Regulation of visfatin by microbial and biomechanical signals in PDL cells

OBJECTIVES This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. MATERIALS AND METHODS PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. RESULTS F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. CONCLUSIONS PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. CLINICAL RELEVANCE Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases.

Soft tissue wound healing around teeth and dental implants.

AIM To provide an overview on the biology and soft tissue wound healing around teeth and dental implants. MATERIAL AND METHODS This narrative review focuses on cell biology and histology of soft tissue wounds around natural teeth and dental implants. RESULTS AND CONCLUSIONS The available data indicate that: (a) Oral wounds follow a similar pattern. (b) The tissue specificities of the gingival, alveolar and palatal mucosa appear to be innately and not necessarily functionally determined. (c) The granulation tissue originating from the periodontal ligament or from connective tissue originally covered by keratinized epithelium has the potential to induce keratinization. However, it also appears that deep palatal connective tissue may not have the same potential to induce keratinization as the palatal connective tissue originating from an immediately subepithelial area. (d) Epithelial healing following non-surgical and surgical periodontal therapy appears to be completed after a period of 7–14 days. Structural integrity of a maturing wound between a denuded root surface and a soft tissue flap is achieved at approximately 14-days post-surgery. (e) The formation of the biological width and maturation of the barrier function around transmucosal implants requires 6–8 weeks of healing. (f) The established peri-implant soft connective tissue resembles a scar tissue in composition, fibre orientation, and vasculature. (g) The peri-implant junctional epithelium may reach a greater final length under certain conditions such as implants placed into fresh extraction sockets versus conventional implant procedures in healed sites.

Effect of pulverized natural bone mineral on regeneration of three-wall intrabony defects. A preclinical study.

AIMS The objective of this study is to evaluate the effects of a paste-like bone substitute material with easy handling properties and improved mechanical stability on periodontal regeneration of intrabony defects in dogs. MATERIALS AND METHODS Mandibular and maxillary first and third premolars were extracted, and three-wall intrabony defects were created on second and fourth premolars. After a healing period of 3 months, acute type defects were filled with a paste-like formulation of deproteinized bovine bone mineral (DBBM) (particle size, 0.125-0.25 mm) in a collagenous carrier matrix (T1), pulverized DBBM (particle size, 0.125-0.25 mm) without the carrier (T2), or Bio-Oss® granules (particle size, 0.25-1.00 mm) as control (C). All defects were covered with a Bio-Gide® membrane. The dogs were sacrificed after 12 weeks, and the specimens were analyzed histologically and histometrically. RESULTS Postoperative healing of all defects was uneventful, and no histological signs of inflammation were observed in the augmented and gingival regions. New cementum, new periodontal ligament, and new bone were observed in all three groups. The mean vertical bone gain was 3.26 mm (T1), 3.60 mm (T2), and 3.81 mm (C). That of new cementum was 2.25 mm (T1), 3.88 mm (T2), and 3.53 mm (C). The differences did not reach statistical significance. The DBBM particles were both incorporated in new bone and embedded in immature bone marrow. CONCLUSIONS The results of this preclinical study showed that the 0.125-0.25-mm DBBM particles in a powder or paste formulation resulted in periodontal regeneration comparable to the commercially available DBBM. Osteoconductivity, in particular, was not affected by DBBM size or paste formulation. CLINICAL RELEVANCE The improved handling properties of the paste-like bone substitute consisting of small DBBM particles embedded in a collagen-based carrier hold promise for clinical applications.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials.

