collagen , one of the major extracellular ( ecm ) components , is critical to the mechanical properties of many types of biological tissues including tendons , ligaments , bones , blood vessels , and skin .
collagen scaffolds have been widely used in tissue engineering , drug delivery , wound healing , and neuroregeneration guide substrate [ 13 ] for its biocompatibility , low toxicity , and well - documented structural , physical , chemical , and immunological properties .
most of these applications require the scaffolds to operate under mechanical stresses , and thus being able to control and tailor the structural - functional integrity becomes crucial .
type i collagen gel prepared from commercially available solutions has been used broadly in biomaterials research .
however , they have extremely poor biomechanical properties compared to the native tissues that they are targeted to mimic or replace .  
aggregation of collagen fibrils forms a collagen fiber , which is the most abundant protein in the body .
collagen can self - assemble through an enzymatic formation of intermolecular crosslinks leading to a head to tail network within the fiber .
the mechanical properties of collagen fibers primarily depend on the formation of intermolecular crosslinks within the fibers to prevent slippage under load .
however in the engineered collagen scaffolds , the density of this type of crosslinking is not large enough for practical applications .
in addition to self - assembly , the overall mechanical strength of collagen fibers can be improved by increasing the density of inter- and intramolecular crosslink with various chemical reagents .  
glutaraldehyde ( ga ) is one of the most common chemical crosslinking reagents for collagen [ 611 ] .
collagen gels crosslinked with ga have already been studied for ocular surfaces , corneal tissue engineering scaffolds , and nanoscale collagen fibril scaffolds [ 8 , 13 ] .
ga helps to retain many of the viscoelastic properties of collagen fibrillar network , and it reacts relatively quickly .
addition of ga will induce covalent bonds between collagen fibrils from aldehyde - amino reactions as well as from aldol condensation .
ga can also lead to intramolecular crosslinks formed between two -chains by aldol condensation . although widely used as a crosslinking reagent for collagen - based biomaterials , the cytotoxicity problem associated with ga is a recognized draw back and prevents its application from in vivo studies .
recently genipin ( gp ) , a compound extracted from the fruit of the gardenia jasminoides , has been shown to effectively crosslink cellular and acellular biological tissues as well as many biomaterials including hydrogels and hydrogel composites .
it was also found that gp is significantly less cytotoxic than ga [ 15 , 16 ] .
similar to ga , gp can also form intramolecular as well as intermolecular crosslinks in collagen .
gp spontaneously reacts with the primary amines , lysine and arginine residues , in collagen to form monomers that further crosslink the collagen .
however , the crosslinking procedure is a complex process , and little is known about the mechanical properties of collagen treated with gp .  
preferred collagen orientation along the dominant physiological loading direction has been observed in many previous studies [ 18 , 19 ] .
assembly of collagen molecules in vitro remains a major challenge for fabricating the next generation of engineered tissues .
molecules can be aligned by flow , microfluidic channels   and the application of external anisotropic mechanical forces [ 2123 ] , electric currents , and magnetic fields .
constant magnetic fields are able to align collagen molecules because the collagen molecules have diamagnetic anisotropy .
barocas et al .   demonstrated the circumferential alignment of the collagen in a tubular mold .
recently , guo and kaufman   utilized the flow of magnetic beads enmeshed in collagen fibrils to align collagen .
the streptavidin - coated ferromagnetic beads ( about 1.5  m in diameter ) were shown to facilitate collagen alignment under magnetic field as low as 10  t. collagen was aligned as the beads move towards the magnetic poles .
the timescales of beads travel and gelation need to be comparable for the alignment to occur properly .   as a network with hierarchical structures ,
the present study was designed to characterize the biaxial tensile and rheological mechanical properties of collagen scaffolds .
different concentrations of gp were used to modify the degree of crosslinking in the collagen scaffolds .
the method of using the flow of magnetic beads enmeshed in collagen fibrils   was adapted to achieve the alignment of fibers in the scaffolds .
the coupled effects of fiber alignment and crosslinking across hierarchies on the mechanical properties of collagen scaffolds were studied .

