in tissue engineering research , it is necessary to develop effective culture substrates for cells to facilitate regeneration of deficient tissue .
it has been shown that the environment surrounding cells has a significant effect on cell behavior .
the physical conditions of any scaffold to which cells adhere are an important determinant of cell growth [ 16 ] , along with biochemical signals .
cells receive various types of information from the culture substrate , such as surface stiffness [ 14 ] or roughness [ 5 , 6 ] .
therefore , a good understanding of the relationship between cellular behavior and the physical characteristics of culture substrates is critical for development of effective scaffolds that allow suitable cell adhesion , proliferation , and differentiation . in a previous study
, we demonstrated that ferric - ion - cross - linked alginate ( fe - alginate ) was an efficient material for use as scaffold , allowing good cell adhesion and proliferation .
alginates are a material widely used in various biomedical fields , and are composed of 1,4-linked -d - mannuronic acid ( m ) and -l - guluronic acid ( g ) residues , forming gels with certain multivalent metal ions [ 8 , 9 ] .
calcium ions are used most frequently as a cross - linking agent for alginates , but calcium - ion - cross - linked alginate ( ca - alginate ) is known to be an unfavorable culture substrate for cells .
fe - alginate is able to overcome the deficiencies of ca - alginate , such as poor protein adsorptive capacity , and has been established as an effective cell culture substrate .
alginates are commonly derived from seaweed , and have widely varying proportions and sequences of two the m and g monomers depending on the source from which they are derived [ 8 , 10 ] .
it is known that the m / g ratio influences the mechanical properties of ca - alginate gels : those with a high g - content have higher mechanical strength than those with a high m - content .
it has also been reported that high - g ca - alginate forms more porous gels , and that the cross - linkages tend to be more stable over a longer period , than is the case for high - m alginate . in the case of fe - alginate
, it is considered that the alginate composition can also influence the condition of the gel .
however , the effects of fe - alginate substrate composition on gel properties and the growth of cultured cells have not been clarified .
the aim of this study was to define the relationship between fe - alginate composition and the growth of cells cultured on fe - alginate films .
we evaluated the efficiency of cell adhesion and proliferation on fe - alginate films with differing m / g ratios , using normal human dermal fibroblasts ( nhdf ) as model cells .
we also focused on the degree of change in the condition of the fe - alginate gel during cell culture . here
we report that favorable cell proliferation was observed on high - g , rather than on high - m fe - alginate gels , and that the stability of fe - alginate gel with firm cross - linkage between alginate and ferric ions seems to be a key supportive element for good cell proliferation .
eagle 's minimum essential medium ( e - mem ) was obtained from nissui pharmaceutical ( tokyo , japan ) , fetal bovine serum ( fbs ) was from tissue culture biologicals ( tulare , ca , usa ) , and trypsin was from becton dickinson ( sparks , md , usa ) .
anti - vitronectin polyclonal antibody was purchased from abd serotec ( oxford , uk ) .
horseradish peroxidase ( hrp)-conjugated anti - rabbit igg was from santa cruz biotechnology ( santa cruz , ca , usa ) .
all other chemicals were purchased from wako pure chemical co. ( tokyo , japan ) .
two types of alginate were used in this study : a high g - content alginate ( m  :  g ratio 34  :  66 ) and a high m - content alginate ( m  :  g ratio 57  :  43 ) . the viscosity ( 1% ( w / v ) solution at 20c ) of both alginates , purchased from kimica corporation ( tokyo , japan ) , was about 100200  mpas .
the procedures for preparation of the fe - alginate gel films have been described previously .
briefly , 3  ml of 1% ( w / v ) alginate solution was added to the wells of 6-well tissue culture plates along with cellulosic sheets ( bemcot , ozu corporation , tokyo , japan ) as frames , and then dried at 60c overnight . after drying ,
3  ml of 500  mm or 50  mm fecl3 was added to each well to induce cross - linkage of the alginate at 25c for 30  min .
