Abstract Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including “anatomical structure development” and “cell adhesion.” Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process “anatomical structure development,” Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies.

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.

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.

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.

O objetivo deste estudo foi avaliar os resultados clínicos e centrados no paciente após abordagem cirúrgica minimamente invasiva (CMI) associada à aplicação das proteínas derivadas da matriz do esmalte (PDE) no tratamento de defeitos infra-ósseos. Doze pacientes apresentando um sítio com profundidade de sondagem >5 mm e sangramento à sondagem , associado à evidência radiográfica de defeito infra-ósseo, foram tratados com CMI e aplicação das PDE. Os parâmetros clínicos foram avaliados imediatamente antes do procedimento e após 3 e 6 meses. A percepção de dor e desconforto do paciente durante o período trans-cirúrgico e ao longo da primeira semana de pós-operatório, bem como a satisfação estética 6 meses após o tratamento, foram avaliadas por meio de questionários. Os resultados mostraram que o uso da CMI associada à aplicação de PDE promoveu melhoras estatisticamente significantes nos parâmetros clínicos, mínima dor e desconforto e máxima satisfação estética aos pacientes. Dentro dos limites do estudo, foi demonstrado que a associação de CMI e PDE, no tratamento de defeitos infra-ósseos, é capaz de promover satisfatórios resultados clínicos e centrados no paciente.

This case series evaluated the clinical performance and patient-centered outcomes after a minimally invasive surgical technique (MIST) associated with enamel matrix protein derivative (EMD), for the treatment of intra-bony defects. Twelve patients presenting teeth with probing depth >5 mm and bleeding on probing associated with radiographic evidence of intra-bony defect were treated by MIST associated with EMD. Clinical parameters were measured at baseline, 3 and 6 months. Patient perception during the intraoperative period and during the first postoperative week was evaluated. The use of MIST with EDM promoted significant improvements in clinical parameters, minimal pain/discomfort and maximum esthetics satisfaction. Within of limits of the present study, it could be shown that MIST combined with EMD for the treatment of intra-bony defects promotes satisfactory clinical and patient-centered outcomes.

O objetivo deste estudo foi avaliar o defeito na superfície radicular produzido por curetas manuais e instrumento ultra-sônico em diferentes potências. Quarenta superfícies radiculares foram divididas de acordo com o tratamento em 4 grupos: a) Curetas Gracey; b) Instrumento ultra-sônico a 10%; c) Instrumento ultra-sônico a 50%; d) Instrumento ultra-sônico a 100%. Cada amostra recebeu 15 movimentos de raspagem e foram divididas ao meio para avaliar: (1) a profundidade do defeito produzido pela instrumentação e (2) a área de contato do instrumento com a superfície. Todas as superfícies foram então avaliadas com o uso de Microscopia Eletrônica de Varredura. Para a análise estatística foi utilizado o teste Tukey/ANOVA. Os resultados (média ± DP) encontrados para a área de contato foram significantemente maiores para a instrumentação manual (2092,9 ± 482,0) que para os instrumentos ultra-sônicos, independentemente da potência utilizada (606,8 ± 283,0; 858,6 ± 422,5; 1212,0 ± 366,7, respectivamente). Os valores para a profundidade do defeito demonstraram numericamente, mas não estatisticamente, diferenças entre a instrumentação manual (66,1 ± 34,0) e ultra-sônica em 10%, 50% ou 100% de potência (52,4 ± 22,1; 72,0 ± 29,9; 77,7 ± 37,7, respectivamente). Os resultados encontrados demonstraram que a instrumentação ultra-sônica produziu defeitos com profundidade similar aos produzidos pela instrumentação manual, com um menor contato do instrumento, independente da potência utilizada.

The aim of this study was to evaluate the root surface defect produced by hand curettes and ultrasonic tips with different power settings. Forty root surfaces were divided into 4 groups according the treatment: Gracey curettes, ultrasonic scaler at 10% power, ultrasonic scaler at 50% power and ultrasonic scaler at 100% power. Each specimen was instrumented with 15 strokes and the and divided in the middle to evaluate: (1) the defect depth produced by the instrumentation and (2) contact area of the instrument tips, which was analyzed by scanning electron microscopy. ANOVA and Tukey's test were used for statistical analysis (a=0.05). The results (mean ± SD) of the contact area showed significantly greater defects (p<0.05) for the hand instrumented groups (2092.9 ± 482) compared to the ultrasonic groups (606.8 ± 283.0; 858.6 ± 422.5; 1212.0 ± 366.7, respectively), independently of the power setting. The values for the defect depth on root surface showed no statistically significant difference (p<0.05) between hand instrumentation (66.1 ± 34.0) and ultrasonic scaling at 10%, 50% or 100% power settings (52.4 ± 22.1; 72.0 ± 29.9; 77.7 ± 37.7, respectively). The findings of this study demonstrate that ultrasonic instrumentation produced a similar defect depth to that of hand instrumentation, with a smaller tip contact area, independently of the power setting used for scaling.