 genipin crosslinked collagen scaffoldsnutragen type i collagen solution ( 6  mg / ml ) was purchased from advanced biomatirx .
collagen was dissolved in 0.01  n hcl with a ph value of approximately 2.0 .
neutralized collagen solution was prepared by quickly mixing nutragen collagen solution , 10x pbs ( fisher scientific ) , and 0.1  m naoh ( fisher scientific ) solution with a ratio of 8  :  1  :  1 at 4c with a final collagen concentration of 4.8  mg / ml . the ph value of the solution was adjusted to be between 7.2~7.4 .
the neutralized solution was transferred into a custom - made square reservoir with a dimension of about 30  mm  30  mm that sits in a petri dish . on each side of the reservoir
, a 15  mm   1  mm notch was cut to fit the loading bars .
four porous polyethylene bars ( 18  mm  3  mm  1.5  mm ) ( fisher scientific ) prethreaded with nylon sutures were placed by the sides of the reservoir . the dimension between polyethylene bars was about 15  mm  15  mm , and the thickness of the gel was about 1  mm .
the solution was firstly kept in an incubator at 37c for 12 hours for gelation [ 4 , 6 , 11 ] . during gelation
, the polyethylene bars were polymerized into the collagen gels   ( figure 1 ) .
the collagen gels were then immersed in 0.03% , 0.1% , and 0.25% gp solutions for another 6 hours in the incubator for crosslinking . 
 
nutragen type i collagen solution ( 6  mg / ml ) was purchased from advanced biomatirx . collagen was dissolved in 0.01  n hcl with a ph value of approximately 2.0 .
neutralized collagen solution was prepared by quickly mixing nutragen collagen solution , 10x pbs ( fisher scientific ) , and 0.1  m naoh ( fisher scientific ) solution with a ratio of 8  :  1  :  1 at 4c with a final collagen concentration of 4.8  mg / ml . the ph value of the solution was adjusted to be between 7.2~7.4 .
the neutralized solution was transferred into a custom - made square reservoir with a dimension of about 30  mm  30  mm that sits in a petri dish . on each side of the reservoir
, a 15  mm   1  mm notch was cut to fit the loading bars .
four porous polyethylene bars ( 18  mm  3  mm  1.5  mm ) ( fisher scientific ) prethreaded with nylon sutures were placed by the sides of the reservoir .
the dimension between polyethylene bars was about 15  mm  15  mm , and the thickness of the gel was about 1  mm .
the solution was firstly kept in an incubator at 37c for 12 hours for gelation [ 4 , 6 , 11 ] . during gelation
, the polyethylene bars were polymerized into the collagen gels   ( figure 1 ) .
the collagen gels were then immersed in 0.03% , 0.1% , and 0.25% gp solutions for another 6 hours in the incubator for crosslinking . 

magnetically aligned collagen gelthe aligned collagen scaffolds were obtained with the aid of a flow of magnetic particles embedded in collagen fibrils .
streptavidin - coated iron oxide magnetic particles ( bangs labs ) of 1.5  m in diameter were added into the neutralized collagen solution at a concentration of 0.1  mg / ml . the solution with beads was then transferred into the reservoir and incubated for 12 hours at 37c , during which a magnetic bar was placed under the petri dish .
after gelation , samples were immersed in 0.03% , 0.1% , and 0.25% gp solutions for further crosslinking . 
 
the aligned collagen scaffolds were obtained with the aid of a flow of magnetic particles embedded in collagen fibrils .
streptavidin - coated iron oxide magnetic particles ( bangs labs ) of 1.5  m in diameter were added into the neutralized collagen solution at a concentration of 0.1  mg / ml . the solution with beads was then transferred into the reservoir and incubated for 12 hours at 37c , during which a magnetic bar was placed under the petri dish .
after gelation , samples were immersed in 0.03% , 0.1% , and 0.25% gp solutions for further crosslinking .
the morphology of the collagen scaffolds was examined using a joel jsm-6100 sem operated at 10  kv . to prepare the sample for sem , crosslinked collagen gels
the fixed samples were dehydrated in a graded distilled water / ethanol series : 30% , 50% , 70% , and 100% for 15 minutes each , followed by washes with a graded ethanol / hmds series : 30% , 50% , 70% , and 100% for 15 minutes each , and finally allowed to dry overnight [ 2931 ] .
sem images were taken at multiple locations across the sample and used to qualitatively assess the alignment of collagen fibers .
the tensile mechanical properties of collagen scaffolds were characterized using a planar biaxial tensile tester . in biaxial tensile testing ,
a roughly square - shaped specimen was mounted so that it could be stretched along both the x and y in - plane directions .
