Fibrillin-1 regulates periostin expression during maintenance of periodontal homeostasis.

Background/purpose: Human periodontal ligament consists of elastic system fibers, mainly fibrillin-1 (FBN1). Periostin (POSTN) maintains periodontal homeostasis. A previous study showed that the expression of Postn in periodontal ligament cells was decreased in mice underexpressing Fbn1. However, the relationship between FBN1 and POSTN is not fully understood in the context of mechanical stress. FBN1 contributes to transforming growth factor ß1 (TGF-ß1) activation; TGF-ß1 upregulates the expression of POSTN in human periodontal ligament cells. This study examined whether FBN1 contributed to the maintenance of periodontal homeostasis in cultured human periodontal ligament cells. Materials and methods: Human periodontal ligament fibroblasts (HPDLFs) were exposed to mechanical force via centrifugation. The expression of POSTN was examined by quantitative reverse transcription polymerase chain reaction. The phosphorylation of Smad2 in the TGF-ß/Smad signaling pathway was monitored by western blotting. Results: The expression levels of FBN1 and POSTN were not significantly decreased by centrifugation. However, the expression of POSTN after centrifugation significantly decreased upon knockdown of FBN1. The phosphorylation of Smad2 after centrifugation was decreased, regardless of FBN1 knockdown. Supplementation with 0.1 ng/ml recombinant human TGF-ß1 rescued POSTN expression after centrifugation in HPDLFs upon knockdown of FBN1. Conclusion: FBN1 regulates the expression of POSTN to maintain periodontal homeostasis via TGF-ß/Smad signaling during centrifugation.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats.

OBJECTIVE: Pain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DESIGN: Expression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. RESULTS: After HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 µM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). CONCLUSIONS: These results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.

Orthodontic force-induced oxidative stress in the periodontal tissue and dental pulp elicits nociception via activation/sensitization of TRPA1 on nociceptive fibers.

Orthodontic patients complain of pain for the first few days after insertion of appliances. Mechanical force has been reported to produce oxidants in periodontal ligament (PDL) cells. It has not been studied whether orthodontic force-induced oxidative stress elicits nociception. Herein, we focused on the role of the oxidant-sensitive channel TRPA1 on nociception in orthodontic pain. In a rat model of loaded orthodontic force between the maxillary first molar and incisor, the behavioral signs of orofacial nociception, facial rubbing and wiping, increased to a peak on day 1 and gradually diminished to the control level on day 5. Administration of free radical scavengers (Tempol and PBN) and TRPA1 antagonist (HC-030031) inhibited nociceptive behaviors on day 1. In the PDL, the oxidative stress marker 8-OHdG was highly detected on day 1 and recovered on day 5 to the sham-operated level. The dental pulp showed similar results as the PDL. TRPA1 mRNA was abundantly expressed in the trigeminal ganglion relative to PDL tissue, and there were TRPA1-immunopositive neuronal fibers in the PDL and pulp. In dissociated trigeminal ganglion neurons, H2O2 at 5 mM induced a Ca2+ response that was inhibited by HC-030031. Although H2O2 at 100 µM did not yield any response, it enhanced the mechanically activated TRPA1-dependent Ca2+ response. These results suggest that oxidative stress in the PDL and dental pulp following orthodontic force activates and/or mechanically sensitizes TRPA1 on nociceptive fibers, resulting in orthodontic nociception. Later, the disappearance of nociception seems to be related to a decrease in oxidative stress, probably due to tissue remodeling.

Maldevelopment of the submandibular gland in a mouse model of apert syndrome.

BACKGROUND: Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome. RESULTS: Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed. CONCLUSIONS: These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc.

Evaluation of tongue volume and oral cavity capacity using cone-beam computed tomography.

The aims of this study were to reveal the usefulness of a newly developed method for measuring tongue volume (TV) and oral cavity capacity (OCC) and to assess the relationship between them. The tongue was coated with a contrast agent, and the TV and OCC were determined using cone-beam computed tomography (CBCT). We enrolled 20 adults who were scheduled to undergo CBCT to evaluate the relationship of the third molar roots to the alveolar nerve before molar extraction. Each participant's tongue was coated with a contrast agent, and CBCT of the tongue and oral cavity was performed. Using computer software, we evaluated reconstructed 3D images of the TV, oral cavity proper volume (OCPV), and OCC. The mean TV was 47.07 ± 7.08 cm3. The mean OCPV and OCC were 4.40 ± 2.78 cm3 and 51.47 ± 6.46 cm3, respectively. There was a significant correlation between TV and OCC (r = 0.920; p < 0.01) but not between TV and OCPV. The mean TV/OCC ratio was 91 ± 5%. The proposed method produced CBCT images that enabled effective measurement of TV and OCC. This simple method of measuring TV and OCC will be useful in the diagnosis on the tongues with abnormal size.

