Research Article Cell Communication in a Coculture System ...downloads.hindawi.com/journals/bmri/2014/320123.pdf · Research Article Cell Communication in a Coculture System Consisting

Research ArticleCell Communication in a Coculture System Consisting ofOutgrowth Endothelial Cells and Primary Osteoblasts

David Paul Eric Herzog Eva Dohle Iris Bischoff and Charles James Kirkpatrick

Institute of Pathology University Medical Center Johannes Gutenberg University Langenbeckstraszlige 1 55101 Mainz Germany

Correspondence should be addressed to Eva Dohle dohleuni-mainzde

Received 30 September 2013 Revised 29 January 2014 Accepted 12 March 2014 Published 22 April 2014

Academic Editor Dominique Shum-Tim

Copyright copy 2014 David Paul Eric Herzog et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Bone tissue is a highly vascularized and dynamic system with a complex construction In order to develop a construct for implantpurposes in bone tissue engineering a proper understanding of the complex dependencies between different cells and cell typeswould provide further insight into the highly regulated processes during bone repair namely angiogenesis and osteogenesis andmight result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task This study is basedon an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used indifferent studies of bone repair processes with special regard to angiogenesis and osteogenesis Coculture systems of OECs andpOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures Although many studies have focused on cell communication there are still numerous aspects which remain poorlyunderstood Therefore the aim of this study is to investigate certain growth factors and cell communication molecules that areimportant during bone repair processes Selected growth factors like VEGF angiopoietins BMPs and IGFs were investigatedduring angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks ofcultivation In addition to gain a better understanding on the origin of different growth factors both direct and indirect coculturestrategies were employed Another important focus of this study was to investigate the role of ldquogap junctionsrdquo small protein poreswhich connect adjacent cells With these bridges cells are able to exchange signal molecules growth factors and other importantmediators It could be shown that connexins the gap junction proteins were located around cell nuclei where they await theirtransport to the cell membrane In addition areas in which two cells formed gap junctions were found

1 Introduction

Cell communication is conducted in five different waysendocrine paracrine autocrine electric signalling anddirectcell-cell contacts A soluble factor or mediator released inthe blood stream can reach cells far away from the sourcewhich is called endocrine signalling When neighbour cellsinteract they often release mediators in a paracrine mannerto influence the surrounding cells A number of releasedmediators can also affect the producing cell itself This istermed autocrine signalling Electric signalling occurs inthe central nervous system and direct cell-cell contacts areformed by so-called gap junctions small protein tunnels [1]which consist of different connexin isoforms providing theexchange of ions smallmolecules or potentials [2ndash4] Several

thousand gap junctions are either located at the same plasmamembrane area called gap junction plaques [5] or are locatedin the endoplasmic reticulum awaiting their transport to theplasmamembrane [6] In vascular cells connexins 43 40 and37 are the most abundant isoforms [7ndash10] whereas connexinisoform 43 clearly dominates bone cells [11ndash15]

The field of tissue engineering aims to provide innovativestrategies to compensate for ldquothe loss or the failure of anorgan or tissue one of the most frequent devastating andcostly problems in human health carerdquo [16] Therefore bonetissue engineered constructs should afford the key elementsof functional and long-lasting bone constructs mechanicalstrength substrates for osteoid formation sufficient porosityto permit angiogenesis and vasculogenesis proper vascularnetwork bone cell migration controlled degradation to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 320123 15 pageshttpdxdoiorg1011552014320123

2 BioMed Research International

nontoxic products to accommodate the expanding tissueand controlled inflammation due to high biocompatibility[17ndash19] Several studies have shown that the combinationof cells of endothelial and osteogenic origin improves bothangiogenesis and osteogenesis [20] It has been controver-sially discussed which type of endothelial cell should beused for in vitro and in vivo experiments Late outgrowthendothelial cells (OECs) can be easily isolated from theperipheral blood mononuclear cell fraction and they possessa high proliferation capacity as well as a strong tendencyto form capillary tubes both in vitro and in vivo [21ndash24] Ithas already been shown that angiogenesis and osteogenesiscan be highly improved in a bidirectional way by combiningOECs with primary osteoblasts (pOBs) [25 26] makingOECs predestined for bone tissue engineering strategies

