Periodontal dental ligament mesenchymal stem cells (PDLMSCs) play a major role in periodontal tissue regeneration by the neoformation of root cementum and alveolar bone. These cells are highly heterogeneous, and many present low potential to renovate the hard tissue damaged by periodontal disease. A previous study found that the low osteoblast/cementoblast (O/C) differentiation potential of PDLMSCs is related to high asporin (ASPN) expression, which was identified as a negative regulator of PDL cells differentiation and mineralization, suppressing BMP-2-induced O/C differentiation. Objective This study aimed to investigate whether 1,25(OH)2D3 treatment could stimulate the O/C differentiation of periodontal ligament mesenchymal progenitor cells characterized as low osteoblast potential (LOP), by asporin and bone morphogenetic protein-2 alteration. Methodology Three LOP cell populations were cultured in standard medium (CONTROL), osteogenic medium (OM), and osteogenic medium associated with 1 nM of 1,25(OH)2D3 (OM + VD). The following assays were performed: 1) MTT to evaluate metabolic activity; 2) gene expression for asporin (ASPN), bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and vitamin D receptor (VDR) using qRT-PCR; 3) BMP-2 extracellular expression; and 4) quantification of mineralized nodule deposition by Alizarin Red Staining. Data were subjected to two-way ANOVA and Tukey’s test (P<0.05). Results The results showed that the 1,25(OH)2D3 treatment did not affect the cell viability, as demonstrated by metabolic activity increase over the 10 days in culture. After 14 days of 1,25(OH)2D3 treatment, the mRNA levels for ASPN and VDR decreased (P<0.05), while BMP-2 transcripts and extracellular expression increased (P<0.05). In parallel, RUNX2, ALP, and OCN gene expression was upregulated by 1,25(OH)2D3 treatment, resulting in an increase of mineral nodule deposition in vitro (P<0.05). Conclusions These data show that 1,25(OH)2D3 improves osteoblast/cementoblast differentiation of low osteoblast potential accompanied by alterations in ASPN and BMP-2 expression. (PDLMSCs heterogeneous disease osteoblastcementoblast cementoblast OC O C (O/C (ASPN mineralization BMP2induced BMPinduced BMP induced 1,25OH2D3 125OH2D3 OHD 1,25 OH 2D3 25 1,25(OH)2D LOP, , (LOP) protein2 protein protein- alteration CONTROL, CONTROL (CONTROL) OM, OM (OM) VD. VD . VD) performed ASPN, BMP2, BMP2 (BMP-2) runtrelated runt RUNX2 RUNX (RUNX2) ALP (ALP) OCN, (OCN) (VDR qRTPCR qRT PCR qRT-PCR 3 BMP- 4 Staining twoway two way Tukeys Tukey s P<0.05. P005 P P<0.05 0 05 (P<0.05) viability culture P<0.05, parallel 25OH2D3 1,25OH2D 125OH2D 125 1,2 2D (LOP (CONTROL (BMP-2 (RUNX2 (ALP (OCN P00 P<0.0 (P<0.05 25OH2D 12 1, (BMP- (RUNX P0 P<0. (P<0.0 (BMP P<0 (P<0. P< (P<0 (P< (P

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.

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.

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 as condições clínicas periodontais de dentes anteriores com migração patológica (MDP) em pacientes com periodontite crônica generalizada e comparar a severidade de destruição periodontal entre dentes migrados e não-migrados. Foram selecionados 32 pacientes, de ambos os sexos, apresentando média de idade de 46,0 anos (± 11,6), com perda clínica de inserção em dentes anteriores e presença de algum tipo de MDP, a saber: vestibularização, diastema, inclinação proximal, giroversão ou extrusão. Os parâmetros avaliados foram a perda clínica de inserção (PIC) e o percentual de perda óssea radiográfica (PO). Os resultados mostraram, em média, uma PIC de 5,50 mm (± 2,20 mm) e uma PO de 41,90% (± 15,40%) do comprimento radicular, em 115 dentes selecionados. Os tipos mais freqüentes de migração foram vestibularização (34,80%) e presença de diastemas (27,00%). A extrusão foi a menos freqüente (4,30%). Maiores valores de PO e PIC foram registrados nos dentes com extrusão (59,44% e 8,42 mm) e vestibularização (45,17% e 6,07 mm). Esses valores de PO foram superiores aos observados nos dentes com giroversão ou inclinação proximal (p < 0,05 - Kruskal-Wallis). A PIC não apresentou diferenças significativas entre os diferentes tipos de migração (p = 0,11). Constatou-se que os dentes anteriores com MDP apresentaram maior PIC e PO (5,1 mm e 40%) quando comparados aos não-migrados (4,1 mm e 31%). Pôde-se concluir também que o tipo de MDP mais prevalente foi a vestibularização, que esteve relacionada a maiores níveis de perda óssea.