Comprehending the three-dimensional mandibular morphology of facial asymmetry patients with mandibular prognathism.

BACKGROUND: The purpose of this study was to elucidate the factors that cause facial asymmetry by comparing the characteristics of the mandibular morphology in patients with mandibular prognathism with or without facial asymmetry using three-dimensional computed tomography (3D-CT). METHODS: We studied 28 mandibular prognathism patients whose menton deviated by ≥ 4 mm from the midline (FA group, n = 14) and those with a < 4-mm deviation (NA group, n = 14). DICOM data from multislice CT images were reconstructed and analysed using 3D image analysing software. Mandibular structures were assessed via linear, angular, or volumetric measurements and analysed statistically. RESULTS: The lengths of the ramal and body components and condylar volume in the FA group were significantly greater on the nondeviated side than those on the deviated side. The mandibular body length of the nondeviated side in the FA group was significantly longer than that of the NA group. Other components of the FA group did not significantly differ from those of the NA group. CONCLUSIONS: Imbalances in the sizes of the ramal and body components as well as the increased body length of the nondeviated side in the FA group compared with that of the NA group may contribute to facial asymmetry in patients with mandibular prognathism.

Orthodontic Treatment and Long-Term Management of a Patient With Marfan Syndrome.

Marfan syndrome (MFS) is caused by abnormal systemic connective tissue. The main clinical manifestations include long limbs, long slender fingers, lens subluxation, abnormal cardiac valves, and aortic aneurysm. We report the case of an 11-year-old patient with MFS who underwent orthodontic treatment and was followed up until the age of 25 years. We found no significant differences in tooth movement between the patient with MFS and healthy subjects. However, because patients with MFS show characteristic facial growth and an increased risk of developing systemic comorbidities, their dental status requires careful observation over time.

Systemic and maxillofacial characteristics of 11 Japanese children with Russell-Silver syndrome.

Russell-Silver syndrome (RSS) is a congenital anomaly characterized by intrauterine and postnatal growth retardation, typical facial features, fifth-finger clinodactyly, and skeletal asymmetry. Although data on intrauterine and postnatal growth retardation have been reported, there are few reports concerning the typical maxillofacial morphology in individuals with RSS. The aim of this study was to describe the details of this systemic condition and to characterize maxillofacial morphology based on cephalograms in 11 Japanese patients (age range, 3.9-12.0 years) with RSS. All 11 individuals had intrauterine and postnatal growth retardation. In addition, most showed mandibular retrognathia and relative macrocephaly. Lateral cephalogram measurements showed that mandibular retrognathia resulted from short mandibular body length, whereas the depth of the cranial base was close to normal. Although asymmetry of hand, foot, and limb length were present in most individuals, obvious facial asymmetry was not common. Differences between left and right skeletal and dental age were not observed, indicating that children with RSS might show asymmetry because of quantitative differences in skeletal growth rather than delayed growth rate. Our findings not only provide important information about the maxillofacial characteristics of RSS, but also help to clarify the association between these characteristics and genetics, which will add to the body of information on clinical symptoms.

Periostin inhibits hypoxia-induced apoptosis in human periodontal ligament cells via TGF-ß signaling.

Periostin (POSTN) is an extracellular matrix protein expressed predominantly in periodontal ligament (PDL) cells. The aim of this study was to investigate the effects of POSTN on human PDL cell apoptosis under hypoxic conditions. The percentage of apoptotic PDL cells under hypoxia was increased significantly when the endogenous POSTN gene was silenced using siRNA, but decreased when cells were treated with recombinant human POSTN (rhPOSTN), or when mouse Postn was overexpressed in vitro. Silencing POSTN during hypoxia decreased the expression of HIF prolyl-hydroxylase 2 (PHD2), but increased HIF-1α protein level. Conversely, treating hypoxic cells with rhPOSTN or overexpressing Postn increased PHD2 expression but decreased HIF-1α levels. The addition of rhPOSTN in the absence of a TGF-ß receptor inhibitor (SB525334) significantly decreased hypoxia-induced apoptosis, while the effects of rhPOSTN were abolished when cells were co-treated with SB525334. Consistent with this, the phosphorylation of SMAD2 was increased in hypoxic PDL cells by the knockdown of POSTN, but decreased by treatment with rhPOSTN. Under normoxia, the PHD2 expression, HIF-1α level, and apoptosis were unaffected by POSTN siRNA, rhPOSTN, or Postn overexpression. These findings suggest that, under hypoxic conditions, POSTN regulates PHD2 expression and HIF-1α levels by modulating TGF-ß1 signaling, leading to decreased apoptosis.

