| Part OneConstruction of abnormal mechanical stimulation induced calcificationmodel by bicuspid tissue engineering heart valveObjective: By constructing tricuspid and bicuspid heart valve models by tissue engineeringtechniques, and applying fluid mechanical stimulations by assembled bioreactor system, anabnormal mechanical stimulation induced valve calcification model was set up. Themodeling efficiency was evaluated and the optimum modeling conditions were determined.Methods: Decellularized porcine aortic valve leaflets were modified with4armspolyethylene glycol (PEG) and bonded covalently with RGD peptide, to manufacture atissue-engineering heart valve. Homo valvular interstitial cells (huVICs) were isolated andidentified from normal aortic valve specimens and seeded on tissue engineering leaflets.Then the leaflets were sewed on a custom titanium-nickel alloy stent to obtain tricuspid andbicuspid heart valve models. BWET artificial heart valves detector was adopted to test thehemodynamic performance of these valve models. Application by pulsating flow metermeasuring sewing tricuspid and bicuspid valve fluid mechanics of the model. A bioreactorsystem was assembled with WT600-1F rolling peristaltic pump and custom reactionchamber. The valve models were separated into4groups: A, tricuspid valve under no flow;B, tricuspid valve under physiologic flow; C, bicuspid valve under no flow; D, bicuspidvalve under physiologic flow. All tissue engineering valves were removed and tested after14days of intervention (scanning electron microscopy detection, ALP contentdetermination, Runx2/Cbfα1immunohistochemical staining, and von Kossa staining)Results: tricuspid tissue engineering valve can open thoroughly and close tightly. However, bicuspid valve’s opening is significantly restricted. In vitro hemodynamic performance testshows that, under the same heart rate and cardiac output, these two models reveal samecharacteristics in mean arterial pressure and regurgitation percentage (P>0.10), whileeffective orifice area of bicuspid valve is much smaller than tricuspid group (P <0.001),and its pressure gradient increased significantly (P <0.001). After14days of mechanicalstimulation by bioreactor, the scanning electron microscopy (sem) test found that, withoutflow stimulation, tissue engineering leaflets surface was covered by monolayer, tightarranged, non-polarized huVICs in group A and C. Under the stimulus of physiologicalflow, leaflets of group B were also covered by monolayer huVICs, however, the cellarranged orderly in parallel to the direction of the flow. In group D, the cell morphologychanged obviously and lost its polarity. ALP content of leaflets in A, B, C groups remainedin low level, and there was no significant difference between them (P>0.05), while ALPcontent in group D increased significantly (P <0.01). Immunohistochemical stainingshowed that monolayer cells attached on the surface of leaflets in all groups, while positivestaining of Runx2only existed in cytoplasm of huVICs in group D. The von Kossa stainingalso confirmed that calcium salt deposition only presented in valve leaflets of group D, butits distribution is rather limited.Conclusion: An abnormal mechanical stimulation induced valve calcification model can besuccessfully constructed by applying bicuspid tissue engineering techniques and bioreactorsystem. Part TwoMechanism research of TGF-β1/BMP-2pathways in abnormal mechanics induced tissue engineering valvular calcificationObjective: To explore the effect of TGF-β1/BMP-2pathway in abnormal mechanicalstimulation induced valvular calcification with the help of bicuspid tissue engineeringleaflets model.Methods: According to the type of working liquid and flow of fluid produced by bioreactor,bicuspid tissue engineering valve models were randomly assigned into3groups: group Afilled with complete culture medium and exerted no flow stimulus; group B filled withcomplete medium and stimulated with physiological flow; group C filled with completemedium containing10ng/ml of reconstructed TGF-β1), and exerted physiological flowstimulus. At the time-points of3d,7d and14d, leaflet samples from each group wereharvested and their calcification degree was analyzed (western blot for Runx2and ALPcontent test). Then RT-PCR and western blot methods were adopted to measure the mRNAand protein expression levels of signal molecules in TGF-β1/BMP-2pathway.Results: Abnormal mechanical stimulus lead to an increase in both ALP content andRunx2protein expression in group B compared to group A (P <0.01). With the addition ofexogenous TGF-β1, the calcification extent of group C is even severer than group B (P <0.05). The results of RT-PCR and western blot test showed that mRNA and protein ofTGF-β1and BMP-2in leaflets of group A were at a low level. Flow dynamic stimulusresulted in an increase in both gene transcription and translation levels of TGF-β1andBMP-2in the bicuspid valves leaflets of group B (P <0.01). In group C, the addition ofexogenous TGF-β1can aggravate the degree of valvular calcification as well asup-regulated transcription and translation of BMP-2significantly (P <0.01).Conclusion: Abnormal mechanical stimulation can cause calcification in tissue engineeringvalve leaflets as well as a raise of transcription and translation levels of TGF-β1andBMP-2. Exogenous TGF-β1has a synergistic effect on both valvular calcification andBMP-2expression. By these findings, it is clear that TGF-β1/BMP-2signaling pathway mediates valvular calcification process induced by abnormal mechanical stimulation. Part ThreeThe effect of BMP-2RNA interference on abnormal mechanicalstimulation induced valvular calcificationObjective: To inhibit the BMP-2expression in huVICs through RNA interference andobserve its effect on abnormal mechanical stimulation induced valve calcification.Methods: Construct plasmids containing BMP-2shRNA, and transfected huVICs with thisplasmids under the mediation of LipofectamineTM2000. Transfection efficiency wasevaluated. The BMP-2silencing huVICs was applied to manufacture tissue engineeringvalve leaflets. According to types of seeded cells and hemodynamic circumstances, sixgroups were separated and intervention respectively. group A: normal huVICs under staticenvironment; group B: normal huVICs under physiological flow; group C: BMP-2silencing huVICs under static environment; Group D: BMP-2silencing huVICs underphysiological flow; group E: blank plasmid transfectional huVICs under static environment;F group: blank plasmid transfection huVICs under physiological flow. At3d,7d and14d,leaflet samples were took from each group to have calcification evaluation (western blot ofRunx2and ALP content test), and detection for mRNA and protein expression level ofmolecular in BMP-2related pathways (BMP-2, Smad1, and Msx2).Results: BMP-2shRNA plasmids can be transfected effectively into huVICs andsignificantly inhibit the BMP-2expression. After the gene intervene, abnormalhemodynamic can still cause an increase of ALP content and Runx2expression (group D, P>0.01), however, the level of these two calcification index decreased remarkably whencompared with group B and F (P <0.001). It was confirmed by RT-PCR and western blotthat abnormal mechanical stimulus can lead to significantly raise of mRNA and proteinlevels of BMP-2, Smad1and Msx2in group B, F (compared with group A, C, E, P<0.01).And BMP-2gene silencing can suppress the elevation effect on transcription andtranslation of BMP-2and Smad1that caused by abnormal fluid dynamics (compared withgroup A, C, E, P>0.05, compared with group B, F, P<0.01).Conclusion: RNA interference can effectively suppress the expression of BMP-2,down-regulate the mRNA and protein expression level of Smad1, and inhibit valvularcalcification induced by abnormal mechanical stimulation. But the expression of Msx2cannot be depressed by BMP-2gene silencing. All these findings suggest that canonicalBMP-2/Smad1pathway plays an important role in abnormal mechanical stimulationinduced valvular calcification. And noncanonical BMP-2/Msx2pathway is not involved inabnormal mechanical stimulation induced calcification progress. |
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