chondrocytes cultured in   monolayer have a tendency to dedifferentiate [ 1 , 2 ] , and serial expanded chondrocytes in monolayer culture gradually dedifferentiated by reducing the production of type ii collagen [ 3 , 4 ] . thus , to maintain the phenotypes of chondrocytes , three - dimensional scaffolds were developed for tissue engineering [ 57 ] . after long - term immobilization culture , rabbit articular chondrocytes in alginate gel still exhibited metabolic activities , patterns of division , as well as synthesis and secretion of extracellular matrix macromolecules such as type ii collagen and proteoglycans . allogenic implants of chondrocytes in alginate gels was tested for the in vivo reconstruction of artificially full - thickness - damaged articular rabbit cartilage . thus , in this study , alginate was selected as a three - dimensional scaffold . currently , basal culture medium supplemented with 10% fbs was used for enhancing cell activities during in vitro three - dimensional culture of chondrocytes . however , it was reported that fbs would result in excessive cell proliferation   or chondrocyte phenotypic instability . studies have shown that insulin - transferrin - selenium ( its ) prevented human chondrocyte dedifferentiation and promoted the formation of high - quality tissue engineering human hyaline cartilage . its was used as a baseline medium supplement for in vitro studies concerning cartilage explants , as well as chondrocyte expansion or redifferentiation [ 13 , 14 ] . however , the optimum combination of insulin - transferrin - selenium and fetal bovine serum for culture of rabbit articular chondrocytes in three - dimensional alginate scaffolds has not been extensively investigated . in this study , it was hypothesized that a small volume of fbs in medium with 1% its can sustain extracellular matrix synthesis and maintain the biological activities of chondrocytes in alginate hydrogels . in order to choose an appropriate fbs concentration , culture medium supplemented with 1% its plus 2% fbs , 1% its plus 4% fbs , 1% its plus 8% fbs , or medium plus 10% fbs were selected for three - dimensional culture of rabbit chondrocytes . the biological , biochemical properties of chondrocytes seeded in alginate hydrogels were evaluated with histological staining , immunohistochemistry , quantitative gene expression analysis . the basal medium was high - glucose dulbecco 's modified eagle 's medium ( dmem ; gibco ) , with ascorbate ( 50 g / ml ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) . basal medium was added with 1% insulin - transferrin - selenium ( its ; gibco ) plus 2% fetal bovine serum ( fbs ; invitrogen ) , 1% its plus 4% fbs , 1% its plus 8% fbs , or 10% fbs for the in vitro culture of chondrocyte - seeded hydrogels . chondrocytes were isolated from 4-week - old male new zealand rabbits . the knee , hip , elbow joint cartilage slices were harvested , minced , and then washed three times in pbs . the fragments were digested in 0.25% trypsin for 30 minutes at 37c , followed by digestion in 0.2% ( w / v ) collagenase ii for 3 hours . the cells released in the supernatant were collected by centrifuging ( 100 g / min , 5 minutes ) . isolated chondrocytes were washed three times and counted in a hemocytometer by trypan blue dye exclusion . chondrocytes were suspended in dmem with 10% fbs and 100 u / ml penicillin and 100 g / ml streptomycin , 50 g / ml ascorbate and cultured in flasks at an initial density of 4  10 cells / cm . the chondrocytes were incubated at 37c , 5%   co2 , and 95% humidity , and the medium was changed every three days . a 1.5% ( w / v ) alginate ( sigma ) solution in 0.01 m pbs was prepared and sterilized by filtering through a 0.22 m pore size polyethersulfone filter ( millipore ) . the second - generation chondrocytes were used for seeding in the 1.5% alginate solution at a density of about 5  10 cells / ml . for alginate scaffold encapsulation , isolated chondrocytes the chondrocyte - alginate suspension was pipetted into 8 mm diameter silanized plastic rings ( 150 l / ring ) in 24-well plates and gelled for 10 minutes by adding an excess volume of 102 mm cacl2 , and then the ring and the cacl2 solution were removed . as designed , various media were added to the 