Dissertations / Theses on the topic 'Periodontal cells'

Background: Directing autogenous Mesenchymal Stem Cell (MSC) to defect sites has a great promise in bone regeneration. We designed a MSC specific, bone affinity peptide (E7HA7) by conjugating E7 with a polyglutamate hydroxyapatite (HA) binding motif. We sought to characterize the in-vivo releasing pattern and bioactivity of E7HA7. Methods: HA discs were coated with fluorescent labeled peptides E7HA7, E7HA2 or E7 were subcutaneously implanted in Sprague Dawley rats. In an ectopic bone formation model was used to test the in-vivo bioactivity of E7HA7 conjugated to DBM. Results: E7HA7 showed slower peptide release from scaffolds in comparison to other groups, being statistically significant at week 2 compared to E7, and to E7HA2 at week 4 and 8. In ectopic model, the medians for new bone formation in each group were: iDBM=0.041mm3, iDBM-E7=0.071mm3, aDBM=0.138mm3, and aDBM-E7=0.192mm3. Conclusions: Conjugation of E7 to polyglutamate bone binding domain showed slow releasing kinetics and osteoinductive potential.

Introduction: Periodontal disease (PD) is the most common bone destructive chronic inflammatory disease in humans. Severe PD affects 8-15% of the population and impacts on the ability to chew and appearance, reduces quality of life, and is responsible for a substantial proportion of dental care costs. A dysbiotic oral biofilm is necessary but insufficient for development of PD. Rather, a dysregulated immune response to the disease-associated biofilm results in destruction of tooth supporting structures and eventual tooth loss. Despite the apparent involvement of the immune system in PD, clinical management focuses solely on the mechanical removal of the oral biofilm – with partial success and frequent recurrence. Therefore, a better understanding of the immune response in PD could highlight potential novel preventative and therapeutic strategies. T cells are present at sites of PD; however, there remains ambiguity regarding whether these T cells are protective or destructive in PD. The aim of these studies was to characterize CD4+ T cells in a P. gingivalis-induced murine model of PD. Results: P. gingivalis-infected mice displayed subtle changes in their CD4+ T cell compartment, predominantly in the draining lymph nodes (dLNs). Such changes included a suggested increase in T follicular helper cells, a trend towards a decrease in regulatory T cells and a trend towards increased production of IFN-γ. Elevated levels of IFN-γ were also noted in gingival CD8+ T cells and splenocytes, with similar trends in CD8+ T cells from dLNs. The transcriptome of CD4+ T cells isolated from gingivae and dLNs of P. gingivalis–infected suggested minimal changes in gene expression following infection; however, identified a profile of the mucosal oral CD4+ T cell compared with CD4+ T cells of the dLN. To investigate the response of CD4+ T cells specific for P. gingivalis, the bacteria were genetically manipulated to express ovalubumin (OVA) peptide 323-339. However, these OVA peptide expressing P. gingivalis failed to induce a response in OVA-specific T cells, both in vitro and in vivo. Conclusion: These data imply that CD4+ T cells do not substantially change upon P. gingivalis infection in a murine model. IFN-γ production, however, was elevated both locally and systemically. Together, the data presented in this thesis and data previously published warrant further investigations into the role of IFN-γ in PD and may point to IFN-γ as a biomarker or biological target for adjunctive PD therapy.

Periodontal disease is an inflammatory disorder affecting the supporting tissues of the tooth. The inflammation triggers a destruction of the connective- and bone tissue surrounding the tooth, a process that is not fully elucidated. It is known that periodontitis shares features with other inflammatory disease like Crohn's disease and rheumatoid arthritis. The cell surface proteins signal regulatory protein alpha (SIRPα) and cluster of differentiation 47 (CD47) are important for the progression of the inflammation in rheumatoid arthritis and Crohn's disease. It has also been shown that lack of SIRPα or CD47 render in reduced number of osteoclast. The aim of this study was to investigate if cells from the periodontium (human gingival fibroblasts) from periodontally healthy individuals express SIRPα and CD47 and if the expression of these membrane proteins is regulated under inflammatory conditions.   We demonstrate, by using quantitative rt-qPCR, that human gingival fibroblasts express both CD47 and SIRPα mRNA. The expression of SIRPα was positively regulated (2-fold) by the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1-beta (IL-1β). TNF-α caused a 2-fold up-regulation of CD47 in human gingival fibroblasts. Neither CD47 nor SIRPα were time-dependently regulated by the two pro-inflammatory cytokines.   We here conclude that SIRPα and CD47 gene expression are up-regulated in human gingival fibroblasts cultured under inflammatory conditions. These findings indicate that SIRPα and CD47 may play a role in periodontal disease.

Periodontitis is a chronic inflammatory disease initiated by gram negative anaerobic bacteria. These bacteria possess pathogen-associated molecular patterns (PAMPs) that interact with various receptors including Toll like receptors (TLRs). Bacterial DNA (bDNA) is one of the PAMPs mainly recognized by TLR9. Interaction of bDNA and its receptors leads to activation of inflammatory signaling pathways potentially resulting in periodontal bone destruction. The aim of this study was to determine the production of IL- 6 and IL-8 in response to periodontal bDNA from human osteoblastic cells (MG-63). MG- 63 cells were stimulated in duplicate for 20 hours with 100ng/μl of bDNA from various pathogens including Porhyromonas gingivalis, Esherichia coli, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans as well as heat killed whole bacteria (1:100). E.coli LPS (10ng/μl) was used as a positive control in each experiment. To block TLR9 signaling, further experiments were carried out by treating MG-63 cells with chloroquine (10ng/μl) for 2 hours at 37ºC prior to stimulations. Cytokine levels were determined using enzyme linked-immunosorbent assay. Although IL-6 and IL-8 production was increased in response to periodontal bDNA in MG-63 cells, the results were not significant compared to unstimulated controls. As expected, E.coli DNA, E.coli LPS and heat killed whole bacteria stimulated significantly increased cytokine production (p<0.05). Blocking TLR9 with chloroquine did not affect the amount of cytokine production in bDNA stimulated cells suggesting that TLR9 may not be operant in triggering IL-6 and IL-8 production from MG- 63 cells. In conlusion, periodontal bDNA did not trigger significantly increased IL-6 and IL-8 production from MG-63 cells. Considering the involvement of several inflammatory mediators in periodontal bone destruction, further studies are warranted to assess the production of other cytokines in response to periodontal bDNA in human osteoblastic cells.

Background and objectives: The most widely accepted tooth movement model is defined by the pressure-tension hypothesis. An orthodontic force applied to a tooth generates areas of compression and tension in the periodontal ligament (PDL), which are transmitted to the alveolar bone. Areas of tissue exposed to tensile strain undergo bone deposition, whereas areas of tissue exposed to compressive strain undergo bone resorption. We propose that human PDL cells in monolayer culture exposed to tensile mechanical strain would express multiple genes involved in osteogenesis. Materials and Methods: Human PDL cells were isolated and cultured from premolar teeth that were extracted for orthodontic reasons. These cells were plated on control and experimental Uniflex[TM] plates. Using a Flexercell FX4000 strain unit, PDL cells on experimental plates were exposed to a 12% uni-axial cyclic strain for 6 seconds out of every 90 seconds over a 24 hour period. RNA was extracted from the PDL cells at 6 hours, 12 hours and 24 hours. The differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism was analysed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) array technology. Results: Of the 78 genes tested, sixteen genes showed statistically significant (p<0.05) changes in expression in response to the mechanical strain regime. Eight genes were up-regulated (ALPL, BMP2, BMP6, COL2A1, ICAM1, PHEX, SOX9, and VEGFA) and eight genes were down-regulated (ANXA5, BMP4, COL11A1, COL3A1, EGF, ITGB1, MSX and SMAD1). Conclusions: This study has demonstrated that cultured human PDL cells express multiple osteogenic genes under tensile strain, which suggests that PDL cells may have a potential role in osseous remodeling during tooth movement. Key Words: Tooth movement, human PDL cells, tensile mechanical strain, osteogenic genes, real-time RT-PCR array, and Flexercell FX4000.

Orientadores: Pedro Luiz Rosalen, Gilson Cesar Nobre Franco
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-17T12:39:43Z (GMT). No. of bitstreams: 1 Almeida_LucianaSallesBrancode_D.pdf: 18183834 bytes, checksum: 7e68ca8a08aa330701eecf3a55349a69 (MD5) Previous issue date: 2011
Resumo: A fluoxetina é um droga inibidora seletiva da recaptação de serotonina que apresenta propriedades imunomoduladoras e antiinflamatórias. O objetivo desse estudo foi avaliar os efeitos da fluoxetina sobre a resposta imuno-inflamatória relacionada à doença periodontal (DP). In vitro, avaliou-se a influência da fluoxetina sobre a capacidade das células dendríticas (DCs) em apresentar antígeno aos linfócitos T. As DCs foram obtidas da medula óssea de camundongos C57BL/6 e diferenciadas utilizando-se GM-CSF (20 ng/mL). As DCs foram tratadas com a fluoxetina (concentrações: 0,01, 0,1 ou 1 ?M) para análise da produção de citocinas e quimiocinas, bem como da expressão de MHC-II e moléculas co-estimuladoras (CD80, CD86, PD-L1, ICOS-L) aos linfócitos T, utilizando-se ensaios de ELISA e citometria de fluxo respectivamente. Culturas de DCs e linfócitos T reativos ao Aggregatibacter actinomycetemcomitans (×Aa-T) foram utilizadas para avaliação de proliferação/ativação de linfócitos T. A desipramina, um inibidor seletivo da recaptação de norepinefrina, também foi incluída nos ensaios in vitro para comparação. In vivo, avaliou-se os efeitos da fluoxetina sobre a resposta inflamatória e a destruição tecidual utilizando-se modelo de DP induzida por ligadura. Ratos Wistar machos (SPF) foram submetidos à colocação de ligadura em torno dos primeiros molares inferiores e divididos em 3 grupos experimentais (n=10 animais/grupo): 1) ratos sem ligadura e sem tratamento (grupo controle); 2) ratos com ligadura e tratados com solução salina (grupo ligadura); 3) ratos com ligadura e tratados com a fluoxetina (20 mg/kg/dia, grupo ligadura + fluoxetina). Análises de reabsorção óssea na região de furca (lâminas coradas com H&E) e de colágeno no tecido conjuntivo da mesial dos primeiros molares (coloração de picrosirius) foram realizadas nos ratos submetidos a 15 dias de indução da DP. Tecidos gengivais de ratos submetidos a 3 dias de indução da DP foram submetidos às seguintes análises: expressão de IL-1?, COX-2, MMP-9 e iNOS utilizando-se RT-PCR e atividade da MMP-9 utilizando-se zimografia. Como resultados, a fluoxetina diminuiu a produção de IL-12, IL-1?, TNF-?, RANTES e MIP-1??pelas DCs estimuladas com LPS (P < 0,05, ANOVA, teste t de Student), bem como diminuiu significativamente a expressão de ICOSL. Além disso, reduziu a proliferação de ×Aa-T estimulados com Aa pelas DCs. A serotonina (5- HT) aumentou a proliferação de ×Aa-T, indicando que os efeitos da fluoxetina são independentes da 5-HT. A desipramina apresentou perfil semelhante à fluoxetina nos ensaios in vitro. No estudo in vivo, a fluoxetina reduziu a perda óssea em região de furca quando comparada ao grupo ligadura (P < 0,05 ANOVA, teste t de Student) e manteve a porcentagem de fibras colágenas com níveis similares ao grupo controle (P > 0,05). Ainda, a fluoxetina reduziu a expressão de IL-1??e COX-2 e a atividade da MMP-9 quando comparada ao grupo ligadura (P < 0,05). Em conjunto, os dados demonstram que a fluoxetina diminuiu a capacidade de apresentação de antígeno das DCs, bem como a resposta inflamatória, a reabsorção óssea e a perda de colágeno na DP, indicando que ela pode constituir uma abordagem terapêutica promissora como moduladora da resposta do hospedeiro na DP
Abstract: Fluoxetine is a selective serotonin reuptake inhibitor presenting immunomodulatory and anti-inflammatory properties. The aim of this study was to evaluate the fluoxetine effects on immunoinflammatory response associated with periodontal disease (PD). The in vitro study evaluated the effects of fluoxetine on antigen-presentation capacity of dendritic cells (DCs). Bone marrow DCs obtained from C57BL/6 wild type mice were differentiated using GM-CSF (20 ng/mL). DCs were treated with fluoxetine (concentrations of 0.01, 0.1 or 1 ?M) for subsequent cytokine/chemokine assays (ELISA) and analysis of expression of MHC-class II and co-stimulatory molecules (CD80, CD86, PD-L1, ICOS-L) to T cell activation using flow cytometry. Fluoxetine was also applied to cultures with both DCs and Aggregatibacter actinomycetemcomitans (Aa)-reactive T cells (×Aa-T), which were used for analysis of T cells proliferation/activation using thymidine and ELISA assays. Desipramine, a selective norepinephrine reuptake inhibitor, was also tested in vitro for comparison to fluoxetine. In vivo, male Wistar rats received ligature placement around mandibular first molars and were randomly assigned into three experimental groups (n=10/group): 1) Control rats (without ligature); 2) rats with ligature + placebo (saline; oral gavage); 3) rats with ligature + fluoxetine (20 mg/kg/day in saline; oral gavage). Histometric and histological analyses were performed for measurement of loss of bone in furcation region (H&E stain) and collagen fibers (picrosirius red stain) in the connective tissue of rats submitted to 15 days of PD induction. Gingival tissues were collected from animals submitted to 3 days of PD induction for analyses of mRNA expression of IL-1?, COX-2, MMP-9 and iNOS using RT-PCR, measurement of total protein concentration and MMP-9 activity using zymogram. Fluoxetine suppressed IL 12, IL-1?, TNF-?, RANTES and MIP-1??production by LPS-stimulated DCs (P < 0.05, ANOVA, Student's t test), as well as significantly reduced the expression of ICOS-L. Fluoxetine suppressed the proliferation of ×Aa-T stimulated with Aa-antigen presentation by DCs from co-cultures. When applied to ×Aa-T/DCs co-cultures, serotonin (5-HT) increased T cell proliferation, indicating that fluoxetine effects are independent of 5-HT. Desipramine effects were similar to those of fluoxetine. In the in vivo study, fluoxetine reduced alveolar bone loss as compared to ligature group (P < 0.05, ANOVA, Student's t test) and maintained collagen fibers levels similarly to control group (P > 0.05). Fluoxetine reduced IL-1??and COX-2 expression, as well as MMP-9 activity, from gingival tissues when compared to ligature group (P < 0.05). Altogether, data showed that fluoxetine can modulate the antigenpresentation capacity of DCs and reduce inflammatory response and loss of bone and collagen associated with PD. In conclusion, fluoxetine can modulate both immune and inflammatory responses on PD, suggesting that it may constitute a new therapeutic approach for modulation of host response in periodontal therapy
Doutorado
Farmacologia, Anestesiologia e Terapeutica
Doutor em Odontologia

