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The Effects of Chitin and Its Derivatives on the Proliferation of NIH3T3 Cells and Human Periodontal Ligament Cells in Vitro

Author: LiYan
Tutor: XueYi
School: Hebei Medical University
Course: Clinical Stomatology
Keywords: chitin chitosan cell proliferation flow cytometry(FCM) proliferative cell nuclear antigen(PCNA)
CLC: R780.2
Type: Master's thesis
Year: 2005
Downloads: 75
Quote: 1
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Abstract

Chitin((1→4)-2-acetamido-2-deoxy-β-D-glucan) andits derivatives are a new kind of bioactive polysaccharideswhich are widely used in agriculture, industry and medicine.In the last ten years, the medicine authors focused on chitinand its detrivatives.A great deal of experiments indicated that chitin and itsderivatives, such as chitosan,possess the same potential asproteoglycans of conducting cell attachment and promotingfunction and stimulating attachment , proliferation anddifferentiation of fibroblast cells. It is possible that chitin andits derivatives can stimulate the proliferation of humanperiodontal ligament cells(HPDLC). Periodontal disease isa kind of common oral disease whose characteristic is thedistruction of periodontal tissues. So the stimulation of theHPDLC’s proliferation activity and the restoration ofperiodontium is the aim of therapy. The mini grain sizemakes it possible to act adequately.Objective: Microparticle chitin, chitosan of differentmocular weights and water soluted carboxymethyl-chitosanwere choosed to the study. To investigate the effects of thesedifferent biomaterials with different concentrations on theproliferation of HPDLC,selecting and recommending thebetter biomaterials with higher biocompatibility, supplyingbasic experimental data for further clinic application inperiodontal treatment.Methods: There are two parts in the study.1 Effects of chitin and its derivatives on the proliferation ofNIH3T3 cellsNIH3T3 cells were seeded on 24-well culture plate witha start cell density of 2×104/ml cells(200μl per well). Cellnumbers were counted every 24 hours and the growth curveof NHI3T3 cells was drawn, getting the redouble time in thelogarithmic period of growth phase.2% microparticle chitin/chitosan in 1% acetic acid, or2% carboxymethyl-chitosan in double-distilled water, thesolution was cast on a plastic board and dried at roomtemperature. The membranes were cut into 1×1cm2 smallpieces. The membrane was immersed in 10ml serum-freeRPMI1640 medium at 37℃for 24 hours. Medium withextract on different concentrations was prepared. NIH3T3cells at their logarithmic phase were seeded in 96-wellculture plate and incubated with these mediums for 48 hours,the effects of chitin and its derivatives on the proliferation ofNIH3T3 cells with MTT assay to find the concentrations ofmedium that affected the proliferation most significantly.Cells at their logarithmic phase seeded in 25cm2 cultureflasks incubated in medium containing chitin withconcentrations that affected the proliferation mostsignificantly for 48 hours. Then the cells were collected andfollowed with other experiments. (1) After they werecentrifuged to get cell-coated slices, proliferative cell nuclearantigen ( PCNA ) activity was assayed by PCNAimmunohistochemical staining. 500 cells were countedrandomly on every visual field for calculating the percentageof PCNA positive cell. (2) The cells suspension wereadjusted at a cell density of 106/ml, fixed with 70% ethanol,stained with PI, cell cycle was determined by flow cytometry.2 Effects of chitin and its derivatives on the proliferation ofHPDLCFresh premolars of 10-16 years old clinic patients weresoaked into D-Hanks solution (with 1000U/ml penicillin andstreptomycin) for 5 to 10 minutes at 4℃under asepsiscondition, and then the premolars were rinsed twice toremove blood. Middle 1/3 of periodontal tissues of the toothwas scrached and cut into 1mm3 for spreading evenly on thebottom of the culture bottle pretreated with serum. The bottlewas left up side down and DMEM solution with 20%newborn calf serum (CS) was added. The specimen wascultured for 2-6 hours under standard environment ofsaturated humidity, 50ml/L CO2, 950ml/L air and 37℃. Thebottle was turn over, observe under inversion microscopeeveryday. Replaced the medium if necessary until thegeneration of HPDLC was collected by 0.0625% trypin and0.0125% EDTA. Keratin and vimentin protein was detectedby immunhistochemical SP method.The 5th generation of HPDLC were seeded on 96-wellculture plate with a start cell density of 3×104/ml cells and200μl each well. Cell density was assayed with MTT every24 hours and the growth curve of the 5th generation of PDLCwas drawn, the redouble time in the logarithmic period ofgrowth phase was calculated.Effects of chitin and its detrivatives on the proliferationof HPDLC were tested with MTT assay as that of NIH3T3cells.Results1 Effects of chitin and its derivatives on the proliferation ofNIH3T3 cellsChitin and its derivatives in lower concentration(2.5%-15%) can stimulate the proliferation of NIH3T3 cells,the higher concentration ( ≥20%) ones inhibit theproliferation by contrary.2 Effects of chitin and its derivatives on the proliferation ofHPDLCIn vitro, HPDLC were in shuttle or polygon shape withseveral umbos. The karyon was oval in the center of the cellwith 2-3 nucleoluses. The cell showed acidophilia staining.The cells arrayed radially in regularity.Mesoblast-origination of HPDLC was proved by the

The Effects of Chitin and Its Derivatives on the Proliferation of NIH3T3 Cells and Human Periodontal Ligament Cells in Vitro

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