BACKGROUND
mesenchymal stromal cells  have been successfully extracted from bone marrow, muscle, adipose tissue, placenta, and umbilical cord blood  <cit> .
mscs have also been found in both human adult dental pulp and exfoliated deciduous teeth  <cit> .
bone marrow-derived mscs are characterized by a high proliferative capacity ex vivo and can differentiate into a wide variety of mesenchymal progenitor cells under defined culture conditions  <cit> .
the potential therapeutic value of mscs in regenerative medicine, tissue engineering, and cellular therapy has attracted substantial interest in recent years  <cit> .
because mscs can be induced to differentiate into osteogenic tissues  <cit> , their application to modern dentistry and orthopedic surgery as an alternative to autologous bone grafts has been explored  <cit> .
accordingly, most studies focused on using mscs for in vitro or in vivo tissue regeneration in the setting of osseointegration of dental implants or bone grafts for either alveolar bone or long bone reconstruction  <cit> .
however, the human oral cavity contains a high density of microflora, and dental implants grafted with mscs are hence continuously exposed to different bacterial species and their toxins.
in addition, msc-aided dental implants or bone grafts might encounter periodontopathic bacteria-causative agents of periodontitis.
the feasibility of using mscs in osseointegration by virtue of their differentiation into osteoblasts depends on whether the growth and osteogenic differentiation of mscs is affected by bacterial stimuli.
yet, little is known about the interactions between bacteria and mscs.
most studies on this subject focused on the effects of periodontopathic bacteria on osteoclasts, a key cell population involved in bacteria-induced bone destruction.
only a few studies have been done on osteoblasts  <cit>  or its progenitors  <cit> , or mscs  <cit> .
therefore, this study aimed to investigate the effect of purified bacterial toxins on the proliferation and osteogenic differentiation of mscs.
streptococcus pyogenes is a gram-positive bacterium that is commonly found in the human oral cavity; and escherichia coli is a gram-negative bacterium that is commonly associated with clinical sepsis or occassionally osteomyelitis  <cit> .
lipopolysaccharide  and lipoteichoic acid  are bacterial cell-wall components that are derived from these gram-negative and gram-positive bacteria, respectively.
lps triggers immune responses such as proliferation and release of proinflammatory cytokines from human immune cells.
lta shares with lps many of its pathophysiological properties and can thus be considered as the gram-positive counterpart of lps but with a lower mitogenicity  <cit> .
the signaling pathways of these two bacterial cell-wall components are initiated through the toll-like receptors  on the surface of immune cells such as macrophages and dendritic cells.
tlrs are specific receptors that respond to specific pathogen-associated molecular patterns on microbial pathogens by activating innate immune responses and triggering host defense mechanisms  <cit> .
there are at least  <dig> different tlrs found in mammals  <cit> ; among them, tlr <dig> is the major lps receptor  <cit> , and tlr <dig>  is predominantly responsible for recognizing gram-positive cell-wall structures such as lta  <cit> .
although the expression of tlrs has been thoroughly investigated in immune cells, only several studies in recent  <dig> years studied about the gene or protein expression of tlrs in human bone marrow-derived mscs  <cit> .
previous studies demonstrated that osteoblastic cells or mscs express tlr <dig>  tlr <dig>  and other molecules involved in lps signaling pathways .
+, constitutively expressed; -, not detected; na, not available.
saos- <dig>  hos, mg-63: human osteosarcoma cell-lines; mc3t3-e1: mouse osteoblastic cell-line; st-2: mouse stromal cell-line; sam-1: human osteoblastic cell-line from periosteum
tlr2: toll-like receptor 2; tlr4: toll-like receptor  <dig>
a protein expression of tlr <dig> was also observed for saos- <dig> only.
in this study, we examined the effects of lps and lta on the proliferation and osteogenic differentiation of mscs.
then, we explored tlr <dig> and tlr <dig> gene expression profiles during the osteogenic differentiation of mscs with and without the presence of relevant bacterial toxins.
