BACKGROUND
the increasing epidemic of obesity within the united states has been associated with a higher risk of developing co-morbidities that are categorized as part of the metabolic syndrome that include dyslipoproteinemia , diabetes mellitus, and cardiovascular and coronary artery diseases.
obesity has also been identified as a risk factor for an increased incidence of several forms of cancer, including colon and breast cancers  <cit> .
a high incidence of these morbidities exists in obese individuals, both adolescents and adults  <cit> .
recent studies have also investigated the effects of obesity on the musculoskeletal system  <cit> , and have identified obesity as an independent risk factor for increased bone fracture risk  <cit>  and clinical implant failure following total joint replacement  <cit> .
obese individuals with higher body mass indices  to height ) exhibit lower relative bone area and bone mass compared to non-obese individuals  <cit> .
while various explanations have been widely suggested, our knowledge relating the pathogenesis of obesity at the cellular level and its potential impact in tissue engineering and regenerative medicine applications is very limited.
adipose tissue is a complex, highly active endocrine organ that secretes bioactive peptides, or adipocytokines, that are known to affect a number of physiological functions in the reproductive system, neuroendocrine system, rennin-angiotensin system, and in bone metabolism.
<cit> .
adipose tissue contains mature adipocytes, endothelial cells, cells of the immune system, and a small percentage of the adipocyte precursors termed adipose tissue-derived stem cells .
the ability of ascs to differentiate into cells of the endodermal, mesodermal, and ectodermal lineages makes ascs optimal candidates for applications in cellular therapies  <cit> , including tissue engineering and regenerative medicine  <cit> .
these applications potentially involve the repair of the musculoskeletal and other biological systems  <cit> .
however, the growing interest in ascs for cell therapeutics has led to questions about donor physiological conditions on asc functionality .
recent reports have positively correlated bmi with asc yields from adipose tissue and inversely correlated bmi with adipocyte size  <cit> .
bmi has also been inversely correlated with bone-marrow derived mesenchymal stem cell  cyclic tensile strain capacity, or mechano-response, and alkaline phosphatase activity in vitro, which suggested a possible compromise in the osteogenic differentiation potential of bmscs from individuals with higher bmis  <cit> .
similar studies on ascs have not been conducted.
the current study investigated the proliferation ability, in vitro differentiation potential, relative cell volume and complexity, and colony forming potential of ascs isolated from patients having different bmis, within the ranges of  <dig> - <dig>  kg/m <dig>  , and 25– <dig>  kg/m <dig>  .
it was hypothesized that overweight bmi-ascs would be more compromised in the ability to proliferate, differentiate, and form colonies in vitro, thereby contributing to the problematic obesity-associated pathologies, and therefore bmi should be considered when using ascs for regenerative medicine applications.
RESULTS
percent serum and bmi inversely correlated with asc growth
cryopreserved ascs isolated from lipoaspirates of women with different body mass indices , were cultured for up to 72 hours in asc culture medium supplemented with  <dig> to 10% fbs.
cell growth was measured by mtt and cyquant cell proliferation assays.
growth data reflected an inverse relationship between bmi and asc growth in vitro .
the largest effect was observed in 2% serum for 48 hrs ; however, growth was also compromised when ascs were cultured 10% serum .
mtt data also revealed a time-dependent biphasic response in cell growth in which full recovery and maximum growth occurred at 72 hrs following a decline in higher bmi-asc growth at 24 hrs and 48 hrs .
non linear regression analyses using least fit ordinary squares supported the strong inverse relationship between both bmi  and serum  on asc growth , where culture in the lowest percent serum  reflected the strongest determination coefficient.
quadratic equations were used for nonlinear regression analyses, curve-fitting and subsequent r <dig> values .
all ascs were obtained from subcutaneous abdominal adipose tissue of female, caucasian donors of ages between  <dig> and  <dig> with a mean ± sd of  <dig>  ±  <dig>  years.
ascs isolated from patients having different body mass indices  were designated bmi-ascs.
bmi-ascs used in the study were grouped into categories and identified as either ‘lean bmi-ascs’  or ‘overweight bmi-ascs’ .
the patients displayed a mean body mass index   of  <dig>  ±  <dig> .
bmi negatively correlated with colony-forming unit potential
to further examine differences in bmi-asc growth, colony-forming unit  assays were performed on the bmi-asc donors .
these donors were grouped as follows: lean , and overweight .
