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Biologicals 39 (2011) 217e223

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Biologicals

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Effect of bone sialoprotein on proliferation and osteodifferentiation of humanbone marrow-derived mesenchymal stem cells in vitro

Bing Xia a,*,1, Jie Wang a,1, Lida Guo a, Zujun Jiang b

aDepartment of Medical Research, Guangzhou General Hospital of Guangzhou Military Command, 111 Liuhua Road, Guangzhou 510010, ChinabDepartment of Hematology, Guangzhou General Hospital of Guangzhou Military Command, 111 Liuhua Road, Guangzhou 510010, China

a r t i c l e i n f o

Article history:Received 16 September 2009Received in revised form9 August 2010Accepted 18 April 2011

Keywords:Bone sialoproteinHuman bone marrow-derivedmesenchymal stem cellProliferationOsteodifferentiationSTRO-1Alkaline phosphataseBone tissue engineering

* Corresponding author. Tel.: þ86 20 3665 3433; faE-mail address: xiab1972@163.com (B. Xia).

1 These authors contributed equally to this work.

1045-1056/$36.00 � 2011 The International Alliancedoi:10.1016/j.biologicals.2011.04.004

a b s t r a c t

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on theproliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into4 groups: control group, 10�10 M BSP group (BSP group), osteogenic medium group (10 nM dexameth-asone, 10 mM b-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP þ OM group (OMplus10�10 M BSP). Compared with the control group, cell growth of the other three groups slowed down,while fluorescence at the G0/G1 phase increased. After 28 days, in the OM group and the BSP þ OM group,the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50%and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNAlevels increased in the OM and BSP þ OM groups, and the mRNA level change was greater in theBSP þ OM group. After 28 days, the number of nodules in the BSP þ OM group was 112.5% more than thatin the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP iscapable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and miner-alization in the presence of OM.

� 2011 The International Alliance for Biologicals. Published by Elsevier Ltd. All rights reserved.

1. Introduction

The tissue engineering approach for bone regeneration andrepair is a new challenge in surgery [1e3]. The bone tissue engi-neering paradigm typically incorporates three componentseadegradable support or scaffold material, bioactive factors such asgrowth factors or other pharmaceuticals, and cells such as osteo-blasts or osteoblast progenitors [4e6].

Bone marrow-derived mesenchymal stem cells (BMSCs), whichare capable of differentiating into multiple mesodermal tissuetypes, including bone, cartilage, tendon, muscles, cardiomyocytes,fat, brain, and marrow stromal connective tissue [7,8], have recentlybeen identified as promising precursor cells in bone tissue engi-neering [9,10].

In order to induce precursor cells to differentiate into adultosteoblasts, bioactive factors are needed. Many factors have beenfound, such as dexamethasone, humanparathyroid hormone (PTH),1,25-dihydroxy vitaminD3 (1,25-(OH)2D3) and bonemorphogenetic

x: þ86 20 3665 3434.

for Biologicals. Published by Elsevi

protein (BMP) [11,12]. However, to improve the osteogenic efficency,novel factors need to be identified. We wondered if bone sialopro-tein(BSP) is such a factor.

BSP is a highly sulfated, glycosylated and phosphorylatedprotein with a high content of sialic acid (15% of glycosaminogly-cans). As one of the noncollagenous proteins in the extracellularbone matrix, BSP plays an important role in the mineralization andadhesion of osteoclasts to the bone surface [13e18], and is alsoa marker of osteoblast differentiation [19e21]. BSP belongs to theSmall Integrin-binding Ligand N-linked Glycoprotein (SIBLING)family of RGD (Arg-Gly-Asp)-containing matrix proteins, severalmembers of which have been shown to affect cell differentiation.Moreover, it was reported that BSP directly influenced differenti-ation of osteoblasts. In the mouse osteoblastic MC3T3E1 cell line,the overexpression of BSP or supplementation of cultures withrecombinant BSP increased osteoblast-related gene expression,calcium incorporation and nodule formation. Conversely, suppres-sion of BSP expression by RNA interference inhibited the expres-sion of osteoblast markers and nodule formation. Together, theseresults demonstrated that BSP might serve as a matrix-associatedsignal which directly promotes osteoblast differentiation resultingin the increased production of a mineralized matrix [22]. Inaddition, BSP stimulated site-specific in vivo calcification andosteogenesis of a rat calvarial defect and a thoracic subcutaneous

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B. Xia et al. / Biologicals 39 (2011) 217e223218

pouch model [23]. So BSP is indeed capable of promoting osteo-blast differentiation.

