Human Bone Marrow Stem Cell Characteristics And Electro-acupuncture To Induce Their Differentiation To Osteoblasts Experimental Study

1 ObjectiveThis study focuses on tissue engineering of the actual clinical need.To explore and compare of density gradient centrifugation using percoll and the whole bone marrow method as solutes respectively to separate human mesenchymal stem cells(MSCs)from bone marrow.To determinate and analysis the biological characteristics of MSCs from second to eleventh of generation.We hope to provide a theoretical basis and technical foundation.To explore and compare through electric acupuncture induction of bone marrow-derived mesenchymal stem cells to transfer to osteoblasts and the traditional method of chemical-induced differentiation of bone mesenchymal stem cells to transfer to osteoblasts.Through determinate two methods of induced osteoblast biological characteristics prove the electric acupuncture method has been applied to human bone mesenchymal stem cells induced osteoblasts and bone tissue engineering.2 MethodsMSCs were isolated from bone marrow of voluntary normal adults by methods mentioned above respectively.To determinate of the generation of human bone marrow mesenchymal stem cell biological characteristics.The third of generation of human bone mesenchymal stem cells were randomly divided into three groups,divided into general media group,chemically induced and electric acupuncture -induced group.To determinate and analysis the biological characteristics of cells afer 28 days.Separation from the two cultivate method on the cell morphology and growth rate were compared.Observation of the generation of MSCs morphological changes,MTT determined cell activity,fluorescence activated cell sorotor division period and the proportion of apoptotic cells.Division induced by human bone marrow mesenchymal stem cells to osteoblast differentiation,observing changes in their cell morphology,histochemical staining,respectively,induced by 14d and 28d in the determination of cells alkaline phosphatase levels, induced by 28d using RT-PCR determination of cells osteocalcin mRNA, induced by 28d using western-blot method cells osteocalcin protein content.3 ResultsExperimental results show that MSCs of the whole bone marrow method proliferate faster than percoll method.And the whole bone marrow method passage of time early 2-3d than percoll method.Through second to eleventh of generation MSCs by MTT colorimetric test and fluorescence activated cell sorotor showed that:MTT values gradually increased,when the cell proliferate to ninth MTT values reach the highest level.When the cell proliferate to tenth MTT values started decline.At the same time,fluorescence activated cell sorotor analysis showed that although cell proliferation to the eleventh generation,the number of split-phase cells is still an upward trend, but beginning from the sixth generation of the number of apoptotic cells increased significantly,eighth after the quantity of apoptotic cells reached more than 60%,and from the morphological observation found in the ninth after the cell growth slowed,and a cytoplasmic vacuolization,cell bodies become larger and so on basically the same as the aging phenomenon.Osteogenic induction experiments was found from the morphology induced by the initial stage of electric acupuncture-induced cell morphological changes induced by slower than the chemical group.The chemical-induced group gathered monolayer cells after 5 days to 7 days which cell connected and showed triangular in shape,polygonal or scale-shaped.Also the cytoplasm contained more matrix components and granular material,and overlapping growth and did not happen between cell contact inhibition phenomenon,but at this time induced by electric acupuncture-induced cell group still spindle-shaped cells.After 9 days to 10 days electric acupuncture stimulation group showed same phenomenon of the chemical-induced group.However,the morphological changes induced by the latter no significant difference between the two groups,20 days to 24 days are formed nodular cells, collagen accumulation and calcium deposition,the formation of the mineralized nodule opacity.After 28 days we observed thatchemically induced and electric acupuncture stimulation group are positive by Yin Su Hong staining while the ordinary cultivate cells is negative. Determination of alkaline phosphatase found in MSCs in vitro for induction electric acupuncture stimulation group and chemical-induced alkaline phosphatase content increase by time,and this corresponds to cultivate group of ordinary alkaline phosphatase activity gradually over time reduce the general group and cultivate electric stimulation group and the group of chemical-induced alkaline phosphatase levels in 14 days and 28 days the difference has statistical significance(t=57.857,112.505,47.098,108.808,P＜0.05); at the same time comparison of chemical groups and electric acupuncture stimulation of alkaline phosphatase levels in 14 days the difference has statistical significance,but 28 days there was no significant difference(t=15.807,1.357,14 days P＜0.05,28 days P=0.212＞0.05).After 28 days,using RT-PCR determination of cells osteocalcin mRNA,we found that there was no significant difference between chemical groups and electric acupuncture stimulation,but compare ordinary cultivate group there was singnificant difference. After 28 days,using western-blot determination of cells osteocalcin protein content,we found that there was no significant difference between chemical groups and electric acupuncture stimulation,but compare ordinary cultivate group there was singnificant difference.4 ConclusionsWhole bone marrow method isolated MSCs have a shorter incubation time,cell nember more than percoll method though the purity is relatively low,it is also a good method for isolation of MSCs.In addition,the whole bone marrow method is simple and less polluted,and just a small amount of bone marrow MSCs can obtain a lot of cells, reducing the work load of clinical samples of bone marrow puncture and patients'injury,it can be used to comfort clinical needs.It is recommended the use of whole bone marrow method cultivate,when we cultivate MSCs to meet the substantial clinical transplantation necessarily rather than to obtain high purity of MSCs.Generations of biological characteristics of MSCs analysis,recommend the use of the third to the sixth generation of the MSCs for clinical theray.To compare chemical-induced methods,electric acupuncture stimulation can be successfully induced MSCs transfer to osteoblasts, even though stage of the early induction of electric acupuncture induced slower than chemical-induced methods.But when induced after 28 days induced method,there is not significant difference between the two groups cells in morphology,histochemistry and the cell components.Therefore,we can conclude that electric acupuncture stimulation method can be successfully induced MSCs into osteoblasts.It is the physical effect so it has smaller potential toxicity than chemical-induced methods.And it is a new MSCs osteogenic induction method used in bone tissue engineering.