Effect of cell culture density on dental pulp-derived mesenchymal stem cells with reference to osteogenic differentiation.

Dental pulp stem cells (DPSCs) are a good source for tissue regeneration, however, the number of DPSCs in the pulp tissue is limited. Cell propagation is essential for tissue engineering using DPSCs and the cell culture conditions may affect the properties of DPSCs. The purpose of this study was to analyze the effect of cell culture condition, especially dense culture condition, on the property and differentiation pathway of DPSCs. We cultured DPSCs under sparse (sDPSCs; 5 × 103 cells/cm2) or dense (dDPSCs; 1 × 105 cells/cm2) conditions for 4 days and compared their properties. The populations of CD73+ and CD105+ cells were significantly decreased in dDPSCs. Both groups showed multi-differentiation potential, but mineralized nodule formation was enhanced in dDPSCs. The phosphorylation of focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) proteins was promoted in dDPSCs, and alkaline phosphatase (ALP) mRNA expression in dDPSCs was abolished in the presence of pan-PI3K and FAK inhibitors. dDPSCs implanted into mouse bone cavities induced more mineralized tissue formation than sDPSCs and control. These findings indicate that dense culture conditions modified the properties of DPSCs and gave rise to osteogenic-lineage commitment via integrin signaling and suggest that dense culture conditions favor the propagation of DPSCs to be used for mineralized tissue regeneration.

Properties of Dental Pulp-derived Mesenchymal Stem Cells and the Effects of Culture Conditions.

Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration. The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods. DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition. The ability of DPMSCs to differentiate into hard tissue-forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy.

Periostin is a negative regulator of mineralization in the dental pulp tissue.

The dental pulp tissue is encased in hard tissue and surrounded by hard tissue-forming cells, but remains in a non-mineralized state itself, suggesting the presence of regulatory mechanisms precluding pulp mineralization. This study aimed to reveal the regulatory function of periostin (Postn), which is essential for osteoblast differentiation, for odontoblast differentiation/mineralization. We evaluated the effects of Postn overexpression and RNAi-mediated suppression in mouse dental papilla cells (MDPs) on the expression of odontoblastic markers and Notch signaling molecules, and on the formation of mineralized nodules. Localization of Postn in the dental pulp tissue of normal and cavity-prepared molars was observed immunohistologically. Enforced overexpression of Postn in MDPs induced down-regulation of odontoblastic markers and in vitro mineralization. Conversely, silencing of Postn mRNA in MDPs induced up-regulation of odontoblastic markers and ALP activity. Up- and down-regulation of Postn caused increased and decreased expression, respectively, of Notch signaling molecules. Postn expression was minimal in normal dental pulp, but was rapidly and globally increased in the whole pulp tissue of molar teeth at 1 day after cavity preparation, decreasing thereafter. These results indicate that Postn may be a negative regulator of odontoblast differentiation/mineralization, and that may exert its actions via Notch signals.

Down-regulation of inflammatory mediator synthesis and infiltration of inflammatory cells by MMP-3 in experimentally induced rat pulpitis.

INTRODUCTION: Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. METHODS: Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. RESULTS: NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. CONCLUSIONS: These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells.

OBJECTIVE: Three-dimensional (3D) spheroid culture is a method for creating 3D aggregations of cells and their extracellular matrix without a scaffold mimicking the actual tissues. The aim of this study was to evaluate the effects of 3D spheroid culture on the phenotype of immortalized mouse dental papilla cells (MDPs) that have the ability to differentiate into odontoblasts. METHODS: We cultured MDPs for 1, 3, 7, and 14 days in 96-well low-attachment culture plates for 3D spheroid culture or flat-bottomed plates for two-dimensional (2D) monolayer culture. Cell proliferation and apoptosis were detected by immunohistochemical staining of Ki67 and cleaved caspase-3, respectively. Hypoxia was measured by the hypoxia probe LOX-1. Odonto/osteoblastic differentiation marker gene expression was evaluated by quantitative PCR. We also determined mineralized nodule formation, alkaline phosphatase (ALP) activity, and dentine matrix protein-1 (DMP1) expression. Vinculin and integrin signalling-related proteins were detected immunohistochemically. RESULTS: Odonto/osteoblastic marker gene expression and mineralized nodule formation were significantly up-regulated in 3D spheroid-cultured MDPs compared with those in 2D monolayer-cultured MDPs (p<0.05). Histologically, 3D spheroid colonies consisted of two compartments: a cell-dense peripheral zone and cell-sparse core zone. Proliferating cells with high ALP activity and DMP1 expression were found mainly in the peripheral zone that also showed strong expression of vinculin and integrin signalling-related proteins. In contrast, apoptotic and hypoxic cells were detected in the core zone. CONCLUSION: 3D spheroid culture promotes odonto/osteoblastic differentiation of MDPs, which may be mediated by integrin signalling.

