Experimental Study Of Ionic Dissolution Products Of Dicalcium Silicate Coating On Osteogenic Differentiation In Human Mesenchymal Stem Cells

PartⅠIsolation, culture and identification of human mesenchymal stem cellsObjective To isolate and culture human mesenchymal stem cells (hMSCs) from bone marrow in vitro and observe their biological characteristics. Methods Bone marrow aspirates were obtained from the posterior iliac crest of healthy human donors under aseptic conditions. hMSCs were isolated from the marrow by Percoll density gradient centrifugation method. The nucleated cells were plated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). hMSCs were highly purified and expanded through adherence filtration. Cell morphology and proliferation status were observed by phase contrast microscope. Standard flow cytometry techniques were used to determine the cell surface epitope profile and cell cycle of hMSCs. Results The cells exhibited a fusiform shape and swirled when they assembled together. The amplification speed of hMSCs became slow and cell morphology changed after passage 8. The adherent cells did not express hematopoietic lineage markers CD34 and CD11b, while hMSCs collected from passage 3 were strongly positive for CD90 and CD105 markers. The growth curves of hMSCs from the first, third and fifth passage were S-shaped. The cell cycle analysis showed that the percentage of G0/G1 phase of cells from passage 3 was 77.42% and the percentage of S+G2/M phase was 22.58%. Conclusion The density gradient centrifugation technique in combination with adherence filtration is a simple, economical and efficient method to isolate and purify hMSCs. PartⅡEffect of ionic dissolution products of dicalcium silicate coating on human mesenchymal stem cells proliferation and apoptosisObjective To explore the influence of ionic dissolution products of dicalcium silicate coating on proliferation, cell cycle and apoptosis in human mesenchymal stem cells. Methods Ten pieces of plasma sprayed dicalcium silicate (DS) coatings were soaked in 200 ml DMEM for 72 h at 37℃in static conditions, then inductively coupled plasma atomic emission spectroscopy (ICP-AES) was employed to measure the concentrations of silicon (Si), calcium (Ca) and phosphorus (P). hMSCs were cultured in four media (OS-DS-, OS-DS+, OS+DS-, OS+DS+). The proliferation of hMSCs was determined using MTT assay. Cell cycle and apoptosis were analyzed by flow cytometry on days 4, 7 and 14. Results The concentration of Si in OS-DS+ and OS+DS+ was significantly higher than OS-DS-, Ca concentration decreased slightly, and P content of osteogenic medium (OS+DS-, OS+DS+) was higher than OS-DS- medium. Cell proliferation process in OS-DS+ and OS+DS+ was consistently higher than that in OS-DS- in the first 4 days of culture, and significant difference was seen at 2 and 3 days, respectively. Throughout the assay period, the number of hMSCs exposed to OS+DS- was generally lower than that in OS-DS-, and significant decrease did not become apparent until day 7 of culture. The percentage of G0/G1 phase of cells cultured in OS-DS+ and OS+DS+ was lower than that in OS-DS-, while the percentage of G2/M phase was higher on day 4. Smaller percentage of S phase was detected in hMSCs exposed to OS+DS- at 4 days, whereas bigger percentage of S phase was observed in OS+DS+ and OS-DS+ at 4 and 14 days, respectively. Apoptosis was markedly increased in OS+DS- and OS+DS+ on days 7 and 14, and in OS-DS+ on day 14 when compared to OS-DS-. Conclusion The ionic dissolution products of dicalcium silicate coating can promote the early proliferation of hMSCs by accelerating cell cycle, which may be attributed to the high concentration of silicon ion. PartⅢStudy of ionic dissolution products of dicalcium silicate coating on osteogenic differentiation in human mesenchymal stem cellsObjective To investigate the effect of ionic dissolution products of dicalcium silicate coating on human mesenchymal stem cells osteogenic differentiation. Methods hMSCs were cultured in four media (OS-DS-, OS-DS+, OS+DS-, OS+DS+). Alkaline phosphatase (ALP) activity was assessed at the protein level. Calcium deposition was analyzed by Alizarin Red S staining. Reverse transcription-polymerase chain reaction (RT-PCR) was used for analysis of mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Col-I), osteonectin (ON) and osteocalcin (OC). Results Throughout the cultivation period, hMSCs exposed to osteogenic medium (OS+) had significantly higher ALP activity than did cells exposed to growth medium (OS-). ALP activity of hMSCs cultured in OS+DS+ was significantly higher than that of cells cultured in OS+DS- on days 7, 14 and 21. The level of ALP activity was appreciably higher in hMSCs exposed to OS-DS+ when compared to OS-DS- on day 14. The optical density (OD) value in hMSCs exposed to OS-DS+ was higher than that of cells exposed to OS-DS- at 14 days. Treatment in OS+DS- resulted in a significant increase in OD, and hMSCs in OS+DS+ further increased the OD value. The hMSCs grown in OS-DS- or OS-DS+ medium failed to form any detectable calcium nodules after 28 days of culture. Cells cultured in OS+DS- medium exhibited some small mineralized nodules, while a greater extent of mineralization with more detectable bone nodules was observed in hMSCs exposed to OS+DS+ medium. The mRNA levels of osteogenic markers investigated were higher in hMSCs exposed to osteogenic medium (OS+) than cells exposed to growth medium (OS-) throughout the assay period. hMSCs grown in OS-DS+ expressed higher levels of Runx2 and ON mRNA than did cells exposed to OS-DS- on days 7 and 14, respectively. The osteogenic markers were expressed at higher level in the (OS+DS+)-group at 14 days when compared to the (OS+DS-)-group. Conclusion The ionic dissolution products of dicalcium silicate coating alone partly induce hMSCs differentiation towards the osteoblastic lineage. The cooperation between ionic dissolution products of dicalcium silicate coating and osteogenic supplements can fully induce osteoblast-specific gene expression and significantly promote osteogenic activity of hMSCs.PartⅣStudy of the mechanisms of dicalcium silicate coating ionic dissolution products on human mesenchymal stem cells osteogenic differentiationObjective To explore the possible mechanisms of ionic dissolution products released from dicalcium silicate coating in promoting osteogenic differentiation of hMSCs. Methods hMSCs were cultured in OS+DS- medium, OS+DS+ medium or OS+DS+ medium supplemented with PD98059. The levels of extracellular signal-regulated kinase (ERK) phosphorylation were examined by Western blot. PD98059, a specific inhibitor of ERK, was added into OS+DS+ medium to investigate the changes in alkaline phosphatase activity and calcium deposition. Results ALP activity of hMSCs cultured in OS+DS+ was significantly higher than that of cells cultured in OS+DS- on days 7, 14 and 21, but the increase was inhibited by PD98059. Calcium deposition in OS+DS+ was greater than that in OS+DS- at 14 days, and the increase was also suppressed by PD98059. ERK was activated at 10 min after treatment in OS+DS+ medium. The ERK activation was maintained at higher level to 60 min, and then declined to the baseline. hMSCs in OS+DS- medium expressed a moderate level of ERK phosphorylation, and treatment with ionic dissolution products of dicalcium silicate coating further increased the level of ERK phosphorylation, but the increase was blocked by pre-treating cells with PD98059. Conclusion The ionic dissolution products from the dicalcium silicate coating may promote osteogenic differentiation of hMSCs mainly through ERK signal transduction pathway.