BMP-2 and asporin expression regulate 5-aza-dC-mediated osteoblast/cementoblast differentiation of periodontal dental ligament mesenchymal progenitor cells.

Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 µM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Novel rare frameshift variation in aggressive periodontitis: Exomic and familial-screening analysis.

BACKGROUND: Aggressive periodontitis (AgP), currently periodontitis grade C, presents early onset, rapid progression, and a poorly established genetic association. Thus, this study aimed to identify genetic variants associated with AgP via whole exome sequencing (WES) through a familial screening approach. METHODS: WES was performed in two nuclear families, including a proband and a parent affected by AgP and an unaffected parent and sibling. Common variants among affected individuals, excluding those common to healthy people, from each family, composed the data set associated with AgP. In silico analysis evaluated the impact of each variant on protein structure and protein-protein interactions. Moreover, identified deleterious variants were validated in a populational analysis (n = 96). RESULTS: The missense single nucleotide variations (SNVs) rs142548867 in EEFSEC (c.668C>T), rs574301770 in ZNF136 (c.466C>G), and rs72821893 in KRT25 (c.800G>A) and the frameshift indels rs37146475 in GPRC6A (c.2323-2324insT) and c.1366_1372insGGAGCAG in ELN were identified in AgP and have a predicted functional impact on proteins. In silico analysis indicated that the indel in GPRC6A generates a loss of the C-terminal tail of the Gprca protein. Furthermore, this SNV was significantly associated with AgP in a population-based investigation. CONCLUSION: Novel frameshift variation in GPRC6A (c.2323-2324insT) was identified as a potential genetic alteration associated with AgP occurrence.

Local IL-10 level as a predictive factor in generalized aggressive periodontitis treatment response.

Generalized aggressive periodontitis (GAgP) presents a reduced response to non-surgical therapy. However, it is not clear if the initial clinical, microbiological or immunological characteristics are impacting the worse response to treatment. This study aimed to identify the predictive value of clinical, microbiological and immunological patterns on the clinical response to therapy in GAgP patients. Twenty-four GAgP patients were selected, and gingival crevicular fluid (GCF) and subgingival biofilm were collected. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia levels were evaluated by qPCR, and IL-1ß and IL-10 concentration by ELISA. Twelve patients were treated with SRP (scaling and root planning), and twelve with SRP plus 375 mg amoxicillin and 250 mg metronidazole (8/8 hours, 7 days) (SRP + AM). The clinical changes (Probing Pocket Depth [PPD] reduction and Clinical Attachment Level [CAL] gain) 6 months post-treatment were correlated to the initial clinical, inflammatory and microbiological variables using stepwise logistic regression (α = 5%). CAL gain at 6 months was 1.16 ± 0.77 for SRP and 1.74 ± 0.57 mm for SRP + AM (P > .05). PPD reduction was 1.96 ± 0.82 for SRP and 2.45 ± 0.77 mm for SRP + AM (P < .05). In the SRP group, IL-10 showed a predictive value for clinical response. The higher the IL-10 concentration at baseline, the higher the reduction in PPD at 6 months (P = .01, r = .68). However, when antimicrobials were administered, no significant influence was detected (P > .05). It can be concluded that the IL-10 levels in GFC act as a predictor of clinical response to GAgP. Moreover, the intake of antimicrobials appears to overlap the influence of the inflammatory response on clinical response to treatment. Clinical trial registration number: NCT03933501.

Experimental and clinical studies on regenerative periodontal therapy.

The recognition of a periodontal therapy as a regenerative procedure requires the demonstration of new cementum, periodontal ligament, and bone coronal to the base of the defect. A diversity of regenerative strategies has been evaluated, including root surface conditioning, bone grafts and bone substitute materials, guided tissue regeneration, enamel matrix proteins, growth/differentiation factors, combined therapies and, more recently, tissue-engineering approaches. The aim of this chapter of Periodontology 2000 is to review the research carried out in Latin America in the field of periodontal regeneration, focusing mainly on studies using preclinical models (animal models) and randomized controlled clinical trials. This review may help clinicians and researchers to evaluate the current status of the therapies available and to discuss the challenges that must be faced in order to achieve predictable periodontal regeneration in clinical practice.

RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are important for tissue regeneration but their epigenetic regulation is not well understood. Here we investigate the ability of a non-nucleoside DNA methylation inhibitor, RG108 to induce epigenetic changes at both global and gene-specific levels in order to enhance mesenchymal cell markers, in hBMSCs. hBMSCs were treated with complete culture medium, 50 µM RG108 and DMSO for three days and subjected to viability and apoptosis assays, global and gene-specific methylation/hydroxymethylation, transcript levels' analysis of epigenetic machinery enzymes and multipotency markers, protein activities of DNMTs and TETs, immunofluorescence staining and western blot analysis for NANOG and OCT4 and flow cytometry for CD105. The RG108, when used at 50 µM, did not affect the viability, apoptosis and proliferation rates of hBMSCs or hydroxymethylation global levels while leading to 75% decrease in DNMTs activity and 42% loss of global DNA methylation levels. In addition, DNMT1 was significantly downregulated while TET1 was upregulated, potentially contributing to the substantial loss of methylation observed. Most importantly, the mesenchymal cell markers CD105, NANOG and OCT4 were upregulated being NANOG and OCT4 epigenetically modulated by RG108, at their gene promoters. We propose that RG108 could be used for epigenetic modulation, promoting epigenetic activation of NANOG and OCT4, without affecting the viability of hBMSCs. DMSO can be considered a modulator of epigenetic machinery enzymes, although with milder effect compared to RG108.

Evaluation of peri-implant marginal tissues around tissue-level and bone-level implants in patients with a history of chronic periodontitis.

OBJECTIVE: To evaluate clinical and radiographic characteristics in peri-implant marginal tissues in patients with a history of chronic periodontitis, rehabilitated using tissue-level or bone-level implants. MATERIAL AND METHODS: Using a split-mouth design, 20 patients with a history of chronic periodontitis were selected and received two different implants, tissue-level group (n = 20) and the bone-level group (n = 20). Peri-implant probing depth, relative peri-implant mucosal margin position, relative peri-implant clinical attachment level, peri-implant plaque index and peri-implant bleeding on probing were evaluated at prosthesis installation, 1, 3, 6, 12 and 24 months after implant loading. Radiographic marginal bone level was evaluated at implant insertion, prosthesis installation, 6 and 24 months after implant loading. RESULTS: The mean difference of peri-implant marginal bone resorption from implant installation to 24 months in function was 0.75 ± 1.12 mm for the tissue-level group and 0.70 ± 0.72 mm for the bone-level group. No statistically significant difference was found between groups at all assessment periods for clinical and radiographic peri-implant evaluation. CONCLUSION: Under a rigid supportive therapy, both approaches performed likewise regarding clinical and radiographic parameters for rehabilitation of patients with a history of chronic periodontitis.

Furcation Therapy With Enamel Matrix Derivative: Effects on the Subgingival Microbiome.

BACKGROUND: Although enamel matrix derivative (EMD) has been used to promote periodontal regeneration, little is known of its effect on the microbiome. Therefore, this investigation aims to identify changes in periodontal microbiome after treatment with EMD using a deep-sequencing approach. METHODS: Thirty-nine patients with mandibular Class II buccal furcation defects were randomized to beta-tricalcium-phosphate/hydroxyapatite graft (BONE group), EMD+BONE, or EMD alone. Plaque was collected from furcation defects at baseline and 3 and 6 months post-treatment. Bacterial DNA was analyzed using terminal restriction fragment length polymorphism and 16S pyrotag sequencing, resulting in 169,000 classifiable sequences being compared with the Human Oral Microbiome Database. Statistical comparisons were made using parametric tests. RESULTS: At baseline, a total of 422 species were identified from the 39 defects, belonging to Fusobacterium, Pseudomonas, Streptococcus, Filifactor, and Parvimonas. All three regenerative procedures predictably altered the disease-associated microbiome, with a restitution of health-compatible species. However, EMD and BONE+EMD groups demonstrated more long-term reductions in a higher number of species than the BONE group (P <0.05), especially disease-associated species, e.g., Selenomonas noxia, F. alocis, and Fusobacterium. CONCLUSIONS: EMD treatment predictably alters a dysbiotic subgingival microbiome, decreasing pathogen richness and increasing commensal abundance. Further investigations are needed to investigate how this impacts regenerative outcomes.