Submitted by GUSTAV GUIMARÃES (DINTER) null (gustav@saolucas.edu.br) on 2016-11-25T15:10:43Z No. of bitstreams: 1 TESE GUSTAV Final.pdf: 701691 bytes, checksum: 5a36e117e77c5b5164011367f92fe48f (MD5)
Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-30T10:55:34Z (GMT) No. of bitstreams: 1 guimaraes_g_dr_araca.pdf: 701691 bytes, checksum: 5a36e117e77c5b5164011367f92fe48f (MD5)
Made available in DSpace on 2016-11-30T10:55:34Z (GMT). No. of bitstreams: 1 guimaraes_g_dr_araca.pdf: 701691 bytes, checksum: 5a36e117e77c5b5164011367f92fe48f (MD5) Previous issue date: 2016-11-22
O objetivo deste trabalho foi investigar a influência da extensão da doença periodontal no perfil sanguíneo de ratos Wistar. Foram utilizados 30 ratos divididos em 3 grupos de 10 animais: C- ratos controle; DP1- ratos com doença periodontal em 3 molares superiores; DP2 – ratos com doença periodontal em 3 molares superiores e 3 inferiores. A doença periodontal foi induzida por meio da confecção de ligadura em torno do colo dentário dos molares selecionados de acordo com cada grupo. Após 30 dias, os animais foram anestesiados e, por meio de uma punção cardíaca, foi coletado 5ml de sangue para as análises dos parâmetros do hemograma, creatinina, triglicérides e colesterol. Em seguida, os animais foram sacrificados e as maxilas removidas e processadas para análise histopatológica em coloração de H.E. para caracterizar o perfil da doença periodontal em cada animal. Os resultados obtidos a partir do tecido hematológico foram analisados estaticamente por meio dos testes de ANOVA e Tukey, com nível de significância de 5%. Pode-se observar nos grupos DP1 e DP2 presença de infiltrado inflamatório crônico, perda de inserção conjuntiva, perda de estrutura óssea cortical e alveolar, presença de reabsorções dentárias, presença de biofilme e sequestro ósseo. Quanto ao perfil sanguíneo, pode-se observar diferença estatisticamente significante entre o grupo controle e os grupos DP1 e DP2 em relação à quantidade de leucócitos e linfócitos (p<0,05). Ainda, o grupo DP2 apresentou maior quantidade de leucócitos e neutrófilos em relação ao grupo DP1 e controle (p<0,05). Pode-se concluir que a presença da doença periodontal eleva a quantidade de leucócitos, neutrófilo e linfócitos no sangue de ratos Wistar, e que, a extensão da doença periodontal influencia na quantidade de leucócitos e neutrófilos.
The aim of this study was to evaluate the influence of the periodontal disease extension on the blood profile of Wistar rats. Thirty rats were divided into 3 groups of 10 animals each: C control rats; DP1- rats with periodontal disease in 3 upper molars; DP2 - rats with periodontal disease in 3 upper and 3 lower molars. Periodontal disease was induced by ligature around the tooth according to each group. After 30 days, the animals were killed and 5ml of blood was collected for analyzes of blood count parameters, creatinine, triglycerides and cholesterol. Then the animals were sacrificed and the jaws removed and processed for histopathological analysis to characterize, in H.E. staining, the profile of periodontal disease in each animal. The results from the hematologic tissue were analyzed statistically by ANOVA and Tukey test, with 5% significance level. It can be observed in groups DP1 and DP2 presence of chronic inflammatory infiltrates, connective tissue attachment loss and alveolar bone cortical structure loss, presence of resorptions, presence of biofilm and bone sequestration. As for the blood profile, it is can observe a statistically significant difference between the control group and the groups DP1 and DP2 to the amount of leukocytes and lymphocytes (p <0.05). Further, DP2 group had a higher amount of leukocytes and neutrophils compared to DP1 and control group (p <0.05). It can be concluded that the presence of periodontal disease increases the amount of leukocytes, neutrophils, and lymphocytes in the blood of Wistar rats, and the extent of periodontal disease affects the quantification of leukocytes and neutrophils.

Thesis (D.Clin.Dent.)--University of Adelaide, Dental School, 2001.
Second copy has title: Prevalence and determination of mast cell type within the periodontal ligament of the developing rat tooth. Bibliography: leaves 109-119.

The design of a tissue engineered pulmonary valve (TEPV) involves cells source(s), scaffold, in vitro conditioning system and the functional stability of the TEPV in vivo. Vascular cells (pulmonary artery smooth muscle (SMCs) and endothelial cells (ECs)) and periodontal ligament derived stem cells (PDLSCs) are relevant sources for the designing of TEPVs. In this study, labeling of these cell populations with super paramagnetic iron oxide microparticles along with concomitant usage of transfection agents was followed by visualization using magnetic resonance, while Intracellular iron oxide was confirmed by prussian blue staining and fluorescence microscopy. Also, the potential of PDLSC as a feasible source for TEPVs was investigated, expressing differentiative capacity to both SMC and EC phenotypes by a combination of biochemical and mechanical stimulation. Flow conditioning in a u-shaped bioreactor augmented collagen production in SMC-EC (99.5% for n=3) and PDLSC (93.3% for n=3) seeded scaffolds after a 3-week culturing period (P<0.05).

Orientador: Karina Gonzales Silvério Ruiz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-27T02:48:19Z (GMT). No. of bitstreams: 1 Oliveira_GuilhermeHenriqueCosta_M.pdf: 1623320 bytes, checksum: af95c8df2b95e735fcae950c2260eb9e (MD5) Previous issue date: 2015
Resumo: Células mesenquimais isoladas do ligamento periodontal (PDLSCs) de dentes humanos mostraram ser multipotentes e possuem a capacidade de se diferenciar em adipócitos osteoblastos in vitro. Além disso, possuem a capacidade de formar tecido semelhante ao cemento e ao ligamento periodontal quando transplantadas para animais imunossuprimidos. Por outro lado foi mostrado na literatura uma grande heterogeneidade destas linhagens de células e muito tem sido feito na tentativa de isolar grupos com fenótipos mais favoráveis a diferenciação em tecido mineralizado. A literatura aponta que existe uma modulação na expressão do marcador de superfície CD166 durante a diferenciação osteoblástica e a partir destes achados levanta-se a hipótese de sua participação neste processo. Em acréscimo, células positivas para este marcador mostraram alta capacidade de diferenciar-se em fenótipo osteoblástico. Deste modo, este estudo tem como objetivo avaliar se um grupo de células enriquecido para o marcador CD166 através de separação magnética (PDL/CD166+) apresenta maior potencial de diferenciação osteoblástica/cementoblástica quando comparado ao pool de células não separadas (PDL) e ao grupo de células que não foram retidas na coluna magnética (PDL/CD166-). Este estudo ainda se propõe a avaliar se o marcador de superfície CD166 é regulado no processo de diferenciação osteoblástica. Após a separação magnética de três populações celulares, os grupos PDL/CD166+ foram submetidos a citometria de fluxo para que fosse comprovado a alta expressão de CD166. Em seguida os três grupos, PDL, PDL/CD166+ e PDL/CD166- das três populações, foram submetidos aos ensaio de Alizarina para avaliar a capacidade formação de nódulos minerais in vitro, foram ainda caracterizados por real-time PCR para avaliação da expressão de genes relacionados ao fenótipo osteoblástico (fator de transcrição relacionado a Runt-2 -Runx-2; a fosfatase alcalina ¿ ALP ¿ e a osteocalcina ¿ OCN). Nos grupos PDL e PDL/CD166+ também foi avaliado a expressão de CD166 por qPCR e citometria de fluxo. Os grupos enriquecidos apresentaram uma expressão média de 91,5% (±4,3%) de CD166. Apenas um grupo PDL/CD166- de uma população não apresentou depósitos minerais in vitro e foi identificado diferença estatisticamente significante entre os grupos PDL/CD166+ e PDL/CD166- (p<0,05). Quanto a expressão dos marcadores biológicos do tecido ósseo, foi identificado uma tendência a aumento da expressão destes marcadores quando as células foram cultivadas sob indução e ao longo do tempo de cultura. Os grupos cultivados em meio osteogênico apresentaram maior expressão de CD166 por PCRq e também foi identificado que o grupo PDL/CD166+ mostrou uma expressão significativamente maior que o PDL, ambos sob indução (p<0,05). Por citometria de fluxo, foi identificado um acréscimo na expressão de CD166 no terceiro dia de diferenciação nas células PDL/CD166+ e foi estatisticamente significativo comparado ao meio padrão (p<0,05). Ao longo do tempo de cultivo sob indução, a expressão deste marcador foi diminuída. Através dos resultados deste estudo é possível concluir que o grupo enriquecido na expressão de CD166 apresenta um alto potencial de formação de nódulos minerais in vitro. Além disso, foi notado que os três grupos apresentam uma tendência a maior expressão de Runx-2, ALP e OCN quando cultivados em meio osteogênico e que a grande heterogeneidade destas três populações dificulta a identificação de diferenças significativas tanto na análise intergrupo como intragrupo e compromete a identificação de um subgrupo com fenótipo mais favorável a diferenciação osteoblástica. A expressão gênica de CD166 é aumentada sob indução e ao longo do tempo de cultura e por citometria de fluxo observa-se que as células cultivadas em OM tendem a reduzir a expressão deste marcador
Abstract: Mesenchymal stem cells from periodontal ligament (PDLSCs) isolated from human teeth were shown to be multipotent and have the capacity to differentiate into adipocytes and osteoblasts in vitro. Moreover, they have the ability to form cement and periodontal ligament-like tissues when transplanted into immunosuppressed animals. On the other hand, it has been shown in literature a great heterogeneity of these cell lines and much has been done in the attempt to isolate groups with a phenotype more prone to differentiate into mineralized tissues. The literature suggests that there is a modulation of the CD166 surface marker expression during osteoblast differentiation and from these findings it is hypothesized of its participation in this process. In addition, cells positive for this marker have shown strong ability to differentiate into osteoblastic phenotype. Thus, this study aims to assess whether an enriched cell group for the CD166 marker (PDL/CD166+) has greater potential for osteoblastic/cementoblastic differentiation when compared to the not separated cell pool (PDL) and cell group which are not enriched (PDL/CD166-). Then, the three groups PDL, PDL/CD166+ and PDL/CD166- from three populations were submitted to Alizarin red-based assay to evaluate the ability of mineral nodule formation in vitro, groups were further characterized by qPCR for the evaluation of the expression of genes related to the osteoblastic phenotype (transcription factor related to Runt-2 -Runx-2; alkaline phosphatase - ALP - and osteocalcin - OCN). In the PDL and PDL/CD166+ groups the CD166 expression was also assessed by qPCR and flow cytometry. Enriched groups showed an average expression of 91.5% (± 4.3%) of CD166. Only a PDL/CD166- group from one population showed no mineral deposits in vitro and it was identified statistically significant difference between the groups PDL/CD166+ and PDL/CD166- (p <0.05). Regarding the expression of bone tissue biomarkers, it was noted a tendency to increased expression of these markers when cultured under osteogenic induction and along time in culture. Groups cultured in osteogenic medium showed higher expression of CD166 by qPCR and it was also identified that the PDL/CD166+ group showed a significantly higher expression than the PDL group, both under osteogenic induction (p <0.05). By flow cytometry, it was noticed an increase in CD166 expression on the third day of differentiation in PDL/CD166+ cells and it was statistically significant compared to the standard medium (p <0.05). Along time in culture, the expression of this marker was decreased. Through the results of this study it is possible to conclude that the group enriched in CD166 expression has a high potential to form mineral nodules in vitro. In addition, it was noted that the three groups have a tendency to increased Runx-2, ALP and OCN expression when cultured in osteogenic medium and the great heterogeneity of these three populations difficult to identify significant differences both intra and inter-group analysis what may jeopardize the identification of a subgroup with a phenotype more prone to osteoblastic differentiation. Gene expression for CD166 is increased under induction e through culture time and by flow cytometry it is observed that cells cultured in OM tend to reduce the expression of this marker
Mestrado
Periodontia
Mestre em Clínica Odontológica

The application of cellular therapies for the treatment of myocardial infarction has provided encouraging evidence for the possibility of cellular therapies to restore normal heart function. However, questions still remain as to the optimal cell source, pre-conditioning methods and delivery techniques for such an application. Here I propose the use of a unique population of stem cells arising from the embryonic neural crest. These cells were shown to express neural crest markers as well as pluripotency-associated markers. Furthermore, the cells were shown to express proteins essential to the formation of gap junctions and to possess a cardiomyogenic differentiation potential by several means. Furthermore, I explore the use of mechanical strain as an inducer of cardiomyogenesis and possibly pre-conditioning stimulus for the better engraftment of the cells while in the heart. Mechanical strain was shown to elicit a cardiomyogenic response from the cells following just a couple of hours of stimulation. The mode in which mechanical strain elicited these responses was demonstrated to be via the mediation of the reactive oxygen species (ROS) pathways. Given the results presented here, the use of these periodontal ligament-derived stem cells (PDLSC) in combination with mechanical strain preconditioning of the cells prior to their delivery into the heart may pose a valuable alternative for the treatment of myocardial infarction and merits further exploration for its capacity to augment the already observed beneficial effects of cellular therapies.

ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården.
ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.

Submitted by VICTOR GUSTAVO BALERA BRITO (victor_balera@hotmail.com) on 2018-07-03T00:06:47Z No. of bitstreams: 1 Dissertação Mestrado (Victor G B Brito) (FINAL).pdf: 4446716 bytes, checksum: 348cbec88d5fb2161f9d4aaa27992ac2 (MD5)
Approved for entry into archive by Ana Paula Rimoli de Oliveira null (anapaula@foa.unesp.br) on 2018-07-03T12:24:17Z (GMT) No. of bitstreams: 1 brito_vgb_me_araca_int.pdf: 4446716 bytes, checksum: 348cbec88d5fb2161f9d4aaa27992ac2 (MD5)
Made available in DSpace on 2018-07-03T12:24:17Z (GMT). No. of bitstreams: 1 brito_vgb_me_araca_int.pdf: 4446716 bytes, checksum: 348cbec88d5fb2161f9d4aaa27992ac2 (MD5) Previous issue date: 2018-06-13
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Introdução: A doença periodontal (DP) é uma desordem inflamatória dos tecidos de suporte dos dente, iniciada pelo acumulo de biofilme bacteriano, apresentando consequências locais e sistêmicas. A coexistência de doenças sistêmicas, como a hipertensão, pode levar a uma inflamação exacerbada, maior reabsorção óssea e dano sistêmico pronunciado. Além disso, células imunes residentes têm importante papel na progressão da DP, porém, a participação dos mastócitos (MC) ainda não é bem compreendida. Objetivos: Avaliar o papel dos MC sobre o metabolismo ósseo local (mandíbula) e sistêmico (fêmur), em modelo animal normotenso (ratos Wistar) e hipertenso (ratos SHR) com DP. Métodos: Ratos machos Wistar e SHR (10 semanas) foram utilizados. A depleção de MC foi conduzida pelo pré-tratamento com o composto 48/80 e a DP foi induzida por ligadura bilateral nos primeiros molares inferiores, mantida por 15 dias. Foi realizada a identificação de MC no tecido gengival, por coloração com Azul de Toluidina. A perda óssea alveolar e parâmetros de arquitetura óssea na mandíbula e fêmur foram avaliados por microtomografia computadorizada, a expressão gênica de marcadores de formação, remodelamento e reabsorção na mandíbula e fêmur foram avaliada por RT-PCR em tempo real, e a produção de citocinas nos tecidos de interesse foi avaliada por ELISA. Principais resultados: Observamos significativa perda óssea induzida por DP, principalmente no SHR, em comparação ao Wistar, e a depleção de MC foi capaz de prevenir esta perda. A DP levou a expressão aumentada de Opn, Opg, Rankl e Rank, Trap, Ctsk, Vtn, Itga5 e Itgb5, além de aumento na produção de TNF-α, IL-6, IL-10 e CXCL3 na mandíbula, o que foi reduzido em animais depletados de MC. No fêmur, a DP não levou a alterações da arquitetura óssea, mas foi capaz de aumentar a expressão de Runx2, Opn, Opg, Rankl, Rank, Trap, Mmp2, Mmp9, Vtn, Itga5 e Itgb5, o que também foi significativamente reduzido pela depleção de MC. Conclusões: Nossos dados sugerem um importante papel dos MC nas consequências ósseas locais (mandíbula) da DP, especialmente nos animais SHR, além dos efeitos sistêmicos (fêmur) onde os MC possivelmente têm participação na modulação da expressão de marcadores ósseo, alterados pela DP, o que foi mediado por vias diferentes das observadas na resposta local.
Introduction: Periodontal disease (PD) is an inflammatory disorder of the tissues supporting the teeth, which start from the bacterial biofilm accumulation, and results in local and systemic damage. The coexistence of systemic diseases, such as hypertension, can lead to exacerbated inflammation, increased alveolar bone resorption and increased systemic damage. In addition, resident immune cells have an important role in PD progression, however the mast cells (MC) participation is still not well understood. Aims: To evaluate the role of MC in the local (mandible) and systemic (femur) bone metabolism in a normotensive (Wistar) and hypertensive (SHR) rats with PD. Methods: Males Wistar and SHR rats (10-week old) were used. MC depletion was conducted by compound 48/80 treatment and PD was induced by bilateral ligature placed in the lower first molars, which was maintained for 15 days. The identification of MC in the gingival tissue was done by Toluidine Blue staining, alveolar bone loss and bone architecture parameters of mandible and femurs were evaluated by micro-computed tomography, bone markers gene expression on the mandible and femur were evaluated by real time RT-PCR, and the cytokines production was evaluated by ELISA. Key results: We observed a more significant PD-induced bone loss in SHR, compared to Wistar, and MC depletion was able to prevent this loss. PD increased the expression of Opn, Opg/ Rankl/Rank, Trap, Ctsk, Vtn, Itga5 and Itgb5, as well as increased production of TNF-α, IL-6, IL-10 and CXCL3 in the mandible, what was prevented by MC depleted animals. In the femur, PD did not change bone architecture parameters, but was able to increase expression of Runx2, Opn, Opg/Rankl/Rank, Trap, Mmp2, Mmp9, Vtn, Itga5 and Itgb5, which also was significantly reduced by MC depletion. Conclusions: Our data suggest an important role of MC in the local bone consequences (mandible) of PD, especially in SHR, as well the systemic effects (femur), where MC may have a role in bone dynamics by modulating the expression of bone markers, altered by PD, through different mechanism from those observed in the local response.
15/03965-2