Angiogenesis and osteogenesis are prerequisites for theformation of a functional and perfused tissue engineeredconstruct Therefore involved growth factors and mediatorsduring these processes are very important In our coculturesystem essential growth factors were analysed in order togain better understanding of their contribution to angio-genesis and osteogenesis Well-known proangiogenic andosteogenic growth factors like vascular endothelial growthfactor A (VEGF-A) angiopoietins platelet-derived growthfactor B (PDGF-B) and transforming growth factor 120573 (TGF-120573) as well as insulin-like growth factors (IGFs) and bone mor-phogenetic proteins (BMPs) were examined during the courseof cocultivation by comparing two different cultivation timepoints 1 and 4weeks of cocultivation In addition by using anindirect coculture system consisting of pOBs and OECs thisstudy should also evaluate which cell type produces whichgrowth factor An additional focus of the study was to analysedirect cell-cell interaction via connexins and gap junctionsAs well as localizing gap junctions we hoped to establishhow connexins behave in the course of coculture growth anddevelopment

2 Materials and Methods

21 Isolation of Outgrowth Endothelial Cells (OEC) Out-growth endothelial cells are isolated from the mononu-clear cell fraction of peripheral blood buffy coats byficollhistopaque separation [26] which is used to separateblood into its components Therefore peripheral blood wasdiluted 1 2 in phosphate-buffered saline (PBS) containing05 fetal calf serum (FCS) and 2mM ethylene diaminetetra-acetic acid (EDTA) to prevent cells from clotting andsubsequently centrifuged for 35min at 400 g (without brak-ing) with histopaque placed at the bottom of a 50mL tubeAfter centrifugation 3 different components become visiblefrom the bottom-up erythrocytes mononuclear cells andplasmaThemononuclear cell fractionwas separated washedseveral times in PBS and cultured in endothelial cell growthmedium-2 (EGM-2) with supplements from the kit 5fetal calf serum and 1 penicillinstreptomycin on collagen-coated plates (35 120583gmL) A total number of 5 lowast 106 cellsper well were seeded on a 24-well plate After three to fourweeks the first OECs with their typical endothelial cell-like

cobblestone morphology and expansion capacity appearedin the culture OECs were then trypsinized and expandedon fibronectin-coated 24-well plates (10 120583gmL) over severalpassages at a splitting ratio 1 2 Passage numbers of OECsused for this study ranged from passage 8 to 18

22 Isolation of Human Primary Osteoblasts Human pri-mary osteoblasts (pOBs) were isolated from human bonefragments from healthy donors according to an establishedprotocol approved by the responsible Ethical Commission[27] Collagenase type IV at a concentration of 1mgmL wasadded severalwashing stepswith PBS followed and the enzy-matically digested bone fragments were subsequently placedon 6-well plates in Dulbeccorsquos modified Eagle medium con-taining 20 FCS and 1 penicillinstreptomycin (DMEM-Ham F12) During the first two to four weeks cells werefed every day until the plates reached subconfluence Thecells were then transferred to T75 culture flasks and culturedwith DMEM-Ham F12 containing only 10 FCS and 1penicillinstreptomycin Cells were passaged in a ratio 1 2using accutase The present study used cells from differentdonors up to the third passage

23 Coculture Consisting of Primary Osteoblasts and Out-growth Endothelial Cells Cocultures of OECs and pOBswere seeded on Thermanox coverslips (12mm in diameter)Primary osteoblasts were seeded first at a density of 300000cellswell in a fibronectin-coated 24-well plate followed byseeding of 200000 OECwell 24 hours later Cells werecocultivated in EGM-2 with supplements from the kitincluding low VEGF concentrations (2 ngmL according tothe manufacturer) 5 FCS and 1 penicillinstreptomycinfor different time periods For each coculture experimentat least three different donors were used Control pOB-and OEC-monocultures were cultivated in EGM-2 withsupplements from the kit in addition to 5 FCS and 1penicillinstreptomycin

