Impact of a modified implant macrogeometry on biomechanical parameters and bone-related markers in rats.

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, ß-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.

Impact of a modified implant macrogeometry on biomechanical parameters and bone-related markers in rats

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.

Obesity affects the proteome profile of periodontal ligament submitted to mechanical forces induced by orthodontic tooth movement in rats.

The prevalence of obesity has increased significantly worldwide. Therefore, this study aimed to evaluate the influence of obesity on the proteomic profile of periodontal ligament (PDL) tissues of rat first maxillary molars (1 M) submitted to orthodontic tooth movement (OTM). Ten Holtzman rats were distributed into two groups (n = 5): the M group (OTM), and the OM group (obesity induction plus OTM). Obesity was induced by a high-fat diet for the entire experimental periods After that period, the animals were euthanized and the hemimaxillae removed and processed for laser capture microdissection of the PDL tissues of the 1 M. Peptide extracts were obtained and analyzed by LC-MS/MS. Data are available via ProteomeXchange with identifier PXD033647. Out of the 109 proteins with differential abundance, 49 were identified in the OM group, including Vinculin, Cathepsin D, and Osteopontin, which were selected for in situ localization by immunohistochemistry analysis (IHC). Overall, Gene Ontology (GO) analysis indicated that enriched proteins were related to the GO component cellular category. IHC validated the trends for selected proteins. Our study highlights the differences in the PDL proteome profiling of healthy and obese subjects undergoing OTM. These findings may provide valuable information needed to better understand the mechanisms involved in tissue remodeling in obese patients submitted to orthodontic treatment. SIGNIFICANCE: The prevalence of obesity is increasing worldwide. Emerging findings in the field of dentistry suggest that obesity influences the tissues around the teeth, especially those in the periodontal ligament. Therefore, evaluation of the effect of obesity on periodontal tissues remodeling during orthodontic tooth movement is a relevant research topic. To our knowledge, this is the first study to evaluate proteomic changes in periodontal ligament tissue in response to the association between orthodontic tooth movement and obesity. Our study identified a novel protein profile associated with obesity by using laser microdissection and proteomic analysis, providing new information to increase understanding of the mechanisms involved in obese patients undergoing orthodontic treatment which can lead to a more personalized orthodontic treatment approach.

Obesity influences the proteome of periodontal ligament tissues following periodontitis induction in rats.

BACKGROUND AND OBJECTIVES: Many studies have been conducted to better understand the molecular mechanism involved with periodontitis progression. There has been growing interest in the potential impact of obesity on periodontitis onset and progression, but the mechanisms involved remain to be elucidated. The present study was designed to determine the impact of obesity on experimentally induced periodontitis in rats and identify novel pathways involved. METHODS: Sixteen Holtzman rats were distributed into two groups (n = 8): ligature-induced periodontitis (P) and obesity plus ligature-induced periodontitis (OP). Obesity was induced by a high-fat diet for 70 days, whereas periodontitis was induced for 20 days, with a cotton thread placed around the upper first molars bilaterally. Alveolar bone loss was measured by microtomographic analysis and histologically by histometry on the hemimaxillae. The protein composition of the periodontal ligament was evaluated by proteomic analysis. RESULTS: Data analysis (body weight, adipose tissue weight, and blood test) confirmed obesity induction, whereas bone loss was confirmed by micro-CT and histologic analyses. Proteome analysis from the periodontal ligament tissues (PDL) identified 819 proteins, 53 exclusive to the P group, 28 exclusive to the OP group, and 738 commonly expressed. Validation was performed by immunohistochemistry for selected proteins (spondin1, vinculin, and TRAP). CONCLUSION: Histologically, it was found that obesity did not significantly affect bone loss resulting from periodontitis. However, the present study's findings indicated that obesity affects the proteome of PDL submitted to experimental periodontitis, allowing for identifying potential targets for personalized approaches.

Novel LRAP-binding partner revealing the plasminogen activation system as a regulator of cementoblast differentiation and mineral nodule formation in vitro.