Strontium (Sr2+) är det aktiva ämnet i läkemedel som används för att reducera frakturrisken hos patienter som lider av osteoporos. På senare tid har Sr2+ kombinerats med olika biomaterial i syfte att gynna benbildning, emellertid är ämnet vagt studerat avseende effekten på parodontala vävnader. Trots omfattande användning, är verkningsmekanismer av Sr2+ inte helt klarlagda. Denna studie syftar därmed till att utvärdera effekten av Sr2+ på humana parodontalligament-celler (PDL-celler) och osteoblaster avseende proliferation och osteogen aktivitet. Odlade humana PDL celler och osteoblastcellinjerna MG63 och hFOB 1.19 behandlades med SrCl2 (0,1-10 mM) eller vehikel under 72 h. Manuell cellräkning utfördes med en Bürker kammare. Det totala proteininnehållet fastställdes med en kolorimetrisk mätning genom användning av Bio-Rad proteinanalys. Aktiviteten av alkaliskt fosfatas bestämdes enzymatiskt och normaliserades till totalt proteininnehåll. SrCl2 hade ingen signifikant effekt på PDL celler (p> 0.05), däremot observerades en tendens till inducerade osteoblastiska egenskaper. I motsats till det, ökade 5 mM SrCl2 den totala proteinhalten i MG63 celler med 37% (p <0,01) jämfört med vehikel, medan en lägre koncentration (0,1 mM) inte hade någon påverkan. 5 mM SrCl2 ökade MG63 cellantalet med 38% (p <0,001), medan en högre koncentration (10 mM) inte uppvisade en signifikant ökad effekt jämfört med 5 mM (+54%, jämfört med vehikel, p<0.05). Resultaten visar att 72 h administrering av ≥ 5 mM SrCl2 ger en pro-proliferativ effekt på humana osteoblastliknande MG63 celler och uppvisar en tendens till att stimulera osteogena egenskaper hos primära humana PDL-celler.
Strontium (Sr2+) is the active substance of pharmaceuticals used for reducing fracture risk in osteoporotic patients. Lately, Sr2+ is combined with biomaterials to enhance osteogenesis, which has been vaguely studied considering periodontal tissue regeneration. Despite extensive use, the mechanisms of action of Sr2+ are not fully understood. The present study assesses the impact of Sr2+ on primary human periodontal ligament cells (PDL cells) and human osteoblasts in regard to proliferation and pro-osteogenic activity. Cultured human PDL cells and osteoblast cell lines MG63 and hFOB 1.19 were treated with SrCl2 (0.1-10 mM) or vehicle for 72 h. Cells were counted manually using a Bürker chamber. Total protein content was determined by colorimetric analysis using Bio-Rad protein assay. Alkaline phosphatase activity was determined enzymatically and normalized to total protein content. SrCl2 had no significant effect on PDL cells (p>0.05), but a tendency towards induced osteogenic characteristics was observed. In contrast, 5 mM SrCl2 enhanced total MG63 cell protein content by 37% (p<0.01), compared to vehicle, whereas a lower concentration (0.1 mM) did not. 5 mM SrCl2 increased MG63 cell number by 38% (p<0.001), while a higher concentration (10 mM) did not have a significant additional effect over the 5 mM (+54%, compared to vehicle, p<0.05). The results demonstrate that 72 h administration of ≥ 5 mM SrCl2 exerts a pro-proliferative effect on human osteoblast-like MG63 cells and display a tendency to induce osteogenic characteristics in primary human PDL cells.

El tratamiento de los defectos infraóseos es uno de los objetivos de la terapia de la enfermedad periodontal. La literatura actual reporta ciertas limitaciones en cuanto a la predictibilidad de las técnicas, por consiguiente, siguen siendo necesarios modelos predictivos y estrategias regenerativas más precisas. Uno de los problemas actuales desde un punto de vista clínico, es el desconocimiento estructural de los tejidos que deseamos regenerar. Definir las proteínas expresadas en el periodonto podría ayudar a establecer estrategias clínicas regenerativas más fiables. El presente estudio tiene como objetivo evaluar la composición del ligamento periodontal y el hueso alveolar de diferentes donantes usando análisis de cromatografía líquida conjuntamente con espectrometría de masas (LC-MS/MS) para identificar los marcadores específicos de cada tejido. En paralelo, nuestro estudio pretende investigar el potencial regenerativo de las DPPSCs (dental pulp pluripotent-like stem cells), para formar tejido periodontal nuevo in vitro usando dientes humanos sanos y estériles como soporte. Posteriormente, estudiar a nivel proteico la estructura formada por las DPPSCs in vitro después de diferenciarlas a tejido periodontal y tejido óseo y compararlas con el proteoma del ligamento periodontal y hueso alveolar. Y finalmente, con el objetivo de conseguir una superfície radicular adecuada para la posterior agregación y diferenciación celular, se estudió varias técnicas de instrumentación radicular (ultrasonido, cureta, perioset  y combinación de ambos) y varias técnicas de acondicionamiento radicular con agentes químicos (ácido fosfórico, EDTA, ácido cítrico y tetraciclina). A posteriori, se realizó un análisis de microscopía electrónica de las diferentes muestras. En conclusión, el análisis proteómico de nuestro estudio clasificó las proteínas del ligamento periodontal y hueso alveolar en diversas categorías de ontología genética: 1) Marcadores de remodelación de cromatina 2) Marcadores de desarrollo 3) Marcadores de diferenciación celular 4) Marcadores de expresión génica 5) Marcadores de matriz extracelular 6) Marcadores de migración celular 7) Marcadores de células madre 8) Marcadores estructurales. Los resultados demostraron un grado de similitud entre los tejidos del ligamento y del hueso alveolar. Se identificaron 319 proteínas exclusivas en el ligamento periodontal, 118 proteínas exclusivas en el hueso alveolar y 562 proteínas comunes a ambos tejidos. Las DPPSCs mostraron una posible capacidad de diferenciación a tejido periodontal y tejido óseo por la expresión de marcadores específicos de ligamento periodontal y hueso alveolar a los 21 días de la diferenciación que no se identificaron en el día 0. La utilización de las DPPSCs en medicina regenerativa permitiría realizar un tipo de terapia celular más individualizada gracias a su fácil acceso, su presencia en todas las edades y a su capacidad de diferenciación. Sin embargo, faltaría por confirmar nuestros resultados en un buen modelo animal de la enfermedad periodontal. Y finalmente, para futuros estudios in vitro nuestros resultados demostraron que existía agregación celular en todas las condiciones de instrumentación y acondicionamiento radicular analizadas. Asimismo sería necesario realizar estudios del efecto microbiológico de las técnicas tras la terapia de desbridamiento para verificar la eliminación del biofilm bacteriano y asegurar una superficie radicular compatible con la salud y estabilidad de la condición periodontal.

The periodontium is a complex structure that is comprised of soft tissue components the gingiva and periodontal ligament, as well as hard tissue components in the form of alveolar bone and cementum. Human periodontal ligament cells (HPDLCs) and their extracellular matrix are regarded as essential components to achieve successful periodontal regeneration when treating periodontal lesions. In the present study, cell sheet technology was utilized to fabricate periodontal ligament cell sheets. These cell sheets were subsequently decellularized to isolate and preserve the extracellular component structural and functional characteristics, and were evaluated in vitro and in vivo. In all experiments that were undertaken in this study, melt electro-spun polycaprolactone (PCL) scaffolds were used as a carrier for the cell sheets, in order to support their fragile nature during the processes of decellularization. The combined cell sheet-PCL scaffold structure is referred to as a decellularized cell sheet construct.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Dentistry and Oral Health
Griffith Health
Full Text

Orientador: Karina Gonzáles Silvério Ruiz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-22T14:42:06Z (GMT). No. of bitstreams: 1 Saito_MikiTaketomi_M.pdf: 1292081 bytes, checksum: 368c2e71f9e5362b6da282ee020ff058 (MD5) Previous issue date: 2013
Resumo: Vários estudos têm sido conduzidos com o intuito de isolar e caracterizar células com fenótipo mesenquimal indiferenciado a partir do ligamento periodontal de humanos e avaliar o seu potencial em promover a neoformação dos tecidos periodontais. A partir destes estudos, sabe-se que há uma grande heterogeneidade celular no ligamento periodontal. Contudo, ainda não está claro na literatura se apenas um tipo de célula progenitora é capaz de se diferenciar em todos os tecidos presentes no periodonto ou se há um fenótipo celular mais favorável à regeneração periodontal. Neste contexto, o objetivo deste estudo foi isolar e caracterizar subclones celulares que apresentam maior comprometimento para aquisição do fenótipo osteoblástico/cementoblástico a partir de uma população de células do ligamento periodontal caracterizadas como mesenquimais indiferenciadas (CD105+ CD34- CD45-). Utilizando-se a técnica do cilindro de clonagem, subclones celulares foram isolados e avaliados quanto ao seu potencial de diferenciação osteoblástica/cementoblástica (ensaio de Von Kossa), à capacidade proliferativa (ensaio de MTS), e expressão da proteína STRO-1 pela técnica da imunofluorescência. Adicionalmente, os subclones celulares que apresentaram potencial de diferenciação osteoblástica/cementoblástica foram caracterizados pelo PCRq quanto à expressão de genes relacionados ao fenótipo osteoblástico (fator de transcrição relacionado à Runt- 2 - RUNX2 - e fosfatase alcalina - ALP), e modulação dos marcadores específicos para células mesenquimais indiferenciadas (CD105, CD166 e OCT-4) durante o processo de indução osteogênica. Os resultados mostraram que dos seis subclones isolados, três apresentavam potencial de diferenciação osteoblástica/cementoblástica, (grupo C-O), e os outros três não possuíam capacidade de formar matriz mineralizada (grupo C-F). O grupo C-O apresentou capacidade proliferativa significativamente menor comparada ao grupo C-F (p?0,05) e ambos os grupos apresentaram marcação positiva para proteína STRO-1. Durante o processo de indução osteogênica do grupo C-O, foi observado um aumento significativo (p?0,05) da expressão de RUNX2 e CD166, mas não dos outros marcadores avaliados (ALP, CD105 e OCT-4). Os achados deste estudo mostraram que as células mesenquimais indiferenciadas do ligamento periodontal de humanos CD105+ CD34- CD45- constituem uma população celular heterogênea, compreendendo um grupo de células mais proliferativas, mas sem potencial para depositar matriz mineralizada (grupo C-F), e outro grupo de células com menor potencial proliferativo, mas que possuem capacidade de diferenciação osteoblástica/cementoblástica (grupo C-O). Adicionalmente, foi observado que a expressão do marcador de superfície CD166 é modulada durante o processo de indução à diferenciação osteoblástica/cementoblástica no grupo C-O
Abstract: Several studies have been conducted in order to isolate and characterize cells from human periodontal ligament with mesenchymal stem cell phenotype, and evaluate its potential to promote periodontal tissues neoformation. From these studies, it was observed that there is a great heterogeneity in periodontal ligament cells. However, it is not clear in the literature whether only one type of progenitor cell is able to differentiate into all tissues of the periodontium or whether there is a cell phenotype more favorable for periodontal regeneration. In this context, the aim of this study was to isolate and characterize subclones that show greater commitment to acquisition of an osteoblastic/cementoblastic phenotype from a population of periodontal ligament cells characterized as mesenchymal stem cells (CD105+ CD34- CD45-). Cell subclones were isolated by ring-cloning technique and evaluated for their osteoblastic/cementoblastic differentiation potential (Von Kossa assay), proliferative capacity (MTS assay), and expression of STRO-1 protein by immunofluorescence technique. Additionally, the subclones that showed potential to osteoblastic/cementoblastic differentiation were characterized by qPCR for expression of genes related to osteoblastic phenotype (runt-related transcriptor factor-2 - RUNX2 and alkalin phosphatase - ALP) and modulation of specific markers of undifferentiated mesenchymal cells (CD105, CD166 and OCT-4) during osteogenic induction. Six subclones were isolated, and three of them presented osteoblastic/cementoblastic differentiation potential (C-O group), and the other three did not present this potential (C-F group). The C-O group showed significantly lower proliferative capacity compared to the C-F group (p ?0.05), and both groups were positively stained for protein STRO-1. During the osteogenic induction of C-O group, there was a significant increase in the expression of RUNX2 and CD166 (p ?0.05), but not in the other assessed markers (ALP, CD105 and OCT-4). The findings of the present study showed that CD105+ CD34- CD45- mesenchymal stem cells from human periodontal ligament are a heterogeneous cell population, comprising a group of more proliferative cells without potential to deposit mineralized matrix (C-F group), and another group of cells with lower proliferative potential with capacity of osteoblastic/cementoblastic differentiation (C-O group). Additionally, it was observed that the expression of CD166 is modulated during osteoblastic/cementoblastic induction process in C-O group
Mestrado
Periodontia
Mestra em Clínica Odontológica

Trabalho apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
Nos últimos anos têm-se realizado pesquisas sobre a utilidade de células estaminais em diversas áreas da saúde. Estes avanços científicos na área da bioengenharia tecidual têm levado ao desenvolvimento de novas terapias e curas de doenças que, até aos dias de hoje, não teriam cura. Recentes pesquisas têm revelado que os dentes são ótimas fontes de células estaminais. Tanto os dentes decíduos como permanentes têm células estaminais com grande potencial proliferativo e apresentam as caraterísticas de células estaminais: capacidade de autorrenovação e diferenciação em várias linhagens celulares. Mediante este contexto, o objetivo geral desta monografia foi realizar uma revisão de literatura em foco para atualizar o conhecimento acerca das células estaminais e a sua aplicação em medicina dentária. Tendo como objetivos específicos: analisar as células estaminais dentárias, ampliando o conhecimento sobre as possibilidades do seu potencial regenerativo, como fonte de células estaminais e sistematizar os conhecimentos, avanços científicos, limitações e perspetivas relativas à aplicação de células estaminais dentárias. Com esta revisão bibliográfica, é possível esclarecer determinadas dúvidas sobre células estaminais. O futuro passará em usar estas células como tratamento terapêutico, de maneira a regenerar muitos tecidos, fazendo com que os órgãos lesados possam voltar à sua vitalidade completamente saudável. As células estaminais são, assim, de uma enorme utilidade. Stem cells have been the target of abundant research focusing their potential in regenerative medicine. Scientific advances in bioengineered tissue has allowed the development of new therapies for diseases that, until recently, would not be possible. Recent researches have revealed teeth as great sources of stem cells. Both deciduous or permanent teeth have stem cells with high proliferative potential, exhibiting the characteristics of other studied stem cells: self-renewal capacity and differentiation into various cell lines. Under this context, the general objective of this thesis was to conduct a literature review focused on the stem cells knowledge and its application in dentistry. Having specific objectives: the study the dental stem cells, considering their regenerative potential as a source of stem cells and systematize the knowledge, scientific advances, limitations and prospects for the application of stem cells decay. This literature review, enables the clarification of certain doubts about stem cells. The future use of these cells as a therapeutic treatment in order to regenerate different tissues, causing the damaged organs can return to its vitality completely healthy. The stem cells are therefore extremely useful.