when grouped, the lean bmi-ascs formed a significantly higher percentage of colonies  compared to the overweight bmi-ascs ; p <  <dig>  .
representative photomicrographs of cfus are shown in figure 2c.
annexin-v/pi staining and fluorescence-activated cell sorting  was used to determine whether the compromised growth in higher bmi-ascs was accompanied by elevated apoptosis.
there was no significant difference in apoptosis between lean and overweight bmi-ascs at  <dig> and 48 hrs of culture with 2% or 10% fbs .
the percent early apoptotic cells did not exceed  <dig>  ±  <dig> %, and the percent late apoptotic cells did not exceed  <dig>  ±  <dig> % .
bmi did not affect late time point adipogenic differentiation in vitro
to investigate the effect of bmi on asc differentiation, adipogenesis was induced by culturing ascs in differentiation induction medium for three days followed by culture in maintenance medium until day  <dig>  differentiation was induced in both 3% and 10% serum.
lipid formation was assessed by percent intracytoplasmic incorporation of oil red-o  into monolayers at days  <dig> and  <dig> of adipogenesis.
oil-red-o staining at day  <dig> revealed a positive correlation between bmi and adipogenesis at early time points .
grouping of bmi-ascs revealed that overweight bmi-ascs had significantly higher oil-red-o staining  compared to the lean bmi-ascs ; p =  <dig>  at day  <dig> .
staining at day  <dig> revealed that bmi has no significant effect on asc adipogenesis during late time points .
to further investigate the correlation between bmi and adipogenesis, nonlinear regression analyses were applied to adipogenesis data from days  <dig> and  <dig>  r <dig> values reflected a correlation between bmi and adipogenesis at day  <dig> , and no correlation at day  <dig> .
representative photomicrographs of oro staining in bmi-ascs at day  <dig> are shown in figure 3e.
to determine whether cryopreservation was a factor in the observed absence of bmi effects on adipogenic differentiation, we induced adipogenesis in freshly isolated ascs and cryopreserved ascs at passage  <dig> from two of the same donors .
oro staining  demonstrated no significant difference in intracytoplasmic lipid accumulation prior to or following the freeze-thaw process.
bmi negatively correlated with extracellular matrix mineralization
the effects of bmi and percent fbs on asc osteogenesis at early and late time points was investigated by inducing osteogenesis using an osteogenic cocktail medium for 16 days, as described in the materials and methods.
differentiation was induced in both 3% and 10% serum.
calcium deposition was assessed by alizarin red staining  at days  <dig> and  <dig>  ars revealed an inverse correlation  between bmi and asc osteogenic potential at both time points; this was most pronounced at day  <dig> .
non linear regression analyses of the ars data revealed that% fbs had no significant effect on bmi-asc osteogenic potential, similar to that observed with adipogenesis .
to evaluate the correlation between bmi and osteogenesis, nonlinear regression analyses were performed on osteogenesis data from days  <dig> and  <dig> using the least squares ordinary fit.
r <dig> values reflected a strong inverse correlation at both time points .
representative photomicrographs of ars in bmi-ascs at day  <dig> are shown in figure 4e.
to determine whether cryopreservation was a factor in the observed effects on osteogenic differentiation, we induced osteogenesis in freshly isolated ascs and cryopreserved ascs from two of the same donors at passage  <dig> .
ars  demonstrated no significant difference prior to or following the freeze-thaw process.
bmi negatively correlated with alkaline phosphatase mrna expression
to confirm the results of figure  <dig> demonstrating a significant effect of bmi on asc osteogenesis, alkaline phosphatase mrna expression was measured in additional asc cultures after induction of osteogenesis.
alkaline phosphatase mrna results confirmed that when grouped, overweight bmi-ascs were significantly more compromised in osteogenic differentiation potential  compared to the lean bmi-ascs .
nonlinear regression analyses were performed on osteogenesis rt-pcr data using least squares ordinary fit.
the r <dig> value again reflected a strong inverse correlation  between bmi and osteogenic potential .
bmi did not affect asc relative cell size and complexity in vitro
recent studies indicated that mature adipocytes from overweight and obese individuals were smaller in size compared to adipocytes from lean individuals  <cit> .
to determine whether ascs might exhibit similar relationships between bmi and cell size and complexity, forward-scatter flow cytometry , and side- scatter flow cytometry  was performed on bmi-ascs following culture in 2% or 10% fbs for  <dig> and 48 hrs.
