of epithelial differentiation by Foxi3 is essential for molar crown patterning.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here, we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium in mice. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops and in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling, we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium, was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified keratin 10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

Biology Explaining Tooth Repair and Regeneration: A Mini-Review.

The tooth is an intricate composition of precisely patterned, mineralized matrices and soft tissues. Mineralized tissues include enamel (produced by the epithelial cells called ameloblasts), dentin and cementum (produced by mesenchymal cells called odontoblasts and cementoblasts, respectively), and soft tissues, which include the dental pulp and the periodontal ligament along with the invading nerves and blood vessels. It was perceived for a very long time that teeth primarily serve an esthetical function. In recent years, however, the role of healthy teeth, as well as the impact of oral health on general well-being, became more evident. Tooth loss, caused by tooth decay, congenital malformations (tooth agenesis), trauma, periodontal diseases, or age-related changes, is usually replaced by artificial materials which lack many of the important biological characteristics of the natural tooth. Human teeth have very low to almost absent regeneration potential, due to early loss of cell populations with regenerative capacity, namely stem cells. Significant effort has been made in recent decades to identify and characterize tooth stem cells, and to unravel the developmental programs which these cells follow in order to generate a tooth.

Intracellular autofluorescence: a biomarker for epithelial cancer stem cells.

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.

Mesenchymal Wnt/ß-Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10.

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/ß-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/ß-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no ß-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/ß-catenin. Stem Cells 2015;33:1670-1681.

Stem cells as the root of pancreatic ductal adenocarcinoma.

Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.

The genetic aspects of hidradenitis suppurativa.

Genetic aspects have a substantial role in hidradenitis suppurativa (HS) pathogenesis. A positive family history of HS occurs in about one-third of HS cases and is significantly higher in patients with early onset of the disease. Recent twin studies have shown a high heritability in HS, fortifying the importance of genetic factors in disease pathogenesis. Based on existing knowledge on the genomics of HS, the disease can be categorized as familial HS, sporadic, syndromic HS, and "HS plus" associated with other syndromes. In familial HS, autosomal dominant transmission is proposed, and monogenic inheritance is rare. This monogenic trait is related to mutations of γ-secretase component genes and Notch signaling or defects in inflammasome function. With newly discovered gene mutations, such as those related to innate and adaptive immunity, skin microbiome, inflammasome, epidermal homeostasis, and keratinization pathway, we can define HS as a polygenic, multifactorial, autoinflammatory disease. To fully elucidate the genetic aspects of HS, we need extensive, long-term global collaborations.

Extracellular matrix remodelling in dental pulp tissue of carious human teeth through the prism of single-cell RNA sequencing.

Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.

Dermoscopy of Zosteriform and Swirling Pattern Type 1 Segmental Darier Disease.

Dear Editor, Segmental Darier disease (DD) is a rare disease with around 40 described English literature cases. It is hypothesized that one of the causes of the disease is a post-zygotic somatic mutation for the calcium ATPase pump, only present in lesional skin. There are two types of segmental DD: type 1, where lesions follow Blaschko's lines unilaterally, and type 2, characterized by focal areas of increased severity in patients with generalized DD (1). Type 1 segmental DD is not easily diagnosed due to the lack of positive family history, the late onset of the disease in the third or fourth decade of life, and lack of DD-associated features. The differential diagnosis of type 1 segmental DD includes acquired papular dermatoses distributed in linear or zosteriform fashion, such as lichen planus, psoriasis, lichen striatus, or linear porokeratosis (2). We report two cases of segmental DD, of which the first case was a 43-year-old woman who presented with pruritic skin changes five years in duration and a history of seasonal aggravation. On examination, light brownish to reddish keratotic small papules were observed on the left abdomen and inframammary area, arranged in a swirling pattern (Figure 1, a). Dermoscopy showed polygonal or roundish yellowish/brown areas surrounded with whitish structureless areas (Figure 1, b). The histopathological correlations for dermoscopic brownish polygonal or round areas are hyperkeratosis, parakeratosis, and dyskeratotic keratinocytes, which were present in the biopsy specimen (Figure 1, c). The patient was prescribed 0.1% tretinoin gel, which led to marked improvement (Figure 1, d). The second case was a 62-year-old woman who presented with a flare of small red-brown papules, eroded papules, and some yellowish crusts arranged in a zosteriform pattern on the right side of the upper abdomen (Figure 2, a). Dermoscopy showed polygonal, roundish, yellowish areas surrounded with whitish and reddish structureless areas (Figure 2, b). Histopathology mainly revealed compact orthokeratosis and small foci of parakeratosis, marked granular layer with dyskeratotic keratinocytes, and foci of suprabasal acantholysis consistent with the diagnosis of DD (Figure 2, d, d). The patient was prescribed topical steroid cream and 0.1% adapalene cream, which also led to improvement. In both of our cases, a final diagnosis of type 1 segmental DD was established based on clinico-histopathologic correlation, since acantholytic dyskeratotic epidermal nevus could not have been ruled out only based on the histopathology report as it is clinically and histologically indistinguishable from segmental DD. However, the late age of onset and aggravation resulting from external factors such as heat, sunlight, and sweat supported the diagnosis of segmental DD. Although the final diagnosis of type 1 segmental DD is typically established based on clinico-histopathological correlation, we find dermoscopy particularly useful in aiding the diagnosis by eliminating differential diagnoses and being aware of their well-known dermoscopic patterns.

Nogo-A Regulates the Fate of Human Dental Pulp Stem Cells toward Osteogenic, Adipogenic, and Neurogenic Differentiation.