Dental and maxillofacial characteristics of six Japanese individuals with ectrodactyly-ectodermal dysplasia-clefting syndrome.

Objective : Ectrodactyly-ectodermal dysplasia-clefting syndrome is a congenital anomaly characterized by ectodermal dysplasia, ectrodactyly, cleft lip and palate, and lacrimal duct anomalies. Because this syndrome is frequently accompanied by a congenital lack of teeth, narrow palate, and malocclusion, comprehensive orthodontic intervention is required. Design : To highlight the specific dental and maxillofacial characteristics of ectrodactyly-ectodermal dysplasia-clefting syndrome, six Japanese individuals diagnosed with the syndrome are described here. Patients : The subjects consisted of two boys and four girls (age range, 6.0 to 13.9 years) diagnosed with ectrodactyly-ectodermal dysplasia-clefting syndrome by medical and dental specialists. Their conditions included ectodermal dysplasia (hypodontia, microdontia, enamel hypoplasia, and abnormalities in hair and nails), cleft lip and/or palate, and ectrodactyly. Cephalograms, panoramic x-rays, and dental casts were taken; systemic complications were recorded at the first visit to our dental hospital. Results : All individuals had severe oligodontia with 9 to 18 missing teeth. The missing teeth were mainly maxillary and mandibular incisors and second bicuspids, arranged in a symmetrical manner. Cephalometric analysis showed retruded and short maxilla due to cleft lip and/or palate. It is interesting that all individuals showed a characteristically shaped mandibular symphysis with a retruded point B. It is likely that this unusual symphyseal morphology is due to the lack of mandibular incisors. Conclusions : This study demonstrates the presence of severe oligodontia in the incisal and premolar regions and describes a characteristic maxillary and mandibular structure in Japanese individuals with ectrodactyly-ectodermal dysplasia-clefting syndrome.

Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts.

Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with ß-tricalcium phosphate (ß-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome.

Marfan syndrome and its disorder in periodontal tissues.

Elastic system fibers are composed of two distinct elements, elastin, which is an amorphous component crosslinked in the core, and microfibril, localized in the periphery of elastin. As microfibrillar proteins, fibrillins, microfibril-associated glycoproteins (MAGPs), latent TGF-beta-binding proteins (LTBPs), microfibril-associated proteins (MFAPs), and fibulins are known. Fibrillin-1 is a major microfibrillar protein and characterized by calcium binding EGF-like (cbEGF) domain. Association between fibrillin-1 and TGF-beta is a recent topic of this field and this interaction is known to inactivate and target TGF-beta action. FBN1 encoding fibrillin-1 is a responsible gene for Marfan syndrome type 1 (MIM #154700), characterized by increased height and long limbs, ectopia lentis, and cardiovascular disorders, such as mitral valve prolapse and aortic dilation and regurgitation. Animal models suggest that the abnormal TGF-beta signaling is underlying as the pathogenesis of these conditions. Besides skeletal, ocular and cardiovascular conditions, severe periodontitis is frequently seen in affected patients. To clarify the unknown function of elastic system fibers in the periodontal ligament (PDL), PDL-cells were isolated from a Marfan syndrome type 1 patient who was with the severe periodontitis and had a mutation in one of the cbEGF domain of fibrillin-1. These results suggested that wild-type fibrillin-1 was required for the normal cell alignment and tissue architecture of PDLs. Evidences are now accumulated to suggest that fibrillin-1 is one of the molecule involved in the interaction between cell and extracellular matrix.

Case report of de novo dup(18p)/del(18q) and r(18) mosaicism.

This is a report of a 27-year-old woman with an unusual de novo chromosomal abnormality. Mosaicism was identified in peripheral blood cells examined by standard G-bands by trypsin using Giemsa (GTG) analysis and fluorescence in situ hybridization (FISH) analysis with chromosome-18 region-specific probes, 46,XX,del(18)(pter --> q21.33:)[41], 46,XX,r(18)(::p11.21 --> q21.33::)[8], and 46,XX,der(18)(pter --> q21.33::p11.21 --> pter)[1]. On the other hand, the karyotype of periodontal ligament fibroblasts was nonmosaic, 46,XX, der(18)(pter --> q21.33::p11.21 --> pter)[50]. All cell lines appeared to be missing a portion of 18q (q21.33 --> qter). The pattern of the dup(18p)/del(18q) in the rod configuration raises the possibility of an inversion in chromosome 18 in one of the parents. However, no chromosomal anomaly was detected in either parent. The most probable explanation is that de novo rod and ring configurations arose simultaneously from an intrachromosomal exchange. The unique phenotype of this patient, which included primary hypothyroidism and primary hypogonadism, is discussed in relation to her karyotype.

Characteristic phenotype of immortalized periodontal cells isolated from a Marfan syndrome type I patient.

The periodontal ligament (PDL) is situated between the tooth root and alveolar bone, thereby supporting the tooth, and is composed of collagen and elastic system fibers. Marfan syndrome type I (MFS1, MIM #154700) is caused by mutations in FBN1 encoding fibrillin-1, which is a major microfibrillar protein of elastic system fibers. MFS1 is characterized by tall stature, aortic/mitral valve prolapse, and ectopia lentis and is occasionally accompanied by severe periodontitis. Since little is known about the biological functions of elastic system fibers in PDLs and the pathogenesis of the periodontitis in MFS1, PDL cells were isolated from an MFS1 patient with a heterozygous missense mutation in a calcium-binding epidermal-growth-factor-like domain of FBN1. Isolated PDL cells were immortalized by transducing a retrovirus carrying genes for the human Polycomb group protein, Bmi-1, and human telomerase reverse transcriptase. Immortalized PDL cells from the MFS1 patient (termed M-HPL1) and those of a healthy volunteer (termed HPDL2) both expressed various PDL-related genes. The growth and attachment of M-HPL1 and HPDL2 to hydroxyapatite particles were comparable. However, when M-HPL1 were transplanted with hydroxyapatite particles into immunodeficient mice, disorganized cell alignment and irregular microfibril assembly were noted. The activation of the signaling of transforming grwoth factor-beta (TGF-beta) is thought to cause the pathogenesis for lung and cardiovascular abnormalities in MFS1. Interestingly, M-HPL1 shows a higher level of activated TGF-beta than HPDL2. Thus, M-HPL1 represent a powerful tool for clarifying the biological roles of elastic system fibers in PDL and the pathogenesis of periodontitis in MFS1. Our findings also suggest that FBN1 regulates cell alignment and microfibril assembly in PDLs.

Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants.

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without beta-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or beta-estradiol (20 ng/ml) or relaxin plus beta-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and beta-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants--a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or beta-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and beta-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and provide evidence for a link between relaxin, MMPs, and matrix degradation.

Ascorbic acid induces collagenase-1 in human periodontal ligament cells but not in MC3T3-E1 osteoblast-like cells: potential association between collagenase expression and changes in alkaline phosphatase phenotype.

Ascorbic acid (AA) enhances osteoblastic differentiation by increasing collagen accumulation, which in turn, results in increased alkaline phosphatase (AP) expression in some osteogenic cells. However, in other cells, including human periodontal ligament (PDL) cells, additional osteoinductive agents are required for this response. To understand the potential basis for the maintenance of the AP phenotype of PDL cells exposed to AA, we examined the modulation of the tissue-degrading matrix metalloproteinases (MMPs) and their inhibitors by AA in short-term cell cultures. Early passage PDL cells in serum-free medium were exposed to AA for 5 days. The samples were analyzed for MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), AP, collagen I(alpha1), and osteocalcin. We found that AA dose-dependently increased the expression of collagenase-1, and minimally TIMP-1, but not stromelysin-1 or TIMP-2. Additionally, AA caused substantial increases in levels of type I collagen. AA was unable to increase AP activity or osteocalcin messenger RNA in PDL cells. However, the cells retained the ability to show a significantly greater AP expression in high- versus low-density cultures, and increased osteocalcin as well as AP levels when cultured in the presence of dexamethasone. Moreover, in cells exposed to dexamethasone, increases in AP and osteocalcin were accompanied by a repression of collagenase-1 expression. In contrast to PDL cells, AA did not induce collagenase but produced a significant increase in AP expression in MC3T3-E1 cells. These findings provide the first evidence that AA, by modulating both collagen and collagenase-1 expression in PDL cells, most likely contributes to a net matrix remodeling response in these cells. Furthermore, the relationship between changes in collagenase expression and alterations in AP activity in PDL and MC3T3-E1 cells suggests a potential role for collagenase in modulating the AP phenotype of cells with osteoblastic potential.