Amelogenin isoforms, including full-length amelogenin (AMEL) and leucine-rich amelogenin peptide (LRAP), are major components of the enamel matrix, and are considered as signaling molecules in epithelial-mesenchymal interactions regulating tooth development and periodontal regeneration. Nevertheless, the molecular mechanisms involved are still poorly understood. The aim of the present study was to identify novel binding partners for amelogenin isoforms in the cementoblast (OCCM-30), using an affinity purification assay (GST pull-down) followed by mass spectrometry and immunoblotting. Protein-protein interaction analysis for AMEL and LRAP evidenced the plasminogen activation system (PAS) as a potential player regulating OCCM-30 response to amelogenin isoforms. For functional assays, PAS was either activated (plasmin) or inhibited (ε-aminocaproic acid [aminocaproic]) in OCCM-30 cells and the cell morphology, mineral nodule formation, and gene expression were assessed. PAS inhibition (EACA 100 mM) dramatically decreased mineral nodule formation and expression of OCCM-30 differentiation markers, including osteocalcin (Bglap), bone sialoprotein (Ibsp), osteopontin (Spp1), tissue-nonspecific alkaline phosphatase (Alpl) and collagen type I (Col1a1), and had no effect on runt-related transcription factor 2 (Runx2) and Osterix (Osx) mRNA levels. PAS activation (plasmin 5 µg/µl) significantly increased Col1a1 and decreased Bglap mRNA levels (p < .05). Together, our findings shed new light on the potential role of plasminogen signaling pathway in the control of the amelogenin isoform-mediated response in cementoblasts and provide new insights into the development of targeted therapies.

Cemento dental: aspectos atuais de interesse para o clínico / Dental cementum: current aspects to the clinicians

Embora tenha havido avanço no entendimento da homeostase do cemento dental, o papel deste tecido e sua biologia permanecem não completamente elucidados. Este estudo buscou fornecer informações sobre os conhecimentos mais recente relacionados à biologia do cemento dental, com o objetivo de discutir o papel exercido por este tecido em condições não fisiológicas nos tecidos periodontais. Devido aos avanços na exploração do tecido ósseo, que compartilha diversas características similares, a pesquisa abrangente sobre o cemento dental tem sido encorajada, a fim de esclarecer a função completa deste tecido na homeostase periodontal e regeneração. Desta forma, no presente trabalho, sempre que possível será feito um paralelo entre osso alveolar e cemento dental. O desenvolvimento de metodologias e técnicas celulares e moleculares avançadas possibilitou um melhor entendimento do comportamento do cemento em situações diversas, como quando em situações patológicas, como a doença periodontal, e até mesmo frente à regeneração tecidual. Ademais, estudos clínicos e em modelo animal demonstraram resultados em relação à formação de cemento em abordagens regenerativas. No entanto, sugere-se que estudos posteriores possam contribuir para um melhor conhecimento sobre o cemento e o perfil celular dos cementoblastos e cementócitos, bem como suas interações para fornecer novos insights para o desenvolvimento de terapias eficientes e mais previsíveis para regeneração dos tecidos periodontais. Apesar dos avanços dos estudos clínicos e laboratoriais, pôde-se concluir que inúmeras questões referentes à biologia do cemento permanecem não esclarecidas.

Although some progress has been made to understand dental cementum homeostasis, its role and biology remains not completely elucidated. This study aimed to provide information on the recent knowledge related to the dental cementum biology, in order to discuss the role of this tissue in physiological and non-physiological conditions in the periodontal tissues. Due to advances in the exploration of bone tissue, which shares several similar features, comprehensive research on dental cementum has been encouraged in order to clarify the complete function of this tissue in periodontal homeostasis and regenerative approach. Novel methodologies and advanced cellular and molecular techniques provided better understanding of cementum in different circumstances, as pathological situations such as periodontal disease and even tissue regeneration. In addition, clinical and animal model designs show positive outcomes to cementum formation in regenerative approaches, however, it is suggested that further studies may contribute to better understand cementum tissue and cementoblasts and cementocytes profile, as well as their interactions, providing new insights to develop efficient and more predictable therapies for periodontal tissue regeneration. Despite advances in clinical and laboratory studies, it can be concluded that many questions regarding the cementum biology remain unclear.

Gene expression analysis in microdissected samples from decalcified tissues.

