Study On The Mechanotransduction Through P38MAPK/NF-κB Signal Pathway During Chondrogenic Differentiation Process Of BMSCs In BMSCs/PRF Construct Under Hydrolic Pressure

With the increase of the incidence of articular cartilage injury, how to repair theinjured articular cartilage has been a difficult clinical problem. In recent years, tissueengineering cartilage brings new hope to repair cartilage defects. Seed cell, scaffold,growth factor and bioreactor are four factors in tissue engineering cartilage. However, howto integrate them and how to apply the integrated unit in clinical work are still unsolved.As BMSCs (bone marrow mesenchymal stem cells) can be obtained widely and hasthe potential of chondrogenesis in mechanical environment, it can be used as seed cell torepair the articular cartilage. Also, the transplanted BMSC could be maintained well aftershaping them with cell sheets technology. As a rich source of scaffold material of platelets and white blood cells, PRF (plaletet-rich fibrin) can not only release some growth factorssuch as TGF-, PDGF-AB, VEGF, etc, but also have the ability to promote tissueregeneration. We composited PRF membrane and BMSCs cell sheets into BMSCs/PRFconstruct as transplanted material according to our patented technology. In addition, wefound the chondrogenic differentiation of the composite under suitable pressure conditions.But it is a pity that the related mechanism is unclear. P38MAPK is distributed in thecytoplasm and having serine and tyrosine protein kinase double phosphorylation ability. Itcan be activated by a variety of stimuli, and participated in cell formation, growth, anddifferentiation and other physiological processes. Studies have shown that p38MAPKinvolves in the process of BMSCs chondrogenic differentiation post mechanicalstimulation, but it is unknown whether p38MAPK has involved in the process of BMSCscell membrane film chondrogenic differentiation post mechanical stimulation. NF-kappaBis one of the important signaling pathway in the nucleus. It could be activated by variousstimuli and involve in lots of cell physiological responses. Nevertherless, it is not clearwhether NF-kappaB is involved in the cartilage formation. Its upstream and downstreamrelationship with P38MAPK in the process of mechanical signal transduction is also indispute. Therefore, from the perspective of molecular biology, we want to evaluate theexpressions of P38MAPK and NF-kappa B signal related molecules when the theBMSCs/PRF double membrane complex in rabbit is stimulated by pressure in thisexperiment. Exploring what roles the P38MAPK and NF-kappaB signaling pathway playsin the chondrogenic differentiation of pressure-regulating rabbit BMSCs/PRF doublemembrane complex and evaluating its signal transduction pathway are also the objectivesof this experiment.There are four parts in this experiment:In the first study, BMSCs were separated and purified from the bone marrowobtained from the femur of New Zerland white rabbits by density gradient centrifugation.After subculture and expansion, we corfirmed that the isolated cells were BMSCsby flow cytometry, osteogenic differentiation and adipogenic differentiation. In the secondand third studies, PRF gel was obtained from the10ml blood extracted from rabbit ears central arterial by3000r/min centrifugal for10min. Then, the PRF gel was wrapped bysterile gauze and made into PRF membrane after extrusion. In accordance with ourprevious established technology (a stem cell patch fragment the composite richplatelet-fibrin film double-membrane complex graft material preparation methods), theautologous BMSCs membrane and the autologous PRF were composited intoBMSCs/PRF double-membrane complex. According to our previous results, the suitablebiomechanical stimulus conditions to promote the cultured BMSCs/PRF doublemembrane complex in vitro (cultured in the cell hydraulic loading device developed byour group) to chondrogenic differentiation is120KPa,1h/d for4consecutive days.Western blot was used to determine the protein expression of P38MAPK and NF-kappaBsignal pathway in BMSCs cell patch and BMSCs/the PRF double membrane complexwith120KPa at different tine points. The experimental results are as follows:(1) P38MAPK and NF-κB pathways were unactived when rabbit’s BMSCs sheetswere induced by PRF in0-120min.(2) The expression of phosphorylated protein levels of p38MAPK increased whenBMSCs sheets and BMSCs/PRF construct were under120KPa for15minutes, while itwas not for the expression of NF-kappaB singal pathway related IκB, p-IκB, p-p65andp65, indicating us that only P38MAPK pathway was activated under120KPa for15minutes. The expression of IκB, p-IκB, p-p65and p65increased when BMSCs sheetsand BMSCs/PRF construct were under120KPa for30minutes, indicating us thatNF-kappaB pathway was activated under120KPa for30minutes. We also found that theexpressions of P38MAPK and NF-kappaB signal pathwas related proteins were highestunder120KPa for60minutes but gradually returned to normal level even for longer time.Based on the previous findings, we chose120KPa for60min as the experimentalcondition, and investigated the relationship between P38MAPK and NF-kappaB singalpathways in BMSCs sheets and BMSCs/PRF construct under mechanical stimulation. Wefound that SB203580which is the inhibitor of P38MAPK could inhibit P38MAPK andNF-kappaB pathways,but PDTC which is the inhibitor of NF-kappaB only inhibitedNF-kappaB pathway and had no effects to P38MAPK. It means that maybe P38MAPK is an upstream signaling molecule of NF-kappaB. Realtime-PCR and Western blot wereused to assess P38MAPK and NF-kappaB signal pathways during chondrogenicdifferentiation process of BMSCs in BMSCs/PRF construct under hydrolic pressure. Itshowed that when BMSCssheets and BMSCs/PRF construct were under pressure of120KPa for60min, the proteinand gene expressions of specifical index of chondrocytes(Sox-9,Aggrecan and Col-II) elevated.When SB203580or PDTCwere added,the protein and gene expressions of Sox-9,Aggrecan and Col-II were decreased.Summary: In the present study, we found that BMSCs sheets and BMSCs/PRFconstruct stimulated by the hydrolic pressure of120KPa for60min showed higher geneand protein expression of P38MAPK and NF-kappaB pathway relative signal molecules,as well as higher chondrogenic markers including Sox-9, Aggrecan and Col-II. WhenP38MAPK or NF-kappaB pathway were blocked, the promotion effects of hydrolicpressure to the chondrogenic differentiation of BMSCs in BMSCs sheets or BMSCs/PRFconstruct also vanished correspondingly. That is to say, P38MAPK and NF-kappaB signalpathways play an important role in promoting BMSCs/PRF construct to the chondrogenicdifferentiation under the given pressure condition (120KPa,60min).