24 Indirect Cocultures Seeded on Transwells The use ofa Transwell filter system allows different cell types in acoculture system to take up and secrete growth factors andsignalling molecules on both sides of a transmembrane filtertheir basal and their apical surface It is thus possible topromote themetabolic activities of these two cell types withina coculture system 100000 primary osteoblasts were seededat the lower surface and 66000 OECs were seeded at theupper surface of a polycarbonate transmembrane filter in aTranswell filter system in a 24-well plate (pore size 04 120583m0588 cm2filter) coated with fibronectin (10 120583gmL) to gaininsight into the origin of several growth factors producedby the different cell types in the coculture Cells were fedwith EGM-2 with supplements from the kit 5 FCS and1 penicillinstreptomycin and cultured for different timeperiods Culture supernatants as well as cell lysates of OECand pOB were collected separately from the Transwell filtersystem and then used for further experiments

BioMed Research International 3

25 Cryostat Sectioning Cell layers of cocultures consistingof OEC and pOB were snap frozen in liquid nitrogen andsectioned at a thickness of 10 120583m using a cryostat (LeicaMicrosystems Wetzlar Germany) Samples were stored atminus20∘C until use for immunohistochemical analysis Forimmunofluorescent staining the sections were first thawed atroom temperature before performing the staining procedureas described in the following section

26 Paraffin Sectioning Cocultures of primary osteoblastsand outgrowth endothelial cells were seeded on the uppersurface of a fibronectin-coated transmembrane filter in aTranswell filter system After 14 days of cultivation mem-branes were fixed with 37 PFA for 10 minutes and sub-sequently washed with PBS The fixed membranes were cutinto appropriate sizes and placed in embedding cassettesThe samples were dehydrated for paraffin embedding in anascending alcohol series (70 80 95 and 100) each for1 hour After an incubation step in xylene for an additionalhour membranes were embedded in paraffin blocks andcut into 4 120583m sections and stained for Hematoxylin andeosin (HE-stain) For immunofluorescent staining of paraffinsections the sections were rehydrated in a descending ethanolseries (100 95 80 and 70) and rinsed in distilledwaterbefore staining according to the protocol described below

27 3D Matrigel-Based Angiogenesis Assay Matrigel Base-ment Membrane Matrix (Becton Dickinson Labware Bed-ford UK) was thawed on ice and diluted 1 2 in cold EBM-2 cell culture medium with supplements from the kit and5 FCS 100000 primary osteoblasts (pOBs) and 66000outgrowth endothelial cells (OECs) were resuspended in50120583L liquid MatrigelEBM-2 mixture and added to the wellof a 96-well plate The Matrigel Basement Membrane Matrixwas allowed to solidify for 30min at 37∘C Subsequently100 120583L EBM-2 cell culture medium was added After 20 h or40 h of incubation at 37∘C in an atmosphere of 5 CO

2and

95 air the cocultures were imaged using a phase-contrastmicroscope (Biozero Keyence Neu-Isenburg Germany)

28 Immunofluorescent Staining Immunofluorescent stain-ing cells were fixed with 37 paraformaldehyde (PFA)(Merck Darmstadt Germany) washed three times withPBS and then permeabilized for 5 minutes using 01Triton-X in PBS Cells were washed again with PBS beforebeing incubated with different primary antibodies dilutedin 1 bovine serum albumin (BSA)PBS for 45 minutes atroom temperature CD31 (dilution 1 50 Dako HamburgGermany) and Cx43 (dilution 1 250 (mouse)1 400 (rabbit)Millipore Australia) After washing three times with PBScells were incubated with fluorescently labelled secondaryantibodies (Alexa Molecular probes MoBiTec GottingenGermany) diluted 1 1000 in 1 BSA in PBS for 45 minutesin darkness at room temperature Finally cell nuclei werecounterstainedwith 1120583gmLHoechst and cells weremountedwith Gelmount (Biomeda Foster City CA)The stained sam-ples or frozen sections were examined using a confocal laser

scanning microscope (LeicaTCS-NT) (Leica MicrosystemsWetzlar Germany)