As for human BMSCs (hBMSCs), the osteoblast precursors, canBSP also regulate their osteodifferentiation? Up to now, we havefound only one report about the effects of BSP on hBMSCs. Itshowed that BSP coating of various substrates could not enhancein vitro osteodifferentiation or in vivo bone formation capacity ofhBMSCs, and that BSP on polymeric materials was not sufficient toprime BMSC functional osteoblastic differentiation in vivo [24].That is to say, BSP itself might have no ability to induce BMSCs todifferentiate into osteoblasts. However, to draw that conclusion,more experiments are needed. So here, we investigated the influ-ences of recombinant human BSP (rhBSP) on the proliferation andosteodifferentiation of hBMSCs cultured in vitro. Our data supportthat BSP alone has no significant effects on osteodifferentiation ofthese stem cells, but we also demonstrate that BSP can inhibithBMSCs proliferation and enhance osteogenic medium(OM)-induced osteodifferentiation.

2. Materials and methods

2.1. Human bone marrow-derived mesenchymal stem cell harvestand isolation

To isolate hBMSCs, bone marrow aspirates of 10 mL were takenfrom the iliac crest of normal donors ranging in age from 25 to 34years old under an Institutional Review Broad approved protocol.All bone marrow cells derived from one donor were plated in 5 mLGrowth Medium [LG-DMEM (Gibco, Grand Island, N.Y., USA), 10%fetal bovine serum (Hyclone, Logan, UT), 100 units/mL of peni-cillin, 100 mg/mL of streptomycin, 2 mM L-glutamine (Invitrogen,Carlsbad, CA)] at 1 � 106 cells/cm2 in a 25-cm2 culture flask.Usually 8 � 106 w 5 � 107 cells were obtained by aspiration of oneindividual before plating. At 5 w 7 d after plating, hematopoieticand other nonadherent cells were washed away during mediumchanges. The remaining purified BMSC population was expandedin culture. All cells were harvested when they reached 80e90%confluence at passage 2. BMSCs were put into a cryopreservationsolution consisting of 10% Dimethyl Sulfoxide and 90% fetal bovineserum. Aliquots of about 1.8 mL each were slowly frozen andstored in liquid nitrogen. Using trypan blue staining, the viabilityof all thawed BMSC lots were verified to be >85% before use in thestudy. Because no significant differences in the following cultureswere found between the aspirates of different donors, all theBMSCs were pooled. Individual cell colonies were isolated andcells were further cloned at 0.3 cell/well in 96-well plates. Theproliferation potential of the BMSC was quantified by counting thetotal number of colony-forming unit fibroblasts (CFU-F) ofadherent cells (>50 cells). Clonal BMSCs were expanded andtested for their adipogenic, osteogenic and chondrogenic differ-entiation potential.

2.2. Cell culture and experimental groups

The BMSCs were seeded at a density of 1.0 � 104 cells/cm2 in96-well, 24-well or 6-well culture plates in Growth Medium. After24 h, the medium was changed with different media and thecultures were divided into the following four groups: the Osteo-genic Medium group in which 10 nM dexamethasone, 10 mMb-glycerophosphate, and 50 mg/L ascorbic acid was added toGrowth Medium, the BSP group in which 10�10 M recombinanthuman sialoprotein II (rhBSP, Cat. No. 4014-SP, RD systems Inc) wasadded to Growth Medium, the BSP þ OM group in which 10�10 MBSP was added to the OM culture and the control group in whichthe above supplements were omitted.

2.3. Measuring cell growth

Cell growth was measured by counting cells. Briefly, cells wereplated on 24-well culture plates at a density of 5 � 103 cells in 1 mLof Growth Medium per well. Cell counting was conducted in tri-placate every 24 h for the next 13 days. At indicated times, the cellswere gently washed 3 times with phosphate-buffered saline (PBS)and then harvested with 2.5 g/L trypsin and 0.2 g/L EDTA. Aftercentrifugation 500 � g for 5 min, the cell pellets were resuspendedin Growth Medium and dripped onto the blood counting chamber.The cells were counted with an inverted microscope (Axiovert 40,Carl Zeiss, Berlin, Germany).