RESULTS
lps and lta did not induce msc proliferation
the multipotency of mscs used in this investigation was confirmed by standard differentiation assays along three lineages including osteogenic, adipogenic and chondrogenic.
all the mscs from the three donors showed similar differentiating functions.
we then tested whether the mscs responded to bacterial toxins lps or lta.
as shown in figure 1a, lps  and lta  did not affect the proliferation of mscs during either the 3- or 7-day period, even after incubation with a relatively high dose of toxin .
as expected, these toxins evoked a marked increase in proliferation of peripheral mononuclear cells  in compared to untreated controls  .
for the response of pbmcs to bacterial toxins,  <dig> ng/ml lps was sufficient to induce a significant increase in proliferation on day  <dig> and day  <dig>  since lta is less potent as compared to lps, higher dosages of lta,  <dig> ng/ml and  <dig> ng/ml, were needed to generate a significant response on day  <dig> and day  <dig> respectively.
prolonged lps challenge enhanced osteogenic differentiation of mscs
to study the effect of bacterial toxins on the osteogenic differentiation of mscs, we exposed mscs with osteogenesis-induction medium and toxin for a short-term of  <dig> days, or prolonged period of either  <dig> or  <dig> days as time points for respective quantitative assays.
alkaline phosphatase  activity was used as an early marker of osteogenic differentiation  <cit> .
a period of  <dig> days was chosen as the duration for the short-term challenge because a significant difference in alp activity between mscs with and without osteogenic induction has been reported previously on day  <dig> after osteogenic induction  <cit> .
prolonged toxin challenge lasted  <dig> to  <dig> days to allow optimal quantification of msc osteogenic differentiation by alp and calcium assays respectively based on our preliminary studies.
under short-term toxin challenge, neither lps at concentrations of  <dig> ,  <dig>  and  <dig> μg/ml  nor lta at  <dig> μg/ml  had a significant effect on alp activity or calcium deposition.
after prolonged lps challenge of higher doses , alp activity and calcium levels were significantly higher than those in untreated controls  .
in contrast, prolonged lta challenge had no effect on alp activity or calcium level .
the increased calcium deposition after prolonged lps challenge was further confirmed semi-quantitatively by von kossa staining .
basal levels of tlr <dig> and tlr <dig> expression in mscs were low
because only the osteogenic function but not the proliferation of mscs was affected by lps, we hypothesized that msc may not express tlr <dig>  until osteogenic differentiation is triggered.
we thus evaluated tlr <dig> and tlr <dig> mrna levels by quantitative pcr.
the constitutive gene expressions of tlr <dig> and tlr <dig> on mscs were determined using pbmcs as a positive control because these cells express all tlrs constitutively.
as expected, the basal level of tlr <dig> mrna in mscs was much lower than that of pbmcs  .
a similar result was observed for the tlr <dig> mrna level, which was virtually undetectable in mscs  .
msc-derived osteoprogenitors expressed tlr <dig> and tlr <dig> during osteogenesis
when we monitored the expression of tlr <dig> and tlr <dig> during msc osteogenic differentiation, tlr <dig> mrna levels were low during the initial  <dig> days but increased sharply on day  <dig>  by about 9-fold compared to control  .
a similar expression pattern was observed for tlr <dig> mrna levels, with an increase of about 48-fold.
.
without osteogenic induction, tlr <dig> and tlr <dig> expression were very low.
lps challenge downregulated tlr <dig> and tlr <dig> expression in msc-derived osteoprogenitors
to determine the effect of lps on tlr <dig> expression during msc osteogenic differentiation, we exposed mscs to lps  under osteogenic conditions.
lps was chosen for we did not find any effect induced by lta based on our result mentioned.
the tlr <dig> and tlr <dig> mrna levels were then determined by quantitative pcr on day  <dig>   <dig>   <dig>  and  <dig> after osteogenic induction.
under both short-term  and prolonged lps challenge, tlr <dig> expression of msc-derived osteoprogenitors was downregulated significantly, by about 75%, on day  <dig>  .
surprisingly, even the level of tlr <dig> expression was also suppressed by prolonged lps challenge on day  <dig>  by about 67%  .
discussion
the increasing interest in using mscs as a source of cellular therapy means that the potential impact of bacterial toxins on the growth and differentiation of mscs will be a growing concern.
exposure to endotoxin-enriched environment may affect many aspects of mscs properties such as self-renewal, differentiation potential, production of cytokines & ecm compound.
our study focused on the investigation of the effects of two purified bacteria-derived toxins on the growth and osteogenic differentiation of human mscs.
cell proliferation was not affected by lps or lta, and osteogenic differentiation was promoted by prolonged exposure to lps but not to lta.
