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pgs membranes ( ~250 m thick ) on silicon wafers16 were situated on the x - y stage ( accuracy  1 m ) of a rapid x 1000 system ( resonetics , nashua , nh ) with a 248 nm krypton fluoride lpx200 excimer laser ( coherent - lambda physik , santa clara , ca ) . pores were patterned via g - code programs . for highly cross - linked pgs ( 16 h/160c ) , a power of 350 mj , burst frequency of 500/sec and burst count of 1000 were used . for partially cross - linked pgs ( e.g. , 7.5h/160c ) ,
scaffolds were loosened from wafers in deionized water ( 24 h ) and 70% ethanol ( 24 h ) .
bilaminar scaffolds ( ~400 m ) with 3-d pore networks were fabricated by microablating a lateral array of troughs in one pgs lamina ( burst spacing of 0.01 sec ; table speed of 0.5 mm / sec ) , overlaying a second pgs lamina , microablating top - to - bottom pores through both lamina ( burst frequency of 500/sec ; burst count of 2000 ) , and then stabilizing the resultant scaffold by thermally cross - linking17 via autoclaving ( 121c for 30 min ) .
cells were obtained from 2-day old neonatal rat hearts via an institute - approved protocol as described8 ( see supplementary information , methods ) . in studies of pgs scaffolds with varying effective stiffness and prototype bilaminar scaffolds ( 5 mm  5 mm for imaging ; 10 mm  5 mm for mechanics ) ,
scaffolds were autoclave - sterilized and seeded with freshly harvested , unseparated neonatal rat heart cells at ~36  10 cells / cm . in brief , 10 ml of cell suspension were added to a vent - capped 50 ml tube ( product # 91253 ; tpp , trasadingen , switzerland ) containing one scaffold , and tubes were placed in a 37c/5% co2 humidified incubator and gently mixed ( 8 rpm ) using a rotisserie ( labquake , barnstead - thermolyne , dubuque , ia ) .
after 4 days , culture media were replaced and culture was continued statically for 3 more days . in studies of 16h/160c pgs scaffolds with varying pore structures , scaffolds ( 5 mm  5 mm ) were autoclave - sterilized and sequentially seeded30 first with cardiac fibroblasts at ~1  10 cells / cm , followed by a heart cell population enriched for cardiomyocytes at ~36  10 cells / cm . in brief , 10 ml of cardiac fibroblast cell suspension was added to a 50 ml tissue culture tube containing a single scaffold and cultured with gentle mixing as described above .
after 5 days , cardiomyocyte - enriched heart cells were added , and after an additional 3 days , grafts were transferred to petri dishes ( costar ultra low attachment ; corning , corning , ny ) ( one per 35 mm well ) and cultured statically for 4 more days . culture media ( 6 ml / well ) were replaced every 2 days .
specimens were assessed using our previous method , wherein an electrical pulse generator applied biphasic square waveforms at 1 , 2 , 3 , and 4 hz to a specimen positioned between two carbon rod electrodes within an environmentally - controlled , perfused test chamber34 , 35 .
excitation threshold ( et ) was defined as the minimum voltage observed to initiate synchronous contractions .
video recordings made at twice the et used a 30 frame / s video camera ( sony xcd - x710 ) and nikon diaphot microscope at 10x magnification .
cell orientation was evaluated using confocal laser microscopy with staining of filamentous actin ( f - actin)39 and fast fourier transform ( fft)-based image analysis40 , 51 . in brief , specimens were rinsed , fixed in 10% neutral buffered formalin ( sigma ) for 2 h , rinsed , extracted in 0.2% ( v / v ) triton x-100 ( sigma ) in pbs for 2 h , rinsed , pre - incubated in 1% ( w / v ) bovine serum albumin ( sigma ) in pbs ( bsa buffer ) for 2 h , incubated in alexa fluor 488-phalloidin ( 2:150 ( v / v ) dilution ; molecular probes ) for 3 h , rinsed , and then incubated in draq5(25 m in pbs ; biostatus limited , leicestershire , uk ) for 30 min , all at room temperature .
specimens were then rinsed , bulk mounted on glass slides in vectashield medium ( vector laboratories , burlingame , ca ) , and cover - slipped .
specimens were imaged with 25x and 100x oil immersion objectives on a zeiss lsm 510 laser scanning confocal microscope , with f - actin and nuclei pseudo - colored green and blue , respectively .
micrographs of f - actin filaments ( i.e. , green channel ) were analyzed for cell orientation using a matlab program .
fft image analysis was similar to our previous study40 , and adapted from ng et al.51 .
see descriptions of fft in quantifying 2-d fiber network orientation52 and comparisons of fft to morphometry53 .
micrographs taken at ~30 m from the graft and rv epicardial surfaces were analyzed ( n=6 pores ; one representative sample per group ) .
orientation index ( oi ) was defined as the angular increment centered about the distribution mean encompassing 50% of the population41 .
for random cell orientations oi approaches 90 degrees ; for highly aligned cells oi approaches zero .
comparisons were made by one - factor ( anisotropy ratio ) or two - factor ( other parameters ) anova with tukey s post - hoc test ( statistica v7 , statsoft , tulsa , ok ) .
pgs membranes ( ~250 m thick ) on silicon wafers16 were situated on the x - y stage ( accuracy  1 m ) of a rapid x 1000 system ( resonetics , nashua , nh ) with a 248 nm krypton fluoride lpx200 excimer laser ( coherent - lambda physik , santa clara , ca ) . pores were patterned via g - code programs . for highly cross - linked pgs ( 16 h/160c ) , a power of 350 mj , burst frequency of 500/sec and burst count of 1000 were used . for partially cross - linked pgs ( e.g. , 7.5h/160c ) ,
scaffolds were loosened from wafers in deionized water ( 24 h ) and 70% ethanol ( 24 h ) .
