Comparison Of The Effects Of HMSCs Cultured By Autologous Serum Into Osteoblasts By Extracorporeal Shock Waves And Dexamethasone

Objective:Human mesenchymal stem cells (hMSCs)is a pluripotent stem cells isolated from bone marrow.It possessing of self-replication, self-renewal, multi-differentiation and the potential to become bone, cartilage, nerves, muscle, skin and liver and many other types of mature cells .So it is an ideal seed cell in bone tissue engineering. However, due to the limited number of hMSCs, we have to expanse before transplant into vivo . At present the main expansion of hMSCs in medium is suppled with fetal bovine serum(FBS) ,but it has a potential risk of transmitting viral and prion diseases and causing immunological rejection. This greatly hindered the clinical application of hMSCs. The autologous serum (AS) can avoid the above problems.The induction of hMSCs to osteoblasts is major applicated of traditional dexamethasone, but the concentration of dexamethasone have a great impact on the proliferation of hMSCs.With the concentration increased slightly, it has the trend of induced hMSCs to adipocyte differentiation.This limited the effect of dexamethasone on osteogenic. In recent years ,extracorporeal shock wave therapy (ESWT) for its ability to significantly promote the proliferation of osteoblasts, its treatment of avascular necrosis, nonunion, and delayed fracture healing achieved good efficacy. Therefore,we compared the effect of hMSCs cultured by autologous serum into osteoblasts by ESWT and dexamethasone. There were no reports of this study at home and abroad.Methods:1. Collecting bone marrow and separated autologous serum:Collecting 60mL iliac crest bone marrow and 100ml peripheral blood from each of 10 volunteers(none had documented metabolic disease),and then separated autologous serum from peripheral blood(about 55ml). 2.Observe the effects of AS on the proliferation of hMSCs:Then cultured the hMSCs with 10% AS and 10% FBS. Then cultivating both the experiment group and control group in the same condition.During cultivation observing the cell morphology,24-hour cell adhesion rate, cell doubling time with inverted microscope, detecting cell proliferation curve by the method of CCK-8,detecting cellular surface antigen,measuring generation cycle by flow cytometry and observing immunocytochemical staining.3.Compare of the effects of hMSCs cultured by AS into osteoblasts by ESWT and dexamethasone:Then we applied ESWT and dexamethasone to induct hMSCs cultured by AS into osteoblasts.After inducion,observing ALP staining and Alizarin Red staining,decting the activity of ALP .Using RT-PCR to analyze the expression differences of oteogenic gene(ALP,OP,OC,ON,colⅠ) exppress between two groups.Using a statistical method to analyse the part of corresponding data.Results:1.The cell morphology was observated by the inverted microscope. The AS group has no significant deviations compared with the FBS group in cell morphology.With the passages increased,the spindle cells were growing into spacious flat-zoster or multilateral.There were some floating cells. Part of the cells have a strong increase secretion, and some cell surface would be dirty. Cell proliferation rate significantly decreased.This phenomenon was particularly evident after the seven-generation. The percent of the 24-hour cell adhesion rate of passage 3 is (94.3±1.7)% in the AS group compared with (93.0±1.6) % in the FBS group(P>0.05).The data don't exist statistics distinction. The percent of the cell doubling time of passage 3 is 53h(an average of 41-54h) in the AS group compared with 84h(an average of 76-89h)in the FBS group(P<0.01).The data exist statistics distinction.The data of growth curve detecting by CCK-8 exist statistics distinction between two groups.The AS group has a higher peak value which means the reproductive activity is enhanced. AS group was significantly better than the FBS group.2.The cellular surface antigen of hMSCs were detected by flow cytometry.Cell surface antigen CD44(+)of AS group and FBS group were 99.96% and 99.30%,which suite the character of hMSCs. The AS group has no significant deviations compared with the FBS group(P>0.05). The result of immunocytochemical staining showed CD44, CD105 were positive expression in two groups and green, red fluorescence throughout the cytoplasm.3.Detecting the cell cycle with flow cytometry: The results showed that hMSCs have a typical stem cell characteristics cycle. Most cells are in the G0 / G1 phase and only a few in S phase. With the passages increased,the percentage of G0 / G1 phase increased and S phase decreased.The result is coincidence with the observating of the cell morphology, which reflects the process of cell senescence. The AS group has no significant deviations compared with the FBS group.(P>0.05).4.The results showed that at each time point ALP activity of ESWT group was significantly higher than dexamethasone group.The contents of ALP of the two groups begin increase at 4th day and continued to increase. At the 14th days to reach peak. The ESWT group has significant deviations compared with the dexamethasone group.(P<0.05).The two groups were cultured for 14 days after osteogenic induction, disposable medium, wash two times with PBS, fixed with 95% alcohol for 10 minutes. Carried out by ALP staining kit instructions. The results showed that ESWT group of ALP staining intensity was higher than dexamethasone group.5.The two groups were stained by Alizarin red after 28 days inducted by ESWT and dexamethasone which show that there are red calcifiied nudules.Then selected three vision randomly and counted mineralized nodule 100 times light microsope and the result is 7.5±1.2 in ESWT group and 5.0±0.8 in dexamethasone group. ESWT group is a large number of calcified nodules than dexamethasone group.6.Rt-PCR products reavealed that ALP,OC,OP,ON, ColⅠexpression of the two group can be detected at the 28th d after induction.The ESWT group was stronger than dexamethasone group significantly.Conclusion: 1. Thus compared with autologous serum,the fetal calf serum is more favorable on the proliferation of hMSCs. So we have chosen AS to conduct hMSCs into osteogenic culture.2. The effects of ESWT are more obvious than dexamethasone.Thus the ESWT is a better way to stimulate osteoblastic differentiation.