2.4. Morphological observations

The morphology of the cell cultures was observed with aninverted microscope (Axiovert 40, Carl Zeiss, Berlin, Germany) andmicrophotographs were taken.

2.5. Cell cycle analysis

At indicated times, the cells were harvested as described in 2.3.After centrifugation 500 � g for 5 min, 2 � 105 w 1 �106 cells wereresuspendedwith PBS, and then fixed in 2mL of 70% cold ethanol at4 �C for 24 h. After washing in cold PBS three times, the cells wereresuspended in 0.8 mL of PBS with 40 mg of propidium iodide and0.1 mg of RNase A for 30 min at 37 �C in the dark. Samples wereanalyzed for DNA content with FACS Calibur (Becton Dickinson,San Jose, CA) using the blue 488-nm laser for excitation and the fl2detector 585 � 21 nm for emission.

2.6. Analysis of STRO-1 expression by flow cytometry

Expression of Stro-1 was determined at day 6, 9, and 12 usingflow cytometry. Briefly, 1 � 105 cells were labeled with 0.5 mg ofmouse anti-human STRO-1 monoclonal antibody (R&D SystemsCompany, Minneapolis, MN) in Dulbecco’s phosphate-bufferedsaline (DPBS) containing 10% FBS (DPBS þ S) for 40 min at 4 �C.After that, the cells were incubated with goat anti-mouse IgM (m)conjugated to fluorescein (UK KPL,Wembley, UK) for 30min at 4 �C.Stained cells were fixed and 1 � 104 viable cells were analysed byflow cytometry using standard settings. Staining was comparedwith an isotype control antibody (purified mouse IgM, USA BioL-egend, San Diego, CA) to correct for non-specific binding.

2.7. Measurement of alkaline phosphatase (ALP)

ALP activity was assessed at day 6, 9, and 12 after treatment. Atindicated times, the cultures grown in 96-well plates were washedthree times in PBS, and then 50 mL 0.2% Triton X-100 was added at4 �C overnight to lyse the cells. On the next day, ALP activity wasassessed using a biochemical kit (Nanjing Jiancheng BioengineeringInstitute, Nanjing, China), following the manufacturer’s instruc-tions. The kinetic measurements of the enzyme were measuredbased on hydrolysis of p-nitrophenyl phosphate at 520 nm using2-methyl-1-propanol at pH 10.3 as a buffer. ALP activity wascalculated using a p-nitrophenol standard curve and was normal-ized to total protein content across treatments. The total proteincontent of each culture was determined using the BCA assay(Thermo Fisher Scientific Inc., Rockford, IL, USA). Cell lysates wereanalyzed in sextuplicate. Enzyme activity was expressed as nano-moles of p-nitrophenol produced per hour per milligram of totalcellular proteins.

B. Xia et al. / Biologicals 39 (2011) 217e223 219

2.8. von Kossa staining

Mineralization of extracellular matrix was visualized by vonKossa staining at day 21 and 28 after treatment [25]. Briefly, cellsgrown in 6-well plates were fixed with 3.6% formaldehyde for60 min at room temperature. After being rinsed with distilledwater, cells were overlaid with 2% weight by volume (w/v) silvernitrate solution (SigmaeAldrich, St. Louis, MO) under direct UVlight exposure for 20 min. Excess silver staining was removed byincubation with 5% sodium thiosulfate solution (SigmaeAldrich)for 5min, and then the samples were observed. Calcium-phosphatedeposits stained black. Calcium nodules with a diameter more than0.5 mm at 6-well plates were counted and compared for all the 4groups (n ¼ 6 wells per group).