bilaminar scaffolds ( ~400 m ) with 3-d pore networks were fabricated by microablating a lateral array of troughs in one pgs lamina ( burst spacing of 0.01 sec ; table speed of 0.5 mm / sec ) , overlaying a second pgs lamina , microablating top - to - bottom pores through both lamina ( burst frequency of 500/sec ; burst count of 2000 ) , and then stabilizing the resultant scaffold by thermally cross - linking17 via autoclaving ( 121c for 30 min ) .
cells were obtained from 2-day old neonatal rat hearts via an institute - approved protocol as described8 ( see supplementary information , methods ) . in studies of pgs scaffolds with varying effective stiffness and prototype bilaminar scaffolds ( 5 mm  5 mm for imaging ; 10 mm  5 mm for mechanics ) ,
scaffolds were autoclave - sterilized and seeded with freshly harvested , unseparated neonatal rat heart cells at ~36  10 cells / cm . in brief , 10 ml of cell suspension were added to a vent - capped 50 ml tube ( product # 91253 ; tpp , trasadingen , switzerland ) containing one scaffold , and tubes were placed in a 37c/5% co2 humidified incubator and gently mixed ( 8 rpm ) using a rotisserie ( labquake , barnstead - thermolyne , dubuque , ia ) .
after 4 days , culture media were replaced and culture was continued statically for 3 more days . in studies of 16h/160c pgs scaffolds with varying pore structures , scaffolds ( 5 mm  5 mm )
were autoclave - sterilized and sequentially seeded30 first with cardiac fibroblasts at ~1  10 cells / cm , followed by a heart cell population enriched for cardiomyocytes at ~36  10 cells / cm . in brief , 10 ml of cardiac fibroblast cell suspension was added to a 50 ml tissue culture tube containing a single scaffold and cultured with gentle mixing as described above .
after 5 days , cardiomyocyte - enriched heart cells were added , and after an additional 3 days , grafts were transferred to petri dishes ( costar ultra low attachment ; corning , corning , ny ) ( one per 35 mm well ) and cultured statically for 4 more days . culture media ( 6 ml / well ) were replaced every 2 days .
specimens were assessed using our previous method , wherein an electrical pulse generator applied biphasic square waveforms at 1 , 2 , 3 , and 4 hz to a specimen positioned between two carbon rod electrodes within an environmentally - controlled , perfused test chamber34 , 35 . excitation threshold ( et )
video recordings made at twice the et used a 30 frame / s video camera ( sony xcd - x710 ) and nikon diaphot microscope at 10x magnification .
cell orientation was evaluated using confocal laser microscopy with staining of filamentous actin ( f - actin)39 and fast fourier transform ( fft)-based image analysis40 , 51 . in brief , specimens were rinsed , fixed in 10% neutral buffered formalin ( sigma ) for 2 h , rinsed , extracted in 0.2% ( v / v ) triton x-100 ( sigma ) in pbs for 2 h , rinsed , pre - incubated in 1% ( w / v ) bovine serum albumin ( sigma ) in pbs ( bsa buffer ) for 2 h , incubated in alexa fluor 488-phalloidin ( 2:150 ( v / v ) dilution ; molecular probes ) for 3 h , rinsed , and then incubated in draq5(25 m in pbs ; biostatus limited , leicestershire , uk ) for 30 min , all at room temperature .
specimens were then rinsed , bulk mounted on glass slides in vectashield medium ( vector laboratories , burlingame , ca ) , and cover - slipped .
specimens were imaged with 25x and 100x oil immersion objectives on a zeiss lsm 510 laser scanning confocal microscope , with f - actin and nuclei pseudo - colored green and blue , respectively .
micrographs of f - actin filaments ( i.e. , green channel ) were analyzed for cell orientation using a matlab program .
fft image analysis was similar to our previous study40 , and adapted from ng et al.51 .
see descriptions of fft in quantifying 2-d fiber network orientation52 and comparisons of fft to morphometry53 .
micrographs taken at ~30 m from the graft and rv epicardial surfaces were analyzed ( n=6 pores ; one representative sample per group ) .
orientation index ( oi ) was defined as the angular increment centered about the distribution mean encompassing 50% of the population41 .
for random cell orientations oi approaches 90 degrees ; for highly aligned cells oi approaches zero .
comparisons were made by one - factor ( anisotropy ratio ) or two - factor ( other parameters ) anova with tukey s post - hoc test ( statistica v7 , statsoft , tulsa , ok ) .
tissue engineered grafts may be useful in myocardial repair , however previous scaffolds have been structurally incompatible with recapitulating cardiac anisotropy . 
 utilizing microfabrication techniques , a novel accordion - like honeycomb microstructure was rendered in poly(glycerol sebacate ) to yield porous , elastomeric 3-d scaffolds with controllable stiffness and anisotropy . 
 accordion - like honeycomb scaffolds with cultured neonatal rat heart cells demonstrated utility via : ( 1 ) closely matched mechanical properties compared to native adult rat right ventricular myocardium , with stiffnesses controlled by polymer curing time ; ( 2 ) heart cell contractility inducible by electric field stimulation with directionally - dependent electrical excitation thresholds ( p<0.05 ) ; and ( 3 ) greater heart cell alignment ( p<0.0001 ) than isotropic control scaffolds . 
 prototype bilaminar scaffolds with 3-d interconnected pore networks yielded electrically excitable grafts with multi - layered neonatal rat heart cells . 
 accordion - like honeycombs can thus overcome principal structural - mechanical limitations of previous scaffolds , promoting the formation of grafts with aligned heart cells and mechanical properties more closely resembling native myocardium .