Notch-1/2 receptors and Jagged-1 ligand, but not HERP-1 transcription factor, are immunohistochemically expressed in the epithelial lining of periapical cysts.

Background: To further understand the involvement of Notch pathway signaling in the pathogenesis of periapical cyst the immunohistochemical expression of Notch-1 and Notch-2 receptors, Jagged-1 ligand, and HERP-1 transcription factor in the lining epithelium of periapical cysts was investigated. Material and Methods: Thirty human periapical cysts were immunohistochemically stained with antibodies against Notch-1, Notch-2, Jagged-1, and HERP-1. Epithelial expression of each antibody was correlated with the presence of inflammation in the connective tissue of the cystic wall. Results: Notch-1 was identified in the basal and suprabasal epithelial cells of 30/30, Notch-2 in 19/24, and Jagged-1 in 27/30 cysts. HERP-1 was detected in scattered subepithelial inflammatory cells, but not in the lining epithelium of cysts. There was no significant correlation between the immunohistochemical expression of each antibody and the presence of inflammation in the connective tissue of the cystic wall. Conclusions: This immunohistochemical study showed expression of Notch-1/2 and Jagged-1 in periapical cysts that combined with the expression of HES1/5 found in a previous report, are indicative of the activation of Notch an endocrine-paracrine mechanism. Further research on the activity of Notch and other pathways in periapical cysts may contribute both to elucidate their pathogenesis and select molecular targets for future novel treatments. Key words:Odontogenic cyst, radicular cyst, etiology, epithelial cells, Notch, Jagged, HERP.

Assessing the fate and contribution of Foxd1-expressing embryonic precursors and their progeny in palatal development, homeostasis and excisional repair.

Oral mucosal tissues heal rapidly with minimal scarring, although palatal mucosa can be associated with excessive fibrosis in response to injury. Investigations on the balance between neovascularization and tissue repair suggests regulation of angiogenesis is an important determinant of repair versus scarring. Associated with pericyte mediated fibrosis in kidney injury, FoxD1 is implicated in growth centres during cranio-facial development, although which cell lineages are derived from these embryonic populations in development and in adult animals is unknown. Using a lineage tracing approach, we assessed the fate of embryonic Foxd1-expressing progenitor cells and their progeny in palatal development and during wound healing in adult mice. During palatal development as well as in post-natal tissues, Foxd1-lineage progeny were associated with the vasculature and the epineurium. Post-injury, de novo expression of FoxD1 was not detectable, although Foxd1-lineage progeny expanded while exhibiting low association with the fibroblast/myofibroblast markers PDGFα, PDGFß, vimentin, α-smooth muscle actin, as well as the neuronal associated markers S100ß and p75NTR. Foxd1-lineage progeny were primarily associated with CD146, CD31, and to a lesser extent CD105, remaining in close proximity to developing neovascular structures. Our findings demonstrate that FoxD1 derived cells are predominantly associated with the palatal vasculature and provide strong evidence that FoxD1 derived cells do not give rise to populations involved directly in the scarring of the palate.

Initial assessment of gingival biotype as a potential source of variability in the migration, contraction and gene expression of fibroblasts.

OBJECTIVE: Gingival biotype refers to the clinical classification of gingiva based on the thickness of the tissue, with thick gingival tissues more resistant to trauma and recession than the thin variant. However, to date there has never been an analysis of whether fibroblasts isolated from different biotypes possess inherent phenotypic differences. We hypothesized that gingival fibroblasts from thick and thin biotype would exhibit differences in migration, contraction and gene expression in vitro in the presence of either transforming growth factor beta one (TGF-ß1) or tumor necrosis factor alpha (TNFα), two major cytokines involved in wound repair. DESIGN: Migration was quantified using closure of scratch wound assays, contraction was assessed using attached and detached collagen lattices and extracellular matrix related gene expression using Taqman Realtime polymerase chain reaction. RESULTS: Human gingival fibroblasts isolated from both biotypes showed similar rates of closure of scratch wounds, which was not influenced by the addition of TGF-ß1 or TNFα. Fibroblasts from both biotypes contracted detached, but not attached, collagen gels to 50 % of their original weight although this contraction was not associated with incorporation of α-smooth muscle actin into stressfibres under any tested culture condition. Analysis of gene expression showed that POSTN, and ACTA2 mRNA levels did not significantly change, but CCN2 and COL1A2 mRNA levels were significantly higher in thick compared to thin fibroblasts in response to TGF-ß1. CONCLUSION: While supra-cellular factors influence the healing, esthetic outcomes and recession in thin gingival biotypes, differences in gingival fibroblast gene expression in response to growth factors may also play a role and warrants further investigation.

Functions of Matricellular Proteins in Dental Tissues and Their Emerging Roles in Orofacial Tissue Development, Maintenance, and Disease.

