Coordination Of FGF-2and BMP-2for Osteogenic Differentiation Of Low-population Density HMSCs

In the modern design, most delivery systems for bone regeneration focus on a single growth factor (GF) or a simple mixture of multiple GFs, overlooking the coordination of proliferation and osteogenesis induced by various factors. In this study, core/shell microspheres with poly-L-lactide core-poly(lactic-co-glycolic acid) shell were fabricated, and two GFs, basic fibroblast growth factor2(FGF-2) and bone morphogenetic protein2(BMP-2) were encapsulated into the core or/and shell. The effects of different release patterns (parallel or sequential manners) of FGF-2and BMP-2from these core-shell microspheres on the osteogenic differentiation of low-population density human mesenchymal stem cells (hMSCs) were investigated and the temporal organization of GF release was optimized. In vitro experiments suggested that induction of osteogenic differentiation of low-population density hMSCs by the sequential delivery of FGF-2followed by BMP-2from the core-shell microspheres (group S2) was much more efficient than that by the parallel release of the two factors from uniform microspheres (group U). The osteogenic induction by the sequential delivery of BMP-2followed by FGF-2from core-shell microspheres (group S1) was even worse than that from microspheres loaded with BMP-2in both core and shell (group B), although comparable to the cases of parallel delivery of dual GFs (group P). Animal experiments (critical-sized mouse tibia bone defect model) proved that core-shell microspheres sequentially releasing FGF-2and BMP-2would heal bone defect and remodel bone graft the most efficiently, which confirmed the advantages of group S2microspheres in osteogenesis and bone repair, and the necessity of time sequence studies in tissue engineering while multiple GFs are involved.Chapter Ⅰ Biological effects of FGF-2and BMP-2on low-density human bone mesenchymal stem cellsExperiment1The effects of FGF-2and BMP-2on proliferation of low-density human bone mesenchymal cellsObjective:To evaluate the effects of FGF-2and BMP-2on proliferation of of low-density mesenchymal stem cells(hMSCs) by using them alone, associatedly.Methods:Cell isolation and culture technique and CyQUANT cell proliferation assay were used to evaluate the proliferation of hMSCs on3rd day by exogenously adding different concentration of BMP-2or FGF-2or combination.Results:Statistical analysis showed that FGF-2exhibited a close-to-linear stimulation on hMSCs proliferation when the concentration was lower than40ng/mL, but the stimulation weakened substantially while the concentration was higher. Specifically,20ng/mL of FGF-2stimulated the proliferationby-60%compared with control, and100ng/mL of FGF-2increased the proliferation by-67%. The effect of BMP-2on hMSCs growth was in a dose-dependent manner. At low concentrations (0.1-1.0ng/mL), BMP-2showed a nonsignificant stimulatory or inhibitory effect on cells, whereas at higher concentrations (10-200ng/mL) it inhibited the proliferation in a dose-dependent manner. Specifically,20ng/mL of BMP-2inhibited the proliferation by-6%compared with control, and100ng/mL of BMP-2inhibited theproliferation by-15%. Under identical experimental conditions, when BMP-2was added in conjunction with FGF-2(20ng/mL, fixed), a substantial inhibitory effect of BMP-2on FGF-2-induced proliferation was detected.Conclusion:Different growth factor has different effect on proliferation of hMSCs by adding them on different ways. The proliferation of hMSCs was inhibited by adding the combinations of FGF-2and BMP-2. A substantial inhibitory effect of BMP-2on FGF-2-induced proliferation was detected.Experiment2The effects of BMP-2and FGF-2on differentiation of bone mesenchymal stem cellsObjective:To evaluate the effects of BMP-2and FGF-2on differentiatin of hBMSCs by using them alone, associatedly.Methods:The ALP activity of cells after different treatments was determined by biochemical colorimetric assays with Sigma ALP assay kit (104-LS),and the calcium accumulation was determined by spectrophotomety with Sigma calcium assay kit587-A. Results Satistical analysis showed time-course ssay of ALP activity of cells treated by different agents through a quantitative biochemical colorimetric method displayed the effects of GFs on ALP activity. ALP activity levels in FGF-2-treated cell cultures did not change significantly over a period of4weeks. BMP-2treatment resulted in a significant increase in ALP activity even higher lever at week4, comparable to the level induced by Dex. Particularly, about4-fold decrease in ALP activity was noticed in FGF-2plus BMP-2-treated cultures compared with BMP-2alonetreated cultures at week4.Conclusion:Different growth factor has different effect on