Effects of Collagen Grafting on Cell Behaviors in …1) 01-07.pdfRESEARCH ARTICLE Open Access...

RESEARCH ARTICLE Open Access

Effects of Collagen Grafting on CellBehaviors in BCP Scaffold withInterconnected Pore StructureDong-Jun Yang1 Jae-Hui Jeon2 Sun-Young Lee1 Hyun-Wook An1 Keun Oh Park1 Kwang-Bum Park1

and Sukyoung Kim2

Abstract

Background This study was to investigate the effect of collagen grafted porous biphasic calcium phosphate (BCP)on cell attachment proliferation and differentiation Porous BCP scaffolds with interconnected micropore structurewere prepared with were prepared and then grafted with a collagen type I The hydroxyapatite (HA) and β-tricalcium phosphate (TCP) ratio of the TCP scaffolds was about 6040 and the collagen was crosslinked on the TCPscaffold surface (collagen-TCP)

Results The sintered BCP scaffolds showed fully interconnected micropore structures with submicron-sized grainsThe collagen crosslinking in the scaffolds was conducted using the the N-(3-Dimethylaminopropyl)-Nprime-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (NHS) crosslinking method The cell proliferation ofcollagen-BCP scaffolds showed a similar result to that of the BCP scaffolds However osteoblastic differentiation andcell attachment increased in the collagen-BCP scaffolds

Conclusions Collagen-BCP scaffold improved the cell attachment ability in early phase and osteoblasticdifferentiation

Keywords Hydroxyapatite (HA) Tricalcium phosphate (TCP) Biphasic calcium phosphate (BCP) Collagen

BackgroundAutograft xenograft and synthetic grafting bone substi-tutes with diverse chemical compositions are widelyused as an alternative to autogenous grafting material torepair osseous defects in dentistry [1ndash5] The calciumphosphate such as hydroxyapatite (HA) β-tricalciumphosphate (β-TCP) and biphasic calcium phosphate(BCP) are commonly used as a bone substitute due totheir excellent biocompatibility The most synthetic BCPbone substitute consists of a mixture of HA and β-TCPwith various ratio HA and β-TCP are very different interms of the solubility or dissolution rate which reflectstheir bioreactivity β-TCP resorbs more quickly than HA[6] Therefore the bioreactivity of BCP can be controlledby changing the ratio of HA and β-TCP [7]

The structural characteristics and chemical compos-ition of BCP scaffolds play a critical role in osteoconduc-tivity of bone substitute In structural aspects the poresize at both macro- and micro-levels porosity and theinterconnection of microspores are important factors forbone healing [8ndash13] In recent study donut shape BCPbone substitutes made of a central macro-pore (about300 ~ 400 μm) and micro-pores (about 20ndash60 μm)showed greater new bone formation when comparedwith similar BCP composition with micro-pores [14]In general BCP with various HATCP ratio showsgreater new bone formation when compared with HAand β-TCPCollagen which is one of extracellular components of

bone tissue promotes osteogenic differentiation of osteo-blast and mesenchymal stem cells in vitro [15ndash18] It isknown that HA functionalized with collagen I affects thecell adhesion and mineralization of mesenchymal stemcells [19] And collagen-TCP porous ceramics are used

Correspondence sykimynuackr2School of Materials Science amp Engineering Yeungnam UniversityGyeongsan Gyeongbuk 712-749 KoreaFull list of author information is available at the end of the article

copy 2016 Yang et al Open Access This article is distributed under the terms of the Creative Commons Attribution 40International License (httpcreativecommonsorglicensesby40) which permits unrestricted use distribution andreproduction in any medium provided you give appropriate credit to the original author(s) and the source provide a link tothe Creative Commons license and indicate if changes were made The Creative Commons Public Domain Dedication waiver(httpcreativecommonsorgpublicdomainzero10) applies to the data made available in this article unless otherwise stated

Biomaterials

Research

The Korean Society for Biomaterials

Biomater Res (2016) 20(1) 1-7

DOI 101186s40824-016-0049-3

ISSN 1226-4601(Print) ISSN 2055-7124(Online)

Special Issue Ceramics in bio-application

1

in human extraction socket healing and forms sufficientamounts of vital bone [20]This study aimed to investigate the cell behaviors such

as cell attachment proliferation and differentiation inporous BCP ceramics Especially the effect of collagencrosslinked on BCP ceramic surface was examined Inorder to compare the cell behaviors between pure BCPand collagen grafted BCP ceramics (collagen-BCP) withinterconnected micropore structures collagen-BCP sam-ples were prepared by crosslinking the N-(3-Dimethyla-minopropyl)-Nprime-ethylcarbodiimide hydrochloride andN-hydroxysuccinimide (NHS) on pure BCP ceramics Itis known that the compound of EDC and NHS is acoupling agent and efficient and non-toxic crosslinkingmaterial [21ndash23]

MethodsPreparation of BCP scaffoldsBCP powder was synthesized by a precipitation methodusing 1417 g of Ca (NO3)24H2O (Duksan Pure Che-micals Gyunggi-do Korea) and 511 g of (NH4) 2HPO4

(Duksan Pure Chemicals Gyunggi-do Korea) First Ca(NO3) 24H2O and (NH4) 2HPO4 were dissolved in dis-tilled water and (NH4) 2HPO4 solution was added dropby drop to the Ca (NO3) 24H2O solution The pH ofthe solution was adjusted to 85 with ammonium hy-droxide (Duksan) after dissolved completely at 80 degCAnd the solution was stirred for 1 h washed with dis-tilled water to remove ammonium hydroxide and filteredwith 02 μm membrane filter The filter cake wascrushed and dried in a drying oven for 12 h The as-dried powder was then calcined at 900 degC for 1 h Thedonut shape porous BCP samples were produced withthe calcined powder

Collagen crosslinkingThe collagen on the BCP scaffold surface was chemicallycrosslinked First 5 collagen was dispersed in 1 acetic acid at 0 ~ minus5 degC for 6 ~ 12 h A mixture of005 g N-(3-dimethylaminopropyl)-Nrsquo-ethylcarbodiimidehydrochloride (EDC Sigma-Aldrich Canada Ltd Oak-ville Canada) and 005 g N-hydroxysuccinimide (NHSSigma-Aldrich Canada Ltd Oakville Canada) was pre-pared in distilled water as described previously [21ndash23]Carbodiimide crosslinking in collagen solution by usingEDC and NHS was performed by reacting the two solu-tions at 0 ~ minus5 degC for 24 h in ice bath In order to cross-link the collagen on BCP surface the BCP scaffolds wereimmersed in 10 3-aminopropyltriethoxysilane (3-APTES) at 95 degC for 2 h washed three times with dis-tilled water and dried in a drying oven The crosslinkingof amino group on the scaffold surface was performedvia the 3-APTES terminal amino group The 3-APTEStreated BCP scaffolds with amino groups reacted with

the prepared collagen solution at room temperature for6 h Collagen treated BCP samples (collagen-TCP) werewashed three times with distilled water and dried

X-ray diffraction (XRD)Both BCP scaffolds before and after collagen crosslink-ing (TCP and collagen-TCP) were analyzed to examinethe crystalline phases (HA and TCP) with X-ray diffract-ometer (DMAX-2500 RIGAKU Japan) The diffractom-eter was operated at 40 kV and 30 mA employing a stepsize of 1degmin

Scanning electron microscopy (SEM)Surface morphology of both scaffolds was observedusing scanning electron microscope (SEM) equippedwith energy dispersive X-spectroscope (EDS) (Hitachi S-4200 Tokyo Japan) Accelerating voltage was set as15 kV

X-ray photoelectron spectroscopyIn order to confirm the collagen crosslinked on BCPsurface X-ray photoelectron spectroscopy (XPS Quan-tera SXM ULVAC-PHI Japan) was used

Coomassie brilliant blue stainingScaffolds were stained in 01 Coomassie brilliant blueR250 for 20 min and destined in 45 methanol and10 glacial acetic acid until the background of the gelwas removed

Cell attachmentThe MC3T3-E1 cells (2 times 104 cells) a mouse calvaria-derived osteoblast-like cell line and implants in α-modified Eaglersquos medium (α-MEM) were repeatedly ro-tated by using a rotation plate (2 rpm) in a flat-bottomtube at 37 degC for 3 h [24] The cells on three samples

Fig 1 X-ray diffraction of BCP scaffolds and collagen BCP scaffolds

2 Dong-Jun Yang Jae-Hui Jeon Sun-Young Lee Hyun-Wook An Keun Oh Park Kwang-Bum Park and Sukyoung Kim

Biomaterials Research 2016

(control HA pure BCP and collagen-BCP) were incu-bated in a 5 CO2 incubator at 37 degC for 3 h After in-cubation the scaffolds were washed twice withphosphate buffered saline (pH 74) Fixation was carriedout for 30 min in 2 glutaraldehyde The scaffold sam-ples were then washed twice with 01 M sodium cacody-late buffer (pH 74) dehydrated sequentially in 25 50 75 95 and 100 ethanol for 5 min eachand dried with tetramethylsilane The scaffold specimenswere coated with gold examined and photographed

using a SEM equipped with an EDS (SEMEDS S-4800Hitachi Tokyo Japan)

Cell proliferationThe MC3T3-E1 cells were seeded into 24-well plates ata density of 2 times 104 cells per well After 24 h controlpure BCP and collagen-BCP scaffolds were added intoeach well The cells on three samples were incubated ina 5 CO2 incubator at 37 degC for 1 4 and 7 days MTT(3-(45-dimethylthiazol-2yl)-25-diphenyl tetrazolium

Fig 3 X-ray photoelectron spectroscopy spectra of BCP scaffolds and collagen BCP scaffolds XPS of BCP scaffolds (a) and collagen BCPscaffolds (b)

Fig 2 Scanning electron microscope image of BCP scaffolds and collagen BCP scaffolds SEM images of BCP scaffolds (a b c) and collagen BCPscaffolds (d e f) at magnifications of times 1000 (a d) times10000 (b e) and times 20000 (c f)

Effects of Collagen Grafting on Cell Behaviors in BCP Scaffold with Interconnected Pore Structure 3

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bromide) assay was performed for the cell proliferationat 1 4 and 7 days 05 mgml of MTT solution wasadded to each well After 3 h the MTT solution was as-pirated and the dimethylsulfoxide was added tosolubilize the formed formazan The optical density wasmeasured at a wavelength of 570 nm using an ELISAreader (PowerWave XS Bio-Tek Winooski USA) Cellcounting was performed for the cell quantification for 35 and 7 days The cells were detached from cultureplate and washed with PBS The cells were counted byusing a haemocyotometer

Alkaline phosphate (ALP) stainingThe MC3T3-E1 cells were seeded into 24-well plates ata density of 2 times 104 cells per well After 24 h the mediawas changed with osteogenic medium and then pureBCP and collagen-BCP scaffolds were added into eachwell The cells were incubated at 37 degC in a humidifiedatmosphere of 5 CO2 for 7 days The cells werewashed PBS and the ALP staining was performed using

alkaline phosphatase (ALP) Kit (SIGMA-ALDRICHINC St Louis MO USA)

Statistical analysisStatistical analysis was performed by a using SPSS 110statistical system (SPSS Inc Chicago IL USA) Thepaired Student t-test was performed to compare the sig-nificance of the differences in cell proliferation Valuesof p were statistically significant at lt 005

Results and discussionCharacterization of BCP and collagen-BCP ScaffoldsThe crystallinity and phase composition in pure BCPand collagen-BCP scaffolds were investigated by usingXRD X-ray diffraction patterns of pure BCP andcollagen-BCP scaffolds are consisted of two phases (HAand β-TCP) and is shown in Fig 1 The ratio of HA andβ-TCP phases calculated by Rietveld method in bothBCP scaffolds was 6040 and did not show any changeof crystallinity

Fig 5 Cell morphology of MC3T3-E1 on BCP scaffolds and collagenBCP scaffolds SEM images of MC3T3-E1 cultured on BCP scaffolds (a c) andcollagen-BCP scaffolds (b d) for 24 h at magnifications of times 500 (a b) and times 10000 (c d)

Fig 4 Coomassie brilliant blue staining of collagen BCP scaffolds



The surface topography in both BCP and collagen-BCP scaffolds was observed by using SEM (Fig 2) SEMimages show submicron-sized grains with intercon-nected micropore structures in the BCP and collagen-BCP scaffold The collagen-BCP scaffold had a similarsurface morphology to the BCP scaffold at low magnifi-cation images but showed the collagen on the surface athigher magnificationsIn order to confirm the collagen grafting XPS analysis

was conducted (Fig 3) The N1s (nitrogen peak) on thecollagen-BCP scaffold was observed in the XPS pattern(Fig 3-b) An observation of N1s signal in the XPS pat-tern means the presence of amino group of collagen andthe crosslinking of collagen on BCP scaffoldsThe presence of collagen on collagen-BCP scaffold

was also observed by using Coomassie brilliant bluestaining (Fig 4) Coomassie brilliant blue staining is gen-erally used for detection of protein on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)and widely used in a various area The color of BCP scaf-folds is purple if the collagen is present on the surfaceThe Coomassie brilliant blue is binding with collagenThis method has an advantage that the presence of

collagen on specimen can distinguish with the naked eyewithout using equipment such as SEM and XPS etc Thepurple color on collagen-BCP scaffold was observed onall surfacesTherefore it is demonstrated that the collagen was

crosslinked efficiently on BCP scaffolds using EDCNHS method and the crosslinking of collagen did notaffect overall structure of scaffolds

Behaviors of osteoblastic cells on collagen-BCP ScaffoldTo evaluate the effects of collagen on cell attachmentMC3T3-E1 cells were cultured on the BCP scaffold andthe collagen-BCP scaffold for 24 h and then the cellmorphology was observed by using SEM The cells onthe collagen-BCP scaffold were more spread comparedwith the cells on the BCP scaffold (Fig 5) This resultcoincides with the result of previous study The cells oncollagen grafted HA were more spread than those onpure HA [25] Therefore it is demonstrated that the col-lagen in BCP scaffold enhanced the cell attachment abil-ity in early phaseAfter incubation for 1 4 7 days the cell proliferation

in control pure BCP and collagen-BCP scaffolds was

Fig 7 ALP staining of differentiating MC3T3-E1 cultured on BCP scaffolds and collagenBCP scaffolds MC3T3-E1 cells were cultured in osteogenicmedia and then ALP staining was performed ALP staining image of BCP scaffolds (a) and collagenBCP scaffolds (b)

Fig 6 Cell proliferation of MC3T3-E1 cultured on BCP scaffolds and collagen BCP scaffolds Proliferation of MC3T3-E1 cells were determined byMTT assay (a) and cell counting (b) Data are expressed as the mean plusmn SD (n = 3)


Vol 20 No 1

analyzed by MTT assay The cell counting in mouseosteoblastic cells (MC3T3-E1 cells) for cultured sampleswas conducted in terms of incubation periods (3 57 days) The collagen-BCP scaffold showed similar ab-sorbance and cell number with that of cells on the BCPscaffold for the incubation time (Fig 6) There was nostatistical difference in cell proliferation between thecollagen-BCP and pure BCP scaffold (P gt 01)To evaluate the effects of collagen on osteoblastic dif-

ferentiation MC3T3-E1 cells were cultured in osteo-genic media and then ALP staining was performed(Fig 7) ALP-positive cells on the collagen-BCP scaffoldwere increased compared with the cells on the pure BCPscaffold ALP positive cells are shown in red Even ex-ogenous type I collagen facilitated osteogenic differenti-ation and acts as a substrate for mineralization [18]It is believed that the collagen in BCP scaffold en-

hanced the cell attachment ability in early phase andosteoblastic differentiation That is collagen which isbone extracelluar matrix protein may play a criticalrole in osteoblastic differentiation and phenotypicexpression

ConclusionsBCP scaffolds were HAβ-TCP phase ratio of 6040 andhad porous microstructure with submicron-sized grainsThe collagen was successfully crosslinked into the BCPscaffolds by the EDCNHS crosslinking method The cellproliferation of collagen-BCP scaffolds showed a similarpattern to those of the BCP scaffolds However cell at-tachment and osteolbastic differentiation were improvedin the collagen-BCP scaffolds The collagen in thecollagen-BCP scaffold was effective in osteoblastic differ-entiation and phenotypic expression These results indi-cate that the collagen-BCP scaffolds with interconnectedmicropore structures is a good candidate as an osteo-conductive bone substitute for the repair of bonedefects

Availability of supporting dataThere was no available supporting data

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsDJY designed the experiments JHJ and HWA carried out characterizations ofBCP fabricated by precipitation method KOP participated in the crosslinkingof collagen and in vitro assays SYL drafted the manuscript and helped tointerpret data KBP and SYK participated in its design and coordination andhelped to draft the manuscript All authors read and approved the finalmanuscript

AcknowledgementsThis work was supported by the Yeungnam University and and Dae-GyeongLeading Industry Office through the Leading Industry Development forEconomic Region

Author details1Department of Institute of Science amp Technology Megagen ImplantJain-myeon Gyeongsan Gyeongbuk 712-852 Korea 2School of MaterialsScience amp Engineering Yeungnam University Gyeongsan Gyeongbuk712-749 Korea

Received 24 September 2015 Accepted 4 January 2016

References1 Moskow BS Lubarr A Histological assessment of human periodontal defect

after durapatite ceramic implant Report of a case J Periodontol198354455ndash62

2 Kenney EB Lekovic V Han T Carranza Jr FA Dimitrijevic B The use of aporous hydroxyapatite implant in periodontal defects I Clinical results aftersix months J Periodontol 19855682ndash8

3 Yukna RA Yukna CN A 5-year followup of 16 patients treated with corallinecalcium carbonate (Biocoral) bone replacement grafts in infrabony defectsJ Clin Periodontol 1998251036ndash40

4 Zerbo IR Zijderveld SA De Boer A Bronckers AL De Lange G TenBruggenkate CM et al Histomorphometry of human sinus flooraugmentation using a porous betandashtricalcium phosphate a prospectivestudy Clinical Oral Implants Research 200415724ndash32

5 Simion M Fontana F Rasperini G Miorana C Vertical ridge augmentationby expanded-polytetrafluoroethylene membrane and a combination ofintraoral autogenous bone graft and deproteinized anorganic bovine bone(Bio-Oss) Clinical Oral Implants Research 200718620ndash9

6 Lu J Descamps M Dejou J Koubi G Hardouin P Lemaitre J et al Thebiodegradation mechanism of calcium phosphate biomaterials in boneJ Biomed Mater Res 200263408ndash12

7 LeGeros RZ Lin S Rohanizadeh R Mijares D LeGeros JP Biphasic calciumphosphate bioceramics preparation properties and applications J Mater SciMater Med 200314201ndash9

8 Hulbert SF Young FA Mathews RS Klawitter JJ Talbert CD Stelling FHPotential of ceramic materials as permanently implantable skeletalprostheses J Biomed Mater Res 19704433ndash56

9 Tsuruga E Takita H Itoh H Wakisaka Y Kuboki Y Pore size of poroushydroxyapatite as the cell-substratum controls BMP-induced osteogenesisJ Biochem 1997121317ndash24

10 Gauthier O Bouler JM Aguado E Pilet P Daculsi G Macroporous biphasiccalcium phosphate ceramics influence of macropore diameter andmacroporosity percentage on bone ingrowth Biomaterials 199819133ndash9

11 Kuboki Y Jin Q Kikuchi M Mamood J Takita H Geometry of artificial ECMsizes of pores controlling phenotype expression in BMP-inducedosteogenesis and chondrogenesis Connect Tissue Res 200243529ndash34

12 Lecomte A Gautier H Bouler JM Gouyette A Pegon Y Daculsi G et alBiphasic calcium phosphate a comparative study of interconnectedporosity in two ceramics J Biomed Mater Res B Appl Biomater 2008841ndash6

13 Walsh WR Vizesi F Michael D Auld J Langdown A Oliver R et al Beta-TCPbone graft substitutes in a bilateral rabbit tibial defect model Biomaterials200829266ndash71

14 Park JW Kim ES Jang JH Suh JY Park KB Hanawa T Healing of rabbitcalvarial bone defects using biphasic calcium phosphate ceramics made ofsubmicron-sized grains with a hierarchical pore structure Clin Oral Impl Res201021268ndash76

15 Andrianarivo AG Robinson JA Mann KG Tracy RP Growth on type Icollagen promotes expression of the osteoblastic phenotype in humanosteosarcoma MG-63 cells J Cell Physiol 1992153256ndash65

16 Lynch MP Stein JL Stein GS Lian JB The influence of type I collagen onthe development and maintenance of the osteoblast phenotype in primaryand passaged rat calvarial osteoblasts modification of expression of genessupporting cell growth adhesion and extracellular matrix mineralizationExp Cell Res 199521635ndash45

17 Mizuno M Fujisawa R Kuboki Y Type I collagen-induced osteoblasticdifferentiation of bone-marrow cells mediated by collagenalpha2beta1integrin interaction J Cell Physiol 2000184207ndash13

18 Kihara T Hirose M Oshima A Ohgushi H Exogenous type I collagenfacilitates osteogenic differentiation and acts as a substrate formineralization of rat marrow mesenchymal stem cells in vitro BiochemBiophys Res Commun 20063411029ndash35



19 Teixeira S Fernandes MH Ferraz MP Monteiro FJ Proliferation andmineralization of bone marrow cells cultured on macroporoushydroxyapatite scaffolds functionalized with collagen type I for bone tissueregeneration J Biomed Mater Res A 2010951ndash8

20 Brkovic BM Prasad HS Rohrer MD Konandreas G Agrogiannis G AntunovicD et al Beta-tricalcium phosphatetype I collagen cones with or without abarrier membrane in human extraction socket healing clinical histologichistomorphometric and immunohistochemical evaluation Clin OralInvestig 201216581ndash90

21 Wissink MJ Beernink R Pieper JS Poot AA Engbers GH Beugeling T et alImmobilization of heparin to EDCNHS-crosslinked collagenCharacterization and in vitro evaluation Biomaterials 200122151ndash63

22 Wissink MJ Beernink R Poot AA Engbers GH Beugeling T Van Aken WG et alImproved endothelialization of vascular grafts by local release of growth factorfrom heparinized collagen matrices J Control Release 200064103ndash14

23 Wissink MJ Beernink R Scharenborg NM Poot AA Engbers GHM BeugelingT et al Endothelial cell seeding of (heparinized) collagen matrices effects ofbFGF pre-loading on proliferation (after low density seeding) and pro-coagulant factors J Control Release 200067141ndash55

24 van den Dolder J Vehof JW Spauwen PH Jansen JA Bone formation by ratbone marrow cells cultured on titanium fiber mesh Effect of in vitro culturetime J Biomed Mater Res 200262350ndash8

25 Lee DW Lee EJ Chum SS Ahn MW Song IW Kang IK et al Characterizationof bone cell behaviors on collagen grafted hydroxyapatite surfaces Key EngMater 2008361ndash3631143ndash6

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Vol 20 No 1

in human extraction socket healing and forms sufficientamounts of vital bone [20]This study aimed to investigate the cell behaviors such

as cell attachment proliferation and differentiation inporous BCP ceramics Especially the effect of collagencrosslinked on BCP ceramic surface was examined Inorder to compare the cell behaviors between pure BCPand collagen grafted BCP ceramics (collagen-BCP) withinterconnected micropore structures collagen-BCP sam-ples were prepared by crosslinking the N-(3-Dimethyla-minopropyl)-Nprime-ethylcarbodiimide hydrochloride andN-hydroxysuccinimide (NHS) on pure BCP ceramics Itis known that the compound of EDC and NHS is acoupling agent and efficient and non-toxic crosslinkingmaterial [21ndash23]

MethodsPreparation of BCP scaffoldsBCP powder was synthesized by a precipitation methodusing 1417 g of Ca (NO3)24H2O (Duksan Pure Che-micals Gyunggi-do Korea) and 511 g of (NH4) 2HPO4

(Duksan Pure Chemicals Gyunggi-do Korea) First Ca(NO3) 24H2O and (NH4) 2HPO4 were dissolved in dis-tilled water and (NH4) 2HPO4 solution was added dropby drop to the Ca (NO3) 24H2O solution The pH ofthe solution was adjusted to 85 with ammonium hy-droxide (Duksan) after dissolved completely at 80 degCAnd the solution was stirred for 1 h washed with dis-tilled water to remove ammonium hydroxide and filteredwith 02 μm membrane filter The filter cake wascrushed and dried in a drying oven for 12 h The as-dried powder was then calcined at 900 degC for 1 h Thedonut shape porous BCP samples were produced withthe calcined powder

Collagen crosslinkingThe collagen on the BCP scaffold surface was chemicallycrosslinked First 5 collagen was dispersed in 1 acetic acid at 0 ~ minus5 degC for 6 ~ 12 h A mixture of005 g N-(3-dimethylaminopropyl)-Nrsquo-ethylcarbodiimidehydrochloride (EDC Sigma-Aldrich Canada Ltd Oak-ville Canada) and 005 g N-hydroxysuccinimide (NHSSigma-Aldrich Canada Ltd Oakville Canada) was pre-pared in distilled water as described previously [21ndash23]Carbodiimide crosslinking in collagen solution by usingEDC and NHS was performed by reacting the two solu-tions at 0 ~ minus5 degC for 24 h in ice bath In order to cross-link the collagen on BCP surface the BCP scaffolds wereimmersed in 10 3-aminopropyltriethoxysilane (3-APTES) at 95 degC for 2 h washed three times with dis-tilled water and dried in a drying oven The crosslinkingof amino group on the scaffold surface was performedvia the 3-APTES terminal amino group The 3-APTEStreated BCP scaffolds with amino groups reacted with

the prepared collagen solution at room temperature for6 h Collagen treated BCP samples (collagen-TCP) werewashed three times with distilled water and dried

X-ray diffraction (XRD)Both BCP scaffolds before and after collagen crosslink-ing (TCP and collagen-TCP) were analyzed to examinethe crystalline phases (HA and TCP) with X-ray diffract-ometer (DMAX-2500 RIGAKU Japan) The diffractom-eter was operated at 40 kV and 30 mA employing a stepsize of 1degmin

Scanning electron microscopy (SEM)Surface morphology of both scaffolds was observedusing scanning electron microscope (SEM) equippedwith energy dispersive X-spectroscope (EDS) (Hitachi S-4200 Tokyo Japan) Accelerating voltage was set as15 kV

X-ray photoelectron spectroscopyIn order to confirm the collagen crosslinked on BCPsurface X-ray photoelectron spectroscopy (XPS Quan-tera SXM ULVAC-PHI Japan) was used

Coomassie brilliant blue stainingScaffolds were stained in 01 Coomassie brilliant blueR250 for 20 min and destined in 45 methanol and10 glacial acetic acid until the background of the gelwas removed

Cell attachmentThe MC3T3-E1 cells (2 times 104 cells) a mouse calvaria-derived osteoblast-like cell line and implants in α-modified Eaglersquos medium (α-MEM) were repeatedly ro-tated by using a rotation plate (2 rpm) in a flat-bottomtube at 37 degC for 3 h [24] The cells on three samples

Fig 1 X-ray diffraction of BCP scaffolds and collagen BCP scaffolds









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Effects of Collagen Grafting on Cell Behaviors in …1) 01-07.pdfRESEARCH ARTICLE Open Access...

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