four carbon dots markers forming a 5  mm  5  mm square were placed in the center of the testing specimen , and a ccd camera was used to track the position of markers from which the tissue strains in both directions can be determined throughout the deformation .
tensile tension was applied to the specimen and the load was measured using load cells during the loading and unloading processes .
samples were preconditioned equibiaxially for 8 cycles with a load of 10  g to achieve a repeatable material response .
the samples were then tested under load control method and subjected to a set of equibiaxial loads with the maximum loads varying from 70  g to 100  g. cauchy stress and logarithm strain were calculated   and used for the description of the biaxial tensile mechanical behavior of collagen gel .
the mechanical properties of collagen gel were also assessed using a parallel plate rheometer ( ar2000 , ta instrument ) . neutralized collagen solution was prepared as stated above with a final collagen concentration of 4.8  mg / ml . the solution was transferred to p60 petri dishes and incubated at 37c for 12 hours .
the collagen gels were then crosslinked with 0.03% , 0.1% , and 0.25% gp for 6 hours . before rheometry tests ,
the gel samples of 60  mm in diameter and 2  mm in thickness were carefully removed from the petri dish and transferred to the bottom plate of the rheometer .
the dynamic storage and loss moduli were evaluated at 1% shear strain amplitude at frequencies ranging from 0.1 to 10  hz . for strain sweep test
, the moduli were evaluated at shear strain ranging from 0 to about 10% at 5  hz .   in order to capture the characteristics of the stress - strain behavior exhibited by collagen gels ,
the strain energy function was originally introduced by holzapfel et al .   for the description of the passive mechanical response of arterial tissue , in which each layer is treated as a fiber - reinforced material with the fibers corresponding to the collagenous component distributed helically around the arterial wall .
the structurally based constitutive model has material parameters possessing physical meanings that can be related to the structure and components of the material being studied .
the constitutive model was chosen in this study as it has been successfully used in previous studies for collagenous cardiovascular tissue and elastin - degraded arteries [ 3537 ] .
the general form of the strain energy function is 
 ( 1)u = c10(i13)+k12k2=1n{exp  [ k2e2]1 }  + 1d((jel)212ln  jel ) . 
							 in ( 1 ) , e=def(i1 - 3)+(1 - 3)(i4()-1 ) , where  = ( 1/4)0()sind. the response of the model is governed by two parts , the isotropic and anisotropic components .
the first term in ( 1 ) captures the isotropic behavior of the matrix material .
i1 is the first invariant of tensor c , where c  is the modified counterparts of right cauchy - green tensor .
c10  is a stress - like material parameter associated with the isotropic part of the overall response of the tissue .
the anisotropy of the tissue is captured by the second term in ( 1 ) .
an assumption in this model is that the collagen fibers are only active in extension and not in compression .
k1 is another stress - like parameter , and k2 is a dimensionless constant related to the collagen fibers .
n is the number of families of fibers , and  is a measure of the dispersion of the fiber orientation around a mean direction , as shown in figure 2(a ) .
the mean direction of the fibers ,  , is defined as the angle between the circumference direction of the tissue and the mean direction of the fibers .
( ) is the orientation density function and gives the normalized number of fibers within a certain orientation with respect to the mean direction .
i4( ) are squares of the stretches in the direction of the  family of fibers . finally , d is the inverse of the bulk modulus and is set to zero as the material is assumed to be incompressible .
interested readers are referred to gasser et al .   for more detailed explanations of the model . due to the symmetric loading conditions in biaxial tensile test
, a quarter of the collagen sample was modeled in abaqus 6.8 - 4 with loading and boundary conditions shown in figure 2(b ) .
shell edge loading x- and y - symmetry boundary conditions were applied in order to simulate biaxial tensile testing experimental settings .
general - purpose shell elements ( s4r ) with inherent plane stress assumption were used in the finite element model .
material parameters were adjusted to fit the simulation results to experimental stress - strain curves .
thermally crosslinked collagen gels at 37c without any crosslinking reagents have a whitish color and are semitransparent .
collagen gels cross - linked with gp turn into bluish color and become opaque , as shown in figure 1 .
figure 3 shows the representative stress - strain responses of collagen scaffolds crosslinked with different concentration of gp .