in addition, brief exposure to lps during osteogenic differentiation reduced the expression of tlr <dig>  whereas prolonged exposure reduced the expression of both tlr <dig> and tlr <dig>
most previous related studies have been conducted on osteoclasts  <cit> , than osteoblasts  <cit>  or their committed progenitors  <cit> .
among the studies related to the bacterial effects on the osteogenic progenitors, they used either a 6-day chick periosteal osteogenesis model or rat calvaria cells, which contain a subpopulation of osteogenic precursors.
the objectives of these studies were to find out the possible adverse effects of bacterial toxin on osteogenesis and they did show bacterial toxin impaired the osteogensis process.
we studied the effect of bacterial toxins on osteogenic differentiation using human mscs from bone marrow aspirate, which is an enriched source of multipotential stem cells.
in addition, it is a novel model comparing to the chick or rat models for it allowed us to study how bacterial toxins might affect osteogenic differentiation from mscs to osteogenic progenitors.
our study showed that neither lps nor lta affected the proliferation of mscs, even at relative high endotoxin doses.
this is in line with the findings of previous study by hwa cho et al  <cit> .
we then explored whether these responses pattern may be related to the expression levels of tlr <dig> and tlr <dig> genes in mscs.
this hypothesis was partly supported by our observation that very low level of tlr <dig> nor tlr <dig> mrnas expression levels were detectable in mscs.
these low tlr <dig> and tlr <dig> expression profiles may serve as a protective mechanism to maintain stem cell survival from the effect of bacterial toxins, even at high concentrations.
for cellular therapy purpose, this suggested that mscs could be safely used in clinical settings even in the microenvironment with bacteria such as oral surgical sites.
tlr <dig> and tlr <dig> expressions in msc-derived osteoprogenitors were minimal during early osteogenic induction but emerged by day  <dig>  the expression of these two genes seems to be a natural part of osteoblast differentiation in the absence of external bacterial stimuli.
this finding agrees with those of previous studies in which osteoblastic cells and osteoblasts constitutively express tlr <dig>  tlr <dig>  and other lps-signaling molecules .
this is slightly different from what was observed previously which showed tlr- <dig>   <dig>   <dig>   <dig> had a higher expression as compared to tlr- <dig>   <dig>   <dig>  but such differences may be related to different type of methodology being adopted  <cit> .
another study on murine bm-derived mscs also showed tlr <dig> to tlr <dig> but not tlr <dig> mrna was expressed  <cit> .
whether such variations in tlrs expression profile has something to do with the state of differentiation require further investigation.
although previous studies examined the basal tlr expression level in mscs and how they affect msc differentiation as we did, our study provided information on the real time dynamic changes in the tlr <dig> and tlr <dig> mrna expression profile as they underwent osteogenic differentiation from mscs under the influence of lps or lta.
interestingly, lps but not lta could also downregulate the tlr <dig> and  <dig> expressions on day  <dig> of differentiation, no matter it was under either short or prolonged lps exposure.
our findings suggested that the tlr <dig> and  <dig> expressions could be negatively regulated by endotoxin and such phenomenon has been described in some immune cells such as monocytes.
when testing whether lps and lta affect the osteogenic differentiation of human mscs, we found that short-term challenge by either toxin had no effect, but prolonged lps challenge upregulated both alp activity and calcium deposition.
this is in line with the findings of hwa cho et al.
who found an increase in the osteogenic marker genes expression such as the alp, osteopontin and bmp <dig> at the 5th and 10th day after induction  <cit> .
we confirmed such findings by measuring the alp protein level on day  <dig>
documented effects of e. coli lps on osteoblastic cells are mixed.
no inhibitory effect of e. coli lps were found on osteoblastic cell line mc3t3-e <dig> and rat calvaria cells  <cit> .
however, shjoi et al.
found an inhibitory effect on alp activity of saos- <dig>  an osteoblast-like osteosarcoma cell line  <cit> .
factors contributing to discrepancies in results may include heterogeneity of the cell source, the experimental conditions, and the culture system used.
for tlr <dig> ligands, it has been shown that pgn stimulated the osteogenic differentiation of human adipose-derived mscs in a dose-dependent manner similar to that of lps  <cit> .
however, no such effect was found concerning lta used in our study on bm-derived mscs.
why lps and lta affect the msc differentiation differently in our study remains to be answered, but the specificity and potency of different ligands on the tlrs and also the involvement of different signaling pathways may account for such variations  <cit> .
in this study, the effects of lps exposure during msc osteogenic differentiation depended on the duration of exposure.
