Relevance of Caspase-1 and Nlrp3 Inflammasome on Inflammatory Bone Resorption in A Murine Model of Periodontitis.

This study investigates the role of NLRP3 inflammasome and its main effector Caspase-1 in inflammation and alveolar bone resorption associated with periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans (Aa) was injected 3x/week (4 weeks) into gingival tissues of wild-type (WT), Nlrp3-KO and Caspase1-KO mice. Bone resorption was measured by µCT and osteoclast number was determined by tartrate-resistant acid phosphatase (TRAP) staining. Inflammation was assessed histologically (H/E staining and immunofluorescence of CD45 and Ly6G). In vitro studies determined the influence of Nlrp3 and Caspase-1 in Rankl-induced osteoclast differentiation and activity and on LPS-induced expression of inflammation-associated genes. Bone resorption was significantly reduced in Casp1-KO but not in Nlrp3-KO mice. Casp1-KO mice had increased in osteoclast numbers, whereas the inflammatory infiltrate or on gene expression were similar to those of WT and Nlrp3-KO mice. Strikingly, osteoclasts differentiated from Nlrp3-deficient macrophages had increased resorbing activity in vitro. LPS-induced expression of Il-10, Il-12 and Tnf-α was significantly reduced in Nlrp3- and Casp1-deficient macrophages. As an inceptive study, these results suggest that Nlrp3 inflammasome does not play a significant role in inflammation and bone resorption in vivo and that Caspase-1 has a pro-resorptive role in experimental periodontal disease.

Short Peptides Protect Oral Stem Cells from Ageing.

Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs). Cells at the 25th passage were divided into 3 groups: 1 - control (without adding peptide), 2 - treated with AEDG peptide, 3 - treated with KED peptide. Cell cultures were analyzed by an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16 and p21 mRNA expression by 1.56-2.44 times in comparison with the control group. KED peptide decreased p16 and p21 mRNA expression by 1.82-3.23 times in comparison with the control group. These results were confirmed by immunofluorescent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG and KED peptide, which was shown early in animal and cells models.

Targeting macrophages and their recruitment in the oral cavity using swellable (+) alpha tocopheryl phosphate nanostructures.

The phosphorylation of (+) alpha tocopherol produces adhesive nanostructures that interact with oral biofilms to restrict their growth. The aim of this work was to understand if these adhesive (+) alpha tocopheryl phosphate (α-TP) nanostructures could also control macrophage responses to the presence of oral bacteria. The (+) α-TP planar bilayer fragments (175 nm ±â€¯21 nm) formed in a Trizma®/ethanol vehicle swelled when exposed to the cell lines (maximum stabilized size = 29 µm). The swelled (+) α-TP aggregates showed selective toxicity towards THP-1 macrophages (LD50 = 304 µM) compared to human gingival fibroblasts (HGF-1 cells; LD50 > 5 mM), and they inhibited heat killed bacteria stimulated MCP-1 production in both macrophages (control 57.3 ±â€¯18.1 pg/mL vs (+) α-TP 6.5 ±â€¯3.2 pg/mL) and HGF-1 cells (control 673.5 ±â€¯133 pg/mL vs (+) α-TP - 463.9 ±â€¯68.9 pg/mL).

Multi-species oral biofilm promotes reconstructed human gingiva epithelial barrier function.

Since the oral mucosa is continuously exposed to abundant microbes, one of its most important defense features is a highly proliferative, thick, stratified epithelium. The cellular mechanisms responsible for this are still unknown. The aim of this study was to determine whether multi-species oral biofilm contribute to the extensive stratification and primed antimicrobial defense in epithelium. Two in vitro models were used: 3D reconstructed human gingiva (RHG) and oral bacteria representative of multi-species commensal biofilm. The organotypic RHG consists of a reconstructed stratified gingiva epithelium on a gingiva fibroblast populated hydrogel (lamina propria). Biofilm was cultured from healthy human saliva, and consists of typical commensal genera Granulicatella and major oral microbiota genera Veillonella and Streptococcus. Biofilm was applied topically to RHG and host-microbiome interactions were studied over 7 days. Compared to unexposed RHG, biofilm exposed RHG showed increased epithelial thickness, more organized stratification and increased keratinocyte proliferation. Furthermore biofilm exposure increased production of RHG anti-microbial proteins Elafin, HBD2 and HBD3 but not HBD1, adrenomedullin or cathelicidin LL-37. Inflammatory and antimicrobial cytokine secretion (IL-6, CXCL8, CXCL1, CCL20) showed an immediate and sustained increase. In conclusion, exposure of RHG to commensal oral biofilm actively contributes to RHG epithelial barrier function.

