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[(For)-735(practical)-740.6(application,)-742(the)-743.5(adipose-)]TJ
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[(derived)-453.4(stem)-454.5(cell)-455.2(\(ASC\))-456.2(is)-453.8(increasingly)-456.9(favored)-456(over)-453(bone)]TJ
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[(marrow-derived)-349.2(MSC)-351.5(due)-345.7(to)-351(the)-345.2(relative)-349.7(ease)-348.4(of)-351(tissue)-349.3(har-)]TJ
T*
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[(commission)-588.4(of)-591.3(ASC-derived)-593(osteoprogenitor-enriched)-597.2(cell)]TJ
T*
[(\(OPEC\))-678.7(and)-676.1(endothelial)-680.5(progenitor-enriched)-684.8(cell)-682.8(\(EPEC\))]TJ
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[(neous)-584.1(cell)-581.6(population)-582.8(comprising)-586.9(of)-578.6(a)-581.2(spectrum)-583.7(of)-578.6(cells)]TJ
T*
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T*
[(mesenchymal)-458.7(cells)-462.8(to)-458.5(fully)-462.2(end-differentiated)-461.3(cells,)-459.4(includ-)]TJ
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[(ing)-479.2(a)-480.1(signicant)-483.4(population)-481.6(of)-477.5(osteoprogenitor)-488.8(and)-480.1(EPCs.)]TJ
0 -1.2203 TD
[(We)-435.2(investigate)-434.5(eight)-435.3(coculture)-438.6(combinations)-432.9(to)-439.5(learn)-430.7(whe-)]TJ
T*
[(ther)-466(optimal)-464(cell)-467.8(combinations)-464.6(exist)-468.9(for)-465.5(vascularized)-467.1(bone)]TJ
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(engineering.)Tj
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27.9978 74.5598 TD
[(Materials)-339(and)-331.9(Methods)]TJ
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0 -1.8336 TD
[(ASC)-340.1(isolation)-334.1(and)-335.7(expansion)]TJ
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[(Stromal)-376.1(stem)-372.3(cells)-374.3(were)-375.2(isolated)-374.4(and)-372.6(expanded)-378.9(from)-368.2(hu-)]TJ
-1.1128 -1.2203 TD
[(man)-500(lipoaspirate)-504.4(as)-494(previously)-500.2(described.)]TJ
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(27,33)Tj
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[(Cell)-494(culture)]TJ
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[(media)-256.4(for)-256.9(all)-259.5(experiments)-256.8(are)-253(summarized)-256.9(in)-259.8(Table)-257(1.)-249(A)-260.6(stem)]TJ
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[(cell)-448.9(growth)-443.3(medium)-445.5(includes)-445.3(the)-446.4(Dulbeccos)-445.1(modied)-442.7(Ea-)]TJ
T*
[(gles)-428.4(medium)-426.6(\(DMEM\))-426.6(and)-423.2(50%)-424.3(fetal)-427.5(bovine)-425.7(serum)-424.5(\(FBS\).)]TJ
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T*
[(expansion)-327.1(medium)-312.7(with)-318.8(0.01)-165.8(nM)-318.3(dexamethasone)-322.6(and)-322(20)-163.4(nM)]TJ
T*
[(ascorbate)-279.8(added,)-270.6(and)-277.8(aims)-267.5(to)-275.1(direct)-278.8(cell)-271.8(differentiation)-277.9(to)-275.1(the)]TJ
0 -1.2266 TD
[(osteogenic)-460.4(route.)-457(The)-455.2(vasculogenic)-455.3(differentiation)-461.3(medium)]TJ
0 -1.2203 TD
[(\(VDM\))-358(includes)-350.4(components)-360(from)-349.3(the)-351.5(EGM-2)-354.6(MV)-349.4(BulletKit)]TJ
T*
[(in)-557(proprietary)-559.2(concentrations)-563.9(\(Lonza,)-561.7(Basel,)-553.9(Switzerland\),)]TJ
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[(and)-499.1(aims)-495.1(to)-496.4(direct)-500.1(cell)-493.1(differentiation)-505.6(to)-496.4(the)-496.9(vasculogenic)]TJ
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[(route.)-362.2(Briey,)-352.6(human)-353(lipoaspirate)-358.9(was)-356.5(obtained)-352.1(from)-355.6(seven)]TJ
T*
