briefly , poly(-caprolactone ) ( pcl ) were dissolved at 143 mg / ml in equal parts tetrahydrofuran and n , n - dimethylformamide , then extruded at 2.5 ml / h through a spinneret charged to + 13 kv .
the resulting nanofibrous jet was collected on a grounded mandrel rotating at 10 m / s and located 20 cm from the spinneret .
aluminum shields on either side of the spinneret were charged to + 9 kv to focus the jet . the spinneret was fanned back and forth to ensure uniform fiber deposition .
mscs were isolated from femoral and tibial bone marrow of 3 - 6 month old calves and expanded to passage 2 as described previously .
scaffolds were hydrated by sequential washes in 100 % , 70 % , 50 % and 30% ethanol and finally phosphate buffered saline ( pbs ) . before seeding ,
50 l of cell solution ( 1  10 cells / ml ) were applied to one side , followed by incubation at 37c for one hour .
scaffolds were then turned and an additional 50 l of cell solution applied to the other side .
after two hours further incubation , samples were transferred to chemically defined media ( dmem , 0.1m dexamethasone , 40 g / ml lproline , 100 g / ml sodium pyruvate , 1% insulin , transferrin , selenium / premix , and 1% penicillin , streptomycin and fungizone supplemented with 10 ng / ml transforming growth factor 3 ) . media was replaced twice weekly for the duration of the study . after measuring cross - sectional area using a custom laser device , samples ( n=5 ) were clamped with serrated grips and loaded into an instron 5542 testing device .
all testing was performed in a pbs bath . the mechanical testing protocol consisted of : ( 1 ) a nominal tare load of 0.1n applied at 0.1% strain / sec , followed by stress relaxation for 5 minutes , ( 2 ) 15 preconditioning cycles to 0.1% strain at 0.05% strain / sec , and ( 3 ) a quasi - static elongation at 0.1% strain / sec until failure .
strain was determined as extension normalized to gauge length ; stress was computed as the load normalized to initial cross - sectional area .
modulus was computed as the slope of the stress - strain plot , determined by regression to the linear portion of the curve . for biochemical analyses ,
samples were digested for 16 hours in papain at 60c , then analyzed for s - gag content using the 1,9-dimethylmethylene blue dye - binding assay , for orthohydroxyproline ( ohp ) content ( after acid hydrolysis ) to determine collagen content by reaction with chloramine t and dimethylaminobenzaldehyde , and for dna content using the picogreen dsdna quantification kit .
ohp content was converted to collagen with a ratio of 1 : 7.14 ( ohp : collagen ) .
paraformaldehyde - fixed sections were stained for cell nuclei ( 40,6-diamidino-2-phenylindole , dapi ) , glycoasaminoglycans ( alcian blue ) and collagen ( picrosirius red ) .
dapi - stained sections were visualized at 20 on a nikon t30 inverted fluorescent microscope .
quantitative polarized light microscopy was performed on picrosirius red stained sections to quantify collagen alignment as described previously .
briefly , grayscale images were collected ( 20 ) at 10 increments using a green bandpass filter ( bp 546 nm ) with crossed analyzer and polarizer coordinately rotated through 90. this was repeated with the filter replaced by a  compensator .
custom software was then used to determine collagen fiber orientations for a series of nodes within the central portion of each region of interest .
nanofibrous scaffolds of approximately 1 mm thickness were prepared as above and excised along the fiber direction .
two weeks of after seeding with mscs , samples ( n=5 ) were placed in apposition with a 20 mm overlap and secured with porous polypropylene and foil and cultured as above .
lap tests were performed by gripping the overhang on either end of the bilayer and extending to failure at 0.2 mm / s .
aligned nanofibrous scaffolds of approximately 1 mm thickness were excised at 30 from the fiber direction .
agarose was dissolved in pbs at 2 , 4 , 5 , and 6% w / v and melted by autoclaving .
molten agarose was applied between layers of scaffold , and allowed to set at room temperature .
no significant difference was observed in cross sectional area across all concentrations , indicating controlled , reproducible interface formation .
significance was established by p  0.05 as determined by two - way anova with a tukey 's post hoc test for independent variables of bilayer orientation ( parallel / opposing ) and culture duration ( or agarose concentration ) .
briefly , poly(-caprolactone ) ( pcl ) were dissolved at 143 mg / ml in equal parts tetrahydrofuran and n , n - dimethylformamide , then extruded at 2.5 ml / h through a spinneret charged to + 13 kv .
the resulting nanofibrous jet was collected on a grounded mandrel rotating at 10 m / s and located 20 cm from the spinneret .
aluminum shields on either side of the spinneret were charged to + 9 kv to focus the jet . the spinneret was fanned back and forth to ensure uniform fiber deposition .
