The Role Of Mechanical Stimulation On Bone Marrow Mesenchymal Stem Cells To Chondrocytes

Objectives：Discussion on how different centrifugal forces play an important rolein stimulating Bone marrow mesenchymal stem cells into chondrocytes and giveinfluence on the change in the micro-mass culture conditions. Obtain the centrifugalforce strength which is the most suitable for differentiation of bone marrowmesenchymal stem cells into chondrocytes through the experiment.Methods：Isolate the femurs and tibias from the6-week-old SD rats in sterileconditions to extract primary BMSCs through density gradient centrifugal process andobserve changes of cell morphology through microscope every day. Gain the purifiedBMSCs by repeated changes of medium and passage. Measure the levels of CD29,CD90and CD45using flow cytometry to identify the purity of the cells. The sixthsubculture (P6) of BMSCs is introduced to proceed with three-dimensional culture bymicro-mass culture, during the process of which BMMY (induction medium) mainlycomposed of TGF-β1and ITS needs replacing and cells are divided into four groups onthe basis of different strength of centrifugal forces as follows: unstressed control group(0g group),100g stress group,200g stress group and300g stress group. Identify the cellmass after28days’ culture involving such indicators as morphology and glossiness ofcell mass in visual study, testing of cell activity in Trypan blue staining, testing ofsynthesis of Proteoglycans in toluidine blue staining, and content testing of type IIcollagen using type II collagen immunohistochemistry theory.Results:1. The cells are shown as quasi-circular when primary cells are earlyinoculated. Several cells are attached to the surface and gradually extended to longspindle after48hours. We can see the obvious formation of colonies and cell fusiontogether with other colonies around them after7days. After two weeks, cells are closelyand orderly arranged and the fusion rate is up to90%. The cell morphology of the fifth subculture (P5) has become narrowly spindle, closely and orderly arranged and thecenter of colonies has a vortex structure.2. Testing results of flow cytometry: CD29and CD90expression rate are stronglypositive, separately as97.36％and99.61％while CD45expression rate is negativeshown as0.44%. These show that BMSCs cell is highly purified and consistent withexperimental requirements.3. After28days’ induction, the silvery white cell mass at the bottom of centrifugetube through visual study. It is spherical, slightly shiny and poorly tough. The size andcolor of cell mass are no significant differences in each group.4. After28days’ induction，results of Trypan blue staining: there were noobvious variations on cell activity among0g,100g and200g groups while those in300ggroup are distinct from others, sharply reduced.(p <0.05)5. After28days’ induction,results of toluidine blue staining: there is more visiblepurplish red metachromatic in the extracellular matrix of200g group while100g and300g groups follow and0g group ranks last with the weakest metachromasia.6. After28days’ induction,results of type II collagen immunohistochemistry:200g group showed a strong positive reaction with large amounts of brownish yellowchondrocyte specificity matrix.100g and300g groups had small amounts of lightlybrownish yellow chondrocyte specificity matrix and0g group was the least one.Conclusions：The force of100g is not enough to make the cells translatedfully,while300g is too large to make the cells injured.200g is the most suitablecentrifugal force for the bone marrow mesenchymal stem cells into chondrocytes.