Matricellular proteins (MCPs) are defined as extracellular matrix (ECM) associated proteins that are important regulators and integrators of microenvironmental signals, contributing to the dynamic nature of ECM signalling. There is a growing understanding of the role of matricellular proteins in cellular processes governing tissue development as well as in disease pathogenesis. In this review, the expression and functions of different MP family members (periostin, CCNs, TSPs, SIBLINGs and others) are presented, specifically in relation to craniofacial development and the maintenance of orofacial tissues, including bone, gingiva, oral mucosa, palate and the dental pulp. As will be discussed, each MP family member has been shown to have non-redundant roles in development, tissue homeostasis, wound healing, pathology and tumorigenesis of orofacial and dental tissues.

Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing.

Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn-/-) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and ßigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn-/-, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn-/- fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn-/- fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn-/- cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn-/- cells. Quantification of immunostaining for vinculin and integrinß1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinß1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.

Wound healing and fibrosis: a contrasting role for periostin in skin and the oral mucosa.

Both skin and oral mucosa are characterized by the presence of keratinized epithelium in direct apposition to an underlying collagen-dense connective tissue. Despite significant overlap in structure and physiological function, skin and the oral mucosa exhibit significantly different healing profiles in response to injury. The oral mucosa has a propensity for rapid restoration of barrier function with minimal underlying fibrosis, but in contrast, skin is associated with slower healing and scar formation. Modulators of cell function, matricellular proteins have been shown to play significant roles in cutaneous healing, but their role in restoration of the oral mucosa is poorly defined. As will be discussed in this review, over the last 12 years our research group has been actively investigating the role of the profibrotic matricellular protein periostin in tissue homeostasis and fibrosis, as well as healing, in both skin and gingiva. In the skin, periostin is highly expressed in fibrotic scars and is upregulated during cutaneous wound repair, where it facilitates myofibroblast differentiation. In contrast, in gingival healing, periostin regulates extracellular matrix synthesis but does not appear to be associated with the transition of mesenchymal cells to a contractile phenotype. The significance of these findings will be discussed, with a focus on periostin as a potential therapeutic to augment healing of soft tissues or suppress fibrosis.

JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing.

In healthy individuals, the healing of soft tissues such as skin after pathological insult or post injury follows a relatively predictable and defined series of cell and molecular processes to restore tissue architecture and function(s). Healing progresses through the phases of hemostasis, inflammation, proliferation, remodeling, and concomitant with re-epithelialization restores barrier function. Soft tissue healing is achieved through the spatiotemporal interplay of multiple different cell types including neutrophils, monocytes/macrophages, fibroblasts, endothelial cells/pericytes, and keratinocytes. Expressed in most cell types, c-Jun N-terminal kinases (JNK) are signaling molecules associated with the regulation of several cellular processes involved in soft tissue wound healing and in response to cellular stress. A member of the mitogen-activated protein kinase family (MAPK), JNKs have been implicated in the regulation of inflammatory cell phenotype, as well as fibroblast, stem/progenitor cell, and epithelial cell biology. In this review, we discuss our understanding of JNKs in the regulation of cell behaviors related to tissue injury, pathology, and wound healing of soft tissues. Using models as diverse as Drosophila, mice, rats, as well as human tissues, research is now defining important, but sometimes conflicting roles for JNKs in the regulation of multiple molecular processes in multiple different cell types central to wound healing processes. In this review, we focus specifically on the role of JNKs in the regulation of cell behavior in the healing of skin, cornea, tendon, gingiva, and dental pulp tissues. We conclude that while parallels can be drawn between some JNK activities and the control of cell behavior in healing, the roles of JNK can also be very specific modes of action depending on the tissue and the phase of healing.

Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling.

During skin healing, periostin facilitates myofibroblast differentiation through a ß1 integrin/FAK dependent mechanism and continued expression is associated with scarring. In contrast to skin, gingival tissue does not typically scar upon injury, but the role of periostin in gingival healing has never been investigated. Using a rat gingivectomy model, we show that the gingival architecture is re-established within 14 days of wounding. Periostin mRNA levels peak at day 7 post-wounding, with persistence of periostin protein in the connective tissue through day 14. Collagen type I and lysyl oxidase mRNA levels peak at day 7 post wounding, which corresponded with the peak of fibroblast proliferation. Although α-smooth muscle actin mRNA levels increased 200-fold in the tissue, no myofibroblasts were detected in the regenerating tissue. In vitro, human gingival fibroblast adhesion on periostin, but not collagen, was inhibited by blocking ß1 integrins. Fibroblasts cultured on periostin exhibited similar rates of proliferation and myofibroblast differentiation to cells cultured on collagen only. However, human gingival fibroblasts cultured in the presence of periostin exhibited significantly increased fibronectin and collagen mRNA levels. Increases in fibronectin production were attenuated by pharmacological inhibition of FAK and JNK signaling in human gingival fibroblasts. In vivo, mRNA levels for fibronectin peaked at day 3 and 7 post wounding, with protein immunoreactivity highest at day 7, suggesting periostin is a modulator of fibronectin production during gingival healing.

