Abstract This study investigated the influence of resveratrol on peri-implant repair and its effects on bone-related markers in ovariectomy-induced osteoporosis in rats. Animals were divided into: OVX+PLAC (n = 10): ovariectomized animals treated with placebo; OVX+RESV (n = 10): OVX treated with resveratrol; OVX+PLAC+ZOL (n = 10): OVX treated with PLAC and zoledronate; OVX+RESV+ZOL (n = 10): OVX treated with RESV and ZOL; and SHOVX+PLAC (n = 10): sham ovariectomy treated with PLAC. RESV and PLAC were administrated after ovariectomy and ZOL after six weeks after OVX, until the end of experiment. One implant was inserted in each tibiae of animals 18 weeks after ovariectomy. After 4 weeks, one implant was removed for counter-torque, and peri-implant tissue was collected for mRNA quantification of several osteogenic markers by PCR. The other tibia was submitted to micro-computed tomography analysis. Reduced counter-torque values, bone-implant contact (BIC) and bone volume fraction (BV/TV), and higher bone porosity (BP) were detected in OVX+PLAC group when compared to SHOVX+PLAC (p < 0.05). OVX+RESV rats presented lower BIC, BV/TV, and trabecular number (Tb.N), and augmented BP and trabecular spacing (Tb.Sp) when compared to SHOVX+PLAC (p < 0.05). Higher Tb.N and connectivity density (Conn.Dn) and reduced Tb.Sp were observed in OVX rats treated with ZOL, independently of RESV, when compared to OVX+PLAC and OVX+RESV groups (p < 0.05), whereas the combination ZOL+RESV promoted lower BP when compared to OVT+PLAC and OVX+RESV (p < 0.05). Gene expression was not influenced by RESV (p > 0.05), whereas ZOL promoted up-regulation of BMP-2 (p<0.05). RESV did not improve peri-implant bone repair in rats with ovariectomy-induced osteoporosis.

Abstract This study investigated the impact of a modified implant macrogeometry on peri-implant healing and its effect on bone-related molecules in rats. Eighteen rats received one implant in each tibia: the control group received implants with conventional macrogeometry and the test group received implants with modified macrogeometry. After 30 days, the implants were removed for biomechanical analysis and the bone tissue around them was collected for quantifying gene expression of OPN, Runx2, β-catenin, BMP-2, Dkk1, and RANKL/OPG. Calcein and tetracycline fluorescent markers were used for analyzing newly formed bone at undecalcified sections of the tibial implants. These fluorescent markers showed continuous bone formation at cortical bone width and sparse new bone formed along the medullary implant surface in both groups. However, higher counter-torque values and upregulation of OPN expression were achieved by test implants when compared to controls. The modified macrogeometry of implants optimized peri-implant healing, favoring the modulation of OPN expression in the osseous tissue around the implants.

Abstract Periodontitis Stage III-IV, Grade C (PerioC) is a severe form of Periodontitis. The individual genetic background has been shown to be an important etiopathogenic factor for the development of this disease in young, systemically healthy, and non-smokers patients. Recently, after exome sequencing of families with a history of the disease, PerioC was associated with three single nucleotide variations (SNVs) – rs142548867 (EEFSEC), rs574301770 (ZNF136), and rs72821893 (KRT25) – which were classified as deleterious or possibly harmful by prediction algorithms. Objective Seeking to validate these findings in a cohort evaluation, this study aims to characterize the allele and genotypic frequency of the SNVs rs142548867, rs574301770, and rs72821893 in the Brazilian population with PerioC and who were periodontally healthy (PH). Methodology Thus, epithelial oral cells from 200 PerioC and 196 PH patients were harvested at three distinct centers at the Brazilian Southern region, their DNA were extracted, and the SNVs rs142548867, rs574301770, rs72821893 were genotyped using 5′-nuclease allelic discrimination assay. Differences in allele and genotype frequencies were analyzed using Fisher’s Exact Test. Only the SNV rs142548867 (C > T) was associated with PerioC. Results The CT genotype was detected more frequently in patients with PerioC when compared with PH subjects (6% and 0.5% respectively), being significantly associated with PerioC (odds ratio 11.76, p=0.02). Conclusion rs142548867 represents a potential risk for the occurrence of this disease in the Brazilian population.