[(female)-260.1(donors)-254.2(\(ages)-258.5(1855\))-250.9(\(Institutional)-260.3(Review)-256.5(Board)-255.6([IRB])]TJ
0 -1.2266 TD
[(approval)-483.7(#H12756-01b\).)-478.6(Each)-479.4(patient)-482.8(received)-481.2(information)]TJ
0 -1.2203 TD
[(about)-255.1(the)-250.3(scope)-257.5(and)-252.5(goals)-251.8(of)-249.8(this)-249(study.)-253.8(According)-258(to)-249.8(the)-250.4(IRB)]TJ
T*
[(protocol,)-377(verbal)-369.2(informed)-378.1(consent)-370.9(was)-369.2(obtained)-377.4(before)-368.6(sur-)]TJ
T*
[(gery.)-309.5(The)-303.5(specimens)-311.1(were)-311.9(collected)-308.8(as)-304.3(discarded)-304.1(tissue)-311.4(from)]TJ
0 -1.2266 TD
[(patients)-275.5(undergoing)-273.5(elective)-279.1(lipoaspiration.)-270.5(After)-274.8(harvest,)-274.5(all)]TJ
0 -1.2203 TD
[(samples)-339.6(were)-337.2(de-identied)-339.2(before)-336.9(processing.)-337(There)-335.7(was)-337.6(no)]TJ
T*
[(collection)-296.7(of)-287.8(personal)-290.7(health)-288.4(information,)-292.9(demographic)-293.6(data,)]TJ
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[(or)-338.6(other)-336(identifying)-333.5(data.)]TJ
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[(Each)-605.9(triplicate)-606.6(experiment)-609.5(utilized)-609.2(cells)-601.9(from)-608.5(one)-606.7(do-)]TJ
-1.1128 -1.2203 TD
[(nor.)-490.3(Crude)-485.7(lipoaspirate)-491.7(was)-489.3(washed)-489.3(three)-491.2(times)-483.3(with)-489.6(the)]TJ
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[(Hanks)-421.5(balanced)-423.5(saline)-415.8(solution)-423.8(\(HBSS\))-424.5(\(Invitrogen/Gibco,)]TJ
ET
313.058 385.852 239.131 .22675 re
f
313.058 113.953 239.131 .28348 re
f
313.058 366.86 239.131 .28348 re
f
BT
/F7 1 Tf
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(Table)Tj
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3.0603 0 TD
(1.)Tj
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1.2456 0 TD
[(Cell)-333.8(Culture)-336.9(Media)]TJ
/F4 1 Tf
-10.5529 -2.0423 TD
[(Cell)-337.5(culture)-340.1(medium)-9266.1(Composition)]TJ
/F3 1 Tf
0 -2.0929 TD
[(Stem)-337.8(cell)-335(growth)-335.8(medium)-2121.1(DMEM)-330.8(\(Fisher\))]TJ
13.6828 -1.1128 TD
[(50%)-329.4(FBS)-334.7(\(Invitrogen/Gibco\))]TJ
T*
[(1%)-329.9(antibioticantimycotic)]TJ
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(\(Sigma-Aldrich\))Tj
-14.6818 -1.4416 TD
[(Stem)-337.8(cell)-335(expansion)-339.8(medium)-869.2(DMEM)]TJ
/F8 1 Tf
17.0972 0 TD
(+)Tj
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[(10%)-329.4(FBS)]TJ
-17.818 -1.4416 TD
[(Osteogenic)-340.5(differentiation)]TJ
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[(medium)-331.7(\(ODM\))]TJ
12.6774 1.1128 TD
(DMEM)Tj
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[(10%)-329.4(FBS)]TJ
T*
[(0.01)-165.8(mM)-331.5(dexamethasone)]TJ
T*
[(20)-163.4(mM)-337.8(ascorbate)]TJ
-13.6828 -1.4416 TD
[(Mineralization)-338.2(medium)]TJ
1.0053 -1.1128 TD
[(\(for)-333.2(Von)-337.8(Kossa)-330.2(assay\))]TJ
12.6774 1.1128 TD
(DMEM)Tj
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[(10%)-329.4(FBS)]TJ
T*
[(20)-163.4(mM)-337.8(ascorbate)]TJ
0 -1.1065 TD
[(10)-163.4(mM)]TJ
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3.3132 0 TD
(b)Tj
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(\(Sigma-Aldrich\))Tj
-14.6818 -1.4416 TD
[(Vasculogenic)-343.6(differentiation)]TJ
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[(medium)-331.7(\(VDM\))]TJ
12.6774 1.1128 TD
[(From)-334.2(EGM-2)-335.6(MV)-330.4(BulletKit)]TJ
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(\(Lonza\))Tj
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(a)Tj
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[(:)-338(Endothelial)-335.9(Basal)]TJ
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[(Cell)-335.9(Medium-2)]TJ
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(+)Tj
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[(5%)-329.9(FBS)]TJ
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(hFGF-)Tj
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2.7568 0 TD
(b)Tj
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-2.7568 -1.1065 TD
(R)Tj
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(3)Tj
8.9663 0 0 8.9663 445.0959 163.1622 Tm
(-IGF-1)Tj
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(VEGF)Tj
T*
(Ascorbate)Tj
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(Dual-differentiation)Tj
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[(medium)-331.7(\(DDM\))]TJ
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[(50:50)-329.8(mixture)-340.8(of)-332(ODM)]TJ
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[(and)-334.7(VDM)]TJ
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(a)Tj
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[(Lonza)-377.2(BulletKit)-376.6(includes)-374.1(standard)-372(proprietary)-372.6(concentratio)7.6(ns)-384.8(of)]TJ
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[(listed)-331(substrates.)]TJ
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[(DMEM,)-556.5(Dulbecco)10.3(s)-565.9(modied)-554.1(Eagles)-558.1(media;)-557.2(FBS,)-556.5(fetal)-561(bovine)]TJ
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[(serum;)-356.2(EGM,)-358.3(endothelial)-352.4(growth)-351.6(medium;)-357.5(hFGF,)-351.3(human)-352.1(broblast)]TJ
T*
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[(endothelial)-331.1(growth)-323.1(factor.)]TJ
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(1374)Tj
44.3868 0 TD
[(VALENZUELA)-338.1(ET)-338.7(AL.)]TJ
ET
endstream
endobj
96 0 obj
<>stream
/GS1 gs
BT
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0 Tc
0 Tw
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[(1%)-652.4(collagenase)-662(at)-651.2(37)]TJ
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10.1356 0 TD
()Tj
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[(C)-651.4(for)-655.2(90)-163.4(min.)-652.3(After)-654.2(centrifugation)]TJ
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T*
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11.8997 0 TD
()Tj
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[(cell)-531(layer)-526.5(was)-527.3(isolated)-526.2(by)-527.3(density)-528.2(gradient)-527.1(centrifugation)]TJ
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T*
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(10)Tj
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(3)Tj
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(cells/cm)Tj
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(2)Tj
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[(courage)-431.6(adherence.)]TJ
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(34)Tj
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T*
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T*
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T*
[(VECs\))-342.9(\(Lonza/Clonetics\))-342.6(were)-337.2(cultured)-344.7(in)-335.7(ODM)-338.4(and)-341(VDM,)]TJ
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(respectively.)Tj
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[(vasculogenic)-334.7(differentiation)]TJ
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.0143 Tc
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T*
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[(du)-6.3(al-diff)-5.4(e)1.5(r)-5.7(e)1.5(nt)-5.4(ia)-4.5(tion)-539.5(media)-535.6(\()-5.4(DDM\).)-539(Ce)-4.9(lls)-537.4(w)-6.6(ere)-542.3(h)-2(arv)-5.7(e)1.5(st)-5.4(ed)]TJ
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[(at)-564.1(3,)-560.2(6,)-566.5(9)5.9(,)-566.5(12)5.9(,)-566.5(o)2.7(r)-564.3(1)-.4(5)-563.1(d)2.5(a)5.9(y)1(s)-564.9(o)2.7(f)-570.4(d)8.8(iff)5(e)-.8(r)4.8(e)5.6(ntia)5.9(ti)8.2(on)-567(for)-564.3(f)5(ur)4.8(t)5(h)2.1(er)]TJ
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[(exper)3.9(i)1(me)4.7(nt)4.1(s.)]TJ
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0 Tc
[(Fluorescent)-339.2(labeling)-335.5(and)-329.4(coculture)-335.5(of)-336.7(EPECs)]TJ
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[(and)-335.7(OPECs)]TJ
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[(EPECs)-287.1(and)-290.4(OPECs)-287.1(were)-286.7(differentiated)-296.5(as)-285.4(described)-293.3(above)]TJ
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[(for)-484.5(15)-479.6(days.)-479.1(OPECs)-483.1(were)-482.7(stained)-479(with)-483.2(3-3)]TJ
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[(-d)21.8(io)19(c)13.4(t)0(a)19(d)0(e)22.4(c)0(y)21.6(l)0(-)]TJ
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[(3,)-8.8(3,)-8.8(3)]TJ
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[(C,)-477.3(and)]TJ
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[(then)-544.6(washed)-546.2(in)-538(phosphate-buffered)-555.3(saline)-542.3(\(PBS\).)-544.1(Labeled)]TJ
T*
[(OPECs,)-403.8(EPECs,)-403.8(and)-404.2(HUVECs)-407(were)-406.8(trypsinized)-406.8(and)-397.9(centri-)]TJ
0 -1.2266 TD
[(fuged)-407.5(at)-398.3(1300)-162.4(rpm)-404.3(for)-402.3(10)-163.4(min)-402.8(at)-398.3(37)]TJ
/F8 1 Tf
15.7504 0 TD
()Tj
/F3 1 Tf
.3983 0 TD
[(C,)-401.4(the)-402.1(supernatant)-403.4(was)]TJ
-16.1487 -1.2203 TD
[(removed,)-380.5(and)-379(cells)-380.6(were)-381.5(resuspended)-381.9(in)-380(a)-378.9(medium.)-378.9(Subse-)]TJ
28.0042 51.3357 TD
[(quently,)-551.5(a)-549.6(50:50)-544.7(ratio)-550.8(of)-553.3(OPECs:EPECs)-551.8(was)-546.2(seeded)-550.8(onto)]TJ
0 -1.2203 TD
[(polystyrene)-268.8(plates)-265.3(and)-265.1(cultured)-262.5(in)-266.2(DDM.)-263.8(At)-267.4(day)-259.8(15,)-267.5(imaging)]TJ
0 -1.2266 TD
[(was)-394.5(performed)-392.1(\(20)]TJ
/F8 1 Tf
8.6687 0 TD
()Tj
/F3 1 Tf
.7145 0 TD
[(\))-398.8(with)-394.7(an)-388.6(Eclipse)-397.6(TE300)-390.2(inverted)-397.3(uo-)]TJ
-9.3832 -1.2203 TD
[(roscope)-381.1(\(Nikon,)-384.2(Melville,)-383.1(NY\),)-380.6(using)-380.7(a)-378.9(rhodamine)-380.5(lter)-387(for)]TJ
T*
[(diD)-374.7(and)-379(diI)-375.4(\()]TJ
/F4 1 Tf
6.0004 0 TD
(red)Tj
/F3 1 Tf
1.2709 0 TD
[(\),)-376.4(and)-372.6(a)-378.9(uorescein)-381.9(isothiocyanate)-378.4(lter)-380.7(for)]TJ
-7.2714 -1.2266 TD
[(diO)-578.6(\()]TJ
/F4 1 Tf
2.5671 0 TD
(green)Tj
/F3 1 Tf
2.213 0 TD
[(\).)-578.8(Spot)-584.8(Software)-592(4.1.1)-579.8(\(Diagnostic)-589.2(Instruments,)]TJ
-4.7801 -1.2203 TD
[(Sterling)-488.6(Heights,)-490.1(MI\))-494.6(was)-483(used)-492.4(for)-490.8(image)-485.5(processing.)-495.1(La-)]TJ
T*
[(beled)-440.7(HUVECs)-438.6(were)-444.7(plated)-437.7(alone)-445.4(or)-439.8(in)-436.9(a)-442.1(50:50)-437.2(ratio)-436.9(with)]TJ
0 -1.2266 TD
[(labeled)-380.5(OPECs)-381.9(according)-388.9(to)-382.6(the)-383.1(Matrigel)-383.9(tubularization)-384.5(as-)]TJ
0 -1.2203 TD
[(say)-334(protocol)-336.1(described)-337.5(below.)]TJ
/F9 1 Tf
0 -2.4406 TD
[(Quantitative)-332.7(real-time)-339.1(polymerase)-331.7(chain)-332.2(reaction)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(For)-241.8(quantitative)-251.3(real-time)-245.8(polymerase)-251.2(chain)-243.1(reaction)-249.8(\(qRT-)]TJ
-1.1128 -1.2266 TD
[(PCR\))-505.7(experiments,)-512.6(total)-506.3(RNA)-505.1(was)-502(isolated)-507.2(from)-507.3(the)-509.6(pro-)]TJ
0 -1.2203 TD
[(cessed)-282.9(lipoaspirate)-289.4(cells)-279.4(at)-284.5(each)-286.6(time)-279.7(point)-283.4(using)-285.9(the)-282(RNeasy)]TJ
T*
[(Mini)-407.6(Kit)-405.2(and)-404.2(the)-408.4(RNase-Free)-415(DNase)-406.8(Kit)-405.2(\(Qiagen,)-409.2(German-)]TJ
0 -1.2266 TD
[(town,)-390.5(MD\).)-389.3(The)-392(RNA)-391.3(purity)-397.5(and)-385.3(concentration)-398.2(was)-394.5(evalu-)]TJ
0 -1.2203 TD
[(ated)-584.6(with)-584.4(a)-587.5(NanoDrop)-587.3(1000)-579.8(spectrophotometer)-596.8(\(Thermo)]TJ
T*
[(Scientic,)-292(Waltham,)-290.5(MA\).)-288.1(Concentrations)-292.9(of)-294.1(total)-285(RNA)-290.1(were)]TJ
0 -1.2266 TD
[(equalized)-488.9(before)-495(reverse)-490(transcription)-494.6(with)-489.6(the)-490.6(Quantitect)]TJ
0 -1.2203 TD
[(Reverse)-240.6(Transcription)-244.3(Kit)-240.8(\(Qiagen\).)-239(18s)-234.3(rRNA)-240.3(was)-242.7(utilized)-236.1(as)]TJ
T*
[(the)-389.5(normalizer)-384.2(gene.)-389.9(Primers)-390.8(were)-387.8(designed)-385.2(to)-388.9(span)-384.9(an)-388.6(in-)]TJ
T*
[(tron)-291.9(whenever)-287.7(possible)-296.3(\(Table)-289(2\).)-287.4(Oligonucleotide)-293.7(sets)-293.8(are)-291(of)]TJ
0 -1.2266 TD
[(original)-539.9(design,)-536.9(except)-540.5(for)]TJ
/F4 1 Tf
12.7407 0 TD
(BGLAP)Tj
/F3 1 Tf
3.7305 0 TD
(\(osteocalcin\),)Tj
6.7246 0 0 5.9768 509.6125 472.2518 Tm
(35)Tj
/F4 1 Tf
8.9663 0 0 8.9663 521.1779 468.2267 Tm
(SPARC)Tj
/F3 1 Tf
-23.458 -1.2203 TD
(\(osteonectin\),)Tj
6.7246 0 0 5.9768 363.2314 461.2534 Tm
(36)Tj
/F4 1 Tf
8.9663 0 0 8.9663 372.5857 457.2849 Tm
(SPP1)Tj
/F3 1 Tf
2.5481 0 TD
(\(osteopontin\),)Tj
6.7246 0 0 5.9768 449.9149 461.2534 Tm
(37)Tj
8.9663 0 0 8.9663 459.2692 457.2849 Tm
(and)Tj
/F4 1 Tf
1.9664 0 TD
(SP7)Tj
/F3 1 Tf
1.9475 0 TD
(\(osterix\).)Tj
6.7246 0 0 5.9768 528.6046 461.2534 Tm
(38)Tj
8.9663 0 0 8.9663 537.9589 457.2849 Tm
(All)Tj
-25.3296 -1.2203 TD
[(samples)-478.7(were)-482.7(run)-479.7(in)-481.1(triplicate)-486.5(using)-481.9(the)-484.3(Quantitect)-485.6(SYBR)]TJ
0 -1.2266 TD
[(Green)-552.7(PCR)-555.8(Kit)-550.6(\(Qiagen\))-552.2(with)-552.8(a)-555.9(DNA)-552.9(Engine)-555.1(Opticon)-551.3(2)]TJ
0 -1.2203 TD
[(thermal)-574.1(cycler)-569.9(\(Bio-Rad,)-576.3(Hercules,)-577.9(CA\).)-574(Conditions)-574.4(were)]TJ
T*
(95)Tj
/F8 1 Tf
.999 0 TD
()Tj
/F3 1 Tf
.392 0 TD
[(C)-316.3(for)-313.8(15)-169.7(min,)-310.9(followed)-314.8(by)-312.3(35)-315.2(cycles)-317.6(of)-313.1(94)]TJ
/F8 1 Tf
18.0456 0 TD
()Tj
/F3 1 Tf
.3983 0 TD
[(C)-310(for)-313.8(15)-169.7(s,)-314.1(53)]TJ
/F8 1 Tf
5.7286 0 TD
()Tj
/F3 1 Tf
.392 0 TD
(C)Tj
-25.9556 -1.2266 TD
[(for)-370.7(30)-163.4(s,)-371(and)-372.6(72)]TJ
/F8 1 Tf
6.8667 0 TD
()Tj
/F3 1 Tf
.392 0 TD
[(C)-373.2(for)-370.7(30)-163.4(s.)-371(Each)-371.9(set)-368.4(of)-376.3(primers)-371.8(was)-369.2(used)-372.3(to)]TJ
-7.2587 -1.2203 TD
[(amplify)-345.5(a)-340.9(standard)-352.3(template)-344.1(series)-348.1(of)-344.7(pooled)-348.4(sample)-344.6(cDNA)]TJ
T*
[(spanning)-519.8(a)-511.7(1000-fold)-520.5(concentration)-518.4(range,)-516.7(and)-518.1(all)-512.4(sets)-521.5(of)]TJ
T*
[(primers)-371.8(had)-372.6(an)-369.6(efciency)-373.6(of)-370(greater)-372.3(than)-373.5(90%,)-370.3(and)-372.6(a)-372.6(preci-)]TJ
0 -1.2266 TD
[(sion)-409.3(of)-414.2(r)]TJ
6.7246 0 0 5.9768 345.5999 362.6078 Tm
(2)Tj
/F8 1 Tf
8.9663 0 0 8.9663 350.4755 358.6393 Tm
()Tj
/F3 1 Tf
.7208 0 TD
[(0.97.)-409(Amplicon)-411.2(size)-408.9(and)-410.6(purity)-416.5(were)-406.8(conrmed)]TJ
-5.1405 -1.2203 TD
[(with)-369.4(melting)-379.5(curves,)-372.9(and)-372.6(an)-376(H)]TJ
6.7246 0 0 5.9768 435.5715 346.2235 Tm
(2)Tj
8.9663 0 0 8.9663 438.9164 347.6975 Tm
[(O)-369.8(negative)-378.6(control)-373.3(was)-375.5(run)]TJ
-14.2835 -1.2203 TD
[(with)-331.5(each)-337.2(plate.)]TJ
/F9 1 Tf
0 -2.4596 TD
[(Fluorescence-activated)-337.2(cell)-333(sorting)-331.5(\(FACS\))]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(Cells)-526.9(cultured)-528.1(in)-525.4(ODM)-528(or)-528.3(VDM)-521.5(were)-533.2(trypsinized)-526.9(and)]TJ
-1.1128 -1.2203 TD
[(washed)-432.4(in)-430.6(HBSS.)-439.1(About)-434.1(1)]TJ
/F8 1 Tf
12.14 0 TD
()Tj
/F3 1 Tf
.7208 0 TD
(10)Tj
6.7246 0 0 5.9768 435.118 291.2881 Tm
(5)Tj
8.9663 0 0 8.9663 442.3747 287.3196 Tm
[(cells)-431.2(were)-438.4(then)-430.8(blocked)-434.6(in)]TJ
-14.6692 -1.2203 TD
[(10%)-342.1(goat)-342.3(serum)-342.3(\(Gibco\))-344.8(with)-344.1(PBS)-344.3(for)-345.4(30)-163.4(min)-345.9(and)-341(stained)-346.2(at)]TJ
0 -1.2266 TD
(4)Tj
/F8 1 Tf
.4995 0 TD
()Tj
/F3 1 Tf
.392 0 TD
[(C)-366.9(for)-364.3(45)-169.7(min)-364.9(with)-363.1(relevant)-368.5(mouse)-363.3(anti-human)-373(antibodies:)]TJ
ET
59.754 214.696 490.167 .22676 re
f
59.754 57.373 490.167 .22681 re
f
59.754 195.704 490.167 .22676 re
f
BT
/F7 1 Tf
8.9663 0 0 8.9663 184.1385 220.3653 Tm
(Table)Tj
/F3 1 Tf
3.0603 0 TD
(2.)Tj
/F7 1 Tf
1.2456 0 TD
[(Polymerase)-337.9(Chain)-332.5(Reaction)-335.1(Primer)-339(Sequences)]TJ
/F4 1 Tf
-18.1784 -2.0423 TD
[(Gene)-4441(Location)-2770.8(GeneID)-4986.6(Sense)-333.4(sequence)-336.3(\(5)]TJ
/F8 1 Tf
28.0421 0 TD
()Tj
/F4 1 Tf
.2466 0 TD
(3)Tj
/F8 1 Tf
.999 0 TD
()Tj
/F4 1 Tf
.2529 0 TD
[(\))-6974.6(Antisense)-339.3(sequence)-330(\(5)]TJ
/F8 1 Tf
16.2562 0 TD
()Tj
/F4 1 Tf
.2466 0 TD
(3)Tj
/F8 1 Tf
.999 0 TD
()Tj
/F4 1 Tf
.2529 0 TD
[(\))-3471.7(Size)-335.1(\(bp\))]TJ
-47.2954 -2.0992 TD
(PECAM1)Tj
/F3 1 Tf
6.9173 0 TD
[(17q23)-3764.6(5175)-2040.3(TCATTGCTCTCTTGATCATTGC)-2010.6(AGCTTCCATATTGGGATCTGAC)-2545.5(115)]TJ
/F4 1 Tf
-6.9173 -1.1065 TD
(CD34)Tj
/F3 1 Tf
6.7908 0 TD
[(1q32.2)-3887.7(947)-2293.8(ACTCCAGAGACAACCTTGAAGC)-920.6(CAGCTGGAGGTCTTATTTTGCT)-2825.5(212)]TJ
/F4 1 Tf
-6.7908 -1.1128 TD
(ENG)Tj
/F3 1 Tf
5.855 0 TD
[(9q33-q34.1)-2702.9(2022)-2040.3(ATACCACTAGCCAGGTCTCGAA)-1086.6(TTTACACTGAGGACCAGAAGCA)-2047.5(175)]TJ
/F4 1 Tf
-5.855 -1.1128 TD
(MSX2)Tj
/F3 1 Tf
6.2281 0 TD
[(5q34-q35)-3079.8(4488)-2040.3(TCGGAAAATTCAGAAGATGGAG)-806.6(TGGAGAGGTACTGTTTCTGACG)-2451.5(190)]TJ
/F4 1 Tf
-6.2281 -1.1128 TD
(BGLAP)Tj
/F3 1 Tf
6.2281 0 TD
[(1q25-q31)-3326.4(632)-2293.8(ATGAGAGCCCTCACACTCCTC)-1970.6(GCCGTAGAAGCGCCGATAGGC)-2577.5(294)]TJ
/F4 1 Tf
-6.2281 -1.1065 TD
(SPARC)Tj
/F3 1 Tf
5.855 0 TD
[(5q31.3-q32)-2702.9(6678)-2040.3(GGCTCAAGAACGTCCTGGT)-3292.6(CTGCTTGATGCCGAAGCAG)-4367.5(374)]TJ
/F4 1 Tf
-5.855 -1.1128 TD
(SPP1)Tj
/F3 1 Tf
6.2281 0 TD
[(4q21-q25)-3079.8(6696)-2040.3(AGTACCCTGATGCTACAGACG)-1804.6(CAACCAGCATATCTTCATGGC)-3087.5(321)]TJ
/F4 1 Tf
-6.2281 -1.1128 TD
(SP7)Tj
/F3 1 Tf
6.2913 0 TD
[(12q13.13)-2641.1(121340)-1539.9(GCCAGAAGCTGTGAAACCTC)-2408.6(GCTGCAAGCTCTCCATAACC)-3753.5(161)]TJ
/F4 1 Tf
-6.2913 -1.1065 TD
(RUNX2)Tj
/F3 1 Tf
7.1449 0 TD
[(6p21)-4242.6(860)-2293.8(CCTTGACCATAACCGTCTTCA)-2230.6(GAGGCGGTCAGAGAACAAAC)-3047.5(146)]TJ
/F4 1 Tf
-7.1449 -1.1128 TD
(SOX9)Tj
/F3 1 Tf
5.229 0 TD
[(17q24.3-q25.1)-2078.8(6662)-2040.3(GACTCGCCACACTCCTCCT)-3562.6(AGGTCTCGATGTTGGAGATGA)-2993.5(238)]TJ
/F4 1 Tf
-5.229 -1.1128 TD
(FLT1)Tj
/F3 1 Tf
6.9173 0 TD
[(13q12)-3764.6(2321)-2040.3(TACTTGGATTTTACTGCGGACA)-1574.6(TCTAAAGTGGTGGAACTGCTGA)-2275.5(282)]TJ
/F4 1 Tf
-6.9173 -1.1128 TD
(KDR)Tj
/F3 1 Tf
6.2281 0 TD
[(4q11-q12)-3079.8(3791)-2040.3(TCATCCTTACCAATCCCATTTC)-1834.6(ACTGGTAGGAATCCACAGGAGA)-1839.5(117)]TJ
-6.2281 -1.1065 TD
[(18s)-335.5(rRNA)-796.7(Multiple)-334.9(sites)-796.9(100008588)-792.3(CATTCGAACGTCTGCCCTAT)-2948.6(GCCTTCCTTGGATGTGGTAG)-4043.5(131)]TJ
/F5 1 Tf
0 76.5072 TD
[(VASCULARIZED)-341.6(BONE)-335.4(ENGINEERING)]TJ
52.4612 0 TD
(1375)Tj
ET
endstream
endobj
100 0 obj
<>stream
/GS1 gs
BT
/F3 1 Tf
8.9663 0 0 8.9663 62.022 725.7825 Tm
0 0 0 1 k
0 Tc
0 Tw
[(allophycocyanin-conjugated)-641.6(anti-ENG)-635.1(\()]TJ
/F4 1 Tf
18.1341 0 TD
(CD105)Tj
/F3 1 Tf
2.9338 0 TD
[(\),)-635.7(FITC-con-)]TJ
-21.0679 -1.2203 TD
[(jugated)-437.2(anti-)]TJ
/F4 1 Tf
5.7033 0 TD
(CD34)Tj
/F3 1 Tf
2.4343 0 TD
[(,)-432.9(phycoerythrin-conjugated)-445.6(anti-)]TJ
/F4 1 Tf
14.4984 0 TD
(PECAM1)Tj
/F3 1 Tf
-22.636 -1.2266 TD
(\()Tj
/F4 1 Tf
.3225 0 TD
(CD31)Tj
/F3 1 Tf
2.4406 0 TD
[(\),)-534.5(and)-537(phycoerythrin-conjugated)-546.7(anti-)]TJ
/F4 1 Tf
17.2363 0 TD
(BGLAP)Tj
/F3 1 Tf
3.7305 0 TD
(\(osteo-)Tj
-23.7299 -1.2203 TD
[(calcin\))-420(\(BD)-415.9(Biosciences,)-418.5(San)-412.4(Jose,)-418.4(CA/Miltenyi)-420.5(Biotec,)-416.5(Ber-)]TJ
T*
[(gisch)-637.9(Gladbach,)-634(Germany\).)-633.6(Staining)-636.4(for)]TJ
/F4 1 Tf
19.424 0 TD
(FLT1)Tj
/F3 1 Tf
2.839 0 TD
(\()Tj
/F4 1 Tf
.3225 0 TD
(VEGFR1)Tj
/F3 1 Tf
3.7621 0 TD
(\))Tj
-26.3476 -1.2266 TD
(and)Tj
/F4 1 Tf
2.1308 0 TD
(KDR)Tj
/F3 1 Tf
2.5545 0 TD
(\()Tj
/F4 1 Tf
.3225 0 TD
(VEGFR2)Tj
/F3 1 Tf
3.7621 0 TD
[(\))-449.4(was)-451.4(performed)-455.3(using)-456.6(the)-452.7(monoclonal)]TJ
-8.7699 -1.2203 TD
[(mouse)-401.2(anti-human)-410.9(VEGF-R)-399.4(PE)-402.7(sampler)-402.2(kit)-400.4(\(R&D)-405.8(Systems,)]TJ
T*
[(Minneapolis,)-460.2(MN\).)-451.1(Cells)-457.4(were)-451(costained)-455.8(with)-451.6(7-aminoacti-)]TJ
0 -1.2266 TD
[(nomycin)-488.6(D)-488.3(\(Invitrogen\))-490.3(to)-490.1(detect)-489.9(nonviable)-489.1(cells.)-484.7(Positive)]TJ
0 -1.2203 TD
[(staining)-304.1(was)-306(measured)-300.4(using)-304.9(a)-303(FACSCaliber)-300.8(ow)-304.6(cytometer)]TJ
T*
[(\(BD)-314.7(Biosciences\),)-324.1(and)-315.7(analyses)-316.4(were)-318.3(performed)-316.2(with)-318.8(FlowJo)]TJ
T*
[(8.0)-299.1(software)-310.3(\(TreeStar,)-303.2(Ashland,)-303(OR\).)-301.5(NHOsts)-309.6(and)-303.1(HUVECs)]TJ
0 -1.2266 TD
[(served)-239.5(as)-241.1(representative)-245.7(controls)-241.9(for)-244.2(terminally)-237.8(differentiated)]TJ
0 -1.2203 TD
[(osteoblastic)-341.8(and)-328.4(ECs,)-336.2(respectively.)]TJ
/F9 1 Tf
0 -2.7758 TD
[(von)-330.8(Kossa)-334.6(mineralization)-337.4(assay)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(ASCs,)-269.8(EPECs,)-271(OPECs,)-271(HUVECs,)-270.8(and)-271.5(NHOsts)-271.7(were)-267.7(plated)]TJ
-1.1128 -1.2266 TD
[(in)-272.5(triplicate)-277.8(wells)-269.1(at)-271.9(a)-271.4(density)-275.3(of)-275.1(100,000)-271.9(cells/well)-272.1(\(50,000)-272.8(of)]TJ
0 -1.2203 TD
[(each)-488.9(cell)-480.5(type)-486.7(in)-487.5(coculture\))-489.7(and)-480.1(incubated)-489.1(at)-486.8(37)]TJ
/F8 1 Tf
22.3262 0 TD
()Tj
/F3 1 Tf
.392 0 TD
[(C)-487(i)0(n)-487.5(ap-)]TJ
-22.7182 -1.2203 TD
[(propriate)-263.2(media)-262.7(for)-263.2(3)-258.7(days)-261.2(\(stem)-259(cell)-265.5(growth)-260(medium,)-258.8(VDM,)]TJ
0 -1.2266 TD
[(ODM,)-505.7(VDM,)-505.4(and)-499.1(ODM,)-505.7(respectively\).)-505.3(The)-505.8(mineralization)]TJ
0 -1.2203 TD
[(medium)-249.5(was)-249(used)-252.2(for)-250.5(18)-251.9(additional)-249.9(days,)-251.4(and)-252.5(it)-250.6(is)-251.4(comprised)]TJ
T*
[(of)-382.6(the)-376.8(DMEM)-381.4(with)-382.1(10%)-380(FBS,)-381.9(20)-163.4(mM)-382.1(ascorbate,)-377.5(and)-378.9(10)-169.7(mM)]TJ
/F10 1 Tf
T*
(b)Tj
/F3 1 Tf
.5501 0 TD
[(-glycerophosphate)-357.7(\(Sigma-Aldrich\).)-351.5(Twenty-one)-348.1(days)-343.4(after)]TJ
-.5501 -1.2266 TD
[(initial)-725.7(plating,)-721(cells)-722(were)-722.9(xed)-722.5(in)-721.4(methanol)-725(at)]TJ
/F8 1 Tf
23.8437 0 TD
(-)Tj
/F3 1 Tf
.7208 0 TD
(20)Tj
/F8 1 Tf
.999 0 TD
()Tj
/F3 1 Tf
.3983 0 TD
(C)Tj
-25.9619 -1.2203 TD
[(for)-364.3(2)-163.9(min)-364.9(and)-360(subsequently)-367.2(exposed)-364.5(to)-363.6(2%)-361.5(silver)-363.8(nitrate)-368.4(for)]TJ
T*
[(1)-170.2(h)-920.2(under)-918.8(a)-922.7(50-watt)-924.7(lamp.)-921.7(Representative)-925.8(polarized-)]TJ
0 -1.2266 TD
[(light)-331.5(photomicrographs)-341.4(of)-332(low-power)-337.2(elds)-326.9(\(LPF,)-334.6(4)]TJ
/F8 1 Tf
23.1419 0 TD
()Tj
/F3 1 Tf
.7145 0 TD
[(\))-335.6(were)]TJ
-23.8563 -1.2203 TD
[(taken.)-440.6(Bone)-439(nodules)-438.9(were)-438.4(counted)-440.1(by)-438.8(computer-aided)-442.8(im-)]TJ
T*
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(39)Tj
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[(using)-488.2(the)-496.9(object)-492.3(counting)-492.8(function)-495.2(in)-487.5(NIS-)]TJ
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[(.)-540.4(This)-548.7(area)-543.4(was)-539.9(chosen)-547.5(to)-547(exclude)-546.6(cellular)]TJ
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T*
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T*
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[(,)-451.8(95%)-449.6(humidied)-454.6(air)-452.7(for)-459.2(up)-451.6(to)]TJ
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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T*
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[(\()-89(MSC\))-668.1(to)-673.5(well-differentiated)-678.6(osteoblasts)-675.4(\(OB\))-670.9(and)-676.1(endothelial)-674.2(cells)-671.5(\(EC\),)-670.9(via)-674(osteoprogenitor)-678.5(cells)-671.5(\(OPC\))-669.4(and)-669.8(en-)]TJ
T*
[(dothelioprogenitor)-690.6(cells,)-680.7(respectively.)-688.2(We)-681.8(used)-682.1(adipose-derived)-686.5(stem)-682.1(cells)-684.1(\(ASC\))-683.9(to)-679.8(yield)-684.6(osteoprogenitor-rich)-692.2(en-)]TJ
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[(dothelioprogenitor-rich)-312.3(cell)-303.4(populations)-299.6(\(OPECs)-306.5(and)-296.8(EPECs,)-302.6(respectively\).)]TJ
/F6 1 Tf
33.151 0 TD
(\(B\))Tj
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1.6123 0 TD
[(ASCs)-304.9(cultured)-300.4(in)-297.8(50:50)-298.1(mixture)-302.9(of)-300.4(osteogenic)]TJ
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[(differentiation)-347.5(medium)-344.4(\(ODM\):vasculogenic)-348.6(differentiation)-347.5(medium)-338(\(VDM\))-345.4(media)-338.6(\(i.e.,)-341.4(dual-differentiation)-349.1(media)-338.6([DDM]\))]TJ
T*
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32.0698 0 TD
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(\(C\))Tj
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1.6187 0 TD
[(Predifferentiated)-260.5(OPECs)-255.5(and)-252.5(EPECs)-255.4(grew)-253.2(in)]TJ
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T*
[(markers)-642.7(OPECs)-634.9(\(3-3)]TJ
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9.6045 0 TD
()Tj
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[(-dioctadecyloxacarbocyanine)-644.4(perchlorate)-643.8([diO],)-639.4(green\))-641.1(and)-638.2(EPECs)-634.8(\(1,1)]TJ
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(-dioctadecyl-3,3,3)Tj
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45.5376 0 TD
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[(VASCULARIZED)-341.6(BONE)-335.4(ENGINEERING)]TJ
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