Phenytoin activates Smad3 phosphorylation and periostin expression in drug-induced gingival enlargement.

Drug-induced gingival enlargement (DIGE) is a fibrotic condition associated with systemic administration of the anti-epileptic drug, phenytoin. We have previously demonstrated that periostin, which is transforming growth factor-beta (TGF-ß) inducible gene, is upregulated in various fibrotic conditions including gingival enlargement associated with nifedipine. The objective of this study was to assess periostin expression in phenytoin-induced gingival enlargement (PIGE) tissues and to investigate the mechanisms underlying periostin expression. Human PIGE tissues were assessed using Masson's trichrome, with cell infiltration and changes in extracellular matrix composition characterized through labeling with antibodies to periostin, phospho-SMAD 3, TGF-ß, as well as the macrophage markers CD68 and RM3/1. Using human gingival fibroblasts (HGFs) in vitro we examined the pathways through which phenytoin acts on fibroblasts. In PIGE tissues, which demonstrate altered collagen organization and increased inflammatory cell infiltration, periostin protein was increased compared with healthy tissues. p-SMAD2/3, the transcription factor associated with canonical TGF-ß signaling, is localized to the nuclei in both gingival fibroblasts and oral epithelial cells in PIGE tissues, but not in healthy tissue. In vitro culture of HGFs with 15 and 30 µg/ml of phenytoin increased periostin protein levels, which correlated with p-SMAD3 phosphorylation. Inhibition of canonical TGF-ß signaling with SB431542 significantly reduced phenytoin induction of SMAD3 phosphorylation and periostin expression in HGFs. Analysis of PIGE tissues showed a subset of CD68 stained macrophages were TGF-ß positive and that RM1/3 regenerative macrophages were present in the tissues. Our results demonstrate that phenytoin up-regulates periostin in HGFs in a TGF-ß-dependent manner.

Emergency care provided in a Greek dental school and analysis of the patients' demographic characteristics: a prospective study.

OBJECTIVES: The aim of this study was to evaluate the incidence of pain of endodontic origin and its relationship with socio-economic and demographic factors among patients seeking unscheduled urgent dental care. METHODS: Patients attending the Emergency Clinic of Athens Dental School, Greece, between November 2011 and June 2012, were evaluated to determine their socio-economic profile, dental problem and treatment required. The facility operated from Monday to Friday, from 8.30 am to 1.00 pm, excluding the 4 weeks encompassing the Christmas and Easter holidays. In total, 533 patients were assessed regarding gender, age, ethnicity, occupation, reason for visiting, diagnosis and treatment provided. The data obtained were recorded, reviewed, coded and analysed using Poisson regression models. RESULTS: Mondays and Wednesdays were the busiest days of the week. The most common occupation among the patients was 'unemployed'. Pain of endodontic origin (reversible or irreversible pulpitis, or acute apical periodontitis) was the prevailing reason for the visit. The most frequent treatments administered were pulpectomy and drainage. Prescriptions for medications were rare. CONCLUSION: Services were requested primarily by individuals who were experiencing acute pain of endodontic origin, had low or no income and were available during morning hours, probably because of the service's low cost and operational hours. Prospective studies, such as the present investigation, can provide epidemiological evidence and indicate areas in the infrastructure of emergency services which may be improved. Additionally, such studies can provide rationale for public insurance programs and can generate profiles of the patients who utilise these low-cost public services.

Evaluation of the Root and Canal Morphology of Maxillary Permanent Molars and the Incidence of the Second Mesiobuccal Root Canal in Greek Population Using Cone-beam Computed Tomography.

OBJECTIVES: Cone-Beam Computed Tomography is an alternative imaging technique which has been recently introduced in the field of Oral & Maxillofacial Radiology. It has rapidly gained great popularity among clinicians due to its ability to detect lesions and defects of the orofacial region and provide three-dimensional information about them. In the field of Endodontics, CBCT can be a useful tool to reveal tooth morphology irregularities, additional root canals and vertical root fractures. The objective of this study is to evaluate the root and root canal morphology of the maxillary permanent molars in Greek population using Cone-Beam Computed Tomography. MATERIALS AND METHODS: 273 cone-beam computed tomography (CBCT) images were examined. The number of roots and root canals of the first and second maxillary molars were evaluated. Root canal configuration was classified according to Weine's classification by two independent examiners and statistical analysis was performed. RESULTS: A total of 812 molars (410 first and 402 second ones) were evaluated. The vast majority of both first and second molars had three roots (89.26% and 85.07%, respectively). Most first molars had four canals, while most second molars had three. In the mesiobuccal roots, one foramen was recorded in 80.91% of all teeth. Other rare morphologic variations were also found, such as fusion of a maxillary second molar with a supernumerary tooth. CONCLUSION: Within the limitations of this study, it can be concluded that more attention should be given to the detection of additional canals during root canal treatment in maxillary permanent molars. Towards this effort, CBCT can provide the clinician with supplemental information about the different root canal configurations for successful Root Canal Treatment.