Abstract The presence of a tooth-surface defect, such as a non-carious cervical lesion (NCCL), associated with sites of gingival recession (GR) defects creates a combined soft tissue/tooth defect (CD) that requires a different treatment plan. This study aimed to critically review the literature regarding the available treatment protocols for CDs and suggest a new decision-making process. NCCLs were classified as Class A-: the cementoenamel junction (CEJ) was visible and the root surface discrepancy was < 0.5 mm (no step); Class A+: CEJ was visible and the root surface discrepancy was > 0.5 mm (with a step); Class B-: unidentifiable CEJ without a step; Class B+: unidentifiable CEJ with a step. NCCLs affecting both root and crown surfaces (Class B) lead to CEJ destruction and consequently eliminate an important landmark used before and after root coverage procedures. The depth of the root surface discrepancy is vital owing to its possible impact on soft tissue adaptation after healing, which, in turn, may influence the treatment options, namely the use of graft and/or composites to compensate for the discrepancy. Clinically, a step with horizontal depth greater than 0.5 mm should be recognized as the minimum threshold value to define this condition. Extremely deep defects tend to assume a V-shaped topography. Therefore, extremely deep V-shaped defects were classified into subclasses A+V, a V-shaped defect, and B+V, a V-shaped defect with loss of CEJ, for management considerations. The treatment options, supported by the literature, and a decision-making process to deal with each condition are presented.

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Resumo O presente estudo teve como objetivo avaliar os efeitos clínicos e microbiológicos de terapia periodontal cirúrgica e não cirúrgica no tratamento da periodontite agressiva generalizada (PAgG). Dezesseis pacientes portadores de PAgG foram incluídos neste estudo clínico, prospectivo, randomizado, de boca dividida. Os quadrantes superiores de cada paciente foram alocados em dois grupos: um grupo de terapia não-cirúrgica (NST) e um grupo de terapia cirúrgica (ST). Os parâmetros clínicos avaliados foram: índice de placa (PI), sangramento à sondagem índice (BoP), profundidade de sondagem (PD), nível clínico de inserção (CAL) e posição da margem gengival (GMP). Também foram determinadas as concentrações de Porphyromonas gingivalis (Pg) e Aggregatibacter actinomycetemcomitans (Aa) no biofilme subgengival. Os parâmetros clínicos e microbiológicos foram avaliados no início, 3, 6 e 12 meses após o tratamento. A terapia cirúrgica foi capaz de promover maior redução de PD em comparação com NST em bolsas profundas aos 12 meses (p<0,05) e em dentes posteriores aos 6 meses (p<0,05). Além disso, houve maior recessão gengival nos dentes posteriores do grupo ST no 6° mês (p<0,05). Entretanto, ST não promoveu ganho adicionais de inserção (CAL) em nenhum período do avaliação. A avaliação microbiológica não mostrou diferença estatística nos níveis de Aa e Pg, para ambos os grupos, em todos os períodos de acompanhamento. O tratamento cirúrgico promoveu benefícios clínicos similares ao tratamento não cirúrgico em pacientes com PAgG. Além disso, ambas as terapias não conseguiram reduzir os níveis Aa e Pg após terapia.

Abstract The present study aimed to evaluate clinical and microbiological effects of surgical and nonsurgical periodontal therapy in generalized aggressive periodontitis (GAgP) treatment. Sixteen GAgP patients were included in this randomized split-mouth design clinical trial. Maxillary quadrants were allocated into two groups: Nonsurgical Therapy (NST) and Surgical Therapy (ST). The following clinical parameters were assessed: plaque index (PI), bleeding on probing index (BoP), probing depth (PD), clinical attachment level (CAL) and gingival margin position (GMP). Concentrations of Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) in the subgingival biofilm were also determined. Clinical and microbiological parameters were assessed at baseline (n=16), 3 (n=15), 6 (n=15) and 12 months (n=8) after treatment. ST was able to promote higher PD reduction compared to NST in deep pockets at 12 months (p<0.05) and in posterior teeth at 6 months (p<0.05). In addition, higher gingival recession was observed in posterior teeth of the ST group at the 6th month (p<0.05). However, ST failed to promoted additional CAL gain in any timepoint (p>0.05). Moreover, microbiological evaluation showed no statistical difference in levels of Aa and Pg for both groups at all follow-up periods. Surgical therapy promoted similar clinical benefits to GAgP therapy. Moreover, both therapies failed to reduce Aa and Pg levels at different follow-up times.

