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Objective: To provide the reference for the stentless aortic valve design with the study of the inner configuration of porcine aortic root., Methods: The orifice areas of porcine aortic root at 4 levels (OA1 to 4), the average area of leaflets (Sa), the area analogue of leaflets (AA, AA = 1/2PH), the average area analogue of leaflets (AAa), the value PH of the left, right, non coronary leaflets (PHl, PHr, PHn) and the sums of PHs of the left and non-coronary leaflets (PHln) in the fresh and glutaraldehyde and epichlorohydrin-treated porcine aortic valves (20 respectively) were measured and calculated. The linear correlation and regression analysis by SPSS 12.0 was used to analyze the correlation between Sa and AAa, OA and Sa, OA and AAa, PHl, PHr and PHn, PHln and PHr in both groups., Results: The coefficient correlation between Sa and AAa in fresh and treated groups were 0.886 and 0.872 respectively (P < 0.05). The coefficient correlation between OA1 to 4 and AAa were 0.810, 0.851, 0.900, and 0.815 respectively in fresh group (P < 0.05), and were 0.852, 0.888, 0.836, and 0.817 respectively in treated group (P < 0.05). This showed that the degree of correlation between the average area analogue of leaflets and the average area of leaflets, the orifice areas of aortic root were relatively large. Additionally, the equation of linear regression existed between PHln and PHr in treated group as follows: PHr = -1.665 + 0.688 PHln (r = 0.907, P < 0.05), thereby PHr could be predicted by PHln., Conclusion: The value of PH of leaflets could represent the spatial configuration of the aortic root, which provided a referred index for the stentless bioprostheses design.

Objective: To prepare a porcine aortic valve (PAV) free of the cellular components., Methods: The cellular components of porcine PAV were completely removed using trypsin and Triton X-100, and the acellular PAV was examined microscopically with HE staining with its physical and chemical properties assessed. Transmission electron microscopy was used to observe the integrity of the collagen and elastin and the DNA contents in the PAV was detected to confirm the total removal of the cellular components. With the fresh PAV as the control, small pieces of the acellular PAV were implanted into the subcutaneous tissues of 4 rabbits, and 4 weeks after the implantation, the implants were harvested for microscopic observation., Results: The cellular components were effectively removed from the cusps and roots of the PAV by trypsin and TritonX-100, with marked soluble protein loss [(0.24-/+0.04)% vs (0.48-/+0.12)%] and significantly increased water content [(92.2-/+1.5)% vs (89.2-/+1.6)%]. The acellular PAV still maintained good fibrous scaffold structure and the shrinkage temperature and tension at fracture underwent no significantly changes [(67.9-/+1.0) degrees celsius; vs (68.8-/+0.8) degrees celsius; and (489.3-/+19.0) g/mm2 vs (540.7-/+19.5) g/mm2, respectively]. The PAVs implanted in rabbits showed only mild tissue reaction with a few infiltrating neutrophils, lymphocytes and plasmocytes observed 4 weeks later. The accelular PAV caused obviously milder inflammatory reactions than fresh PAV., Conclusions: The acellular PAV prepared by treatment with trypsin and Triton X-100 retains good fibrous scaffold structure and mechanical strength with low antigenicity.

Objective: to study the effect of modified acellularization process on porcine endogenous retrovirus (PERV) in porcine aorta valves (PAVs)., Methods: Twenty aortic valves of pig were put into 0.1% trypsin solution, hypotonic and hypertonic TritonX-100, DNAse solution, RNAse solution, and Hanks solution in succession so as to remove the cells. The specimens of PAV were to undergo gross observation and microscopy before and after the acellularization procedure. Fracture test was made. Primers specific for the conservative gag gene of PERV were designed PCR and RT-PCR were used to detect the expression of gag. In addition, 20 samples of native PAV were collected. Peripheral mononuclear cells (PBMCs). Were isolated from 20 samples of porcine peripheral blood. Ten dogs underwent acellularized PAV replacement; 3 months later, samples of the dogs' peripheral blood were collected. Porcine kidney cells of the line PK15 were used as positive controls., Results: Microscopy showed that all the cells were removed from the acellularized PAVs. Histological analysis showed that the major structural components were maintained. There was no significant difference in fracture strength between the native and acellularized PAVs (P > 0.05). PCR and RT-PCR showed a PERV 219 bp DNA fragment, 90%-95% homologous with the published PERV gene, in the genomic DNA of all native PAVs, pig PBMCs, and PK15 cells, but not in the acellularized PAVs and dog PBMCs., Conclusion: PERV exists in all native PAVs. The modified acellularization process succeeds in removing all the cell component and PERV in the PAVs, thus preventing cross-species transmission of PERV.

