| Part One Primary Study on Decelluarization Rate of Poly Ethylene Glycol in preparing porcine Tssue Egineering Hart VlvesObjective: To study the feasibility of a new decellularization method in preparing porcine tissue engineering heart valves by poly ethylene glycol, determine the decelluarity efficiency and the preservation of the ECM by the method. Methods: The porcine aortic valves were harvested from slaughterhouse and divided into control group and PEG treated group. The specimens in control group were sink in phosphate buffer saline (PBS), while that in PEG group were decellularized with poly ethylene glycol and DNAse I. The PEG group was subdivided into 20min, 35min and 45min subgroup based on the time in PEG. Histochemical studies were performed on sequential valve sections dyed with hematoxylin and eosin. Specimens also were observed by scanning electron microscope (SEM). The content of DNA was detected by photometer. And the thickness and amount of water in specimens were measured in each group. The change in tissue protein was evaluated by SDS-PAGE electrophoresis. Results: In the light microscope, the control group has typical tri-layer structure as native valves. The cells were separated in all three layers and were predominant in spongiosa and fibrosa; the PEG group also has tri-layer structure and the extra cellular matrix (ECM) was well reserved. The collagen was compact and no apparent split. Cells could be seen in 20min and 35min subgroup, but no signs in 45min subgroup. The decellularity percentage could be calculated by DNA concentration in PEG subgroups, which were 28.17±9.58%,40.62±7.94%,95.32±3.61% respectively. The difference between 45min and 20min,35min was significant, which was not between 20min and 35min. the thickness of specimens in four groups were 0.39±0.05mm,0.41±0.06mm,0.43±0.05mm,0.44±0.07mm, and the amount of water in valves were 88.37±4.27%,90.81±2.21%,91.47±2.63%,91.89±3.01%, respectively. The difference has no significance. Extracted protein of nature and PEG treated porcine aortic valves were 0.46±0.07 mg/mL and 0.15±0.04 mg/mL. However, within the examined molecular weight range, protein bands were still detectable by SDS-PAGE within the decellularized porcine aortic valve. Conclusion: Sinking in PEG for 45min could remove celluar components effectively; the extra cellular matrix (ECM) was well reserved; the thickness and amount of water in valves had no significant change. The decellularization procedure removes considerable amounts of proteins within the porcine aortic valves. The method has value in preparing porcine decelluarization heart valves.Part Two Primary Study on plane Biaxial Mechanical Properties of the PEG Treated and Natural Porcine Aortic Valve CuspObjective: To validate the reliability of the method of preparing porcine decellularization tissue engineering heart valves by poly ethylene glycol, determine and compare the biaxial mechanical properties of PEG treated and natural porcine aortic valve. Methods: The porcine aortic valves were divided into control group and PEG group. The specimens in control group were sink in phosphate buffer saline (PBS), while that in PEG group were decellularized with poly ethylene glycol and DNAse I. Histochemical studies were performed on sequential valve sections dyed with hematoxylin and eosin. The content of DNA was detected by photometer. Rectangle specimens were dissected from the central belly region of the leaflet in either radial or circumferential directions. After the thickness of the specimens was measured in each group, the specimens were mounted onto the AGS-J device and got tensile test. The stress-strain curve, max-stress, max-load, max-strain and Elastic modulus were got from the experimental data. Results: in the light microscope, the control group has typical tri-layer structure as native valves. The cells were separated in all three layers and were predominant in spongiosa and fibrosa; the PEG group also has tri-layer structure and the extra cellular matrix (ECM) was well reserved, no cellular signs could be seen. The decellularizaty percentage was 94.48±4.32%. The thickness of specimens in two groups was 0.39±0.05mm and 0.41±0.05mm. The two groups had similar stress-strain curve, and there was difference in mechanical properties between radial and circumferential directions. The max max-stress, max-strain and elastic modulus of the two groups were 2.831±1.036MPa,2.496±1.251MPa;0.387±0.127,0.573±0.143;17.671±0.957 MPa,16.552±1.038 MPa in radial direction and 5.103±1.078 MPa,4.897±0.989 MPa ; 0.207±0.059,0.219±0.214 ; 36.854±1.566 MPa,35.741±1.207MPa in circumferential direction, respectively. Conclusion: Sinking in PEG for 45min could remove celluar components effectively; the thickness of valves had no significant change. The two groups had character of mechanical