| Purposes:1.This research tried to prepare cell microcarriers with different proportions of PLGA/HA by electrostatic instillation method and find the best preparation conditions.2.Rabbit bone marrow mesenchymal stem cells cultured in the third generation were co-cultured with cell microcarriers to detect the changes in proliferation and osteogenic activity.3.Common growth factors and special binding growth factors were added in the co-culture system of rBMSCs and cell microcarriers to detect the changes in osteogenic activity.Methods:1.Rabbit bone marrow mesenchymal stem cells were isolated and purified using whole bone marrow adherence culture method and its cell growth curve was detected by CCK-8 assay.Osteogenic induction and adipogenesis induction were performed on third-generation rBMSCs to examine their multilineage differentiation potential.2.The microcarriers with suitable shape and size were made by adjusting the voltage,needle diameter and distance conditions.3.The cell microcarriers prepared under the optimal conditions with different PLGA/HA ratios were co-cultured with the third-generation rBMSCs,and the cell proliferation ability was detected by the CCK-8 assay.After osteogenic induction,osteogenic activity was detected by ALP assay and osteogenic related gene expression.4.In the above co-culture system,IGF-1(ordinary growth factor)and DOPAIGF1(special binding growth factor)were added.After osteogenic induction,osteogenic activity was detected by ALP assay and osteogenic related gene expression.Results:1.The rabbit bone marrow mesenchymal stem cells were isolated and cultured to the third generation.Under the observation of the inverted microscope,rBMSCs were spindle-shaped and adherently growing.Its cell growth curve showed a linear upward trend.After the osteogenic induction,calcium nodule deposition was found by alizarin red staining.After adipogenesis induction,lipid droplets were found by oil red O staining.2.By varying preparation conditions,spherical micro-carriers with uniform diameter and size were made.The preparation conditions were as follow: voltage was 3.5 kV,syringe needle type was 27 G,distance between the syringe and the receiving device was 80 mm,and the receiving device solution was 60% ethanol.3.After rBMSCs were co-cultured with cell microcarriers,the cell proliferation activity was significantly increased(*P<0.05).With the increase of HA ratio,cell proliferation activity increased in a positive proportion.ALP assay showed that the osteogenic activity of the composite cell microcarriers with HA was higher than that of PLGA microcarriers alone(*P<0.05).With the increase of HA ratio,the expression of osteogenic related genes increased,but there was no significant difference among the groups(*P>0.05).4.ALP assay showed that the osteogenic activity was enhanced(*P<0.05)and the expression of osteogenic related genes was greater in the co-culture group with DOPA-IGF1 than in the group with cell microcarriers alone and in the co-culture group with IGF-1.But there was no significant difference among the groups(*P>0.05).Conclusions:1.Rabbit bone marrow mesenchymal stem cells were successfully isolated,purified and cultured.And their biological characteristics were confirmed to be in accordance with the characteristics of stem cells.2.Cell microcarriers with uniform spherical shape and diameters were made successfully,and the optimal conditions for preparation were confirmed.3.The co-culture of rBMSCs with cell microcarriers proved that it promoted the proliferation of rBMSCs.With the increasing proportion of HA,the proliferation activity of rBMSCs was enhanced.But some of its effects on osteogenic activity needs to be further studied.4.The osteogenic activity of rBMSCs could be influenced by adding special binding growth factor,but it needs to be further studied. |