Neste estudo foram avaliados a morfologia, o tamanho e a composição química dos seguintes substitutos ósseos: osso bovino orgânico cortical e esponjoso com micropartículas medindo entre 0,25 e 1,0 mm e macropartículas medindo entre 1,0 e 2,0 mm; osso bovino cortical inorgânico com partículas medindo entre 0,25 e 1,0 mm; hidroxiapatita com partículas medindo entre 0,75 e 1,0 mm; e osso humano descalcificado, congelado e seco medindo entre 0,25 a 0,5 mm. Para a analise da morfologia e tamanho das partículas, as amostras foram preparadas em porta-espécime, metalizadas em ouro e analisadas a vácuo em microscopia eletrônico de varredura (MEV). Para a análise da composição química, as partículas não foram metalizadas e foram analisadas por microanálise por espectroscopia por dispersão de energia (EDS). A análise em MEV, demonstrou que as partículas substitutos ossos apresentaram formato irregular e tamanho variável, maior do que o mencionado pelo fabricante. A microanálise por EDS detectou a presença de elementos como sódio, cálcio e fósforo, que são comuns à composição do tecido ósseo, porém revelaram a presença de elementos químicos nas partículas de osso bovino orgânico, exceto para a macropartícula de osso bovino orgânico esponjoso. Esses resultados sugerem que o osso bovino orgânico não pode ser considerado um material orgânico puro.

This study evaluated the morphological and chemical composition of the following bone substitutes: cancellous and cortical organic bovine bone with macro and microparticle size ranging from 1.0 to 2.0 mm and 0.25 to 1.0 mm, respectively; inorganic bovine bone with particle size ranging from 0.25 to 1.0 mm; hydroxyapatite with particle size ranging from 0.75 to 1.0 mm; and demineralized freeze-dried bone allograft with particle size ranging from 0.25 to 0.5 mm. The samples were sputter-coated with gold in an ion coater, the morphology was observed and particle size was measured under vacuum by scanning electron microscopy (SEM). The chemical composition was evaluated by spectroscopy of dispersion energy (EDS) microanalysis using samples without coating. SEM analysis provided visual evidence that all examined materials have irregular shape and particle sizes larger than those informed by the manufacturer. EDS microanalysis detected the presence of sodium, calcium and phosphorus that are usual elements of the bone tissue. However, mineral elements were detected in all analyzed particles of organic bovine bone except for macro cancellous organic bovine bone. These results suggest that the examined organic bovine bone cannot be considered as a pure organic material.

O objetivo do presente estudo foi avaliar histometricamente a influência da nicotina sobre a regeneração óssea de defeitos criados cirurgicamente em rebordos alveolares edêntulos de cães. Defeitos ósseos foram criados cirurgicamente em um dos lados da mandíbula de dezesseis cães e foram deixados para que curassem espontanea-mente. Os animais foram aleatoriamente designados para um dos seguintes grupos: Grupo 1 - controle (n = 8) e Grupo 2 - administração subcutânea de nicotina (2 mg/kg) duas vezes ao dia durante 4 meses (n = 8). Os animais foram sacrificados, e secções semi-seriadas descalcificadas, obtidas. Os parâmetros histométricos avaliados foram altura, largura, área e densidade do tecido ósseo neoformado. A análise intergrupos (Mann-Whitney "rank sum test") demonstrou que a administração de nicotina não influenciou altura, largura e área de tecido ósseo neoformado (p > 0,05). Entretanto, a administração de nicotina influenciou significativamente a densidade do tecido ósseo neoformado (p < 0,001). Dentro dos limites do presente estudo, pode-se concluir que a nicotina pode afetar, mas não impedir a regeneração de defeitos ósseos criados cirurgicamente em mandíbulas edêntulas de cães.

The objective of this study was to investigate the histometric impact of nicotine on bone regeneration of surgically created alveolar ridge defects in dogs. Sixteen mongrel dogs were used. One defect was surgically created unilaterally in the mandible, and left to heal spontaneously. The animals were randomly assigned to one of the following groups: Group 1 - control (n = 8) and Group 2 - subcutaneous nicotine administration (2 mg/kg) twice a day (n = 8). After 4 months, the animals were sacrificed and the specimens routinely processed for semi-serial decalcified sections. Bone height (BH), bone width (BW), bone density (BD), and bone area (BA) of the newly-formed bone were evaluated. Intergroup analysis (Mann-Whitney rank sum test) showed that regardless of the presence of nicotine, no significant differences were observed regarding bone width (BW), bone area (BA) and bone height (BH) (p > 0.05). On the other hand, it was demonstrated that nicotine administration significantly influenced the proportion of mineralized tissue within the limits of the newly-formed bone (BD) (p < 0.001). Within the limits of the present study, it can be concluded that nicotine might affect but not prevent bone healing in defects left to heal spontaneously.