the resulting gel films were washed with deionized water to remove unreacted ions on the surface , and sterilized with 70% ( v / v ) ethanol . before seeding of cells on the films , the films were stabilized by immersion in e - mem at 37c under a 5% co2 atmosphere for 72  h , and the medium was replaced every 24  h. nhdf were cultured in e - mem with 10% ( v / v ) fbs at 37c under a 5% co2 atmosphere . to detach cells by trypsinization
, the cells were incubated with 0.25% ( w / v ) trypsin and 0.02% ( w / v ) edta in ca- , mg - free phosphate - buffered saline ( pbs ) at 37c for 10  min .
e - mem containing 10% ( v / v ) fbs was subsequently added to terminate the enzyme reaction .
the cell suspension was centrifuged at 1000  g for 5  min and resuspended in e - mem containing 10% ( v / v ) fbs .
the cells were counted with a coulter counter ( beckman coulter corporation , fl , usa ) and seeded onto fe - alginate films in 6-well tissue culture plates at a density of 4000  cells / cm in 3  ml of culture medium .
samples were incubated at 37c under a 5% co2 atmosphere and the medium was replaced every day .
after 1 , 5 , and 8 days , the cells were observed using a phase - contrast microscope and photographed , and then cell counting was performed to quantify the degree of cell proliferation . to count the attached cells ,
the cells were detached by trypsinization at 37c for 10  min , and then resuspended in e - mem containing 10% ( v / v ) fbs .
cells were also cultured on high m - content fe - alginate films after immersion in e - mem at 37c under a 5% co2 atmosphere for 8 days to stabilize the gel condition .
alginate films were immersed in fbs ( 0.2  ml / cm ) in a 6-well tissue culture plate at 37c for 2  h. after incubation , the films were washed with deionized water , and transferred to new tissue culture plates .
then 1% triton x-100 ( 0.1  ml / cm ) was added to desorb the proteins on the film surfaces at 37c for 2  h. protein concentrations of the desorption fraction were determined using a bicinchoninic acid ( bca ) protein assay reagent kit ( thermo fisher scientific pierce biotechnology , il , usa ) in accordance with the manufacturer 's instructions .
the solutions were also collected to separate the proteins in the solutions using sds - page on 10% polyacrylamide gel .
protein bands in the gel were transferred to immobilon membranes ( millipore , bedford , ma , usa ) for immunoblot analysis .
the blotted membranes were treated with 5% ( w / v ) non - fat dried milk in pbs for blocking , and then incubated with anti - vitronectin antibody diluted with the blocking solution at room temperature for 60  min .
after washing with 0.05% ( v / v ) tween 20 in pbs , the membranes were incubated with hrp - conjugated secondary antibodies at room temperature for 60  min , and the immunospecific bands on the membranes were visualized by the dab color reaction method .
quantitative analysis of adsorbed vitronectin was performed using image j 1.40  g ( http://rsb.info.nih.gov/ij ) .
alteration of alginate gel conditions during the culture period was evaluated by measuring the degree of swelling and polymer content of the gels .
the assessments were conducted at 1 , 5 , 8 , and 16 days after the start of incubation . to investigate the degree of gel swelling ,
the thickness of the alginate films was determined using a dial thickness gauge ( g-7c ; ozaki mfg .
co. , ltd . ) . for estimation of polymer content , dry weight was measured after cultivation by weighing the gel that had been dried at 60c for 2  h. statistical analysis was performed using kaleidagraph version 4.0 software .
the adequacy of high g- and m - content fe - alginate films as culture substrates was investigated by cultivation of nhdf on the respective gels . in the initial stage of culture ,
the number of attached cells and cell morphology showed little difference between the two fe - alginates ( figures 1 and 2 ) .
however , as culture time progressed , the number of attached cells on the high - g fe - alginate gel was notably higher than that on the high - m gels ( figure 1 ) .
cells on the high - g fe - alginate film showed good spread morphology throughout the culture period ( figures 2(a)2(c ) ) .
conversely , on the high - g fe - alginate film , many cells became detached from the substrate , while maintaining cell - cell association , after 8 days of culture ( figure 2(f ) ) .
the protein adsorption ability of high - g and high - m alginate was investigated .
quantitative analysis demonstrated that both types of gel had an equivalent ability to adsorb serum proteins ( figure 3(a ) ) .
moreover , immunoblot analysis revealed the same degree of adsorption of vitronectin , the major cellular adhesion - related serum protein , on both alginate gels ( figure 3(b ) ) .