Objectives: To analyse the changes in gene expression in a population of human periodontal ligament (HPDL) cells exposed to cyclic tensile strain in a three dimensional culture system. Results: Cells derived from extracted teeth were shown to represent a mixed population with greater than 95% of cells expressing markers characteristic of periodontal ligament fibroblasts with an oestobastic phenotype with a smaller sub-population (2-3%) of stem cells. Cells from several different donors were subjected to an applied tensile strain (5%) and compared to unstressed controls. RNA extracted immediately post-stress was analysed to determine changes in gene expression. Microarray data indicated a total of 164 genes responding to the applied strain, 38% of which have no known function. qRT-PCR analysis of HAS1, NR4A2, FOS, INHBA, FOSL2, RUNX2, ATF4, FOSL1, SP7, COL1A1, COL3A1, MMP1, MMP2, MMP3 and TIMP3 expression levels confirmed the microarray data. Further analysis of the data highlighted changes in expression of CLDN4, BCOR, PROK2 and INHBA genes known to be associated with inherited orofacial disorders. Conclusion: This study has shown that the periodontal ligament fibroblasts derived from extracted teeth and cultured in vitro in 3D 'in-vivo-like' constructs can be induced to respond to mechanical stimuli. The molecular events detected in periodontal ligament cells subjected to cyclic tensile strain direct the cell population to remodel the entire periodontium (cells and matrices) in a process involving cell migration. This study presents several new findings including the first report of the involvement of HAS1, NR4A2 and FOSL2 in the response of HPDL cells to mechanical stimulation, the first report for the of modulation CLDN4, BCOR, and PROK2 genes by mechanical perturbation, and the differential induction of SP7 isoforms by mechanical stimulation in HPDL.

Periodontitis is one of the most prevalent bacterial diseases affecting man with up to 90% of the global population affected. Its severe form can lead to the tooth loss in 10-15% of the population worldwide. The disease is caused by a dysbiosis of the local microbiota and one organism that contributes to this alteration in the bacterial population is Prophyromonas gingivalis. This organism possesses a range of virulence factors that appear to contribute to its growth and survival at a periodontal site amongst which is its ability to invade oral epithelial cells. Such an invasion strategy provides a means of evasion of host defence mechanisms, persistence at a site and the opportunity for dissemination to other sites in the mouth. However, previous studies have demonstrated that invasion of the mammalian cells in a population by P. gingivalis is heterogenous, with some cells becoming heavily invaded while others harbour no or only a few bacteria. An understanding of this heterogeneity may throw light on the mechanisms involved and we hypothesised that the phase of the host cell cycle may explain this phenomenon. In an attempt to study the factors influencing P. gingivalis invasion and the cell response to that invasion, a standard antibiotic protection assay was employed and an oral keratinocyte cell line, H357. The results showed that P. gingivalis NCTC 11834 invasion was significantly increased with increasing time of exposure to the cells and the cell density. This may reflect an increased host cell surface area available for bacterial attachment. No effect on invasion of P. gingivalis invasion was observed by the bacterial growth phase, H357 cell passage number or whether cells were pre-incubated with P. gingivalis lipopolysaccharide. Epithelial cells did, however, respond to the presence of P. gingivalis in a number of ways. For example, the mRNA expression of endothelin-1 and urokinase receptor were upregulated with increasing P. gingivalis infection time, suggesting that these proteins could act as inflammatory mediators and possibly as useful markers of the severity of periodontal disease or in the diagnosis and treatment of periodontitis. iii Secondly, in an attempt to investigate the reason for the observed heterogeneous P. gingivalis invasion of H357 cell populations, the effect of cell cycle phase on P. gingivalis invasion was investigated. H357 cells were synchronized by serum starvation. On re-introduction of serum, characterisation of cell cycle phase distribution was performed by flow cytometry following staining with propidium idodide (PI) or by immunofluorescence using bromodeoxyuridine (BrdU), which specifically identifies cells in S-phase. The effect of cell cycle phases on P. gingivalis invasion was measured using the antibiotic protection assay, immunofluorescence and flow cytometry and these were correlated with gene and surface expression of the urokinase receptor and the α5-integrin subunit, which is thought to mediate P. gingivalis invasion. Results showed that the percentage invasion was enhanced with increasing serum re-introduction time, and positively correlated with the number of cells in S-phase. In addition, flow cytometry data showed that the highest association of fluorescent P. gingivalis was with PI positive S-phase cells. Moreover, BrdU positive S-phase cells were 3 times more likely to be invaded and contained 10 times more P. gingivalis than cells in other phases. Also, α5-integrin was more highly expressed in cells in S-phase than other phases, which could explain the mechanism underlying this enhanced invasion. Data presented here have suggested that P. gingivalis targeting of cells in S- phase could, in vivo, allow preferential invasion of the junctional epithelial cells which turns over rapidly. The data presented in this thesis suggest that P. gingivalis invasion is greatly dependent on several factors attributed to the host, the bacteria itself, and to the environment which the bacteria reside in. The invasion occurs within a population of host cells in a heterogeneous fashion, and is dependent on the cell cycle phase, specifically S-phase. This novel finding, in addition to the previously reported mechanisms of P. gingivalis invasion, increases our understanding of this virulence trait and suggests that such a strategy is a highly organised process which the bacteria can follow to ensure its survival within the host. Furthermore, knowledge of these mechanisms could provide novel approaches to treatment of periodontal diseases.

The goal of this study is to assess the osteogenic potential of two types of dental stem cell lines within a tissue engineering application. More specifically, the goal of this study is to find a readily abundant cell source with capacity to express an osteogenic phenotype. There are two parameters utilized to evaluate tissue engineering potential of cells: proliferation rate and differentiation potential. Briefly, proliferation rate is the speed at which cells divide and differentiation potential determines if cells are capable of committing towards specific lineages (e.g. osteogenic). These components are important, because if cells are not expanding at a specific rate and are not differentiating towards the lineage desired, the tissue engineered will not mirror the characteristics of native tissue. Therefore, both components are necessary for osteogenic tissue engineering applications. Several stem cell lines have been isolated from different sources (e.g. umbilical, bone marrow) and characterized for their proliferative capacity and their potency. Among these progenitor or stem cell lines, are those isolated from human dental tissue. Due to the similarities between teeth and bone, this specific cell line may be useful in osteogenic tissue engineering applications. In this study, stem cells extracted from human exfoliated deciduous teeth (SHEDs) and periodontal ligament stem cells (PERIOs), were evaluated and compared. Briefly, to evaluate the proliferation rate an ex-vivo expansion study was conducted. This experiment found that both SHEDs and PERIOs were proliferative lines with doubling times of 23 hours and 19 hours respectively. Subsequently, osteogenic differentiation of SHEDs and PERIOs was assessed utilizing a 3-D fibrin gel suspension treated with osteogenic media containing either dexamethasone (DEX) or Retinoic Acid (RA) for 28 days. At day 28, osteogenic markers for collagen 1 (Col1), osteocalcin (OCN), and alkaline phosphatase (ALP) were evaluated using qPCR. Results demonstrated both SHEDs and PERIOs exhibited significant (p<0.05) increases in osteogenic gene expression under the influences of DEX and RA. However the most significant increases were expressed by the SHEDs that received the DEX treatment. Additionally, the synergistic ability of TGF-beta 3 on the osteogenic differentiation of the stem cells was evaluated. Cells were cultured in a 3-D fibrin gel suspension and allowed to differentiate in DEX osteogenic media with and without the supplementation of TGF-beta 3 for 21 days. Using qPCR the cells were evaluated for expression of Col1, OCN, and ALP. In both the SHEDs and PERIOs, the samples treated with TGF-beta 3 the osteogenic gene expression increased in reference to the control, but had a hindering effect compared to cells treated in DEX without the TGF-beta 3. These results from this study suggested, SHED cells grown in 3-D fibrin gel suspension, may be better than PERIO cells for osteogenic tissue engineering applications when treated with DEX media without the supplementation of TGF-beta 3.

Made available in DSpace on 2014-06-11T19:27:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-13Bitstream added on 2014-06-13T20:36:16Z : No. of bitstreams: 1 freire_ir_me_araca.pdf: 351696 bytes, checksum: 8de9c54d57cecaf8154b65ccf640d4b9 (MD5)
O objetivo do presente estudo foi investigar o papel dos Mastócitos (MAST) na resposta tecidual de camundongos com Diabetes Mellitus (DM) submetidos a Doença Periodontal (DP). Os camundongos foram pré-tratados com uma dose única de estreptozotocina (STZ) para indução do DM. Para avaliar o papel dos MAST no controle da DP, os camundongos foram depletados de MAST pelo tratamento com composto 48/80. Subsequentemente foi realizada a indução da DP nos camundongos com DM e controles pela ligadura dos primeiros molares homólogos. Após um período de 7 e 14 dias os animais foram sacrificados para coleta das amostras para ensaios subseqüentes. Os níveis de reabsorção óssea dos camundongos diabéticos e normais foram avaliados radiograficamente para confirmação da presença da DP. O recrutamento de Neutrófilos (NE) foi avaliado pela produção da enzima Mieloperoxidase (MPO) no tecido gengival. Os níveis de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Linfotactina/ XCL1 nos tecidos gengivais e IFN-g e IL-4 no plasma foram avaliados pelo método imunoenzimático (ELISA). Os resultados mostram que animais diabéticos com DP apresentaram após 14 dias da indução da DP uma perda óssea significativa quando comparado ao grupo controle, diferentemente do grupo de 7 dias. Esta perda foi potenciada nos animais diabéticos com DP depletados de MAST. Verificou-se elevados níveis de MPO nos animais normais e com DM após 14 dias da indução da DP. Nos animais com DM e com DP tratados com 48/80 foi observada uma redução parcial dos níveis de MPO. A produção de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Lin-fotactina/XCL1 foi observada nos animais diabéticos independente da indução da DP após o período 7 e 14 dias. Conclui-se então que o DM favoreceu o aumento da perda óssea e o recrutamento de neutrófilos na DP. Como também induziu a produção de altos níveis dos mediadores...
The aim of this study was to investigate the role of Mast cells (MAST) on the tissue response in mice with Diabetes Mellitus (DM) submitted to Periodontal Disease (PD). The mice were pretreated with a single dose of streptozotocin (STZ) for the induction of DM. To evaluate the role of MAST in the PD, the mice were depleted of MAST by a pretreatment with compound 48/80. Subsequently, PD was induced in diabetic and normoglycemics mice by using a ligature around the first molars homologous. Seven and fourteen days after the surgery, the animals were sacrificed and the samples were collected for the subsequent experiments. The levels of bone resorption in diabetic and normoglycemic mice with PD were radiographically evaluated to confirm the presence of the PD. Neutrophil migration (NE) was quantified by the presence of the MPO enzyme in the gingival tissue. The levels of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphotactin/XCL1 from gingival tissues and plasma were evaluated by Enzyme- Linked Immunosorbent Assay (ELISA). The results showed that diabetic mice had a significant bone resorption 14 days after the induction of PD when compared to normoglycemics mice. This bone resorption was higher in the diabetic MAST-cell depleted mice with PD. The level of MPO was higher in diabetic and normoglycemic mice 14 days after the induction of PD. Furthermore, it was observed a partial reduction of MPO levels in diabetic mice with PD treated with compound 48/80. The level of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL was observed in diabetic mice independently of the induction of PD after 7 or 14 days. In conclusion, DM increased bone resorption and neutrophil recruitment in the PD mice, as well as it induced the production of high levels of IFN-g, IL -4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL1. The MAST depletion increased bone resorption and reduced NE recruitment... (Complete abstract click electronic access below)