The aim of the present study was to evaluate the periodontal conditions of anterior teeth that presented pathologic migration in patients with chronic periodontitis and to compare periodontal destruction in migrated versus non-migrated teeth. The sample included 32 patients of both sexes (mean age: 46.0 ± 11.6 years) diagnosed with generalized chronic periodontitis and selected on the basis of the presence of pathologic migration in one or more anterior teeth. This migration was classified according to the following categories: facial flaring, diastema, proximal tilting, rotation or extrusion. The periodontal parameters recorded were clinical attachment loss (CAL) and percentage of radiographic bone loss (BL). Mean CAL of 5.50 ± 2.20 mm and mean BL of 41.90 ± 15.40% were found in 115 teeth assessed. The most frequent type of migration was facial flaring (34.80%), followed by diastema (27.00%). Extrusion was hardly observed in the sample (4.30%). However, greater severity of BL and CAL were observed in teeth with this type of migration (59.44% and 8.42 mm, respectively), and in teeth with facial flaring (45.17% of BL and 6.07 mm of CAL). Kruskal-Wallis test indicated that BL presented by teeth with extrusion or facial flaring was greater than that observed in rotated or tilted teeth (p < 0.05), while there was no difference between groups regarding CAL (p = 0.11). It was observed that anterior teeth with pathologic migration presented greater CAL and BL (5.1 mm and 40%) than non-migrated teeth (4.1 and 31%). The study indicated that the most prevalent kind of pathologic migration is facial flaring, which was associated to higher level of bone loss.

El objetivo del estudio fue evaluar "in vitro" el efecto de la cotinina en la viabilidad celular utilizando un linaje continuo de fibroblastos. Fueron formados grupos experimentales según las concentraciones de cotinina : 0 (control), 50ng, 100ng, 500ng, 1ug/ml. y tiempo de condicionamento (1, 24, 48 horas). Cada uno de los 12 orificios de una placa para cultivo celular recibió 1 mL de medio de Eagle y 1mL de cotinina en las diferentes concentraciones, inmediatamente fue acrescentado 1ml de medio de cultivo conteniendo 1x10(5) cels/mL. Después del condicionamento con la droga, en los 3 períodos ensayados, las células fueron teñidas con azul de tripan 0,4% y observadas en un microscopio invertido, por un examinador ciego para los grupos experimentales. Los experimentos fueron repetidos 5 vezes. Los resultados mostraron que las dos concentraciones mayores de cotinina presentaron los menores porcentajes de células viables, sin embargo, esta diferencia fue reducida. La viabilidad celular presentó una pequeña disminución con el aumento del tiempo de condicionamiento. Concluimos que la cotinina puede afectar la viabilidad celular solo en las dos mayores concentraciones ensayadas.

The aim of this study was to evaluate, in vitro, the effect of cotinine on the viability of fibroblasts from a continuous lineage. Experimental groups were formed according to drug dosage: 0 (control), 50ng, 100ng, 500ng, 1ug/ml, and time of exposure: (1, 24, 48 horas).Twelve wells microplates were used. Each well received 1ml of Eagle medium and 1ml of a solution containing 1x10(5) cells/ml. Cotinine at the tested concentrations was then added to the wells. After the incubation period, cells viability was assessed by using trypan blue 0,4%. Cell viabylity were assessed on a inverted micorscope, by the single examiner who was blend to the experimentals groups. The experiment was repeated 5 times. Results demostrated that the two greater concentrations presented the more non viable cells, but these diferences were minimal. The cells viability presented minimal decreased with the increased of time exposure. Cotinine affect fibroblasts for the high tested dose.

O objetivo do estudo foi avaliar in vitro o efeito da nicotina sobre a viabilidade e a morfologia celular utilizando-se uma linhagem contínua de fibroblastos. Para tal, foram formados dois grupos experimentais segundo a dose (0 - controle, 10 mig, 100 mig, 0,5 mg, 1 mg) e o tempo de condicionamento (1 e 24 horas). Cada um dos 12 orifícios de uma placa para cultura celular recebeu 2 ml de meio de Eagle, e 1 ml de suspensão de meio de cultura contendo aproximadamente 1 × 10(5) células/ml. Foi, então, acrescentada a solução de nicotina nas diferentes concentrações. Após o condicionamento com a droga, nos dois períodos testados, as células foram coradas com azul de trypan 0,4%, e observadas em microscópio invertido por um examinador cego para os grupos experimentais, que avaliou a viabilidade e a morfologia segundo o índice de Gamal. Os experimentos foram repetidos 5 vezes. Quanto à morfologia, os resultados obtidos demonstraram, no grupo condicionado por 1 h, que os controles apresentaram diferenças estatisticamente significantes em relação apenas à maior dose de nicotina; no entanto, foram encontradas diferenças estatisticamente significantes entre o controle e todas as concentrações após 24 horas de condicionamento. Na viabilidade celular, um maior número de células não viáveis foi observado nas diferentes concentrações de nicotina em comparação aos controles tanto após 1 quanto 24 horas de condicionamento (p < 0,05). Em ambos períodos existiu uma tendência significativa de aumento do número de células não viáveis com o aumento da dose de nicotina (p = 0,0053; p = 0,00001 após 1 e 24 h respectivamente). Portanto, conclui-se que a nicotina pode alterar, in vitro, a viabilidade e a morfologia de fibroblastos de forma proporcional à dose e ao tempo de exposição.