grouping of forward and side scatter analyses revealed no significant difference in cell size or complexity between lean and overweight bmi-ascs following 24 hrs of culture in both 2% and 10% serum .
discussion
the potential use of ascs in tissue engineering and regenerative medicine has been well demonstrated in multiple pre-clinical animal models  <cit> .
although ascs have been characterized based on immunophenotype, cell yield and differentiation properties  <cit> , the effects of bmi on asc functionality have not been fully elucidated.
the present study demonstrated that cryopreserved human ascs from patients with higher bmis were compromised in colony-forming potential and growth under exposure to low serum concentrations.
overweight bmi-ascs were also compromised in osteogenic differentiation capacity when compared to lean bmi-ascs.
cryopreservation was not a factor in the observed differences, as both freshly isolated bmi-ascs and cryopreserved bmi-ascs were comparable in both osteogenic and adipogenic differentiation capacities.
moreover, in vitro adipogenesis data suggested that early time point intracytoplasmic lipid uptake was positively correlated to bmi.
exposure of ascs to low serum uncovered differences among bmi-ascs that were less apparent at higher serum concentrations.
although one individual donor exhibited variation in the trend of bmi and proliferation , the determination coefficients were sufficient to form conclusions about the correlation between proliferation and bmi.
overweight bmi-asc osteogenesis was reduced independent of serum levels.
these results were similar to a published report that the bmsc osteogenic alkaline phosphatase response to mechanical strain was inversely correlated to donor bmi  <cit> .
these findings paralleled clinical data demonstrating that obese patients possessed lower relative bone area and bone mass compared to lean individuals, when adjusted for body weight,  <cit> , and this may contribute to an increased risk for fractures  <cit> .
other clinical studies have demonstrated an inverse correlation between body weight and bone density and peak bone mass.
in addition, there is evidence of increased risk of implant failure following total joint knee replacement in obese patients  <cit> .
an understanding of the impact of fat metabolism on bone precursors and osteogenic capacity will be necessary to advance asc use in bone repair applications.
the present data suggests that overweight bmi dependent increase in early adipogenesis may be associated with a decrease in osteogenic capacity.
multiple studies suggest that when asc fate is committed to the adipogenic pathway, osteogenesis is concomitantly down regulated, consistent with observations in bmscs  <cit> .
this balance is suggested to be partly modulated by the presence and amount of glucocorticoid, the glucocorticoid receptor pathway activity, crosstalk with the jak/stat <dig> pathway, and the subsequent activation of the ap- <dig> pathway  <cit> .
other studies have suggested that an inverse relationship exists between donor age and asc osteogenesis  <cit> , and that ascs from male donors exhibit increased osteogenic potential compared to ascs from female donors in vitro <cit> .
however, conflicting outcomes were reported in studies comparing donor age and bmsc osteogenic potential in vitro with one study demonstrating an inverse correlation  <cit>  whereas other studies reported a positive correlation  <cit> .
ascs from overweight bmi patients  showed an increase in the intracytoplasmic oil-red-o staining at early adipogenesis time points, but no significant difference at later time points.
although an increasing trend was observed in early adipogenic potential that correlates to bmi, an individual donor effect may have contributed to the low adipogenesis in donor l <dig> .
genes that are induced during both early and late stage adipogenesis have been extensively described  <cit> .
yu et.
al.,  <dig>  <cit>  performed time-dependent rt-pcr analyses of genes involved in adipogenesis in  <dig> donors, bmi  <dig>  ±  <dig> , age 39 ± 6 years.
the adipogenic transcription factor c/ebpα, the adipokines adiponectin and leptin, and lipoprotein lipase  all displayed a time-dependent increase during adipogenesis, with the greatest percent increase occurring within the initial 3-day period; however bmi had no significant effect on the mrna expression of the representative genes that were upregulated on day  <dig> of adipogenesis .
this suggests that bmi has no overall effect on adipogenesis in vitro, and supports our data demonstrating no correlation between bmi and late adipogenesis.
however, schipper et.
al.
<cit>  reported age-dependent differences in asc adipogenic potential and proliferative capacity in vitro.
further investigation is needed relating the impact of age and bmi on early and late asc functionality.
it is widely accepted that hyperplasia follows adipocyte hypertrophy in patients during fat mass expansion which requires a gradual replenishment of the pool of adipocytes in obese individuals  <cit> .
one study proposes that bmscs are recruited and give rise to bone, muscle, and both white and brown adipose tissues in response to the appropriate developmental cues in vivo <cit> .
however, the ability of ascs to differentiate into terminally differentiated cells of both white and brown adipose tissues adds further complexity to the interesting dynamic.
the formation of newer, small mature adipocytes is thought to occur via differentiation of preadipocytes; however whether the preadipocyte originates from the fat mass, or from the recruitment of the circulating bone-marrow progenitor cells  <cit>  remains to be determined.
in this regard, it should be noted that koh et.
al.