29 Quantitative Real-Time Polymerase Chain Reaction(Qreal-Time PCR) RNA isolation was performed usingRNeasy Mini Kit according to the manufacturerrsquos protocol(Qiagen Hilden Germany) One 120583g of extracted RNA wastranscribed into complementary DNA (cDNA) according toa standard protocol using Omniscript Reverse TranscriptionKit (Qiagen Hilden Germany) Quantitative real-time PCRenabling the quantification of relative gene expression wasperformed using SYBR green DNA-binding fluorescent dye125 120583L of QuantiTect SYBRGreen PCRMasterMix 25120583L ofQuantiTect SYBRGreen primer assay (VEGF-A Ang1 Ang2PDGF-BB TGF-120573 IGF-1 IGF-2 ALP BMP-2 BMP-4 Cx37Cx40 Cx43 all provided by Qiagen Hilden Germany) 6 120583Lof RNase free water and 4 120583L of cDNA (1 ng120583L) were usedfor one reaction Quantitative real-time PCR was performedin triplicate with the following cycler program 95∘C 15mindenaturation step 94∘C 15 sec annealing step 55∘C 30 secelongation step 72∘C 35 sec and dissociation 95∘C 15 sec60∘C 1min and 95∘C 15 sec 40 cycleswere performed in totalGlycerin-aldehyde-3-phosphate (GAPDH) or ribosomal pro-tein 13A (RPL13A) was taken as endogenous standards andrelative gene expression was determined using the ΔΔCtmethod Gene expression was compared by setting controlcultures to 1 (reference value) as indicated in the relevantfigures

210 RT2 Profiler PCR Array System Polymerase Chain Reac-tionArray (PCRArray) TheRT2 profiler PCR array system isa pathway-focused gene expression screening method usingquantitative real time PCR RT2 profiler PCR arrays containa panel of 96 primer sets per single 96-well plate for aresearched set of 84 relevant pathway-focused genes plus 5different housekeeping genes Angiogenesis and osteogenesisRT2 profiler PCR array systems were performed with 2 ngcDNA from different experiments according to the manufac-turerrsquos protocol For one reaction 125 120583L 2x SABiosciencesRT2 qPCR Master Mix 1 120583L cDNA and 115 120583L RNase freewater were used The following cycler program was applied95∘C 10min denaturation step 95∘C 15 sec annealing step60∘C 1min elongation step 72∘C 35 sec dissociation 95∘C15 sec 60∘C 1min and 95∘C 15 sec 40 cycles were performedin total Ribosomal protein 13A (RPL13A) was taken as anendogenous standard and relative gene expression was deter-mined using ΔΔCt method Gene expression was comparedby setting control cultures to 1 (reference value) as indicatedin the relevant figures

211 Enzyme-Linked Immunosorbent Assay (ELISA) Culturesupernatants from differently treated cells were collectedand the concentration of different growth factors was mea-sured using ELISA DuoSets (RampD Systems Wiesbaden Ger-many) ELISA was performed in triplicate according to themanufacturerrsquos protocol A streptavidin-HRP (horseradish-peroxidase) colorimetric reaction was used to visualize pro-tein concentrations The optical density of each well was


measured using a microplate reader (GENios plus TECANCrailsheim Germany) at a wavelength of 450 nm Results aredepicted as absolute values as indicated in the relevant figures