samples were under equibiaxial tensile test with a maximum load of 70100  g. the biaxial tensile test revealed that the stiffness of collagen gel increases with higher concentrations of gp .
it is also noted that all collagen scaffolds exhibit isotropic mechanical behavior as seen from the equibiaxial testing results . to further compare the stiffness of crosslinked collagen gels ,
the tangent modulus et was obtained for each sample by differentiating the stress - strain curves and was estimated from et  0.5  d/d .
averaged tangent moduli were then obtained for collagen scaffolds with 0.03% , 0.1% , and 0.25% gp .  
figure 4 shows that the initial tangent moduli of collagen scaffold increase with the concentration of gp .
the tangent moduli also increase with strain . for strain less than 2% , the tangent moduli of the 0.03% and 0.1% gp crosslinked collagen scaffolds increase faster than the 0.25% gp crosslinked one .
however , the tangent modulus of the 0.25% gp crosslinked collagen gel remained the highest .
when strain is higher than 2% , the tangent moduli of the 0.1% gp crosslinked scaffolds are about the same as the 0.25% gp crosslinked scaffolds . as the strain further increases , the tangent moduli increase at about the same rate for all the samples .  
rheological testing with a parallel plate rheometer demonstrate that the storage ( g ) and loss ( g ) moduli of gp - crosslinked collagen scaffolds both increase with gp concentration , as shown in figures 5(a ) and 5(b ) .
the g increases slightly with frequency , while the g decreases with frequency initially followed by a slight increase .
the g of collagen gels is about 10 times greater than the g which suggests that collagen gel is a predominantly elastic material with small viscosity .
the variation of storage and loss modulus with shear strain amplitudes are presented in figures 5(c ) and 5(d ) .
there is a slight decrease in the storage moduli and an increase in the loss moduli with increasing strain amplitude .
overall , the storage and shear moduli do not vary significantly within the shear strain range applied in this study .  
the nonaligned collagen gel was also examined for comparison . as shown in figure 6(a ) , the fibers in the nonaligned collagen gel distribute randomly and there is no preferred fiber distribution . on the other hand , the fibers in the aligned collagen gel show an overall fiber alignment in a particular direction , as shown in figure 6(b ) .
the mechanical behavior of the aligned collagen scaffolds were tested using a biaxial tensile tester and compared with the results from the nonaligned ones .
the samples were subjected to equibiaxial tensile test with one loading axis parallel to the fiber alignment direction while the other loading direction perpendicular to the fiber alignment direction .
the nonaligned collagen scaffolds with magnetic beads imbedded were also tested to validate that the presence of beads would not affect the mechanical property of collagen scaffolds ( results now shown ) .
the stress - strain responses of the aligned collagen scaffolds are shown in figure 7 . compared with the nonaligned scaffolds ,
the aligned ones demonstrate obvious anisotropic mechanical behavior as manifested by one direction being stiffer than the other . as expected , the scaffolds are stiffer in the direction parallel to fiber alignment than in the direction perpendicular to the fiber alignment .
the nonaligned collagen scaffolds appear to be isotropic with the stress - strain curves falling in between those from the aligned scaffolds . 

 was set to 45 and  was set to 0.333 to represent an isotropic material .
the c10 , k1 , and k2 values were then chosen in order to fit the model to experimental data .
for the aligned scaffolds , c10 was kept the same as the corresponding nonaligned one and appropriate choices of k1 , k2 ,  , and  were made to fit the experimental data .
the change of color in collagen scaffolds crosslinked with chemical reagents indicates microstructure changes during crosslinking . for gp
it was proposed that the reaction between gp and an amino acid in collagen molecule will form a monomer and further radical reaction will cause dimerization .
previous studies suggested that gp may form intramolecular and intermolecular crosslinks within collagen fibers in biological tissue .
various methods have been developed to improve the mechanical properties of collagen scaffolds including physical crosslinking through exposure to a radiation sources such as uv light [ 41 , 42 ] and chemical crosslinking by chemical reactions with various crosslinking reagents [ 6 , 43 ] .
it has been shown that the stiffness of collagen gel increases with higher ga concentration . however , this trend stops when the ga concentration is higher than a threshold value .
previous study using dermal sheep collagen   showed that collagen network treated with 0.08% and 0.5% ga had a similar modulus .