anisotropy. There was no significant difference in max-stress and elastic modulus in radial or circumferential direction between the two groups. The PEG trated porcine arotic valve has biaxial mechanical properties and could be an alternative for tissue engineering heart valve scaffold.Part Three Evaluation on the immunogenic property of PEG treated porcine tissue engineering valvesObjective: To compare and evaluate the immunogenic property of nature porcine aortic valve and PEG treated porcine decellularated tissue engineering heart valves. Methods: The nature porcine aortic valves were treated with PEG as the method we used previously. The nature valves and PEG treated valves were cut into 1×1cm2. The BALB/C mice were divided into two groups: control and PEG groups, which were then subcutaneous embedded with nature and PEG treated valves in nape, respectively. On 5d, 10d, 20d after valves were subcutaneous embedded, six mice in each group were executed and the valves and spleen were explanted and dyed with hematoxylin and eosin. The serum was got from the whole blood and then the content of IL-2, IL-10 in serum was measured by ELISA assay. The transcription activities of IL-2, IL-10 in mice spleen were self-quantity measured by RT-PCR. The result of them was compared between the two groups. Results: There were no deaths between the two groups in the time of experiment. The histology examination showed that the rejection happened in control group was earlier and severer than PEG group. And the tissue construction was destroyed badly in control group than that in PEG group. The result of ELISA assay showed that at each sample time point, the content of IL-2 in control group was higher than PEG group; and the curve of IL-2 was wave like, up from 5d, max at 10d, down but still high at 20d. There no significant difference between the two groups in the content of IL-10 at each time point. the curve of IL-10 was escalated. The result of RT-PCR of IL-2 and IL-10 got the same trend as that of ELISA. Conclusion: PEG treated porcine heart valves get rid of the immunogenicity thoroughly. The rejection happened after subcutaneous embedded is moderate and transitory. There have little tissue construction destroyed, and the fibroblast could adhere and live in easily. The decelluarationed valve is suit for TEHV scsffold.Part Four Evaluation on the cell adhesion efficiency of PEG treated porcine tissue engineering valves and the impact of surface modificationObjective: To get in sight of the cell adhesion efficiency of PEG treated porcine decellularated tissue engineering heart valves, and evaluate the impact of surface modification on cell adhesion efficiency of PEG treated valves. Methods: The thoracic aorta of kunming mouse was separated carefully and cut into small tissue pieces. The explants were seeded onto culture flasks. Cell was observed through phase contrast microscope and fibroblasts from 5-8 generations are applied to cell adhesion experiment. All the valve specimens were cut into 1cm×1cm and divided into PEG group, PEG + gelatin group, PEG + Poly-L-Lysine (PLL) group, PEG + fetal bovine serum (FBS)group. The specimens in PEG group were PEG treated valves with no surface modification, while that in PEG + gelatin group, PEG + PLL group, PEG + FBS group were PEG treated valves pre-coated with gelatin, poly-l-lysine and fetal bovine serum before experiment, respectively. These specimens were put into 24 cell culture medium. 5×105 fibroblasts were added into every cell and culture for 1 h. After that the valves were took out and washed out carefully to get the fibroblasts that did not adhere to the valve could stay in the culture medium. The number of cells in culture medium was got by MTT assay, so the number of cells adhere on valve can be calculated. Some of the valves in PEG group were transferred into culture flasks and static culture for two weeks, and then the valves were checked by histology. Results: The fibroblast can be culture and harvest from the method of tissue pieces culture effectively. The rate of cell adhere to the valves in PEG group, PEG + gelatin group, PEG + PLL group and PEG + FBS group was 42.63%±5.19%,69.22%±6.63%,65.78%±4.45%,85.32%±3.61%, respectively. The valves in surface modification groups has significant higher cell adhere rate than that in PEG group. After two weeks static culture, the histology examination showed that the valve preserved tri-layer structure as native valves. The cells were separated in all three layer and the number of them in superficial was more than that in deeper. The continued cell line could be seen in the surface. Conclusion: PEG treated porcine heart valves have excellent in vitro biocompatibility; the valve scaffold can obviously improve cell adhere efficiency after surface modification.
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