Preliminary Studies On Muscle Engineering By Using Myogenic Differentiated Bone Marrow Derived Mesenchymal Stem Cells And Alginate

OBJECTIVESSUI is a kind of disease which commonly occurs among adult females.The symptoms increase with age,seriously affecting the physical and mental health of older women.We intend to use bone marrow derived mesenchymal stem cells(BMSCs) which is induced by myogenic differentiation and alginate gel for transurethral injection in the mouth,on the one hand to increase the urethral resistance,on another hand,the use of myogenic differentiation induced by bone marrow-derived mesenchymal stem cells to replace the relevant parts of reverse and degenerative changes in cells to repair pelvic floor muscles,fascia and intrinsic urethral sphincter the physiological functions.This experiment,acting as part of the research,focuses on the isolation and cultivation of the BMSCs.We hypothesized that BMSCs isolated from rat are induced into myogenic cells and compounded with Alginate gel to enhance the stability of Alginate gel.The way the diversification of the BMSCs and the myogenic cells in vivo is ensured.MATERIALS and METHODSPartⅠ,studies on the isolation of rat bone marrow derived mesenchymal stem cells(BMSCs) BMSCs were isolated from newborn rats,which were 1 week old,by using percoll and anchoring methods,and analyzing the surface antigens with the help of Flow Cytometer.Acridine Orange(AO) was used as a differentiating material preparing for the following step.PartⅡ,studies on the differentiation of rat bone marrow derived mesenchymal stem cells into myogenic cells The BMSCs were inducted with 10 mol/L 5-azacytidine for 24 hours.After 3 weeks,the expression of desminandα-SMA in the cells was studied with immunohistochemistry.PartⅢ,studies on the preparation of Alginate gel Mixing 2%sodium alginate with different chroma calcium chloride for volume at the rate of 1:1,5:1,10:1,analyzing the character of the Alginate gel at 4℃,room temperature and 37℃.PartⅣ,studies on the Cells compounded with Alginate gel for tissue engineering Mixing 2%sodium alginate with 1%chroma calcium chloride for volume at the rate of 5:1 made up the injectable material. Alginate gel was used as the frame of BMSCs and myogenic cells were injected into the rat(weight 200～250g) After 4 weeks and 8 weeks, the gel was analyzed according to the change of the volume of the mass under the skin and the expression of desmin andα-SMA in the cells with immunohistochemistry.RESULTSPartⅠ,studies on the isolation of rat bone marrow derived mesenchymal stem cells(BMSCs) BMSCs could be harvested effectively and safely by using percoll and anchoring methods from rats' bones.After 2 days, BMSCs anchored and grew like spindle,and the cells were round or ellipse.After 5～10 days,BMSCs' inosculation rate reached 90%, and the form changed into as star shape,and the rate of the growing slowed down.0.25%Trypsinprotease plus 2g/L EDTA were used to assimilate the BMSCs to impress at the rate of 1:3.We could get 3～5×10~6 cells per 10cm culture medium.After being passed for 2～3 times, BMSCs were almost pure cells.The expression of CD29 and CD105 were 89.4%and 46.0%,CD34 and CD45 were negative.Acridine Orange(AO) could make the BMSCs express green fluorescence.PartⅡ,studies on the differentiation of rat bone marrow derived mesenchymal stem cells into myogenic cells After 24 hours of induction directly with 10μmol/L 5-azacytidine and being cultivated for 3 weeks,the ceils grew into coarctation.The inducted cells were polygonal,we could see fascicle-liking shape in the cells.BMSCs,cells inducted 1 weeks and cells inducted 3 weeks were analyzed by immunohistochemistry.There were no differences among them in H&E.The desmin andα-SMA in the cells with immunohistochemistry were no color. Cells inducted for 1 weeks were cambridge blue as in desmin andα-SMA,in the same case,was brown.Cells inducted 3 weeks looked strong blue and brown.PartⅢ,studies on the preparation of Alginate gel The 2%sodium alginate was snuff color ropy.Alginate gel was no color transparent or white. The gel which was formed by mixing the volume at the rate of 1:1 with calcium chloride was stable and hard.And the gel formed by adding calcium chloride at the rate of 5:1 was a little flowing and uniform. After 24 hours,the 1%chroma calcium chloride group was suitable for the injectable material.After the calcium chloride was added at the rate of 10:1,there were more sodium alginate,and the gel could not be formed immediately.The gel became frangible and its volume was smaller than the one before.The temperature ranging from 4 to 37℃did not affect the form of the gel.After drying,the gel's volume reduced into 5～10%of the one before.The observation with the scan electron microscope showed that the higher the chroma of the calcium was,the less holes on the gel's surface there are.PartⅣ,studies on the Ceils compounded with Alginate gel for tissue engineering The scan electron microscope found the cells spread uniformly in the small interstices of the Alginate gel.The volume of the gel mass reduced 50%in 1 month,and 80%in 2 month.After 4 weeks there was envelope formed outside the gel mass.Analyzing them in the way of immunohistochemistry,that there was inflammatory reaction around the mass,and new blood vessel grew into the mass. BMSCs and myogenic cells survived,but the color of the immunohistochemistry is so feebleness,so the question,is there new born muscle come from the cells which we put in,still need us to do more research. CONCLUSIONSBMSCs could be harvested effectively and safely by using percoll and anchoring methods from rats' bones.The BMSCs expanded rapidly in the culture concentration.After 3 weeks of induction with 5-azacytidine,BMSCs can express characteristic desmin andα-SMA.But the consistency and the affecting time must be limited.Sodiumalginate,mixed with calcium chloride of different consistency,produce the different gel.It was the higher the consistency,he harder the gel.The consistency needs to be controlled to some exten,which we need is injectable alginate gel.The BMSCs and the myogenic cells compounded with alginate gel can grow up in vivo.But there were no obvious evidence to say that the cells we put in grew into muscle.