24-well plates and placed in a cell culture incubator . the morphology and proliferation of chondrocytes in alginate hydrogels cultured under various media were examined using an inverted phase contrast microscope ( olympus ) . the cellular activity of chondrocytes in the cultured scaffolds was estimated by mtt ( 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide ) assay . in this study , a modified mtt assay was employed according to zund et al . report . in brief , at different time points , 800 l serum - free medium and 80 l mtt solution ( 5 mg / ml in pbs ) were added to each sample , followed by incubation at 37c for 4 hours for mtt formazan formation ; and then the medium was removed and the converted dye was dissolved with 800 l 10% sodium dodecyl sulfate ( sds ) in 0.01 m hcl . after all crystals were solubilized , the optical density of the solution was determined at 570 nm against sds solution as blank using an elisa plate reader . the harvested samples were fixed in 4% paraformalydehyde in pbs overnight at 4c , then washed , dehydrated in graded ethanol , cleared in xylene , and embedded in paraffin . sections of 6 m were made through the cross - section of the sample and prepared for histological and immunohistochemical examination . , immunohistochemistry was carried out to detect   the   expression of   chondrocyte marker type ii collagen . sections were treated with 0.4% pepsin for 30 minutes at 37c , washed with pbs , treated with 0.6%   h2o2 in methanol , and rinsed again with pbs . the immunohistochemical staining for collagen was then performed with rabbit type ii collagen antibody ( calbiochem , 1 : 1000 in pbs ) over night at 4c . the following manipulations were carried out using streptavidin/ peroxidase ( sp ) staining kit ( sp-9002 , zhongshan golden bridge biotechnology co. , ltd ) according to the manufacturer 's instructions and finally the color was developed with diaminobenzidine ( dab - kit , vector ) . cell nuclei were counterstained with mayer 's hematoxylin ( sigma ) and embedded on microscopic slides using neutral balsam ( sigma ) for microscopic examination . during the whole procedure of the experiment , the manipulations were strictly controlled and the time of staining was same for each sample . semiquantitative real - time pcr was performed to determine the gene expression for chondrocyte differentiation ( type ii collagen and aggrecan core protein ) , and dedifferentiation ( type i collagen ) . at week 1 and week 3 , total rna in the chondrocyte - seeded hydrogels was extracted by trizol reagent ( gibco ) according to the manufacture 's instruction . the rna sample was converted to a complimentary dna ( cdna ) with revertaid first strand cdna synthesis kit ( fermentas ) according to the manufacture 's recommendation . primers of rabbit glyceraldehyde-3-phosphatase dehydrogenase ( gapdh , housekeeping gene ) , collagen type i , collagen type ii , and   aggrecan core protein were designed with primer express v 3.0 software ( applied biosystems ) and blasted with genebank database sequences to get highly specific primers . the primers sequences were shown as follows :    forward : 5-caggatgtccaggaggct-3 , reverse : 5-gcagtggcgaggtcagtag-3 ;    forward : 5-gcaacttgaacaaggctgtc-3,reverse : 3-caaggaagggcaaacgag-5 ;    forward :   5-cgagaatcaaatggagccg-3,reverse : 3-cacaacacctttcaccacgac-5 ;     forward : 5-atgtttgtgatgggcgtgaa-3,reverse : 5-tggaggcagggatgatgtt-3. semiquantitative real - time pcr was performed in a 25 l reaction system , and was programmed to run 35 cycles . the end products were separated with 1.5% agarose gel electrophoresis and the results were analyzed with uvi image software after capturing by a gel image system ( bio - rad ) . all numerical data were presented as mean  standard error of the mean ( sem ) . in order to reveal the morphology of cells cultured on three - dimensional alginate hydrogels under different media , cells cultured for 7 , 14 , and 21 days were examined . no significant differences were observed between chondrocytes cultured under the four kind of tested media . it seems that the growth pattern , cell aggregations , and the rates of colonization between the tested groups were similar from the very beginning to the end of the culture