Abstract Considering the absence of predictable and effective therapeutic interventions for the treatment of peri-implantitis, scientific evidence concerning the host response profile around dental implants could be important for providing in the future a wider preventive and/or therapeutic window for this peri-implant lesion, indicating biomarkers that provide quantifiable measure of response to peri-implant therapy. Moreover, a better knowledge of pattern of host osteo-immunoinflammatory modulation in the presence of peri-implantitis could either benefit the early diagnostic of the disease or to cooperate to prognostic information related to the status of the peri-implant breakdown. Finally, new evidences concerning the host profile of modulators of inflammation and of osseous tissue metabolism around dental implants could explain the individual susceptibility for developing peri-implant lesions, identifying individuals or sites with increased risk for peri-implantitis. The focus of this chapter was, based on a systematically searched and critically reviewed literature, summarizing the existing knowledge in the scientific research concerning the host osteo-immunoinflammatory response to the microbiological challenge related to periimplantitis.

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

RESUMO A engenharia de tecidos é um campo contemporâneo da ciência, que visa criar condições baseadas em princípios de biologia celular e molecular, bioengenharia e biomateriais para regenerar tecidos. As células tronco mesenquimais apresentam altas taxas de proliferação e são capazes de se diferenciar, sob certas condições, em multi-linhagens, sugerindo que elas têm grande potencial para atuar no campo da regeneração. As células tronco derivadas de tecidos dentais são uma fonte alternativa adequada de células mesenquimais uma vez que são de fácil acesso e têm baixa morbidade para o doador. Estudos demonstraram que elas já foram isoladas e caracterizadas a partir de diversos tecidos tais como polpa dentária, dentes decíduos esfoliados, ligamento periodontal, gengiva, folículo dental e papila apical. Entretanto, os estudos demonstram que há heterogeneidade entre essas populações e não existe um método padrão para selecionar as células-tronco dentais mais apropriadas para procedimentos regenerativos. O objetivo desta revisão é apresentar o conhecimento atual dos vários tipos de células-tronco derivadas de dentes e discutir as novas perspectivas para seu uso na engenharia de tecidos periodontais.

ABSTRACT Tissue engineering is a contemporary field of science, which aims to create conditions based on principles of cell and molecular biology, bioengineering and biomaterials to regenerate tissues. Mesenchymal stem cells present high proliferation rates and are able to differentiate into multilineages under certain conditions, suggesting that they have great potential to act in regeneration field. Tooth derived stem cells are a suitable alternative source of mesenchymal cells once they are easily accessible and have poor morbidity to the donor. Studies showed that they have been isolated and characterized from diverse tissues such as dental pulp, exfoliated deciduous teeth, periodontal ligament, gingiva, dental follicle and apical papilla. However studies show that there is heterogeneity among these populations and there is no standard method to select the most appropriate tooth derived stem cells for regenerative procedures. The aim of this review is to present the current perspective of the multiple types of tooth-derived stem cells and to discuss the basis for their use in periodontal tissue engineering.

Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs) phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS) on mesenchymal phenotype and osteoblast/cementoblast (O/C) potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2) expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i) cell metabolic activity using MTS; (ii) gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR); (iii) flow cytometry for STRO-1 and CD105, and (iv) osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.