.   suggested that a complete crosslinking in collagen gel is reached when the ga concentration is 0.12% .
after fully crosslinking , no more amino groups will be available to react with the aldehydes from ga .
lacking of amino groups in the collagen gel will lead to self - polymerization of ga , which may adversely affect the mechanical property of the entire collagen gel [ 9 , 14 ] .
although ga crosslinking greatly improved the mechanical strength of collagen gel , the potential toxic effect has been a vital drawback of this commonly used chemical reagent for biological tissues .
a few studies have demonstrated that gp has the potential to be used as a substitute crosslinking reagent [ 7 , 15 , 16 , 44 ] . among these studies , gp has been found to be significantly less cytotoxic than ga [ 15 , 44 ] . from the mechanical perspective
, previous studies found that gp can be used to stiffen collagen gels in a relatively short - time frame .
it is also noted that the mechanical properties of collagen gel can be controlled by varying the concentration of gp .
sundararaghavan et al .   found that the degree of crosslinking and the storage modulus of gp - crosslinked collagen gel increased with higher gp concentration .
our results from both biaxial tensile testing ( figures 3 and 4 ) and rheometry measurements ( figure 5 ) show that the tangent modulus , the storage , and loss modulus of collagen scaffolds increase with gp concentration . although genipin can be used to tune the overall stiffness of collagen scaffolds , higher concentrations of genipin can lead to significant cell death , which needs to be considered in applications of cell - populated collagen scaffolds .
mechanical properties of collagen scaffolds are not only affected by the degree of crosslinking , but also highly correlated with the orientation of collagen fibers in the scaffolds .
preferred fiber orientation along the dominant physiological loading direction has been observed in many previous studies on various tissues [ 4547 ] .
collagen fiber orientation plays important roles in determining the mechanical functionalities of collagen - based native and engineered biological material . in the present study ,
the alignment of collagen fibers was achieved via the flow of streptavidin - coated magnetic beads bond with collagen fibrils proposed previously by guo and kaufman , to which interested readers are referred for greater detail .
briefly , the streptavidin coating contains an arg - tyr - asp ( ryd ) amino acid sequence similar to the rgd receptor domain of fibronectin , which has a function of binding with collagen fibrils .
our results demonstrated that , with this simple technique , the resulted scaffolds possess obvious anisotropic mechanical properties as manifested by the stress - strain responses from equibiaxial tensile test ( figure 7 ) .
such kind of preliminary results are usefully for future quantitative investigation of the structure - function relation of collagen scaffolds .
the anisotropic scaffolds are stiffer in the direction that the fibers are aligned than in the direction perpendicular to the alignment .  
our results also demonstrated that crosslinking of the scaffolds can be used to tune the overall stiffness of the scaffolds without affecting the existence of anisotropy in the collagen gel .
previous study by sung et al .   found that fixation of the aortic valves using gp and ga did not alter the mechanical anisotropy observed in fresh valve leaflets .
they concluded that the intramolecular and intermolecular crosslinks introduced into the collagen fibrils during fixation are of secondary importance to the presence of structural and mechanical anisotropy in fresh leaflets .
studied the effect of nitrite on the mechanical properties of uniaxially and biaxially constrained collagen gels .
they quantitatively demonstrated that the crosslinking agent did not alter the fiber distribution in collagen gels significantly .
their study also suggested that the stiffening of collagen fibers might be another source of the anisotropic mechanical behavior seen in the uniaxially constrained collagen gel , in addition to the reorientation of fibers in the constraint direction . in the present study , the reorientation of collagen fibers induced by magnetic flow plays a dominant role in controlling the anisotropic mechanical behavior of collagen scaffolds .  
planar biaxial tensile testing was used in this study to characterize the tensile mechanical properties of collagen scaffolds .
planar biaxial tensile test with independent control of load in both perpendicular loading directions has been used broadly to study the mechanical behavior of various native and engineered biological tissues [ 28 , 32 , 49 , 50 ] .
although it can not replicate the physiological loading conditions , biaxial tensile test is sufficient on elucidating the anisotropic mechanical properties of biological tissues with plane stress assumptions . in the present study
, equibiaxial tensile testing was performed to study the effects of crosslinking and fiber - alignment on the mechanical properties of collagen scaffolds .
rheological testing has been applied to study the mechanical properties of polymeric and biological materials [ 29 , 51 , 52 ] .