2.9. Quantitative real-time RT-PCR

Cells grown on 6-well plates were harvested using 0.25% trypsinand 1 mM EDTA (Gibco). Total RNA was prepared using the TRIzol(Invitrogen, Carlsbad, CA). The 260-nm optical density of theprepared total RNAwasmeasured in a spectrophotometer and usedto determine RNA concentration. The ratio of 260-nm opticaldensity and 280-nm optical density was used to determine RNApurity. Then, the RNA was subjected to quantitative real-timeRT-PCR using the SYBR� PrimeScript� RT-PCR kit (TaKaRa, Dalian,China), following the instructions of the manufacturer. To performRT, each 10 mL RT mix contained 2 mL 5 � Primer Script Buf-fer(TaKaRa), 0.5 mL Primer Script RT Enzyme Mix I (TaKaRa), 0.5 mLOligo dT Primer(50 mM), 0.5 mL Random 6 mers(100 mM), 500 ngtotal RNA template, and RNase-free water. To perform real-timePCR, each 25 mL RT-PCR mix contained 12.5 mL of 2 � SYBRPremix Ex Taq (TaKaRa), 200 nM forward and reverse primers,10 ngof total RNA template, and distilled water. Conditions for RTwere asfollows: reverse transcription at 37 �C for 15 min, followed by 85 �Cfor 5 s to inactivate the RTenzyme. For PCR, hold stagewas 95 �C for30 s, cycling was 40 cycles of 95 �C for 5 s, 55 �C for 30 s, and 72 �Cfor 30 s. GADPH was used to control the amount of template foreach sample. Primers used in this study are shown in Table 1.Standard curves for each amplification product were generatedfrom 10-fold dilutions of pooled total RNA to determine primerefficiency. Raw PCR values were determined by plotting signalsgenerated from individual wells against standard curves. Eachreaction was run in triplicate with the results averaged. Annealingcurves were performed to ensure the absence of primer dimmers.Amplification products were run on 1% agarose gels to ensure onesingle band was generated for each of the tested genes.

2.10. Statistical analysis

Differences were analyzed using one-way ANOVA and Bonfer-roni post-test by SPSS 13.0 software (Chicago, IL). p values less than0.05 were considered statistically significant.

Table 1Sense and antisense primer sequences used in quantitative real-time RT-PCR.

Gene of interest Oligonucleotide sequence

GADPH sense 50-ctcatgaccacagtccatgc-30

GADPH antisense 50-cacattgggggtaggaacac-30

cbfa1 sense 50-gccgggaatgatgagaacta-30

cbfa1 antisense 50-ggaccgtccactgtcacttt-30

CD271 sense 50-agagaagcatcggagggaat-30

CD271 antisense 50-cacactggcccacttttctt-30

Osteocalcin sense 50-aagcaggagggcaataaggt-30

Osteocalcin antisense 50-ttagggcagcacaggtccta-30

Osterix sense 50-caagaagggggaagtccaat-30

Osterix antisense 50-agaatccctttccctctcca-30

3. Results

3.1. Characterization of hBMSCs

Adherent colonies of cells derived from bone marrowmorphologically resembling fibroblasts were obtained betweenday 8 and day 16. On day 16, the number of colony-forming units(CFU-F) obtained from 1.2 � 106 plated cells was 47.2 � 3.6 (n ¼ 5).Phenotypically, these clonally expanded cells expressed allmarkers of MSC (CD90þCD73þCD105þCD45e, not shown). Next,they were tested for their differentiation potential. Culturestreated with adipogenic supplements showed cytoplasmic lipiddroplets visualized by staining with Oil-red O. Induced by theosteogenic medium, cells broadened and formed mineralizedmatrix as evidenced by von Kossa staining. Furthermore, cellcultures maintained in chondrogenic medium were composed ofchondrocyte-like cells surrounded by a sulfated proteoglycan-richAlcian blue-positive extracellular matrix (not shown). Thus, thefibroblast-like cells, which can be clonally expanded in vitro andretain the capacity to differentiate into at least 3 mesodermallineages, were identified as BMSCs.

3.2. Effect of BSP on the morphology of hBMSCs

The untreated hBMSCs grew into tiered clones, and most ofthem were fibroblast-like (Fig. 1A). BSP alone had no obviousinfluence on the morphology of BMSCs (Fig. 1B). After beingcultured in OM with or without BSP (10�10 M), the cells showeda completely different morphology, compared to the untreatedcontrol group. Gradually they became large and flat, and the clonesdispersed (Fig. 1C and D).

3.3. Inhibitory effect of BSP on cell growth rate of hBMSCs

The cultures of hBMSCs had an initial lag phase of about 3 days,during which the colonies grew from single cells. Then the cellswent into a logarithmic growth phase, from about day 5 to day 13.After that, the cell number did not increase any more unless theywere passaged. Compared to the control group, BSP(10�13 M w 10�7 M) inhibited the growth rate of hBMSCs ina concentration- dependent manner (Fig. 2A). Similar to BSP(10�10 M), the osteogenic supplements also inhibited growth. Also,the effect of OM þ BSP was stronger than either of them alone(Fig. 2B). Based on the above results, for the following experiments,the BSP concentration and treatment duration was set as 10�10 Mand 12 days, respectively.

