, 3. EXPER IM ENTAL STUDY OF XENOGENE IC HEART VAL VE M ATER IAL gs H I K ai2hu, ZH A N G X i, ZH A N G

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1 # , 4 : 1 (GA ), 2 (EC), 3 + (EC+ L 2GA ), (EC+ L 2 GA + CE) (6 ), M 199 (10% + 20% + + ),, g : 1, 2, 3 4 (P < 0. 01) : 1 ; 2 ; 3 ; 4,,,,,,, EXPER IM ENTAL STUDY OF XENOGENE IC HEART VAL VE M ATER IAL gs H I K ai2hu, ZH A N G X i, ZH A N G J ing 2f ang. D ep artm ent of Card iovasic S u rg ery of the F irst A f f iliated H osp ital, S un Y at2s en U niversity. Guang z hou Guang d ong, P. R. Ch ina Abstract Objective To exp lo re the po ssibility of imp roving the perfo rm ance of tissue engineering valve by m eans of p reendo thelialization w ith cultured hum an um bilical vein endo thelial cell(huv EC) and to develop a new xeno2 geneic biop ro sthesis valve m aterial. M ethods T he po rcine ao rtic valves treated by use of glutaraldehyde (GA ), epoxy2 ch lo rop ropane (EC ), L 2glutam ic acid (L 2GA ) and cellular extraction (CE) respectively w ere divided into four group s: group 1 (GA ), group 2 (EC), group 3 (EC+ L 2GA ), and group 4 (EC+ L 2GA + CE). T he cultrued huv EC s w ere seed2 ed onto the treated po rcine ao rtic valve, then that stuff w ere exam ined by m eans of EC g facto r staining, living cells counting and m icro scopy. Results T he cultured huv EC could adhere to culturing bo ttle w all an hour later, and p ropa2 gated to tw o passages after seven dayṡ T he cells increased w ith serial passage at a 72day interval. But the huv EC grew slow ly w hen seeded onto the treated valve m aterial excep t group 4. T he cells in group 4 covered the surface of valve comp letely seven days later, w h ich could also be seen in group 3 but no t comp letely. T here w as no cell grow ing in group 1, and only few er in group 2. T he living cell in group s 3 and 4 w ere significantly mo re than in group s 1 and 2 on the 3rd, 7th and 14th days (P < 0. 01), m eanw h ile, the num ber of cells in group 4 w ere also significantly mo re than that in group 3 (P < 0105). T he covering area of cultured cell on the valve m aterial in group s 3 and 4 w as significantly larger than that in group s 1 and 2. T he covering area of cell in group 4 w as over 95%, and h igher than that in group 3 (60% 2 70% ). T he huv EC of group 4 arranged in pattern of th ree dim ension. So it could resist rising of fo reign pow er from the cardiac cavity of h igh p ressure and flow ing vo lum e. T here w as no cell on the leaflet surface in group 1, and only a few p inch of cells could be seen in group 2. Conclusion T he po rcine ao rtic valve can be used to be an ideal xenogeneic valve scaffo ld; the scaffo ld of po rcine ao rtic valve should be treated by use of epoxy2ch lo rop ropane, L 2glutam ic acid and cellular extraction, so that a best grow ing environm ent to the huv EC w ould be given; the cultured huv EC s used to be source of seed living cell had a boundless p ro spects; the grow ing velocity of cultured huv EC w as contro llable, w h ich facilitated clin ical app lication; and the endo thelial cells of xenogeneic valve m aterialw h ich grew com pactly on to the scaf2 fo ld can resist rising of fo reign pow er from the cardiac cavity itselḟ Key words T issue engineering valve Xenogeneic heart valve H um an um bilical vein endo thelial cells, 3 : 1 (, ) ; 2 #,,,

2 Fo rm a23548 CO 2 ( ), O lym 2 Pu s21x70 ( ), O lym Pu s2cx40 ( ), (1 250 D ibco), (J r Scien tific Inc, ),M 199 (D ibco ), 10% ( ), g (San ta C ruz, ) ,, PBS : 1 (Glu taraldehyde, GA ) : 0. 5% GA (ph 7. 4, 4 ) 72, 4 ; 2 ( Epoxy2ch lo rop ropane, EC ) : 4% PC 48 ; 3 + GA (EC + L 2glu tam ic acid, EC+ L 2GA ) : 4% PC 48, 8% L 2(pH 3. 5) 72 ; ( EC + L 2GA + cellu lar ex traction, EC + L 2GA + CE ) : 0. 25% 37 20, 4% PC 48, 8% L 2GA (ph 3. 5) 72 ( 1 2) 1 ( 200) 2(SEM 500) F ig. 1 The f ibrous scaffold of porc ine aortic valve treated by cellular extraction ( 200) F ig. (SEM 500) 2 The acellular f ibrous scaffold of heart valve ( 6 ), PBS,, 0125% 37 PBS 20, 1 2 m l 10% (1 000 rgm in) 10 ;, M 199 ( 10% 2 m l+ 20 m ggl ), U gl 100 m ggl T 225 (25 cm 2 ), gm l; 12,, 1, 6 1, 3, 01125% % ED TAN a2 PBS, ( gm l) M 199 (10% + 20% + + ), gcm 2 7, M 199 1, g, 1. 6 SA S,, ς 2, t, P < , 7, 1 2 7,