212 Statistical Analysis Data are represented as meanvalues plusmn standard deviation of the mean Data distributionwas checked with the Shapiro Wilk test Statistical signifi-cance was assessed using the paired Students t-test (bilateral119875 value lowast119875 lt 005) and MS-Excel (Microsoft OfficeMicrosoft Munchen Germany) when data could be assessedas normally distributed Nonnormally distributed data werestatistically analysed using the nonparametric Wilcoxon test

3 Results

31 Cocultures of OECs and pOBs Revealed a Positive Effecton the Cellular Organization of OECs into Angiogenic Struc-tures To verify the angiogenic potential of the coculturemodel outgrowth endothelial cells were cocultured withprimary osteoblasts and analysed using immunofluorescentstaining for the endothelial marker PECAM (CD31) after4 weeks of cocultivation and compared to the 1 weekcoculture (Figure 1(a)) After 4 weeks in coculture OECsformed numerous microvessel-like structures reminiscentof a prevascular network as depicted in Figure 2(b) Inthe coculture system with pOBs endothelial cells appearedelongated and formed intercellular contacts and luminarstructures as demonstrated by CD31 immunofluorescentstaining of cryostat sections (Figure 1(c)) This formation ofa vascular lumen by OECs in the coculture could also beobserved in paraffin sections of cocultures seeded on a trans-membrane filter in a Transwell filter system (Figure 1(d)) Incontrast OECs inmonoculture did not formmicrovessel-likestructures either after 1 week or after 4 weeks of cultivation(Figures 1(e) and 1(f)) In addition a 3D angiogenesis assay(Figures 1(m)ndash1(o)) confirmed this positive angiogenic effectof the coculture system when compared to the endothelialmonoculturing alone (Figure 1(l)) Due to the fact that theformation of angiogenic structures formed by OECs clearlyincreased during the course of cocultivation from 1 weekto 4 weeks a human angiogenesis RT2 profiler array (PCRarray) was used to compare the expression of angiogenesis-related factors after 1 and 4 weeks of cocultivation (Figures1(e) and 1(f)) The aim was to gain insight into the molecularmechanisms and possible factors that might be responsiblefor the proangiogenic effect of the coculture on the OECIn total 60 genes involved in the process of angiogenesiswere upregulated after 4 weeks of cocultivation compared to1 week as depicted in the diagram (Figure 1(g)) and listed indetail in the table in Figure 1(h) In addition quantificationof relative gene expression of well-known angiogenic growthfactors such as VEGF Ang1 Ang2 IGF-1 IGF-2 TGF-1205731and PDGF-BB was determined using quantitative real timePCR (Figures 1(i)ndash1(k)) The expression level of mRNA afterone week was set as control (=10) The data clearly showan upregulation of all tested growth factors after 4 weeksof cocultivation compared to 1 week in coculture In accor-dance with a higher formation of angiogenic structures after

4 weeks of cocultivation the expression of proangiogenicfactors for example VEGF Ang1 and Ang2 increases after4 weeks of cocultivation Additionally PDGF-BB mRNAan important pericyte recruiter protein and TGF-1205731 mRNAwere upregulated Moreover IGF mRNAs were also found tobe upregulated

32 Cocultures of OECs and pOBs Promote Osteogenic Differ-entiation Calcification and mineralization level were testedusing alizarin red staining and quantitative real-timeRT-PCRof osteogenic and bone formation markers including ALPBMP-2 and BMP-4 in 4-week cocultures and were comparedwith the 1-week cultures (Figure 2) The longer cultivationtime resulted in higher alizarin red levels in cocultures andpOBmonocultures (Figure 2(a)) In general it is evident thatcocultures revealed a higher amount of alizarin red thanthe monocultures after both one and four weeks of cultiva-tion Accordingly relative expression of alkaline phosphatase(ALP) an important enzyme of bone tissue metabolismand marker of osteogenic activity and differentiation wasalso higher in the long-term cocultures (Figure 2(b)) BMP-2 and BMP-4 are important during bone formation andbone development The expression of these molecules wasexamined on the mRNA level setting the one week coculturelevel as control (=10) BMP mRNA was affected by a longercultivation time as well relative expression of BMP-2 andBMP-4 was in part significantly higher (BMP-4) after fourweeks of cocultivation time (Figure 2(c)) Additionally aPCR array system was used to detect other osteogenesis-related genes (Figure 2(d)) Their relative expression after acultivation period of four weeks compared to a cultivationperiod of only one week is depicted in Figure 2(e)