Made available in DSpace on 2014-12-17T15:43:54Z (GMT). No. of bitstreams: 1 DiegoMS_DISSERT.pdf: 1792102 bytes, checksum: 305ff686e7a1606e27026b528fd1d091 (MD5) Previous issue date: 2013-01-25
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Low level laser irradiation (LLLI) has been used in Dentistry to promote wound healing and tissue regeneration. The literature shows a positive effect of LLLI on cell proliferation, but little is known about their effectiveness in promoting stem cells proliferation. The aim of this study was to evaluate the effect of LLLI on the proliferative rate of human periodontal ligament stem cells. Extracts of periodontal ligament were isolated from two third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were grown in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were irradiated with a InGaAlP-diode laser, using two different energy densities (0,5J/cm 2 - 16 seconds and 1,0J/cm? - 33 seconds), with wavelength of 660nm and output power of 30mW. A new irradiation, using the same parameters, was performed 48h after the first. A control group (non irradiated) was kept under the same experimental culture conditions. The Trypan blue exclusion test and the mitochondrial activity of the cells measured by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] essay were performed to assess the cell proliferation in the intervals of 0, 24, 48 e 72 h after irradiation. The data of cell counts were submitted to nonparametrical statistical tests (Kruskal-Wallis and Mann-Whitney), considering a confidence interval of 95%. DAPI (4 -6-Diamidino-2-phenylindole) staining of the cells was performed at 72h interval to evaluate possible nuclear morphological changes induced by LLLI. The results of this study show that the energy density of 1,0 J/cm? promoted greater cell proliferation compared to the other groups (control and 0,5 J/cm?) at intervals of 48 and 72h. The mitochondrial activity measured by MTT essay showed similar results to the Trypan blue cell counting test. The group irradiated with 1,0J/cm? exhibited a significantly higher MTT activity in the intervals of 48 and 72h, when compared to the group irradiated with 0,5J/cm?. No nuclear morphological change was observed in the cells from the three groups studied. It is concluded that LLLI has stimulatory effects on the proliferation of human periodontal ligament stem cells. Therefore, the use of laser irradiation in this cell type may be important to promote future advances in periodontal regeneration
O laser de baixa intensidade (LBI) tem sido utilizado na Odontologia com a finalidade de promover cicatriza??o e regenera??o dos tecidos. A literatura mostra um efeito positivo do LBI na prolifera??o celular, por?m pouco se sabe sobre a sua efic?cia na prolifera??o de c?lulas-tronco. O objetivo deste estudo foi avaliar o efeito da irradia??o do LBI na taxa proliferativa de c?lulas -tronco do ligamento periodontal humano. Extratos de ligamento periodontal foram isolados de dois terceiros molares h?gidos removidos por indica? ?o cir?rgica e/ou ortod?ntica. Ap?s a digest?o enzim?tica, as c?lulas foram cultivadas em meio de cultura α-MEM suplementado com antibi?ticos e 15% de soro fetal bovino. No terceiro subcultivo, as c?lulas foram irradiadas com um laser diodo InGaAlP, utilizando-se duas diferentes densidades de energia (0,5J/cm 2 - 16 segundos e 1,0J/cm? - 33 segundos), comprimento de onda de 660nm e pot?ncia de 30mW. Uma nova irradia??o, utilizando os mesmos par?metros, foi realizada 48 h ap?s a primeira. Um grupo controle (n?o irradiado) foi mantido nas mesmas condi??es experimentais de cultivo. O m?todo de exclus?o por azul de Tripan e a atividade mitocondrial das c?lulas medida atrav?s do ensaio de MTT [brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetraz?lio], nos intervalos de 0, 24, 48 e 72 h p?s-irradia??o, foram utilizados a fim de avaliar a prolifera??o celular. Os dados das contagens celulares foram submetidos a testes estat?sticos n?o param?tricos de Kruskal-Wallis e Mann-Whitney, considerando um intervalo de confian?a de 95%. Com o objetivo de verificar poss?veis altera??es morfol?gicas nucleares induzidas pelo laser, as c?lulas foram submetidas ? marca??o com DAPI (4 -6-Diamidino-2-phenylindole) no intervalo de 72 h. Os resultados do presente estudo mostraram que a densidade de energia de 1,0 J/cm? promoveu maior prolifera??o das c?lulas em compara??o com os outros grupos (controle e laser 0,5 J/cm?) nos intervalos de 48 e 72 h. A atividade mitocondrial, medida pelo ensaio de MTT, apresentou resultados semelhantes ?s contagem celulares com azul de Tripan, com o grupo irradiado com 1,0 J/cm? exibindo uma atividade significativamente maior do MTT nos intervalos de 48 e 72 h, quando comparado com o grupo irradiado com 0,5 J/cm?. Nenhuma altera??o morfol?gica nuclear foi observada, tanto das c?lulas do grupo controle quanto nas c?lulas irradiadas. Conclui-se que o LBI apresenta efeitos estimulantes sobre a prolifera??o de c?lulas-tronco do ligamento periodontal humano. Portanto, a aplica??o da laserterapia neste tipo celular pode ser importante para futuros avan?os na regenera??o periodontal

As doenças periodontais (DP) afetam os tecidos de suporte dos dentes e são desencadeadas por micro-organismos gram-negativos anaeróbios presentes no biofilme periodontal. A evolução da doença é influenciada pela resposta inflamatória e imunológica do hospedeiro e envolve a participação de diversos tipos celulares, que atuam no micro ambiente local modulando a resposta do hospedeiro em busca do controle da infecção. Acredita-se que citocinas inflamatórias, quimiocinas e seus receptores estão envolvidos na migração celular para os tecidos periodontais, contudo, pouco se sabe sobre os mecanismos de determinação de resistência ou susceptibilidade às DP; ou no desencadeamento do dano tecidual decorrente da resposta. Neste projeto, avaliou-se o papel das células CCR5+ na DP experimental induzida pela inoculação oral de Aggregatibacter actinomycetemcomitans em camundongos C57BL/6 wild type e camundongos CCR5-knockout. Os resultados mostram que a maioria das células CCR5+ possuem fenótipo compatível com células T do subtipo Th1, devido a co-expressão de CD3 e CXCR3; além de co-expressarem RANKL. Na ausência das células CCR5+, houve uma significativa diminuição da migração de células inflamatórias totais e RANKL+ para os tecidos periodontais, diminuição da reabsorção óssea alveolar, diminuição dos níveis de expressão de citocinas pró-inflamatórias TNFα-, IL-1β e IFN-γ, assim como diminuição na expressão de MMP-1, MMP-2 e MMP-13. Sua ausência não interferiu no controle da infecção periodontal apesar da diminuição dos níveis de iNOS. Estes resultados conduzem à conclusão de que a maioria das células CCR5+ são células T do subtipo Th1, que atuam como importantes moduladoras das citocinas TNFα-, IL-1β e IFN-γ, das metaloproteinases de matriz MMP-1, MMP-2 e MMP-13, e que também expressam e modulam a expressão de RANKL, tendo participação importante na imunopatogenese da DP experimental, sem interferir no controle da infecção periodontal. Estes fatos tornam as células CCR5+ potenciais alvos para intervenção terapêutica visando ao controle das doenças periodontais.
The periodontal diseases (PD) affect the supportive tissues of the teeth and are triggered by periodontopathogens present in the dental biofilm. The clinical outcome is highly influenced by the host inflammatory and immune response with participation of many cellular types, that act in the local microenvironment modulating the host response to control the infection. Inflammatory cytokines, chemokines and its receptors are thought to be involved in the cellular migration to the periodontal tissues, but there is little knowledge about the mechanisms of determination of resistance or susceptibility to the PD and in the triggering of tissue damage by immune response components. This study evaluated the role of CCR5+ cells in the experimental PD induced by oral inoculation of Aggregatibacter actinomycetemcomitans in C57BL/6 wild type mice and CCR5-knockout mice. The phenotypic analysis of inflammatory infiltrate demonstrated that the most of CCR5+ cells coexpress CD3 and CXCR3, suggesting a phenotype compatible with Th1-type cells, and also co-express RANKL. In the absence of CCR5+ cells there was a significant overall reduction of inflammatory cells and RANKL+ cells influx to the periodontal tissues, reduction in the alveolar bone resorption, reduction in the levels of pro-inflammatory cytokines TNFα-, IL-1β and IFN-γ expression, as a reduction in the expression of MMP-1, MMP-2 and MMP-13. The absence of CCR5+ cells did not impair the control of periodontal infection, despite the reduction of iNOS levels. In conclusion, these data demonstrate that the most of CCR5+ cells are Th1 cells, which act as important modulators of TNFα-, IL-1β and IFN-γ, MMP-1, MMP- 2 and MMP-13 levels, and which also express and modulate the expression of RANKL, playing an important role in the immunopathogenesis of experimental PD, without impairing the control of periodontal infection. These facts point to CCR5+ cells as potentials targets to therapeutic interventions aimed to control periodontal diseases.

Orientador: Sandra Helena Penha de Oliveira
Banca: João Eduardo Gomes Filho
Banca: Ana Paula Campanelli
Resumo: O objetivo do presente estudo foi investigar o papel dos Mastócitos (MAST) na resposta tecidual de camundongos com Diabetes Mellitus (DM) submetidos a Doença Periodontal (DP). Os camundongos foram pré-tratados com uma dose única de estreptozotocina (STZ) para indução do DM. Para avaliar o papel dos MAST no controle da DP, os camundongos foram depletados de MAST pelo tratamento com composto 48/80. Subsequentemente foi realizada a indução da DP nos camundongos com DM e controles pela ligadura dos primeiros molares homólogos. Após um período de 7 e 14 dias os animais foram sacrificados para coleta das amostras para ensaios subseqüentes. Os níveis de reabsorção óssea dos camundongos diabéticos e normais foram avaliados radiograficamente para confirmação da presença da DP. O recrutamento de Neutrófilos (NE) foi avaliado pela produção da enzima Mieloperoxidase (MPO) no tecido gengival. Os níveis de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Linfotactina/ XCL1 nos tecidos gengivais e IFN-g e IL-4 no plasma foram avaliados pelo método imunoenzimático (ELISA). Os resultados mostram que animais diabéticos com DP apresentaram após 14 dias da indução da DP uma perda óssea significativa quando comparado ao grupo controle, diferentemente do grupo de 7 dias. Esta perda foi potenciada nos animais diabéticos com DP depletados de MAST. Verificou-se elevados níveis de MPO nos animais normais e com DM após 14 dias da indução da DP. Nos animais com DM e com DP tratados com 48/80 foi observada uma redução parcial dos níveis de MPO. A produção de IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 e Lin-fotactina/XCL1 foi observada nos animais diabéticos independente da indução da DP após o período 7 e 14 dias. Conclui-se então que o DM favoreceu o aumento da perda óssea e o recrutamento de neutrófilos na DP. Como também induziu a produção de altos níveis dos mediadores... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this study was to investigate the role of Mast cells (MAST) on the tissue response in mice with Diabetes Mellitus (DM) submitted to Periodontal Disease (PD). The mice were pretreated with a single dose of streptozotocin (STZ) for the induction of DM. To evaluate the role of MAST in the PD, the mice were depleted of MAST by a pretreatment with compound 48/80. Subsequently, PD was induced in diabetic and normoglycemics mice by using a ligature around the first molars homologous. Seven and fourteen days after the surgery, the animals were sacrificed and the samples were collected for the subsequent experiments. The levels of bone resorption in diabetic and normoglycemic mice with PD were radiographically evaluated to confirm the presence of the PD. Neutrophil migration (NE) was quantified by the presence of the MPO enzyme in the gingival tissue. The levels of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphotactin/XCL1 from gingival tissues and plasma were evaluated by Enzyme- Linked Immunosorbent Assay (ELISA). The results showed that diabetic mice had a significant bone resorption 14 days after the induction of PD when compared to normoglycemics mice. This bone resorption was higher in the diabetic MAST-cell depleted mice with PD. The level of MPO was higher in diabetic and normoglycemic mice 14 days after the induction of PD. Furthermore, it was observed a partial reduction of MPO levels in diabetic mice with PD treated with compound 48/80. The level of IFN-g, IL-4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL was observed in diabetic mice independently of the induction of PD after 7 or 14 days. In conclusion, DM increased bone resorption and neutrophil recruitment in the PD mice, as well as it induced the production of high levels of IFN-g, IL -4, RANTES/CCL5, KC/CXCL1 and Lymphofotactin/XCL1. The MAST depletion increased bone resorption and reduced NE recruitment... (Complete abstract click electronic access below)
Mestre

Le rachitisme vitamino-résistant hypophosphatémique (RVRH) est une maladie génétique rare causée par des mutations du gène PHEX. La perte de fonction de la protéine PHEX conduit à l’augmentation du FGF23, une hormone circulante qui agit sur le rein et entraîne une perte systémique de phosphate. Le squelette rachitique des patients atteints de RVRH présente des déformations osseuses et une ostéomalacie. La dentine hypominéralisée des patients est à l’origine d’abcès dentaires fréquents, mais le statut parodontal des patients RVRH est mal connu, de même que leur risque de développer une parodontite pouvant aboutir à la perte des dents. La fonction et le substrat de la protéine PHEX ne sont pas identifiés avec exactitude. Il a été montré in vitro que PHEX avait la capacité d’interagir et de dégrader des protéines membres de la famille des SIBLINGs comme MEPE ou OPN, toutes les deux impliquées dans la régulation de la minéralisation des tissus osseux et dentinaires, mais on ne sait pas si in vivo les défauts de minéralisation observés résultent principalement de l’hypophosphatémie systémique ou bien également des effets directs de l’absence de PHEX sur les protéines régulatrices de la minéralisation. L’objectif de cette thèse a consisté à s’intéresser à la physiopathologie du parodonte dans le RVRH ainsi qu’à déterminer quel était l’impact de la mutation de PHEX dans un modèle de biominéralisation humaine où les conditions de concentration en phosphate pouvaient être ajustées et normalisées. Nous avons d’abord analysé le statut parodontal de 34 patients RVRH dans une étude clinique cas-témoins et ainsi montré que les malades dont la supplémentation en phosphate et vitamine D était tardive ou incomplète présentaient une fréquence et une sévérité accrues de maladie parodontale. Le phénotype parodontal du RVRH a alors été étudié sur des échantillons humains et sur le modèle murin du RVRH, la souris HYP. Nous avons réalisé un modèle d’égression dentaire de façon à permettre une apposition du cément cellulaire, ainsi qu’un modèle de résorption et de réparation osseuses parodontales afin de caractériser l’impact du RVRH sur la physiopathologie parodontale. Nos résultats ont montré que le phénotype parodontal et sa physiopathologie étaient très perturbés dans le rachitisme vitamino-résistant hypophosphatémique et chez la souris HYP, nous avons aussi pu mettre en évidence que le rôle pathologique majeur joué par l’ostéopontine dans le tissu osseux au cours du RVRH ne pouvait pas être généralisé aux autres tissus minéralisés du parodonte. De façon à identifier le rôle de PHEX dans la minéralisation matricielle locale indépendamment de la phosphatémie systémique, nous avons ensemencé des matrices de collagène dense avec des cellules primaires humaines issues de patients RVRH comparés à des contrôles que nous avons cultivés pendant 24 jours en conditions ostéogéniques avec des concentrations en phosphate identiques. Nos résultats ont montré que malgré une concentration normale en phosphate, la perte de fonction de la protéine PHEX entraînait une diminution de la quantité et de la qualité de la phase minérale et une accumulation et une dégradation pathologiques de la protéine OPN. Les contributions originales de ce travail de thèse doctorale ont consisté à démontrer sur le plan clinique et biologique la susceptibilité accrue du rachitisme hypophosphatémique lié à l’X quant au risque de développer une maladie parodontale, ainsi qu’à apporter la preuve d’un rôle pathologique de l’absence de PHEX indépendant de la phosphatémie sur des cultures primaires humaines
X-linked hypophosphatemia (XLH) is a rare X-linked dominant disorder caused by inactivating mutations in the PHEX gene. The impairment of PHEX protein leads to an increase in FGF23, a circulating factor that causes systemic loss of phosphate. The rachitic skeleton of patients with XLH displays short stature and osteomalacia. Dental defects include poorly mineralized dentin and spontaneous dental abscesses. Little is known about the periodontal condition of XLH and if patients are more prone to develop periodontitis, eventually leading to tooth loss. Although the exact function and substrate of PHEX are not known, it has been shown in vitro that PHEX could interact with SIBLING proteins such as MEPE or OPN, both involved in the regulation of bone and dentin mineralization, but it is not yet clear if the defects in the calcified extracellular matrices of XLH are caused by systemic hypophosphatemia only, or also by local consequences of the absence of PHEX. The aim of this doctoral dissertation was to explore the pathobiology of the XLH periodontium and to determine the impact of PHEX deficiency at the local level in a model of human biomineralization where phosphate supply could be adjusted and normalized. We first examined 34 adults with XLH in a case-control study and observed that periodontitis frequency and severity were increased in individuals with late or incomplete supplementation in phosphate and vitamin D analogs. The periodontium was then analyzed in XLH dental roots and further characterized in the Hyp mouse, the murine model of XLH. We performed a model of tooth movement adaptation leading to the formation of cellular cementum and a model of periodontal breakdown and repair to investigate the impact of XLH on the pathobiology of periodontal tissues. Our results showed strongly affected XLH/Hyp periodontal phenotype and impaired pathobiology and suggested that the key role played by OPN in bone could not be generalized to other periodontal mineralized tissues. In order to determine the role of PHEX in local human mineralization, dense collagen gels were seeded with primary human dental pulp cells harvested from XLH patients displaying PHEX mutations and age-matched healthy individuals. Cell-seeded gels were cultured up to 24 days under osteogenic conditions and controlled phosphate medium concentrations. Our results showed that despite normal phosphate concentrations, PHEX deficiency led to decreased quantity and quality of the mineral phase and a pathologic accumulation and processing of OPN. Overall the original contributions of this doctoral dissertation consist in the demonstration of a higher susceptibility of XLH patients to periodontitis and in the evidence of a local effect of PHEX deficiency in the pathologic intrinsic mineralization from XLH osteogenic cells