this finding might be explained partly by the kinetics of tlr <dig> gene expression: the lack of an effect from short-term lps challenge might be due to the delay in expression of tlr <dig>  therefore, the timing of the activation of tlr genes may play an essential role in altering the osteogenic activity of msc-derived osteoprogenitors.
this finding may explain why studies using osteoblasts and osteogenic precursor cells as effector cells can yield different results after bacterial challenge.
on the other hand, since osteogenic differentiation of mscs was promoted by prolonged lps challenge, we further examined whether constitutive tlr gene expression on osteoprogenitors was also affected by lps.
interestingly, we found that expression of both tlr <dig> and tlr <dig> genes was downregulated in osteoprogenitors after continuous lps exposure.
little has been reported on how bacterial toxins regulate the expression of tlrs on osteoblastic cells.
one study  <cit>  showed an immediate effect of lps challenge on tlr <dig> and tlr <dig> expression of primary murine osteoblasts and mc3t3-e <dig> cells; unexpectedly, tlr <dig> but not tlr <dig> mrna was upregulated within two hours of e. coli lps exposure.
this raised a question about the specificity of tlrs and some degree of crosstalk between tlr <dig> and tlr <dig> may actually exist.
in our experiments, prolonged toxin exposure was designed to mimic the situation in which mscs interact continuously with oral microflora or with bacterial toxins during chronic inflammatory bone disease.
our results suggested that msc-derived osteoprogenitors could adapt to continuous lps challenge by reducing tlr <dig> and tlr <dig> expression, thereby they can be spared from the toxic effect of these toxins, leading to a paradoxical upregulation of osteogenic activities.
a similar adaptive response phenomenon, known as "endotoxin tolerance," can be found in monocytes and macrophages  <cit> .
endotoxin tolerance has been well documented as a cell-desensitizing phenomenon that results from sustained exposure of sublethal doses of lps, which leads to a reduced capacity of the host  or of macrophages  to further respond to lps  <cit> .
furthermore, tlr <dig> expression on the surface of lps-tolerant macrophages has been shown to be downregulated  <cit> , which may account for the molecular mechanism of endotoxin tolerance.
therefore, we suggest that msc-derived osteoprogenitors acquire an adaptive tolerance under continuous lps exposure in order to prevent excessive bone and tissue destruction, thereby protecting or preserving the host organ tissues.
endotoxin tolerance can be found in oral mucosa cells and is associated with chronic periodontitis  <cit> .
in addition, tlr <dig> and tlr <dig> mrnas are significantly downregulated in the gingival tissue of patients with chronic periodontitis as compared to healthy persons  <cit> , suggesting that the oral mucosa develops endotoxin tolerance in chronic periodontitis.
although experimental endotoxin tolerance involves prior incubation of lps before subsequent challenge, a study of human gingival fibroblasts-a major constituent of gingival connective tissue that interacts directly with bacteria in periodontitis-has shown that tlr <dig> expression can be downregulated by lps without prior lps incubation  <cit> .
whether osteoprogenitors or osteoblasts indeed display endotoxin tolerance remains to be confirmed.
interestingly, we also observed downregulation of tlr <dig> gene expression after osteogenic induction in mscs that had undergone short-term lps challenge.
these data may suggest that gene might be switched off in the early phase of osteogenic differentiation.
finally, the paradoxical puzzle of inhibition of the tlr receptor expression by lps was not contradictory to the positive effect on osteogenic differentiation remained to be solved.
possible explanation may include the presence of alternated receptor and signaling pathway for lps induced osteogenesis.
CONCLUSIONS
in conclusion, we found that mscs initially express very low levels of tlr <dig> and tlr <dig>  but msc-derived osteoprogenitors acquire these receptors.
such dynamic changes may at least partly explain why osteoblasts become responsive to lps after an early critical stage of osteogenic differentiation.
more importantly, msc-derived osteoprogenitors adapt by reducing tlr <dig> and tlr <dig> expression after exposure to lps during osteogenic differentiation.
this adaptive response suggests that an intrinsic regulatory mechanism maintains homeostasis in tissues that persistently interact with microbial flora such as in the oral mucosa or infected tissue.
these findings support the potential of using human mscs as a biological graft, even in a bacterial toxin-rich environment.
however, it is still not clear from our data that whether other properties of mscs such as cytokine release, cell adhesion, etc.
are affected by exposure to endotoxins and further works are needed to clarify on these aspects.