33 Cell-Cell Communication inCocultures of pOBs andOECsOrigin of Different Growth Factors The next object was todetermine the source of the involved growth factors VEGF-A is one of the most important proangiogenic proteins HighVEGF protein levels could be found in direct cocultures aswell as in monocultures of primary osteoblasts after bothcultivation time points (Figure 3(a)) After four weeks ofcultivation more VEGF-A could be found compared to oneweek in vitro Practically no VEGF-A could be detected inOEC monocultures This finding was confirmed by the dataof the indirect cocultures in which the different cell typeswere separated through a Transwell membrane Osteoblastsproduced significantly (lowast119875 lt 005) more VEGF-A thanOECsin indirect cocultures (Figure 3(a1015840)) Ang1 is an importantprotein involved in stabilizing and maturing vessels Ang1levels correlate with anatomically normal and nonleakyvessel structures Four-week direct cocultures containedmore Ang1 than one-week cocultures and monoculturesof pOBs and OECs (Figure 3(b)) Ang1 concentration inboth monocultures tended to decrease after four weeks ofcultivation compared to one week Lower levels of Ang1could be found in OEC monocultures and in OECs of theindirect cultures whereas pOBs produced high amountsof Ang1 (Figure 3(b1015840)) PDGF-BB is an important proteinduring pericyte chemotaxis It is released by ECs and attracts


(a) (b) (c)

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Akt1 1781 FGF2 6104 PGF 6968ANGPT2 37305 FGFR3 28705 PLAU 31175ANGPTL4 4051 HIF1A 4262 PLXDC 40845ANPEP 34175 ID1 39545 SERPIN 45425CCL11 53905 ID3 26685 SPHK1 1606CCL2 1257 IFNA1 72635 STAB1 2345CDH5 23585 IFNB1 2051 TGFB1 14345COL4A3 4395 IFNG 22695 TGFBR 6836CXCL1 2142 IL1B 58755 THBS1 1746CXCL3 63355 ITGAV 22775 THBS2 26205CXCL5 8175 ITGB3 2084 TIMP1 2998CXCL6 72845 KDR 1053 TIMP2 5281ECGF1 1444 LAMA5 4664 TIMP3 7212EDG1 719 LECT1 17375 TNF 36865EFNA1 5236 LEP 942 TNFAI2 1838EFNB2 54855 MDK 35285 VEGFA 5411ENG1 2212 MMP2 5197 VEGFC 6892EPHB4 36795 Notch4 354 COL18 220803EREG 35655 PDGFA 2227 FIGF 108783FGF1 4211 PECAM 19755 TGFB2 410711

Coculture 4 weeks Coculture 4 weeks Coculture 4 weeks

(h)

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0123456789

VEGF Ang1 Ang2

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101214161820

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(j)

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IGF-1 IGF-2


(k) (l)

Figure 1 Continued


(m) (n) (o)

Figure 1 Angiogenesis in cocultures OECpOB cocultures were cultivated for one week (a) and four weeks (b) and for four weeks (cd)mdashusing cryostat sectionsmdashrevealing several vessel-like angiogenic structures Cultures were stained with CD31 to detect endothelium andvessel wall OECmonocultures were cultivated for one week (e) and four weeks (f) revealing the absence of angiogenic structures A relevantupregulation of several genes associated with angiogenesis could be detected using a PCR array system (g h) The relative mRNA expressionof the most important growth factors involved in angiogenesis (indashk) was examined and compared after two time points It was shown that alonger cultivation time led to a better established microvessel system and an upregulation of growth factor mRNA One week values were setas control (=10) (lndasho) 3D-Matrigel angiogenesis assay comparing angiogenic behaviour OEC monoculture (L) with cocultures consisting ofpOB and OEC (mndasho) at different cultivation time points Scale bars (andashc) = 150 120583m (dndashf) = 75 120583m (lndasho) = 50 120583m 119899 = 3