Orientador: Karina Gonzales Silvério Ruiz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-22T13:33:07Z (GMT). No. of bitstreams: 1 Albiero_MayraLaino_M.pdf: 1767389 bytes, checksum: 14c8bc6c823503f78c08850abc5ed95b (MD5) Previous issue date: 2013
Resumo: O objetivo do presente estudo foi avaliar se a exposição das células mesenquimais indiferenciadas do ligamento periodontal (PDLMSCs) ao lipopolissacarídeo da Porphyromonas gingivalis (Pg) e da Escherichia coli (Ec), levaria a alterações biológicas que comprometessem as propriedades relacionadas ao fenótipo mesenquimal indiferenciado. Para testar essa hipótese, inicialmente foi verificado se as cinco populações de células mesenquimais indiferenciadas purificadas para o antígeno de superfície CD105 (células CD105+) expresssavam os receptores Toll-like 2 e 4 (TLR2 e 4). Em seguida, as células foram cultivadas na presença do LPS da Pg e da Ec, e avaliadas quanto a sua viabilidade e proliferação pelo ensaio do MTS, expressão dos genes para citocinas inflamatórias IL-1?, IL-6, IL-8 e TNF-? (técnica do PCRq), imunomarcação para STRO-1 e expressão do gene identificador de pluripotencialidade - Oct-4, e capacidade de diferenciação osteoblástica/cementoblástica através dos ensaios para identificação de nódulos minerais e expressão dos genes para RUNX2, ALP e OCN. Os resultados mostraram que todas as populações celulares apresentaram fenótipo mesenquimal indiferenciado com marcação positiva para STRO-1, além de se mostrarem positivas para os receptores TRL2 e 4. O ensaio de MTS revelou que a exposição às três concentrações de LPS da Pg e da Ec (100 ng, 1 ?g e 10 ?g/ml) não comprometeu a viabilidade celular, mantendo todas as populações de PDLMSCs proliferativas ao longo dos 10 dias de cultivo. Em paralelo, verificou que LPS da Pg não alterou os níveis de RNAm para citocinas estudadas enquanto que, o LPS da Ec promoveu um aumento significativo na expressão de IL-6 e IL-8, quando aplicado nas concentrações de 100 ng e 1 ?g/ml. Adicionalmente, os resultados revelaram que a exposição celular aos LPS bacterianos, ambos na concentração de 1?g/ml, não alterou o fenótipo mesenquimal indiferenciado destas populações, uma vez que a expressão do gene OCT-4 e a marcação positiva para STRO-1 mantiveram-se semelhantes às células do grupo controle. Além disso, as células mantiveram a capacidade de diferenciação em fenótipo osteoblástico/cementoblástico, confirmado pela produção de nódulos minerais (ensaio de vermelho de alizarina) e expressão dos genes para RUNX-2, ALP e OCN semelhante ao grupo controle (células cultivadas em meio osteogênico). Somente foi observado um aumento significativo (p<0,05) na produção de nódulos minerais quando as células foram cultivadas na presença de 1?g/ml do LPS de Ec. Contudo, esse aumento da matriz mineral não está relacionado ao aumento nos níveis de RNAm para os genes relacionados ao fenótipo osteogênico comparado ao grupo controle. Dentro das condições experimentais avaliadas, concluí-se que a exposição das PDLMSCs ao LPS da Porphyromonas gingivalis e da Escherichea coli não alterou as propriedades biológicas destas células, mantendo assim, as características que conferem a estas a condição de mesenquimal indiferenciada
Abstract: The aim of this study was to evaluate if the exposure of mesenchymal stem cells of the periodontal ligament (PDLMSCs) to lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) and Escherichia coli (Ec), would lead to biological changes that compromised the properties related to undifferentiated mesenchymal phenotype. To test this hypothesis, initially it was checked whether the five populations of mesenchymal stem cells purified by surface antigen CD105 (CD105+ cells) expressed the Toll-like receptors 2 and 4 (TRL2 and 4). Then, cells were cultured in the presence of LPS from Pg and Ec, and evaluated for viability and proliferation by the MTS assay, expression of proinflammatory cytokines IL-1?, IL-6, IL-8 and TNF-? (PCRq technique), immunostaining for STRO-1 and OCT-4 gene expression, an identifier of pluripotency, and osteoblast/cementoblastic differentiation capacity through assays to identify minerals nodules, as well as the gene expression of RUNX2, ALP and OCN. All these assays were carried out also in the presence of LPS from Escherichia coli (Ec), which is not considered a periodontal pathogen. The results showed that all cell populations with undifferentiated mesenchymal phenotype had positive staining for STRO-1, and also showed to be positive for the receptors TLR2 and 4. The MTS assay revealed that exposure to the three concentrations of Pg and Ec LPS (100 ng, 1?g and 10?g/ml) did not affect cell viability, keeping all PDLMSCs populations proliferative over 10 days of culture. In parallel, it was found that the LPS Pg did not alter mRNA levels for the cytokines studied, while the Ec LPS caused a significant increase in IL-6 and IL-8, when applied concentrations of 100ng/ml and 1?g/ ml. Additionally, the results revealed that cellular exposure to both LPS in the concentration of 1?g/ml, did not alter the undifferentiated mesenchymal phenotype of these populations, since the expression of gene OCT-4 and positive STRO-1 staining remained similar to cells in the control group. In addition, these cells retained their ability to differentiate into osteoblastic/cementoblastic phenotype confirmed by production of mineral nodules (alizarin red assay) and expression of the genes for RUNX-2, ALP and OCN similar to cells control group (cultured in osteogenic medium only). It was only observed a significant increase (p<0.05) in the production of mineral nodules when cells were cultured in the presence of 1?g/ml Ec LPS. However, this increase in the mineral matrix it is not related to increased levels of mRNA for genes related to osteogenic phenotype compared to the control group. Within the experimental conditions, it can be concluded that exposure of PDLMSCs to LPS of Porphyromonas gingivalis and Escherichia coli did not alter the biological properties of these cells thereby, maintaining the characteristics that confer to these cells the condition of mesenchymal stem cells
Mestrado
Periodontia
Mestra em Clínica Odontológica

The pressure-tension hypothesis is the governing dogma of orthodontic tooth movement. This theory proposes that the application of loads to the crown of a tooth during orthodontic mechano-therapy results in differential site-specific reactionary strains in the para-dental tissues. Briefly, following the application of orthodontic load the bone and periodontal ligament (PDL) on one side of the tooth is placed in compression favoring bone resorption, while on the other side of the tooth they are placed in tension favoring osteogenesis The present in vitro model provides a surrogate for the PDL on the tension side of the tooth during orthodontic tooth movement and aims to identify mechanically induced changes in the expression of osteo-regulatory cytokines in human PDL cell cultures in response to tensile mechanical strain. Materials and Methods: PDL explants were obtained from pathology free bicuspids of two human subjects following extraction of the teeth for orthodontic purposes. Following serial passage, cells were plated on Uniflex� plates and consigned to either the experimental or control groups. Experimental cells were exposed to a cyclic uniaxial tensile mechanical strain for 6,12 or 24 hours using the Flexercell FX 4000 strain unit. Total RNA was extracted using a two-step procedure and samples were analysed using real-time RT-PCR assays for a range of osteo-regulatory cytokines. Results: Human PDL cells expressed mRNA for a range of cytokines of known significance to osteogenesis and osteoclastogenesis in response to mechanical stimulation. Conclusions: The production of osteo-regulatory cytokines by PDL cells in response to mechanical strain suggests that these cells have the potential to contribute to the osseous modeling of orthodontic tooth movement. The presence of osteogenic signalling drive in response to tensile strain tends to support the basic assertions of the pressure-tension hypothesis.

Mesenchymal stem cells (MSCs) are uniquely capable of crossing germinative layers borders (these cell populations are able to differentiate towards ectoderm-, mesoderm- and endoderm- derived lineages) and are viewed as promising cells for regenerative medicine approaches in several diseases. Some undoubtedly limiting factors for the clinical use of MSCs, i.e. for the repair of bone defects, are related to many different problems, that only partially today can be overcome through ex-vivo expansion and cells modification strategies. Considering MSCs sources, the use of fetal annexes-derived MSCs, such as MSCs from Whartonʼs jelly of umbilical cord (WJMSCs), or the recruitment of MSCs from “stem cell niches” in adult discard tissues, like periodontal ligament (PDL) of extracted teeth (PDLMSCs), could be promising in compensating limits of MSCs traditional sources, i.e. Bone Marrow, like small cells number, high harvesting technique morbility, difficult cells commitment due to early senescence. Uniforming and investigating best cells culture conditions with in vitro different experimental strategies is useful in our job to understand and control differentiation mechanisms and finally to influence the yield and proliferation rate of these MSCs populations, together with their osteogenic potential. The aim of our study is briefly sumarized: - To isolate and culture MSCs cells from human Umbilical Cord Whartonʼs Jelly and Periodontal Ligament; - To compare characteristics between all samples recruited and to link them to clinical aspects of tissue donors; - To characterise both MSCs population in regard to their proliferation and differentiation potential; - To investigate their functional characteristics before and after alginate microbeads encapsulation; - To investigate effects of three-dimensional systems and microgravity on MSCs before and after differentiation. WJMSCs and PDLSCs were analyzed for the expression of MSC markers, and then committed to osteogenic differentiation. Before and after differentiation, alkaline phosphatase (ALP) activity, the expression level of a specific osteoblast transcription factor (Runx2), and mineralization status were evaluated. The performance of WJMSCs and PDLSCs was then compared in 3-D culture systems (alginate beads and bioreactor system) in terms of viability, proliferation, secretive profile, expression of markers and effectiveness of phenotype modulation. Characterization of MSCs population was succesfull for all samples investigated, and for largest samples cohort (WJMSCs) it was possible to correlate cells biochemical parameters to clinical donors features. These findings may help during selection of best donors to combine them with scaffolds and biomaterials to promote tissue regeneration. For both MSCs populations, we demonstrated that cells can live and grow in 3- D systems. All MSCs samples analyzed showed a substantial osteogenic potential, before and after encapsulation in alginate microbeads. Modulation of cells culture conditions, with the use of nanotechnologies strategies or the use of bioreactors, like Rotary Cell Culture SystemTM (RCCS-4TM bioreactor, SyntheconTM, Inc., Houston, TX, U.S.A.) with High Aspect Ratio Vessel (HARVTM) has been shown to be a useful and promising approach to investigate characteristics and environmental effects of cells/ biomaterials combinations, in order to predict their effect and potential for regenerative medicine strategies.

Estudos clínicos tem mostrado resultados satisfatórios na aplicação da matriz derivada de esmalte (MDE) nas superfícies radiculares. Porém, a completa regeneração de todos os tecidos periodontais perdidos permanece como um desafio. Na tentativa de alcançar esse objetivo o uso de células tronco mesenquimais derivadas de medula óssea (CTM-MO) em um veículo apropriado na engenharia tecidual vem sendo bastante estudada. Uma interessante aplicação da MDE seria como um mediador biológico específico para CTMMO no intuito de promover a diferenciação e proliferação celular e auxiliar nos processos de regeneração periodontal. Sendo assim, o objetivo do presente estudo é avaliar a influência do Emdogain (EMD) e seu veículo alginato de propileno glicol (APG) na proliferação, diferenciação e mineralização de CTM-MO CD 45-90+. Células estromais de medula óssea (CE-MO) foram coletadas da crista ilíaca de ratos Wistar-Kyoto, isoladas pelo método Ficoll e separadas em CTM-MO CD 45-90+ por citometria de fluxo. A caracterização dessas células como células tronco se deu por meio de análise da presença de marcadores de células tronco (gene REX-1 e antígeno de superfície CD 90), habilidade de formação de colônias e capacidade de expressar fenótipo osteogênico e adipogênico. Após a caracterização, as CTM-MO CD 45-90+ foram plaqueadas com um dos seguintes tratamentos: Grupo Controle Positivo - meio contendo 10% de soro fetal bovino (SFB); Grupo Controle Negativo - meio contendo 2% de SFB; Grupo APG meio contendo 2% de SFB associado a 25 μg/ml de APG; Grupo EMD meio contendo 2% de SFB associado a 25 μg/ml de EMD. A avaliação da proliferação celular foi realizada por ensaio de MTT em 3, 7, 10 e 14 dias. Após diferenciação osteoblástica, a atividade de fosfatase alcalina (ALP) e a avaliação da capacidade de diferenciação celular feita por análise genética (PCR) dos genes osterix (OSX), osteopontina (OPN), ALP e RUNX2 foram mensuradas em 7, 10 e 14 dias. A mineralização de matriz extracelular foi avaliada qualitativamente por meio de ensaios de Von Kossa e Alizarina Red e quantitativamente pela mensuração do teor de cálcio. CTM-MO CD 45-90+ apresentaram maior expressão de REX-1, maior quantidade de antígeno de superfície CD 90, e mais unidades formadoras de colônia do que CE-MO. Além disso, foram capazes de expressar fenótipos osteoblástico e adipogênico. EMD e APG não influenciaram a proliferação de CTM-MO CD 45-90+. A atividade de ALP não foi influenciada pelo EMD e foi reduzida no grupo APG. Ambos EMD e APG não influenciaram a expressão dos genes OSX e RUNX2 em 7 e 10 dias e reduziram em 14 dias. O EMD não influenciou a expressão gênica de ALP e OPN, mas a presença do gel de alginato de propileno glicol pareceu prover melhores resultados, com maiores expressões de OPN e ALP. Os grupo EMD e APG apresentaram menor mineralização de matriz extracelular em 28 dias comparados aos grupos controles. Conclui-se que o EMD não influenciou a proliferação, atividade de ALP e diferenciação de CTM-MO CD 45-90+, mas a presença do gel (APG) parece otimizar a diferenciação dessas células. Ambos, APG e EMD reduziram a mineralização de matriz extracelular.
Clinical studies have shown satisfactory results in the application of the enamel matrix derivate (EMD) on root surfaces. However, the complete regeneration of all lost periodontal tissues remains a challenge. To achieve this aim the use of bone marrow mesenchymal stem cells (BM-MSCs) in an appropriate vehicle in tissue engineering has been extensively studied. An interesting application of the EMD would be like a specific biological agent for BM-MSCs in order to promote cell differentiation and proliferation and assist in periodontal regeneration processes. Therefore, the aim of this study is evaluate the influence of Emdogain (EMD) and its vehicle (Propylene Glycol Alginate - PGA) in proliferation, differentiation and mineralization of CD 45-90+ bone marrow mesenchymal stem cells (BM-MSCs). Rat bone marrow stromal cells (BMSCs) were colleted from iliac crest of rat Wistar-Kyoto, isolated by Ficoll method and separated into CD 45-90+ BM-MSCs by flow cytometer. For cellular characterization the presence of stem cells markers (Rex-1 gen and surface antigen CD 90), capacity to form colonies (CFU) and to express osteoblast and adipogenic phenotype was assessed. Four experimental groups was plated with CD 45-90+ BM-MSCs: Positive Control group: media containing 10% of fetal bovine serum (FBS); (2) Negative Control Group: media containing 2% FBS; (3) EMD Group: 25 μg/ml EMD in 2% FBS media; (4) PGA Group: 25 μg/ml PGA in 2% FBS media. Cellular proliferation was assed in 3, 7, 10 and 14 days with MTT assay. After osteogenic induction, alkaline phosphatase (ALP) activity and gene expression of osterix (OSX), osteopontin (OPN), ALP and RUNX2 by RT-PCR was assessed at 7, 10 and 14 days. Extracellular matrix mineralization was evaluated at 21 and 28 days qualitatively by Von-kossa and Alizarina Red staining, and quantitatively by calcium content. CD 45-90+ BM-MSCs presented higher Rex-1 gen expression, more surface antigen CD 90 and more CFU/well than BMSCs, and expressed osteoblast and adipogenic phenotype. EMD and PGA not influenced proliferation of CD45-90+ BM13 MSCs. ALP activity was not influenced by EMD and was reduced in PGA group. EMD and PGA not influenced expression of OSX and RUNX2 in 7 and 10 days and reduced in 14 days. EMD not influenced ALP and OPN expression, but the presence of the gel provided better results, with higher expressions of ALP and OPN genes. Both PGA and EMD groups presented less mineralized ECM in 28 days. It concludes that EMD not influenced the proliferation, differentiation and ALP activity of CD 45-90+ BM-MSCs, but the presence of the gel (PGA) seems optimize the differentiation of these cells. Both reduced ECM mineralization.