mscs were isolated from femoral and tibial bone marrow of 3 - 6 month old calves and expanded to passage 2 as described previously .
scaffolds were hydrated by sequential washes in 100 % , 70 % , 50 % and 30% ethanol and finally phosphate buffered saline ( pbs ) . before seeding ,
50 l of cell solution ( 1  10 cells / ml ) were applied to one side , followed by incubation at 37c for one hour .
scaffolds were then turned and an additional 50 l of cell solution applied to the other side .
after two hours further incubation , samples were transferred to chemically defined media ( dmem , 0.1m dexamethasone , 40 g / ml lproline , 100 g / ml sodium pyruvate , 1% insulin , transferrin , selenium / premix , and 1% penicillin , streptomycin and fungizone supplemented with 10 ng / ml transforming growth factor 3 ) . media was replaced twice weekly for the duration of the study .
after measuring cross - sectional area using a custom laser device , samples ( n=5 ) were clamped with serrated grips and loaded into an instron 5542 testing device .
all testing was performed in a pbs bath . the mechanical testing protocol consisted of : ( 1 ) a nominal tare load of 0.1n applied at 0.1% strain / sec , followed by stress relaxation for 5 minutes , ( 2 ) 15 preconditioning cycles to 0.1% strain at 0.05% strain / sec , and ( 3 ) a quasi - static elongation at 0.1% strain / sec until failure .
strain was determined as extension normalized to gauge length ; stress was computed as the load normalized to initial cross - sectional area .
modulus was computed as the slope of the stress - strain plot , determined by regression to the linear portion of the curve . for biochemical analyses ,
samples were digested for 16 hours in papain at 60c , then analyzed for s - gag content using the 1,9-dimethylmethylene blue dye - binding assay , for orthohydroxyproline ( ohp ) content ( after acid hydrolysis ) to determine collagen content by reaction with chloramine t and dimethylaminobenzaldehyde , and for dna content using the picogreen dsdna quantification kit .
ohp content was converted to collagen with a ratio of 1 : 7.14 ( ohp : collagen ) .
paraformaldehyde - fixed sections were stained for cell nuclei ( 40,6-diamidino-2-phenylindole , dapi ) , glycoasaminoglycans ( alcian blue ) and collagen ( picrosirius red ) .
dapi - stained sections were visualized at 20 on a nikon t30 inverted fluorescent microscope .
quantitative polarized light microscopy was performed on picrosirius red stained sections to quantify collagen alignment as described previously .
briefly , grayscale images were collected ( 20 ) at 10 increments using a green bandpass filter ( bp 546 nm ) with crossed analyzer and polarizer coordinately rotated through 90. this was repeated with the filter replaced by a  compensator .
custom software was then used to determine collagen fiber orientations for a series of nodes within the central portion of each region of interest .
nanofibrous scaffolds of approximately 1 mm thickness were prepared as above and excised along the fiber direction .
two weeks of after seeding with mscs , samples ( n=5 ) were placed in apposition with a 20 mm overlap and secured with porous polypropylene and foil and cultured as above .
lap tests were performed by gripping the overhang on either end of the bilayer and extending to failure at 0.2 mm / s .
aligned nanofibrous scaffolds of approximately 1 mm thickness were excised at 30 from the fiber direction .
agarose was dissolved in pbs at 2 , 4 , 5 , and 6% w / v and melted by autoclaving .
molten agarose was applied between layers of scaffold , and allowed to set at room temperature .
no significant difference was observed in cross sectional area across all concentrations , indicating controlled , reproducible interface formation .
significance was established by p  0.05 as determined by two - way anova with a tukey 's post hoc test for independent variables of bilayer orientation ( parallel / opposing ) and culture duration ( or agarose concentration ) .
 successful engineering of load - bearing tissues requires recapitulation of their complex mechanical functions . given the intimate relationship between function and form , biomimetic materials that replicate anatomic form are of great interest for tissue engineering applications . 
 however , for complex tissues such as the annulus fibrosus , scaffolds have failed to capture their multi - scale structural hierarchy . 
 consequently , engineered tissues have yet to reach functional equivalence with their native counterparts . here 
 we present a novel strategy for annulus fibrosus tissue engineering that replicates this hierarchy with anisotropic nanofibrous laminates seeded with mesenchymal stem cells . 
 these scaffolds directed the deposition of organized , collagen - rich extracellular matrix that mimicked the angle - ply , multi - lamellar architecture and achieved mechanical parity with native tissue after 10 weeks of in vitro culture . 
 further , we identified a novel role for inter - lamellar shearing in reinforcing the tensile response of biologic laminates , a mechanism that has not previously been considered for these tissues . 