periods . chondrocytes in alginate hydrogel cultured under different media could be seen to form cell islets and cell aggregates with spherical shape , which maintained typical chondrocyte appearance ( data not shown ) . figure 1 shows the cell viability of chondrocytes cultured with different media at various time points , there was no significant difference ( p > .05 ) between four groups . in addition , the cell viabilities of chondrocytes in all groups increased with culture time and the chondrocytes began to enter log phase at about day 6 . production of glycosaminoglycans ( gags ) by chondrocytes in alginate hydrogels cultured under various media was confirmed by toluidine blue staining ( figure 2 ) . for chondrocyte - seeded alginate hydrogels cultured in medium supplemented with 1% its plus 2% fbs for 21 days , significant staining for clusters of chondrocytes and their extracellular matrix was noted , indicating large amounts of gag accumulation in the matrix surrounding chondrocytes . the groups cultured in the medium with 8% and 10% showed the lowest staining , which appeared to be significantly different from the others . immunohistochemical staining of chondrocytes in alginate hydrogels cultured under various media is seen in figure 3 . staining for collagen type ii revealed significant pericellular matrix staining for chondrocyte - seeded alginate hydrogels cultured in medium supplemented with 1% its plus 2% fbs and in medium with 1% its plus 4% fbs . whereas , for the groups ( figures 3(c )   and 3(d ) ) cultured in medium supplemented with 1% its plus 8% fbs and in medium with 10% fbs , the staining was less positive . the data show that the medium supplemented with 1% its plus lower fbs is more propitious to chondrocyte production and deposition of extracellular matrix molecules such as type ii collagen and gag - containing proteoglycans . semiquantitative rt - pcr analysis showed collagen ii , collagen i , and aggrecan expression of chondrocytes in alginate hydrogels cultured with different media ( figure 4 ) and the data were representative of five experiments .   at week 1 , the aggrecan expression of groups cultured in medium with 1% its plus 2% fbs ,   medium with 1% its plus 4% fbs was greater than the other tested groups ; at week 3 , aggrecan expression of the groups cultured in medium with 1% its plus 2% fbs , medium with 1% its plus 4% fbs , and medium with 1% its plus 8% fbs was slightly higher than that in medium with 10% fbs . at week 1 , chondrocyte - seed alginate hydrogels cultured in medium with 1% its plus 2% fbs expressed the highest levels of type ii collagen compared to the other three groups , and at week 4 , the group cultured in medium with 10% fbs had the lowest expression level of type ii collagen whereas those cultured in medium with 1% its plus 2% fbs , medium with 1% its plus 4% fbs , and medium with 1% its plus 8% fbs maintained a relatively high level of type ii collagen expression . besides , at week 3 , chondrocyte - seeded hydrogels cultured in medium with 1% its plus 8% fbs and medium with 10% fbs expressed relatively high levels of type i collagen , indicating a loss of chondrocyte phenotype . by contrast , those cultured in medium with 1% its plus 2% fbs maintained low levels of type i collagen expression . these results suggested that medium with 1% its plus 2% fbs was sufficient to maintain a chondrocyte phenotype in alginate hydrogel . the final goal of this study was to find an optimum combination of its and fetal bovine serum for culture of rabbit articular chondrocytes in three - dimensional alginate scaffolds . serum can provide nutrients for cell growth and promote cell synthesis and fetal bovine serum was frequently selected element for the culture of chondrocytes [ 1518 ] . however , the addition of serum would cause chondrocytes to change its morphological features to become more fibroblastic and dedifferentiate in the monolayer culture [ 19 , 20 ] . moreover , medium supplemented with serum resulted in excessive tissue swelling and significant degradation of explant properties . recently , several reports [ 14 , 22 ] have shown the potential of chondrocytes cultured either serum free or with a small volume of serum . the commercially available its was used in this study to support cell growth in low serum culture medium . it was demonstrated that medium supplemented with 1% its plus 2% fbs was able to enhance chondrocyte growth and reduced the dedifferentiation process of cultured chondrocytes .   