The Induction Of Vascular Endothelial Differentiation Of Encapsulated Mesenchymal Stem Cells In Calcium Alginate Microfibers

The construction of bioartificial blood vessels possesses great value in the fields of tissue engineering,vascular bypass therapy as well as physiology and pathology.Attaching vascular cells to the surfaces of biomaterialsor encapsulating them in the matrix environment and then promoting their growth toward vascular tissue has increasingly becoming a technical strategy for the construction of biological blood vessels.Yet these strategies often face certain challenges when they attempt to buildvascular graftsin the microscale range.Currently the biological materials that used for the formation of blood vessel prosthesiswere mainly the synthetic materials,which possessed superior mechanical properties while poor biocompatibilities.Meanwhile the vascular grafts often led the intimal hyperplasia,thrombosis or even pseudoaneurysm in vivo.Several techniques in present studies have been applied to fabricate the tubular vascular prosthesis to date,such as electrospinning and phase inversion.But no ideal technique exists because most of them required complicated devices with high voltages or temperatures,which would easily cause damage to the generated vascular prosthesis and also made it difficult to load cells directly into them during the fabrication process.Although techniques like cell sheet technology?CST?have created the possibilies on the cultivation of mature vascular cells?like vascular endothelial cells,VECs?,their complete formation of endothelium remained limited for the inabilities to differentiation and the difficulties in obtaining autologous cells.It was definite that the bone marrow mesenchymal stem cells?MSCs?held mulit-differentiation and immunoregulation abilities,and recently their antithrombotic potential have drew attention on resereachers in the field of vascular engineering.Systematic investigations are needed to address on the MSCs serving as the seeding cells in the bioartificial blood vessels construction.In this study we firstly synthetisis alginate hydrogel microfibers with superior mechanical properties as well as superior biocompatibility by crosslinking sodium alginate and the CaCl₂ inthe microfluidic device,on this basis we encapsulated MSCs together with the vascular endothelial growth factor?VEGF?and fibroblast growth factor?FGF?in alginate hydrogel microfibers and then studyed the their proliferation and VECs differentiation abilities.After that we transplantated microfiber-VECs composites into mice abdomens to study the actue toxicity of the implant grafts on cardiovascular system in vivo.The main research contents and results are as follows:?1?A simple perspex microfluidic device with a‘T-shape'cylindrical-flow channel was developed through precision engraving lathe,and tapered capillaries with precise diameters in their tips were prepared by capillary puller,the microchannels of the microfluidic device were connected with the external nuts and were effectively sealed by the silicone rubber,the alginate hydrogel microfibers generation platform was constructed by linking the microfluidic device to the terufusion syringe pump.Alginate hydrogel microfibers with diameters<600?m and 0.3 m-0.5 m long were synthetised by introducing the sodium alginate as the core sample flow and the calcium chloride as the sheath sample flow into microfluidic system to form the cross linking reaction,microfibers had regular shapes with symmetric characteristics,numerous porosa areas were seen on the surfaces of the microfibers,The diameters of the microfibers were linearly related to the core sample flow rates,the suture retention strengths of the microfibers with diameters about 500?m and 300?m were comparable with those of microfibers synthesized by other natural biomaterials.?2?The proliferation and the viability of MSCs in the alginate microfibers was detected by CCK-8 assay,DiO cell staining and the Live/Dead cell staining.The released VEGF concentration from the microfibers were determined with the ELISA Kit;The endothelial differentiation of MSCs was examined by immunofluorescence staining,RT-PCR,Western blot and Flow cytometry;Results showed that the proliferation and the viability of MSCs in the alginate microfibers were similar with those in the traditional two-dimensional culture medium,the cell density of MSCs reached the highest at day 7 and the cumulative releasing of VEGF in growth factor group accounted for more than 85.10%of the total releasing.The growth of MSCs tended to apoptosis beyond one week.Meanwhile the endothelial cell markers CD31,VE-cadherin and vWF were expressed after 10 days when MSCs cultured with VEGF and FGF in alginate microfibers,and the synergy endothelial inducing effect of VEGF and FGF were better than that of single growth factor,suggesting the cell-encapsulating composite microfibers hold the potential application on the construction of artificial blood vessels;?3?microfiber-cells composites were intraperitoneal transplantated into mice abdomens,then the actue toxicity of the implant grafts on cardiovascular system of mice were detected by the histopathological assessment.Results showed that no apparent clinical abnormalities in transplanted mice with alginate microfiber-cells composites,the vascular endothelial cells and myocardial cells of the mice were clear and arranged in good order at 7 and 14 days after the transplantion.Indicating the alginate microfiber-cells composites were non-toxic to the organism and had excellent biocompatibilities with antithrombotic potential in vivo.In conclusion,in this study we synthetisis alginate hydrogel microfibers with superior mechanical properties as well as superior biocompatibility through the microfluidic approaches,theangiogenesis effects of VEGF and FGF could efficiently promote proliferation and VECs differentiation abilities of encapsulated MSCs in the microfibers.Meanwhile the alginate microfiber-cells composites were non-toxic to the organism,indicating microfluidic system was conducive to fabricate the initial form of bioartificial blood vessels.This study provided a novel and feasible method for the synthesis of artificial blood vessels and their subsequent treatment of vascular diseases.