Study Of Biological Behaviors Of Bovine Periodontal Ligament Cells And Effects Of Shuanghuangbu On The Cell's Proliferation In Vitro

Objective: The primary goal of periodontal treatment isto regenerate the supporting periodontal structure, i.e, to formnew bone, cementum and new periodontal ligament. Successfulperiodontal regeneration may be achieved by proliferation anddifferentiation of the periodontal ligament cells. Cell culture is amain method to researches of periodontal ligament biologicalcharacters and implantment. Because of the unique structure ofthe periodontal ligament and quantity limit during extraction,gaining abundant experiment cells or seed cells cell remainsvery difficult. This study observed the biological behaviors ofnew-born bovine periodontal ligament cells for the purpose ofestablishing continuous periodontal ligament cell lines andsearching suitable seed cells for future investigations.Successful periodontal regeneration depends on themigration, proliferation and differentiation of periodontalligament cells in periodontal defects. Researches have shownmarvelous effects of Shuanghuangbu on elimimatinginflammation and inducing bone formation. Now we prepare toinvestigate the effect of Shuanghuangbu on bovine periodontalligament cells proliferation in different period and to selectoptimal density. In this way we evaluate the feasibility ofcombining Shuanghuangbu, periodontal ligament cells andmacromolecule material together and planting the combinationinto periodontal defects.Methods: Isolated periodontal ligament cells fromnew-born bovine were cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum(FBS). The biological behaviors of the cells were investigatedby measuring the cell's attachment efficiency, growth curve andmitotic index. And the effects of Shuanghuangbu on theproliferation of bovine periodontal ligament cells wereevaluated by MTT method.1 Isolation and culture of new-born bovine periodontalligament cellsA new-born cattle sacrificed within 12 hours after deliverywas selected in this study. Eight mandible incisor teeth whichhad already erupted were extracted. The periodontal ligamenttissue was mechanically removed under sterile condition byscraping the middle third of the root surface with a sharp blade,according to the method of Situ.2 Measuring the attachment efficiency of bovine periodontalligament cellsThe third passage cells were enzyme-digested andsuspended in DMEM solution. Then adjusted to 1×106/ml andincubated in 24-well plate, calculated in 2, 4, 6, 8, 10, 12, 14 hrespectively.3 Drawing the growth curve of bovine periodontal ligamentcellsThe third passage cells were enzyme-digested andsuspended in DMEM solution. Then adjusted to 5×103/ml andincubated in 24-well plate, calculated per day continuously in 7days.4 Calculating the mitotic index of bovine periodontal ligamentcellsThe fourth passage cells were enzyme-digested andsuspended in DMEM solution. Then adjusted to 1×104/ml andincubated in 6-well plate with a glass slide being placed on theplate floor beforehand. Took out one slide each 24 hours anddyed it. Calculated the mitotic cells under the light microscopein 6 days.5 Shuanghuangbu decoction preparationA mixture of Rhizoma coptidis, Radix scutellariae andRhizoma drynariae was soaked in water, boiled twice andfiltered, and the concentration of 1g/ml Shuanghuangbudecoction was made. And then Shuanghuangbu decoction wasfiltered and diluted with serum free Dulbecco's modified eagle'smedium(DMEM) to final concentrations (10, 50, 100, 500, 1000μg/ml )respectively. The pH of final decoction was adjusted to7.0～7.1, then the decoction was divided and stored at 4℃.6 Effects of Shuanghuangbu on the proliferation of bovineperiodontal ligament cellsThe fourth passage cells were enzyme-digested andsuspended in DMEM solution. Then adjusted to 1×104/ml andincubated in 96-well plate, cultured for 24 hours. After that,randomly divided those wells into 1 control group and 5experiment groups, added Shuanghuangbu decoctions in therange of 0 to 1000μg/ml to each well, respectively. The optimalconcentration was gained and then checked the time effect inthis density. Cell proliferation was studied by MTT colorimetricmethod.Results1 The periodontal ligament cells were successfully cultured inall biopsies of periodontal ligament ( success rate 100% ).Under the inverted microscope, cells emigrated from theisolated tissue in 3 or 5 days. Cultured cells were stellate orspindle-shaped, radiating from the tissue edge. The periodontalligament cells were grown to confluence at the day 15.2 After passage, the new-born bovine periodontal ligamentcells were firstly suspended in the DMEM, in spherical shape.Then the cells stretched in 15 to 30 minutes. The attachmentefficiency reached 25.8% in 6 h and 80.6% in 14 h, respectively.In 24 h the cells connected like a net.3 The bovine cells showed "S"growth curve. One day afterpassage, the cells number declined a little. And then it increasedand reached its peak in 5 days. After that, it turned to stablestate.4 The bovine cells showed various mitoses rates in 6 daysfrom 2.89% to 11.66%.