our results show that higher gp concentration will generate a collagen gel with higher dynamic storage and loss moduli , which is consistent with previous study by sundararaghavan et al . .
although biaxial tensile testing results show a similar trend , the tangent stiffness from biaxial tensile test is two to three orders of magnitude higher than the storage moduli from rheological measurements , which is much greater than 3  :  1 for ideal elastic networks .
higher ratio of tangent modulus to shear modulus has been observed in previous studies on biomaterials and hydrogel system . using a three - point bending test , spatz et al .  
reported that the ratio of young 's modulus to shear modulus of cortical bone is in the order of 20  :  1 .
they suggested that materials comprising stiff fillers embedded in a compliant matrix could have a rather low shear modulus .
such kind of properties allow hollow bones to react smoothly to local impacts , which otherwise may lead to failure .
study by richter   on poly(vinyl methylether ) hydrogels showed that the ratio of compressive to shear moduli varied from 4.8 to 10.9 , which was contributed to the inhomogeneity of the sample .  
it is known that collagen gel is a biphasic system consisting of a fibrillar network structure filled with a large excess of interstitial fluid .
tensile tests on collagen gels probe the tensile behavior of the fibrillar network , and the interstitial flow resistance is negligible in extension tests .
results from our biaxial tensile testing on collagen scaffolds give a nonlinear stress - strain response with a compliant  toe  region , followed by a stiff region .
region is due to the network orientation changes toward the loading axis , while the stiff region corresponds to resistance from fibril extension . in rheological test , the fluids or soft solids flow rather than
the top plate of the rheometer compresses the collagen gel , oscillates , and exerts a dynamic torsional force on the sample . during the unconfined compression fluid , escape may not be trivial anymore .
another reason which may contribute to the low - measured shear modulus is that , during rheological test , fluid running out of the collagen gel can act as a lubricant between the gel surface and top plate .
thus the gel might in fact experience smaller shear than that applied by the top plate .
this lubricant effect may dominate the increase in storage and loss moduli associated with dehydration .
results from our study suggest that , in rheological test , the movement of the interstitial fluid play a significant role in determining the overall mechanical properties of a collagen gel .
it is important to understand the different mechanical properties that a biphasic collagen gel can exhibit under specific loading conditions , as such understandings are necessary for mechanobiological studies in which ecm mechanics play critical roles . the anisotropic constitutive model in ( 1 )
was proposed originally for simulating arterial tissue in which elastin is the primary means of support at low stress - loading regions and then collagen being the major load - bearing component at higher stresses [ 33 , 34 ] . in an earlier study by holzapfel et al .
their study using the constitutive model by holzapfel et al .   was successful in modeling the intimal and adventitial layers that lack two distinct structural components as the medial layer . regarding the parameters in the model with respect to our collagen gels
, there was no elastin or other ecm ground substance material that support the initial loading in this experiment .
the c10 parameter is likely more representative of some other phenomenon such as uncoiling of collagen fibers .
parameters  and  can vary the amount of anisotropy in the stress - strain curves , and  has a greater effect on the amount of curvature in the stress - strain curve [ 33 , 59 ] .
finally , the most significant effect of increasing / decreasing k1 and k2 is in translating the stress - strain curves to lower / higher strains , respectively .
this inverse relationship in the material is logical as they describe the properties of the collagen fibers .  
examining the parameters for the aligned and nonaligned collagen gels , we initially attempted to take the parameters from the nonaligned simulations and only change  and  to create a best fit of the aligned collagen gel , since  controls the orientation of the fibers , and  determines the fiber dispersion ( amount of fiber alignment ) .
fitting of  and  has generally been done phenomenologically instead of based on histology studies .
however , it is interesting to point out that our results indicate a correlation between the degree of anisotropy demonstrated by stress - strain curves and . among the three aligned collagen scaffolds shown in figure 8 , the collagen scaffolds aligned with 0.03% gp possessing the highest amount of anisotropy has the smallest model parameter  correspondingly . unfortunately , a good fit of the aligned collagen gel was not possible without modification of the k1 and k2 as well .
this seems to indicate that the alignment process is affecting the material properties of the collagen fibers and not just their orientation / dispersion .   in the nonaligned and aligned collagen scaffolds , c10 values
were increased with higher gp concentrations , which suggest higher c10 values for stiffer tissues resulted from increased crosslinking .