O objetivo deste estudo foi verificar se o tratamento periodontal não cirúrgico exercia alguma influência sobre o perfil lipídico, os elementos celulares das séries banca e vermelha do sangue, plaquetas e VHS de pacientes portadores de periodontite crônica generalizada. Dezoito pacientes, com média de idade de 50,6 anos ( 7,6), foram submetidos, previamente ao tratamento periodontal e 30 dias após o mesmo, à coleta de 10ml de sangue periférico, através do qual foram analisados o perfil lipídico, os elementos celulares das séries branca e vermelha, o número de plaquetas e VHS. Destes 18 pacientes, 7, com média de idade de 47,4 anos ( 5,9) também foram reavaliados 90 dias após o término do tratamento. Os parâmetros clínicos utilizados, previamente ao tratamento e nas reavaliações, foram o Índice de Placa (IP), de Silness e Löe (1964), o Índice Gengival (IG), de Löe (1967), Sangramento na Sondagem (SS), Profundidade de Bolsa à Sondagem (PBS) e Nível de Inserção (NI). Foram ainda registrados e classificados os sítios com envolvimento de furca. O tratamento periodontal consistiu de terapia básica não cirúrgica. Após 30 dias do término do tratamento periodontal todos os pacientes foram reavaliados sendo verificada melhora significativa (P<0,05) dos valores de IP, IG, SS e PBS e de NI ≥ 6mm (P=0,05). Sítios com envolvimento de furca classes II e III apresentaram também diminuição significativa (P=0,01). Os 7 pacientes submetidos às reavaliações de 30 e 90 dias pós-tratamento também mostraram melhora significativa (P<0,05) dos valores de IP, IG, SS e PBS entre estas fases. Já o NI entre 4-5mm aumentou de forma significativa (P=0,04) entre o pré-tratamento e 90 dias após o mesmo, enquanto que o NI ≥ 6mm diminui significativamente entre as reavaliações de 30 e 90 dias (P=0,01 e P=0,02, respectivamente). Quando comparados os valores de 30 com os de 90 dias resultados semelhantes aos supracitados foram observados, inclusive o aumento do NI entre 4-5mm (P=0,02). É verificado também entre estas fases um IG aumentado (P=0,07). Quanto aos valores hematológicos ocorreu uma diminuição significativa dos níveis de bastões (P=0,05) e de monócitos (P=0,03) após o tratamento periodontal (30 dias), enquanto que o colesterol total e o LDL apresentaram uma tendência ao aumento (P=0,09 para ambos). Já nos sete pacientes submetidos às duas reavaliações o colesterol total apresentou aumento significativo entre as fases pré-tratamento, 30 (P=0,04) e 90 dias (P=0,02) após terapia, assim como o LDL (P=0,04 e P=0,03, respectivamente). Quando comparados os valores plaquetários entre as fases 30 e 90 dias pós-tratamento, verifica-se uma tendência a sua diminuição (P=0,09). O Índice de Castelli II (relação colesterol/HDL) apresenta entre as fases pré e 30 dias pós-tratamento tendência a aumento (P=0,09). Através desses resultados é possível concluir que o tratamento periodontal exerceu influência sobre bastões e monócitos do sangue, caracterizada pela diminuição dessas células, e sobre o colesterol total e o LDL, representada pelo aumento de seus valores.
The aim of this study was to verify whether non-surgical periodontal treatment had any influence on lipid profiles, white and red blood cell elements, platelets and ESR of patients with generalized chronic periodontitis. Eighteen patients average aged 50,6 years ( 7,6) had 10ml of peripheral blood collected prior to the periodontal treatment and 30 days after it to be analyzed for lipidic profile, white and red blood cell elements and ESR. Seven of those patients average aged 47,4 years ( 5,9) were also reassessed 90 days after the end of the treatment. The clinical parameters used prior to the treatment and on the reassessment were Plaque Index (PI), Silness and Löe (1964), Gengival Index (GI), Löe (1967), Bleeding On Probing (BOP), Pocket Probing Depth (PPD), and Attachment Level (AL). Furcation involvement sites were also recorded and classified. The periodontal treatment consisted of non-surgical basic therapy. Thirty days after the end of the treatment all patients were reassessed and a significant improvement (P<0,05) in PI, GI, BOP, PPD values and in AL ≥6mm (P=0,05) was observed. Class I and II furcation involvement sites also showed a significant decrease. The seven patients reassessed at 30 days and at 90 days pos-treatment also showed significant (P<0,05) PI, GI, BOP and PPD value improvement between those phases. The 4-5 mm AL increased significantly (P=0,04) between pre-treatment and 90 days pos-treatment, while AL ≥6mm decreased significantly between the 30-day and 90-day follow-up reassessments (P=0,01 and P= 0,02 respectively). When the values at 30 days and those at 90 days were compared results similar to the ones described above were observed, including the 4-5 mm AL increase (P=0,02). An increased GI (P=0,07) is also verified. As for hematological values there was a significant decrease in band cell levels (P=0,05) and monocyte levels (P= 0,03) after the periodontal treatment (30 days), while both total cholesterol and LDL showed a trend toward an increase (P=0,09). The seven patients who underwent two follow-up reassessments showed a significant increase in total cholesterol when pre-treatment, 30 days (P=0,04) and 90 days (P=0,02) phases were compared as well as in LDL (P=0,04 and P=0,03, respectively ).When the 30-day and 90-day pos-treatment platelets values were compared, a trend toward a decrease (P =0,09) was observed. Castelli Index II (cholesterol/ HDL) shows a trend toward an increase (P=0,09) between the pre-treatment and the 30-day pos-treatment phases. These results allow us to conclude that the periodontal treatment influenced band cells and monocytes, by decreasing them, as well as on total cholesterol and LDL as shown by their value increase.

Articular cartilage lines the surfaces of load bearing joints and has limited capabilities for self-repair due to its alymphatic and avascular structure. Attempts at making repairs to this tissue has resulted in substandard materials and/or causing further injury to the patient making this tissue a prime candidate for tissue engineering studies incorporating adult stem cells. These studies have given rise to some answers and many more questions including a search for alternative stem cell sources and what biochemical changes the cells undergo during the differentiation of these stem cells into chondrocytes, the cells which make up articular cartilage. Recently, periodontal dental ligament stem cells (PDLs) have come to the forefront as a practical alternative to other adult stem cells as well as the involvement of the mitogen-activated protein kinases (MAPKs) in stem cell differentiation via mechanical stimulation. During dynamic unconfined compression, levels of p42/44 MAPK increased by 50% (p<0.05). Additionally, the expression of the chondrogenic differentiation factor SRY (sex determining region Y)-box 9 (SOX-9) increased by 3-fold (p<0.05) as well as the chondrocyte marker aggrecan by over 2-fold after 4h of dynamic unconfined compression. Addition of the p42/44 phosphorylation inhibitor PD98059, along with compression, yielded no change in SOX-9 or aggrecan expression levels from basal levels in uncompressed controls. Inhibition of p38 MAPK or JNK phosphorylation during unconfined compression had no effect on the elevated expression of SOX-9 and aggrecan as compared to compressed cells without the addition of an inhibitor. It is therefore the overall findings of this study that PDLs possess the ability to differentiate into chondrocytes by mechanical compression and this differentiation is mediated by the p42/44 MAPK cascade.

Master of Science
Periodontal disease is one of the most widespread diseases in humans and is characterised by chronic gingival inflammation and B cell accumulation and resorption of the crest of alveolar bone with subsequent loss of teeth. Porphyromonas gingivalis has been identified as a putative aetiological agent for periodontitis. The aim of the research presented in this thesis was to investigate, using in vitro systems, the responses of autoreactive B-1 and B-2 cells to enterobacterial and nonenterobacterial lipopolysaccharide (LPS) to shed light on the pathogenesis of chronic periodontitis and other diseases involving B cell accumulation and autoantibody production. The hypotheses tested were: (1) B cells respond differently to enterobacterial and non-enterobacterial LPS. (2) B-1 cells are activated by a lower concentration of LPS than B-2 cells. (3) LPS stimulation results in preferential accumulation of B-1 cells. Findings consistent with these hypotheses would provide new evidence for different roles for B-1 and B-2 cells in immune responses and that LPS stimulation could lead to B-1 cell accumulation in diseases thus characterised. Initial experiments investigated the responses of representative B-1 (CH12) and B-2 (WEHI-279) cell lines to preparations of P. gingivalis and Salmonella enteritidis LPS utilising flow cytometric and quantitative molecular methods. The cell lines responded differently to the two LPS preparations. There were significant but limited effects on viability and proliferation in the WEHI-279 cell line, but no significant changes in mRNA expression levels for genes including Toll-like receptors (TLR2, TLR4, RP105), immunoglobulin (IgM), cytokines (IL-6, IL-10), co-stimulatory molecules (CD80, CD86), and regulators of apoptosis (Bcl-2, Bax). In the CH12 cell line however, LPS stimulation had greater effect. Addition of S. enteritidis LPS from a threshold level of 100ng/mL was found to rescue the cells from death, reflected by the percentage viability and proliferation. Stimulation of CH12 cells with S. enteritidis LPS also led to a decrease in expression of RP105 mRNA, which may be part of a negative feedback loop. Interestingly, stimulation with low concentrations P. gingivalis LPS appeared to inhibit proliferation but high LPS concentrations stimulated proliferation of CH12 cells, although no further significant effects were noted in other analyses. Evidence was found that CH12 cells have a high basal level of activation. This suggests that this line is constitutively activated. Stimulation with P. gingivalis or S. enteritidis LPS did not affect the level of CD80 mRNA expression. It is possible that the CH12 line constitutively expresses a maximal level of CD80 (and possibly CD86) and further stimulation will not cause any increase. Since S. enteritidis LPS appeared to have more pronounced effects on both B cell populations, this LPS was used to further investigate B cell subset responses in a mixed splenocyte culture system. Experiments examining percentage viability and number of viable cells indicated that B-1 and B-2 B cells responded differently to LPS stimulation. A threshold level for B-2 cell response (significant increase in cell number) was found to be 100ng/mL LPS, in contrast to the B-1 B cell subset which were only significantly different to the unstimulated cells when stimulated with 50μg/mL LPS. By examining the expression of CD80, the majority of murine splenic B-1 cells were found to activated prior to any LPS stimulation in vitro. In contrast, the B-2 subset showed significant increase in CD80 expression only at high (≥10μg/mL) LPS concentrations. Studies of the division index of B-1 and B-2 cells showed a significant response in both subsets following stimulation with 1μg/mL and 10μg/mL LPS. However, overall, the results are inconsistent with LPS driving the preferential accumulation of B-1 cells in disease states. These experiments provided useful evidence that supported the idea that B-1 and B-2 cells respond differently to LPS. However, these studies were unable to directly address the role of P. gingivalis LPS in periodontitis. It may be that P. gingivalis LPS could have different effects to S. enteritidis LPS on primary B cells. It is still possible that B-1 cells may be more sensitive to P. gingivalis, as opposed to S. enteritidis LPS. Studies by other groups have suggested that the TH1/TH2 profile is skewed towards TH2 in chronic periodontitis and that P. gingivalis may drive this shift via its ability to signal through TLR2 (and modulate TLR4 signalling). Further, recent studies in our laboratories have found that P. gingivalis gingipains are able to polyclonally activate B cells and to break down both IFNγ and IL-12. Future studies should further examine the effects of B-1 and B-2 interactions in the mixed lymphocyte system together with subsequent studies utilising human periodontitis biopsies. The results presented in this thesis, together with work undertaken by other investigators, suggests that LPS could perturb the normal homeostatic mechanisms of the B-1 B cell-subset and increase polyclonal activation therefore contributing to the genesis of pathologies such as chronic periodontitis.

Master of Science
Periodontal disease is one of the most widespread diseases in humans and is characterised by chronic gingival inflammation and B cell accumulation and resorption of the crest of alveolar bone with subsequent loss of teeth. Porphyromonas gingivalis has been identified as a putative aetiological agent for periodontitis. The aim of the research presented in this thesis was to investigate, using in vitro systems, the responses of autoreactive B-1 and B-2 cells to enterobacterial and nonenterobacterial lipopolysaccharide (LPS) to shed light on the pathogenesis of chronic periodontitis and other diseases involving B cell accumulation and autoantibody production. The hypotheses tested were: (1) B cells respond differently to enterobacterial and non-enterobacterial LPS. (2) B-1 cells are activated by a lower concentration of LPS than B-2 cells. (3) LPS stimulation results in preferential accumulation of B-1 cells. Findings consistent with these hypotheses would provide new evidence for different roles for B-1 and B-2 cells in immune responses and that LPS stimulation could lead to B-1 cell accumulation in diseases thus characterised. Initial experiments investigated the responses of representative B-1 (CH12) and B-2 (WEHI-279) cell lines to preparations of P. gingivalis and Salmonella enteritidis LPS utilising flow cytometric and quantitative molecular methods. The cell lines responded differently to the two LPS preparations. There were significant but limited effects on viability and proliferation in the WEHI-279 cell line, but no significant changes in mRNA expression levels for genes including Toll-like receptors (TLR2, TLR4, RP105), immunoglobulin (IgM), cytokines (IL-6, IL-10), co-stimulatory molecules (CD80, CD86), and regulators of apoptosis (Bcl-2, Bax). In the CH12 cell line however, LPS stimulation had greater effect. Addition of S. enteritidis LPS from a threshold level of 100ng/mL was found to rescue the cells from death, reflected by the percentage viability and proliferation. Stimulation of CH12 cells with S. enteritidis LPS also led to a decrease in expression of RP105 mRNA, which may be part of a negative feedback loop. Interestingly, stimulation with low concentrations P. gingivalis LPS appeared to inhibit proliferation but high LPS concentrations stimulated proliferation of CH12 cells, although no further significant effects were noted in other analyses. Evidence was found that CH12 cells have a high basal level of activation. This suggests that this line is constitutively activated. Stimulation with P. gingivalis or S. enteritidis LPS did not affect the level of CD80 mRNA expression. It is possible that the CH12 line constitutively expresses a maximal level of CD80 (and possibly CD86) and further stimulation will not cause any increase. Since S. enteritidis LPS appeared to have more pronounced effects on both B cell populations, this LPS was used to further investigate B cell subset responses in a mixed splenocyte culture system. Experiments examining percentage viability and number of viable cells indicated that B-1 and B-2 B cells responded differently to LPS stimulation. A threshold level for B-2 cell response (significant increase in cell number) was found to be 100ng/mL LPS, in contrast to the B-1 B cell subset which were only significantly different to the unstimulated cells when stimulated with 50μg/mL LPS. By examining the expression of CD80, the majority of murine splenic B-1 cells were found to activated prior to any LPS stimulation in vitro. In contrast, the B-2 subset showed significant increase in CD80 expression only at high (≥10μg/mL) LPS concentrations. Studies of the division index of B-1 and B-2 cells showed a significant response in both subsets following stimulation with 1μg/mL and 10μg/mL LPS. However, overall, the results are inconsistent with LPS driving the preferential accumulation of B-1 cells in disease states. These experiments provided useful evidence that supported the idea that B-1 and B-2 cells respond differently to LPS. However, these studies were unable to directly address the role of P. gingivalis LPS in periodontitis. It may be that P. gingivalis LPS could have different effects to S. enteritidis LPS on primary B cells. It is still possible that B-1 cells may be more sensitive to P. gingivalis, as opposed to S. enteritidis LPS. Studies by other groups have suggested that the TH1/TH2 profile is skewed towards TH2 in chronic periodontitis and that P. gingivalis may drive this shift via its ability to signal through TLR2 (and modulate TLR4 signalling). Further, recent studies in our laboratories have found that P. gingivalis gingipains are able to polyclonally activate B cells and to break down both IFNγ and IL-12. Future studies should further examine the effects of B-1 and B-2 interactions in the mixed lymphocyte system together with subsequent studies utilising human periodontitis biopsies. The results presented in this thesis, together with work undertaken by other investigators, suggests that LPS could perturb the normal homeostatic mechanisms of the B-1 B cell-subset and increase polyclonal activation therefore contributing to the genesis of pathologies such as chronic periodontitis.

