Construction Of Vascular Bioreactor And In Vitro Biosafety Ealuation Of Degradable PLLA And PLCL Stents

The mortality rate of cardiovascular diseases is increasing year by year,which promotes the development of stents.In recent years,the research on degradable polymer stents is very hot.The material of degradable stent affects the choice of coating drugs,so the evaluation of vascular stent materials is of great significance.For the biosafety evaluation of vascular stents,the methods previously used are in vitro cell experiment and in vivo animal experiment.With the advancement of the research,the evaluation method of in vitro organ culture has been proposed.Objective: In this paper,an in vitro stress culture bioreactor for blood vessels was constructed to simulate the vascular mechanical environment in vivo from the aspects of heart rate,vascular inner wall shear force,vasoconstriction and diastolic pressure,liquid laminar flow,etc.,making it a kind of experimental means to evaluate the biosafety of vascular stents in vitro.Vessels implanted with L-polylactic acid(PLLA)stent and Polycaprolactone(PLCL)stent were cultured in that in vitro reactor.Based on the culture results,these two stents were evaluated and some references were provided for the optimization of degradable stent materials.Methods:(1)Construction of the in vitro stress culture bioreactor for blood vessels: the mechanical environment of blood vessels in vivo was simulated from the aspects of heart rate,vascular wall shear force,vasoconstriction and diastolic pressure,laminar flow of fluid,etc.The overall morphological changes of blood vessels were observed by H&E staining.Masson and EVG staining were used to observe the collagen and elastic fibers of blood vessels,and to evaluate the activity of blood vessels.The permeability and integrity of the intima were observed by Evans blue staining to evaluate the success and effectiveness of the constructed bioreactor in vitro culture.(2)In vitro culture after vascular implantation of PLLA and PLCL stents: no stent and static culture were set as controls.After 3,7,10 and 14 days of vascular culture,H&E staining was performed on the vessels to observe the overall morphological changes of the vessels.Masson and EVG staining were used to observe the collagen and elastic fibers of blood vessels to evaluate the status of blood vessels.CD31 immunohistochemical staining was used to observe the growth of vascular endothelial cells.Phenotypic changes of vascular smooth muscle cells were observed by α-SMA and OPN immunohistochemical staining to determine the hyperplasia of vascular smooth muscle cells.The i NOS immunohistochemical staining was used to evaluate the inflammatory response of blood vessels.Results:(1)The influence of stress culture on blood vessels in vitro was analyzed from the overall morphology of blood vessels,elastic fibers and collagen fibers.The results showed that vascular endothelial cells were damaged under high stress,which resulted in smooth muscle cell proliferation.The overall change of elastic fiber was little,but the loss of collagen fiber was more serious.(2)The results of Evans blue staining showed that the permeability and integrity of the intima under stress culture were well restored.(3)H&E,Masson,and EVG staining showed that after stent implantation,the vascular wall was squeezed by the stent and gradually fitted to the shape of the stent,and the elastic fibers were stretched.After dynamic culture,vascular elastic fibers and collagen fibers degraded to different degrees.(4)The results of CD31 immunohistochemical staining showed that the two groups had similar changes in the stent.After 3 days of culture,there was almost no positive expression in the contact site between the vessels and the stent.After continuous culture,the intima gradually repaired itself and the positive expression increased,but the average optical density of PLCL group was higher than that of PLLA group.(5)i NOS immunohistochemical staining results showed that the positive expression of vascular inflammatory factors after dynamic culture of the two scaffolds was firstly decreased and then increased,mainly expressed in the smooth muscle cells of the middle membrane.The average optical density of PLCL group was higher than that of PLLA group.(6)The results of α-SMA immunohistochemical staining showed that the positive expression level of PLLA stent group increased gradually,while the positive expression level of PLCL stent group decreased first and then increased gradually.(7)The results of OPN immunohistochemical staining showed that the expression level of PLLA stent group was increased at the beginning and then became stable,while that of PLCL group was decreased at first and then increased.Conclusion:(1)This paper successfully established a new in vitro stress culture bioreactor for blood vessels,which simulated the vascular mechanical environment in vivo from the aspects of heart rate,vascular wall shear force,vasoconstriction and diastolic pressure,laminar flow of liquid,etc.,providing a new method for the evaluation of the biosafety of vascular stents in vitro.(2)PLLA and PLCL stents were evaluated in terms of biosafety by using vascular stress culture bioreactor in vitro.The results of H&E,Masson,and EVG staining showed that the implantation of different stents would change the vascular morphology,and the radial support force of the two stents had similar effects on elastic fibers and collagen fibers.The results of CD31,i NOS,α-SMA and OPN immunohistochemical staining showed that PLCL had lower damage and inhibition on endothelial growth than PLLA.The vascular inflammatory response caused by PLCL stent implantation was more intense.PLCL could inhibit the proliferation of smooth muscle cells.