1w1w

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ALPL 1213 COMP 3414 PHEX 18531 AMELY 84052 CTSK 6063 RUNX2 4102 ANXA5 2353 EGFR 6185 SERPIn 312 BGN 1226 FGF1 1525 SMAD1 7 BMP1 13635 FGF2 15657 SMAD3 7425 BMP4 200071 FN1 5165 SMAD4 130959 CALCR 2603 ICAM1 337 SOX9 28019 CD36 38723 IGF2 6664 STATH 20632 CDH11 26 ITGA3 4223 TGFB2 99978 COL10A1 22794 ITGB1 1233 TNF 11978 COL11A 58 MINPP1 14156 TWIST1 2542 COL12A 1207 MMP10 175517 VDR 37905 COL14A 32383 MMP2 4356 VEGFA 3254 COL3A1 1121 PDGFA 4134 VEGFB 13813


(e)

Figure 2 Calcification and osteogenesis (a) Cocultures (co) and monocultures (pOB and OEC) were compared using alizarin red staining(120583Mmg protein) at two different time points (one week versus four weeks) revealing a higher amount in the longer cultivated cocultures andpOBs ALP (b) BMP-2 and BMP-4 (c) mRNA expression was determined via quantitative real-time RT PCR in cocultures after a cultivationtime of one week and four weeks A relevant upregulation of several genes connected with osteogenesis could be detected using a PCR arraysystem (d e) It was shown that cocultures after four weeks express a higher amount of ALP BMP-2 and BMP-4 mRNA than cocultures afterone week One week values were set as control (=10) (lowast119875 lt 005) 119899 = 3


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Figure 3 Continued


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Figure 3 Protein concentration of growth factors contributing to angio- and osteogenesis Supernatants of direct cocultures monocultures(left) and four-week cultivated indirect cocultures (right) were collected and VEGF-A (aa1015840) Ang1 (bb1015840) PDGF-B (cc1015840) BMP-4 (dd1015840) andIGF-1 (ee1015840) protein concentrationsmeasured to give the distribution throughout the different culture typesThe indirect cocultures confirmedthe sources of the examined growth factors known from the literature (lowast119875 lt 005) 119899 = 3

pericytes and smooth muscle cells to establish a mural layerThe protein was detected inOECmonocultures (Figure 3(c))PDGF-BB concentration was higher after four weeks ofcultivation compared to one week In addition indirectcocultures confirmed that PDGF-BB is mainly producedby OECs (Figure 3(c1015840)) Neither pOB monocultures nordirect cocultures showed any protein concentration TGF-1205731 is a very important growth factor involved in differentcell activities including vessel structure maturation andosteogenic cell proliferation Unfortunately monoculturesand direct cocultures did not produce an appropriate proteinconcentration but indirect cocultures showed that pOBsreleased significantly more TGF-1205731 than OECs (lowast119875 lt 005)(data not shown) Furthermore the concentration of BMP-4 an important osteogenic growth factor was examined inthe supernatants of the different cell cultures (Figures 3(d)and 3(d1015840)) One-week and four-week cocultures and pOBmonocultures released high amounts of BMP-4 whereasOECs did not The indirect cocultures revealed that pOBsclearly produced more BMP-4 than OECs (lowast119875 lt 005)(Figure 3(d1015840)) In the end IGF-1 is a protein which hasmarked effects on nearly all cell types for example tissuerepair and EC enhancement The amount of IGF-1 washigher in supernatants of OEC monocultures compared tothe cocultures (Figure 3(e)) In addition IGF-1 concentra-tion decreased in coculture supernatants after four weekscompared to the one-week concentration Indirect coculturesshowed that OECs produced more IGF-1 than pOBs (lowast119875 lt005) (Figure 3(e1015840))