3.4. Analysis of cell cycle perturbations induced by BSP in hBMSCs

Next, we explored the mechanism underlying the growth inhi-bition induced by BSP. The lack of growth in BSP-treated cells mighthave resulted from cell cycle perturbations. The cell cycle distri-bution of cultures was determined by FACS analysis of propidiumiodideestained cells to measure DNA content. As shown in Fig. 3,compared with the control group, fluorescence at G0-G1 phaseincreased by 11.22%, 16.49% and 18.36%, in BSP, OM, and BSP þ OMgroups, respectively. Meanwhile, all of the treated groups showeda higher proportion of cells in G0-G1 phase than the control groupand there was a concomitant decrease in the proportion of cells in Sphase by 49.88%, 70.20% and 74.61%, in the BSP, the OM and theBSP þ OM groups, respectively, compared with the control group.Thus, among those three treated groups, the effect of BSP was theweakest, while that of BSP þ OM was the strongest.

Fig. 1. Effect of BSP on growth morphology of hBMSCs. BMSCs at passage 2 were treated with 10�10 M rhBSP (B), OM (C), or 10�10 M BSP plus OM (D) for 12d, and the untreated cellswere control (A).

B. Xia et al. / Biologicals 39 (2011) 217e223220

3.5. Effect of BSP on expression of STRO-1

The effect of BSP on expression of STRO-1 was examined usingFACS analysis. Compared with the control, the proportion of STRO-1-positive cells decreased by 22.7% (p < 0.05) and 38.4% (p < 0.01)in the OM group and BSPþ OM group, respectively, with no changein the BSP group (p > 0.05) (Fig. 4).

3.6. Effect of BSP on ALP activity of the hBMSCs

Compared with control, the ALP activity did not change in BSPgroup, whereas increased in OM and OM þ BSP groups. As thetreatment time became longer, the ALP activity increased propor-tionally in the two groups. After treatment for 12 days, the ALPactivity of the OM group and OM þ BSP group increased by 50%(p < 0.01) and 71.43% (p < 0.01), respectively, compared to thecontrol (Fig. 5).

3.7. Effects of BSP on expression of Cbfa1, CD271, osteocalcin andosterix in hBMSCs

When treated with OM for 28 days, CD271 mRNA cell expressiondecreased by 65% (p < 0.01) (Fig. 6A), while Cbfa1, osteocalcin andosterix mRNA levels increased by 4.22 (p < 0.01), 1.86 (p < 0.05) and3.16 times (p < 0.01), respectively (Fig. 6BeD), as compared to thecontrol group.When treatedwithBSPþOMfor28days,CD271mRNAexpression decreased by 74% (p < 0.01) (Fig. 6A), while Cbfa1, osteo-calcin and osterix mRNA levels increased by 6.32 (p < 0.01), 2.92(p < 0.01) and 4.34 times (p < 0.01), respectively (Fig. 6BeD), ascompared to the control group. Obviously, the changes of the mRNAlevels in BSP þ OM group were more than those in BSP group(p < 0.05). BSP alone had no significant influence on the mRNA

expressions. Furthermore,weperformedadifferentiationexperimentwhere BSP was added at 7d, 14d and 21d, respectively. Our datashowed that it was the addition of OM, not BSP, that coincided withthe appearance of the abovementioned differentiationmarkers (datanot shown).

3.8. Effect of BSP on mineralization of hBMSCs

The mineralized nodule was a mixture of silver and calcium salt,which was stained black by von Kossa staining (Fig. 7A). The resultsshowed that the stain was negative in both the control and BSPgroups, while positive in the OM and BSP þ OM groups. Further-more, the number of nodules in the BSPþ OM groupwas 70.0% and112.5% more than that in OM group on day 21 and day 28,respectively (Fig. 7B).

4. Discussion

When searching for more bioactive factors to modulate andoptimize bone formation in tissue engineering, BSP gained ourinterest. Since it can directly promote osteoblast differentiation, canit also enhance osteogenic differentiaton of hBMSCs? And can itaffect proliferation of those stem cells? To answer these questions,we performed the present study.