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Neuropilin Controls Endothelial Differentiation by Mesenchymal Stem Cells From the Periodontal Ligament.

BACKGROUND: Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105(+)-enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)-like cells and to give insights into the mechanism involved. METHODS: CD105(+)-enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM-2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real-time polymerase chain reaction; 2) Western blotting; 3) fluorescence-activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays. RESULTS: Data analyses demonstrated that EGM-2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain-containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube-like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105(+)-enriched PDLSCs resulted in increased cord/tube-like structures in a matrigel assay. CONCLUSION: These findings demonstrated the potential of CD105(+)-enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.

Enamel matrix protein derivative and/or synthetic bone substitute for the treatment of mandibular class II buccal furcation defects. A 12-month randomized clinical trial.

OBJECTIVE: This study aims to clinically evaluate the treatment of mandibular class II furcation defects with enamel matrix derivative (EMD) and/or a bone substitute graft made of ß-tricalcium phosphate/hydroxyapatite (ßTCP/HA). MATERIALS AND METHODS: Forty-one patients, presenting a mandibular class II buccal furcation defect, probing pocket depth (PPD) ≥4 mm and bleeding on probing, were included. They were randomly assigned to the groups: 1-EMD (n = 13); 2-ßTCP/HA (n = 14); 3-EMD + ßTCP/HA (n = 14). Plaque index (PI), gingival index (GI), relative gingival margin position (RGMP), relative vertical and horizontal attachment level (RVCAL and RHCAL), and PPD were evaluated at baseline and 6 and 12 months. The mean horizontal clinical attachment level gain was considered the primary outcome variable. RESULTS: No significant intragroup differences were observed for RGMP, but significant changes were observed for RVCAL, RHCAL, and PPD for all groups (p < 0.05). After 12 months, the mean horizontal clinical attachment level gain was 2.77 ± 0.93 mm for EMD, 2.64 ± 0.93 mm for ßTCP/HA, and 2.93 ± 0.83 mm for EMD + ßTCP/HA, with no significant differences among the groups. At the end of the study, 85.3 % of the sites were partially closed; however, no complete closure was observed. CONCLUSION: EMD + ßTCP/HA does not provide a significant advantage when compared to the isolated approaches. All three tested treatments promote significant improvements and partial closure of class II buccal furcation defects. Based on its potential to induce periodontal regeneration, EMD may be considered an attractive option for this type of defect, but complete closure remains an unrealistic goal. CLINICAL RELEVANCE: The partial closure of buccal furcation defects can be achieved after the three tested approaches. However, the combined treatment does not provide a significant benefit when compared to the isolated approaches.

DNA methylation analysis of SOCS1, SOCS3, and LINE-1 in microdissected gingival tissue.

OBJECTIVES: DNA methylation plays a critical role in the regulation of the transcription of the suppressors of cytokine signaling (SOCS) 1 and SOCS3, which are modulators in the inflammation. We hypothesized that the methylation status of SOCS1, SOCS3, and long interspersed nuclear element (LINE)-1 in gingival tissues previously inflamed would be similar to that found in gingival tissues without clinical inflammation in the period studied. MATERIALS AND METHODS: Laser capture microdissection was performed to isolate epithelial and connective gingival tissues. The groups were comprised by ten patients without history of periodontitis and absence of clinical signs of inflammation in the gingiva during the study (healthy group) and ten patients with history of periodontitis, presenting inflammation in the gingival tissue at the first examination of the study (controlled chronic periodontitis group). The gingival biopsies from the controlled chronic periodontitis group were collected after controlling the inflammation. DNA methylation patterns were analyzed using methylation-specific high-resolution melting and combined bisulfite restriction analysis. RESULTS: DNA methylation levels for SOCS1 and SOCS3 did not differ between groups or tissues; likewise, no differences were observed in total LINE-1 methylation or at specific loci. CONCLUSION: At 3 months following control of inflammation in gingival tissues, the methylation profile of SOCS1, SOCS3, and LINE-1 is similar between connective and epithelial tissues from patients that were previously affected or not by chronic inflammation. CLINICAL RELEVANCE: Clinical results of a successful treatment are observed after inflammation control and the molecular findings illustrate local and general methylation patterns in recovering tissues toward health conditions and might help to understand events that are occurring in oral cells.

