| OBJECTIVE:This study is to investigate the pluripotency of human gingival fibroblasts(h GFs) for osteogenic, chondrogenic and adipogenic differentiation, furthermore, to study the proliferation and osteogenic differentiation of h GFs stimulated by bone morphogenetic protein-2(BMP-2) and dexamethasone(DEX).METHODS:Volunteers(age ranges from 18 to 25 years) were selected from Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Tianjin Medical University. With informed consent, fresh and healthy gingiva was isolated and collected from impact teeth extraction. HGFs was obtained from the gingiva by tissue culture. The third passage of h GFs was applied in the following study. The study was divided into two parts. One was to study pluripotency differentiation of h GFs. The control group was cultured by DMEM(Low glucose) and 3% fetal bovine serum(FBS). Osteogenic medium contained DMEM(Low glucose), 3% FBS, 10-2 mol/L β-glycerophosphate disodium salt hydrate, 5 mg/L L-Ascorbic acid and 2×10-3 mol/L L-Glutamine and10-7 mol/L DEX. Chondrogenic medium contained DMEM(Low glucose), 3% FBS,50mg/L L-ascorbic acid, 6.25mg/L insulin and 10μg/L transforming growth factor β1(TGF-β1). Adipogenic medium contained DMEM(Low glucose), 3% FBS, 10-6mol/L DEX, 10-5 mol/L insulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine(IBMX) and2×10-4 mol/L indometacin. Cells were examined by alkaline phosphatase(ALP)staining, calcium nodulus staining, oil red O staining and Alcian blue staining for the differentiation. The other part was set up by five groups. Group A was cultured by DMEM(Low glucose) containing 3% FBS; group B was cultured by basal osteogenic medium which contained DMEM(Low glucose), 3% FBS, 10-2 mol/Lβ-glycerophosphate disodium salt hydrate, 5 mg/L L-ascorbic acid and 2×10-3mol/L L-glutamine; group C was cultured by basal osteogenic medium plus 10-7 mol/L DEX;group D was cultured by basal osteogenic medium plus 50 μg/L BMP-2; group E was cultured by basal osteogenic medium plus 10-7 mol/L DEX and 50 μg/L BMP-2.Cell proliferation was detected by MTT. ALP activity, calcium nodulus staining and reverse transcription polymerase chain reaction(RT-PCR) were applied to examine for the differentiation.RESULTS:(1) HGFs cultured by osteogenic medium were found massive violet deposit at day 7and calcium nodulus at day 28. Chondrogenic group were found blue deposit at day14. Adipogenic group were found lipid-filled droplets in cells that accumulated oil red O at day 14. However, h GFs in control group had no any positive staining.(2) MTT data showed that h GFs had a stable growth in all culture conditions. No significant difference in cell proliferation was among the groups(P>0.05).(3) ALP activity showed that no staining was found in group A. A little violet deposit could be seen in group B and group D, massive violet deposit was seen in group C and group E. In addition, the violet deposit in group E was more than that in group C.(4) Alizarin red staining results showed that no calcium nodulus was detected in group A, while a mass of calcium nodulus was seen in other groups. The number of calcium nodulus reduced from group E, group C, group D to group B.(5) The results of RT-PCR showed that group E had the highest expression of ALP,Col1 and Runx2 among all groups at the same time(P<0.0001), followed by group C(P<0.0001). Except for group E, the expression of Runx2 had no significant difference in all other groups at day 7. At day 14, group D had second highest expression of Runx2. than that of group D at day 21(P<0.05). At day 21, group C had a higher expression of Runx2 than that of group D at day 21(P<0.05).CONCLUSION:(1) HGFs have the pluripotency of osteogenic, chondrogenic and adipogenic differentiation.(2) DEX and BMP-2 have no significant influence on the proliferation of h GFs.(3) Alkaline phosphatase activity and calcium deposition were promoted by DEX or BMP-2, especially by both together. DEX and BMP-2 could promote the expression of osteogenic-related genes, which enhanced the osteogenic differentiation of h GFs. |
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