<cit>  demonstrated that bmscs in bone marrow transplant mice became resident as phagocytic macrophages in adipose tissues and resembled trans-differentiated adipocytes, but did not express adipocyte markers.
the present study used cryopreserved ascs that were passaged in vitro, not freshly isolated ascs.
several studies have investigated the cryopreservation characteristics of ascs and examined different methods of cryopreservation on asc biology in vitro <cit> .
thirumala et al.,  <dig>  <cit> , showed that the post-thaw viability of ascs differed significantly based on the method of freezing and rate of thaw, and determined that using an ethanol enclosed jacketed container, with 10% dmso and 80% fbs in the cryopreservation medium, yielded the highest post-thaw viability.
other studies have examined the effects of various cryoprotective agents on cell viability, proliferation, and osteo- and adipogenic differentiation  <cit> .
these studies indicated that proliferation and osteo- and adipogenic differentiation of ascs could be maintained in vitro post-thaw.
our studies also demonstrated that cryopreservation did not significantly affect asc adipogenic or osteogenic potential .
reports on the proliferation index of freshly plated ascs versus cryopreserved ascs are conflicting.
james et.
al.,  <dig>  <cit>  compared ascs freshly harvested from lipoaspirate to cryopreserved ascs, and measured growth using trypan blue dye exclusion assay.
their results demonstrated cryopreservation significantly affected in vitro proliferative capacity.
however, studies by deng et.
al.,  <dig>  <cit> , and gonda et.
al.,  <dig>  <cit>  suggest that asc proliferative capacity following cryopreservation is maintained.
calculations of asc population doubling times in our studies coincide with reports by deng et.
al.,  <dig>  and gonda et.
al.,  <dig>  <cit> , as there was no significant difference observed between the doubling times of freshly isolated ascs and cryopreserved ascs .
james et.
al.,  <dig>  <cit>  also compared ascs freshly harvested from lipoaspirate to cryopreserved ascs, and measured osteogenic differentiation , and adipogenic differentiation .
the results demonstrated cryopreservation significantly affected osteogenic differentiation, both in vitro and in vivo.
the use of recombinant proteins such as igf and bmp, however, were used to mitigate the deleterious effects of the freeze–thaw process on osteogenic potential.
it should be noted that bmi was not a tested factor, and no cytokines or signaling factors were added to their differentiation medium.
a handful of cytokines and other factors are known to stimulate osteogenic differentiation in ascs, including vitamin d, which was added in the differentiation medium used in the present study.
in addition, the strong determination coefficients derived from performing the nonlinear regression analyses of the proliferation  and osteogenic differentiation studies  suggest donor metabolic status may also have an effect on asc functionality.
in vivo, near complete wound healing was observed in mice with calvarial defects that were engrafted with fresh human ascs as compared to groups engrafted with cryopreserved ascs which exhibited reduced healing  <cit> .
although freshly isolated ascs may exhibit beneficial properties for wound healing and other applications, the majority of applications for ascs will likely use cryopreserved ascs, therefore it is important to understand the characteristics and limitations of cryopreserved ascs.
future studies comparing the functionality of freshly isolated ascs to cryopreserved ascs within different bmi groupings are needed to determine whether the observed differences exist in freshly isolated cells.
other possible contributing factors to the outcomes of this study are patient clinical characteristics that were unavailable from patient records, such as donor hormonal status, smoking and other undisclosed patient parameters.
regression analyses were conducted to determine whether a correlation exists between age, passage and the measured endpoints within the study.
all r <dig> values were less than  <dig>  and as such reflect no correlation between the known factors and the measured endpoints.
CONCLUSIONS
while ascs are proving to be promising candidates for many applications in regenerative medicine and tissue engineering, understanding the phenotypic characteristics of these cells is essential to ensure product consistency and suitability towards specific applications.
the present study demonstrated that in comparison to ascs from lean individuals, ascs that originate from individuals with higher bmis had comparable adipocyte differentiation but reduced proliferation and osteogenic potential.
thus, ascs derived from donors with overweight bmi may be less than optimal for applications that involve usage in biocompatible scaffolds for bone grafting.