since chondrocytes cultured with only 1% its medium had a relatively poor growth rate , we did not present these data . as to serum - free or low - serum culture , most previous reports focused on monolayer . since three - dimensional culture could maintain the phenotype for a long culture period , it is more practical to investigate the influence of serum chondrocytes cultured in three - dimensional scaffolds . it was proved that alginate scaffold biomaterial has a great potential in cartilage tissue scaffolds for cartilage tissue engineering [ 5 , 8 , 9 , 23 ] , thus we adopted alginate hydrogel as a three - dimensional scaffold . in alginate hydrogel , chondrocytes distributed throughout the network of the matrices and maintained spherical morphology . for chondrocytes grown under the various culture media , significant differences were observed in the staining of toluidine blue and type ii collagen immunohistochemistry . compared with other tested groups , denser staining was shown for chondrocytes cultured in medium supplemented with 1% its plus 4% fbs as well as for the 1% its plus 2% fbs group . these results indicated that reducing the percent fbs may promote the maintenance of the chondrocyte phenotype to some extent . results from semi - quantitative reverse transcriptase - polymerase chain reaction analysis also support this suggestion . at 7 days after cultivation , the mrna for type ii collagen and aggrecan was well expressed in all specimens and type i collagen was poorly expressed . however , after longer periods of culture , differences among the four groups were revealed ; expression of type i collagen become evident for chondrocytes cultured under medium with 1% its plus 4% fbs , medium with 1% its plus 8% fbs , and medium with 10% fbs , while the expression level remained low for chondrocytes cultured in medium with 1% its plus 2% fbs . in brief , 1% its supplementation combined with 2% fbs resulted in higher differentiation ability while high volume of fbs led to less extracellular matrix accumulation during long culture period . the reduction of the amount of animal serum used in the medium from 10% to 2% provided valuable implications on three - dimensional cartilage engineering . however , further work needs to be done to investigate the mechanical change on alginate hydrogel under this culture medium . this study demonstrated that medium supplemented with 1% its plus 2% fbs could stimulate chondrocyte proliferation and increase cartilage matrix synthesis . chondrocytes seeded in alginate hydrogel could maintain their chondrocyte phenotype for long - period culture under medium supplemented with 1% its plus 2% fbs , which may be widely used in three - dimensional chondrocytes culture for tissue engineering .
fetal bovine serum ( fbs ) has been reported to affect chondrocyte biosynthesis in monolayer culture . 
 insulin - transferrin - selenium ( its ) was investigated as a partial replacement for fbs during in vitro culture of rabbit articular chondrocytes in three - dimensional alginate scaffold . 
 chondrocyte - seeded alginate hydrogels were cultured in dulbecco 's modified eagle 's medium plus 10% fbs , 1% its plus 2% fbs , 1% its plus 4% fbs , or 1% its plus 8% fbs . at designed time point , the chondrocyte - seeded alginate hydrogels were harvested and evaluated with histological staining , immunohistochemistry , and quantitative gene expression analysis . 
 viable cell density and cell division were also evaluated . 
 chondrocytes biosynthesis and cell division in 1% its with 2% fbs medium were similar to that in medium added with 10% fbs . for a total culture of 3 weeks , phenotypic gene expression in chondrocyte - seeded hydrogels was maintained at high levels in medium with 1% its plus 2% fbs , while it was decreased to varying degrees in the other groups . in conclusion , with 1% its , medium with 2% fbs could promote chondrocyte biosynthesis and cell division , and prevented cell dedifferentiation in three - dimensional alginate scaffolds .