In the proliferation experiments, our results showed that BSPinhibited proliferation of hBMSCs. After exposing BMSC to BSP thecell growth rate slowed down. Moreover, BSP-treated cellsdemonstrated a higher proportion of cells in G0-G1 phase anda lower proportion of cells in S phase, indicating that to some extentcell cycle progression was blocked. Thus we conclude that BSPinhibits the proliferation of hBMSC by holding back the transitionfrom G0-G1 phase to S phase. Furthermore, we could see that BSP

A

B

Fig. 2. Effect of BSP on hBMSC growth rate. (A) Effect of different concentrations of BSPon hBMSC growth rate. BMSCs at passage 2 were plated at a density of 1.0 � 104 cells/cm2 in 24-well plates. After 24 h, cells were treated with BSP of various concentrations(10�13 w 10�7 mol/L). At defined time points, cells were trypsinized and counted byhemocytometer. CONT, control group; 13 w 7, 10�13 w 10�7 M BSP groups. Values aremean � SD (n ¼ 3). (B) Effect of BSP and OM alone or combination on hBMSC growthrate. BMSCs in 24-well plates were treated with 10�10 M BSP, OM, OM plus10�10 M BSP,or not treated. At defined time points, cells were counted. Values are mean � SD(n ¼ 3). *p < 0.05, **p < 0.01, experimental groups vs matched control; #p < 0.05,BSP þ OM group vs matched BSP group.

Fig. 3. Effect of BSP on cell cycle distribution of hBMSCs. Human BMSCs were treatedwith 10�10 M BSP, OM, 10�10 M BSP þ OM, or untreated for 12 d. Propidium iodidestaining followed by flow cytometry was used to analyze cell cycle distribution. BSP,OM and OM þ BSP resulted in an increased proportion of cells in the G0/G1 phase anda decreased proportion of cells in the S phase. Results shown are representative ofthree separate experiments. Values are mean � SD (n ¼ 3). As for G0/G1 phase,*p < 0.05, vs control; #p < 0.05, vs BSP group.

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Fig. 4. Effect of BSP on STRO-1 expression in hBMSCs detected by FACS. The hBMSCswere seeded at a density of 1.0 � 104 cells/cm2 in 6-well plates in Growth Medium.After 24 h, the cultures were treated with 10�10 M BSP, OM, or 10�10 M BSP þ OM, andthen subjected to STRO-1 expression analysis by FACS at indicated times. The cellscultured in Growth Mediumwere control group. CONT, control group; BSP, 10�10 M BSPgroup; OM, OM group; OM þ BSP, OMþ10�10 MBSP group. *p < 0.05, **p < 0.01, vsControl; #p < 0.05, vs OM group.

B. Xia et al. / Biologicals 39 (2011) 217e223 221

and OM affected hBMSC proliferation synergistically. Dexametha-sone, b-glycerophosphate and ascorbic acid are widely used asosteodifferentiation inducers in vitro [26e28]. They also have aninhibitory effect on the proliferation of hBMSCs. When BSP wasadded to OM, the inhibitory effect of OM on cell proliferationbecame even stronger, suggesting that BSP and OM are coordinatedin this regard.

In the osteodifferentiation experiments, our data indicatedthat BSP significantly enhanced the osteogenic effect of OM onhBMSCs, although BSP alone had no significant influence on thedifferentiation.

First,we determined STRO-1 expression andALP activity. STRO-1is amarker ofmesenchymal progenitor cells [29], and its expressionin vitro remains a characteristic of less well-differentiated cells ofthe osteoblast lineage [30]. In 1991, Simmons and Torok-Storbidentified stromal cell precursors in human bone marrow usingthe novel monoclonal antibody STRO-1 [31]. The STRO-1 antigen ispresent only on the fibroblast colony-forming unit (CFU-F) cells inadult human bonemarrow, and potentially defines aMSC precursorsubpopulation. The STRO-1þ marrow cell population is multipo-tential, capable of differentiating into multiple mesenchymal line-ages including hematopoiesis-supportive stromal cells with

a vascular smooth muscle-like phenotype, adipocytes, osteoblastsand chondrocytes [29e34]. The work of Gronthos et al provideddirect evidence that osteoprogenitors derived from human bonemarrow were present in the STRO-1þ population [35]. Conversely,ALP is one of the markers of osteoblast differentiation. There is aninverse association between the expression of STRO-1 and ALP incultures of BMSCs, and dual labeling of BMSCs with monoclonalantibodies recognizing STRO-1 and ALP permits the identificationand isolation of osteoblast lineage cells at different stages of differ-entiation [30]. In this study,wedetermined the activityof ALP, not itsexpression levels, because we had found that ALP activity wasproportional to mRNA expression level (data not shown) and moreconvenient to quantify. In the present study, after exposure toOMorBSP þ OM, STRO-1 expression of hBMSCs decreased while ALP

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Fig. 5. Effect of BSP on ALP activity of the hBMSCs. ALP activity was assessed at day 6,9, and 12 after treatment. Enzyme activity was expressed as nanomoles of p-nitro-phenol produced per hour per milligram of total cellular proteins. Results shown arerepresentative of three separate experiments. Values are mean � SD (n ¼ 6). CONT,control group; BSP, 10�10 MBSP group; OM, OM group; OM þ BSP, OMþ10�10 M BSPgroup. *p < 0.05，**p < 0.01，vs control；#p < 0.05，##p < 0.01，vs OM group.

B. Xia et al. / Biologicals 39 (2011) 217e223222

activity increased, indicating that the stem cellswere differentiatinginto adult osteoblasts.

Secondly, we quantified the mRNA levels of Cbfa1, osteocalcin,osterix and CD271. CD271 is a marker of mesenchymal stem cells

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Fig. 6. Effects of BSP on mRNA expressions of CD271, Cbfa1, osteocalcin and osterix in hBM10�10 M BSP þ OM. Real-time PCR was performed as described in Materials and methods. In eCbfa1, osteocalcin and osterix mRNA levels were all upregulated (BeD), compared to the c

[36,37], while Cbfa1, osteocalcin and osterix are three key osteo-genic markers [38,39]. Consistent with the above observations,their expressions had no significant changes in the BSP group, butCD271 mRNA expression was downregulated and the other 3molecules’ expressions were upregulated in the OM and BSP þ OMgroups. Moreover, the mRNA level changes were greater in theBSP þ OM group than in OM group. Also, for the cells cultured inOM, it was more effective to add the BSP at an earlier point in thedifferentiation process.

Thirdly, we observed the mineralization of hBMSCs with thedata showing that BSP enhanced the mineralization stage ofhBMSCs induced by OM. The cells cultured in inductive mediumsecreted extracellular matrix, and the presence of mineralizedmatrix was confirmed after 3 weeks by von Kossa staining, which isable to stain extracellular matrix containing calcium deposits. Blackmineralized nodules appeared in the OM and BSPþ OM groups, butnot in the control and BSP groups. Furthermore, the number ofnodules in the BSP þ OM group was significantly more than that inthe OM group, indicating that the effect of BSP þ OM was signifi-cantly stronger than OM alone.

In summary, the data presented here showed that BSP inhibitsthe proliferation of hBMSCs, and that BSP alone has no osteogeniceffect on hBMSCs, but it can enhance osteodifferentiation in thepresence of OM. It is known that stem cells are likely to differentiatewhen their proliferation is suppressed, so BSP might promoteosteodifferentiation by inhibiting cell proliferation of hBMSCs. Ofcourse the exact mechanism needs to be explored in the future.Also, we got an idea from this study that combining BSP with otherosteogenic inducers may improve the osteogenic efficiency in bonetissue engineering.

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SCs. Total RNA was collected at 0, 7, 14, 28 days after exposure of 10�10 M BSP, OM orither of OM or BSP þ OM group, CD271 mRNA expressionwas downregulated (A), whileontrol. The result was the representative of three separate experiments.

Fig. 7. Effect of BSP on calcium nodules formation in the hBMSCs. The cells weregrown in 6-well plates, and the mineralization of extracellular matrix was visualizedby von Kossa staining at 21d and 28d after treatment. Calcium-phosphate depositsstained black (A). The nodules were counted and compared between the OM groupand OM þ BSP group (n ¼ 6) (B). *P < 0.05, **P < 0.01, vs matched OM group.

B. Xia et al. / Biologicals 39 (2011) 217e223 223

Acknowledgments

Wewould like to give our thanks to all the other members in ourlaboratory for their technical assistance, and to Mr. Phillip Connolly,Universityof TexasM.D.AndersonCancerCenter, forhisEnglishedits.This work was supported by the research grant from the NaturalScience Foundation of Guangdong Province of China 06104396.

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