The aim of this study was to evaluate, in vitro, the effect of nicotine on the viability and morphology of fibroblasts from a continuous lineage. Two experimental groups were prepared, with different drug dosages (0 - control, 10 mug, 100 mug, 0.5 mg, 1 mg) and conditioning time (1 and 24 hours). Twelve-well microplates were utilized. Each well received 2 ml of fresh culture medium and 1 ml of a solution containing 1 × 10(5) cells/ml. Nicotine was then added to the wells, at the tested concentrations. After the incubation period, cell viability was assessed by means of 0.4% trypan blue staining. Cell viability and morphology were assessed in an inverted microscope, by a single examiner, who was blind as to the experimental groups. The experiment was repeated 5 times. Regarding morphology, in the 1-hour conditioned group there was statistically significant difference between the control group and the group with the greatest dose of nicotine. These differences were also observed between the control group and all nicotine groups after 24 hours. The results of the Kruskal-Wallis test revealed that more unviable cells were found in the groups exposed to nicotine, in comparison with the control group, both after 1 and 24 hours of conditioning (p < 0.05). Moreover, with increasing doses of nicotine there was a directly proportional increase in the number of unviable cells, both after 1 and 24 hours of exposure (p = 0.0053 and p = 0.00001, respectively). The conclusion of this study is that nicotine can alter, in vitro, the viability and morphology of fibroblasts in a manner proportional to the dose and time of exposure.

Medicamentos homeopáticos como o Symphytum officinalle e a Calendula officinallis são dotados de propriedades anti-sépticas, antiinflamatória, cicatrizantes e também agem como promotores da consolidação de fraturas ósseas. Neste trabalho, uniram-se esses dois medicamentos similares em um complexo para verificar o seu efeito no reparo em feridas de extração dentária em camundongos. O complexo Symphytum officinalle e Calendula officinallis nas potências de 6CH e 3CH, respectivamente, foi ministrado por via oral ao grupo tratado durante 5 dias antes e após a extração do incisivo superior direito. No grupo controle, administraram-se 5ml de álcool etílico a 70% diluídos em 30 ml de soro fisiológico. Após a proservação, os animais foram sacrificados, a maxila direita separada da esquerda, fixada e processada para inclusão em parafina. Após a microtomia, os cortes obtidos foram corados pela H/E. A análise histológica mostrou que, tanto no grupo controle como no tratado, o alvéolo dentário estava preenchido por tecido de granulação e tecido ósseo neoformado, com graus variáveis de maturação, rico em osteócitos. No entanto, nos animais tratados, o processo de reparo em feridas após extração dentária do incisivo superior direito mostrou um avanço progressivo de neoformação óssea mais acentuado quando comparado ao grupo controle, em tempos equivalentes. Estes resultados enfatizam as propriedades biológicas do complexo Symphytum officinalle e Calendula officinallis e sua possível utilização como recurso terapêutico na Odontologia.

Homeopathic medicines as Symphytum officinale and Calendula officinallis are endowed with antiseptic, antiphlogistic and cicatrizant properties and promoter of the consolidation of bone fracture. This research combined these two similar medicines in a compound to examine its action in the repair of tooth extraction sores in mice. The compound Symphytum offic. and Calendula offic. at the respective potencys of 6CH and 3CH was orally administered to the treated group during 5 days before and after the extraction of the rigth upper incisor. To the control group were administered 5 ml of ethylic alchol 70% diluted in 30 ml of physiologic serum. After a period of expectation, the animals were sacrificed, the right maxila was separated of the left maxila, this was fixed and the laboratories techniques were realized for inclusion in paraffin. After that, the piece was cut in the microtome, and the laminas were dyed by H/E. The analysis showed that the control and treated group exhibited the dental alveolus fulfilled with granulation tissue and neoformed bone tissue with variable degrees of maturation, abundant in osteocites. However, at the treated animal the healing process of the sore after the extraction of the rigth upper incisor showed a bone neoformation very pronounced when compared with the control group at equivalent times. Those results showed the biological properties of the compound Symphytum offic. and Calendula offic. and its utilization as a therapeutical help in Odontology.