Study Of Biological Activity Of Gingival Fibroblasts And Periodontal Ligament Cells

Objective: The periodontal tissue is composed of gingiva, periodontal ligament,alveoler bone and cementum and its chief effect is to support teeth. This diverse composition makes peri- odontal wound healing a more complex process than general so- ft tissue healing.Fibroblasts are the predominant cell type residi- ng in the soft connective tissue and play important roles in deve- lopment, regeneration, function and pathological alteration.Alt- hough the morphologies of both types of cells cultured in vivo have been reported to be similar when examined by phase- con- trast and scanning electron microscope,other studies suggest that heterogeneous subsets of cells are present which have different characteristic.While gingival fibroblasts maintain the synthesis and integrity of gingival connective tissue,periodontal ligament cells with specialized functions are considered to be responsible not only for the formation and maintenance of the PDL but also for repair,remodeling and regeneration of the adjacent alveolar bone and cementum in vivo. Furthermore,the exact cell markers betweenthe two cell linages are unknown.Periodontitis is one of the two disease which do harm to human oral health with the incidence above 80%,and induce to destruction of the periodontal tissue, loss of periodontal attach- ment, formation of periodontal pocke, and tooth mobility even falling off.It is the main reason of clinical adult tooth- missing.It has been proposed that only cells from periodontal ligament have the potential to create new connective tissue attachment to root surface. The goal of periodontal therapy is to regenerate the soft and hard tissues including cementum, alveolar bone and insertion of collagen fibre. It is important that periodontal ligament cells and gingival fibroblasts are regulated to regenerate.This study compared the human periodontal ligament cells and gingival fibroblast for the purpose of proliferation, Alkaline phosphatase(ALP) activity,expression of collagenⅠ,collagenⅢand bone morphogenetic proteins(BMP) by immunohistoc- hemistry dyeing , and the synthesis of BMP using flow cytometry(FCM),to obtain advantage and disadvantage of the two kinds of cells to be seeded cells.In this way the two kinds of cells will be made to ideal seeded cells by gene technology in the future.Methods: Isolated human periodontal ligament cells and gingival fibroblast were cultured according to the method of tissue-explant, detected their proliferation and ALP activity, synthesis of collagenⅠ,collagenⅢand BMP by immuno- histochemistry dyeing and FCM, in purpose of comparing char- acteristics, proliferation and differentiation of the two kinds of cells.1 Primary culture and passage Human periodontal ligament cells were isolated from the mid-root of 2 premolars extracted due to orthodontic therapy from 1 adolescent patient (12 years of age) with clinically healthy periodontium. The periodontal ligament tissue was mechanically removed under sterile conditions by scraping the middle third of the root surface with a sharp blade, according to the method of Situzhenqiang. When the cells grew out from the explants and reached 80% of the culture flask'area, they were separated with trypsin. Using the passage second to identify the mesoderm quality.Gingival fibroblast were isolated from gingival papilla of the same tooth,the same method as above.2 Comparisn of proliferationThe fourth passage cells were enzyme-digested and suspended in DMEM solution. Then adjusted to 2×104/ml and incubated in three 96-well plate, cultured for 7 days with 10% DMEM. At the third day,fifth day and seventh day,cell proliferation was studied by MTT colorimetric method.3 Comparisn of ALP activityThe fifth passage cells were enzyme-digested and suspended in DMEM solution. Then adjusted to 1×105/ml and incubated in one 24-well plate, cultured for 7 days with 10% DMEM. Triton X-100 was put in every well, then the plate stayed overnight at 4℃and carried out the experiment according these steps:obtained the above-mentioned liquid in every well and distilled water, then put them in the other 96-well plate. Added the buffer solution, ground substance solution in order according to the description of ALP, aqueous bath, then added the developer. OD values were detected by immunodetection, reflected the activity of ALP indirectly.4 ImmunohistochemistryThe fifth and six passage human dental pulp cells were seeded in culture flask with a coverslip in every well at a density of 2×10~5/ml cells, cultured for 7 days with 10% DMEM. These coverslips were fixed with alcohol of 95 percent, stained with immunohistochemistry and observed under the fiberscope.5 FCM detectionThe sixth passage cells were enzyme-digested and suspended in DMEM solution. Then adjusted to 1×106 and centrifuged to collect cells.After D-Hank's having washed two times, Cells were fixed to be unicell suspension with alcohol of 70 percent,sended to be detected.Results1 The periodontal ligament cells were successfully cultured with success rate 10%,while Gingival fibroblast were cultured in all biopsies of gum( success rate 100% ). Under the inverted microscope, cells emigrated from the isolated tissue in 5 or 10 days. Cultured cells were stellate or spindle-shaped, radiating from the tissue edge. The cells were grown to confluence at the day 20. the morphologies of both types of cells cultured in vivo have been reported to be similar,having a spindle-shaped, elongated appearance characteristic of fibroblast-like cells. Immunohistochemistry identified them to be mesoderm quality.2 After having added MTT for 4 hours, violet crystallization was formed in every well. The A value of Gingival fibroblast was larger than of periodontal ligament cells.The difference was significance(P<0.01).3 The OD values of ALP in periodontal ligament cells were higher than that of gingival fibroblast (P<0.01).4 Immunohistochemistry indicated, positive expression of collagen I, III and BMP in gingival fibroblast,while Strong positive expression of collagen I, III and BMP in periodontal ligament cells.5 FCM detection indicated that the fluorescence intensity values of BMP in periodontal ligament cells were higher than that of gingival fibroblast (P<0.01).Conclusions1 Compared with periodontal ligament cells, gingival fibroblast obtained easily, had high success rate and cell proliferation activity in primary culture, so they could be made to ideal seeded cells in periodontal tissue engineering after further study.2 Immunohistochemistry indicated, the expression of collagen I and III in periodontal ligament cells was higher than in gingival fibroblast. Collagen I and III could be used for the marker of periodontal ligament cells and gingival fibroblast.3 Bone formation ability of periodontal ligament cells was stronger than gingival fibroblast. ALP and BMP could be used for the marker of periodontal ligament cells and gingival fibroblast.