OBJECTIVE: The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples. DESIGN: Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase. RESULTS: Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified. CONCLUSIONS: Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.

MT1-MMP expression in the odontogenic region of rat incisors undergoing interrupted eruption.

MT1-MMP (membrane type matrix metalloproteinase-1) has been considered an important membrane-type matrix metalloproteinase involved in the remodeling process in tissue and organ development, including the processes of the tooth and root growth and dental eruption. Therefore, the aims of this study were to evaluate MT1-MMP expression in the odontogenic region, as well as the eruption rate and morphology of the lower-left rat incisor, where the eruption process was interrupted for 14 days by a steel wire attached from the center of the incisor labial face and braced to the first molar. In the interrupted eruption group, the eruption rate was significantly reduced, producing drastic morphological alterations in the tooth germ and socket area. The MT1-MMP expression was widespread in the dental follicle, in both groups studied (normal and interrupted eruption groups); however a significant decrease in immunostaining was observed in the interrupted eruption group. Results indicate that MT1-MMP may have an important role in the process of dental eruption.

Caso familiar de periodontite agressiva: achados clínicos, microbiológicos e genéticos / A familial case of aggressive periodontitis: clinical, microbiological and genetic findings

This study reports the clinical, microbiological and genetic findings in members of a Brazilian family with Aggressive Periodontitis (AgP). After periodontal exams in eleven members of the family, microbiological samples were collected from subgingival plaque and DNA were obtained from epithelial buccal cells. Polymerase Chain Reaction (PCR) was utilized to detect five species of periodontopathogens. PCR-RFLP (Restriction Fragment Length Polymorphism) and sequencing were used to investigate human polymorphisms in interleukin genes (IL4, IL10). Six members of the family showed Generalized AgP, four had Localized AgP and only one was considered unaffected by AgP. Aggregatibacter actinomycetemcomitans was the most prevalent pathogen (72.7%). The presence of Porphyromonas gingivalis was correlated with clinical findings (visible plaque, bleeding on probing and probing depth, p = 0.03). The genetic analyses revealed that 60% of the kindred affected by AgP showed specifc IL4/ IL10 haplotypes combinations. The predominance of AgP in this family could be influenced by the amount of plaque and subgingival Aggregatibacter actinomycetemcomitans. Indeed, infection by Porphyromonas gingivalis was associated with clinical parameters of periodontitis. Although the majority of AgP family members presented at least one TTD/ ATA (IL4/ IL10) haplotype combination, it was unable to demonstrate an association with AgP.

Este estudo relata achados clínicos, microbiológicos e genéticos em indivíduos de uma família brasileira com Periodontite Agressiva (PA). Após exames periodontais realizados em onze membros da família, foram coletadas amostras microbiológicas de placa subgengival e DNA de células da mucosa oral. A Reação em Cadeia da Polimerase (do inglês, PCR) foi utilizada para detectar cinco espécies de periodontopatógenos. As técnicas do Polimorfismo por Comprimento de Fragmento de Restrição (RFLP) e sequenciamento foram usados para investigar polimorfismos humanos em genes da interleucina (IL4, IL10). Seis membros da família apresentaram PA Generalizada, quatro PA Localizada e apenas um indivíduo foi considerado não afetado pela PA. Aggregatibacter actinomycetemcomitans foi o patógeno prevalente (72,7%). A presença de Porphyromonas gingivalis foi relacionada com achados clínicos (placa visível, sangramento à sondagem e profundidade de sondagem, p = 0,03). A análise genética revelou que 60% dos membros da família afetados pela PA carregavam o mesmo haplótipo formado por uma combinação de alelos dos genes IL4/ IL10. O predomínio de PA nesta família pode ter sido influenciada pela quantidade de placa bacteriana e prevalência de Aggregatibacter actinomycetemcomitans presente no sulco gengival. A infecção por Porphyromonas gingivalis pode ser associada com parâmetros clínicos relacionados a periodontite. Embora a maioria dos membros afetados pela PA tenha apresentado pelo menos uma combinação de haplótipos TTD/ ATA (IL4/ IL10), não foi possível demonstrar estatisticamente uma associação com PA