Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis

Abstract Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. Objective Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. Methodology hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. Results Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine-cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). Conclusions The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.

High glucose affects proliferation, reactive oxygen species and mineralization of human dental pulp cells

Abstract Diabetes is a group of metabolic disorders that can lead to damage and dysfunction of many organs including the dental pulp. Increased inflammatory response, reduction of dentin formation and impaired healing were reported in diabetic dental pulp. Hyperglycemia, which is a main characteristic of diabetes, was suggested to play a role in many diabetic complications. Therefore our aim was to investigate the effects of high glucose levels on proliferation, reactive oxygen species (ROS) production and odontogenic differentiation of human dental pulp cells (HDPCs). HDPCs were cultured under low glucose (5.5mM Glucose), high glucose (25 mM Glucose) and mannitol (iso-osmolar control) conditions. Cell proliferation was analyzed by MTT assay for 11 days. Glutathione and DCFH-DA assay were used to assess ROS and antioxidant levels after 24 h of glucose exposure. Odontogenic differentiation was evaluated and quantified by alizarin red staining on day 21. Expression of mineralization-associated genes, which were alkaline phosphatase, dentin sialophosphoprotein and osteonectin, was determined by RT-qPCR on day 14. The results showed that high glucose concentration decreased proliferation of HDPCs. Odontogenic differentiation, both by gene expression and mineral matrix deposit, was inhibited by high glucose condition. In addition, high DCF levels and low reduced glutathione levels were observed in high glucose condition. However, no differences were observed between mannitol and low glucose conditions. In conclusion, the results clearly showed the negative effect of high glucose condition on HDPCs proliferation and differentiation. Moreover, it also induced ROS production of HDPCs.

Resumo O diabetes abrange um grupo de distúrbios metabólicos que podem levar a danos e disfunções de muitos órgãos, incluindo a polpa dentária. Aumento da resposta inflamatória, redução da formação de dentina e comprometimento da cicatrização foram relatados na polpa dentária diabética. A hiperglicemia, que é uma característica determinante do diabetes, desempenha um papel importante em muitas complicações diabéticas. Portanto, nosso objetivo foi investigar os efeitos dos altos níveis de glicose na proliferação, produção de espécies reativas de oxigênio (ROS, em inglês) e diferenciação odontogênica das células da polpa dental humana (HDPCs, em inglês). As HDPCs foram cultivadas em condições de baixa glicose (glicose 5,5 mM), alta glicose (glicose 25 mM) e manitol (controle iso-osmolar). A proliferação celular foi analisada pelo ensaio MTT por 11 dias. Glutationa e DCFH-DA foram utilizados para avaliar os níveis de ROS e antioxidantes após 24 h de exposição à glicose. A diferenciação odontogênica foi avaliada e quantificada pela coloração com vermelho de alizarina no dia 21. A expressão de genes associados à mineralização, que eram fosfatase alcalina, sialofosfoproteína de dentina e osteonectina, foi determinada por RT-qPCR no dia 14. Os resultados mostraram que a alta concentração de glicose diminuiu a proliferação de HDPCs. A diferenciação odontogênica, tanto pela expressão gênica quanto pelo depósito da matriz mineral, foi inibida pela condição de alta glicose. Além disso, altos níveis de DCF e níveis reduzidos de glutationa foram observados na condição de alta glicose. No entanto, não foram observadas diferenças entre o manitol e as condições de baixa glicose. Em conclusão, os resultados mostraram claramente o efeito negativo da condição de alta glicose na proliferação e diferenciação de HDPCs. Além disso, essa condição também induziu a produção de ROS em HDPCs.

Translocation of Porphyromonas gingivalis infected monocytes and sssociated cellular responses.

Porphyromonas gingivalis (P.gingivalis), an important periodontal pathogen in adult chronic periodontitis, has been reported to co-localize in human atheromatous lesions. We have studied the phagocytosis and survival of P.gingivalis in human monocytes, together with the cellular responses of infected human monocytes. Human monocytes were co-cultured with P.gingivalis and the external bacteria were killed with metronidazole and gentamycin. Localization of P.gingivalis in cells was studied by transmission electron microscopy (TEM). The survival of P. gingivalis was determined by lysing the monocytes and plating on blood agar under anaerobic conditions. Interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were determined using specific enzyme-linked immunosorbent assays (ELISAs) kits. The transwell chamber system was used to investigate the chemotactic response of the infected cells. TEM showed that P.gingivalis organisms were localized within the autophagosome-like structure of monocytes. No significant difference on the survival of P.gingivalis at 0, 4 and 8 h after infection was found. IL-1b and TNF-a were present in the cell culture media in response to bacterial challenge. The infected monocytes showed a normal chemotactic response to monocyte chemotactic protein-1 (MCP-1). The number of monocyte cells migrating through membrane in the presence and absence of P.gingivalis were 18.64±2.33´104 cells and 19.11±1.76´104 cells respectively. The number of viable P.gingivalis per monocyte following translocation in response to the chemotactic gradient was 5.83±1.45´10-3 CFU/cell. The results indicate that P.gingivalis can stimulate cytokine production and survive in monocytes without affecting cell migration.

The molecular basis of mucopolysaccharidosis type I in two Thai patients.

Two Thai patients diagnosed with Hurler syndrome (mucopolysaccharidosis type 1, MPS I) were found to have no detectable alpha-iduronidase (E.C. 3.2.1.76) activity in leukocytes, while normal Thai children all had significant activity, with a mean of 135 +/- 30 nmol/mg/18h. One patient was heterozygous for A75T (311G>A) and S633L (1986C>T) mutation, previously reported to cause MPS I, together with 9 other heterozygous polymorphisms also found in normal controls. The other patient had the previously described frameshift mutation 252insert C and a new nonsense mutation E299X (983G>T).

Effects of lead on the proliferation, protein production, and osteocalcin secretion of human dental pulp cells in vitro.

Teeth have been recognized as providing a useful long-term record of lead (Pb2+) uptake. However, information regarding the effects of lead on dental pulp tissue cells that foster dentinogenesis is scarce. This study investigated the effects of lead on dental pulp tissue using human dental pulp fibroblasts in vitro. Dental pulp cells from the teeth of young patients (aged 17-24 years) were cultured and subsequently treated with lead glutamate. It was shown that, in serum-free conditions, all the tested concentrations of lead (4.5 x 10(-5) M, 4.5 x 10(-6) M, and 4.5 x 10(-7) M) significantly increased pulpal cell proliferation. In the presence of 2% fetal bovine serum, increasing cell proliferation was observed only after exposure to a lead concentration of 4.5 x 10(-5) M. However, protein, procollagen type I, and osteocalcin productions were significantly decreased. The alteration of cell population and protein production of affected human dental pulp shown in this study are toxic effects of the lead.