A novel de novo heterozygous ALPL nonsense mutation associated with adult hypophosphatasia.

Using genetic, clinical, biochemical, and radiographic assessment and bioinformatic approaches, we present an unusual case of adult HPP caused by a novel de novo heterozygous nonsense mutation in the alkaline phosphatase (ALPL). INTRODUCTION: Hypophosphatasia (HPP) is caused by genetic alterations of the ALPL gene, encoding the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Here, the purpose was to perform clinical and molecular investigation in a 36-year-old Caucasian woman suspected to present adult HPP. METHODS: Medical and dental histories were obtained for the proposita and family members, including biochemical, radiographic, and dental assessments. ALPL mutational analysis was performed by the Sanger sequencing method, and the functional impact prediction of the identified mutations was assessed by bioinformatic methods. RESULTS: We identified a novel heterozygous nonsense mutation in the ALPL gene (NM_000478.6:c.768G>A; W[TGG]>*[TGA]) associated with spontaneous vertebral fracture, severe back pain, musculoskeletal pain, low bone density, and short-rooted permanent teeth loss. Functional prediction analysis revealed that the Trp256Ter mutation led to a complete loss of TNSALP crown domain and extensive loss of other functional domains (calcium-binding domain, active site vicinity, and zinc-binding site) and over 60% loss of homodimer interface residues, suggesting that the mutant TNSALP molecules are nonfunctional and form unstable homodimers. Genotyping of the ALPL in the proposita's parents, sister, and niece revealed that in this case, HPP occurred due to a de novo mutation. CONCLUSION: The present study describes a novel genotype-phenotype and structure-function relationship for HPP, contributing to a better molecular comprehension of HPP etiology and pathophysiology.

Genetic and pharmacologic modulation of cementogenesis via pyrophosphate regulators.

Pyrophosphate (PPi) serves as a potent and physiologically important regulator of mineralization, with systemic and local concentrations determined by several key regulators, including: tissue-nonspecific alkaline phosphatase (ALPL gene; TNAP protein), the progressive ankylosis protein (ANKH; ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1; ENPP1). Results to date have indicated important roles for PPi in cementum formation, and we addressed several gaps in knowledge by employing genetically edited mouse models where PPi metabolism was disrupted and pharmacologically modulating PPi in a PPi-deficient mouse model. We demonstrate that acellular cementum growth is inversely proportional to PPi levels, with reduced cementum in Alpl KO (increased PPi levels) mice and excess cementum in Ank KO mice (decreased PPi levels). Moreover, simultaneous ablation of Alpl and Ank results in reestablishment of functional cementum in dKO mice. Additional reduction of PPi by dual deletion of Ank and Enpp1 does not further increase cementogenesis, and PDL space is maintained in part through bone modeling/remodeling by osteoclasts. Our results provide insights into cementum formation and expand our knowledge of how PPi regulates cementum. We also demonstrate for the first time that pharmacologic manipulation of PPi through an ENPP1-Fc fusion protein can regulate cementum growth, supporting therapeutic interventions targeting PPi metabolism.

Loss of Discoidin Domain Receptor 1 Predisposes Mice to Periodontal Breakdown.

The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors and tyrosine kinases. DDRs regulate cell functions, and their extracellular domains affect collagen fibrillogenesis and mineralization. Based on the collagenous nature of dentoalveolar tissues, we hypothesized that DDR1 plays an important role in dentoalveolar development and function. Radiography, micro-computed tomography (micro-CT), histology, histomorphometry, in situ hybridization (ISH), immunohistochemistry (IHC), and transmission electron microscopy (TEM) were used to analyze Ddr1 knockout (Ddr1-/-) mice and wild-type (WT) controls at 1, 2, and 9 mo, and ISH and quantitative polymerase chain reaction (qPCR) were employed to assess Ddr1/DDR1 messenger RNA expression in mouse and human tissues. Radiographic images showed normal molars but abnormal mandibular condyles, as well as alveolar bone loss in Ddr1-/- mice versus WT controls at 9 mo. Histological, histomorphometric, micro-CT, and TEM analyses indicated no differences in enamel or dentin Ddr1-/- versus WT molars. Total volumes (TVs) and bone volumes (BVs) of subchondral and ramus bone of Ddr1-/- versus WT condyles were increased and bone volume fraction (BV/TV) was reduced at 1 and 9 mo. There were no differences in alveolar bone volume at 1 mo, but at 9 mo, severe periodontal defects and significant alveolar bone loss (14%; P < 0.0001) were evident in Ddr1-/- versus WT mandibles. Histology, ISH, and IHC revealed disrupted junctional epithelium, connective tissue destruction, bacterial invasion, increased neutrophil infiltration, upregulation of cytokines including macrophage colony-stimulating factor, and 3-fold increased osteoclast numbers (P < 0.05) in Ddr1-/- versus WT periodontia at 9 mo. In normal mouse tissues, ISH and qPCR revealed Ddr1 expression in basal cell layers of the oral epithelia and in immune cells. We confirmed a similar expression pattern in human oral epithelium by ISH and qPCR. We propose that DDR1 plays an important role in periodontal homeostasis and that absence of DDR1 predisposes mice to periodontal breakdown.