An In Vitro Comparative Study of Multisource Derived Human Mesenchymal Stem Cells for Bone Tissue Engineering.

Mesenchymal stem cells (MSCs) have been considered promising tools for tissue engineering and regenerative medicine. However, the optimal cell source for bone regeneration remains controversial. To better identify seed cells for bone tissue engineering, we compared MSCs from seven different tissues, including four from dental origins, dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), gingival MSCs (GMSCs), and dental follicle stem cells (DFSCs); two from somatic origins, bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs); and one from birth-associated perinatal tissue umbilical cord (UCMSCs). We cultured the cells under a standardized culture condition and studied their biological characteristics. According to our results, these cells exhibited similar immunophenotype and had potential for multilineage differentiation. MSCs from dental and perinatal tissues proliferated more rapidly than those from somatic origins. Simultaneously, DPSCs and PDLSCs owned stronger antiapoptotic ability under the microenvironment of oxidative stress combined with serum deprivation. In respect to osteogenic differentiation, the two somatic MSCs, BM-MSCs and ADSCs, demonstrated the strongest ability for osteogenesis compared to PDLSCs and DFSCs, which were just a little bit weaker than the formers. However, GMSCs and UCMSCs were the most pertinacious ones to differentiate to osteoblasts. We also revealed that the canonical intracellular protein kinase-based cascade signaling pathways, including PI3K/AKT, MAPK/ERK, and p38 MAPK, possessed different levels of activation in different MSCs after osteoblast induction. Our conclusions suggest that PDLSCs might be a good potential alternative to BM-MSCs for bone tissue engineering.

Regeneration of gingival tissue using in situ tissue engineering with collagen scaffold.

OBJECTIVE: The aim of the study was to evaluate 2 types of collagen scaffold for gingival regeneration. STUDY DESIGN: Two types of collagen scaffolds, CS-pH7.4 and CS-pH3.0, were prepared by processing atelocollagen at pH 7.4 or 3.0, respectively, followed by dehydrothermal treatment. Gingival wounds with sizes of 4 × 6 mm (rectangle) or 6 mm diameter (circle) were made with buccal incisions in beagle dogs. The defective area was surgically covered with the CS-pH7.4, CS-pH3.0, or no scaffold (control). Gingival regeneration was assessed by monitoring the differences in the lengths of the epithelial and submucosal tissues at the wound site and the normal site. Histopathologic assessments were performed by 4 evaluators independently; statistical significance was evaluated by using the Wald test. RESULTS: Significantly higher recovery of epithelial and submucosal tissues, which, in turn, resulted in recovery of gum thickness, was observed in gingival wounds treated with the CS-pH7.4 compared with that in the control. CS-pH3.0 treatment also resulted in higher gingival regeneration compared with the control; however, the effects were more pronounced in wounds treated with the CS-pH7.4. CS-pH7.4-treated wounds showed better gingival regeneration compared with the control and CS-pH3.0-treated wounds, even after adjusting for interevaluator differences using a linear mixed model. CONCLUSIONS: CS-pH7.4 is a promising scaffold for gingival tissue regeneration.

The antimicrobial peptide, human ß-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases.

Previous studies have demonstrated increased expression and raised levels of human ß-defensin (hBD)-1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri-implantitis, oral bone-resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD-1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD-1 and cell viability was assayed by MTT. PBMCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) in the presence or absence of non-toxic doses of hBD-1. In vitro osteoclastogenesis was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, osteoclast-specific gene expression, and a resorption pit assay. Involvement of mitogen-activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD-1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP-positive multinuclear cells and resorption areas on the dentin slices, and further up-regulated expressions of osteoclast-specific genes compared to those by RANKL treatment (p <0.05). However, hBD-1 treatment without RANKL failed to induce formation of osteoclast-like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD-1 co-treatment (p<0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD-1-enhanced osteoclastogenesis. Collectively, hBD-1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs.

Cytotoxicity and Antimicrobial Activity of Oral Rinses In Vitro.

While oral rinses used for cosmetic purposes only do not necessarily have to be antiseptic, antimicrobial activity is required for medical indications, including oral and periodontal surgery. So the question arises-is the antimicrobial activity of oral rinses associated with any destructive changes in cell viability in vitro? To answer this question, we examined twelve oral rinses with respect to their antimicrobial and cytotoxic activity. Antimicrobial activity was screened against five bacterial strains using disc diffusion. Cytotoxicity was determined by mitochondrial reductase activity with primary gingival fibroblasts, L929 cells, and HSC-2 epithelial cells. Phase contrast microscopy and trypan blue staining were then performed to reveal cell morphology. Cells remained vital after exposure to oral rinses that were only used for cosmetic purposes. Moderate cytotoxic effects were observed for oral rinses containing 0.05% chlorhexidine, ethanol, or pegylated hydrogenated castor oil and sodium dodecyl sulfate. Other oral rinses containing 0.2% chlorhexidine and cocamidopropyl betaine exhibited strong cytotoxic and antimicrobial activity. Strong cytotoxic but moderate antimicrobial activity was observed in oral rinses containing cetylpyridinium chloride. The in vitro data show that oral rinses are heterogeneous with respect to their cytotoxic and antimicrobial effects. Based on their respective properties, oral rinses can be selected either to reduce the microbial load or for cosmetic purposes.

Premolar autotransplantation in juvenile dentition: quantitative assessment of vertical bone and soft tissue growth.

OBJECTIVE: Premolar autotransplantation represents an effective therapeutic option for the treatment of juvenile dentition with either aquired or congenital hypodontia. The objective of this prospective clinical study was to quantitatively assess bone and soft tissue levels after autogenous premolar transplantation by clinical and radiographic parameters. STUDY DESIGN: In the study, 26 premolars were transplanted in 20 patients after traumatic tooth loss (n = 16) or congenital aplasia (n = 10) in the anterior maxilla. Based on standardized photographic documentation, the relative soft tissue level was measured compared to the healthy adjacent teeth. Radiographic findings included evaluation of root resorption, pulp canal obliteration, and relative bone height. RESULTS: Average survival rate of transplanted premolars (n = 26) was 100% over a follow-up period of 29 months (range 10-60 months). The relative soft tissue level significantly increased by +1.1 mm (P < .01). Radiographs showed a tendency toward vertical bone growth. Continuous root development and signs of pulpal healing were observed postoperatively in 18 transplants (69.2%). CONCLUSIONS: Autogenous premolar transplantation represents a safe method to ensure functional and aesthetic rehabilitation in the anterior maxilla irrespective of the nature of tooth loss.

Repeated exposure to whole cigarette smoke promotes primary human gingival epithelial cell growth and modulates keratin expression.

BACKGROUND AND OBJECTIVE: The gingiva is the first oral tissue directly exposed to cigarette smoke (CS). Exposure to CS compromises the structure and function of gingival tissue. Damaging or altering the gingival epithelium leads to a compromised protective barrier of the periodontium, resulting in several diseases. The aim of this study was to assess the effect of repeated exposure to CS on gingival epithelial cell growth and on expression of apoptotic protein and keratin. MATERIAL AND METHODS: Primary human gingival epithelial cells were seeded on a collagen scaffold for 5 d to allow growth and stratification. The cells were then exposed for 5 min to whole CS for 3, 6 and 9 d. At the end of each exposure period, cell proliferation [using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays], gene expression [by quantitative reverse transcription polymerase chain reaction (qRT-PCR)] and protein production (by western blot analysis) were investigated. RESULTS: Higher metabolic activity was found in the CS-exposed cells than in the nonexposed cells, specifically after 3 and 6 d of exposure to CS. At 9 d there was no significant difference between CS-exposed and nonexposed cells. Metabolic activity was supported by the BrdU cell-proliferation analyses, which showed increased cell growth at 3 d compared with the control. However, at 6 and 9 d, cell proliferation in the CS-exposed culture was comparable to that in the nonexposed culture. Interestingly, the Bax/Bcl-2 protein ratios decreased with increased CS exposure, suggesting cell resistance. Moreover, protein analyses showed that CS decreased expression of keratin(K) 5 at 3, 6 and 9 d, and increased expression of K14 at 6 and 9 d. Finally, mRNA analyses showed significant decreases of K1, K6, K10 and K16 in CS-exposed cultures, correlating, at times, with a decrease of protein production. CONCLUSION: CS was shown to increase epithelial cell proliferation, which may involve cell resistance to apoptosis. This is supported by the modulation of expression of different keratin genes and proteins. Altogether, these data may explain the hyperplasia reported in gingival tissue, as well as periodontal disease, in smokers.

Vitamin C stimulates human gingival stem cell proliferation and expression of pluripotent markers.

Gingival stem cells (GSCs) are a novel source of mesenchymal stem cells (MSCs) that are easily accessed from the oral cavity. GSCs were considered valuable autograft MSCs with particular characteristics. However, the limitation in the number of available GSCs remains an obstacle. Therefore, this study aimed to stimulate GSC proliferation by ascorbic acid (AA) and determined the effects of AA on GSC pluripotent potential-related gene expression. GSCs were isolated from gum tissue by explant culture and continuously subcultured before analysis of stemness and effects of AA on pluripotent-related gene expression. GSCs cultured with various concentrations of AA showed increased proliferation in a dose-dependent manner. AA-treated GSCs showed significantly higher expression of SSEA-3, Sox-2, Oct-3/4, Nanog, and TRA-1-60 compared with control cells. More importantly, GSCs also maintained their stemness with MSC phenotypes and failed to cause tumors in nude athymic mice. Our results show that AA is a suitable factor to stimulate GSC proliferation.

Complication After Extraction of Natal Teeth with Continued Growth of a Dental Papilla.

The purposes of this case report were to describe a growing two-cm gingival mass that developed after natal teeth were extracted in a four-month-old female patient, present a review of the literature on the growth of a gingival mass after the extraction of natal teeth, and illustrate the clinical and histological features that differentiate this condition from other types of gingival masses in infants. Histological examination of the excised mass revealed that it contained tooth-like hard tissue (regular and irregular dentin) that intermingled with bone, dental pulp, and fibrous tissue. We found eight cases from 1962 to 2009 in which a soft-tissue mass with dentin-like hard tissue or a tooth-like structure had developed after the extraction of natal teeth. Based on clinical and histological findings, we deduced that the mass was the result of abnormal growth of a residual dental papilla, including mesenchymal stem cells. Consequently, dentists, obstetricians, gynecologists, and pediatricians should be aware of this potential complication and observe caution before they extract natal teeth.

ACELLULAR HUMAN AMNIOTIC MEMBRANE AS A THREE-DIMENSIONAL SCAFFOLD FOR THE TREATMENT OF MUCOGINGIVAL DEFECTS.

This study was conducted to evaluate the usefulness of decellularized and lyophilized extracellular matrix, which was acquired from human amniotic membrane, for surgical closure of the mucogingival defects. Preliminarily, to create a gingival recession defect, silk ligature was applied on the gingival part of the upper incisor in the first (experimental) (n=20) and second (control) (n=20) groups. On the 14th day, the ligature was removed and the damaged gingival tissues were resected. The formed mucogingival defect, in the animals of the first group, was covered with acellular human amniotic three-dimensional scaffold with bone marrow stem cells. Animals with mucogingival defect of the second group were left untreated and served as controls. Unlike the animals from the control group, in animals from the experimental group the mucogingival defect already on the seventh day was completely closed and there was the newly formed epithelial lining, which in shape and color did not differ from the normal. Acellular human amniotic membrane as a three-dimensional scaffold boosts angiogenesis and increases the reparative regeneration of the damaged tissues; and it is well-tolerated by the gingival tissues. Hence, human amniotic membrane might be a suitable alternative to other conventional methods of treating gingival recession.

Comparison of intermaxillary fixation screw versus eyelet interdental wiring for intermaxillary fixation in minimally displaced mandibular fracture: a randomized clinical study.

PURPOSE: The aim of the present randomized study was to evaluate the efficacy of intermaxillary fixation screw (IMFS) versus eyelet interdental wiring for intermaxillary fixation (IMF) in minimally displaced mandibular fractures. MATERIALS AND METHODS: A total of 50 patients with a minimally displaced mandibular fracture were enrolled, with 25 patients randomly selected for each group. In group I (study group, n = 25), the patients were treated using IMFS, and in group II (control group, n = 25), they received eyelet interdental wiring. Both techniques were assessed for the following parameters: time required for placement and removal of each type of IMF technique, time required for placement of IMF wires, postoperative occlusion, stability of the IMF wire, local anesthesia requirement during removal of each fixation type, oral hygiene status, glove perforation rate, and complications associated with both techniques. The collected data were analyzed using Student's unpaired t test or χ2 test. P < .05 was considered significant and the Statistical Package for Social Sciences software, version 10, was used for analysis. RESULTS: The average time required for placement in groups I and II was 17.56 and 35.08 minutes, respectively (P = .000). The time required for placement of the IMF wire in group I was 2.1 minutes and in group II was 6 minutes. The oral hygiene status was assessed, and the mean plaque index score for groups I and II was 1.44 and 2.12, respectively (P = .00). The glove perforation rate was much less in group I than in group II. Finally, the most common complication in both groups was mucosal growth. CONCLUSIONS: The results established the supremacy of IMFS compared with eyelet interdental wiring. Thus, we have concluded that IMFS, in the present scenario, is a safe and time-saving technique. IMFS is a cost-effective, straightforward, and viable alternative to cumbersome eyelet interdental and other wiring techniques for providing IMF, with satisfactory occlusion during closed reduction or intraoperative open reduction internal fixation of fractures. In addition, oral hygiene can be maintained, and the glove perforation rate was very low using IMFS. The relatively small sample size and limited follow-up period were the study limitations.

Effects of whole cigarette smoke on human gingival fibroblast adhesion, growth, and migration.

The aim of this study was to investigate the effects of a single exposure to whole cigarette smoke on human gingival fibroblast behavior. Normal oral mucosa fibroblasts were exposed once to whole cigarette smoke for 5, 15, or 30 min, and then were used to analyze cell adhesion, ß1-integrin expression, cell growth and viability, cell capacity to contract collagen gel, and cell migration following wound infliction. Our findings showed that when gingival fibroblasts were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell adhesion, a decrease in the number of ß1-integrin-positive cells, increased LDH activity in the target cells, and reduced growth. The smoke-exposed fibroblasts were also not able to contract collagen gel matrix and migrate following insult. Overall results demonstrate that a single exposure to whole cigarette smoke produced significant morphological and functional deregulation in gingival fibroblasts. This may explain the higher predisposition of tobacco users to oral infections and diseases such as cancer.

Cyclosporine-induced gingival overgrowth in New Zealand White rabbits (Oryctolagus cuniculus).

A high incidence of gingival overgrowth occurred in a group of New Zealand White rabbits receiving daily cyclosporine (15 mg/kg IM) while on a retinoblastoma study. Over the course of 2 mo, rabbits presented with clinical signs of ptyalism (4 of 18 rabbits), inappetence (3 of 18), or both (3 of 18); facial dermatitis and erythema occurred secondary to ptyalism. Reducing the dose of cyclosporine to 10 mg/kg led to complete resolution of clinical signs in all but 2 rabbits, which then received azithromycin (62.5 mg PO once daily for 7 d), a common treatment for cyclosporine-induced gingival overgrowth in other species. After dose reduction and azithromycin treatment, clinical signs resolved and did not reoccur for the remainder of the study. Fourteen rabbits were necropsied at the end of the study, and gingival width was measured. Although some rabbits were clinically normal, the gingiva in all rabbits was grossly thickened. Rabbits on cyclosporine had molar gingiva that was significantly thicker (4.8 mm) than controls (2.5 mm) not treated with cyclosporine. Histologic analysis of the gingiva revealed mild to moderate gingival epithelial hyperplasia, hyperkeratosis, and mild inflammation. Gingival overgrowth is a known side effect of cyclosporine administration in other species but, to our knowledge, this report is the first description of the condition in rabbits. Because rabbits frequently are used in studies that involve systemic cyclosporine administration, clinicians are advised to include this possibility in their differential list for cases involving hypersalivation, facial dermatitis, or inappetence in rabbits.