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate the function and fate of stem cells. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S, Nile Red and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells toward the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-γ2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in the presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates toward osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in clinics.

Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation.

The disintegrin and metalloproteinase Adam10 is a membrane-bound sheddase that regulates Notch signaling and ensures epidermal integrity. To address the function of Adam10 in the continuously growing incisors, we used Keratin14 Cre/+;Adam10 fl/fl transgenic mice, in which Adam10 is conditionally deleted in the dental epithelium. Keratin14 Cre/+;Adam10 fl/fl mice exhibited severe abnormalities, including defective enamel formation reminiscent of human enamel pathologies. Histological analyses of mutant incisors revealed absence of stratum intermedium, and severe disorganization of enamel-secreting ameloblasts. In situ hybridization and immunostaining analyses in the Keratin14 Cre/+;Adam10 fl/fl incisors showed strong Notch1 downregulation in dental epithelium and ectopic distribution of enamel-specific molecules, including ameloblastin and amelogenin. Lineage tracing studies using Notch1 CreERT2 ;R26 mT/mG mice demonstrated that loss of the stratum intermedium cells was due to their fate switch toward the ameloblast lineage. Overall, our data reveal that in the continuously growing incisors the Adam10/Notch axis controls dental epithelial cell boundaries, cell fate switch and proper enamel formation.

Ptch2 is a Potential Regulator of Mesenchymal Stem Cells.

Ptch receptors 1 and 2 mediate Hedgehog signaling pivotal for organ development and homeostasis. In contrast to embryonic lethal Ptch1 -/- phenotype, Ptch2 -/- mice display no effect on gross phenotype. In this brief report, we provide evidence of changes in the putative incisor mesenchymal stem cell (MSC) niches that contribute to accelerated incisor growth, as well as intriguing changes in the bones and skin which suggest a role for Ptch2 in the regulation of MSCs and their regenerative potential. We employed histological, immunostaining, and computed tomography (µCT) analyses to analyze morphological differences between Ptch2 -/- and wild-type incisors, long bones, and skins. In vitro CFU and differentiation assays were used to demonstrate the MSC content and differentiation potential of Ptch2 -/- bone marrow stromal cells. Wound healing assay was performed in vivo and in vitro on 8-week-old mice to assess the effect of Ptch2 on the wound closure. Loss of Ptch2 causes increases in the number of putative MSCs in the continuously growing incisor, associated with increased vascularization observed in the tooth mesenchyme and the neurovascular bundle. Increased length and volume of Ptch2 -/- bones is linked with the increased number and augmented in vitro differentiation potential of MSCs in the bone marrow. Dynamic changes in the Ptch2 -/- skin thickness relate to changes in the mesenchymal compartment and impact the wound closure potential. The effects of Ptch2 abrogation on the postnatal MSCs suggest a crucial role for Ptch2 in Hedgehog signaling regulation of the organ regenerative potential.

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

Pyogenic Granuloma of the Proximal Part of a Nail Unit in a Child.

Dear Editor, We present a case of proximal pyogenic granuloma in 4-year-old child. The patient presented to our Department due to a fast-growing lesion on the proximal part of the nail unit. The lesion had appeared over several weeks, and it was extremely painful for the child. On the day of the 1st visit, the lesion was not bleeding but was very painful during examination and photo-documentation. Clinically, it presented as an exogenous tumoral lesion of the proximal 1/3 of the nail, partially exulcerated with one part exhibiting coagulated hemorrhage and with uneven coloration (Figure 1). The lesion was not sharply demarcated. Dermoscopically, the majority of the lesion presented an unspecific dermoscopic structure, orange background color, and matched the criteria for a vascular lesion: few unspecific vessels and hemorrhage. The "sticky fiber" sign was also present (Figure 2). Since the lesion was fast-growing and due to the unspecific dermoscopic appearance, the child was referred to a pediatric surgeon and a complete excisional biopsy of the lesion was performed. The dermoscopy of pyogenic granuloma has been already described (1). The histology report confirmed pyogenic granuloma. Pyogenic granulomas of the nail unit are not a common finding, but our case confirms that even this location can be site of this type of benign lesion. It more commonly found in the periungual region and can be expected due to adverse effects of different kinds of systemic therapies. However, due to differential diagnosis that includes different types of tumors occurring at the nail unit, most importantly amelanotic melanoma and SCC, it is suggested to excise or take a biopsy of this type of lesion to be able to exclude aggressive tumor types, which are very rare but not impossible the in pediatric population (2). In cases of unquestionable diagnosis, several local treatments are available. Since the lesion presented a destructive nature in our case, we decided to perform excisional biopsy followed by histology, which in our case was both a diagnostic and therapeutic procedure.

Prx1 and Prx2 cooperatively regulate the morphogenesis of the medial region of the mandibular process.

Mice lacking both Prx1 and Prx2 display severe abnormalities in the mandible. Our analysis showed that complete loss of Prx gene products leads to growth abnormalities in the mandibular processes evident as early as embryonic day (E) 10.5 associated with changes in the survival of the mesenchyme in the medial region. Changes in the gene expression in the medial and lateral regions were related to gradual loss of a subpopulation of mesenchyme in the medial region expressing eHand. Our analysis also showed that Prx gene products are required for the initiation and maintenance of chondrogenesis and terminal differentiation of the chondrocytes in the caudal and rostral ends of Meckel's cartilage. The fusion of the mandibular processes in the Prx1/Prx2 double mutants is caused by accelerated ossification. These observations together show that, during mandibular morphogenesis, Prx gene products play multiple roles including the cell survival, the region-specific terminal differentiation of Meckelian chondrocytes and osteogenesis.