Transcriptomic profiling of tantalum metal implant osseointegration in osteopenic patients.

OBJECTIVES: The long-term success of dental implants is established by literature. Although clinically well defined, the complex genetic pathways underlying osseointegration have not yet been fully elucidated. Furthermore, patients with osteopenia/osteoporosis are considered to present as higher risk for implant failure. Porous tantalum trabecular metal (PTTM), an open-cell porous biomaterial, is suggested to present enhanced biocompatibility and osteoconductivity. The goal of this study was to evaluate the expression patterns of a panel of genes closely associated with osteogenesis and wound healing in osteopenic patients receiving either traditional titanium (Ti) or PTTM cylinders to assess the pathway of genes activation in the early phases of osseointegration. MATERIAL AND METHODS: Implant cylinders made of Ti and PTTM were placed in osteopenic volunteers. At 2- and 4 weeks of healing, one Ti and one PTTM cylinder were removed from each subject for RT-PCR analysis using osteogenesis PCR array. RESULTS: Compared to Ti, PTTM-associated bone displayed upregulation of bone matrix proteins, BMP/TGF tisuperfamily, soluble ligand and integrin receptors, growth factors, and collagen genes at one or both time points. Histologically, PTTM implants displayed more robust osteogenesis deposition and maturity when compared to Ti implants from the same patient. CONCLUSIONS: Our results indicate that PTTM properties could induce an earlier activation of genes associated with osteogenesis in osteopenic patients suggesting that PTTM implants may attenuate the relative risk of placing dental implants in this population.

Osteopontin regulates dentin and alveolar bone development and mineralization.

The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank-/-) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank-/- mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240days postnatal (dpn) indicated normal histological structures in Spp1-/- comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90dpn revealed significantly increased volumes and tissue mineral densities of Spp1-/- mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1-/- mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1-/- vs. WT mice at 26dpn. We genetically deleted Spp1 on the Ank-/- mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank-/- mice. Ank-/-; Spp1-/- double deficient mice did not exhibit greater hypercementosis than that in Ank-/- mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.

Periodontitis in Chédiak-Higashi Syndrome: An Altered Immunoinflammatory Response.