there was also an increase in the values of k1 and k2 with gp concentration .
previous studies have shown that the method of crosslinking can affect how the crosslinks are organized in the collagen fibers .
genipin has been shown to not only cause collagen to become more crosslinked but also cause the fibers to merge together and form thicker structures .
increase in k1 and k2 indicates that stronger fibers present in collagen gels with higher gp concentration .
takiuchi et al .   showed that crosslinking along fibers may happen more often than across fibers .
they showed that during the process of fixing tendon samples in formalin , the stiffness along the fibers increased linearly with fixing time .
this was explained by a difference in the number and formation rate of crosslinks in the direction along the fibers and across the fibers .
the method for collagen fiber alignment is ideal for thin gels with thickness around 10~20  m ; however , thicker gels were necessary for biaxial testing in this experiment .
thus , it might be possible that alignment of collagen fibers is not uniform in such relatively thick gel , which could have also caused the need to change the values of material parameters in the model .
the fiber alignment method was adopted in this study for the easiness of experimental implementation .
the overall research approach can be applied to other studies with more controllable alignment methods in the future .
sem was used in this study to qualitatively access the fiber alignment . due to the higher concentration of collagen , our existing confocal microscopy capability was incapable of providing quantitative information on fiber orientation .
more quantitative correlation between the structure and function of collagen scaffolds through the constitutive model might be possible by applying experimental techniques .
for example , values for mean fiber orientation  and dispersion around the main direction  have been determined previously for collagen fibers in bladder tissue using small angle light scattering . using scanning electron microscopy techniques ,
fiber mechanical properties were investigated using nanoindentation   or micromanipulation / tensile testing of individual fibers .
improving mechanical properties of collagen scaffolds via a controlled collagen assembly process will greatly broaden their application in numerous life science researches and will be a substantial step toward biomaterial research on tissue repair and replacement . in the present study ,
the mechanical properties of collagen scaffolds with the effects of crosslinking and fiber alignment were studied both experimentally and theoretically .
equibiaxial tensile tests were performed to probe the elastic properties of collagen fibrillar network within the collagen scaffolds .
the anisotropic behavior of collagen scaffolds is correlated with the presence of preferred collagen fiber alignment .
our results demonstrated that crosslinking of the scaffolds can be used to tune the overall stiffness of the scaffolds without affecting the presence of anisotropy in the collagen matrix .
the tangent stiffness from biaxial tensile tests is two to three orders of magnitude higher than the storage moduli from rheological measurements .
this suggests that in rheological tests the movement of the interstitial fluid play a significant role in determining the overall mechanical properties of a collagen gel .
therefore , the highly hydrated collagen gel comprising stiff fibrillar network embedded in a very compliant matrix has rather low shear modulus .
the stress - strain responses of both nonaligned and aligned collagen scaffolds were captured well with the anisotropic hyperelastic constitutive model .
results from simulations suggest possible changes of the mechanical property of collagen fiber in the aligned collagen scaffolds due to preferred crosslinking along the fibers .
the structurally based constitutive model is promising in future studies on relating material parameters possessing physical meanings to the microstructure of collagen scaffolds .
 collagen type i scaffolds are commonly used due to its abundance , biocompatibility , and ubiquity . 
 most applications require the scaffolds to operate under mechanical stresses . 
 therefore understanding and being able to control the structural - functional integrity of collagen scaffolds becomes crucial . using a combined experimental and modeling approach , we studied the structure and function of type i collagen gel with the effects of spatial fiber alignment and crosslinking . 
 aligned collagen scaffolds were created through the flow of magnetic particles enmeshed in collagen fibrils to mimic the anisotropy seen in native tissue . inter- and 
 intra- molecular crosslinking was modified chemically with genipin to further improve the stiffness of collagen scaffolds . 
 the anisotropic mechanical properties of collagen scaffolds were characterized using a planar biaxial tensile tester and parallel plate rheometer . 
 the tangent stiffness from biaxial tensile test is two to three orders of magnitude higher than the storage moduli from rheological measurements . 
 the biphasic nature of collagen gel was discussed and used to explain the mechanical behavior of collagen scaffolds under different types of mechanical tests . 
 an anisotropic hyperelastic constitutive model was used to capture the characteristics of the stress - strain behavior exhibited by collagen scaffolds . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions