Research Article Alendronate-Eluting Biphasic Calcium Phosphate (BCP…downloads.hindawi.com/journals/bmri/2015/320713.pdf · 2019-07-31 · Research Article Alendronate-Eluting Biphasic

Research ArticleAlendronate-Eluting Biphasic Calcium Phosphate (BCP)Scaffolds Stimulate Osteogenic Differentiation

Sung Eun Kim1 Young-Pil Yun1 Deok-Won Lee2 Eun Young Kang1 Won Jae Jeong3

Boram Lee4 Myeong Seon Jeong4 Hak Jun Kim1 Kyeongsoon Park4 and Hae-Ryong Song1

1Department of Orthopedic Surgery and Rare Diseases Institute Korea University Medical College Guro Hospital No 80Guro-dong Guro-gu Seoul 152-703 Republic of Korea2Department of Oral and Maxillofacial Surgery Kyung Hee University Dental Hospital at Gangdong Kyung Hee University892 Dongnam-ro Gangdong-gu Seoul 134-727 Republic of Korea3Department of Molecular amp Cell Biology University of California Berkeley 142 LSA No 3200 Berkeley CA 94720-3200 USA4Disease Research Center Korea Basic Science Institute Gangwondaehakgil-1 Chuncheon Gangwon-do 200-701 Republic of Korea

Correspondence should be addressed to Kyeongsoon Park kspark1223hanmailnet and Hae-Ryong Song songhaekoreaackr

Received 3 March 2015 Revised 11 June 2015 Accepted 17 June 2015

Academic Editor Elena Jones

Copyright copy 2015 Sung Eun Kim 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

Biphasic calcium phosphate (BCP) scaffolds have been widely used in orthopedic and dental fields as osteoconductive bonesubstitutes However BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation Toenhance osteogenic differentiation we prepared alendronate- (ALN-) eluting BCP scaffolds The coating of ALN on BCP scaffoldswas confirmed by scanning electron microscopy (FE-SEM) energy-dispersive X-ray spectroscopy (EDS) and attenuated totalreflectance-Fourier transform infrared spectroscopy (ATR-FTIR) An in vitro release study showed that release of ALN fromALN-eluting BCP scaffolds was sustained for up to 28 days In vitro results revealed thatMG-63 cells grown onALN-eluting BCP scaffoldsexhibited increasedALP activity and calciumdeposition andupregulated gene expression of Runx2ALPOCN andOPNcomparedwith the BCP scaffold aloneTherefore this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulateosteogenic differentiation

1 Introduction

Bone tissues possess the intrinsic capacity for regeneration aspart of the repair process in response to injury skeletal devel-opment or continuous remodeling throughout adult life [1]Bone regeneration is a complex andwell-orchestrated processof biological events of bone induction and conduction tooptimize skeletal repair and restore skeletal function [1 2]In complex clinical conditions however bone regeneration isrequired in large quantities for the repair of large bone defectscreated by trauma infection tumor resection and skeletalabnormalities [3] To stimulate or augment bone regenerationof large bone defects bone grafting such as autologousbone grafts and allografts is a commonly performed surgicalprocedure [4 5] However these bone grafts have severallimitations such as limited supply harvest site morbidityadditional blood loss and inflammatory responses [4 5]

Bone graft substitutes have been developed as alternativesto autologous or allogenic bone grafts Such substitutes (iecollagen hydroxyapatite [Hap] 120573-tricalcium phosphate [120573-TCP] calcium-phosphate cements and glass ceramics) aresynthetic or natural biomaterial scaffolds that promote themigration proliferation and differentiation of bone cellsfor bone regeneration [3 6 7] Biphasic calcium phosphate(BCP) ceramics consist of a mixture of hydroxyapatite (HAp)and beta-tricalcium phosphate (120573-TCP) BCP exhibits goodbiocompatibility and bone conduction performance in hardtissue repair because of their similarity to the minerals inhuman bone and their outstanding bioactivity [8 9] AsBCP is resorbed in vivo it releases calcium and phosphateions into the microenvironment of the implant site theseions can be used for new bone formation [10] Also thepresence of porosity and a bioactive surface on BCP scaffoldsfacilitates cell attachment proliferation and differentiation

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 320713 10 pageshttpdxdoiorg1011552015320713

2 BioMed Research International

and consequently favors increased bone formation [11ndash13]However due to lack of inherent osteoinductivity lim-ited new bone formation occurs after osteoconduction isachievedThus conventional BCP requires the additional useof osteoinductive biomolecules such as bone morphogenicproteins (BMPs) for enhanced osteogenesis [9 14 15]

To promote bone formation of scaffolds the use ofbioactive molecules such as growth factors is very attractiveAmong various bioactive growth factors including bonemorphogenic proteins (BMPs) basic fibroblast growth factor(bFGF) platelet-derived growth factor (PDGF) and vascularendothelial growth factor (VEGF) BMPs are well known toinduce osteogenic differentiation of mesenchymal stem cellsand to stimulate ectopic bone formation [16ndash18] Howeverthere are problems associated with its clinical applicationsuch as the large doses of BMPs required (21ndash120mg) a shorthalf-life in vivo and high cost [19ndash21] To overcome theseshortcomings investigators have been looking for alternativedrugs Bisphosphonates have been widely used as potentosteoinductive factors for the treatment of osteoporosis andseveral bone diseases such as bone metastasis hypercalcemiaof malignancy and Pagetrsquos disease [22] As one of the mostcommonly used bisphosphonates ALN suppresses osteoclastactivity by inhibiting the activity of farnesyl pyrophosphatesynthetase [23] Recent studies reported that ALN enhancesthe osteogenesis of osteoblasts bone marrow mesenchymalstem cells (BMSCs) and adipose-derived stem cells (ADSCs)[24ndash26] ALN can also upregulate mRNA expression duringosteogenic differentiation in vitro including that of BMP-2 type I collagen osteocalcin and osteopontin [27 28]Unlike most drugs ALN has strong negative charge on thetwo phosphonate moieties Thus its oral bioavailability isvery low averaging only 06sim07 under fasting conditionsTo overcome the limitation of low bioavailability recentstudies reported that the local delivery systems includingALN-containing titanium microspheres nanoparticles andchitosan scaffolds could improve osteogenesis of osteoblastsfor bone regeneration [27 29 30]