 the increasing epidemic of obesity within the united states has been associated with a higher risk of developing co-morbidities that are categorized as part of the metabolic syndrome that include dyslipoproteinemia, diabetes mellitus, and cardiovascular and coronary artery diseases. obesity has also been identified as a risk factor for an increased incidence of several forms of cancer, including colon and breast cancers. a high incidence of these morbidities exists in obese individuals, both adolescents and adults. recent studies have also investigated the effects of obesity on the musculoskeletal system, and have identified obesity as an independent risk factor for increased bone fracture risk and clinical implant failure following total joint replacement. obese individuals with higher body mass indices to height ) exhibit lower relative bone area and bone mass compared to non-obese individuals. while various explanations have been widely suggested, our knowledge relating the pathogenesis of obesity at the cellular level and its potential impact in tissue engineering and regenerative medicine applications is very limited. adipose tissue is a complex, highly active endocrine organ that secretes bioactive peptides, or adipocytokines, that are known to affect a number of physiological functions in the reproductive system, neuroendocrine system, rennin-angiotensin system, and in bone metabolism.. adipose tissue contains mature adipocytes, endothelial cells, cells of the immune system, and a small percentage of the adipocyte precursors termed adipose tissue-derived stem cells. the ability of ascs to differentiate into cells of the endodermal, mesodermal, and ectodermal lineages makes ascs optimal candidates for applications in cellular therapies, including tissue engineering and regenerative medicine. these applications potentially involve the repair of the musculoskeletal and other biological systems. however, the growing interest in ascs for cell therapeutics has led to questions about donor physiological conditions on asc functionality. recent reports have positively correlated bmi with asc yields from adipose tissue and inversely correlated bmi with adipocyte size. bmi has also been inversely correlated with bone-marrow derived mesenchymal stem cell cyclic tensile strain capacity, or mechano-response, and alkaline phosphatase activity in vitro, which suggested a possible compromise in the osteogenic differentiation potential of bmscs from individuals with higher bmis. similar studies on ascs have not been conducted. the current study investigated the proliferation ability, in vitro differentiation potential, relative cell volume and complexity, and colony forming potential of ascs isolated from patients having different bmis, within the ranges of -  kg/m, and 25–  kg/m. it was hypothesized that overweight bmi-ascs would be more compromised in the ability to proliferate, differentiate, and form colonies in vitro, thereby contributing to the problematic obesity-associated pathologies, and therefore bmi should be considered when using ascs for regenerative medicine applications.  percent serum and bmi inversely correlated with asc growth cryopreserved ascs isolated from lipoaspirates of women with different body mass indices, were cultured for up to 72 hours in asc culture medium supplemented with to 10% fbs. cell growth was measured by mtt and cyquant cell proliferation assays. growth data reflected an inverse relationship between bmi and asc growth in vitro. the largest effect was observed in 2% serum for 48 hrs; however, growth was also compromised when ascs were cultured 10% serum. mtt data also revealed a time-dependent biphasic response in cell growth in which full recovery and maximum growth occurred at 72 hrs following a decline in higher bmi-asc growth at 24 hrs and 48 hrs. non linear regression analyses using least fit ordinary squares supported the strong inverse relationship between both bmi and serum on asc growth, where culture in the lowest percent serum reflected the strongest determination coefficient. quadratic equations were used for nonlinear regression analyses, curve-fitting and subsequent r values. all ascs were obtained from subcutaneous abdominal adipose tissue of female, caucasian donors of ages between and with a mean ± sd of  ±   years. ascs isolated from patients having different body mass indices were designated bmi-ascs. bmi-ascs used in the study were grouped into categories and identified as either ‘lean bmi-ascs’ or ‘overweight bmi-ascs’. the patients displayed a mean body mass index of  ± . bmi negatively correlated with colony-forming unit potential to further examine differences in bmi-asc growth, colony-forming unit assays were performed on the bmi-asc donors. these donors were grouped as follows: lean, and overweight. when grouped, the lean bmi-ascs formed a significantly higher percentage of colonies compared to the overweight bmi-ascs; p < . representative photomicrographs of cfus are shown in figure 2c. annexin-v/pi staining and fluorescence-activated cell sorting was used to determine whether the compromised growth in higher bmi-ascs was accompanied by elevated apoptosis. there was no significant difference in apoptosis between lean and overweight bmi-ascs at and 48 hrs of culture with 2% or 10% fbs. the percent early apoptotic cells did not exceed  ±  %, and the percent late apoptotic cells did not exceed  ±  %. bmi did not affect late time point