Chédiak-Higashi syndrome (CHS), a rare autosomal recessive disorder caused by mutations in the lysosomal trafficking regulator gene (LYST), is associated with aggressive periodontitis. It is suggested that LYST mutations affect the toll-like receptor (TLR)-mediated immunoinflammatory response, leading to frequent infections. This study sought to determine the periodontal status of patients with classic (severe) and atypical (milder) forms of CHS and the immunoregulatory functions of gingival fibroblasts in CHS patients. In contrast to aged-matched healthy controls, atypical (n = 4) and classic (n = 3) CHS patients presented with mild chronic periodontitis with no evidence of gingival ulceration, severe tooth mobility, or premature exfoliation of teeth. As a standard of care, all classic CHS patients had undergone bone marrow transplantation (BMT). Primary gingival fibroblasts obtained from atypical and BMT classic CHS patients displayed higher protein expression of TLR-2 (1.81-fold and 1.56-fold, respectively) and decreased expression of TLR-4 (-2.5-fold and -3.85-fold, respectively) at baseline when compared with healthy control gingival fibroblasts. When challenged with whole bacterial extract of Fusobacterium nucleatum, both atypical and classic CHS gingival fibroblasts failed to up-regulate TLR-2 and TLR-4 expression when compared with their respective untreated groups and control cells. Cytokine multiplex analysis following F. nucleatum challenge showed that atypical CHS gingival fibroblasts featured significantly increased cytokine expression (interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, IL-12, interferon-γ, tumor necrosis factor-α), whereas classic CHS cells featured similar/decreased cytokine expression when compared with treated control cells. Collectively, these results suggest that LYST mutations in CHS patients affect TLR-2 and TLR-4 expression/function, leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients. Furthermore, our results suggest that atypical CHS patients and classic CHS patients who undergo BMT early in life are less susceptible to aggressive periodontitis and that hematopoietic cells play a critical role in mitigating the risk of aggressive periodontitis in CHS. Knowledge Transfer Statement: Results from this study can be used to create awareness among clinicians and researchers that not all CHS patients exhibit historically reported aggressive periodontitis, especially if they have atypical CHS disease or have received bone marrow transplantation. LYST mutations in CHS patients may affect TLR-2 and TLR-4 expression/function leading to dysregulated immunoinflammatory response, which in turn may influence the periodontal phenotype noted in CHS patients.

Impact of smoking on experimental gingivitis. A clinical, microbiological and immunological prospective study.

OBJECTIVE: The present study assessed the effect of smoking on clinical, microbiological and immunological parameters in an experimental gingivitis model. MATERIAL AND METHODS: Twenty-four healthy dental students were divided into two groups: smokers (n = 10); and nonsmokers (n = 14). Stents were used to prevent biofilm removal during brushing. Visible plaque index (VPI) and gingival bleeding index (GBI) were determined 5- on day -7 (running phase), baseline, 21 d (experimental gingivitis) and 28 d (resolution phase). Supragingival biofilm and gingival crevicular fluid were collected and assayed by checkerboard DNA-DNA hybridization and a multiplex analysis, respectively. Intragroup comparison was performed by Friedman and Dunn's multiple comparison tests, whereas the Mann-Whitney U-test was applied for intergroup analyses. RESULTS: Cessation of oral hygiene resulted in a significant increase in VPI, GBI and gingival crevicular fluid volume in both groups, which returned to baseline levels 7 d after oral hygiene was resumed. Smokers presented lower GBI than did nonsmokers (p < 0.05) at day 21. Smokers had higher total bacterial counts and higher proportions of red- and orange complex bacteria, as well as lower proportions of Actinomyces spp., and of purple- and yellow-complex bacteria (p < 0.05). Furthermore, the levels of key immune-regulatory cytokines, including interleukin (IL)-8, IL-17 and interferon-γ, were higher in smokers than in nonsmokers (p < 0.05). CONCLUSION: Smokers and nonsmokers developed gingival inflammation after supragingival biofilm accumulation, but smokers had less bleeding, higher proportions of periodontal pathogens and distinct host-response patterns during the course of experimental gingivitis.

Alcohol intake may impair bone density and new cementum formation after enamel matrix derivative treatment: histometric study in rats.

BACKGROUND AND OBJECTIVE: Alcohol intake may interfere with bone metabolism; however, there is a lack of information about the outcomes of regenerative approaches in the presence of alcohol intake. Enamel matrix derivative (EMD) has been used in periodontal regenerative procedures resulting in improvement of clinical parameters. Thus, the aim of this histomorphometric study is to evaluate the healing of periodontal defects after treatment with EMD under the influence of alcohol intake. MATERIAL AND METHODS: Twenty Wistar rats were randomly assigned to two groups: G1 = alcohol intake (n = 10) and G2 = non-exposed to alcohol intake (n = 10). Thirty days after initiation of alcohol intake, fenestration defects were created at the buccal aspect of the first mandibular molar of all animals from both groups. After the surgeries, the defects of each animal were randomly assigned to two subgroups: non-treated control and treated with EMD. The animals were killed 21 d later. RESULTS: G1 showed less defect fill for non-treated controls. Bone density (BD) and new cementum formation were lower for G1 when compared to G2, for EMD-treated and non-treated sites. EMD treatment resulted in greater BD and new cementum formation in both groups and defect fill was not significantly different between groups in the EMD-treated sites. The number of tartrate-resistant acid phosphatase-positive osteoclasts was significantly higher in G1 when compared to G2 and in EMD-treated sites of both groups. CONCLUSION: Alcohol intake may produce a significant detrimental effect on BD and new cementum formation, even in sites treated with EMD. A limited positive effect may be expected after EMD treatment under this condition.

