the replacement of tissue lost through trauma , disease , or congenital anomalies is a continuing clinical challenge . current reconstructive options , including autologous tissue transfer , allograft , xenograft , and alloplastic implantation , are limited by donor site morbidity , tissue scarcity , disease transmission or antigenic incompatibility , hardware infection , and implant extrusion . ideally , tissue reconstruction should avoid sacrificing healthy tissue or using alloplastic materials by instead engineering autologous replacement tissue de novo . tissue engineers have successfully cultured the cellular constituents necessary to build a variety of tissue types in vitro . however , traditional two - dimensional ( 2d ) cell - culture techniques ( e.g. , petri dishes and culture flasks ) are inadequate for three - dimensional ( 3d ) tissue engineering . in 2d culture , a monolayer of cells is in continuous contact with culture medium , and simple diffusion is sufficient to maintain cell viability . as scaffolds gain 3d volume , however , the central core becomes increasingly separated from the penumbra of fresh medium ; simple diffusion provides inadequate oxygen delivery and waste removal from cells in the core . as a result , only cells in a thin construct ( with a large surface area - to - volume ratio ) survive , and typically only on the peripheral crust of the scaffold ( up to 2  mm deep ) . nature has addressed this problem in native bone by establishing a complex lacunocanalicular network within which a nutrient - rich fluid circulates [ 4 , 5 ] . thus , successful engineering of thick 3d osseous tissue constructs large enough to solve actual clinical problems will require novel tissue - engineering strategies that address chemotransportative requirements in their design and implementation . for example , cell - seeded porous scaffolds have been set upon orbital shakers , hung in spinner flasks [ 6 , 7 ] , continuously perfused through glass columns , or tumbled in rotational bioreactors [ 6 , 9 , 10 ] . these methods increase medium fluid flow across the external surface of the scaffold , offering an incremental improvement over traditional static culture techniques . while these technologies satisfy the external   requirement for medium flow , convection of medium at the external surface does not guarantee chemotransportation within the porous confines of the scaffold interior . in fact , the majority of medium in the systems described above follow the path of least resistance and circumnavigate the scaffold ( figure 1(a ) ) . consequently , convection of medium alone does not result in penetrating flow that perfuses the porous construct to provide effective   chemotransportation . in contrast to technologies like the spinner flask , we designed a flow - perfusion bioreactor to address the internal   requirement for flow within the porous network of the scaffold ( figures 1(b ) and 1(c ) ) . in this system , porous scaffolds are press fit into an experimental chamber , and medium flows by gravity head or by generated hydrostatic pressure through the scaffold . because the fluid path is confined to pass through the scaffold the flow - perfusion bioreactor promises improved chemotransportation to all regions of a 3d scaffold . furthermore , the flow - perfusion bioreactor is , in theory , a scalable technology that should support porous scaffolds of any thickness . nevertheless , to date , most exogenous tissue - engineering research has been constrained to using scaffolds 2  mm in thickness or less , which are readily sustainable by medium convection or static culture methods . therefore , we evaluated the efficacy of a novel flow - perfusion bioreactor in sustaining  thick specifically , as proof of principle , we tested cylindrical scaffolds measuring 24  mm in diameter and 6  mm in thickness . since these scaffolds are thicker than the  critical depth  of 2  mm ( from core to surface ) , without effective fluid flow , they should suffer central core necrosis . we hypothesized that dynamic cell culture with a flow - perfusion bioreactor will provide adequate chemotransportation to the core of a thick scaffold , thereby , maintaining cell viability and activity . the flow - perfusion bioreactor was machined from solid teflon ( sabic polymershape ; jacksonville , fl ) . it contains 8 independent experimental chambers each measuring 24  mm in diameter and able to accommodate scaffolds up to 10  mm in thickness ( figures 1(c ) and 1(d ) ) . the floor of each experimental chamber is tapered to ensure flow from the outer edges of the scaffold as well as the center to the exit port of the chamber ( figure 1(b ) ) . screw caps are fitted with viton-75 o - rings ( mcmaster - carr ; aurora , oh ) to ensure a tight seal and prevent leakage . the bioreactor rests upon an 8-chamber medium reservoir , with each experimental chamber directly overlying its respective medium chamber . the junction between the bioreactor and reservoir is sealed by a gas - permeable membrane ( tegaderm ; 3 m ; st . paul , mn ) . an 8-channel peristaltic roller pump ( manostat - carter ; barnant co. ; barrington , il ) draws medium from the reservoir and administers it to each experimental chamber via 0.89  mm i d platinum - cured silicone tubing ( cole - parmer ; vernon - hills , il ) . equipment was sterilized by plasma - phase hydrogen peroxide ( sterrad ) processing ( bioreactor , screw caps , and reservoir ) and steam autoclave ( tubing ) . the entire bioreactor was placed in a standard cell - culture incubator ( 37c , 95% humidified air , 5% co2 ) . 