adipogenic differentiation in vitro to investigate the effect of bmi on asc differentiation, adipogenesis was induced by culturing ascs in differentiation induction medium for three days followed by culture in maintenance medium until day differentiation was induced in both 3% and 10% serum. lipid formation was assessed by percent intracytoplasmic incorporation of oil red-o into monolayers at days and of adipogenesis. oil-red-o staining at day revealed a positive correlation between bmi and adipogenesis at early time points. grouping of bmi-ascs revealed that overweight bmi-ascs had significantly higher oil-red-o staining compared to the lean bmi-ascs; p =  at day. staining at day revealed that bmi has no significant effect on asc adipogenesis during late time points. to further investigate the correlation between bmi and adipogenesis, nonlinear regression analyses were applied to adipogenesis data from days and r values reflected a correlation between bmi and adipogenesis at day, and no correlation at day. representative photomicrographs of oro staining in bmi-ascs at day are shown in figure 3e. to determine whether cryopreservation was a factor in the observed absence of bmi effects on adipogenic differentiation, we induced adipogenesis in freshly isolated ascs and cryopreserved ascs at passage from two of the same donors. oro staining demonstrated no significant difference in intracytoplasmic lipid accumulation prior to or following the freeze-thaw process. bmi negatively correlated with extracellular matrix mineralization the effects of bmi and percent fbs on asc osteogenesis at early and late time points was investigated by inducing osteogenesis using an osteogenic cocktail medium for 16 days, as described in the materials and methods. differentiation was induced in both 3% and 10% serum. calcium deposition was assessed by alizarin red staining at days and ars revealed an inverse correlation between bmi and asc osteogenic potential at both time points; this was most pronounced at day. non linear regression analyses of the ars data revealed that% fbs had no significant effect on bmi-asc osteogenic potential, similar to that observed with adipogenesis. to evaluate the correlation between bmi and osteogenesis, nonlinear regression analyses were performed on osteogenesis data from days and using the least squares ordinary fit. r values reflected a strong inverse correlation at both time points. representative photomicrographs of ars in bmi-ascs at day are shown in figure 4e. to determine whether cryopreservation was a factor in the observed effects on osteogenic differentiation, we induced osteogenesis in freshly isolated ascs and cryopreserved ascs from two of the same donors at passage. ars demonstrated no significant difference prior to or following the freeze-thaw process. bmi negatively correlated with alkaline phosphatase mrna expression to confirm the results of figure  demonstrating a significant effect of bmi on asc osteogenesis, alkaline phosphatase mrna expression was measured in additional asc cultures after induction of osteogenesis. alkaline phosphatase mrna results confirmed that when grouped, overweight bmi-ascs were significantly more compromised in osteogenic differentiation potential compared to the lean bmi-ascs. nonlinear regression analyses were performed on osteogenesis rt-pcr data using least squares ordinary fit. the r value again reflected a strong inverse correlation between bmi and osteogenic potential. bmi did not affect asc relative cell size and complexity in vitro recent studies indicated that mature adipocytes from overweight and obese individuals were smaller in size compared to adipocytes from lean individuals. to determine whether ascs might exhibit similar relationships between bmi and cell size and complexity, forward-scatter flow cytometry, and side- scatter flow cytometry was performed on bmi-ascs following culture in 2% or 10% fbs for and 48 hrs. grouping of forward and side scatter analyses revealed no significant difference in cell size or complexity between lean and overweight bmi-ascs following 24 hrs of culture in both 2% and 10% serum. discussion the potential use of ascs in tissue engineering and regenerative medicine has been well demonstrated in multiple pre-clinical animal models. although ascs have been characterized based on immunophenotype, cell yield and differentiation properties, the effects of bmi on asc functionality have not been fully elucidated. the present study demonstrated that cryopreserved human ascs from patients with higher bmis were compromised in colony-forming potential and growth under exposure to low serum concentrations. overweight bmi-ascs were also compromised in osteogenic differentiation capacity when compared to lean bmi-ascs. cryopreservation was not a factor in the observed differences, as both freshly isolated bmi-ascs and cryopreserved bmi-ascs were comparable in both osteogenic and adipogenic differentiation capacities. moreover, in vitro adipogenesis data suggested that early time point intracytoplasmic lipid uptake was positively correlated to bmi. exposure of ascs to low serum uncovered differences among bmi-ascs that were less apparent at higher serum concentrations. although one individual donor exhibited variation in the trend of bmi and proliferation, the determination coefficients were sufficient to form conclusions about the correlation between proliferation and bmi. overweight bmi-asc osteogenesis was