In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells.

AIM: To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form. METHODS AND RESULTS: Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD(50) showed that the fibroblasts were able to tolerate up to 80 microg ml(-1) for 24 h, dropping thereafter to 62 mug ml(-1) after 72 h of contact, compared to 160 microg ml(-1) after 24 h, and 80 microg ml(-1) after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25 microg ml(-1)) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition. CONCLUSION: These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.

Effects of the facial osseous defect morphology on gingival dynamics after immediate tooth replacement and guided bone regeneration: 1-year results.

PURPOSE: This article describes different scenarios of facial osseous defects when the osseous-gingival relationship exceeds 3 mm and evaluates the effects of the morphology of the compromised facial bone on gingival dynamics after immediate tooth replacement and guided bone regeneration. The implant success rate and peri-implant bone change were also reported. MATERIALS AND METHODS: Twenty-three patients treated consecutively with the mean age of 39.5 years (range, 25 to 63 years) underwent immediate tooth replacement and guided bone regeneration in sockets with facial bony defects exceeding 3 mm. Facial bony defects were categorized into V-, U-, and Ultra-U (UU)-shaped. The patients were evaluated clinically and radiographically at 1-year after implant placement. RESULTS: At 1-year, the implant success rate was 100% (23/23). No marginal bone change of greater than 1 mm was observed. Greater than 1.5 mm of facial gingival recessions were noted in 8.3% (1/12) of V-shaped, 42.8% (3/7) of U-shaped, and 100% (4/4) of UU-shaped defects. CONCLUSIONS: U- and UU-shaped defects showed significantly higher frequency and magnitude of facial gingival recession (>1.5 mm) when compared with V-shaped defects 1-year after immediate tooth replacement and guided bone regeneration. It is important to identify the type of facial bony defect during diagnosis and treatment planning, so that appropriate treatment can be prescribed. The combination of delayed implant placement after staged reconstruction of unfavorable U- and UU-shaped labial extraction socket defects should be considered in areas of high esthetic concern.

The esthetic challenge of adjacent implants.

Reformation of natural-appearing sulcular and papilla anatomy between adjacent implants in the esthetic zone presents a complex challenge for the implant team. Guidelines for implant placement of adjacent implants and soft tissue development are introduced that optimize esthetic results. The rule of 3x3x3 PIE is a mnemonic, based on biologically and prosthetically driven implant placement, which maximizes the potential for optimal esthetics between adjacent implants. The 4 interdependent principles of the 3x3x3 PIE rule are as follows: 1) the platforms of the implants should be located 3 mm apical to the zeniths of the predetermined facial-gingival margins of the planned restorations, 2) the centers of the implants should be placed at a distance at least 3 mm palatal to the anticipated facial margins, 3) interimplant spacing of 3 mm is required between adjacent implant platforms, and 4) the implants should emerge through the palatal incisal edge of the ensuing crown positions. After placement of adjacent implants by the surgeon according to the rule of 3x3x3 PIE, the restorative dentist must then develop the remaining soft tissue beginning with a provisional restoration. The reformed peri-implant gingiva is then supported by the subgingival contours of the definitive abutments and crowns. If ideal gingival esthetics are not achievable, pontics or gingival-colored porcelain can be acceptable alternatives. New technologies, including platform switching, are enhancing clinicians' abilities to deliver consistent esthetic results with adjacent implants.

Guidelines for flapless surgery.

With the introduction of in-office cone beam computed tomography (CT), improved access to conventional CT scanning, and dental implant treatment planning software allowing on-the-spot 3-dimensional evaluations of potential implant sites, the use of "flapless" implant surgery has gained popularity among surgeons. Although the flapless approach was initially suggested for and embraced by novice implant surgeons, the successful use of this approach often requires advanced clinical experience and surgical judgment. This article reviews the advantages and disadvantages of and indications and contraindications for flapless dental implant surgery, with special emphasis on requirements for establishing or maintaining long-term health and stability of the peri-implant soft tissues. Prerequisites for surgeons wishing to use the flapless tissue punch approach in dental implant surgery are outlined and put into perspective relative to conventional open-flap surgery techniques and other minimally invasive procedures currently used in implant surgery. Procedures for single- and multiple-tooth applications are illustrated.