changes in the conditions of fe - alginate gel in the culture medium were evaluated by measurement of gel thickness and dry weight ( figure 4 ) .
gel thickness gave an indication of the degree of swelling , whereas dry weight reflected the degree of gel disintegration .
the conditions of the high - g fe - alginate gel showed no significant change even after 16 days .
in contrast , the high - m fe - alginate gel became thicker , and the dry weight decreased with time .
the rate of change in the properties of the high - m fe - alginate gel was rapid until around 8 days , but became more stable thereafter .
fe - alginate gel was stabilized by immersion in culture medium for 8 days to prevent any further changes in gel condition .
good cell proliferation was confirmed on stable high - m fe - alginate films at the same level as that on high - g fe - alginate films ( figure 5 ) . to investigate whether the amount of cross - linked ferric ions has a decisive influence on the properties of the two types of alginate , we studied cell growth on fe - alginate films with a low concentration of cross - linked ions .
the concentration of ferric ions for cross - linkage of alginate employed under ordinary circumstances ( 500  mm fecl3 ) was determined previously to obtain sufficient film strength and to avoid excessive inhibition of cell growth , and the minimum required concentration of fecl3 solution to maintain the film strength needed for cell culture using our methodology was 50  mm . in both types of alginate , cell growth on gels with
a low concentration of cross - linked ferric ions was comparable to that on gels with a normal iron concentration ( figure 6(a ) ) .
the same tendency was revealed with regard to gel condition ( figures 6(b ) and 6(c ) ) , although the thickness of both types of alginate gel increased as the concentration of cross - linked ferric ions decreased ( figure 6(b ) ) .
to establish fe - alginate as an effective material for biomedical applications in research and therapeutics , an understanding of how the composition of fe - alginate influences its suitability as a culture substrate for encouraging good cell growth is of considerable significance . in this study , we observed that strong cross - linkage with ferric ions was necessary to ensure good cell proliferation during culture , and that alginate with a high g rather than a high m content showed better conservation of ferric ion cross - linkages . in the case of ca - alginate gels , it is reported that the m / g ratio influences cell compatibility .
for instance , murine insulinoma cells encapsulated in high - m rather than high - g ca - alginate showed rapid growth and good metabolic activities .
conversely , in the case of encapsulated neural stem cells , high - g ca - alginate microcapsules allowed better release of neurotrophic factor .
studies of ca - alginate as a scaffold for cells have revealed that a high g content promptes good cell growth [ 14 , 15 ] .
our present results demonstrated superior nhdf cell growth on high - g rather than high - m fe - alginate films ( figure 1 ) .
additionally , we demonstrated that good cell growth occurred on high - m fe - alginate films under conditions that tended not to result in gel swelling and disintegration ( figure 5 ) .
it is known that cells recognize proteins that are adsorbed onto the surfaces of substrate materials [ 1618 ] . in particular
, vitronectin is a major factor that mediates initial adhesion of nhdf onto fe - alginate films in the presence of serum . in the present study ,
there was little difference in the ability to adsorb serum proteins , including vitronectin , between the two types of fe - alginate gel ( figure 3 ) .
high - m and high - g alginate gels showed comparable efficacy to promote initial cell adhesion ( figures 1 and 2 ) , being consistent with their similar protein adsorption capacities .
the interaction between cells and high - m fe - alginate appeared to be weakened in response to alterations in the condition of the gel .
a reduced degree of cell - substratum interaction probably caused cell detachment , although cell - cell interaction was maintained .
swelling and dissolution of high - m fe - alginate observed in this study may have been caused by loss of cross - linked ferric ions from the gels . under physiological conditions ,
disengagement of cross - linking ions from alginate gels occurs in the presence of chelators , monovalent ions , and non - cross - linking divalent ions [ 19 , 20 ] .
it is also reported that high - m ca - alginate shows loss of mechanical strength during culture .
calcium ions are known to bind to g- and mg - blocks of alginate . however , since interactions between calcium ions and the hydroxyl groups of mg are prevented by the intrinsic configurational characteristics of the mg building blocks , mg sequences in ca - alginate have a more flexible structure than g sequences . therefore the structures formed by mg junctions are likely to disassociate in response to the presence of competing ions or application of stress .