 mesenchymal stromal cells have been successfully extracted from bone marrow, muscle, adipose tissue, placenta, and umbilical cord blood. mscs have also been found in both human adult dental pulp and exfoliated deciduous teeth. bone marrow-derived mscs are characterized by a high proliferative capacity ex vivo and can differentiate into a wide variety of mesenchymal progenitor cells under defined culture conditions. the potential therapeutic value of mscs in regenerative medicine, tissue engineering, and cellular therapy has attracted substantial interest in recent years. because mscs can be induced to differentiate into osteogenic tissues, their application to modern dentistry and orthopedic surgery as an alternative to autologous bone grafts has been explored. accordingly, most studies focused on using mscs for in vitro or in vivo tissue regeneration in the setting of osseointegration of dental implants or bone grafts for either alveolar bone or long bone reconstruction. however, the human oral cavity contains a high density of microflora, and dental implants grafted with mscs are hence continuously exposed to different bacterial species and their toxins. in addition, msc-aided dental implants or bone grafts might encounter periodontopathic bacteria-causative agents of periodontitis. the feasibility of using mscs in osseointegration by virtue of their differentiation into osteoblasts depends on whether the growth and osteogenic differentiation of mscs is affected by bacterial stimuli. yet, little is known about the interactions between bacteria and mscs. most studies on this subject focused on the effects of periodontopathic bacteria on osteoclasts, a key cell population involved in bacteria-induced bone destruction. only a few studies have been done on osteoblasts or its progenitors, or mscs. therefore, this study aimed to investigate the effect of purified bacterial toxins on the proliferation and osteogenic differentiation of mscs. streptococcus pyogenes is a gram-positive bacterium that is commonly found in the human oral cavity; and escherichia coli is a gram-negative bacterium that is commonly associated with clinical sepsis or occassionally osteomyelitis. lipopolysaccharide and lipoteichoic acid are bacterial cell-wall components that are derived from these gram-negative and gram-positive bacteria, respectively. lps triggers immune responses such as proliferation and release of proinflammatory cytokines from human immune cells. lta shares with lps many of its pathophysiological properties and can thus be considered as the gram-positive counterpart of lps but with a lower mitogenicity. the signaling pathways of these two bacterial cell-wall components are initiated through the toll-like receptors on the surface of immune cells such as macrophages and dendritic cells. tlrs are specific receptors that respond to specific pathogen-associated molecular patterns on microbial pathogens by activating innate immune responses and triggering host defense mechanisms. there are at least different tlrs found in mammals; among them, tlr is the major lps receptor, and tlr is predominantly responsible for recognizing gram-positive cell-wall structures such as lta. although the expression of tlrs has been thoroughly investigated in immune cells, only several studies in recent years studied about the gene or protein expression of tlrs in human bone marrow-derived mscs. previous studies demonstrated that osteoblastic cells or mscs express tlr tlr and other molecules involved in lps signaling pathways. +, constitutively expressed; -, not detected; na, not available. saos- hos, mg-63: human osteosarcoma cell-lines; mc3t3-e1: mouse osteoblastic cell-line; st-2: mouse stromal cell-line; sam-1: human osteoblastic cell-line from periosteum tlr2: toll-like receptor 2; tlr4: toll-like receptor a protein expression of tlr was also observed for saos- only. in this study, we examined the effects of lps and lta on the proliferation and osteogenic differentiation of mscs. then, we explored tlr and tlr gene expression profiles during the osteogenic differentiation of mscs with and without the presence of relevant bacterial toxins.  lps and lta did not induce msc proliferation the multipotency of mscs used in this investigation was confirmed by standard differentiation assays along three lineages including osteogenic, adipogenic and chondrogenic. all the mscs from the three donors showed similar differentiating functions. we then tested whether the mscs responded to bacterial toxins lps or lta. as shown in figure 1a, lps and lta did not affect the proliferation of mscs during either the 3- or 7-day period, even after incubation with a relatively high dose of toxin. as expected, these toxins evoked a marked increase in proliferation of peripheral mononuclear cells in compared to untreated controls. for the response of pbmcs to bacterial toxins, ng/ml lps was sufficient to induce a significant increase in proliferation on day and day since lta is less potent as compared to lps, higher dosages of lta, ng/ml and ng/ml, were needed to generate a significant response on day and day respectively. prolonged lps challenge enhanced osteogenic differentiation of mscs to study the effect of bacterial toxins on the osteogenic differentiation of mscs, we exposed mscs with osteogenesis-induction medium and toxin for a short-term