Study On The Biological Modification Of Dentin Matrix By Proanthocyanidins Of Tea Plant

Dentine matrix is constituted of a collagen network composed mainly of type I collagen.The key to Dentine matrix restoration is to improve the mechanical properties and anti enzymolysis capacity of collagen in dentine matrix.Proanthocyanidins can significantly improve the biological properties of dentin collagen fibers,but its biological activity is affected by many factors,such as material sources,extraction methods,and varying compositions.Recent studies have shown that there are abundant proanthocyanidins in tea root.However,the potential therapeutic value of proanthocyanidins from tea plant on dentin has not yet been intensively studied.The biological effects of proanthocyanidins extracted from tea root on demineralized dentine matrix were investigated in this study.Firstly,the tea root was extracted by organic solvents,and the tea root ethyl acetate fraction,n-butanol fraction and aqueous fraction were obtained.These extracts were then separated by column chromatography and qualitatively analyzed and identified by high resolution mass spectrometry(ESI-HR-MS).Then the biological modification of collagen and the mineralization promoting effect of demineralized dentin were compared with these extracts.The most active extract,n-butanol,was used to study the effects on proanthocyanidins(from tea root)on the stability of resin dentin bonding interface and the proliferation and differentiation of human dental pulp stem cells.Finally,the interactive mechanism between the molecular structure of proanthocyanidins and dentin collagen were further dissected with some proanthocyanidins dimer and ester catechins and their derivatives.The purpose of this study was to provide an experimental basis for optimizing selection of the active ingredients of proanthocyanidins for oral clinical applications,and to provide some theoretical basis for resource development of proanthocyanidins extracted from tea plant.The following are the summary of our main research work and achievements in this area.1、The main component of tea root extracted by ethyl acetate is catechin monomer,the main component extracted by the n-butanol is proanthocyanidins with a degree of polymerization of 2～5 degrees,while the main component in the aqueous fraction is phenolic acid.The extracts of ethyl acetate,n-butanol and aqueous fractions were prepared into solution to treat demineralized dentin collagen respectively.The biological properties of cross-linked collagen dentin were studied by means of the ninhydrin test,water absorption and swelling rate test,hydroxyproline kit assay and three-point bending test.The results showed that n-butanol extract yielded the best biological effect,which could significantly improve the cross-linking degree of collagen,reduce the water absorption rate of collagen,and improve the mechanical properties of collagen and its resistance to enzyme degradation.2、By microscopic morphology observation of the mineralization of dentin surface,X-ray diffraction analysis and microhardness testing etc,it was found that tea tree root n-butyl alcohol extract promoted mineralization of dentin matrix better than the fluoride and ethyl acetate extract.Results showed that proanthocyanidins from the tea tree root are more suitable for dentin mineralization,and promote mineralization related to its composition,while polymer is better than haploid proanthocyanidins in promoting mineralization.3、The demineralized dentin pretreated with tea root proanthocyanidins solution from n-butanol extraction before resin bonding,and glutaraldehyde pretreatment was assigned as the positive control.Micro tensile tests were carried out before and after simulated resin aging.The effect of proanthocyanidins on resin bonding stability was studied by micro tensile strength test and scanning electron microscopy.The results showed that demineralized dentin with proanthocyanidins pretreatment had no effect on the immediate bonding strength.Proanthocyanidins in tea root could significantly enhance the resistance of dentin collagen to enzyme degradation,and improve the micro tensile strength of dentin bonding after the aging procedure,as compared with the glutaraldehyde group.4、h DPSCs were cultured and induced by proanthocyanidins extracted from tea root at different concentrations(0,0.1,1,10 μg/m L).CCK-8 reagent was used to assay its effect on cell proliferation.Osteogenic differentiation of cells was evaluated by alkaline phosphatase activity and alizarin red staining.The expression of osteogenic/odontogenic related genes was detected by Q-PCR.The results showed that the tea tree root proanthocyanidins had no obvious inhibitory effect on the proliferation of human pulp stem cells.Tea tree root proanthocyanidins can induce the osteogenic differentiation of Hd PSCs,and up-regulate osteogenic/odontogenic gene marker expressions(Runx-2,Col-1,DSPP and OSX)of Hd PSCs,thereby promoting the differentiation of Hd PSCs towards the osteogenic/odontogenic lineage.5、Proanthodelphinidin B2 3,3’-O-gallate(EGCG-EGCG),procyanidin B2,EGCG and EGCG-3 "Me were used to pretreat dentin collagen.Fourier-transform infrared(FTIR),anti-enzyme degradation ability detection and ultimate tensile strength methods were used to investigate the mechanism and effect of proanthocyanidins dimer and ester catechins and its derivatives on dentin collagen.The results showed that EGCG-EGCG,EGCG and EGCG-3" Me could significantly improve the biological properties of dentin collagen,and EGCG-EGCG yielded the best improvements.6 、 The inhibitory effects of proanthocyanidin dimer,ester catechin and its derivatives on collagenase were detected by the Bio Visions collagenase activity assay kit.The results showed that both proanthocyanidin dimer EGCG-EGCG and EC-EC exerted good inhibitory effects on collagenase activity.Based on the above results,it could be inferred that the number of phenolic hydroxyl groups within the molecular structures of proanthocyanidins and the degree of polymerization could be two important factors that affect the interaction between proanthocyanidins and Collagen in the dentin matrix,while the number of phenolic hydroxyl groups might play the leading role.Proanthocyanidins in tea root showed good biological effects on both Dentine collagen,collagenase and h DPSCs,indicating that proanthocyanidins in tea plant may have potential therapeutic value in dentistry.