Experimental Study On Tissue Engineered Blood Vessel

Objective To study the method and process of obtaining decellularized vascular scaffolds and evaluate its mechanical characterization and biocompatibility.To evaluate the effects of the decellularized vascular scaffolds coated with anti-CD34 antibodies capturing endothelial progenitor cells from the circulation to participate recellularization.Methods Fresh caprine carotids and brachial arteries were treated with repeated frozen/thawing,cold isostatic pressing(5000 MPa,4℃)and 0.125% sodium dodecyl sulfate(SDS) separately for preparation of decellularized vascular scaffolds.Biological characteristics were compared with the raw caprine carotids using hematoxylin and eosin staining,Masson staining,scanning electron microscopy and mechanical test.Using fluorescence staining and DNA remains test to assess the cell extracting results.Biocompatibility were detected using cell adhesion test,MTT assay,and subcutaneously embedding test.After reacted with photochemical crosslinker SANPAH,anti-rabbit CD34 antibody was coated onto the decellularized vascular scaffolds using ultraviolet ray.20 New Zealand white rabbits were performed right lower-abdominal pedicled skin flap which supplyed by branches of femoral artery.Whole defect 1cm in femoral artery were made and end-to-end anastomosis repaired by 10 crosslinked and 10 non-crosslinked scaffolds in experimental group and control group separately.The patency of the pedicle was observed through color and appearance of the flap postoperatively.After 4 weeks of transplantation,patency rate and cell seeding were detected by Color Doppler,DSA and pathological test. Results Hematoxylin and eosin staining and Masson staining revealed that no nucleus was detected in the decellularized vascular scaffolds.Scanning electron microscopy demonstrated that a lot of collagen fibers were preserved which was beneficial for cell adhesion.Fluorescence staining and DNA remains test showed that the cells were removed completely.Mechanical test revealed that the decellularized vascular scaffolds reserved mechanical properties of the raw caprine arteries.Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1,and the decellularized vascular scaffolds had a good compatibility to endothelial cells.After subcutaneously embedding for 8 weeks,negligible moderately lymphocyte infiltration was observed. Flaps of experimental group had good blood supply after transplantation while swelling and necrosis could be found in the control group.Doppler and DSA showed that the 7 of 10 crosslinked scaffolds remained patent for 4 weeks and also no stenosis had been developed,but bare scaffolds were obstructed in the control group 1 week. Hematoxylin and eosin staining revealed that the inner layers of crosslinked scaffolds were partly covered with endothelial cells in one month.In contrast,no cell coverage was observed in the bare scaffolds but thrombosis.Conclusion The cells were removed completely after multistep processes and no debris and DNA residue.The decellularized vascular scaffolds show negligible cell toxicity but favorable bio compatibility.The mechanical characterization of the decellularized vascular scaffolds is well-preserved by the combination of repeated frozen/thawing,ultrahigh pressure treatment and chemical detergent.Anti-CD34 antibody was firmly coated onto the decellularized vascular scaffolds using ultraviolet ray by crosslinker SANPAH.Crosslinked scaffolds with anti-CD34 antibody were superior to bare scaffolds in early postoperative anticoagulation and patency rate.It is not impossible for anti-CD34 antibody crosslinked scaffolds to recruit endothelial progenitor cells from the circulation to participate recellularization.This method simpilify the procedure of tissue engineering blood vessel fabricating in vitro and take the research one step forward.