| Objective：Periodontitis is bacterial infectious diseases for the characteristic,andresult in formation of periodontal pocket and absorption of alveolarbone,leading to lost of teeth.Nowadays,the routine clinical treatments failed toachieve the ideal effect.How to achieve the regeneration of periodontalsupporting tissue,including aveolar bone,cementum,and periodontalligament,is the direction of us. As the progress of technology of tissueengineering,it has been introduced into the field of periodontology,whichprovides a new research space for the treatment of periodontitis.Periodontal tissue engineering involve three basic biological factors,which are seed cells,differentiation inducing factors and matrical materials.Periodontal ligament (PDL) cell is regarded as the ideal cell to regenerateperiodontal tissues,because of their potential of differentiation to fibroblasts,steoblasts,and cementoblasts under appropriate stimulus.However,PDLCs aredifficult to carried out in clinic,and the success rate of cell culture is relativelylow.Nowadays,some researchers has found that gingival fibroblast cells showsome characters of osteoblasts by some bone-inducing factors,easier to beobtained clinically than PDLCs,and have strong proliferation potential.Now,people pay more and more attention to radix scutellariae for its smallstimulations and side effects. Shuanghuangbu as traditional chinese medicinemainly includes Rhizoma coptidis,Radix scutellariae and Rhizona drynariae inthe reasonable proportion.Among them,rhizoma coptidis and radix scutellariaecould inhibit periodontal pathogenic bacteria obviously. Rhizoma drynariaecould promote proliferation and differentiation of human gingival fibroblastcells into osteoblasts.In our study,shuanghuangbu as a growth factor in periodontal tissue engineering,establish the complex of GF-Zein-shuanghuangbu and implant inthe model of periodontal defects,in order to investigate the effect ofshuanghuangbu on periodontal regeneration and provide experimental basisand theoretical basis for clinical treatment of periodontitis.Methods:1The cultivation of gingival fibroblasts and source identificationGingival tissues romoved from buccal gums of beagle dog weretransferred to sterile environment,washed three times with DMEM,and thentransferred to petri dish.The tissue block was cut into1mm×1mm×1mm piecesand used for primary cultures with the tissue block cultivation.Selecting the third passage cells,keratin staining and vimentin staining byimmunohistochemical and ABC staining identified the source.2Preparation of zein scaffoldsZein dissolved in alcohol.Nacl as porogen was added into the solution.Zein scaffolds were prepared by solvent casting/particle leaching, irradiatedfor2hours under UV and stored for using.3Preparation of shuanghuangbuAccording a certain proportion,coptidis rhizome,scutellariae radix andrhizoma drynariae were mixed together.The mixture was soaked in distilledwater for30minutes and then boiled respectively in8volumes and6volumesof water.Shuanghuangbu was prepared to concentration of1g/ml with waterextraction and alcohol precipitation method,discolored by active carbons forthree times,adjusted pH to7.0-7.1,stored at4℃.4Preparation of GF-Zein complexs and GF-Zein-shuanghaungbu complexThe Zein scaffold were soaked in DMEM containing10%FBS and inshuanghuangbu at37℃for24h in a96-well plate.The forth passage cells werecollected and made into cell suspensions at1.5×105/ml to implant into the zeinscaffolds,and then observed under scanning electron microscope.5Animal experimentTow male clean beagle dogs weighted about15kg and aged6-12monthswere selected in this study. Prepare model of periodontal defects at buccal alveolar bone of the4th and5th premolars of double upper jaws and the3rd,4th,7th and8th of double lower jaw.Zein, GF-Zein complex and GF–Zein-shuanghuangbu complex were separately implanted into orrespondingmodel,while the control group did not give any disposition.Then sutured thegingival tissues.3months after surgery, animals were sacrificed.Collect aspecimen of88in each group to be viewed under scanning electronmicroscope,and the other specimens were evaluated by histology observationand histological measurement (experimental defect,new alveolar boneformation, new cementum formation,the lenghth of junctional epithelium) andimmunohistochemical staining quantitative analysis (insulin like growthfactor-1, osteopontin).6Statistical methodsAll datas are expressed byX--±s, Using SPSS13.0statistical software forstatistical analysis. The amount of alveolar bone formation and the positiverate of insulin like growth factor-1(IGF-1) and osteopontin(OPN) inperiodontic area were compared with a single factor analysis of variance.There is statistical significance when probability is less than0.05.Results:1Morphological observation and source identificationThe gingival fibroblasts cultured with tissue bloke cultivation takethe shape of long spindle and the cell body were plump. After immunohisto-chemical staining,the the anti-vimentin staining was positive,and anti-keratinstaining was negative，which confirmed the cells originated from mesoderm.2Observations of GF adhered to zein scaffolds under scanning electronmicroscopeThrough scanning electron microscopy, the number of gingivalfibroblasts adhered to Zein scaffolds was more under the action of shuang-huangbu,while the number was less without the action of shuanghuangbu.3Dontal and periodontal specimen observation3.1Histology observationThree months after surgery, compared with the rest of the three groups， inflammatory cells were not observed in GF-Zein-shuanghuangbu complexgroups,while New bone and cementum was obviously observed.3.2Histological measurementsThree months after surgery,height of NB in GF-Zein-shuanghuangbucomplex group(1.16±0.06)mm was obviously larger than that in other groups（P<0.05）,while Height of NC and the length of JE has no significantdifference in the four groups（P<0.05）.3.3SEM observationThree months after surgery, trabeculae arranged in order and maturehaversian system were viewed in the GF-Zein-shuanghuangbu complexgroups under SEM.3.4Quantative analysis of IGF-l and OPN in periodontal tissueThree months after surgery, the cell positive rate of IGF-1surroundingnew alveolar bone of GF-Zein-shuanghuangbu complex groups(24.32±1.85)%was significantly higher than the rest of the three groups（P0.05）.The cellpositive rate of OPN surrounding new alveolar bone are significant differencebetween the GF-Zein-shuanghuangbu complex groups(12.68±1.25)%and thecontrol groups(P0.05).The cell positive rate of OPN in periodontic surround-ding new alveolar bone didn’t see anything statistically significant among thegroups of Zein,GF-Zein complex and GF-Zein-shuanghuangbu complex (P0.05),but in GF-Zein-shuanghuangbu complex groups,the number of OPNpositive cells was more.Conclusions：1Shuanghuangbu can effectively control the inflammatory reaction inperiodontal tissues, thus providing a well environment for periodontal tissuerepairing in periodontal tissue engineering.2Shuanghuangbu can stimulates gingival fibroblast cells transform intothe osteoblast and increase the expression of IGF-l and OPN in periodontaltissue,which benefit the regeneration of alveolar bone and cementum.3Shuanghuangbu can serve as a growth factor in periodontal tissueengineering, and has a good prospect in clinical application. |
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