ABSTRACT The use of natural substances and micronutritional approaches has been suggested as a therapeutic alternative to benefit the bone healing associated with no side effects. Nevertheless, the influence of micronutritional interventions with therapeutic proprieties on the bone repair has yet to be intensely evaluated, and no evidence is available exploring the impact of micronutrient supplementation on the peri-implant bone healing. Objective This study investigated the effect of micronutrients supplementation on the bone repair around implants. Material and Methods One screw-shaped titanium implant was inserted in each tibia of each rat, which were assigned to: daily administration, for 30 d, of the placebo solution (Placebo group-n:18) or micronutrients supplementation (Micronutrients group-n:18), based on calcium, magnesium, zinc, and vitamin D3 intake. After, the animals were sacrificed. One of the implants was removed by applying a counter-torque force to evaluate the force to rupture the bone-implant interface. The other implant was evaluated by microcomputed tomography (CT) examination to determine the bone-to-implant contact (BIC) and the bone volume (BV/TV). Results No statistically significant differences were observed between the groups for both counter-torque values and microCT parameters (p>0.05). Conclusion Within the limits of this study, micronutrients supplementation did not provide additional benefits to the bone healing around dental implants.

Abstract The objective of this study was to investigate the antibacterial effect of resveratrol against putative periodontal pathogens during the progression of experimental periodontitis in rats. Periodontitis was induced in rats in one of the first molars chosen to receive a ligature. Animals were assigned to one of two groups: daily administration of the placebo solution (control group, n = 12) or 10 mg/Kg of resveratrol (RESV group, n = 12). The therapies were administered systemically for 30 days, for 19 days before periodontitis induction and then for another 11 days. Then, the presence and concentrations of Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans in the cotton ligatures collected from the first molars were evaluated using real-time PCR. Inter-group comparisons of the microbiological outcomes revealed that no differences were detected for P. gingivalis, T. forsythia and A. actinomycetemcomitans levels (p > 0.05). Continuous use of resveratrol did not promote additional benefits in microbiological outcomes during experimental periodontitis in rats.

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix.Objective: This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide fromEscherichia coli(EcLPS).Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR.Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group.Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.

AIM: To investigate the indication of systemic antimicrobial agents used by dental professionals for treatment of patients affected by periodontal diseases. METHODS: Interviews by a questionnaire were held with 225 professionals of different dental specialties and who performed periodontal treatment. RESULTS: Among interviewees, 94% indicated systemic antibiotics as a form of periodontal disease treatment. Their main indication was for periodontal abscesses (80%) followed by aggressive periodontitis (62%) and necrotizing diseases (45%). The most frequently used antibiotics were amoxicillin (81%) and metronidazole (57%). The medications were indicated in association with mechanical therapy by 67% of the professionals. As regards the occasion of indication, 60% indicated systemic antibiotic therapy before and after mechanical periodontal scaling and root planing. Seventy-eight percent of the professionals indicated antibiotics associated with periodontal surgery for access to scaling, and 76% indicated it before and after the surgical procedure. Among the interviewees, 99% took into account systemic involvement for drug administration. CONCLUSIONS: It was concluded that a considerable portion of professionals indicate systemic antibiotic-therapy in an incoherent manner and in situations in which there is no indication for antibiotic use, or with ineffective protocols.

The aim of the present study was to evaluate the frequency of detection of Mogibacterium timidum in subgingival samples of subjects with generalized aggressive periodontitis (GAgP) and uncontrolled diabetic and non-diabetic subjects with generalized chronic periodontitis (GChP). 48 patients with GAgP, 50 non-diabetic and 39 uncontrolled (glycated hemoglobin >7%) type 2 diabetic subjects with GChP were enrolled in this study. Subgingival biofilm were collected from deep pockets (probing depth > 7 mm). After DNA extraction, M. timidum was detected by Nested Polymerase Chain Reaction and chi-square test was used to data analysis (p>0.05). There were no differences in the frequency of detection of M. timidum between subjects with GAgP (35%) and non-diabetic subjects with GChP (40%) (p>0.05). The frequency of detection of M. timidum was significantly higher in deep pockets of diabetic subjects with GChP (56%) when compared to GAgP (p<0.05), but similar to non-diabetic subjects with GChP (p>0.05). The frequency of detection of M. timidum was higher in subjects GChP presenting uncontrolled type 2 diabetes mellitus, when compared to GAgP subjects.