reduced independent of serum levels. these results were similar to a published report that the bmsc osteogenic alkaline phosphatase response to mechanical strain was inversely correlated to donor bmi. these findings paralleled clinical data demonstrating that obese patients possessed lower relative bone area and bone mass compared to lean individuals, when adjusted for body weight,, and this may contribute to an increased risk for fractures. other clinical studies have demonstrated an inverse correlation between body weight and bone density and peak bone mass. in addition, there is evidence of increased risk of implant failure following total joint knee replacement in obese patients. an understanding of the impact of fat metabolism on bone precursors and osteogenic capacity will be necessary to advance asc use in bone repair applications. the present data suggests that overweight bmi dependent increase in early adipogenesis may be associated with a decrease in osteogenic capacity. multiple studies suggest that when asc fate is committed to the adipogenic pathway, osteogenesis is concomitantly down regulated, consistent with observations in bmscs. this balance is suggested to be partly modulated by the presence and amount of glucocorticoid, the glucocorticoid receptor pathway activity, crosstalk with the jak/stat pathway, and the subsequent activation of the ap- pathway. other studies have suggested that an inverse relationship exists between donor age and asc osteogenesis, and that ascs from male donors exhibit increased osteogenic potential compared to ascs from female donors in vitro. however, conflicting outcomes were reported in studies comparing donor age and bmsc osteogenic potential in vitro with one study demonstrating an inverse correlation whereas other studies reported a positive correlation. ascs from overweight bmi patients showed an increase in the intracytoplasmic oil-red-o staining at early adipogenesis time points, but no significant difference at later time points. although an increasing trend was observed in early adipogenic potential that correlates to bmi, an individual donor effect may have contributed to the low adipogenesis in donor l. genes that are induced during both early and late stage adipogenesis have been extensively described. yu et. al., performed time-dependent rt-pcr analyses of genes involved in adipogenesis in donors, bmi  ± , age 39 ± 6 years. the adipogenic transcription factor c/ebpα, the adipokines adiponectin and leptin, and lipoprotein lipase all displayed a time-dependent increase during adipogenesis, with the greatest percent increase occurring within the initial 3-day period; however bmi had no significant effect on the mrna expression of the representative genes that were upregulated on day of adipogenesis. this suggests that bmi has no overall effect on adipogenesis in vitro, and supports our data demonstrating no correlation between bmi and late adipogenesis. however, schipper et. al. reported age-dependent differences in asc adipogenic potential and proliferative capacity in vitro. further investigation is needed relating the impact of age and bmi on early and late asc functionality. it is widely accepted that hyperplasia follows adipocyte hypertrophy in patients during fat mass expansion which requires a gradual replenishment of the pool of adipocytes in obese individuals. one study proposes that bmscs are recruited and give rise to bone, muscle, and both white and brown adipose tissues in response to the appropriate developmental cues in vivo. however, the ability of ascs to differentiate into terminally differentiated cells of both white and brown adipose tissues adds further complexity to the interesting dynamic. the formation of newer, small mature adipocytes is thought to occur via differentiation of preadipocytes; however whether the preadipocyte originates from the fat mass, or from the recruitment of the circulating bone-marrow progenitor cells remains to be determined. in this regard, it should be noted that koh et. al. demonstrated that bmscs in bone marrow transplant mice became resident as phagocytic macrophages in adipose tissues and resembled trans-differentiated adipocytes, but did not express adipocyte markers. the present study used cryopreserved ascs that were passaged in vitro, not freshly isolated ascs. several studies have investigated the cryopreservation characteristics of ascs and examined different methods of cryopreservation on asc biology in vitro. thirumala et al.,, showed that the post-thaw viability of ascs differed significantly based on the method of freezing and rate of thaw, and determined that using an ethanol enclosed jacketed container, with 10% dmso and 80% fbs in the cryopreservation medium, yielded the highest post-thaw viability. other studies have examined the effects of various cryoprotective agents on cell viability, proliferation, and osteo- and adipogenic differentiation. these studies indicated that proliferation and osteo- and adipogenic differentiation of ascs could be maintained in vitro post-thaw. our studies also demonstrated that cryopreservation did not significantly affect asc adipogenic or osteogenic potential. reports on the proliferation index of freshly plated ascs versus cryopreserved ascs are conflicting. james et. al., compared ascs freshly harvested from lipoaspirate to cryopreserved ascs, and measured growth using trypan blue dye exclusion assay. their results demonstrated cryopreservation