Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells.
BACKGROUND
Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.


METHODS
Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).


RESULTS
EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.


CONCLUSIONS
EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.
Fang
Ning-Tao
NT
Laboratory of Molecular Biology, Shanghai Medical College, Fudan University, Shanghai, China.
Xie
Shang-Zhe
SZ
Wang
Song-Mei
SM
Gao
Hong-Yang
HY
Wu
Chun-Gen
CG
Pan
Luan-Feng
LF
D000818:Animals; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006350:Heart Valve Prosthesis; D006351:Heart Valves / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010974:Platelet Aggregation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism; D014470:Umbilical Cord / Q000166:cytology
D052250:Nitric Oxide Synthase Type III; D010959:Tissue Plasminogen Activator