O objetivo deste estudo foi investigar a relação entre medidas clínicas e radiográficas durante a terapia de manutenção periodontal e a confiabilidade de medidas clínicas periodontais repetidas para o diagnóstico precoce de destruição periodontal de lesões de furca classe II. Dezoito lesões de furca classe II em molares inferiores foram incluídas neste estudo. Radiografias padronizadas e medidas do nível clínico de inserção vertical (NCI-v) e da profundidade de sondagem (PS) foram obtidas imediatamente antes do tratamento cirúrgico e aos 6, 12, 18 e 24 meses após a cirurgia. Um total de 72 pares de radiografias foram subtraídos após correção das distorções geométricas e de contraste, e a altura de ganho/perda óssea (AO) (em mm) foi medida. Não houve correlação estatisticamente significante entre NCI-v e AO. Houve uma correlação estatisticamente significante entre redução na PS aos 24 meses e aumento na AO aos 18 meses (r = 0,5, p < 0,05). Os resultados deste estudo sugerem que medidas clínicas e radiográficas refletem diferentes características da reparação e destruição periodontais e medidas clínicas longitudinais repetidas do NCI-v não são confiáveis para o diagnóstico precoce de destruição periodontal em lesões de furca classe II.

The goal of the present study was to investigate the relationship between probing and radiographic parameters, and the reliability of repeated longitudinal periodontal probing measurements for early diagnosis of periodontal breakdown in class II furcation defects. Eighteen class II furcation defects in lower molars were included in this study. Standardized radiographs and clinical measurements, relative vertical clinical attachment level (CAL-v) and probing depth (PD) were obtained immediately before periodontal surgeries and at 6, 12, 18, and 24 months after surgery. A total of 72 pairs of radiographs were subtracted following correction for contrast and planar geometric discrepancies, and the bone loss/gain (in mm) was measured. There was no statistically significant correlation between CAL-v and bone height (BH) measurements. A statistically significant correlation for PD reduction at 24 months and BH increase at 18 months was found (r = 0.5, p < 0.05). These results suggest that clinical measurements and radiographic bone height reflect different features of periodontal destruction and periodontal healing, and repeated longitudinal measurements of vertical clinical attachment level are not reliable for early diagnosis of periodontal breakdown in class II furcation defects.

O uso de agentes imunossupressores tem sido reconhecido como um fator que afeta os tecidos moles do periodonto. Entretanto, pouco se sabe sobre o seu efeito na progressão da periodontite. O objetivo do presente estudo foi investigar a influência da ciclosporina (CsA), associada ou não à nifedipina, na perda óssea resultante da periodontite induzida por ligaduras em ratos. Vinte e quatro ratos Wistar machos, adultos, foram incluídos no estudo. Após anestesia, foram colocadas ligaduras de fio de algodão ao redor do primeiro molar inferior direito ou esquerdo, aleatoriamente escolhido. O dente contralateral foi deixado sem ligadura. Os animais foram aleatoriamente escolhidos para receber um dos seguintes tratamentos: Grupo A - solução salina; Grupo B - CsA (10 mg/kg); Grupo C - nifedipina (50 mg/kg); Grupo D - CsA (10 mg/kg) e nifedipina (50 mg/kg). Após 45 dias, os animais foram sacrificados para a análise histométrica. A análise intergrupos não revelou diferenças significativas quanto ao volume da perda óssea entre os diferentes tratamentos (0,46 ± 0,11; 0,63 ± 0,32; 0,53 ± 0,14; 0,50 ± 0,18, para os grupos A, B, C e D respectivamente - p > 0,05). Entretanto, a análise intragrupo mostrou um maior volume de perda óssea nos dentes com ligadura, quando comparados aos dentes sem ligadura (p < 0,05). Dentro dos limites deste estudo, conclui-se que a administração de CsA, associada ou não à nifedipina, não influenciou a perda óssea alveolar na periodontite induzida por ligaduras em ratos.

Immunosuppressive agents have been recognized as a factor affecting the soft tissues of the periodontium. However, little is known about their effect on periodontitis progression. The aim of the present study was to investigate the influence of cyclosporin A (CsA) administration, associated or not with nifedipine, on the bone loss resulting from a ligature-induced periodontitis in rats. Twenty-four adult male Wistar rats were used. After anesthesia, the mandibular first molar was randomly assigned to receive the cotton ligature in the sulcular area while the contralateral tooth was left unligated. The animals were randomly assigned to one of the following treatments: Group A - saline solution; Group B - CsA (10 mg/kg); Group C - nifedipine (50 mg/kg); Group D - CsA (10 mg/kg) plus nifedipine (50 mg/kg). Forty-five days later, the animals were sacrificed and the specimens routinely processed for serial decalcified sections. Intergroup analysis did not reveal significant differences regarding the bone loss volume in the ligated teeth between the experimental treatments (0.46 ± 0.11, 0.63 ± 0.32, 0.53 ± 0.14, 0.50 ± 0.18, for groups A, B, C and D, respectively - p > 0.05). However, intragroup analysis showed a greater bone loss volume in the ligated teeth than in the unligated ones (p < 0.05). Within the limits of the present study, the conclusion was that the administration of CsA, associated or not with nifedipine, may not influence bone loss in ligature-induced periodontitis in rats.