of days, or prolonged period of either or days as time points for respective quantitative assays. alkaline phosphatase activity was used as an early marker of osteogenic differentiation. a period of days was chosen as the duration for the short-term challenge because a significant difference in alp activity between mscs with and without osteogenic induction has been reported previously on day after osteogenic induction. prolonged toxin challenge lasted to days to allow optimal quantification of msc osteogenic differentiation by alp and calcium assays respectively based on our preliminary studies. under short-term toxin challenge, neither lps at concentrations of, and μg/ml nor lta at μg/ml had a significant effect on alp activity or calcium deposition. after prolonged lps challenge of higher doses, alp activity and calcium levels were significantly higher than those in untreated controls. in contrast, prolonged lta challenge had no effect on alp activity or calcium level. the increased calcium deposition after prolonged lps challenge was further confirmed semi-quantitatively by von kossa staining. basal levels of tlr and tlr expression in mscs were low because only the osteogenic function but not the proliferation of mscs was affected by lps, we hypothesized that msc may not express tlr until osteogenic differentiation is triggered. we thus evaluated tlr and tlr mrna levels by quantitative pcr. the constitutive gene expressions of tlr and tlr on mscs were determined using pbmcs as a positive control because these cells express all tlrs constitutively. as expected, the basal level of tlr mrna in mscs was much lower than that of pbmcs. a similar result was observed for the tlr mrna level, which was virtually undetectable in mscs. msc-derived osteoprogenitors expressed tlr and tlr during osteogenesis when we monitored the expression of tlr and tlr during msc osteogenic differentiation, tlr mrna levels were low during the initial days but increased sharply on day by about 9-fold compared to control. a similar expression pattern was observed for tlr mrna levels, with an increase of about 48-fold.. without osteogenic induction, tlr and tlr expression were very low. lps challenge downregulated tlr and tlr expression in msc-derived osteoprogenitors to determine the effect of lps on tlr expression during msc osteogenic differentiation, we exposed mscs to lps under osteogenic conditions. lps was chosen for we did not find any effect induced by lta based on our result mentioned. the tlr and tlr mrna levels were then determined by quantitative pcr on day and after osteogenic induction. under both short-term and prolonged lps challenge, tlr expression of msc-derived osteoprogenitors was downregulated significantly, by about 75%, on day. surprisingly, even the level of tlr expression was also suppressed by prolonged lps challenge on day by about 67%. discussion the increasing interest in using mscs as a source of cellular therapy means that the potential impact of bacterial toxins on the growth and differentiation of mscs will be a growing concern. exposure to endotoxin-enriched environment may affect many aspects of mscs properties such as self-renewal, differentiation potential, production of cytokines & ecm compound. our study focused on the investigation of the effects of two purified bacteria-derived toxins on the growth and osteogenic differentiation of human mscs. cell proliferation was not affected by lps or lta, and osteogenic differentiation was promoted by prolonged exposure to lps but not to lta. in addition, brief exposure to lps during osteogenic differentiation reduced the expression of tlr whereas prolonged exposure reduced the expression of both tlr and tlr most previous related studies have been conducted on osteoclasts, than osteoblasts or their committed progenitors. among the studies related to the bacterial effects on the osteogenic progenitors, they used either a 6-day chick periosteal osteogenesis model or rat calvaria cells, which contain a subpopulation of osteogenic precursors. the objectives of these studies were to find out the possible adverse effects of bacterial toxin on osteogenesis and they did show bacterial toxin impaired the osteogensis process. we studied the effect of bacterial toxins on osteogenic differentiation using human mscs from bone marrow aspirate, which is an enriched source of multipotential stem cells. in addition, it is a novel model comparing to the chick or rat models for it allowed us to study how bacterial toxins might affect osteogenic differentiation from mscs to osteogenic progenitors. our study showed that neither lps nor lta affected the proliferation of mscs, even at relative high endotoxin doses. this is in line with the findings of previous study by hwa cho et al. we then explored whether these responses pattern may be related to the expression levels of tlr and tlr genes in mscs. this hypothesis was partly supported by our observation that very low level of tlr nor tlr mrnas expression levels were detectable in mscs. these low tlr and tlr expression profiles may serve as a protective mechanism to maintain stem cell survival from the effect of bacterial toxins, even at high concentrations. for cellular therapy purpose, this suggested that mscs could be safely used in clinical settings even in the microenvironment with bacteria such as oral