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

Mechanical Forces Exacerbate Periodontal Defects in Bsp-null Mice.

Bone sialoprotein (BSP) is an acidic phosphoprotein with collagen-binding, cell attachment, and hydroxyapatite-nucleating properties. BSP expression in mineralized tissues is upregulated at onset of mineralization. Bsp-null (Bsp(-/-)) mice exhibit reductions in bone mineral density, bone turnover, osteoclast activation, and impaired bone healing. Furthermore, Bsp(-/-) mice have marked periodontal tissue breakdown, with a lack of acellular cementum leading to periodontal ligament detachment, extensive alveolar bone and tooth root resorption, and incisor malocclusion. We hypothesized that altered mechanical stress from mastication contributes to periodontal destruction observed in Bsp(-/-) mice. This hypothesis was tested by comparing Bsp(-/-) and wild-type mice fed with standard hard pellet diet or soft powder diet. Dentoalveolar tissues were analyzed using histology and micro-computed tomography. By 8 wk of age, Bsp(-/-) mice exhibited molar and incisor malocclusion regardless of diet. Bsp(-/-) mice with hard pellet diet exhibited high incidence (30%) of severe incisor malocclusion, 10% lower body weight, 3% reduced femur length, and 30% elevated serum alkaline phosphatase activity compared to wild type. Soft powder diet reduced severe incisor malocclusion incidence to 3% in Bsp(-/-) mice, supporting the hypothesis that occlusal loading contributed to the malocclusion phenotype. Furthermore, Bsp(-/-) mice in the soft powder diet group featured normal body weight, long bone length, and serum alkaline phosphatase activity, suggesting that tooth dysfunction and malnutrition contribute to growth and skeletal defects reported in Bsp(-/-) mice. Bsp(-/-) incisors also erupt at a slower rate, which likely leads to the observed thickened dentin and enhanced mineralization of dentin and enamel toward the apical end. We propose that the decrease in eruption rate is due to a lack of acellular cementum and associated defective periodontal attachment. These data demonstrate the importance of BSP in maintaining proper periodontal function and alveolar bone remodeling and point to dental dysfunction as causative factor of skeletal defects observed in Bsp(-/-) mice.

Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein.

Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp(-/-) mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp(-/-) mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp(-/-) mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp(-/-) mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp(-/-) mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp(-/-) molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.

Fibromodulin and Biglycan Modulate Periodontium through TGFß/BMP Signaling.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFß/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFß/BMP signaling, matrix turnover, and collagen organization.

Vitamin D represses dentin matrix protein 1 in cementoblasts and osteocytes.

Calcium and phosphorus homeostasis is achieved by interplay among hormones, including 1,25(OH)2D3 (1,25D), parathyroid hormone, and fibroblast growth factor 23 (FGF23), and their interactions with other proteins. For example, mutations in dentin matrix protein 1 (DMP-1) result in increased FGF23 and hypophosphatemic rickets. 1,25D is reported to modulate FGF23; thus, we hypothesized that 1,25D may be involved in modulating DMP-1 in an intermediary step. Murine cementoblasts (OCCM-30) and osteocyte-like cells (MLO-Y4 and MLO-A5), known to express DMP-1, were used to analyze effects of 1,25D on DMP-1 expression in vitro. DMP-1 mRNA levels decreased by 50% (p < .05) in the presence of 1,25D in all cell types, while use of a vitamin D receptor (VDR) agonist (EB1089) and antagonist (23S,25S)-DLAM-2P confirmed that VDR pathway activation was required for this response. Further analysis showed that histone deacetylase recruitment was necessary, but neither protein kinase A nor C pathways were required. In conclusion, our results support the hypothesis that 1,25D regulates DMP-1 expression through a VDR-dependent mechanism, possibly contributing to local changes in bone/tooth mineral homeostasis.

