Study On The Mechanotransduction Through BMPs Signal Pathway During Chondrogenic Differentiation Process Of BMSCs In BMSCs/PRF Construct Under Hydrolic Pressure

Cartilage defect is a common clinical disease. Cartilage damage has always beendifficult to restore since the proliferation and regeneration capability of chondrocytes islimited. Now, tissue engineering technique is expected to solve this problem. Throughchoosing the appropriate seed cells, growth factors, scaffolds, and suitable environment,we already can regenerate cartilage tissue in vitro. Bone marrow mesenchymal stem cells(BMSCs) have multipotential capability and can differentiate into cartilage in certaincircumstances. Therefore, BMSCs are regarded as ideal seed cells for cartilage repair andregeneration. Platelet-rich fibrin is rich in platelets, leucocytes and cytokines that canenhance cellular enmeshment and migration. Our previous experiment successfullyestablished the construct of BMSCs/PRF and demonstrated that it can do a great job in cartilage regeneration and repair in appropriate environment in vitro and in vivo.Furthermore, since mechanical stimulation plays an important role in the growth,development and the maintaining of structure and function of the articular cartilage in vivo,lack of effective biomechanical stimulation will result in irregular cartilage tissue that hasfewer matrix content and weaker collagen fibers in vitro, and in vivo, cartilage mechanicalproperties will not successfully repair the articular cartilage defect. Then, in our previousexperiments, we detected the mRNA expression level of PCNA, Sox-9, Aggrecan, Col-Ⅱ and have selected the appropriate mechanical stimulation loading mode(120KPa,1h/d,continuous4d hydraulic loading). When the mechanical stimulus is loaded on theBMSCs/PRF construct, it could promote BMSCs chondrogenesis significantly in vitro.Through restoring articular defects with BMSCs/PRF construct implantation in nude miceand rabbits, we demonstrated that the mechanical stimulation preconditioning couldpromote the chondrogenesis in vivo significantly. Bone morphogenetic protein (BMPs) isa kind of acidic glycoprotein isolated from bone matrix, which could induce boneformation and chondrogenesis. Did the BMPs signal pathway involve in the transductionof mechanical signals during the chondrogenesis? In this experiment, we established therabbit’s BMSCs/PRF construction and simulated it with appropriate loading mode toinvestigate the role of BMPs pathway in chondrogenic mechanotransduction ofBMSCs/PRF construction under the mechanical pressure from the molecular machineperspective. This paper has three sections.Part one: BMSCs were separatedly isolated by density gradient centrifugation andadherent culture. The morphology of the cells of passage3was observed under invertedphase contrast microscope. Cell phenotype was detected by flow cytometry. Throughidentification, the BMSCs have the ability of osteogenic differentiation and adipogenicdifferentiation, confirming that the experimental rabbit BMSCs has the characteristics ofseed cells of tissue engineering.The P3generation BMSCs can be successfully inducedinto cell sheet structure. We phlebotomized blood10ml from autologous rabbit. The PRF clot was obtained by centrifugation with3000rpm for10min and been pressed betweentwo layers of sterile dry gauze to form a membrane structure. According to our previouspatent of invention, which is a method of preparing a composite with stem cell membranefragments and platelet-rich fibrin membrane, we successfully prepared the BMSCs/PRFconstruct.Part two: Using the controllable cellular hydraulic pressure loading system, we gavethe BMSCs and BMSCs/PRF120KPa pressure for4continous days,1h/d. At the sametime, the BMSCs and BMSCs/PRF were cultivated in α-MEM with no pressurestimulation. Expression of mRNA and protein of BMPs signaling pathway relatedmolecules was examined by Real-time PCR and Western Blot. Results: For the BMSCsand BMSCs/PRF structure, the mRNA and protein expression of both BMP2and BMPssignal pathway related molecules up-regulated at the biomechanical stimulus conditions of120KPa pressure for4continous days,1h/d. However, for the BMSCs/PRF structurecultured inα-MEM without pressure stimulation, the mRNA and protein expression ofBMPs signaling pathway related molecules didn’t changed during the4days.Part three: In order to explore when BMPs signaling related molecules have themost expression, we give the BMSCs and BMSCs/PRF120KPa pressure for120min. Atthe same time, the BMSCs and BMSCs/PRF were cultivated in α-MEM with no pressurestimulation for120min. At0,30,60,90,120min, we detected the expression of mRNAand protein of BMPs signaling related molecules by Real-time PCR and Western Blot.Results: For the BMSCs and BMSCs/PRF, the expression of mRNA and protein of BMP2and BMPs signal pathway related molecules up-regulated the most at60min during the120min continuous pressure loading process. Nevertheless, for the BMSCs/PRF conductwith conventional culturing inα-MEM without pressure stimulation, the expression ofBMPs signal pathway related molecules didn’t changed during the120min continuousculturing process. By and large, the results proved that the pressure we screened out inprevious experiment was suitable. We also proved that the BMPs signaling pathway was one of the important molecular mechanisms in the activation of BMSCs/PRF constructunder pressure regulation.Through Western Blot technology, the BMPs signaling pathway in response topressure regulation in rabbit BMSCs/PRF construct were studied. Results: after loading120KPa pressure for60min on the BMSCs/PRF construct, the expressions of Col-ⅡandAggrecan protein significantly increased. However, exogenous BMPs inhibitor (Noggin)significantly reduced the expression of Col-II and Aggrecan. The results indicate thatBMPs signaling pathway played an important role in the chondrogenic differentiation ofBMSCs/PRF construct under appropriate pressure conditions.Conclusion: The pressure condition (120KPa,1h/d, hydraulic loading for4continuous days) we have screened out could produce the best effect on chondrogenicdifferentiation of BMSCs/PRF construct in our preliminary experiment. Now in the samecondition, we demonstrated that the expression of BMP2, BMPs receptor(BMPRⅠB、BMPR2), Smad1and Smad5increased in the BMSCs/PRF construct while the expressionof BMPs signaling pathway related molecules had no significant changes in BMSCs/PRFconstruct with conventionally culture. This demonstrated that BMPs signaling pathwaywas involved in the BMSCs/PRF chondrogenic differentiation under appropriate pressureconditions. In further experiments, we found that the expression of BMPs signalingpathway related molecules up-regulated the most at60min during the120min continuouspressure loading process. It proved that the pressure we have screened out was suitable.Besides, Noggin can inhibit the activation of Smad1,5or the expression of Col-II andAggrecan. Therefore, this experiment demonstrated that BMPs signaling pathwayinvolved and played an important role in the BMSCs/PRF construct chondrogenicdifferentiation under an appropriate pressure conditions.