Characterization of highly osteoblast/cementoblast cell clones from a CD105-enriched periodontal ligament progenitor cell population.

BACKGROUND: It is known that periodontal ligament (PDL) harbors a heterogeneous progenitor cell population at different stages of lineage commitment. However, characterization of PDL stem cells committed to osteoblast/cementoblast (O/C) differentiation remains to be elucidated. The present study is carried out to isolate single cell-derived, cluster of differentiation (CD)105-positive PDL clones and to characterize the clones that present high potential to differentiate toward O/C phenotype in vitro. METHODS: Isolation of single cell-derived colonies (clones) from a CD105-enriched PDL progenitor cell population was performed by the ring-cloning technique. Cell clones were evaluated for their O/C differentiation potential, metabolic activity, and expression of STRO-1 protein. Additionally, the clones that showed potential to O/C differentiation were characterized by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for expression of runt-related transcriptor factor 2 (RUNX2), alkaline phosphatase, CD105, and CD166 during osteogenic induction. RESULTS: Six PDL-CD105(+) clones were obtained, three being highly O/C clones (C-O) and three others that did not have the ability to produce mineralized matrix in vitro (C-F). The C-O group showed lower metabolic activity compared with the C-F group, and both cell groups were positively immunostained for STRO-1. qRT-PCR analysis demonstrated an increased expression of transcripts for RUNX2 and CD166 during the maturation of C-O cells toward O/C phenotype. CONCLUSIONS: These results provide evidence that PDL-CD105(+) purified progenitor cells comprise a heterogeneous cell population that presents a cell subset with high O/C potential and, further, that surface antigen CD166 is modulated during the O/C maturation of this cell subset.

Photodynamic therapy during supportive periodontal care: clinical, microbiologic, immunoinflammatory, and patient-centered performance in a split-mouth randomized clinical trial.

BACKGROUND: This study investigates the effect of photodynamic therapy (PDT) as monotherapy during supportive periodontal therapy. METHODS: A split-mouth, randomized controlled trial was conducted in patients with chronic periodontitis (N = 22) presenting at least three residual pockets (probing depth [PD] ≥5 mm with bleeding on probing [BOP]). The selected sites randomly received the following: 1) PDT; 2) photosensitizer (PS); or 3) scaling and root planing (SRP). At baseline and 3 and 6 months, clinical, microbiologic (real-time polymerase chain reaction analyses), cytokine pattern (multiplexed bead immunoassay), and patient-centered (regarding morbidity) evaluations were performed. RESULTS: All therapies promoted similar improvements in clinical parameters throughout the study (P <0.05), except that BOP was not reduced in the PS protocol (P >0.05). Lower levels of Aggregatibacter actinomycetemcomitans were observed in the PDT and SRP protocols at 3 months when compared with the PS protocol (P <0.05). An inferior frequency detection of Porphyromonas gingivalis was observed in the PDT protocol at 3 and 6 months and in the SRP protocol at 6 months from baseline (P <0.05). In addition, PDT protocol presented inferior frequency of P. gingivalis at 3 months when compared with the other therapies (P <0.05). Only patients in the PDT protocol exhibited augmented levels of anti-inflammatory interleukin (IL)-4 and reduced proinflammatory IL-1ß and IL-6 throughout the study (P <0.05). Intergroup analyses showed reduced IL-10 and increased interferon-γ and IL-1ß levels in the PS protocol when compared with the other therapies during follow-ups (P <0.05). No differences in morbidity were observed between the therapies (P >0.05), although the need for anesthesia was higher in SRP-treated sites (P <0.05). CONCLUSION: PDT as an exclusive therapy may be considered a non-invasive alternative for treating residual pockets, offering advantages in the modulation of cytokines.