24  6  mm cylindrical polyurethane scaffolds ( biomerix ; somerset , nj ) with an average pore size of 200  m and 100% pore interconnectivity were used in all experiments . scaffolds were sterilized by ethylene - oxide gas sterilization by the manufacturer and sealed in single - use packets . mc3t3-e1 murine preosteoblastic cells ( riken cell bank ; ibaraki , japan ) were expanded by traditional 2d static culture at 37c and 5% co2 . louis , mo ) supplemented with 10% fbs ( gibco invitrogen , carlsbad , ca ) was used for this and all subsequent experiments . at confluence , cells were lifted using trypsin , resuspended with the same medium , and used to seed the scaffolds . each scaffold was placed in a separate well of a 6-well tissue - culture plate for seeding . due their hydrophobic nature , the scaffolds were compressed to allow sponge - like absorption of the cellular suspension upon release of the compressing force . the scaffolds were seeded at a standard concentration of 4  10  cells / cm   ( scaffolds processed for histology 12 hours after seeding confirmed even distribution of cells throughout the scaffold ) . after seeding , the scaffolds were surrounded by medium ( to prevent desiccation ) and placed in the cell - culture incubator overnight to allow cellular adherence . after 24 hours in static culture , cell - seeded scaffolds were either place in flow - perfusion culture or continued in static culture . for static culture controls , seeded scaffolds were maintained in 6-well tissue culture plates with enough media to cover the scaffold in its entirety ( 10  ml ) . medium was changed every other day to remove waste products of cell metabolism and provide fresh growth supplements ( this protocol was selected following optimization experiments in which static culture of scaffolds in larger volumes of media , such as that used in flow - perfusion , without media changes led to accumulation of waste products and lack of growth supplements in the vicinity of the scaffold ) , but otherwise the plates were left undisturbed in the cell - culture incubator . scaffolds were harvested at days 0 , 2 , 4 , 6 , and 8 ( n = 3 per time point ) . for dynamic culture , scaffolds were loaded into the bioreactor experimental chambers . each reservoir chamber was loaded with 80  ml of fresh medium , which was recycled for the duration of the experiment ( maximum 8 days ) at a rate of 1.0  ml / min as it entered the experimental chamber . this rate was sufficient to provide perfusion and permit chemotransportation , but only generated a fluid shear stress of approximately 0.02  dynes / cm . the fluid flow rate was based upon optimization studies in which three - dimensional finite element fluid mechanics and mass transport models were developed ( data not shown ) . scaffolds were harvested at days 0 , 2 , 4 , 6 , and 8 ( n = 3 per time point ) . sections were viewed on an olympus bx51 microscope ( olympus ; center valley , pa ) . the depth below the scaffold edge was measured using an objective micrometer ( olympus ) . sections representing the periphery ( top third and bottom third ) and core ( middle third ) of the scaffold were reviewed . the number of cells per 4   low - power field ( lpf ) was counted ( 3 nonconsecutive sections for each region at each time point ) by two blinded investigators . an mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay was used ( sigma ) to measure cellular activity in the scaffolds . in this assay , the conversion of yellow mtt to a purple formazan crystal by viable , metabolically active cells is measured using spectrophotometry . scaffolds were harvested from static and flow - perfusion culture at 1 , 2 , and 6 days , washed in phosphate - buffered saline ( pbs ) , and homogenized . each homogenate was subjected to 6  ml of 0.5  mg / ml mtt ( in pbs ) solution and allowed to incubate for 4 hours at 37c . excess mtt solution was then decanted and 5  ml of extraction solution ( 5  ml isopranolol containing 0.01 n hcl ) was added to the homogenate and allowed to incubate at 20 minutes at 37c . 