BACKGROUND The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

A aplicação de plasma rico em plaquetas na regeneração dos tecidos periodontais

O periodonto é uma unidade biológica e funcional, constituída pela gengiva, pelo cemento, pelo ligamento periodontal e pelo osso alveolar. O seu processo de cicatrização envolve mecanismos fisiológicos complexos que requerem a ação dos fatores de crescimento, péptidos oriundos da desgranulação das plaquetas. Neste sentido surge o Plasma Rico em Plaquetas como um produto autólogo, obtido a partir da centrifugação do sangue do próprio paciente e que visa melhorar a cicatrização dos tecidos após procedimentos enquadrados na Medicina Dentária. Esta revisão bibliográfica baseou-se numa pesquisa realizada na base de dados MEDLINE, via pubmed. Foram utilizadas as palavras-chave “plasma rich in growth factors”, “platelet-rich plasma”, “oral surgery”, “dental implants”, “sinus lift”, “third molar surgery” e “bone regeneration”. Após leitura de 40 artigos, foram descartados 9 pela sua pouca relevância no contexto. O objetivo é avaliar a efetividade da aplicação de plasma rico em plaquetas na regeneração dos tecidos periodontais em situações clínicas como alvéolos pós-extracionais, cirurgias de implantes, cirurgias de elevação do seio maxilar e na regeneração óssea. A efetividade em tecidos moles parece ser consensual. A efetividade no tecido ósseo é alvo de contradição entre os diversos autores, concluindo-se que há necessidade de mais estudos randomizados e controlados para que se possa responder à questão com evidência científica suficiente.

Considerações anatómicas sobre o envelhecimento do aparelho estomatognático

Introdução: com o crescente aumento da expectativa de vida, o conhecimento das alterações anatómicas e fisiológicas que ocorrem no aparelho estomatognático durante o envelhecimento é de suma importância para a correta avaliação do paciente idoso. Objetivos: descrição e abordagem das principais estruturas anatómicas do indivíduo, adulto e idoso. Estabelece-se uma anatomia comparativa e evolutiva durante o processo de envelhecimento. Pretende-se contribuir para o conhecimento e reflexão sobre o tema em questão e demonstrar a aplicabilidade deste conhecimento em contexto clínico. Métodos: realizou-se pesquisa bibliográfica, nas bases de dados Pubmed, b-on SciElo e Elsevier, no período entre 2006-2016. Resultados: Maxila - ocorre reabsorção óssea, alteração no contorno do arco da maxila, retrusão maxilar, rotação da maxila no sentido horário, diminuição gradual e constante do ângulo maxilar e redução vertical da altura maxilar. Mandíbula - aumento do ângulo da mandíbula, diminuição da densidade e volume ósseo. Articulação gonfose e Articulação Temporo-Mandibular - pode ocorrer tanto anquilose, como perda das estruturas de suporte. Observa-se degeneração e/ou perfuração do disco radicular e alteração do formato do côndilo. Dentes - cáries radiculares, fraturas dentárias e desgaste dentário. Ocorrem modificações histológicas no esmalte, dentina e polpa dentária. Periodonto: reabsorção do osso alveolar, gengiva atrófica com tendência a migração apical, deposição apical das camadas incrementais e desgaste de cemento exposto, ligamento periodontal fino, irregular e diminuição do espaço periodontal. Conclusões: as alterações anatómicas decorrentes do envelhecimento fisiológico são múltiplas. O Médico Dentista diante de um paciente idoso, deverá conhecer e distinguir entre uma alteração decorrente do envelhecimento fisiológico e uma alteração patológica, para o correto diagnóstico clínico e uma excelente decisão terapêutica. O Médico Dentista deverá contribuir para o envelhecimento saudável e para tal deve ser conhecedor em pleno da temática do presente trabalho.