In this study we developed ALN-eluting BCP (ALNBCP) scaffolds as local delivery system for improving boneformation Based on the fact that ALN has a high bindingaffinity to the bonemineral hydroxyapatite (HAp) [31 32] wefabricated ALNBCP scaffolds by simply mixing BCP scaf-folds with ALN We hypothesized that ALNBCP scaffoldsmay enhance the drug efficacy by achieving the sustaineddelivery over a period of time as well as enhancing the osteo-genesis of osteoblasts compared to BCP alone In this studywe aimed to investigate whether ALNBCP scaffolds caneffectively improve in vitro osteoblast activity and osteogenicdifferentiation and to demonstrate whether ALNBCP scaf-folds have great potential for bone regeneration

2 Materials and Methods

21 Materials Biphasic calcium phosphate substrates (BCPHAp = 60 TCP = 40 diameter times length = 3 times 8mmcylinder type) were kindly donated from OssGen Cor-poration (Gyeongbuk Korea) Alendronate was obtainedfrom Samjin Pharmaceutical Corporation (Seoul Korea)

Dulbeccorsquos Modified Eaglersquos Medium (DMEM) phosphatebuffer saline (PBS) fetal bovine serum (FBS) and 1 antibi-otics (100UmM penicillin and 01mgmL streptomycin)were purchased from Life Technologies Corporation (GrandIsland NY USA) 2-(N-Morpholino) ethanesulfonic acid(MES) was supplied by Sigma Chemical Co (St Louis MOUSA)

22 Preparation of Alendronate- (ALN-) Coated BCP Sub-strates To prepare ALN-eluting BCP substrates BCP sub-strates were immersed in 01MMES buffer (pH 56) contain-ing ALN (01 or 1mg) and gently shaken for 24 h The ALN-coated BCP substrates were collected washed with distilledwater (DW) and dried in a vacuum for 1 day

23 Characterization of BCPandALN-Eluting BCP SubstratesSurface morphologies and elemental compositions of BCPandALN-coated BCP substrates were evaluated by a variable-pressure field emission scanning electron microscopy (VP-FE-SEM SUPRA-55VP Carl Zeiss Germany) at the KoreaBasic Science Institute Chencheon Center The chemicaldistribution mapping and compositions of BCP and ALN-coated BCP substrates were determined using an energy-dispersive X-ray spectrometer (EDS) ATR-FTIR (attenuatedtotal reflectance-Fourier transform infrared spectroscopyAvatar 360 Nicolet Instrument Corp Madison WI USA)spectra had a resolution of 4 cmminus1 between 2000 and500 cmminus1 A commercially available powder was used toobtain the ATR-FTIR spectrum of ALN

24 In Vitro Release Study of ALN from ALN-Eluting BCPSubstrates To assess ALN release from ALN (01mg)BCPand ALN (1mg)BCP each substrate was soaked in a 15mLconical tube with 1mL PBS The tubes were then incubatedat 37∘C with shaking at 100 rpm At predetermined timepoints of 1 3 5 and 10 h and 1 3 5 7 14 21 and 28 dayssupernatants were collected and replaced with fresh PBSThecollected supernatants were stored in a deep freezer at minus20∘Cbefore analysis The released ALN was determined using aUVVis spectrophotometer (DU-530 Beckman Coulter) ata wavelength of 293 nm with a complex of alendronate andstandard iron (III) chloride solution

25 Cell Morphology For in vitro cell studies MG-63cells which were isolated from human bone tissue wereused because MG-63 cells are popular cells to demon-strate the osteogenic effects of drugs peptides or proteinsinon various substrates MG-63 cells were maintained withDMEM supplemented including 10 FBS 50120583gmL ascor-bic acid 10 nM dexamethasone 10mM 120573-glycerophosphate100UmL penicillin and 100120583gmL streptomycin

To confirm the morphologies of MG-63 cells (osteoblast-like cells) grown on BCP and ALN-eluting BCP substratesthe cells (1 times 104 cells100 120583L) were carefully seeded on thesurface of BCP and ALN-eluting BCP substrates in a 24-well tissue culture plate incubated for 30min at 37∘C andadded to 1mL of DMEM supplemented with 10 FBS and1 antibiotics in the presence of 50 120583gmL ascorbic acid10 nMdexamethasone and 10mM120573-glycerophosphate After

BioMed Research International 3

24 hours of incubation the cells on substrates were fixed in37 (vv) formaldehyde for 30min For SEM analysis cellson substrates were further fixed with 1 osmium tetroxidedehydrated in a graded series of ethanoldistilled waterranging from 50 to 100 in steps of 10 for 10 minuteseach and then lyophilized for 1 day The cell morphologywas examined with SEM (VP-FE-SEM SUPRA-VP55 CarlZeiss) For F-actin staining the fixed cells were immersed incytoskeleton buffer (5 times 10minus3M NaCl 150 times 10minus3M MgCl

2

5 times 10minus4M Tris-base and 05 Triton X-100) for 5min at4∘C followed by blocking buffer (5 FBS 01Tween-20 and002 sodium azide in PBS) for 30min at 37∘C The cells onsubstrates were stained with rhodamine-phalloidin (1 200)and DAPI (1 10000) and cell morphology was observedunder a confocal laser scanning microscope (LSM700 ZeissOberkochen Germany)

26 Alkaline Phosphatase (ALP) Activity To evaluate earlyosteogenic differentiation MG-63 cells were seeded on BCPALN (01mg)BCP and ALN (1mg)BCP substrates in a24-well tissue culture plate at a concentration of 1 times 105cellssubstrate and incubated for up to 14 days inDMEM sup-plemented with 10 FBS and 1 antibiotics in the presence of50120583gmL ascorbic acid 10 nMdexamethasone and 10mM120573-glycerophosphate At predetermined time points of 3 7 10and 14 days the cells were lysed using 1x RIPA (radioim-munoprecipitation assay) buffer [50mM TrisndashHCl pH 74150mM NaCl 025 deoxycholic acid 1 tergitol-type-40(NP-40) and 1mM ethylenediaminetetraacetic acid (EDTA)including protease and phosphatase inhibitors (1mMphenyl-methylsulfonyl fluoride (PMSF) 1mM sodium orthovana-date 1mM sodium fluoride 1120583gmL aprotinin 1120583gmLleupeptin and 1 120583gmL pepstatin)] The cell lysates were cen-trifuged at 13500 rpm for 3min at 4∘CThe supernatants wereincubated with p-nitrophenyl phosphate solution for 30minat 37∘C The reaction was stopped by addition of 500120583L of1N NaOH ALP activity was determined by measuring theconversion of p-nitrophenyl phosphate to p-nitrophenol [33]Optical density was determined using a microplate reader(Bio-Rad Hercules CA USA) at a wavelength of 405 nm

27 Calcium Content To assess late osteogenic differentia-tion MG-63 cells were seeded at a concentration of 1 times 105cellsmL on BCP ALN (01mg)BCP and ALN (1mg)BCPsubstrates in a 24-well tissue culture plate After 21 days ofculture the cells were washed with PBS treated with 05 NHCl and centrifuged at 13500 rpm for 1min The resultingsupernatant was used for calcium deposition measurementusing a QuantiChromCalciumAssay Kit (DICA-500 BioAs-say Systems Hayward CA USA) according to the manu-facturerrsquos instructions The amount of calcium produced wasdetermined at 612 nm using a microplate reader

28 Gene Expression To determine mRNA expression of theosteogenic differentiation markers runt related transcriptionfactor (Runx2) ALP osteocalcin (OCN) and osteopontin(OPN) we performed real-time PCR MG-63 cells (1 times 105cellsmL) were seeded on BCP ALN (01mg)BCP and ALN(1mg)BCP substrates in a 24-well tissue culture plate After

7 and 21 days of culture cDNA was synthesized with 1120583gtotal RNA and oligo (dT) primer using the Superscript First-Strand Synthesis System (Bioneer Inc Daejeon SouthKorea)according to the manufacturerrsquos instructions The followingoligonucleotide primers were used for real-time PCR Runx-2 (F) 51015840-ATG GCA TCA AAC AGC CTC TTC AGC A-31015840(R) 51015840-CGT GGG TTC TGA GGC GGG ACA CC-31015840 ALP(F) 51015840-GTG GAA GGA GGC AGA ATT GAC CA-31015840 (R) 51015840-AGG CCC ATT GCC ATA CAG GAT GG-31015840 OCN (F) 51015840-TGA GAG CCC TCA CAC TCC TC-31015840 (R) 51015840-ACC TTTGCT GGA CTC TGC AC-31015840 OPN (F) 51015840-GAG GGC TTGGTT GTC AGC-31015840 (R) 51015840-CAA TTC TCA TGG TAG TGAGTT TTC C-31015840 GAPDH (F) 51015840-ACT TTG TCA AGC TCATTT CC-31015840 and (R) 51015840-TGC AGC GAA CTT TAT TGATG-31015840 PCR amplification and detection were carried out onan ABI7300 Real-TimeThermal Cycler (Applied BiosystemsFoster CA USA) with the DyNAmo SYBR Green qPCR Kit(Finnzymes Espoo Finland) The relative mRNA expressionlevels of Runx2 ALP OCN and OPN were normalized tothat of GAPDH All results were confirmed by repeating theexperiment three times

29 Statistical Analysis Quantitative data are presented asmeans plusmn standard deviation and comparisons were carriedout using one-wayANOVA (Systat Software Inc Chicago ILUSA) Differences were considered statistically significant atlowast

119875 lt 005

3 Results

31 Characterization of BCP and Modified BCP ScaffoldsThe surface morphologies of BCP and ALN-BCP scaffoldswere investigated by SEM In SEM images the surfaces ofBCP and ALN (01mg)BCP and ALN (1mg)BCP scaffoldsshowed similar morphologies with open and round-shapedpores ranging in size from 100 to 300 120583m (Figure 1(a) (A)ndash(C)) To confirm the successful preparation of ALN-elutingBCP scaffolds the chemical distribution (ie C N O P andCa) mapping images of BCP and ALN (1mg)BCP scaffoldswere obtained by EDS analysis As shown in representativemapping images of BCP and ALN (01mg)BCP colorsfor C (green) O (blue) N (light blue) and P (red) onALN (01mg)BCP were evenly distributed on the surface ofBCP scaffolds (Figure 1(b)) The surface elemental chemicalcompositions (ie C N O P and Ca) of BCP and ALN-BCP scaffolds were also determined with EDS In particularsuccessful loading of ALN on the surface of BCP scaffoldswas confirmed by an increase in the N content As theamount of added ALN increased N content increased from0 for BCP 460 for ALN (01mg)BCP and 803 for ALN(1mg)BCP scaffolds (Table 1) The loading of ALN on thesurface of BCP scaffolds was also confirmed with ATR-FTIRAs shown in Figure 2 the characteristic absorption peaks oforthophosphate (PO

4

) were observed at 940ndash1120 cmminus1 and566 cmminus1 on BCP and the two ALNBCP scaffolds As theamount of added ALN increased the characteristic peaks foralendronates were observed at 926 cmminus1 (hydroxyl groups)and 1020 cmminus1 and 1050 cmminus1 (P=O stretch) and their peakintensities increased


(A) (B) (C)

(a)

BCP

C O N P Ca

ALN

(01

mg)

BCP

(b)

Figure 1 (a) Scanning electron microscope (SEM) images of (A) BCP (B) ALN (01mg)BCP and (C) ALN (1mg)BCP scaffolds (b) Therepresentative chemical mapping images of BCP and ALN (01mg)BCP scaffolds

Table 1 Surface elemental composition of BCP and ALNBCPscaffolds

Substrate C () O () P () Ca () N ()BCP 1041 5624 100 2335 00ALN (01mg)BCP 2286 5360 402 1492 460ALN (1mg)BCP 1480 5078 572 2068 803

32 In Vitro ALN Release from ALNBCP Scaffolds Figure 3shows the release profiles of ALN from ALN (01mg)BCPand ALN (1mg)BCP scaffolds Sustained release of ALNfrom ALN (01mg)BCP and ALN (1mg)BCP scaffolds wasobserved for up to 28 days On the first day 105plusmn003 120583g and146 plusmn 008 120583g of ALN were released from ALN (01mg)BCPand ALN (1mg)BCP scaffolds respectively SubsequentlyALNwas slowly released from theALNBCP scaffolds By day28 248 plusmn 021 120583g and 331 plusmn 031 120583g of ALN were releasedfrom ALN (01mg)BCP and ALN (1mg)BCP scaffoldsrespectively

33 Cell Morphology Study Figure 4 shows the cell morphol-ogy ofMG-63 cells cultured on BCP and ALNBCP scaffoldsAs shown in Figure 4(a) the MG-63 cells adhered on thesurface of BCP and ALNBCP scaffolds after 24 hours of

incubation The adhered cells were spread on the surface ofthe scaffolds and the number of adhered cells increased withan increase in the amount of ALN coating the BCP scaffolds(Figure 4(b))

34 In Vitro Osteogenic Differentiation Study Figure 5(a)shows the ALP activity of MG-63 cells cultured on BCPALN (01mg)BCP and ALN (1mg)BCP scaffolds at 3 7 10and 14 days The ALP activity of MG-63 cells grown on allscaffolds gradually increased with incubation time up to 14days Also as the coating amount of ALN on BCP scaffoldsincreased the ALP activity of MG-63 cells also increasedALP activity of ALN (1mg)BCP gt ALN (01mg)BCP gtBCP Furthermore the ALP activity of MG-63 cells culturedon ALN (1mg)BCP scaffolds was significantly higher thanthat of cells on ALN (01mg)BCP and BCP at all incubationtime points (lowast119875 lt 005) The amount of calcium depositionwas determined after culture of MG-63 cells for 21 dayson BCP ALN (01mg)BCP and ALN (1mg)BCP scaffolds(Figure 5(b)) The amount of deposited calcium was 808 plusmn018 for BCP 1181 plusmn 132 for ALN (01mg)BCP and 1324 plusmn074 for ALN (1mg)BCP respectively Calciumdeposition byMG-63 cells cultured onALNBCP scaffolds was significantlyhigher than that on BCP scaffolds (lowast119875 lt 005) However theamount of calcium deposited by MG-63 cells cultured on the


600800100012001400

Tran

smitt

ancy

()

0

50

100

150

200 (a)

(b)

(c)

(d)

Wavenumbers (cmminus1)

Figure 2 Attenuated total Fourier-transform infrared spectroscopy (ATR-FTIR) spectra of (a) ALN (b) BCP (c) ALN (01mg)BCP and(d) ALN (1mg)BCP scaffolds The characteristic peaks of alendronate sodium appear at 926 cmminus1 for the hydroxyl group and at 1020 cmminus1and 1050 cmminus1 for P=O (indicated by the arrows) and BCP appears at 940ndash1120 cmminus1 603 cmminus1 and 566 cmminus1 for orthophosphate (PO

4

)(indicated by the black triangles) These experiments were repeated three times

Time (day)0 5 10 15 20 25 30

0

1

2

3

4

Ale

ndro

nate

cum

ulat

ive r

eleas

e (120583

gm

L)

ALN (01mg)BCPALN (1mg)BCP

Figure 3 In vitro release profiles of ALN from ALN (01mg)BCP and ALN (1mg)BCP scaffolds These experiments were repeated threetimes

two ALNBCP scaffolds was not significantly different after21 days of culture

35 In Vitro Expression of Osteogenic Genes To furtherconfirm osteogenic differentiation of MG-63 cells expres-sion of the osteogenic genes Runx2 ALP OCN and OPNin MG-63 cells cultured on BCP ALN (01mg)BCP andALN (1mg)BCP scaffolds was evaluated by real-time PCR(Figure 6) At 7 and 21 days Runx2 ALP OCN and OPNmRNA levels in MG-63 cells grown on ALNBCP scaffoldswere higher than those in cells grown on BCP scaffoldsIn particular Runx2 and ALP mRNA levels in MG-63 cellsgrown on ALNBCP were significantly higher than thosein cells grown on BCP scaffolds at 7 days (lowast119875 lt 005)However there were no significant differences in Runx2 andALPmRNA levels between BCP andALNBCP scaffolds at 21days On the other hand there were no significant differences

in OCN and OPNmRNA levels between BCP and ALNBCPscaffolds at 7 days At 21 days however OCN and OPNmRNA levels in cells grown on ALNBCP scaffolds weremarkedly higher than those in cells grown on BCP scaffolds(lowast119875 lt 005)

4 Discussion

In bone tissue engineering bone graft substitutes have beenused as alternatives to autologous or allogenic bone graftsAmong the choices available BCP substitutes are consideredthe most promising as osteoconductive bone substitutes forbone regeneration because they provide a framework forvascular and cellular infiltration and consequently favor newbone formation [11ndash13] and overcome critical limitationssuch as expense limited supply additional trauma (in thecase of autografts) and risk of disease transmission (in


(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)

Figure 4 (a) SEM images ofMG-63 cells cultured on (A) BCP (B) ALN (01mg)BCP and (C) ALN (1mg)BCP scaffolds (b) F-actin stainingof MG-63 cells cultured on BCP ALN (01mg)BCP and ALN (1mg)BCP after 24 hours of incubation Bar 80120583mThese experiments wererepeated three times


00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast

BCP ALN (01mg)BCPALN (1mg)BCP

(b)

Figure 5 (a) ALP activity of MG-63 cells cultured on BCP ALN (01mg)BCP and ALN (1mg)BCP scaffolds after culture for 3 7 10 and14 days (lowast119875 lt 005) and (b) calcium deposition by MG-63 cells cultured on BCP ALN (01mg)BCP and ALN (1mg)BCP after culture for21 days (lowast119875 lt 005) The error bars represent mean plusmn SD (119899 = 5) These experiments were repeated three times

the case of allografts) [34] However BCP scaffolds do notstimulate osteogenic differentiation of osteoblast-like cells ormesenchymal stem cellsTherefore in this study we preparedALN-eluting BCP scaffolds and evaluated whether they canstimulate osteogenic differentiation of osteoblast-like cells

Bisphosphonate drugs have hydroxyl groups on the P-C-P structure (hydroxyl methylene bisphosphonate) and showbinding affinity to calcium phosphate of bonemineral [31 3235] Based on the binding affinity of bisphosphonate drugsto bone mineral we fabricated ALN-eluting BCP scaffolds bysimply mixing BCP scaffolds and ALN Once ALN is mixedwithBCP scaffolds ALNanions are coordinatedwith calciumcations of the calcium phosphate phase in BCP scaffoldsthrough a bidentate chelation [29] Significant differences insurface morphologies between BCP and ALN-eluting BCPscaffolds were not observed because immobilization of small-molecular weight ALN might be hard to observe on thesurface of subcentimeter-sized BCP scaffolds The successfulloading of ALN on BCP scaffolds was confirmed with EDSand ATR-FTIR which showed an increase in N content andpeak intensities for hydroxyl and P=O groups of ALN onBCP scaffolds ALN-eluting BCP scaffolds showed sustainedrelease over 28 days after a burst release during the initial24 hThe initial burst releasemight be ascribed toALNboundon the surface of BCP scaffolds and the sustained releasekinetics might be induced by calcium phosphate dissolutionfrom BCP scaffolds [29]