3 498, 7, 4 3, 2. 2 g g, ( 3),, ; 4,,,,, ( 4 5) ( 10 4, x θ s) Tab. Group Number of cells in each group ( 10 4, x θ s) 3 3rd day N um ber of cell 7 7th day 14 14th day P < 0101; P < 0105; 7 3 P < 0105 Compared betw een group 2 and group 3, 4, P < 0101; 3 Com 2 pared betw een group 3 and group 4 on the 7th day, P < 0105; Com 2 pared betw een 7th day and 3rd day in the sam e group, P < 0105 F ig. 3 3 (g 300) 3 The cultured human umbilical ve in endothelial cell propagated to three-passage ( 300) , 60% 70%, ; 4 3, 2. 3,, 95%, , 1 ( 6) ; 1 ; 2, 3 4 ( 7) (P < 0. 01) ; ; 3 4 (P > 0. 05), 7 ; 4 4 (P < 0. 05), 14 4,, ; , (P < 0105), ; 2,, ; (g 400) 5 4 ( 300) F ig. 4 The new hybr id inner wall con structed from tabling growth in group 4 ( 400) F ig. 5 Seed cells grow in to the f ibrous scaffold in group 4 ( 300)

4 (SEM 700) 7 1 (SEM 350) F ig. 6 The new hybr id inner wall costructed from the pure acellular f ibrous scaffold in group 4 ( 700) F ig. 7 The new inner wall in group 1 ( 350) [ 1 ],,,,,,, ( ) [ 2 ] 3. 1,,,,, [ 3 ],, 3. 2,,, Po lyu rethane N o2r eact TM A lginate A zide, AA Am inoo leic A cid Epoxy, Epoxy, : Epoxy Grimm [ 4 ] L 2GA EC, L 2GA (P < 0. 01),, 4 3 (P < 0. 05), PBS,,,, 3,, PBS, :, :,,,,,, 3. 3,,,,

5 500, [ 5 ] (hum an um 2 b ilical vein endo thelial cell, huv EC), : ; ; ;,,,, [ 6 ], : huv EC NO CO,,,,, 3. 4,,, 3 4, 4 :, L 2GA 20% 10%,,, 4 1 KonertzW, T andler R, H asfeld M, et al. A o rtic valve rep lacem ent w ith cryop reserved pulmonary allografṫ J Card Surg, 1994; 9 (1) : 43 2 Sh inoka T, Shum 2T im TD, PX M a, et al. T issue engineered heart valves. A uto logous valve leaflet rep lacem ent study in a lam b model. C irculation, 1996; 94 (9 Supp l) : Sung HW, Chang Y, Ch iu CT, et al. M echanical p roperties of a po rcine ao rtic valve fixed w ith a naturally occurring cro sslinking agenṫ B iom aterials, 1999; 20 (19) : Grimm M, Grabemwoger M Eyble E, et al. Imp roved biocompati2 bility of biop ro sthetic heart valves by L 2glutam ic acid treatm enṫ Card Surg, 1992; 7 (1) : 58 5 Simon A, W ilhelm im, Steinhoff G, et al. Cardiac valve endo thelial cells: Relevance in the long2term function of bio logic valve p ro sthe2 ses. J Tho rac Cardiovasc Surg, 1998; 116 (4) : D esai JK, T homp son MM, Eady SL, et al. Imm unomodulation of cultured vascular endo thelial cell o r serial cell passage. J Eur J V asc Endovasc Surg, 1995; 10 (1) : 101 ( : ) , 2,,, 2 1 4,,, 1 2 1, 28 ; 1, 16,, 7 8 cm cm, 10 14,,,,, 10 cm, cm 14 cm,, ( ),, : (, ) 2 1 9,, 3, 5 8 (60% ) 6 9 (40% ),,,, cm, 918 cm ; mm, ( ) cm, ( ) mm1 (88% ),,,,,,, 3 4, ( : : )