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Subgingival biodiversity in subjects with uncontrolled type-2 diabetes and chronic periodontitis.

BACKGROUND AND OBJECTIVE: There is a bidirectional relationship between periodontal disease and type-2 diabetes mellitus (DM). Inflammatory mediators may negatively affect glycemic control, and increased glucose levels and resultant glycation end-products may alter the host response against bacterial infection. However, no agreement has been reached regarding the effect of DM on periodontal subgingival microbiota. Therefore, the purpose of the present study was to compare the subgingival biodiversity in deep periodontal pockets of subjects with chronic periodontitis and either uncontrolled type-2 diabetes or no diabetes using 16S rRNA gene cloning and sequencing. MATERIAL AND METHODS: Twelve subjects with uncontrolled type-2 diabetes (glycated hemoglobin > 8%) and eleven nondiabetic subjects presenting severe and generalized chronic periodontitis were selected. Subgingival biofilm from periodontal pockets > 5 mm were assessed using the 16S rRNA gene cloning and sequencing technique. RESULTS: Significant differences were observed in subgingival microbiota between diabetic and nondiabetic subjects. Diabetic subjects presented higher percentages of total clones of TM7, Aggregatibacter, Neisseria, Gemella, Eikenella, Selenomonas, Actinomyces, Capnocytophaga, Fusobacterium, Veillonella and Streptococcus genera, and lower percentages of Porphyromonas, Filifactor, Eubacterium, Synergistetes, Tannerella and Treponema genera than nondiabetic individuals (p < 0.05). Moreover, some phylotypes, such as Fusobacterium nucleatum, Veillonella parvula, V. dispar and Eikenella corrodens were detected significantly more often in diabetic subjects than in nondiabetic subjects (p < 0.05). CONCLUSION: Subjects with uncontrolled type-2 diabetes and chronic periodontitis presented significant dissimilarities in subgingival biodiversity compared with nondiabetic subjects.

Deficiency in acellular cementum and periodontal attachment in bsp null mice.

Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp (-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp (-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp (-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects.

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Effect of autologous bone marrow-derived cells associated with guided bone regeneration or not in the treatment of peri-implant defects.

This study investigated the effect of bone marrow-derived cells associated with guided bone regeneration in the treatment of dehiscence bone defects around dental implants. Iliac-derived bone marrow cells were harvested from dogs and phenotypically characterized with regard to their osteogenic properties. After teeth extraction, three implant sites were drilled, dehiscences created and implants placed. Dehiscences were randomly assigned to: bone marrow-derived cells, bone marrow-derived cells+guided bone regeneration, and control (no treatment). After 3 months, implants with adjacent tissues were processed histologically, bone-to-implant contact, bone fill within the threads, new bone area in a zone lateral to the implant, new bone height, and new bone weight at the bottom of the defect were determined. Phenotypic characterization demonstrated that bone marrow-derived cells presented osteogenic potential. Statistically higher bone fill within the threads was observed in both bone marrow-derived cells+guided bone regeneration bone marrow-derived cell groups compared with the control group (P<0.05), with no difference between the groups treated with cells (P>0.05). For the other parameters (new bone area, bone-to-implant contact, new bone height and new bone weight), only the bone marrow-derived cells+guided bone regeneration group presented higher values compared with the non-treated control (P<0.05). Bone marrow-derived cells provided promising results for peri-implantar bone regeneration, although the combined approach seems to be relevant, especially to bone formation out of the implant threads.

Wnt/ß-catenin pathway regulates bone morphogenetic protein (BMP2)-mediated differentiation of dental follicle cells.

BACKGROUND AND OBJECTIVE: Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/ßcatenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/ß-catenin pathway. MATERIAL AND METHODS: SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/ß-catenin pathway activation assessed by western blotting, ß-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of ß-catenin. RESULTS: WNT3A induced ß-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/ß-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, ß-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous ß-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/ß-catenin signaling. CONCLUSION: These data suggest that stabilization of ß-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/ß-catenin signaling to promote cell maturation.