significantly affected in vitro proliferative capacity. however, studies by deng et. al.,, and gonda et. al., suggest that asc proliferative capacity following cryopreservation is maintained. calculations of asc population doubling times in our studies coincide with reports by deng et. al., and gonda et. al.,, as there was no significant difference observed between the doubling times of freshly isolated ascs and cryopreserved ascs. james et. al., also compared ascs freshly harvested from lipoaspirate to cryopreserved ascs, and measured osteogenic differentiation, and adipogenic differentiation. the results demonstrated cryopreservation significantly affected osteogenic differentiation, both in vitro and in vivo. the use of recombinant proteins such as igf and bmp, however, were used to mitigate the deleterious effects of the freeze–thaw process on osteogenic potential. it should be noted that bmi was not a tested factor, and no cytokines or signaling factors were added to their differentiation medium. a handful of cytokines and other factors are known to stimulate osteogenic differentiation in ascs, including vitamin d, which was added in the differentiation medium used in the present study. in addition, the strong determination coefficients derived from performing the nonlinear regression analyses of the proliferation and osteogenic differentiation studies suggest donor metabolic status may also have an effect on asc functionality. in vivo, near complete wound healing was observed in mice with calvarial defects that were engrafted with fresh human ascs as compared to groups engrafted with cryopreserved ascs which exhibited reduced healing. although freshly isolated ascs may exhibit beneficial properties for wound healing and other applications, the majority of applications for ascs will likely use cryopreserved ascs, therefore it is important to understand the characteristics and limitations of cryopreserved ascs. future studies comparing the functionality of freshly isolated ascs to cryopreserved ascs within different bmi groupings are needed to determine whether the observed differences exist in freshly isolated cells. other possible contributing factors to the outcomes of this study are patient clinical characteristics that were unavailable from patient records, such as donor hormonal status, smoking and other undisclosed patient parameters. regression analyses were conducted to determine whether a correlation exists between age, passage and the measured endpoints within the study. all r values were less than and as such reflect no correlation between the known factors and the measured endpoints.  while ascs are proving to be promising candidates for many applications in regenerative medicine and tissue engineering, understanding the phenotypic characteristics of these cells is essential to ensure product consistency and suitability towards specific applications. the present study demonstrated that in comparison to ascs from lean individuals, ascs that originate from individuals with higher bmis had comparable adipocyte differentiation but reduced proliferation and osteogenic potential. thus, ascs derived from donors with overweight bmi may be less than optimal for applications that involve usage in biocompatible scaffolds for bone grafting. 
these data demonstrate that elevated bmi resulted in reduced asc proliferation, and potentially compromised osteogenic capacity in vitro; thus bmi is an important criterion to consider in selecting asc donors for clinical applications. adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells. it was hypothesized that overweight bmi-ascs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. ascs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. adipose stromal/stem cells body mass index osteogenesisproliferationcolony formationcell size yet the question of whether asc functionality is affected by the donor’s body mass index still exists. ascs were isolated from patients having different bmis, within the ranges of -. alizarin red staining and rt-pcr for alkaline phosphatase demonstrated that elevated bmi resulted in compromised asc mineralization of extracellular matrix and decreased alkaline phosphatase mrna expression.
 obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells. ascs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. yet the question of whether asc functionality is affected by the donor’s body mass index still exists.  ascs were isolated from patients having different bmis, within the ranges of -. it was hypothesized that overweight bmi-ascs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. bmi was inversely correlated with asc proliferation and colony forming potential as assessed by cyquant proliferation assay, and colony forming assays. bmi was positively correlated with early time point but not later time point intracytoplasmic lipid accumulation as assessed by oil-red-o staining. alizarin red staining and rt-pcr for alkaline phosphatase demonstrated that elevated bmi resulted in compromised asc mineralization of extracellular matrix and decreased alkaline phosphatase mrna expression.  these data demonstrate that elevated bmi resulted in reduced asc proliferation, and potentially compromised osteogenic capacity in vitro; thus bmi is an important criterion to consider in selecting asc donors for clinical applications. adipose stromal/stem cells body mass index osteogenesisproliferationcolony formationcell size 