Fraturas dentárias

Uma lesão traumática é, a seguir à cárie dentária, um dos problemas mais comuns da saúde oral. O traumatismo dentário consiste numa agressão que afeta os dentes ou os tecidos de suporte, podendo levar ao rompimento do ligamento periodontal, fratura dentária, fratura óssea ou alterações pulpares. Este problema causa desconforto físico, emocional e comprometimento estético. Quando os traumatismos afetam os tecidos duros ou polpa denominam-se de fraturas dentárias. Estas podem ser divididas em fraturas coronárias de esmalte; fraturas coronárias de esmalte e dentina sem exposição pulpar; fraturas coronárias de esmalte e dentina com exposição pulpar; fraturas corono-radiculares de esmalte, dentina e cemento sem exposição pulpar; fraturas corono-radiculares com exposição pulpar e fratura radicular envolvendo cemento, dentina e polpa. O objetivo deste trabalho monográfico foi fazer uma revisão bibliográfica de estudos científicos sobre fraturas dentárias. Assim, serão abordados os diversos tipos de fraturas dentárias, as suas etiologias, prevalências e fatores associados, também definir planos de tratamento, técnicas de restauração direta de fraturas coronárias e possíveis medidas de prevenção. A pesquisa bibliográfica para esta revisão foi realizada em bases eletrónicas de referência como Pubmed, Scielo, selecionando artigos entre os anos 2006 e 2016. Foram também utilizados como elementos de pesquisa o site Dental Trauma Guide (DTG), o site International Association of Dental Traumatology (IADT), livros da especialidade existentes na Biblioteca da Universidade Fernando Pessoa que embora com datas de publicação mais antigas (2001) foram imprescindíveis para o desenvolvimento da monografia. A pesquisa foi realizada em duas línguas, português e inglês. Como conclusão pode-se dizer que nos dias de hoje, o médico dentista tem muitas opções de tratamento de dentes fraturados. Para restaurar a fratura o médico pode optar por colar o fragmento, restaurar usando uma matriz guia ou restaurar à mão livre. A escolha da técnica deve ser estudada de acordo com cada paciente e suas expectativas.

Diabetes Mellitus tipo 1 em Odontopediatria

A Diabetes Mellitus é conhecida por uma doença metabólica caracterizada por um défice na ação ou secreção da insulina, na qual a consequência direta é o aparecimento de hiperglicemia, isto é, o nível de glicose apresentar valores elevados (Kidambi, 2008; Silva-Sousa, 2003). A DM1, especificamente, é apresentada como uma doença que é resultado da destruição das células beta do pâncreas, desenvolvendo assim, um défice na produção de insulina (Raymond et al., 2001). As complicações orais da DM1 incluem xerostomia, doença periodontal (gengivite e periodontite), abcessos dentários, perda de dentes, lesões de tecidos moles e síndrome de ardência oral. A complicação oral mais frequente da DM1 nas crianças é o aumento da sensibilidade à doença periodontal. A doença periodontal é caracterizada como uma reação inflamatória infecciosa dos tecidos gengivais (gengivite) ou do suporte dos dentes, ou seja, ligamento periodontal, cemento e osso alveolar (periodontite), podendo induzir um certo grau de resistência à insulina. Ambas as doenças resultam da interação entre microorganismos periodontais patogénicos. A avaliação e influência do controlo da doença é expressa pelos valores médios de hemoglobina glicosada (Hba1c) na saúde oral nas crianças e adolescentes com DM1. Vários estudos demonstraram que o controlo glicémico teve uma influencia sobre a saúde oral de crianças e adolescentes com DM1. Assim uma avaliação oral, deve fazer parte de procedimentos de rotina no atendimento de crianças e adolescentes com DM1. O dentista deve ser parte da equipa multidisciplinar que auxilia os indivíduos com DM1. O tratamento precoce numa população infantil com DM1, pode diminuir a severidade da doença periodontal. O presente trabalho tem por objectivo realizar uma revisão bibliográfica sobre a importância do estudo em crianças e adolescentes portadores de DM1 e doenças da cavidade oral, nomeadamente, a periodontite, e respetivas implicações.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Conditioned Medium of Demineralized Freeze-Dried Bone Activates Gene Expression in Periodontal Fibroblasts in Vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-β signaling in mesenchymal cells. The aim of the study was to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium (DBCM). Changes in the expression of TGF-β target genes were determined. RESULTS: DBCM altered the expression of TGF-β target genes, e.g., adrenomedullin, pentraxin 3, KN Motif And Ankyrin Repeat Domains 4, interleukin 11, NADPH oxidase 4, and BTB (POZ) Domain Containing 11, by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-β receptor type I kinase inhibitor SB431542, but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. CONCLUSION: The findings suggest that the conditioned medium from demineralized bone matrix can activate TGF-β signaling in oral fibroblasts. KEYWORDS: TGF-beta superfamily proteins; bone; bone substitutes; bone transplantation; conditioned media; freeze drying

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.