The Effects Of High Glucose On Pluripotent Differentiation Of Periodontal-associated Cells

Objective:This study is to compare the difference in pluripotent differentiation between human periodontal ligament cells(HPDLCs) and human gingival fibroblasts(HGFs) in vitro, to observe the effects of high glucose on the ability of pluripotent differentiation and explore its mechanism, which provides broader ideas of tissue regeneration and treatment for diabetic periodontitis.Methods:(1) After informed consent, the extracted premolars due to orthodontic requirement and gingiva due to wisdom teeth extraction were collected from healthy patients. HPDLCs and HGFs were obtained via primary culture. HPDLCs and HGFs at 3rd-4th passage were used in the study.(2) HPDLCs and HGFs were cultured in osteogenic, chondrogenic or adipogenic medium. Alizarin red, Alcian blue and oil red O staining were performed to detect osteogenic, chondrogenic and adipogenic differentiation in vitro, respectively. Reverse transcription polymerase chain reaction(RT-PCR) was applied to examine the expression of osteocalcin(OCN), runt-related transcription factor 2(RUNX2) and collagen 1(Col 1), collagen 10(Col 10) and peroxisome proliferator-activated receptor gamma 2(PPAR?2).(3) The effects of glucose(5.5, 15, 25 mmol/l) on osteogenic differentiation of HPDLCs and HGFs were detected. Real-time PCR was applied to detect the osteogenic-related gene expression at day 7 and 14. Alizarin red staining was performed at day 28.(4) The effects of glucose(5.5, 15, 25 mmol/l) on chondrogenic differentiation of HPDLCs and HGFs were detected. Real-time PCR was applied to detect the chondrogenic-related gene expression at day 7 and 14. Alcian blue staining was performed at day 14.(5) The effects of glucose(5.5, 15, 25 mmol/l) on adipogenic differentiation of HPDLCs and HGFs were detected. Real-time PCR was applied to detect adipogenic-related gene expression at day 7 and 14. Oil red O staining was performed at day 21.Results:(1) A few spindle cells migrated from the edge of tissue during the 5-9 days of the culture. The HPDLCs grew fast and well after being passaged. Cells migrated out from the tissue explants when cultured about 3-7 days, which might be mixed with polygonal epithelial cells. After passaging, cells began to converge in the long fusiform.(2) HPDLCs and HGFs cultured in osteogenic medium showed massive calcium nodules at day 28, but HPDLCs formed more than HGFs. The expressions of OCN, RUNX2 and Col 1 in HPDLCs were higher than in HGFs(P<0.01). In chondrogenic medium both cells were found blue deposit at day 14, and the expressions of Col 10 in HGFs was higher than HPDLCs(P<0.01). Furthermore, in adipogenic medium HGFs showed more lipid-filled droplets stained with oil red O than HPDLCs at day 21. The expression of PPAR?2 in HGFs was higher than HPDLCs(P<0.01).(3) With increase concentration of glucose, alizarin red staining significantly weakened at day 28, and the expression of OCN, RUNX2 and Col 1 m RNA significantly reduced(P<0.01). Also, alcian blue staining significantly weakened at day 14, and the expressions of Col 10 and aggrecan(AGR) m RNA significantly reduced(P<0.01). However, oil red O staining significantly strengthened at day 21, and the expression of PPAR ?2 m RNA significantly increased(P<0.01).Conclusion:(1) Both HPDLCs and HGFs have the pluripotency of osteogenic, chondrogenic, adipogenic differentiation.(2) HPDLCs has better potency of osteogenic differentiation than HGFs, however, HGFs has better potency of chondrogenic and adipogenic differentiation.(3) High glucose inhibits osteogenic and chondrogenic differentiation in HPDLCs and HGFs.(4) High glucose promotes adipogenic differentiation in HPDLCs and HGFs.