Influence Of Gallnut Aqueous Extract On Biological Behaviour Of HDPCs Cultured In Vitro

The infection of the dental pulp and surrounding periapical tissues mainly results from dental caries. It is a complicated process which involves bacteria and their metabolites, as well as pulpal immune and inflammatory reactions. Vital pulp therapy for cariously exposed teeth remains one of the most controversial areas in dentistry. It is beneficial to preserve the vitality and health of an exposed pulp rather than replace it with a root filling material. A vital, functioning pulp is capable of initiating several defensive and reparative mechanisms including the multiplication and differentiation of dental pulp cells. As the main active component of gallnut, gallic acid has various action as follows: antibacterial, anti-free radicle, adstringent, hemostatic, and analgesic. It has been reported that gallic acid could inhibit decomposition of collagen, prevent demineralization of enamel,promote remineralization of dentin, and enhance formation of reparative dentin as pulp cap-indirect. However the possible action of gallic acid as pulp cap-direct has not been reported.This study intended to observe the influence of gallnut aqueous extract on biological behaviour of human dental pulp cells (HDPCs) cultured in vitro, in order to explore the possible use as pulp cap-direct in the future. Part I: Comparison of different methods for primary HDPCs cultureThe primary culture methods of HDPCs were compared to seek an ideal isolation method, optimize culture conditions, and increase achievement ratio of cultivation. HDPCs were dissected and cultured separately by means of tissue explant cultivation, enzyme digestion, and tissue explant plus enzyme digestion. Results show the tissue explant plus enzyme digestion is better than tissue explant cultivation or enzyme digestion. It could increase achievement ratio of primary cultivation of HDPCs.Part II: Influence of gallnut aqueous extract on the proliferation and cell cycle of HDPCs1. Influence of gallnut aqueous extract on the proliferation of HDPCsMTT method was used to examine the proliferation of HDPCs treated with gallnut aqueous extract with concentration of 2.5μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml. Results showed that gallnut aqueous extract with concentration of less than 20μg/ml could promote the proliferation of HDPCs, which presented a relation of concentration-dependence. However gallnut aqueous extract with concentration of 40μg/ml had inhibitory effect on that of HDPCs in 72h, which presented a relation of time-dependence.2. Influence of gallnut aqueous extract on the cell circle of HDPCsFlow cytometry was also used to exmine the cell circle of HDPCs treated with gallnut aqueous extract with different concentration. Ratio of Cell as well as proliferation index decreased at G1 stage and incresed at S stage treated with gallnut aqueous extract, which was obvious with the concentration of 10μg/ml. Part III: Influence of gallnut aqueous extract on alkaline phosphatase activity and mineralinzation of HDPCs1. Influence of gallnut aqueous extract on alkaline phosphatase activity of HDPCsThe alkaline phosphatase(ALP) activity of HDPCs was examined treated with gallnut aqueous extract with concentration of 2.5μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml . Results showed no difference was found with concentration of 2.5μg/ml,5μg/ml. However ALP activity of HDPCs was inhibited treated with gallnut aqueous extract with concentration of more than 10μg/ml, which was obvious with the concentration of 40μg/ml.2. Influence of gallnut aqueous extract on mineralization of HDPCsVon Kossa staining was used to assess calcified extracellular matrix of HDPCs treated with gallnut aqueous extrac with concentration of 2.5μg/ml,5μg/ml,10μg/ml,20μg/ml.Results showed that mineralization of extracellular matrix of HDPCs was promoted.Part IV: The effect of gallnut aqueous extract on the production of collagen type I of HDPCsThe immunohistochemical method was used to examine the production of collagen type I of HDPCs treated with gallnut aqueous extract with concentration of 5μg/ml,10μg/ml. Results showed that gallnut aqueous extract can promote production of collagen type I of HDPCs.