100  l aliquots of the resulting supernatant were then added to a 96-well plate and the absorbance at 570  nm was determined using spectrophotometry . cell activity was then expressed as the absorbance at 570  nm per gram of scaffold . data were analyzed with a two - tailed student 's t - test assuming unequal variance using sigmastat ( spss science ; chicago , il ) . scaffolds harvested 12 hours after seeding demonstrated a homogenous distribution of cells in pores throughout the scaffold ( figure 2 ) . at this early time point , cell density in the periphery ( 109.3  5.5  cells / lpf ) and core ( 106.8  3.9  cells / lpf ) was not significantly different ( p = .67 ) . scaffolds in static culture had a slow , but significant decline in the peripheral cell density over the course of the experiment ( figure 3 ) . compared to the initial seeding density ( 109.3  5.5  cells / lpf ) , there were 106.7  2.0  cells / lpf ( 97.6% of the initially seeded cells ) at day 2 ( p = .4 ) , 82.3  3.8  cells / lpf ( 75.2% ) at day 4 ( p = .003 ) , 76.3  5.2  cells / lpf ( 69.8% ) at day 6 ( p = .004 ) , and 72.3  1.2  cells / lpf ( 66.1% ) at day 8 ( p = .001 ) . cell density in the periphery was similar to a depth of approximately 2  mm from the superior and inferior surface of the scaffold . there was no statistical difference in cell density between superior and inferior portions of the periphery at any time point ( data not shown ) . in marked contrast to the peripheral crust ( outer 2  mm ) of the scaffold , the core ( central 2  mm ) of these same scaffolds exhibited a rapid decline in cell density ( figure 4(a ) ) . within the core , cell density was 70.3  5.5  cells / lpf ( 64.3% remaining of the initially seeded cells ) at day 2 ( p = .003 ) , 39.0  4.4 ( 35.7% ) at day 4 ( p = .001 ) , 4.67  1.8 ( 4.2% ) at day 6 ( p < .001 ) , and 0.25  0.25 ( 0.2% ) at day 8 ( p < .001 ) . the cusp of viability for cells in the core was day 4 ; between days 4 and 6 , there was a dramatic 88% reduction ( p = .002 ) in cell density ( figure 4(b ) ) . the metabolic activity of cells in static culture also declined over the course of the experiment ( figure 5 ) . mtt cell assay demonstrated that cellular activity in scaffolds in static culture declined to 74% of initial activity ( 0.39  0.04  od / g ) at day 2 and 70% of initial activity ( 0.37  0.04  od / g ) at day 6 . the change in metabolic activity was significant by day 6 ( p = .025 ) . in static culture , metabolically active cells were limited to the periphery of the scaffold . similar to static culture , scaffolds placed in flow - perfusion had a peripheral cell density of 108.0  5.9  cells / lpf ( 98.9% of the initially seeded cells ) at day 2 , 99.3  6.1 ( 90.9% ) at day 4 , 92.7  1.8 ( 84.9% ) at day 6 , and 100.0  1.5 ( 91.5% ) at day 8 ( figure 3 ) . pairwise comparisons demonstrated a significantly greater number of cells in the periphery of scaffolds placed in flow - perfusion on days 6 ( p = .04 ) and 8 ( p = .0001 ) . similar to static culture samples , there was no difference in cellular density between the inferior and superior thirds of the scaffold ( data not shown ) . in marked contrast to static culture , scaffolds placed in flow - perfusion maintained significantly higher cell density within the core of the scaffold ( figures 6(a ) and 6(b ) ) . core cell density was 103.7  2.3  cells / lpf ( 94.9% of the initially seeded cells ) at day 2 , 80  5.5  cells / lpf ( 73.2% ) at day 4 , 83.5  1.5  cells / lpf ( 76.4% ) at day 6 , and 81.7  6.4  cells / lpf ( 74.7% ) at day 8 ( figure 4(a ) ) . the differences in core cell density between static and flow - perfusion culture were statistically significant at days 2 ( p = .01 ) , 4 ( p < .001 ) , 6 ( p = .006 ) , and 8 ( p = .002 ) . the metabolic activity of cells in flow - perfusion culture significantly increased over the course of the experiment ( figure 5 ) . mtt cell assay demonstrated that cellular activity in scaffolds in dynamic culture increased 125% ( 0.66  0.03 od / g ) at day 2 and 285% ( 0.37  0.04  od / g ) at day 6 compared to the initial activity . the increase in metabolic activity in flow - perfusion was not only significantly greater than the initial activity ( day 2 , p = .035 ; day 6 , p = .018 ) , but it was also significantly greater than the activity in static culture at similar time ( day 2 , p = .002 ; day 6 , p = .006 ) . to account for the possibility of confounding shear stress ( despite subthreshold mechanotransductive flow ) or differences in cell distribution in static versus dynamic culture , the core  :  periphery ratio was calculated ( figure 6(c ) ) . in static culture , the ratio was 0.66  0.05 at day 2 , 0.48  0.07 at day 4 , 0.06  0.02 at day 6 , and 0.004  0.004 at day 8 . in contrast , flow perfusion maintained the core  :  periphery ratio to 0.97  0.07 at day 2 , 0.82  0.10 at day 4 , 0.61  0.30 at day 6 , and 0.81  0.07 at day 8 . the differences in core  :  periphery ratio of static versus dynamic were statistically significant : day 2 ( p = .004 ) , day 4 ( p = .005 ) , day 6 ( p = .01 ) , and day 8 ( p = .004 ) . the ability to maintain cell viability in vitro in thick 3d scaffolds has important implications for tissue engineering . for example , attempts