In the bone tissue regeneration study initial cell adhe-sion on the scaffolds is one of the critical factors forcell differentiation [36] SEM and F-actin staining resultsshowed that the number of adherent MG-63 cells on ALN-eluting BCP scaffolds increased compared to that on BCPscaffolds This result indicates that the released ALN fromALNBCP scaffolds might promote favorable cell functions

such as cell adhesion on ALNBCP scaffolds consistentwith our previous study [37] The enhanced adhesion ofosteoblast-like cells by scaffolds also affects three principalosteoblast differentiation processes (proliferation extracel-lular matrix maturation and mineralization) [38] Duringthese osteogenic differentiation processes each distinctivestage can be characterized by the expression of distinctiveosteogenic markers As a key component of bone matrixvesicles ALP is an early indicator of immature osteoblastactivity and osteogenic differentiation [36 39 40] Afterosteogenic differentiation osteoblastic cells begin to secretea mineral matrix leading to calcium deposition a markerfor mature osteoblasts [36 39 40] As the most promisingbone graft substitutes BCP scaffolds have beenwidely studiedfor bone tissue regeneration However BCP scaffolds stillhave the challenges of insufficient osteogenic differentiationCompared with BCP scaffolds ALN-eluting BCP scaffoldssignificantly enhanced ALP activity and calcium depositionby MG-63 cells in a dose-dependent manner implying thatthe released ALN from BCP scaffolds stimulates osteogenicdifferentiation Consistent with recent studies this study alsodemonstrated that the delivery of ALN using BCP scaffoldsystems stimulates osteogenic activity and differentiation ofosteoblast-like cells for bone tissue regeneration [28 37]

Only cells that have differentiated to a mature osteoblastphenotype express osteogenic markers such as ALP Runx2OCN and OPN [41 42] ALP is observed on the cell surfaceand in matrix vesicles during osteogenic differentiationRunx2 plays a crucial role and regulates skeletogenesis andbone formation during the early stages of embryogenesis[43] OCN and OPN are extracellular matrix proteins andare thought to be important in modulating mineralizationIt is well known that ALP and Runx2 are early genes amongthe osteoblast markers whereas OCN and OPN are late


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30

Runx27 days 21 days 21 days

Nor

mal

ized

fold

expr

essio

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0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

Figure 6 Real-time PCR analysis of mRNA gene expression of Runx2 alkaline phosphatase (ALP) osteocalcin (OCN) and osteopontin(OPN) of MG-63 cells grown on BCP ALN (01mg)BCP and ALN (1mg)BCP scaffolds after incubation for 7 and 21 days respectively(lowast119875 lt 005) The error bars represent mean plusmn SD (119899 = 5) These experiments were repeated three times

genes [42 44ndash46] To further confirm the stimulation ofosteogenic differentiation by ALN-eluting BCP scaffolds weexamined the gene expression ofmultiple osteogenicmarkersincluding ALP Runx2 OCN and OPN In the BCP scaffoldgroup expression of these four genes was not increasedduring the period of cell culture ALN-eluting BCP scaffoldssignificantly increased ALP and Runx2 gene expressioncompared to BCP-only scaffolds at 7 days However ALPand Runx2 mRNA levels were downregulated at 21 dayspossibly as a result of the switch in cellular processes tomineralization steps [36] Also ALN-eluting BCP scaffoldsmarkedly enhanced the gene expression of OCN and OPNin a dose-dependent manner at 21 days Taken togetherthese results demonstrate that ALN-eluting BCP scaffolds can

deliver ALN for a long period and are sufficient to stimulateosteogenic differentiation We expect that long-term localdelivery of ALN using BCP scaffolds will have great potentialfor new bone formation at sites of bone defects

5 Conclusions

We fabricated ALN-eluting BCP scaffolds by simple mixingof ALN and BCP scaffolds and evaluated whether they canstimulate osteogenic differentiation of osteoblast-like cellsALN-eluting BCP scaffolds showed sustained release of ALNfor 28 days Compared to BCP scaffolds ALN-eluting BCPscaffolds significantly enhanced ALP activity and calciumdeposition by osteoblast-like cells In addition theymarkedly


increased gene expression levels of osteoblastmarkers such asALP Runx2 OCN and OPN in a dose-dependent mannerWe demonstrated that ALN-eluting BCP scaffolds effectivelystimulate osteogenic differentiation and have great potentialfor bone regeneration

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Sung Eun Kim Young-Pil Yun and Deok-Won Lee con-tributed equally to this work

Acknowledgment

This study was supported by a grant of the Korean HealthTechnology RampD Project Ministry of Health amp WelfareRepublic of Korea (HI11C0388 and HI13C1501)

References

[1] T A Einhorn ldquoThe cell and molecular biology of fracture heal-ingrdquo Clinical Orthopaedics and Related Research no 355 suppl-ement pp S7ndashS21 1998

[2] T-J Cho L C Gerstenfeld and T A Einhorn ldquoDifferentialtemporal expression of members of the transforming growthfactor beta superfamily duringmurine fracture healingrdquo Journalof Bone and Mineral Research vol 17 no 3 pp 513ndash520 2002

[3] R Dimitriou E Jones D McGonagle and P V GiannoudisldquoBone regeneration current concepts and future directionsrdquoBMCMedicine vol 9 article 66 2011

[4] H Petite V ViateauW Bensaıd et al ldquoTissue-engineered boneregenerationrdquo Nature Biotechnology vol 18 no 9 pp 959ndash9632000

[5] A J Salgado O P Coutinho and R L Reis ldquoBone tissue engi-neering state of the art and future trendsrdquo MacromolecularBioscience vol 4 no 8 pp 743ndash765 2004

[6] P V Giannoudis H Dinopoulos and E Tsiridis ldquoBone sub-stitutes an updaterdquo Injury vol 36 supplement 3 pp S20ndashS272005

[7] C G Finkemeier ldquoBone-grafting and bone-graft substitutesrdquoThe Journal of Bone amp Joint SurgerymdashAmerican Volume vol 84no 3 pp 454ndash464 2002

[8] P Mercier F Bellavance J Cholewa and S Djokovic ldquoLong-term stability of atrophic ridges reconstructed with hydroxy-lapatite a prospective studyrdquo Journal of Oral and MaxillofacialSurgery vol 54 no 8 pp 960ndash969 1996

[9] L Cheng F Ye R Yang et al ldquoOsteoinduction of hydroxy-apatite120573-tricalcium phosphate bioceramics in mice with afractured fibulardquoActa Biomaterialia vol 6 no 4 pp 1569ndash15742010

[10] A Sendemir-Urkmez and R D Jamison ldquoThe additionof biphasic calcium phosphate to porous chitosan scaffoldsenhances bone tissue development in vitrordquo Journal of Biomed-ical Materials Research A vol 81 no 3 pp 624ndash633 2007

[11] R Xin Y Leng J Chen and Q Zhang ldquoA comparative studyof calcium phosphate formation on bioceramics in vitro and invivordquo Biomaterials vol 26 no 33 pp 6477ndash6486 2005

[12] F Barrere CA vanBlitterswijk andK deGroot ldquoBone regene-ration molecular and cellular interactions with calcium phos-phate ceramicsrdquo International Journal of Nanomedicine vol 1no 3 pp 317ndash332 2006

[13] S E Lobo F H LWykrota A CM B Oliveira I Kerkis G BMahecha and H J Alves ldquoQuantification of bone mass gain inresponse to the application of biphasic bioceramics and plateletconcentrate in critical-size bone defectsrdquo Journal of MaterialsScience Materials inMedicine vol 20 no 5 pp 1137ndash1147 2009

[14] B Li X Liao L Zheng et al ldquoEffect of nanostructure on osteo-induction of porous biphasic calciumphosphate ceramicsrdquoActaBiomaterialia vol 8 no 10 pp 3794ndash3804 2012

[15] B H Fellah O Gauthier PWeiss D Chappard and P LayrolleldquoOsteogenicity of biphasic calcium phosphate ceramics andbone autograft in a goat modelrdquo Biomaterials vol 29 no 9 pp1177ndash1188 2008

[16] E A Wang V Rosen J S DrsquoAlessandro et al ldquoRecombinanthuman bone morphogenetic protein induces bone formationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 6 pp 2220ndash2224 1990

[17] S D Cook MWWolfe S L Salkeld and D C Rueger ldquoEffectof recombinant human osteogenic protein-1 on healing of seg-mental defects in non-human primatesrdquo The Journal of Boneamp Joint SurgerymdashAmerican Volume vol 77 no 5 pp 734ndash7501995

[18] S E Kim O Jeon J B Lee et al ldquoEnhancement of ectopic boneformation by bone morphogenetic protein-2 delivery usingheparin-conjugated PLGA nanoparticles with transplantationof bone marrow-derived mesenchymal stem cellsrdquo Journal ofBiomedical Science vol 15 no 6 pp 771ndash777 2008

[19] O P Gautschi S P Frey and R Zellweger ldquoBone mor-phogenetic proteins in clinical applicationsrdquo ANZ Journal ofSurgery vol 77 no 8 pp 626ndash631 2007

[20] L B E Shields G H Raque S D Glassman et al ldquoAdverseeffects associated with high-dose recombinant human bonemorphogenetic protein-2 use in anterior cervical spine fusionrdquoSpine vol 31 no 5 pp 542ndash547 2006

[21] R S Sellers R Zhang S S Glasson et al ldquoRepair of articularcartilage defects one year after treatment with recombinanthuman bonemorphogenetic protein-2 (rhBMP-2)rdquoThe Journalof Bone and Joint SurgerymdashAmerican Volume vol 82 no 2 pp151ndash160 2000

[22] H Fleisch ldquoDevelopment of bisphosphonatesrdquo Breast CancerResearch vol 4 no 1 pp 30ndash34 2002

[23] G A Rodan and H A Fleisch ldquoBisphosphonates mechanismsof actionrdquoThe Journal of Clinical Investigation vol 97 no 12 pp2692ndash2696 1996

[24] Y Inoue I Hisa S Seino and H Kaji ldquoAlendronate inducesmineralization in mouse osteoblastic MC3T3-E1 cells regula-tion of mineralization-related genesrdquo Experimental and ClinicalEndocrinology amp Diabetes vol 118 no 10 pp 719ndash723 2010

[25] F von Knoch C Jaquiery M Kowalsky et al ldquoEffects ofbisphosphonates on proliferation and osteoblast differentiationof human bone marrow stromal cellsrdquo Biomaterials vol 26 no34 pp 6941ndash6949 2005

[26] C-Z Wang S-M Chen C-H Chen et al ldquoThe effect of thelocal delivery of alendronate on human adipose-derived stemcell-based bone regenerationrdquo Biomaterials vol 31 no 33 pp8674ndash8683 2010

[27] H-J Moon Y-P Yun C-W Han et al ldquoEffect of heparin andalendronate coating on titanium surfaces on inhibition of osteo-clast and enhancement of osteoblast functionrdquo Biochemical and


Biophysical Research Communications vol 413 no 2 pp 194ndash200 2011

[28] G-I Im S A Qureshi J Kenney H E Rubash and A SShanbhag ldquoOsteoblast proliferation and maturation by bispho-sphonatesrdquo Biomaterials vol 25 no 18 pp 4105ndash4115 2004

[29] C W Kim Y-P Yun H J Lee Y-S Hwang I K Kwon and SC Lee ldquoIn situ fabrication of alendronate-loaded calcium phos-phate microspheres controlled release for inhibition of osteo-clastogenesisrdquo Journal of Controlled Release vol 147 no 1 pp45ndash53 2010

[30] S E Kim D H Suh Y-P Yun et al ldquoLocal delivery of alen-dronate eluting chitosan scaffold can effectively increase osteo-blast functions and inhibit osteoclast differentiationrdquo Journal ofMaterials ScienceMaterials inMedicine vol 23 no 11 pp 2739ndash2749 2012

[31] K Miller R Erez E Segal D Shabat and R Satchi-FainaroldquoTargeting bone metastases with a bispecific anticancer andantiangiogenic polymer-alendronate-taxane conjugaterdquo Ange-wandte Chemie International Edition vol 48 no 16 pp 2949ndash2954 2009

[32] H Uludag ldquoBisphosphonates as a foundation of drug deliveryto bonerdquoCurrent Pharmaceutical Design vol 8 no 21 pp 1929ndash1944 2002