surgical sites. tlr and tlr expressions in msc-derived osteoprogenitors were minimal during early osteogenic induction but emerged by day the expression of these two genes seems to be a natural part of osteoblast differentiation in the absence of external bacterial stimuli. this finding agrees with those of previous studies in which osteoblastic cells and osteoblasts constitutively express tlr tlr and other lps-signaling molecules. this is slightly different from what was observed previously which showed tlr- had a higher expression as compared to tlr- but such differences may be related to different type of methodology being adopted. another study on murine bm-derived mscs also showed tlr to tlr but not tlr mrna was expressed. whether such variations in tlrs expression profile has something to do with the state of differentiation require further investigation. although previous studies examined the basal tlr expression level in mscs and how they affect msc differentiation as we did, our study provided information on the real time dynamic changes in the tlr and tlr mrna expression profile as they underwent osteogenic differentiation from mscs under the influence of lps or lta. interestingly, lps but not lta could also downregulate the tlr and expressions on day of differentiation, no matter it was under either short or prolonged lps exposure. our findings suggested that the tlr and expressions could be negatively regulated by endotoxin and such phenomenon has been described in some immune cells such as monocytes. when testing whether lps and lta affect the osteogenic differentiation of human mscs, we found that short-term challenge by either toxin had no effect, but prolonged lps challenge upregulated both alp activity and calcium deposition. this is in line with the findings of hwa cho et al. who found an increase in the osteogenic marker genes expression such as the alp, osteopontin and bmp at the 5th and 10th day after induction. we confirmed such findings by measuring the alp protein level on day documented effects of e. coli lps on osteoblastic cells are mixed. no inhibitory effect of e. coli lps were found on osteoblastic cell line mc3t3-e and rat calvaria cells. however, shjoi et al. found an inhibitory effect on alp activity of saos- an osteoblast-like osteosarcoma cell line. factors contributing to discrepancies in results may include heterogeneity of the cell source, the experimental conditions, and the culture system used. for tlr ligands, it has been shown that pgn stimulated the osteogenic differentiation of human adipose-derived mscs in a dose-dependent manner similar to that of lps. however, no such effect was found concerning lta used in our study on bm-derived mscs. why lps and lta affect the msc differentiation differently in our study remains to be answered, but the specificity and potency of different ligands on the tlrs and also the involvement of different signaling pathways may account for such variations. in this study, the effects of lps exposure during msc osteogenic differentiation depended on the duration of exposure. this finding might be explained partly by the kinetics of tlr gene expression: the lack of an effect from short-term lps challenge might be due to the delay in expression of tlr therefore, the timing of the activation of tlr genes may play an essential role in altering the osteogenic activity of msc-derived osteoprogenitors. this finding may explain why studies using osteoblasts and osteogenic precursor cells as effector cells can yield different results after bacterial challenge. on the other hand, since osteogenic differentiation of mscs was promoted by prolonged lps challenge, we further examined whether constitutive tlr gene expression on osteoprogenitors was also affected by lps. interestingly, we found that expression of both tlr and tlr genes was downregulated in osteoprogenitors after continuous lps exposure. little has been reported on how bacterial toxins regulate the expression of tlrs on osteoblastic cells. one study showed an immediate effect of lps challenge on tlr and tlr expression of primary murine osteoblasts and mc3t3-e cells; unexpectedly, tlr but not tlr mrna was upregulated within two hours of e. coli lps exposure. this raised a question about the specificity of tlrs and some degree of crosstalk between tlr and tlr may actually exist. in our experiments, prolonged toxin exposure was designed to mimic the situation in which mscs interact continuously with oral microflora or with bacterial toxins during chronic inflammatory bone disease. our results suggested that msc-derived osteoprogenitors could adapt to continuous lps challenge by reducing tlr and tlr expression, thereby they can be spared from the toxic effect of these toxins, leading to a paradoxical upregulation of osteogenic activities. a similar adaptive response phenomenon, known as "endotoxin tolerance," can be found in monocytes and macrophages. endotoxin tolerance has been well documented as a cell-desensitizing phenomenon that results from sustained exposure of sublethal doses of lps, which leads to a reduced capacity of the host or of macrophages to further respond to lps. furthermore, tlr expression on the surface of lps-tolerant macrophages has been shown to be downregulated, which may account for the molecular mechanism of endotoxin tolerance. therefore, we