at 3d in vitro bone culture without adequate chemotransportation have invariably shown an inverse relationship between construct thickness and cellular survival [ 2 , 12 ] . this inverse relationship is due to a decline in nutrients and accumulation of waste products in the core of the construct during bone matrix deposition and mineralization . traditional cell - culture methods are not well suited to the maintenance of 3d tissue - engineered constructs due to the inherent limitation in chemotransportation . in our study , we demonstrated that , under static culture conditions , cells in the peripheral crust up to 2  mm deep from the external surface were able to survive by static diffusion , a finding that is in agreement with other published accounts describing survival to a depth of 1 - 2  mm [ 3 , 13 ] . however , we noted a linear decline in core cell density over time , with less than 5% of cells remaining after 6 days of static culture . these observed temporospatial differences in cell density with static culture may be explained by two phenomena associated with poor nutrient diffusion : ( 1 ) death of cells in the interior core of the scaffold , and ( 2 ) chemotaxis of cells from the core toward the periphery . in an effort to support 3d tissue - engineered constructs , various bioreactor systems have been designed , including the spinner flask and the rotational bioreactor . however , because these designs simply move fluid across the exterior of the scaffold , chemotransportation to the interior is not guaranteed . the flow - perfusion bioreactor differs in that it ensures nutrient transport by perfusing medium through the interconnected pores of the scaffold . moreover , the design allows the investigator to control mechanical forces ; the pump speed may be set from 3.5 to 200 revolutions per minute for perfusion ranging from 0.26 to 14.8  mls / min , replicating shear stress from 0.0110  dynes / cm - physiologic in vivo range for osteocytes ( 830  dynes / cm ) . we found that dynamic culture using a flow perfusion bioreactor significantly improved core cell activity and density compared to static culture . after a slight initial decrease in cell density between days 2 and 4 , core cell density in flow perfusion was maintained throughout the course of the experiment at approximately 80% of the initial seeding density . likewise , the core  :  periphery ratio , which compensates for differences in cell distribution and potential effects of fluid shear stress between culture systems ,   was steady ( approximately 0.60.8 ) from day 2 to day 8 . while originally designed to improve chemotransportation , the flow - perfusion bioreactor has the ability to generate fluid - shear forces at the cellular level at higher rates of medium flow . although fluid shear forces were intentionally kept subphysiologic ( i.e. , less than 8 dynes / cm ) , to reduce / eliminate the effects mechanical stimulation during this study ( to concentrate on the effect of chemotransportation , and not mechanical stimulation ) , other studies have suggested that fluid shear forces may be useful for bone or vascular tissue engineering [ 11 , 16 , 17 ] . ultimately , in order to solve clinical problems , engineered bony tissue must be fashioned into 3d patient - specific sizes and shapes . custom - printed scaffolds ( e.g. , those designed from actual patient computed tomography data ) are too thick to survive by nonpenetrating chemotransportation . however , the flow - perfusion bioreactor is in theory a scalable technology that should support porous scaffolds of any thickness . this study demonstrates that thick ( > 6  mm ) 3d constructs are sustainable using a flow - perfusion bioreactor . future work will concentrate on computational modeling of the fluid dynamics and mass transport using specific scaffold designs , and on analysis of cellular proliferation , differentiation , and organization within the construct .
background . 
 bone engineering requires thicker three - dimensional constructs than the maximum thickness supported by standard cell - culture techniques ( 2  mm ) . 
 a flow - perfusion bioreactor was developed to provide chemotransportation to thick ( 6  mm ) scaffolds . 
 methods . 
 polyurethane scaffolds , seeded with murine preosteoblasts , were loaded into a novel bioreactor . 
 control scaffolds remained in static culture . 
 samples were harvested at days 2 , 4 , 6 , and 8 and analyzed for cellular distribution , viability , metabolic activity , and density at the periphery and core . 
 results . by day 8 , static scaffolds had a periphery cell density of 67%  5.0% , while in the core it was 0.3%  0.3% . 
 flow - perfused scaffolds demonstrated peripheral cell density of 94%  8.3% and core density of 76%  3.1% at day 8 . 
 conclusions . 
 flow perfusion provides chemotransportation to thick scaffolds . 
 this system may permit high throughput study of 3d tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems .