Research Article Artificial Extracellular Matrices with

Research ArticleArtificial Extracellular Matrices with OversulfatedGlycosaminoglycan Derivatives Promote the Differentiation ofOsteoblast-Precursor Cells and Premature Osteoblasts

Ute Hempel1 Carolin Preissler1 Sarah Vogel1 Stephanie Moumlller2

Vera Hintze3 Jana Becher2 Matthias Schnabelrauch2 Martina Rauner4

Lorenz C Hofbauer4 and Peter Dieter1

1 Institute of Physiological Chemistry Faculty of Medicine Carl Gustav Carus TU DresdenFiedlerstraszlige 42 01307 Dresden Germany

2 Biomaterials Department INNOVENT e V Prussingstraszlige 27 B 07745 Jena Germany3Max Bergmann Center of Biomaterials TU Dresden Budapester Straszlige 27 01069 Dresden Germany4Division of Endocrinology and Bone Diseases Department of Medicine III Faculty of Medicine Carl Gustav CarusTU Dresden Fetscherstraszlige 74 01307 Dresden Germany

Correspondence should be addressed to Ute Hempel utehempeltu-dresdende

Received 13 January 2014 Revised 7 April 2014 Accepted 8 April 2014 Published 28 April 2014

Academic Editor Bernd Stadlinger

Copyright copy 2014 Ute Hempel et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bonetissue with important functions in regulating stem cell lineage commitment and differentiation We anticipated that artificialextracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially thedifferentiation of osteoblast-precursor cells and early osteoblasts A set of gradually sulfated chondroitin sulfate and hyaluronanderivatives was used for the preparation of aECM All these matrices were analysed with human bone marrow stromal cells toidentify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind ofdisaccharide units on the osteogenic differentiation Oversulfated GAG derivatives with a sulfate group at the C-6 position of theN-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphataseactivity and calcium deposition A subset of the aECM was further analysed with different primary osteoblasts and cell linesreflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status ofthe cells It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts

1 Introduction

Extracellular matrix (ECM) is an important component ofthe stem cell niche influencing stem cell fate [1] Bonemarrow stromal cells (BMSC) sense not only neighbouringcells such as hematopoietic stem cells sinusoidal endothelialcells or fat cells but also the chemical composition oftheir microenvironment In the bone marrow niche theBMSC are in contact with several collagen types fibronectinand sulfated glycosaminoglycans (sGAG) mainly heparansulfate [2] There is growing evidence from in vitro studieson the importance of sGAG in facilitating the osteogenic

differentiation route of BMSC [3 4] Heparan sulfate wasidentified as an important factor initiating embryonic stemcells to exit from self-renewal and regulating their lineagefate [5] Kraushaar et al [6] pointed out that embryonicstem cell differentiation is accompanied by structural changesof heparan sulfate for example by increasing degree ofsulfation of N- 3-O- and 6-O-position Mature bone ECMcontains much less sGAG (less than 1 of bone dry weight)than the ECM of bone marrow and consists predomi-nantly of mineralized collagen [7 8] Synthetically sulfatedhyaluronan derivatives (sHA) and oversulfated chondroitinsulfate derivatives (sCS) as components of artificial ECM

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 938368 10 pageshttpdxdoiorg1011552014938368

2 BioMed Research International

(aECM) have been recently described to promote adhesionand proliferation of dermal fibroblasts [9] and to influ-ence osteoclastogenesis [10] aECM with sHA derivativesare known to enhance osteogenic differentiation of hBMSCeven in the absence of dexamethasone [11] which has beendescribed as an established supplement to induce osteogenicdifferentiation in vitro [12]

In this study a set of gradually sulfated hyaluronan andchondroitin sulfate derivatives differing in the number andthe position of sulfate groups was used for the preparationof aECM The aECM were applied as a substrate for severalosteoblast precursor cells and cell lines derived from differentsources and originsWe hypothesized that the response to theaECM will depend on the maturation state of the cell (line)sTNAP activity and calcium deposition were determined asmarkers for osteogenic differentiation

2 Materials and Methods

Unless otherwise mentioned cell culture reagents were fromBiochrom KG (Berlin Germany) fetal calf serum was fromBioWest (via ThGeyer Hamburg Germany) cell cultureplastic ware was from Greiner BioOne (FrickenhausenGermany) and Nunc (via Thermo Scientific Langensel-bold Germany) and biochemical reagents were from Sigma(Taufkirchen Germany) Rat tail collagen I was from BDBioscience (Heidelberg Germany) and chondroitin sulfate(sCS1 bovine trachea) from Sigma

21 Preparation and Characterisation of Artificial Extracellu-lar Matrices (aECM) Table 1 lists the GAG derivatives whichwere used for the preparation of the aECM The synthesisand characterization of (s)GAG derivatives were performedas described earlier [9 11 13] The preparation of the sulfatedHA derivative sHA1Δ6S was previously reported by Becheret al [14] and Schulz et al [15] aECM were prepared fromcollagen I (col) and (s)GAG derivatives as described in [911 13] Briefly 1mg collagen I was dissolved in 1mL of icecold 10mM acetic acid and was mixed with an equal volumeof 1mg (s)GAG derivativemL dissolved in ice cold doubleconcentrated fibrillogenesis buffer (50mM sodium dihydro-genphosphate and 11mM potassium dihydrogenphosphatepH 74) 220120583L of the collagen I(s)GAG derivative mixtureper cm2 was placed onto tissue culture polystyrene plates(TCPS) Fibrillogenesis was performed overnight at 37∘CThe resulting aECM were air-dried washed two times with1mL deionised sterile water and air-dried again Before cellculture experiments aECM were rinsed with sterile PBS for1 h at 37∘C

22 Isolation of Primary Osteoblast-Precursor CellsPrematureOsteoblasts and Cultivation of Cells Human bone marrowstromal cells (hBMSC) were isolated from bone marrowaspirates obtained from Caucasian donors (average age32 plusmn 7 yrs male and female donors) at the Bone MarrowTransplantation Center of the University Hospital Dresdenand were characterized as described in [16] The donors wereinformed about the procedures and gave their full consent

Table 1 Characteristics of the GAG derivatives

Sample DSSa MW

b

[gmol] PDc

sCS1 Chondroitin sulfate 1 17900 14sCS2 Sulfated chondroitin sulfate 2 23200 15sCS3 Sulfated chondroitin sulfate 3 19900 15HA Hyaluronan 0 117 times 106 48sHA1Δ4S Sulfated hyaluronan 1 26400 20sHA1Δ6S Sulfated hyaluronan 1 42600 20sHA2 Sulfated hyaluronan 2 26400 18sHA3 Sulfated hyaluronan 3 47800 17aDSS degree of sulfation (average number of sulfate groups per disacchariderepeating unit)bMW weight-average molecular weight determined by gel permeationchromatography (GPC) using laser light scattering (LLS) detectioncPD polydispersity index determined from the molecular weight distri-bution calculated from the number-average and weight average molecularweights obtained by GPC with refraction index (RI) detection

The study was approved by the Local Ethics Commission(ethic vote No EK114042009) Four hBMSC preparationswere chosen for the experiments according to prior char-acterization of their osteogenic differentiation capacity andaccording to the characteristic ldquosimilar basal TNAP activityin passage 1rdquo hBMSC preparations of individual donors werenot pooled The cells were used in passages 2ndash4

For isolation of primary rat osteoblastsosteoblast-precursors NIH guidelines for the care and use of laboratoryanimals were considered Rat calvarial osteoblasts (rCaOB)were isolated from new-born Wistar rats and characterizedas described [17] Rat bone marrow stromal cells (hBMSC)were isolated from femora and tibiae of adult Wistar rats(2-3-month old) The bones were dissected and the bonemarrow was flushed three times with medium containing10 HI-FCS and antibiotics The cell suspension wasfiltered through a 40 120583m strainer pelleted and treatedfor 4min with ACK solution (155mM NH

4Cl 10mM

KHCO3 110mM Na

2EDTA pH 74) to remove erythrocytes

Subsequently cells were resuspended in medium andplated onto tissue culture polystyrene (TCPS) plates Theosteoblast phenotype was confirmed by determination oftissue nonspecific alkaline phosphatase (TNAP) activity andcalcium phosphate deposits The cell lines MG-63 (CRL-1427) SaOS-2 (HTB-85) and MC3T3-E1 clone 4 (CRL-2593)were obtained from ATCC (American Tissue amp Cell Culturevia LGC Standards GmbH Wesel Germany) and culturedaccording to supplierrsquos recommendations MLO-Y4 cell linewas described in detail by Kato et al [18] and was a kind giftfrom Lynda Bonewald (Kansas City USA)

Cell characteristics plating density and media used forcell expansion are given in Table 2 In order to have thedifferent cell (line)s at day 4 after plating in the same state ofsubconfluency hBMSC were plated with deviant cell densityconsidering that the cells are larger in size [19] Primarycells were used from the second to fourth passage All mediacontained 2mMglutamine and 10000 IE penicillin10000120583gstreptomycinmL For the experiments cells were plated onto

BioMed Research International 3

Table 2 Cell culture conditions

Cells Species Expansion medium Seeding density[cellscm2] Source Characteristics

hBMSC Human DMEM10 HI-FCS 7000 Iliac crestbone marrow Primary cells Osteoblast-precursorsMG-63 Human DMEM10 HI-FCS 12500 Long bonesosteosarcoma Cell line Premature osteoblastsSaOS-2 Human McCoys5A15 HI-FCS 12500 Long bonesosteosarcoma Cell line Mature osteoblastsrCaOB Rat DMEM10 HI-FCS 12500 Skullcalvariae Primary cells Premature osteoblastsrBMSC Rat DMEM10 HI-FCS 12500 Long bonesbone marrow Primary cells Osteoblast-precursorsMC3T3-E1 Mouse 120572-MEM10 HI-FCS 12500 Skullcalvariae Cell line Premature osteoblasts

MLO-Y4 Mouse 120572-MEM10 HI-FCS 12500 Long bonesfemora tibiae Cell line Senescent osteoblasts(osteocytes)

TCPS or aECM-coated TCPS in medium as listed in Table 2(=basal medium BM) At day 4 after plating cells werecultured in BM in BM supplemented with 10mM 120573-glycerolphosphate and 300120583M ascorbate (=osteogenic mediumOM) and in OM supplemented with 10 nM dexamethasone(dex) (=OMD) At day 11 after plating the activity of TNAPwas determined and at day 22 after plating the amount ofdeposited calcium was quantified

23 Determination of TNAP Activity TNAP activity wasdetermined in cell lysates (lysis buffer 15M Tris-HCl (pH10) 1mM ZnCl

2 1 mMMgCl

2and 1 Triton X-100) with p-

nitrophenyl phosphate as a substrate as described previously[20] TNAP activity was calculated from a linear calibrationcurve (119903 = 09979) prepared with p-nitrophenol SpecificTNAP activity is given inmUmg protein Protein concentra-tion of the lysate was determined by Rotiquant assay (RothGmbH Karlsruhe Germany) and calculated from a linearcalibration curve (119903 = 09967) obtained with bovine serumalbumin

24 Determination of Calcium Deposition Calcium deposi-tionwas quantifiedwith the calciumkit (GreinerDiagnosticsBahlingen Germany) as previously described [11] Cell layerswere washed with PBS dried and incubated with 05M HClat 4∘C for 24 h Calcium content in the lysates was quantifiedphotometrically with cresolphthalein complexone at 570 nmfrom a linear calibration curve (119903 = 09978) prepared withcalcium chloride Calcium content is given in 120583molcm2culture area

25 Morphology of hBMSC on aECM At day 11 after platingon the different aECM F-actin arrangement was monitoredby fluorescence staining The cells were washed gently withPBS and incubated with 4 paraformaldehyde (wv) (SigmaTaufkirchen Germany) in PBS for 10min After permeabi-lization with 01 Triton X-100 (Sigma) in PBS for 20minnonspecific binding sites were blocked with 1 bovine serumalbumin (wv) (Sigma) in PBS containing 005 Tween-20 (Sigma) The cells were incubated with 5U AlexaFluor-488 phalloidinmL (Invitrogen Karlsruhe Germany) of PBScontaining 1 bovine serum albumin and 005 Tween-20 at 25∘C for 1 h Subsequently 02 120583g 410158406-diamidino-2-phenylindole (DAPI)mL (Sigma) PBS for nuclei staining

was applied for 15min at 25∘C Afterwards the cells wereembedded in Mowiol 4ndash88 (Sigma) and visualized using anAxioPhot fluorescence microscope (Carl Zeiss OberkochenGermany) For detection of the fluorescence the follow-ing filters were used excitation 450ndash490 nm and emission515ndash565 nm for AlexaFluor-488 an excitation 365 nm andemission 420 nm for DAPI Digital images were obtainedwith an AxioCam MRm camera (Carl Zeiss) by using Axio-Vision software release 46 (Carl Zeiss)

26 Statistical Analysis Each experiment was performedwith nonpooled cells from four different donors (in thecase of primary cells hBMSC rCaOB rBMSC) or fourindependent cell cultures (in the case of cell lines MG-63SaOS-2 MC3T3-E1 and MLO-Y4) each in triplicate Theresults are presented as mean plusmn standard error of the mean(SEM) Statistical significance was analyzed with GraphPadPrism 504 software (Statcon Witzenhausen Germany) byone-way ANOVA (Figures 2 and 3) and two-way ANOVAanalysis (Figure 4 Table 3) with Bonferronirsquos posttest

3 Results and Discussion

31 Effect of aECM with Gradually Sulfated GAG Derivativeson TNAP Activity Calcium Deposition and Morphology ofhBMSC Gradually sulfated GAG derivatives were synthe-sized using natural chondroitin sulfate (C4S = CSA = sCS1)(Figure 1(a)) and natural hyaluronan (Figure 1(b)) as eductsThese derivatives were used to prepare collagen I matriceswith about 2ndash7 (ww) of GAG (for characteristics see [1113]) Using aECMwith gradually sulfated GAG derivatives asa substrate for hBMSC we addressed the effects of the degreeof sulfation the position of sulfate group(s) and the kindof disaccharide unit (HA glucuronic acid (GlcA)-N-acetyl-glucosamine (NAcGlc) CS GlcA-N-acetyl-galactosamine(NAc-Gal)) on TNAP activity and calcium deposition aECMcomposed of col were used as a reference hBMSCwere platedin BM onto aECM differentiated in OMD from day 4 andanalysed at day 11 after plating for TNAP activity and atday 22 after plating for calcium deposition Both parameterswere determined as indicators of osteogenic differentiation[12] aECM containing sCS2 sCS3 sHA1Δ4S sHA2 andsHA3 caused a significant increase of TNAP activity byabout threefold in comparison to col-aECM (Figure 2(a))


whereas aECM containing sCS1 hyaluronan and sHA1Δ6Sdid not alter TNAP activity Once sufficient phosphate wasreleased by TNAP from the in vitro-substrate 120573-glycerolphosphate (component of OMD) the mineralisation tookplace The elevated calcium deposition around hBMSC onaECM containing sCS2 sCS3 sHA1Δ4S sHA2 and sHA3(Figure 2(b)) correlated with increased TNAP activity Thedifferent aECMdid not cause appreciable changes of cellmor-phology as assessed by F-actin staining (Figure 2(c)) A well-organized dense F-actin cytoskeleton forming strong fibreswas seen in hBMSC on col colsCS1 colHA colsHA1Δ4Sand colsHA1Δ6S On colsCS2 colsCS2 colsHA2 andcolsHA3 the F-actin fibres looked slightly tender neverthe-less they formed also a tight cytoskeletal network

Sulfated GAG derivatives per se and as a component ofaECM were shown to enhance osteogenic differentiation ofhBMSC [11 20] however it was not really clear whetherthe effect was dependent on the number andor positionof sulfate groups and was influenced by the kind of disac-charide unit Here we demonstrated with hBMSC that theeffect of colsGAG-aECM on TNAP activity and calciumdeposition was independent of the kind of disaccharide units(GlcA-GlcNac or GlcA-GalNAc) and the absolute numberof sulfated groups This effect was seen to the same extentwith sulfated chondroitin sulfate derivatives sCS2 and sCS3and all sulfated hyaluronan derivatives (besides sHA1Δ6S)respectively An increasing degree of sulfation (sCS3 versussCS2 sHA2 and sHA3 versus sHA1Δ4S) did not significantlyalter the effect on TNAP activity and calcium depositionOther than the number of sulfate groups their position seemsto be important sGAG derivatives without sulfate group inC-6 position but with a sulfate group at C-4 position ofthe N-acetyl-glucosamine such as the native chondroitin-4-sulfate (sCS1) and the sHA1Δ6S derivative did not increaseTNAP activity and calcium deposition suggesting that thesulfation of the hydroxyl group at C-6 position of the N-acetyl-glucosamine is necessary for the proosteogenic effectof sGAG For a further proof of this hypothesis the useof chondroitin-6-sulfate as a component of aECM wouldbe helpful Sufficiently pure chondroitin-6-sulfate howeverwas not available the commercially chondroitin-6-sulfateconsists to more than the half of chondroitin-4-sulfate [11]

32 Influence of aECM on Osteoblast-Precursor Cells Prema-ture and Mature Osteoblasts Various primary cells and celllines of human rat andmouse originwere used to investigatewhether the effect of aECM on osteogenic differentiation isrestricted solely to human BMSC or whether it is obtainedalso for cells of other species To see if the response tothe aECM depends on the maturation stage of the cellsosteoblast-precursor cells premature and mature osteoblastsof human rat and mouse origin were subjected to colsCS3-and colsHA3-aECM in comparison to col- colsCS1- andcolHA-aECMThe in vitro experimentswere performedwithhuman and rat bone marrow-derived stromal cells (BMSC)reflecting osteoblast-precursor cells with human MG-63 cellline primary rat calvaria osteoblasts (rCaOB) and mouseMC3T3-E1 cell line reflecting early premature osteoblasts

andwith humanSaOS-2 cell line andmouseMLO-Y4 cell linereflecting late mature osteoblasts respectively To evaluatewhether 120573-glycerol phosphate and ascorbate are sufficientsupplements for the induction of osteogenic differentiation orwhether dexamethasone ismandatory the cells were culturedon TCPS in basal medium (BM) osteogenic medium (OM)containing 120573-glycerol phosphate and ascorbate and OMD(OM supplemented with dexamethasone) respectively andanalysed for TNAP activity at day 11 (Figure 3) Dexametha-sone (OMD) was essential to induce TNAP activity in bothhuman BMSC and MG-63 (Figure 3(a)) but not in rBMSCand rCaOB (Figure 3(b)) 120573-Glycerol phosphate and ascor-bate (OM)were sufficient for human SaOS-2 to induce TNAPactivity withOMDno further increase of TNAP activity wasseen (Figure 3(a)) In rCaOB an increase of TNAP activitywas seenwithOMwhereas in the presence of dexamethasoneTNAP remained at the level of BM (Figure 3(b)) TNAPactivity of rBMSC (Figure 3(b)) and MC3T3-E1 (Figure 3(c))was not dependent on medium supplements In MLO-Y4 dexamethasone caused a significant decrease of TNAPactivity (Figure 3(c))

Cells were plated on col-aECM as a reference and oncol-aECM containing sCS1 sCS3 HA and sHA3 They werecultured in BM OM and OMD and analysed for TNAPactivity at day 11 (Figure 4) aECM with sCS3 and sHA3induced an increase of TNAP activity in hBMSC MG-63rCaOB and rBMSC this effect was not dependent on theaddition of osteogenic supplements and always present inBM (Figures 4(a) and 4(b)) TNAP activity of SaOS-2 wasrather negatively influenced by all aECM compared to TCPS(dotted line = 100 Figure 4(a)) in comparison to BMthe effect was more pronounced when SaOS-2 were culturedin OMD and OM In MC3T3-E1 the contact to sCS3- andsHA3-containing aECM caused a decrease of TNAP activitythe other aECM did not alter TNAP activity (Figure 4(c))TNAP activity of MLO-Y4 was higher in cells on colsCS1-aECM than in cells on col-aECM (Figure 4(c))

The results of calcium determination on aECM at day22 are summarized in Table 3 In all cell (line)s a markedcalcium deposition was only seen when the cells werecultured inOMandOMD (both containing120573-glycerol phos-phate) In comparison to col-aECM aECM with sCS3 andsHA3 induced a significant increase of calcium depositionin hBMSC MG-63 rCaOB and rBMSC The decrease ofcalciumdeposition inMC3T3-E1 on colsCS3- and colsHA3-aECM correlated to the effect of these aECM on TNAP activ-ity With SaOS-2 cells no significant differences in calciumdeposition between the different aECMwere found in OMDand OM According to the effect on TNAP activity colsCS1-aECM caused in MLO-Y4 cells in OMD a significant highercalcium deposition in comparison to col-aECM

The results indicate that primary osteoblasts-precursorcells as well as premature osteoblasts (BMSC MG-63 andearly osteoblasts isolated from calvariae of newborn rats) areresponsive to aECM with oversulfated GAG derivatives suchas colsCS3 and colsHA3 BMSC are multipotent precursorcells which can be differentiated into fibrogenic myogenicneuronal osteogenic chondrogenic and adipogenic lineagethe signals driving them into a particular differentiation


NaOOC

OO

O

O

O

R998400O

OR998400

OR

NH

NaO3SO

sCS2 R = SO3Na H R998400 = H SO3Na

sCS3 R = SO3Na R998400 = SO3Na H

sCS1 R = R998400 = H

(a)

NaOOC

O OO

O

OR998400O

R998400998400O998400998400OR

OR

NH

sHA1Δ4S R = SO3Na R998400 = R998400998400 = H

sHA1Δ6S R = H R998400 = R998400998400 = H SO3Na

sHA2 R = SO3Na R998400 = H SO3Na R998400998400 = SO3Na H

sHA3 R = SO3Na R998400 =R998400998400 = SO3Na H

HA R = R998400 = R998400998400 = H

(b)

Figure 1 Chemical structure of sGAG derivatives The chemical structure of chondroitin sulfate derivatives (a) and hyaluronan derivatives(b) and the most putative pattern of substituents as determined by [13C] nuclear magnetic resonance analysis are given

0 100 200 300 400

colsHA3colsHA2

colHAcolsCS3colsCS2colsCS1

col

c

c

cc

ccolsHA1Δ4S

colsHA1Δ6S

TNAP activity (mUmg protein)

(a)

colsHA3

colsHA2

colHA

colsCS3

colsCS2

colsCS1

col

c

c

c

cc

colsHA1Δ4S

colsHA1Δ6S

00 05 10 15 20

Calcium (120583molcm2)

(b)

col

colHA

colsCS1

colsHA1Δ4S

colsCS2

colsHA1Δ6S

colsCS3

colsHA2 colsHA3

(c)

Figure 2 Influence of aECM on TNAP activity calcium deposition and morphology of hBMSC 7000 hBMSC were plated in BM onaECM At day 4 after plating BM was replaced with OMD At day 11 after plating TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrate (a) The released p-nitrophenolate was measured photometrically at 405 nm TNAP activity in mUmLwas normalized to protein concentration in mgmL determined with Rotiquant assay At day 22 after plating calcium deposition wasquantified with cresolphthalein complexone at 570 nm (b) Significant differences of colsGAG-aECM versus col-aECM were calculated byone-way ANOVA analysis and indicated with c (119875 lt 0001) 119899 = 4 (c) At day 11 after plating cells were stained for F-actin fibres withAlexa488-phallodin (green fluorescence) and for nuclei with DAPI (blue fluorescence) scale bar = 50 120583m


SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4

TNAP activity (mUmg protein)TNAP activity (mUmg protein)

BM

OM

OMD

aa

0 1000 2000 3000

(c)

Figure 3 Influence of medium supplements on TNAP activity of osteoblast-precursor cells premature and mature osteoblasts Varioushuman (hBMSC MG-63 SaOS-2) (a) rat (rCaOB rBMSC) (b) and mouse cell (line)s (MC3T3-E1 MLO-Y4) (c) were plated in BM onTCPS At day 4 after plating cells were cultured either in BM OM or OMD At day 11 after plating TNAP activity was determined in celllysates with p-nitrophenylphosphate as a substrate The released p-nitrophenolate was measured photometrically at 405 nm TNAP activityin mUmL was normalized to protein concentration in mgmL determined with Rotiquant assay Significant differences were calculated byone-way ANOVA analysis and indicated with a (119875 lt 005) b (119875 lt 001) and c (119875 lt 0001) 119899 = 4


sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash sCS1 sCS3 HA sHA30

20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)

Figure 4 Influence of aECM on TNAP activity of osteoblast-precursor cells premature and mature osteoblasts Various human (hBMSCMG-63 SaOS-2) (a) rat (rCaOB rBMSC) (b) and mouse cell (line)s (MC3T3-E1 MLO-Y4) (c) were plated in BM on aECM composed ofcollagen I and sCS1 sCS3 HA and sHA3 respectively At day 4 after plating cells were cultured either in BM OM or OMD At day 11 afterplating TNAP activity was determined in cell lysates with p-nitrophenylphosphate as a substrateThe released p-nitrophenolatewasmeasuredphotometrically at 405 nm TNAP activity in mUmL was normalized to protein concentration in mgmL determined with Rotiquant assayData are presented as of TCPS values in the corresponding cell culture medium Significant differences of OMD OM and BM on (s) GAGversus col-aECM were calculated by two-way ANOVA and indicated with a (119875 lt 005) b (119875 lt 001) and c (119875 lt 0001) 119899 = 4


Table 3 Influence of aECM on mineralisation of osteoblast-precursor cells premature and mature osteoblasts

aECMcol colsCS1 colsCS3 colHA colsHA3

hBMSCOMD 0540 plusmn 0066 0778 plusmn 0164 1563 plusmn 0086c 0538 plusmn 0082 1405 plusmn 0116b

OM 0286 plusmn 0068 0355 plusmn 0098 0734 plusmn 0135b 0186 plusmn 0049 0724 plusmn 0130b

BM 0093 plusmn 0004 0044 plusmn 0007 0070 plusmn 0007 0074 plusmn 0002 0096 plusmn 0009

MG-63OMD 0043 plusmn 0001 0040 plusmn 0002 0083 plusmn 0002c 0027 plusmn 0002 0080 plusmn 0003c

OM 0019 plusmn 0002 0014 plusmn 0001 0037 plusmn 0003c 0012 plusmn 0001 0031 plusmn 0002c


SaOS-2OMD 6584 plusmn 0510 6360 plusmn 0322 7766 plusmn 0964 6982 plusmn 0689 8495 plusmn 1550OM 6865 plusmn 0639 7652 plusmn 0519 8504 plusmn 0727 7059 plusmn 0393 8466 plusmn 1515BM 0058 plusmn 0010 0050 plusmn 0058 0059 plusmn 0029 0068 plusmn 0055 0035 plusmn 0011

rCaOBOMD 4408 plusmn 0439 5323 plusmn 0526 1109 plusmn 1401c 3937 plusmn 0123 8995 plusmn 0961c

OM 6552 plusmn 0230 6883 plusmn 0244 1570 plusmn 1730c 5135 plusmn 0122 1286 plusmn 0981b


rBMSCOMD 1625 plusmn 0122 1542 plusmn 0129 3296 plusmn 0598c 1444 plusmn 0104 3521 plusmn 0311c



MC3T3-E1OMD 0288 plusmn 0080 0352 plusmn 0101 0200 plusmn 0026a 0384 plusmn 0082 0090 plusmn 0031b

OM 0384 plusmn 0082 0376 plusmn 0098 0062 plusmn 0005b 0279 plusmn 0022 0044 plusmn 0003c


MLO-Y4OMD 2359 plusmn 0064 7462 plusmn 2161c 3045 plusmn 0423 3436 plusmn 0126 1633 plusmn 0132OM 3436 plusmn 0126 7849 plusmn 1348 5271 plusmn 0371 3783 plusmn 0108 4271 plusmn 0108BM 0084 plusmn 0006 0028 plusmn 0007 0050 plusmn 0014 0048 plusmn 0009 0009 plusmn 0007

Calcium amount [120583molcm2] given as mean plusmn SEM significant differences of OMD OM and BM on (s) GAG versus col-aECM were calculated by two-wayANOVA and indicated with a

(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4

route come from intrinsic genetic programming diversesoluble mediators and the ECM [21 22] Both MG-63 andSaOS-2 are human osteosarcoma-derived cell lines MG-63 cells are referred to as premature osteoblasts SaOS-2 cells are characterized as mature osteoblasts [23ndash26] Incontrast to MG-63 SaOS-2 cells did not respond to aECMwith high-sulfated GAG derivatives no differences in TNAPactivity and calcium deposition were determined on all usedaECM TNAP activity and calcium deposition of SaOS-2was increased with all aECM in the presence of osteogenicsupplements (OM and OMD) Both mouse MC3T3-E1which are premature osteoblasts and MLO-Y4 which aresenescent osteoblasts did not response on aECM with high-sulfated GAG derivatives For MC3T3-E1 it was reported thata natural oversulfated chondroitin sulfate derivative isolatedfrom squid cartilage and consisting of GlcA 1rarr 3 GalNAc(C4-sulfate and C6-sulfate) disaccharide units enhancedthe deposition of collagen and calcium phosphate [27] Incontrast our studies showed that aECM with high-sulfatedGAG derivatives (colsCS3 colsHA3) caused in MC3T3-E1 a decrease of TNAP activity compared to col-aECMReasonably this cell line share some but not all of featuresof primary osteoblasts [28] The osteocyte cell line MLO-Y4 responded in OMDmdashunlike the other osteoblastsmdashtocolsCS1 with a significant increase of TNAP activity and

calcium deposition compared to col-aECM MLO-Y4 cellsreflect the most mature late osteoblast phenotype used in thisstudy [29 30] Late osteoblastsosteocytes derive from activeosteoblasts which had synthesized new bone matrix andbecome incorporated therein [31] Bone matrix maturationis associated with altered GAG composition switching fromheparan sulfate-proteoglycans which are mainly responsiblefor interaction with mediator proteins to chondroitin sulfate-proteoglycans which are less potent in mediator bindingbut support calcium accumulation [32 33] In adult bonematrix chondroitin-4-sulfate (=sCS1)was seen to be themostabundant GAG [34] The results of this study suggest thatsynthetically sulfated GAG derivatives could partially reflectthe bone marrow environment and its influence on earlyosteogenic differentiation

4 Conclusion

Artificial ECMwith collagen I and oversulfated GAG deriva-tives provide a cellular microenvironment which facilitatesthe osteogenic differentiation preferentially of osteoblast-precursor cells and early osteoblastsThe sulfate group in C-6position of theN-acetyl- glucosamine seems to bemandatoryfor the proosteogenic effect of sulfated GAG derivatives


Osteoblast-precursor cells and early osteoblasts as hBMSCand MG-63 revealed a pronounced osteogenic differentia-tion on aECM with oversulfated GAG derivatives even inthe absence of dexamethasone The strong osteoinductiveeffect of aECM with oversulfated GAG derivatives (partiallyindependent on dexamethasone) makes them an interestingtool for tissue engineering approachesThey are a structurallywell-characterised alternative to natural GAG to address theeffects of sulfated GAG on early osteogenic differentiation toa defined GAG structure

Abbreviations

(a)ECM (Artificial) Extracellular matrix120572-MEM Minimal essential mediumCS Chondroitin sulfateDex DexamethasoneDMEM Dulbeccorsquos minimal essential

mediumDSS Degree of sulfation per

disaccharide repeating unitHA HyaluronanhrBMSC Humanrat bone marrow

stromal cellsHI-FCS Heat-inactivated fetal calf serumPBS Phosphate-buffered salinerCaOB Rat calvaria osteoblastsCS1 Low-sulfated CS =

chondroitin-4-sulfate =chondroitin sulfate A (DSS sim 1)

sCS2 Medium-sulfated CS derivate(DSS sim 2)

sCS3 High-sulfated CS derivate(DSS sim 3)

(s)GAG (Sulfated) GlycosaminoglycansHA1Δ4S and sHA1Δ6S Low-sulfated HA derivatives

(DSS sim 1)sHA2 Medium-sulfated HA derivative

(DSS sim 2)sHA3 High-sulfated HA derivative

(DSS sim 3)TCPS Tissue culture polystyreneTNAP Tissue nonspecific alkaline

phosphatase

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

The authors are very grateful to Professor Bornhauser andKatrin Muller from the Stem Cell Lab of the UniversityHospital in Dresden for providing the human mesenchymalstromal cells The authors want to thank the members ofLynda Bonewaldrsquos Lab for MLO-Y4 cell line This work wassupported by Grant of Deutsche Forschungsgemeinschaft(SFBTR67 projects B1 B2 A2 and A3)

References

[1] M Watt and T S Huck ldquoRole of extracellular matrix inregulating stem cell faterdquoNature ReviewsMolecular Cell Biologyvol 14 pp 467ndash473 2013

[2] X-D Chen ldquoExtracellular matrix provides an optimal niche forthe maintenance and propagation of mesenchymal stem cellsrdquoBirth Defects Research C vol 90 no 1 pp 45ndash54 2010

[3] T Hoshiba N Kawazoe T Tateishi and G Chen ldquoDevel-opment of stepwise osteogenesis-mimicking matrices for theregulation of mesenchymal stem cell functionsrdquo The Journal ofBiological Chemistry vol 284 no 45 pp 31164ndash31173 2009

[4] C Dombrowski S J Song P Chuan et al ldquoHeparan sulfatemediates the proliferation and differentiation of rat mesenchy-mal stem cellsrdquo Stem Cells and Development vol 18 no 4 pp661ndash670 2009

[5] D C Kraushaar Y Yamaguchi and L Wang ldquoHeparan sulfateis required for embryonic stem cells to exit from self-renewalrdquoThe Journal of Biological Chemistry vol 285 no 8 pp 5907ndash5916 2010

[6] D C Kraushaar S Dalton and LWang ldquoHeparan sulfate a keyregulator of embryonic stem cell faterdquoThe Journal of BiologicalChemistry vol 394 pp 741ndash751 2013

[7] VMMania AG Kallivokas CMalavaki et al ldquoA comparativebiochemical analysis of glycosaminoglycans and proteoglycansin human orthotopic and heterotopic bonerdquo IUBMB Life vol61 no 4 pp 447ndash452 2009

[8] M R van der Harst P A J Brama C H A van de LestG H Kiers J DeGroot and P R van Weeren ldquoAn integralbiochemical analysis of the main constituents of articularcartilage subchondral and trabecular bonerdquo Osteoarthritis andCartilage vol 12 no 9 pp 752ndash761 2004

[9] A van der Smissen V Hintze D Scharnweber et al ldquoGrowthpromoting substrates for human dermal fibroblasts providedby artificial extracellular matrices composed of collagen I andsulfated glycosaminoglycansrdquo Biomaterials vol 32 no 34 pp8938ndash8946 2011

[10] J Salbach-Hirsch J Kraemer M Rauner et al ldquoThe promotionof osteoclastogenesis by sulfated hyaluronan through interfer-ence with osteoprotegerin and receptor activator of NF-120581Bligandosteoprotegerin complex formationrdquo Biomaterials vol34 pp 7653ndash7661 2013

[11] U Hempel S Moller C Noack et al ldquoSulfated hyaluro-nancollagen I-matrices enhance osteogenic differentiation ofhuman mesenchymal stromal cells in vitro even in the absenceof dexamethasonerdquo Acta Biomaterialia vol 8 pp 4064ndash40722012

[12] M F Pittenger ldquoMesenchymal stem cells from adult bonemarrowrdquo Methods in Molecular Biology vol 449 pp 27ndash442008

[13] U Hempel V Hintze S Moller M Schnabelrauch D Scharn-weber and P Dieter ldquoArtificial extracellular matrices composedof collagen i and sulfated hyaluronan with adsorbed trans-forming growth factor 1205731 promote collagen synthesis of humanmesenchymal stromal cellsrdquoActa Biomaterialia vol 8 no 2 pp659ndash666 2012

[14] J Becher S Moller D Weiss J Schiller and M SchnabelrauchldquoSynthesis of new regioselectively sulfated hyaluronans forbiomedical applicationrdquoMacromolecular Symposia vol 296 no1 pp 446ndash452 2010

[15] MC Schulz P Korn B Stadlinger et al ldquoCoatingwith artificialmatrices from collagen and sulfated hyaluronan influences


the osseointegration of dental implantsrdquo Journal of MaterialsScience Materials in Medicine vol 25 no 1 pp 247ndash258 2014

[16] J Oswald S Boxberger B Joslashrgensen et al ldquoMesenchymal stemcells can be differentiated into endothelial cells in vitrordquo StemCells vol 22 no 3 pp 377ndash384 2004

[17] U Geiszligler U Hempel C Wolf D Scharnweber H Worchand K-W Wenzel ldquoCollagen type I-coating of Ti6Al4V pro-motes adhesion of osteoblastsrdquo Journal of Biomedical MaterialsResearch vol 51 pp 752ndash760 2000

[18] Y Kato J J Windle B A Koop G R Mundy and L FBonewald ldquoEstablishment of an osteocyte-like cell line MLO-Y4rdquo Journal of Bone and Mineral Research vol 12 no 12 pp2014ndash2023 1997

[19] U Hempel T Hefti M Kalbacova C Wolf-Brandstetter PDieter and F Schlottig ldquoResponse of osteoblast-like SAOS-2cells to zirconia ceramics with different surface topographiesrdquoClinical Oral Implants Research vol 21 no 2 pp 174ndash181 2010

[20] M Buttner S Moller M Keller et al ldquoOver-sulfated chon-droitin sulfate derivatives induce osteogenic differentiation ofhMSC independent of BMP-2 and TGF-1205731 signallingrdquo Journalof Cellular Physiology vol 228 no 2 pp 330ndash340 2013

[21] B M Abdallah and M Kassem ldquoHuman mesenchymal stemcells from basic biology to clinical applicationsrdquo GeneTherapyvol 15 no 2 pp 109ndash116 2008

[22] C Nombela-Arrieta J Ritz and L E Silberstein ldquoThe elusivenature and function ofmesenchymal stem cellsrdquoNature ReviewsMolecular Cell Biology vol 12 no 2 pp 126ndash131 2011

[23] S B Rodan Y Imai M A Thiede et al ldquoCharacterizationof a human osteosarcoma cell line (Saos-2) with osteoblasticpropertiesrdquoCancer Research vol 47 no 18 pp 4961ndash4966 1987

[24] M Bachle and R J Kohal ldquoA systematic review of the influenceof different titanium surfaces on proliferation differentiationand protein synthesis of osteoblast-like MG63 cellsrdquo ClinicalOral Implants Research vol 15 no 6 pp 683ndash692 2004

[25] C Pautke M Schieker T Tischer et al ldquoCharacterizationof osteosarcoma cell lines MG-63 Saos-2 and U-2 OS incomparison to human osteoblastsrdquoAnticancer Research vol 24no 6 pp 3743ndash3748 2004

[26] S Vohra KMHennessy A A Sawyer Y Zhuo and S L BellisldquoComparison of mesenchymal stem cell and osteosarcomacell adhesion to hydroxyapatiterdquo Journal of Materials ScienceMaterials in Medicine vol 19 no 12 pp 3567ndash3574 2008

[27] T Miyazaki S Miyauchi A Tawada T Anada S Matsuzakaand O Suzuki ldquoOversulfated chondroitin sulfate-E binds toBMP-4 and enhances osteoblast differentiationrdquo Journal ofCellular Physiology vol 217 no 3 pp 769ndash777 2008

[28] R T Ballock and A B Roberts ldquoThe role of TGF-120573 inbone growth and bone repairrdquo in Growth Factors A PracticalApproach I McKay and I Leigh Eds pp 85ndash107 OxfordUniversity Press New York NY USA 1993

[29] L F Bonewald ldquoThe amazing osteocyterdquo Journal of Bone andMineral Research vol 26 no 2 pp 229ndash238 2011

[30] J Rosser and L F Bonewald ldquoStudying osteocyte function usingthe cell lines MLO-Y4 and MLO-A5rdquo Methods in MolecularBiology vol 816 pp 67ndash81 2012

[31] M L Knothe Tate J R Adamson A E Tami and T W BauerldquoThe osteocyterdquo International Journal of Biochemistry and CellBiology vol 36 no 1 pp 1ndash8 2004

[32] K J Manton D F M Leong S M Cool and V NurcombeldquoDisruption of heparan and chondroitin sulfate signaling

enhances mesenchymal stem cell-derived osteogenic differen-tiation via bone morphogenetic protein signaling pathwaysrdquoStem Cells vol 25 no 11 pp 2845ndash2854 2007

[33] R JWaddington H C Roberts R V Sugars and E SchonherrldquoDifferential roles for small leucine-rich proteoglycans in boneformationrdquo European Cells andMaterials vol 6 pp 12ndash21 2003

[34] CW Prince and JMNavia ldquoGlycosaminoglycan alterations inrat bone due to growth and fluorosisrdquo Journal of Nutrition vol113 no 8 pp 1576ndash1582 1983

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


(aECM) have been recently described to promote adhesionand proliferation of dermal fibroblasts [9] and to influ-ence osteoclastogenesis [10] aECM with sHA derivativesare known to enhance osteogenic differentiation of hBMSCeven in the absence of dexamethasone [11] which has beendescribed as an established supplement to induce osteogenicdifferentiation in vitro [12]

In this study a set of gradually sulfated hyaluronan andchondroitin sulfate derivatives differing in the number andthe position of sulfate groups was used for the preparationof aECM The aECM were applied as a substrate for severalosteoblast precursor cells and cell lines derived from differentsources and originsWe hypothesized that the response to theaECM will depend on the maturation state of the cell (line)sTNAP activity and calcium deposition were determined asmarkers for osteogenic differentiation

2 Materials and Methods

Unless otherwise mentioned cell culture reagents were fromBiochrom KG (Berlin Germany) fetal calf serum was fromBioWest (via ThGeyer Hamburg Germany) cell cultureplastic ware was from Greiner BioOne (FrickenhausenGermany) and Nunc (via Thermo Scientific Langensel-bold Germany) and biochemical reagents were from Sigma(Taufkirchen Germany) Rat tail collagen I was from BDBioscience (Heidelberg Germany) and chondroitin sulfate(sCS1 bovine trachea) from Sigma

21 Preparation and Characterisation of Artificial Extracellu-lar Matrices (aECM) Table 1 lists the GAG derivatives whichwere used for the preparation of the aECM The synthesisand characterization of (s)GAG derivatives were performedas described earlier [9 11 13] The preparation of the sulfatedHA derivative sHA1Δ6S was previously reported by Becheret al [14] and Schulz et al [15] aECM were prepared fromcollagen I (col) and (s)GAG derivatives as described in [911 13] Briefly 1mg collagen I was dissolved in 1mL of icecold 10mM acetic acid and was mixed with an equal volumeof 1mg (s)GAG derivativemL dissolved in ice cold doubleconcentrated fibrillogenesis buffer (50mM sodium dihydro-genphosphate and 11mM potassium dihydrogenphosphatepH 74) 220120583L of the collagen I(s)GAG derivative mixtureper cm2 was placed onto tissue culture polystyrene plates(TCPS) Fibrillogenesis was performed overnight at 37∘CThe resulting aECM were air-dried washed two times with1mL deionised sterile water and air-dried again Before cellculture experiments aECM were rinsed with sterile PBS for1 h at 37∘C

22 Isolation of Primary Osteoblast-Precursor CellsPrematureOsteoblasts and Cultivation of Cells Human bone marrowstromal cells (hBMSC) were isolated from bone marrowaspirates obtained from Caucasian donors (average age32 plusmn 7 yrs male and female donors) at the Bone MarrowTransplantation Center of the University Hospital Dresdenand were characterized as described in [16] The donors wereinformed about the procedures and gave their full consent

Table 1 Characteristics of the GAG derivatives

Sample DSSa MW

b

[gmol] PDc

sCS1 Chondroitin sulfate 1 17900 14sCS2 Sulfated chondroitin sulfate 2 23200 15sCS3 Sulfated chondroitin sulfate 3 19900 15HA Hyaluronan 0 117 times 106 48sHA1Δ4S Sulfated hyaluronan 1 26400 20sHA1Δ6S Sulfated hyaluronan 1 42600 20sHA2 Sulfated hyaluronan 2 26400 18sHA3 Sulfated hyaluronan 3 47800 17aDSS degree of sulfation (average number of sulfate groups per disacchariderepeating unit)bMW weight-average molecular weight determined by gel permeationchromatography (GPC) using laser light scattering (LLS) detectioncPD polydispersity index determined from the molecular weight distri-bution calculated from the number-average and weight average molecularweights obtained by GPC with refraction index (RI) detection

The study was approved by the Local Ethics Commission(ethic vote No EK114042009) Four hBMSC preparationswere chosen for the experiments according to prior char-acterization of their osteogenic differentiation capacity andaccording to the characteristic ldquosimilar basal TNAP activityin passage 1rdquo hBMSC preparations of individual donors werenot pooled The cells were used in passages 2ndash4

For isolation of primary rat osteoblastsosteoblast-precursors NIH guidelines for the care and use of laboratoryanimals were considered Rat calvarial osteoblasts (rCaOB)were isolated from new-born Wistar rats and characterizedas described [17] Rat bone marrow stromal cells (hBMSC)were isolated from femora and tibiae of adult Wistar rats(2-3-month old) The bones were dissected and the bonemarrow was flushed three times with medium containing10 HI-FCS and antibiotics The cell suspension wasfiltered through a 40 120583m strainer pelleted and treatedfor 4min with ACK solution (155mM NH

4Cl 10mM

KHCO3 110mM Na

2EDTA pH 74) to remove erythrocytes

Subsequently cells were resuspended in medium andplated onto tissue culture polystyrene (TCPS) plates Theosteoblast phenotype was confirmed by determination oftissue nonspecific alkaline phosphatase (TNAP) activity andcalcium phosphate deposits The cell lines MG-63 (CRL-1427) SaOS-2 (HTB-85) and MC3T3-E1 clone 4 (CRL-2593)were obtained from ATCC (American Tissue amp Cell Culturevia LGC Standards GmbH Wesel Germany) and culturedaccording to supplierrsquos recommendations MLO-Y4 cell linewas described in detail by Kato et al [18] and was a kind giftfrom Lynda Bonewald (Kansas City USA)