Objective: To explore an experimental method for construction of tissue-engineered heart valve (TEHV) in canine abdominal aorta., Methods: The decellular porcine aortic valve (PAV) leaflets seeded with canine vessel interstitial cells and endothelial cells (ECs) were implanted into 6 canine abdominal aortas. Valve specimens were obtained respectively at the end of 4, 6, 8 and 10 weeks after implantation were studied for morphology, histology and immunohistochemistry., Results: (1) After 4 weeks implantation, multiple layers of cells grew into peripheral portion of valve scaffold, while new extracellular matrix appeared, and original scaffold tissue was partially absorbed. (2) At the end of 10th week after implantation, the decellular PAV scaffold disappeared completely and was substituted by recipient cells and new extracellular matrix. The interstitial cells in matrix was mainly consisted of fibroblasts and myofibroblast. The matrix was mainly composed by type I, III collagen, some elastic fibers with neutral and acid mucopolysaccharide. (3) Surface of valve leaflets were covered with endothelial cells., Conclusions: (1) TEHV is primarily constructed with recellularized PAV after implantation into canine abdominal aorta for 10 weeks. (2) Heterotopic implantation into the abdominal aorta is an alternative experimental procedure to study the TEHV.

Objective: To construct decellular porcine aortic valve (PAV) and to observe the existence of porcine endogenous retrovirus (PERV) and valve scaffold structure before and after implantation., Methods: (1) Porcine aortic valve was obtained. The cellular components of PAV were completely removed by using detergent and nucleotidase solution combined with alteration of osmosis. (2) The decellular underwent HE staining and light microscopy and detection of its physical and chemical properties. (3) 20 pieces of decellular PAV were implanted into dogs. On e month later blood samples of the dogs were collected. PCR and RT-PCR were used to detect the PERV expression in 20 samples of pig's peripheral blood, 20 fresh PAVs, cultured pig kidney cells of the PK15 line (as positive control), decellular PAVs implanted into the dogs, and 10 samples of dogs' peripheral blood. (4) Small pieces of decellular PAVs were implanted into the subcutaneous tissues of 6 rabbits at the back, 6 pieces for one rabbit, and then extracted by the ends of the 4th, 6th, 8th and 10th week respectively after implantation to undergo HE staining and light microscopy., Results: (1) Almost all cellular components in the PAVs had been removed after decellularization; the soluble protein contents lost markedly [(0.238 +/- 0.038)% vs. (0.484 +/- 0.116)%]; the water content of the decellular tissues increased significantly [(92.16 +/- 1.48)% vs. (89.2 +/- 1.55)%]; however, the decellular PAVs still maintained their excellent fibrous scaffold structure, and their shrinkage temperature and tension at fracture were not significantly changed [(72.0 +/- 0.7) degrees C vs. (71.2 +/- 0.8) degrees C, and (448.7 +/- 18.65)g/mm2 vs. (540.7 +/- 19.46)g/mm2 respectively]. (2) Agarose gel electrophoresis of all fresh PAVs and porcine peripheral blood samples showed a 219 bp band, which was 90% to 97% homologous with PERV-C gene, and the sequence of which is published in Medline. No 219 bp amplified band was found in all decellular PAVs and the peripheral blood samples of the dogs implanted with decellular PAV one month after the implantation. (3) The PAVs implanted in rabbit body showed very slight tissue reaction. Neutrophil, lymphocyte and plasmacyte infiltration were seen 4 weeks after; such inflammatory cell infiltration decreased markedly and the peripheral portions of the decellular PAVs began to be absorbed by the end of the 6th week after implantation. The decellular PAVs were completely absorbed without fibrosis or scar formation in the implantation area by the end of the 10th week., Conclusion: (1) The cellular components of PAV can be completely removed, the excellent fibrous scaffold structure and mechanical strength of aorta valve can be maintained, and the antigenicity is very weak. Subcutaneous implantation investigation shows that decellular PAV is an absorbable and degradable biological material. (2) There is PERV-C in PAV that can be removed after decellularization. PERV-C reaction is negative in the peripheral blood samples of the recipients implanted with decellular PAV.