Outcome of enamel matrix derivative treatment in the presence of chronic stress: histometric study in rats.

BACKGROUND: Psychologic stress and clinical hypercortisolism have been related to direct effects on bone metabolism. However, there is a lack of information regarding the outcomes of regenerative approaches under the influence of chronic stress (CS). 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 CS in the rat model. METHODS: Twenty Wistar rats were randomly assigned to two groups; G1: CS (restraint stress for 12 hours/day) (n = 10), and G2: not exposed to CS (n = 10). Fifteen days after initiation of CS, 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 euthanized 21 days later. RESULTS: G1 showed less bone density (BD) compared to G2. EMD provided an increased defect fill (DF) in G1 and higher BD and new cementum formation (NCF) in both groups. 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. CONCLUSIONS: CS may produce a significant detrimental effect on BD. EMD may provide greater DF compared to non-treated control in the presence of CS and increased BD and NCF in the presence or absence of CS.

Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype.

Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318_1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggests a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in the tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization.

Photodynamic therapy in the treatment of class II furcation: a randomized controlled clinical trial.

AIM: To investigate the effect of photodynamic therapy (PDT) as adjunct to mechanical therapy in furcations. MATERIALS AND METHODS: A double-blind, parallel, randomized controlled clinical trial was conducted in subjects presenting class II furcations. The subjects were randomly allocated to a test (PDT; n = 16) or control group (non-activated laser/only photosensitizer; n = 21). At baseline, 3 and 6 months, clinical, microbiological and cytokine pattern evaluation was performed. Clinical attachment level was defined as the primary outcome variable. RESULTS: Clinical parameters improved after both therapies (p < 0.05) with no differences between groups at any time point (p > 0.05). At 6 months, real-time PCR evaluation showed a decrease in Porphyromonas gingivalis and Tannerella forsythia only in the PDT group (p < 0.05) with no inter-group differences. Regarding cytokines, IL-4 and IL-10 levels increased in both groups at 6 months. GM-CSF, IL-8, IL-1ß and IL-6 levels decreased only in the PDT group after 3 months (p < 0.05). At 3 months, inter-group analyses showed that GM-CSF, IFN-γ, IL-6 and IL-8 levels were lower in the PDT group. At 6 months, lower IL-1ß levels were also observed in the PDT group (p < 0.05). CONCLUSION: Photodynamic therapy did not promote clinical benefits for class II furcations; however, advantages in local levels of cytokines and a reduction in periodontopathogens were demonstrated.

Hydroxyapatite/ß-tricalcium phosphate and enamel matrix derivative for treatment of proximal class II furcation defects: a randomized clinical trial.

OBJECTIVE: To clinically evaluate proximal furcations treated with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) isolated or combined with enamel matrix derivative (EMD). MATERIAL AND METHODS: Thirty patients, presenting at least one proximal class II furcation defect, probing pocket depth (PPD) ≥5 mm and bleeding on probing, were included. The defects were assigned to the HA/ß-TCP group (n = 15); open-flap debridement (OFD) + HA/ß-TCP filling, or, HA/ß-TCP-EMD group (n = 15); OFD + HA/ß-TCP + EMD filling. Plaque (PI) and gingival index (GI), PPD, relative gingival margin position (RGMP), vertical and horizontal attachment level (RVAL and RHAL), vertical and horizontal bone level (RVBL and RHBL), and furcation diagnosis were evaluated at baseline and at 6 months. RESULTS: Both groups presented improvements after therapies (p < 0.05); however, no inter-group differences could be seen in any single parameter (p > 0.05). At 6 months, the gains in rVCAL in the HA/ß-TCP and HA/ß-TCP-EMD groups were 1.47 ± 0.99 and 2.10 ± 0.87 mm, while the RHCAL gains were 1.47 ± 1.46 and 1.57 ± 1.58 mm (p > 0.05). The RVBL and RHBL gains for the HA/ß-TCP and HA/ß-TCP-EMD group were 1.47 ± 1.13 and 1.70 ± 1.26 mm, and 1.90 ± 1.11 and 1.70 ± 1.37 mm respectively (p > 0.05). The HA/ß-TCP-EMD group showed seven closed furcations versus four in the HA/ß-TCP group (p > 0.05). CONCLUSION: Both treatments lead to improvements in all clinical variables studied in the present trial. However, the closure of proximal class II furcation defects is still unpredictable.