[33] M Morinobu T Nakamoto K Hino et al ldquoThe nucleocy-toplasmic shuttling protein CIZ reduces adult bone mass byinhibiting bone morphogenetic protein-induced bone forma-tionrdquo Journal of Experimental Medicine vol 201 no 6 pp 961ndash970 2005

[34] R Z Legeros C A Garrido S E Lobo and F M TurıbioldquoBiphasic calcium phosphate bioceramics for orthopaedicreconstructions clinical outcomesrdquo International Journal ofBiomaterials vol 2011 Article ID 129727 9 pages 2011

[35] G H Nancollas R Tang R J Phipps et al ldquoNovel insights intoactions of bisphosphonates on bone differences in interactionswith hydroxyapatiterdquo Bone vol 38 no 5 pp 617ndash627 2006

[36] R Ravichandran J R Venugopal S Sundarrajan SMukherjeeand S Ramakrishna ldquoPrecipitation of nanohydroxyapatite onPLLAPBLGCollagen nanofibrous structures for the differen-tiation of adipose derived stem cells to osteogenic lineagerdquoBiomaterials vol 33 no 3 pp 846ndash855 2012

[37] Y-P Yun S-J Kim Y-M Lim et al ldquoThe effect of alendronate-loaded polycarprolactone nanofibrous scaffolds on osteogenicdifferentiation of adipose-derived stem cells in bone tissueregenerationrdquo Journal of Biomedical Nanotechnology vol 10 no6 pp 1080ndash1090 2014

[38] T A Owen M Aronow V Shalhoub et al ldquoProgressivedevelopment of the rat osteoblast phenotype in vitro reciprocalrelationships in expression of genes associated with osteoblastproliferation and differentiation during formation of the boneextracellular matrixrdquo Journal of Cellular Physiology vol 143 no3 pp 420ndash430 1990

[39] F Liu L Malaval and J E Aubin ldquoGlobal amplification poly-merase chain reaction reveals novel transitional stages duringosteoprogenitor differentiationrdquo Journal of Cell Science vol 116no 9 pp 1787ndash1796 2003

[40] C Heinemann S Heinemann A Bernhardt H Worch andT Hanke ldquoNovel textile chitosan scaffolds promote spreadingproliferation and differentiation of osteoblastsrdquo Biomacro-molecules vol 9 no 10 pp 2913ndash2920 2008

[41] D E Hughes K R Wright H L Uy et al ldquoBisphosphonatespromote apoptosis in murine osteoclasts in vitro and in vivordquo

Journal of Bone and Mineral Research vol 10 no 10 pp 1478ndash1487 1995

[42] M Kato M S Patel R Levasseur et al ldquoCbfa1-independentdecrease in osteoblast proliferation osteopenia and persistentembryonic eye vascularization in mice deficient in Lrp5 a Wntcoreceptorrdquo Journal of Cell Biology vol 157 no 2 pp 303ndash3142002

[43] P Ducy R Zhang V Geoffroy A L Ridall and G KarsentyldquoOsf2Cbfa1 a transcriptional activator of osteoblast differenti-ationrdquo Cell vol 89 no 5 pp 747ndash754 1997

[44] T Katagiri A Yamaguchi M Komaki et al ldquoBone morpho-genetic protein-2 converts the differentiation pathway of C2C12myoblasts into the osteoblast lineagerdquo The Journal of CellBiology vol 127 no 6 part 1 pp 1755ndash1766 1994

[45] R T Franceschi ldquoThe developmental control of osteoblast-spe-cific gene expression role of specific transcription factors andthe extracellular matrix environmentrdquo Critical Reviews in OralBiology and Medicine vol 10 no 1 pp 40ndash57 1999

[46] F Lecanda L V Avioli and S-L Cheng ldquoRegulation of bonematrix protein expression and induction of differentiation ofhuman osteoblasts and human bone marrow stromal cells bybone morphogenetic protein-2rdquo Journal of Cellular Biochem-istry vol 67 no 3 pp 386ndash398 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and consequently favors increased bone formation [11ndash13]However due to lack of inherent osteoinductivity lim-ited new bone formation occurs after osteoconduction isachievedThus conventional BCP requires the additional useof osteoinductive biomolecules such as bone morphogenicproteins (BMPs) for enhanced osteogenesis [9 14 15]

To promote bone formation of scaffolds the use ofbioactive molecules such as growth factors is very attractiveAmong various bioactive growth factors including bonemorphogenic proteins (BMPs) basic fibroblast growth factor(bFGF) platelet-derived growth factor (PDGF) and vascularendothelial growth factor (VEGF) BMPs are well known toinduce osteogenic differentiation of mesenchymal stem cellsand to stimulate ectopic bone formation [16ndash18] Howeverthere are problems associated with its clinical applicationsuch as the large doses of BMPs required (21ndash120mg) a shorthalf-life in vivo and high cost [19ndash21] To overcome theseshortcomings investigators have been looking for alternativedrugs Bisphosphonates have been widely used as potentosteoinductive factors for the treatment of osteoporosis andseveral bone diseases such as bone metastasis hypercalcemiaof malignancy and Pagetrsquos disease [22] As one of the mostcommonly used bisphosphonates ALN suppresses osteoclastactivity by inhibiting the activity of farnesyl pyrophosphatesynthetase [23] Recent studies reported that ALN enhancesthe osteogenesis of osteoblasts bone marrow mesenchymalstem cells (BMSCs) and adipose-derived stem cells (ADSCs)[24ndash26] ALN can also upregulate mRNA expression duringosteogenic differentiation in vitro including that of BMP-2 type I collagen osteocalcin and osteopontin [27 28]Unlike most drugs ALN has strong negative charge on thetwo phosphonate moieties Thus its oral bioavailability isvery low averaging only 06sim07 under fasting conditionsTo overcome the limitation of low bioavailability recentstudies reported that the local delivery systems includingALN-containing titanium microspheres nanoparticles andchitosan scaffolds could improve osteogenesis of osteoblastsfor bone regeneration [27 29 30]

In this study we developed ALN-eluting BCP (ALNBCP) scaffolds as local delivery system for improving boneformation Based on the fact that ALN has a high bindingaffinity to the bonemineral hydroxyapatite (HAp) [31 32] wefabricated ALNBCP scaffolds by simply mixing BCP scaf-folds with ALN We hypothesized that ALNBCP scaffoldsmay enhance the drug efficacy by achieving the sustaineddelivery over a period of time as well as enhancing the osteo-genesis of osteoblasts compared to BCP alone In this studywe aimed to investigate whether ALNBCP scaffolds caneffectively improve in vitro osteoblast activity and osteogenicdifferentiation and to demonstrate whether ALNBCP scaf-folds have great potential for bone regeneration

2 Materials and Methods

21 Materials Biphasic calcium phosphate substrates (BCPHAp = 60 TCP = 40 diameter times length = 3 times 8mmcylinder type) were kindly donated from OssGen Cor-poration (Gyeongbuk Korea) Alendronate was obtainedfrom Samjin Pharmaceutical Corporation (Seoul Korea)

Dulbeccorsquos Modified Eaglersquos Medium (DMEM) phosphatebuffer saline (PBS) fetal bovine serum (FBS) and 1 antibi-otics (100UmM penicillin and 01mgmL streptomycin)were purchased from Life Technologies Corporation (GrandIsland NY USA) 2-(N-Morpholino) ethanesulfonic acid(MES) was supplied by Sigma Chemical Co (St Louis MOUSA)

22 Preparation of Alendronate- (ALN-) Coated BCP Sub-strates To prepare ALN-eluting BCP substrates BCP sub-strates were immersed in 01MMES buffer (pH 56) contain-ing ALN (01 or 1mg) and gently shaken for 24 h The ALN-coated BCP substrates were collected washed with distilledwater (DW) and dried in a vacuum for 1 day

23 Characterization of BCPandALN-Eluting BCP SubstratesSurface morphologies and elemental compositions of BCPandALN-coated BCP substrates were evaluated by a variable-pressure field emission scanning electron microscopy (VP-FE-SEM SUPRA-55VP Carl Zeiss Germany) at the KoreaBasic Science Institute Chencheon Center The chemicaldistribution mapping and compositions of BCP and ALN-coated BCP substrates were determined using an energy-dispersive X-ray spectrometer (EDS) ATR-FTIR (attenuatedtotal reflectance-Fourier transform infrared spectroscopyAvatar 360 Nicolet Instrument Corp Madison WI USA)spectra had a resolution of 4 cmminus1 between 2000 and500 cmminus1 A commercially available powder was used toobtain the ATR-FTIR spectrum of ALN

24 In Vitro Release Study of ALN from ALN-Eluting BCPSubstrates To assess ALN release from ALN (01mg)BCPand ALN (1mg)BCP each substrate was soaked in a 15mLconical tube with 1mL PBS The tubes were then incubatedat 37∘C with shaking at 100 rpm At predetermined timepoints of 1 3 5 and 10 h and 1 3 5 7 14 21 and 28 dayssupernatants were collected and replaced with fresh PBSThecollected supernatants were stored in a deep freezer at minus20∘Cbefore analysis The released ALN was determined using aUVVis spectrophotometer (DU-530 Beckman Coulter) ata wavelength of 293 nm with a complex of alendronate andstandard iron (III) chloride solution

25 Cell Morphology For in vitro cell studies MG-63cells which were isolated from human bone tissue wereused because MG-63 cells are popular cells to demon-strate the osteogenic effects of drugs peptides or proteinsinon various substrates MG-63 cells were maintained withDMEM supplemented including 10 FBS 50120583gmL ascor-bic acid 10 nM dexamethasone 10mM 120573-glycerophosphate100UmL penicillin and 100120583gmL streptomycin

To confirm the morphologies of MG-63 cells (osteoblast-like cells) grown on BCP and ALN-eluting BCP substratesthe cells (1 times 104 cells100 120583L) were carefully seeded on thesurface of BCP and ALN-eluting BCP substrates in a 24-well tissue culture plate incubated for 30min at 37∘C andadded to 1mL of DMEM supplemented with 10 FBS and1 antibiotics in the presence of 50 120583gmL ascorbic acid10 nMdexamethasone and 10mM120573-glycerophosphate After



2







119875 lt 005

3 Results


4



(A) (B) (C)

(a)

BCP

C O N P Ca

ALN

(01

mg)

BCP

(b)









600800100012001400

Tran

smitt

ancy

()

0

50

100

150

200 (a)

(b)

(c)

(d)



4


Time (day)0 5 10 15 20 25 30

0

1

2

3

4

Ale

ndro

nate

cum

ulat

ive r

eleas

e (120583

gm

L)






4 Discussion



(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)



00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2







119875 lt 005

3 Results


4



(A) (B) (C)

(a)

BCP

C O N P Ca

ALN

(01

mg)

BCP

(b)









600800100012001400

Tran

smitt

ancy

()

0

50

100

150

200 (a)

(b)

(c)

(d)



4


Time (day)0 5 10 15 20 25 30

0

1

2

3

4

Ale

ndro

nate

cum

ulat

ive r

eleas

e (120583

gm

L)






4 Discussion



(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)



00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B) (C)

(a)

BCP

C O N P Ca

ALN

(01

mg)

BCP

(b)









600800100012001400

Tran

smitt

ancy

()

0

50

100

150

200 (a)

(b)

(c)

(d)



4


Time (day)0 5 10 15 20 25 30

0

1

2

3

4

Ale

ndro

nate

cum

ulat

ive r

eleas

e (120583

gm

L)






4 Discussion



(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)



00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















600800100012001400

Tran

smitt

ancy

()

0

50

100

150

200 (a)

(b)

(c)

(d)



4


Time (day)0 5 10 15 20 25 30

0

1

2

3

4

Ale

ndro

nate

cum

ulat

ive r

eleas

e (120583

gm

L)






4 Discussion



(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)



00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(A) (B) (C)

(a)

DAPI F-actin Merged

BCP

ALN

(01

mg)

BCP

ALN

(1m

g)B

CP

(b)



00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

08

10

12

BCP

7Time (day)

143 10

ALP

activ

ity (120583

Mm

in120583

g pr

otei

n)

lowast

lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast


(a)

0

5

10

15

20

25

Time (day)21

Calc

ium

cont

ent (120583

gm

L)

lowast

lowast


(b)








Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nor

mal

ized

fold

expr

essio

n

0

1

2

3

OPN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

OCN7 days 21 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

30


Nor

mal

ized

fold

expr

essio

n

0

1

2

3

4

ALP7 days

lowastlowast

lowast





lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast




5 Conclusions








Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgment


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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