differentiation of hMSCs by adding them on different ways. The combination effect of FGF-2and BMP-2will inhibite the BMP-2-induced differentiation of hMSCs. Chapter2Biological effects of Dual GFs core/shell controlledrelease microspheres on cultured low density human bone mesenchymal stem cells Experimentl:Fabrication and property evaluation of the Dual GFs core/shell controlled release microspheresObjective:To fabricate the Blank uniform microspheres、Blank core-shell microspheres、Dual GFS evenly distributed in uniform microspheres, FGF-2evenly distributed in uniform microspheres, BMP-2evenly distributed in uniform microspheres, Dual GFs evenly distributed in core and shell of microspheres, FGF-2solely distributed in core of microspheres; BMP-2solely distributed in shell of microspheresand BMP-2solely distributed in core of microspheres; FGF-2solely distributed in shell of microspheres, otally8group microspheres, and to evaluate the propery of the different group of microspheres.Methods:Using CHEDA method to fabricate the different group microspheres by altering the sequence and type of emusion which through the co-exial needls and the type of the co-exial needl. Many other methods were used to evaluate morphologic property, encapsulation rate, GFs release property.Results:It was found that the microspheres are smooth and uniform, and the existence of core/shell structure within the microspheres fabricated from thecore phase/shell phase flow rate ratio of1.5mL/h/2.0mL/h was confirmed by a confocal microscope. The core/shell microspheres can significant enhance the EE of GFs. Due to the uniform structure and even distributionof two GFs in the microspheres, the release of dual GFs from group U was parallel and the space for modifying release profiles is limited. Core-shell double-wall structured microspheres enabled the release of two GFs in different patterns to achieve the control released of different GFS goal.Conclusion:The CHEDA method could fabricate fairly good core/shell double-wall microspheres, and could the control released of different GFs goal. Experiment2Effects of the different microspheres on osteogenic induction of cultured low-population density human bone mesenchymal stem cellsObjective:To compare the effects of different microspheres on osteogenic induction of cultured low-density human bone mesenchymal stem cells to determin the optimize release pattern of BMP-2and FGF-2.Method:A total of1.0×105hMSCs were inoculated in the well of6-well plates with3mL of DMEM containing0.1%FBS at day0. The cells were cultured for12h to allow attachment before the medium was substituted by3mL of DMEM containing0.1%FBS and respective microspheres(microspheres were sterilized using ethylene oxide, and loaded in Spectro/Por membrane bag then inserted inrespective wells. ALP activity and calcium concentration in each well were separately determined after4weeks of incubation.Result:Control, Blank groups1and2, and Group F cultures showed low levels of ALP activity after4weeks of incubation. Group B and S2cultures showed high levels of ALP activity among all the group. Treatment by group S2samples resulted in a sixfold increase in ALP activity compared with groups U and P samples, although the total amounts of FGF-2and BMP-2utilized were exactly the same in the fabrication. Little calcium accumulation was observed in control, blank groups1and2, but calcium concentrations increased in the cultures treated by groups U, F, B, P, S1, and S2compared with control, although to varied contents. Group-S2-treated cultures displayed a significantly higher calcium concentration than the cell cultures treated by group B samples.Conclusion:The osteogenic induction results demonstrated that the release sequence of FGF-2and BMP-2was critical.Chapter3Investigation of FGF-2and BMP-2loaded microspheres in repairing tibia bone defect in a SD rat modelObjective:To investigation of FGF-2and BMP-2loaded microspheres in repairing tibia bone defect in a SD rat model.Method:5-mm bone fragment was cut out from rat tibia, and frozen in liquid nitrogen for30min, then put back along with different microspheres. The regenerative impact of different microspheres on fractured bone was investigated by X-ray observation, HE staining.Results:X-ray observation, HE staining collectively suggested that group S2samples, which released FGF-2and BMP-2sequentially, bridged the bone defects and remodeled the bone grafts the most efficiently.Conclusion:Distribution of dual growth factors in core-shell microspheres and their release can be precisely controlled. This flexibility provided the possibility of mimicking the microenvironment for cell proliferation and differentiations. The microspheres releasing FGF-2and BMP-2sequentially regenerated the large bone defects the most efficiently.