Wnt11 expression in rat dental pulp and promotional effects of Wnt signaling on odontoblast differentiation.

Congenital anomalies of wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt) are frequently accompanied with tooth and dentin abnormality. The aim of this study was to investigate the effects of Wnt signaling on odontoblast differentiation of mouse dental papilla cells (MDPs). Mouse dental papilla cells were cultured in α-modified minimum essential medium containing 10% fetal bovine serum and antibiotics. Odontoblast differentiation was induced by bone morphogenic protein 2 (BMP2), and the expression of odontoblast-specific markers and Wnt-related signaling molecules was analyzed by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Odontoblast differentiation was evaluated by dentin sialophosphoprotein (Dspp) and dentin matrix protein (DMP) 1 expression. Localization of ß-catenin in MDPs was detected by immunocytochemistry using an anti-ß-catenin antibody. Dspp expression in MDPs was upregulated in the presence of BMP2. Wnt5a, Wnt11, Lef1 and Tcf4 expression was upregulated in BMP2-treated MDPs. Wnt11 expression was detected in rat dental pulp in vivo, and particularly strong expression of Wnt11 was detected in odontoblasts. Enhanced Dspp and DMP1 expression and alkaline phosphatase activity induced by BMP2 were completely negated by the Wnt antagonist: IWR-1-endo treatment. Nuclear translocation of ß-catenin observed in BMP2-treated MDPs was also negated by IWR-1-endo treatment. These results indicate that Wnt signaling upregulates odontoblast marker expression in MDPs, suggesting a promoting effect of Wnt signaling on odontoblast differentiation.

CLL: chromosomal abnormalities (FISH) and their relation with clinical stage, CD38 and ZAP-70 / Leucemia linfocítica crônica: anormalidades cromossômicas e a sua relação com o estágio clínico CD38 e o ZAP-70

Chronic lymphocytic leukemia is the most prevalent type of leukemia in the West. It is characterized by an extremely variable clinical course. The aim of the study was to detect the most frequent chromosomal abnormalities in patients with CLL using FISH, and assess them regarding age, gender, clinical stage and CD38 and ZAP-70 expressions. We found 51.7 percent of the patients with chromosome abnormalities. The most frequent one was del 13q14 in 34.5 percent of cases. It was associated to other alterations in 17.2 percent. 17p13 deletions were found in 17.2 percent and trisomy 12 in 13.8 percent (in isolation in 6.9 percent and associated to del 13q14, in 6.9 percent of the cases). An 11q22 deletion was found in one case associated to a 13q14 deletion. To better evaluate the relationship between chromosome aberrations and other prognostic factors in CLL, two cytogenetics groups were considered: favorable (13q deletion in isolation and no alteration) and unfavorable outcomes (trisomy 12, 17p13 deletion, 11q22 deletion and two simultaneous alterations).The unfavorable alterations were more frequently seen among young individuals (<60y). There were more females (70 percent) than males in this group (p=0.04). In relation to the Binet's staging system, patients with unfavorable cytogenetic alterations, tended to be B and C stages, while in the favorable group prevailed patients in stage A. Additionally, patients with poor prognostic cytogenetics tended to express CD38 and ZAP-70 proteins.

A leucemia linfocítica crônica (LLC) é o tipo de leucemia mais prevalente no Ocidente e é caracterizada por curso clínico extremamente variável. O objetivo deste estudo foi detectar as anomalias cromossômicas mais freqüentes em pacientes com LLC, empregando a técnica FISH, e correlacioná-las com idade, sexo, estádio clínico, expressão de CD 38 e ZAP-70. Foram encontradas alterações cromossômicas em 51,7 por cento dos pacientes. A mais freqüente foi a del 13q14, observada em 34,5 por cento dos casos e que esteve associada a outras anomalias em 17,2 por cento. Deleção 17p13 foi encontrada em 17,2 por cento e trissomia 12 em 13,8 por cento (isolada em 6,9 por cento e associada à del 13q14 em 6,9 por cento). Deleção 11q22 foi observada em um caso em concomitância à del 13q14. Para melhor avaliar a relação entre alteração cromossômica e outros fatores prognósticos em LLC, dois grupos citogenéticos foram considerados: favorável (deleção 13q isolada e ausência de alterações) e desfavorável (trissomia 12, deleção 17p13, deleção 11q22 e duas anomalias simultâneas). As alterações desfavoráveis foram mais freqüentemente observadas em indivíduos jovens (<60 anos) e em mulheres (70 por cento)(p=0,04). Em relação ao sistema de estadiamento de Binet, houve tendência dos pacientes com alterações cromossômicas desfavoráveis apresenteram-se nos estágios B e C enquanto no grupo favorável prevaleceram aqueles com estágio A. Em adição, pacientes com achados citogenéticos de prognóstico desfavorável tiveram tendência a expressar proteínas CD 38 e ZAP-70.