Construction Of Vascularized Tissue Engineered Grafts Based On Cell Sheet Technology

Objectives: In this study,vascularized tissue engineered(TE)graft was constructed by using cell sheet technology(CST).The vascular grafts were implanted into subcutaneous of SD rats to observe the anastomosis between the graft and host vascular with an aim to evaluate the angiogenesis ability in vivo,providing a new-type vascularization strategy for solving the problem of insufficient blood supply faced by organ grafts.Materials and Methods:(1)Culture and passage of adult human mesenchymal stem cells(h MSCs):MTT assay was used to observe the morphology of post-thawed h MSCs and analyze the growth trend of h MSCs;(2)Preparation and characterization of h MSCs sheet: h MSCs were cultured in petri dishes for 14 days to construct h MSCs cell sheet.Properties of h MSCs sheet were investigated by H&E staining and SEM observation;(3)Construction and characterization of vascularized cell sheet and vascularized tissue engineered graft: h MSCs sheet was constructed by continuous culture of h MSCs for 14 days,where the HUVECs were seeded to form vascularized cell sheet.The vascularized tissue engineered grafts were constructed using PTFE pipe as a model and characterized by SEM,immunofluorescence staining and tensile test;(4)In-vivo experiments of vascularized tissue engineered graft: h MSC/HUVEC were subcutaneously implanted into the back of SD rats,and h MSCs sheet was used as control groups.HE staining and immunofluorescence staining were used to evaluate angiogenesis and anastomosis with the host after 2 and 4 weeks,respectively.Results :(1)h MSCs presented a spindle-shaped structure observed by the inverted light microscope.MTT assay showed that the proliferation of h MSCs exhibited a S-shaped curve with prolonging the culture time;(2)The results of HE staining showed that h MSCs sheet secreted a great deal of extracellular matrices with the thickness of about 100-150mm.SEM results showed that h MSCs arranged closely with a large amount of extracellular matrix;(3)CD31 immunofluorescence staining results showed that microvessels did not change remarkably at day 1 and 3,but a large number of microvessels appeared at 7 days.The tensile test results showed that the strain and the stress reached 45% and 1.5MPa,respective;(4)In-vivo experiments of vascularized tissue engineered graft: Grafts were implanted subcutaneously in SD rats for 2 and 4 weeks,the extracted tissues were stained with H&E staining.The results showed that there was a lot of angiogenesis in h MSC/HUVEC grafts,but little angiogenesis was observed in h MSCs grafts without addition of HUVECs;Immunofluorescence analyses showed that h MSC/HUVEC grafts significantly enhanced vascular-like structures formation and interconnected complex capillary networks formation.However,the vascular-like structures were unobvious in control group.Conclusions:The results of in vitro and in vivo experiments showed that vascularized stem cell sheet was fabricated based on cell sheet technology to prepare novel h MSC/HUVEC grafts which are composed of the inner layer of HUVECs and the outer layer of h MSCs sheet,and then successfully implanted into SD rats.This graft is functionally anastomosed with the host and produces a large number of vascular-like structures,providing the necessary stem cells and blood supply for tissue growth.As a new-type vascularization strategy,vascularized TE graft will be widely applied in the field of tissue engineering for tissue construction.