O objetivo deste estudo foi analisar a expressão dos fenótipos fibroblástico e osteoblástico em culturas tridimensionais na presença ou não de partículas de vidro bioativo. Fibroblastos derivados do ligamento periodontal humano (hPDLF) e células osteogênicas da calvária de rato recém-nascidos foram plaqueadas em superfícies bidimensionais - lamínulas de plástico ThermanoxTM (controle); superfícies colágenas bidimensionais - ThermanoxTM revestidas por colágeno I sem partículas de vidro bioativo (2D) e com partículas de vidro bioativo (2D+VB); e em gel colágeno tridimensional sem vidro bioativo (3D) e com partículas (3D+VB). Foram avaliados: Viabilidade celular (MTT) nos tempos 3, 7 e 10 dias; Atividade de fosfatase alcalina (ALP) normalizada pelo conteúdo de proteína total em 7 e 14 dias; Immunolocalização de proteínas da matriz não-colágena (ALP e OPN em células hPDLF aos 7 e 14 dias e OPN e BSP em células osteogênicas aos 7 dias) por imunofluorescência indireta; Expressão quantitativa (PCR em tempo real) dos genes Periostina (PRT), Calcium-Binding Protein (S100A4) e Fibromodulina (FBM), marcadores do fenótipo fibroblástico, em células hPDLF e Fosfatase Alcalina (ALP), Osteopontina (OPN), Sialoproteína Óssea (BSP), Osteocalcina (OC), Colágeno I (COL I) e Runx2, marcadores osteoblásticos, em ambos os tipos celulares; e Mineralização (coloração por vermelho de Alizarina). Os resultados obtidos nas culturas de hPDLF mostraram que aos 3 dias a viabilidade celular em 2D+VB foi maior que no controle (p<0,05) e nenhuma diferença significante entre os grupos foi observada aos 7 e 10 dias. O conteúdo de proteína total aos 7 e 14 dias foi maior nas culturas 3D e 3D+VB, sendo aos 7 dias significantemente diferente de 2D+VB (p<0,05) e controle (p<0,05) e aos 14 dias observou-se diferença significativa entre 3D e 2D. A atividade de ALP aos 7 dias foi maior nos grupos 2D e 2D+VB comparados com 3D (p<0,05) e 3D+VB (p<0,05); e em 3D+VB menor que no controle (p<0,05). Entretanto, aos 14 dias 3D e 3D+VB apresentaram maior atividade de ALP que o controle (p<0,05). Imunomarcações para OPN e ALP foram observadas nas células em 3D em ambos os períodos avaliados, e em 2D, 2D+VB, e controle apenas aos 14 dias. A expressão de RNAm para PRT aos 7 dias apresentou um perfil upregulated em 3D e 3D+VB comparados ao controle (p<0,05); para FBM a expressão gênica foi maior em 3D e 2D que em 3D+VB (p<0,05), e menor em 3D+VB comparado ao controle (p<0,05). As células em 2D exibiram maiores níveis de expressão de RNAm para S100A4 que as cultivadas em 2D+VB (p<0,05) e controle (p<0,05). Os níveis de RNAm para COL I e ALP em 2D+VB e 3D, para RUNX2 e OPN em 3D e 2D, e para OC em 2D apresentaram-se upregulated em relação ao controle (p<0,05). Em 2D o nível de expressão de OC foi maior que em 2D+VB (p<0,05). Aos 14 dias houve uma diminuição da expressão de todos os genes analisados em relação às análises de 7 dias. Células em 3D+VB expressaram menores níveis de PRT que em 2D+VB (p<0,05). A expressão de RNAm para FBM foi maior em 2D e 2D+VB e para S100A4 maior em 2D comparado com 3D (p<0,05), nos quais os níveis de S100A4 ficaram downregulated com relação ao controle. COL I e ALP apresentaram-se downregulated em 3D (p<0,05) e 3D+VB (p<0,05) em relação ao controle. Entre 2D+VB e 3D+VB também foi observada diferença significativa para ALP. A expressão de RUNX2 foi maior em 3D que em 3D+VB (p<0,05) e de OC maior no controle. Nos grupos com VB foi observada maior formação de matriz calcificada aos 10 e 14 dias. Aos 10 dias não foram observadas áreas coradas por Alizarina através da microscopia, mas a quantidade de mineralização em 2D+VB e 3D+VB foi significativamente maior que no controle (p<0,05), 2D (p<0,05) e 3D (p<0,05). Aos 14 dias marcações mais extensas foram observadas nas culturas com VB, porém os nódulos mineralizados apresentavam-se independentes das partículas. 2D+VB e 3D+VB foram significativamente diferentes do controle (p<0,05) e 2D (p<0,05). As culturas de células osteogênicas mostraram que aos 7 dias as células crescidas sobre os arcabouços 3D+VB e 3D exibiram menores índices de viabilidade. Diferenças significativas foram observadas quando 3D+VB foi comparado aos grupos 2D+VB (p<0,05) e controle (p<0,05); e entre 2D e 3D (p<0,05). Aos 3 e 10 dias não foram encontradas diferenças significativas na viabilidade celular entre os grupos. O conteúdo de proteína total foi maior em 3D+VB que em 2D+VB (p<0,05) e no controle (p<0,05) aos 7 e 14 dias. Diferenças significantes também foram observadas entre 3D e 2D (p<0,05) aos 14 dias. A atividade de ALP aos 7 dias foi maior em 2D+VB e 3D+VB. Diferenças significantes foram encontradas entre 2D e 2D+VB (p<0,05), 3D e 3D+VB (p<0,05) e entre 3D e controle (p<0,05). Entretanto, aos 14 dias, 3D e 3D+VB apresentaram os menores valores de atividade de ALP, sendo a significantemente diferentes de 2D (p<0,05) e 2D+VB (p<0,05). Imunomarcações para OPN e BSP foram observadas aos 7 dias em 2D, 2D+VB, 3D e controle. Aos 7 dias os níveis expressão do RNAm para ALP, COL I e RUNX2 foram maiores em 3D e 3D+VB. Os genes OPN, OC e BSP exibiram níveis de expressão mais altos em 2D+VB. A expressão de COL I foi maior em 3D+VB que em 2D+VB (p<0,05). As células em 2D+VB apresentaram maiores níveis de expressão de OPN e OC que em 2D (p<0,05) e 3D+VB (p<0,05), nos quais a expressão desses genes e de BSP estavam downregulated em relação ao grupo controle. Aos 10 e 14 dias áreas coradas por vermelho de Alizarina foram observadas em todos os grupos, sendo mais extensas nos grupos que continham VB. Aos 10 dias a quantidade de cálcio em 3D+VB foi maior que no controle (p<0,05); e maior em 2D+VB comparado com 2D (p<0,05) e controle (p<0,05). Aos 14 dias 2D+VB e 3D+VB apresentaram uma quantidade de cálcio significativamente maior que no controle (p<0,05) e em 2D (p<0,05). Em conclusão, este estudo demonstrou que os arcabouços tridimensionais colágenos são capazes de suportar a viabilidade, proliferação e diferenciação celular se in vitro de hPDLF e células osteogênicas derivadas de calvária de rato recém-nascidos e de favorecerem a expressão dos fenótipos fibroblástico e osteoblástico em hPDLF. As partículas de VB em ambos os tipos celulares também contribuiram para viabilidade, diferenciação, formação de matriz mineralizada e expressão fenotípica.
The aim of this study was to analyze the fibroblastic and osteoblastic phenotypes expression on three-dimensional cultures in the presence or not of bioactive glass particles. Fibroblasts derived from human periodontal ligament (hPDLF) and osteogenic cells from newborn rat calvaria were cultured on bi-dimensional surfaces - plastic coverslips ThermanoxTM (control), bi-dimensional collagen surfaces - ThermanoxTM coated with collagen I without bioactive glass particles (2D) and with bioactive glass particles (2D+BG), on three-dimensional collagen gel without bioactive glass (3D) and with particles (3D+BG). Were evaluated: Cell viability (MTT) in 3 days, 7 and 10 days; Phosphatase alkaline activity (ALP) normalized by total protein content at 7 and 14 days; Immunolocalization of non-matrix proteins collagen (ALP and OPN in hPDLF at 7 and 14 days, OPN and BSP in osteogenic cells at 7 days) by indirect immunofluorescence; Genes expression (real-time PCR) for Periostin (PRT), Calcium-Binding Protein (S100A4) and Fibromodulin (FBM): fibroblastic markers in hPDLF, and Alkaline phosphatase (ALP), Osteopontin (OPN), Bone sialoprotein (BSP), Osteocalcin (OC), Collagen I (COL I) and RUNX2: osteoblastic markers in both cell types; and Mineralization (staining with Alizarin red). The results obtained on hPDLF cultures showed that cell viability on 2D+BG was higher than on control at 3 days (p<0.05), and no significant difference between groups was observed at 7 and 10 days. The total protein content at 7 and 14 days was higher on 3D and 3D+BG cultures compared those on 2D+BG (p<0.05) and control (p<0.05) at 7 days. Significant difference was also observed between 3D and 2D at 14 days. The ALP activity at 7 days was higher on 2D and 2D+BG compared with 3D (p<0.05) and 3D+BG (p<0.05), it was also lower on 3D+BG than on control (p<0.05). However, at 14 days 3D and 3D+BG showed higher ALP activity than control (p<0.05). Immunolabeling for OPN and ALP were observed in cells on 3D at both periods and on 2D, 2D+ BG and control only at 14 days. At 7 days, the expression of mRNA for PRT was upregulated on 3D and 3D+BG compared with control (p<0.05), for the FBM it was higher on 3D and 2D than on 3D+BG (p<0,05), but it was lower on 3D+BG than on control (p<0.05). Cells on 2D exhibited higher levels of S100A4 mRNA expression than those grown on 2D+BG (p<0.05) and control (p<0.05). The mRNA levels expression for COL I and ALP on 2D+BG and 3D, and for RUNX2 and OPN on 3D and 2D, and for OC on 2D presented upregulated compared with control (p<0.05). Also, cells on 2D showed the level expression of OC higher than those on and 2D+BG (p<0.05). At 14 days, there was a decrease in all evaluated genes expression compared with 7 days analyses. Cells on 2D+BG expressed higher levels of PRT than on 3D+BG (p<0.05). 2D and 2D+BG showed the highest levels of mRNA expression for FBM. Gene expression of S100A4 on 2D was higher than on 3D (p<0.05) and 3D+BG (p<0.05), in which the levels of S100A4 were downregulated compared to control. COL I and ALP were downregulated on 3D (p<0.05) and on 3D+BG (p<0.05) compared with control. There was also a significant difference between 2D+BG and 3D+BG for mRNA ALP expression. RUNX2 expression was higher on 3D than on 3D+BG (p<0.05), and OC expression was higher on control. Calcified matrix formation was observed on BG cultures at 10 and 14 days. At 10 days, areas stained by Alizarin were no observed by microscopy, but the amount of mineralization on 2D+BG and 3D+BG was significantly higher than on control (p<0.05), 2D (p<0.05) and 3D (p<0.05). At 14 days, more extensive staining was observed on cultures with BG, but the mineralized nodules formation was independent of the particles. Calcium content on 2D+BG and 3D+BG was significantly higher than control (p<0.05) and 2D (p<0.05). Osteogenic cell cultures showed that cells grown on 3D and 3D+BG surfaces exhibited the lowest levels of cell viability. Significant differences were observed when 3D+BG was compared with 2D+BG (p<0.05) and control (p<0.05) and also between 2D and 3D (p<0.05). At 3 and 10 days, there were no significant differences for cell viabililty between the cultures. The total protein content was higher on 3D+BG than on the control (p<0.05) and 2D+BG (p<0.05) at 7 and 14 days. Significant differences were also observed between 3D and 2D (p<0.05) at 14 days. ALP activity at 7 days was higher on 2D+BG and 3D+BG. Significant differences were found between 2D and 2D+BG (p<0.05), 3D and 3D+BG (p<0.05), and between 3D and control (p<0.05). However, at 14 days 3D and 3D+BG had the lowest levels of ALP activity, significantly different from 2D (p<0.05) and 2D+BG (p<0.05). Immunolabeling for OPN and BSP were observed at 7 days on 2D, 2D+BG, 3D and control. At 7 days, the expression levels of mRNA for ALP, COL I and RUNX2 were higher on 3D and 3D+BG. OPN, BSP and OC exhibited higher expression levels on 2D+BG. COL I expression was higher on 3D+BG than on 2D+BG (p<0.05). Cells on 2D+BG showed higher expression levels of OPN and OC than those on 2D (p<0.05) and 3D+BG (p<0.05), in which both of these genes and BSP expression were downregulated compared with control. At 10 and 14 days areas stained with Alizarin red were observed all evaluated groups, especially on BG cultures. At 10 days the amount of calcium on 3D+BG was higher than on control (p<0.05), and it was also higher on 2D+BG compared with 2D (p<0.05) and control (p<0.05). At 14 days, 2D+BG and 3D+BG showed greater calcium amount than control (p<0.05) and 2D (p<0.05). In conclusion, this study demonstrated that in vitro 3D cultures of hPDLF and osteogenic cells from newborn rats calvaria were able of support cell viability, differentiation, and to contribute to expression of fibroblastic and osteoblastic phenotype in hPDLF. The BG particles also favored the viability, differentiation, mineralized matrix formation and phenotypic expression in both cell types.