suggest that msc-derived osteoprogenitors acquire an adaptive tolerance under continuous lps exposure in order to prevent excessive bone and tissue destruction, thereby protecting or preserving the host organ tissues. endotoxin tolerance can be found in oral mucosa cells and is associated with chronic periodontitis. in addition, tlr and tlr mrnas are significantly downregulated in the gingival tissue of patients with chronic periodontitis as compared to healthy persons, suggesting that the oral mucosa develops endotoxin tolerance in chronic periodontitis. although experimental endotoxin tolerance involves prior incubation of lps before subsequent challenge, a study of human gingival fibroblasts-a major constituent of gingival connective tissue that interacts directly with bacteria in periodontitis-has shown that tlr expression can be downregulated by lps without prior lps incubation. whether osteoprogenitors or osteoblasts indeed display endotoxin tolerance remains to be confirmed. interestingly, we also observed downregulation of tlr gene expression after osteogenic induction in mscs that had undergone short-term lps challenge. these data may suggest that gene might be switched off in the early phase of osteogenic differentiation. finally, the paradoxical puzzle of inhibition of the tlr receptor expression by lps was not contradictory to the positive effect on osteogenic differentiation remained to be solved. possible explanation may include the presence of alternated receptor and signaling pathway for lps induced osteogenesis.  in conclusion, we found that mscs initially express very low levels of tlr and tlr but msc-derived osteoprogenitors acquire these receptors. such dynamic changes may at least partly explain why osteoblasts become responsive to lps after an early critical stage of osteogenic differentiation. more importantly, msc-derived osteoprogenitors adapt by reducing tlr and tlr expression after exposure to lps during osteogenic differentiation. this adaptive response suggests that an intrinsic regulatory mechanism maintains homeostasis in tissues that persistently interact with microbial flora such as in the oral mucosa or infected tissue. these findings support the potential of using human mscs as a biological graft, even in a bacterial toxin-rich environment. however, it is still not clear from our data that whether other properties of mscs such as cytokine release, cell adhesion, etc. are affected by exposure to endotoxins and further works are needed to clarify on these aspects. 
this study investigated whether proliferation and osteogenic differentiation of mscs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. we selected lipopolysaccharide from escherichia coli and lipoteichoic acid from streptococcus pyogenes as our toxins of choice. our findings showed both lps and lta did not affect msc proliferation, but prolonged lps challenge upregulated the osteogenic differentiation of mscs, as assessed by alkaline phosphatase activity and calcium deposition. in conclusion, our findings support the potential of using human mscs as a biological graft, even under a bacterial toxin-rich environment. however, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human mscs remains unknown. because toll-like receptors, in particularly tlr and tlr are important for the cellular responsiveness to lps and lta respectively, we evaluated their expression profiles serially from mscs to osteoblasts by quantitative pcr. we found that during osteogenic differentiation, msc-derived osteoprogenitors gradually expressed tlr and tlr by day but under prolonged incubation with lps, msc-derived osteoprogenitors had reduced tlr and tlr gene expression. human mesenchymal stromal cells are multipotent cells with potential therapeutic value. owing to their osteogenic capability, mscs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect.
 human mesenchymal stromal cells are multipotent cells with potential therapeutic value. owing to their osteogenic capability, mscs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. however, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human mscs remains unknown. this study investigated whether proliferation and osteogenic differentiation of mscs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.  we selected lipopolysaccharide from escherichia coli and lipoteichoic acid from streptococcus pyogenes as our toxins of choice. our findings showed both lps and lta did not affect msc proliferation, but prolonged lps challenge upregulated the osteogenic differentiation of mscs, as assessed by alkaline phosphatase activity and calcium deposition. because toll-like receptors, in particularly tlr and tlr are important for the cellular responsiveness to lps and lta respectively, we evaluated their expression profiles serially from mscs to osteoblasts by quantitative pcr. we found that during osteogenic differentiation, msc-derived osteoprogenitors gradually expressed tlr and tlr by day but under prolonged incubation with lps, msc-derived osteoprogenitors had reduced tlr and tlr gene expression. this peculiar response to lps suggests a possible adaptive mechanism when mscs are subjected to continuous exposure with bacteria.  in conclusion, our findings support the potential of using human mscs as a biological graft, even under a bacterial toxin-rich environment. 