although the blocks of alginate involved in binding to ferric ions are unknown , the present study suggested that the binding of ferric ions to alginate might occur through a mechanism similar to that for calcium ions .
it can be inferred that ferric ions combine with not only g - blocks but also mg- and/or m - blocks in alginate , because gel swelling and breakage were observed in high - m rather than high - g alginate under physiological conditions ( figure 4 ) . further clarification of the interaction between ferric ions and alginate may lead to better understanding of the properties of fe - alginate affecting cell culture . it has been reported that high - m ca - alginate shows no further changes in material properties after 7 days of exposure to nacl . in the present study ,
high - m fe - alginate gels immersed in culture solution for 8 days also showed little change in gel conditions , such as swelling or polymer content ( figure 4 ) . on high - m alginate gels under the same conditions ,
nhdf were able to proliferate at the same level as that on high - g alginate gels ( figure 5 ) .
there is some question as to whether cell growth on high - m alginate films is inhibited by elution of substances from the gels .
however , no harmful effects on cell growth were evident when cells were cultured on the surface of tissue culture plates in medium together with high - m alginate gel film ( data not shown ) , thus suggesting that the eluted materials did not influence cell growth appreciably .
reduction of the concentration of ferric ions cross - linked with alginate did not affect the growth of nhdf seeded on both types of alginate gel ( figure 6 ) .
in fact , cells proliferated well on highly swollen high - g alginate with a decreased amount of cross - linked ferric ions .
thus , swelling of fe - alginate gels was not directly linked to scanty growth of cells cultured on gel surfaces . in the case of high - m alginate ,
gel conditions were changed during immersion in culture medium , even though the concentration of cross - linked ferric ions was low .
it is considered that the percentage of firm cross - linkage in high - m alginate gels is unrelated to the amount of cross - linked ferric ions . in the case of high - m alginate gels under physiological conditions ,
strong cross - linkage may be maintained , while weak cross - linkage may occur as a result of exchange of cross - linked ferric ions for other competing ions .
in this study , we have revealed for the first time the effects of fe - alginate composition on the properties of the gels and their suitability as a culture substrate for cells .
fe - alginate gel with a high g content showed stable cross - linkage with ferric ions , and supported good cell proliferation . in the case of high - m alginate , although partial weak cross - linkage was observed during culture and the cell growth rate was suboptimal , the effectiveness of the gel for supporting cell growth was recognized after it had been stabilized by immersion in culture medium for an appropriate period .
in fact , the protein adsorption abilities of the two types of fe - alginate gels ware similar .
therefore , it is considered that the major factor influencing cell growth on two types of fe - alginate films is the stability of the gel during culture , and that the mode of cross - linkage between ferric ions and alginate differs according to alginate composition .
these results suggest that the composition of fe - alginate gels could be altered according to their intended purpose .
it is anticipated that our findings will be useful for extending the application of fe - alginate to diverse biomedical fields .
 we investigated the suitability of ferric - ion - cross - linked alginates ( fe - alginate ) with various proportions of l - guluronic acid ( g ) and d - mannuronic acid ( m ) residues as a culture substrate for human dermal fibroblasts . 
 high - g and high - m fe - alginate gels showed comparable efficacy in promoting initial cell adhesion and similar protein adsorption capacities , but superior cell proliferation was observed on high - g than on high - m fe - alginate as culture time progressed . during immersion in culture medium , high - g fe - alginate showed little change in gel properties in terms of swelling and polymer content , but the properties of high - m fe - alginate gel were altered due to loss of ion cross - linking . 
 however , the degree of cell proliferation on high - m fe - alginate gel was improved after it had been stabilized by immersion in culture medium until no further changes occurred . 
 these results suggest that the mode of cross - linkage between ferric ions and alginate differs depending on alginate composition and that the major factor giving rise to differences in cell growth on the two types of fe - alginate films is gel stability during culture , rather than swelling of the original gel , polymer content , or protein adsorption ability . 
 our findings may be useful for extending the application of fe - alginate to diverse biomedical fields . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussions
5. Conclusions