Cell characteristics plating density and media used forcell expansion are given in Table 2 In order to have thedifferent cell (line)s at day 4 after plating in the same state ofsubconfluency hBMSC were plated with deviant cell densityconsidering that the cells are larger in size [19] Primarycells were used from the second to fourth passage All mediacontained 2mMglutamine and 10000 IE penicillin10000120583gstreptomycinmL For the experiments cells were plated onto








2 1 mMMgCl


















NaOOC

OO

O

O

O

R998400O

OR998400

OR

NH

NaO3SO



sCS1 R = R998400 = H

(a)

NaOOC

O OO

O

OR998400O

R998400998400O998400998400OR

OR

NH

sHA1Δ4S R = SO3Na R998400 = R998400998400 = H

sHA1Δ6S R = H R998400 = R998400998400 = H SO3Na



HA R = R998400 = R998400998400 = H

(b)


0 100 200 300 400

colsHA3colsHA2


col

c

c

cc

ccolsHA1Δ4S

colsHA1Δ6S


(a)

colsHA3

colsHA2

colHA

colsCS3

colsCS2

colsCS1

col

c

c

c

cc

colsHA1Δ4S

colsHA1Δ6S

00 05 10 15 20


(b)

col

colHA

colsCS1

colsHA1Δ4S

colsCS2

colsHA1Δ6S

colsCS3

colsHA2 colsHA3

(c)



SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4


BM

OM

OMD

aa

0 1000 2000 3000

(c)



sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










2 1 mMMgCl


















NaOOC

OO

O

O

O

R998400O

OR998400

OR

NH

NaO3SO



sCS1 R = R998400 = H

(a)

NaOOC

O OO

O

OR998400O

R998400998400O998400998400OR

OR

NH

sHA1Δ4S R = SO3Na R998400 = R998400998400 = H

sHA1Δ6S R = H R998400 = R998400998400 = H SO3Na



HA R = R998400 = R998400998400 = H

(b)


0 100 200 300 400

colsHA3colsHA2


col

c

c

cc

ccolsHA1Δ4S

colsHA1Δ6S


(a)

colsHA3

colsHA2

colHA

colsCS3

colsCS2

colsCS1

col

c

c

c

cc

colsHA1Δ4S

colsHA1Δ6S

00 05 10 15 20


(b)

col

colHA

colsCS1

colsHA1Δ4S

colsCS2

colsHA1Δ6S

colsCS3

colsHA2 colsHA3

(c)



SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4


BM

OM

OMD

aa

0 1000 2000 3000

(c)



sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials












NaOOC

OO

O

O

O

R998400O

OR998400

OR

NH

NaO3SO



sCS1 R = R998400 = H

(a)

NaOOC

O OO

O

OR998400O

R998400998400O998400998400OR

OR

NH

sHA1Δ4S R = SO3Na R998400 = R998400998400 = H

sHA1Δ6S R = H R998400 = R998400998400 = H SO3Na



HA R = R998400 = R998400998400 = H

(b)


0 100 200 300 400

colsHA3colsHA2


col

c

c

cc

ccolsHA1Δ4S

colsHA1Δ6S


(a)

colsHA3

colsHA2

colHA

colsCS3

colsCS2

colsCS1

col

c

c

c

cc

colsHA1Δ4S

colsHA1Δ6S

00 05 10 15 20


(b)

col

colHA

colsCS1

colsHA1Δ4S

colsCS2

colsHA1Δ6S

colsCS3

colsHA2 colsHA3

(c)



SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4


BM

OM

OMD

aa

0 1000 2000 3000

(c)



sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




NaOOC

OO

O

O

O

R998400O

OR998400

OR

NH

NaO3SO



sCS1 R = R998400 = H

(a)

NaOOC

O OO

O

OR998400O

R998400998400O998400998400OR

OR

NH

sHA1Δ4S R = SO3Na R998400 = R998400998400 = H

sHA1Δ6S R = H R998400 = R998400998400 = H SO3Na



HA R = R998400 = R998400998400 = H

(b)


0 100 200 300 400

colsHA3colsHA2


col

c

c

cc

ccolsHA1Δ4S

colsHA1Δ6S


(a)

colsHA3

colsHA2

colHA

colsCS3

colsCS2

colsCS1

col

c

c

c

cc

colsHA1Δ4S

colsHA1Δ6S

00 05 10 15 20


(b)

col

colHA

colsCS1

colsHA1Δ4S

colsCS2

colsHA1Δ6S

colsCS3

colsHA2 colsHA3

(c)



SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4


BM

OM

OMD

aa

0 1000 2000 3000

(c)



sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




SaOS-2

BM

OM

OMD

c

c

0 2000 4000 6000


cc

9 12

MG-63

BM

OM

OMD

0 3 6


hBMSC

BM

OM

OMD

a

b

0 50 100 150 200


(a)

rBMSC

BM

OM

OMD

0 50 100 150 200 250


rCaOB

BM

OM

OMD

c

c

0 500 1000 1500


(b)

MC3T3-E1

BM

OM

OMD

0 20 40 60 80 100

MLO-Y4


BM

OM

OMD

aa

0 1000 2000 3000

(c)



sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




sCS1 sCS3 HA sHA30

100

200

300

400

c b b b b b

hBMSC

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

sCS1 sCS3 HA sHA30

100

200

300

400

c

c c

cc c

MG-63

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col


20406080

100SaOS-2

TNA

P ac

tivity

( v

ersu

s TCP

S)aECM col

mdash

(a)

sCS1 sCS3 HA sHA30

100

200

300

400c c c

c b c

rCaOB

TNA

P ac

tivity

( v

ersu

s TCP

S)

aECM col

mdash

TNA

P ac

tivity

( v

ersu

s TCP

S)

sCS1 sCS3 HA sHA30

200

400

600 c cb

cb

a

rBMSC

aECM col

mdash

(b)

sCS1 sCS3 HA sHA30

50

100

150

200

b a c a

MC3T3-E1

TNA

P ac

tivity

( v

ersu

s TCP

S)

mdash

OMDOMBM

aECM col

TNA

P ac

tivity

sCS1 sCS3 HA sHA30

50

100

150

200c

OMDOMBM

MLO-Y4

( v

ersu

s TCP

S)

aECM col

mdash

(c)























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
























(119875 lt 005) b(119875 lt 001) and c(119875 lt 0001) 119899 = 4



4 Conclusion




Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Abbreviations












phosphatase



Acknowledgment


References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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