Histometric analysis of the effect of enamel matrix derivative on the healing of periodontal defects in rats with diabetes.

BACKGROUND: Diabetes mellitus (DM) involves metabolic changes that can negatively influence periodontal tissues, resulting in impaired periodontal repair. There is a lack of information about the outcomes of regenerative approaches under the influence of DM. Enamel matrix derivatives (EMDs) have 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 DM. METHODS: Twenty Wistar rats were randomly assigned to two groups: group 1 (G1): DM was induced with a single intraperitoneal injection of streptozotocin (STZ) (n = 10); group 2 (G2): rats were not exposed to STZ (n = 10). Seven days after DM induction, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. After the surgeries, the defects of each animal were randomly assigned to two subgroups: non-treated (control) and treated with EMD. The animals were euthanized 21 days later, and the percentage of defect fill (DF), newly formed bone density (BD), and new cementum formation (NCF) were histometrically assessed. The number of osteoclasts was determined by tartrate-resistant acid phosphatase. Weight and blood glucose were also analyzed. Mann-Whitney U test was used for comparison among groups and Wilcoxon test for comparison between the start and end times (weight and blood glucose) and between treatments (NCF and number of osteoclasts). One-way analysis of variance was used to assess DF and BD. Tukey test was used when the analysis of variance test detected significant differences (α = 5%). RESULTS: G1 (DM) showed less DF and BD compared with G2. EMD provided an increased DF in both groups and enhanced BD and NCF only in G2. The number of TRAP-positive osteoclasts was significantly higher in EMD-treated sites of G1. CONCLUSIONS: DM may produce a significant detrimental effect on BD. EMD may provide greater DF under diabetic or normal conditions; however, it may not significantly increase NCF in animals with DM.

Clinical and microbiological changes after minimally invasive therapeutic approaches in intrabony defects: a 12-month follow-up.

OBJECTIVES: This 12-month randomized, controlled trial evaluated the clinical effects and microbiological changes of minimally invasive nonsurgical and surgical approaches for the therapy of intrabony defects. MATERIALS AND METHODS: Twenty-nine subjects with intrabony defects in single-rooted tooth were randomly assigned to; (1) minimally invasive nonsurgical technique (MINST) or (2) minimally invasive surgical technique (MIST). Quantities of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Porphyromonas gingivalis, determined by using real-time PCR, were evaluated at baseline, 3, 6, and 12 months after the treatments. Clinical recordings--probing depth (PD), position of the gingival margin (PGM), and relative clinical attachment level (RCAL)--were obtained at baseline and 12 months post-therapy. The primary outcome variable of the study was RCAL. RESULTS: Both treatment modalities resulted in an improvement in all clinical recordings, with significant PD reductions (p < 0.05), RCAL gains (p < 0.05), and no change in the PGM (p > 0.05) after 12 months in both MINST and MIST groups. No clinical differences were observed between groups (p > 0.05). Regarding the microbiological outcomes, at the re-examinations, a significant decrease was observed for T. forsythia and P. gingivalis when compared with baseline (p < 0.05) for both treatments. The amount of A. actinomycetemcomitans did not reduced decrease throughout the study (p > 0.05). Intergroup differences in the microbiological assay were not found at any time point (p > 0.05). CONCLUSIONS: Both MINST and MIST provided comparable clinical results and microbiological changes in the treatment of intrabony defects over 12 months follow-up. CLINICAL RELEVANCE: This randomized, controlled, parallel trial revealed that both therapeutic modalities may promote clinical and microbiological benefits at 12 months post-therapy.