34 Direct Communication Connexin Expression in Cocul-tures of pOBs and OECs Connexins are either stored invesicles around the cell nucleus near to the ER or in theplasma membrane of the cell contributing to gap junc-tional intercellular communication Cx43 expression wasanalysed in cocultures forming angiogenic structures andstained to give red fluorescence (Figures 4(a)ndash4(d)) CD-31 was used as endothelial marker and Hoechst dye for

visualization of nuclei A monoclonal Cx43 antibody wasselected to reveal connexin localization In cocultures con-sisting of pOBs and OECs few perinuclear Cx43 spots weredetectable after one week of coculturing (Figure 4(a)) Afterfour weeks in coculture when angiogenic structures havebeen established many OECs showed typical perinuclearCx43 conglomerates (Figure 4(b)) In additionOECs showedCx43-positive spots at locations where two cells are adja-cent seen between the nuclei of two cells More matureand expanded microvessels appeared to contain even moregap junctional communication-positive areas in the plasmamembrane of the cells Large connexin conglomerates werelocated around several nuclei especially in locations whereOECs were merging to some kind of network Interestinglythere were also a number of nonendothelial cells whichcontainedCx43-positive areasThe relative gene expression ofthe three important connexin isoforms during angiogenesisand osteogenesis namely Cx37 Cx40 and Cx43 was alsoassessed in this study After 4 weeks of cocultivation anupregulation of all three tested connexin isoforms could bedetected Cx37 expression rose to a 4-fold level and Cx40and Cx43 to a 25-fold level (Figure 4(c)) Although statisticalsignificance was not achieved a clear trend could be detectedIndirect cocultures might provide the right platform todetermine the origin of the connexin isoforms and to obtainsome indication of the source of transcription In general itwas established that connexin mRNA expression is higher inpOBs than in OECs (Figure 4(d)) This difference is relevantin all three connexin isoforms tested Cx40 expression is 3-fold and Cx43 expression 35-fold higher in pOBs comparedto OECs

4 Discussion

Cocultures of outgrowth endothelial cells and primaryosteoblasts benefit immensely from each other regarding


1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)

1 week 1 week 1 week

4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)

Figure 4 Gap junctional communication in the coculture model system Several Cx43-positive areas could be detected in our coculturemodel system after a cultivation time of one week (a) and four weeks (b) Areas such as those shown were scaled up and presumptive gapjunctions were found where cells were adjacent A longer cultivation time led to an upregulation of relative connexin mRNA expression (c)and it seemed that pOBs expressed more connexin mRNA than did OECs (d) One week values (c) were set as control (=10) for coculturesand OEC values (d) were set as control (=10) to compare pOB and OEC monocultures Scale bars 75 120583m 119899 = 3

angiogenesis and osteogenesis After four weeks of cocul-turing extensive microvessel networks could be detectedcompared to a cultivation period of only one week Thepromoting effect of osteoblasts on the cellular organizationof endothelial cells into tube-like structures in the coculturesystem is in accordance with reports in the literature [28ndash31]Although the detailed mechanisms of the ldquocellular crosstalkrdquothat controls the angiogenic activation of endothelial cellsinduced by the coculture are still under investigation itseems that the coculturewith primary osteoblasts provides onthe one hand proangiogenic matrix components and on theother hand angiogenic growth factors to promote angiogenicactivation of OECs [20 32] To analyse the molecular basis ofthis angiogenic activation of OECs in the coculture system ahuman angiogenesis RT2 profiler array (PCR array) screeningthe expression of more than 80 genes involved in the processof angiogenesis was performed after one and four weeks ofcocultivation In total 60 angiogenesis-related genes wereupregulated after four weeks of cocultivation which was in

accordance with the increase in the formation of angiogenicstructures in a time-dependent manner as also reportedby Fuchs et al [32] The upregulated genes in long termcocultures of OECs and pOBs included growth factors theirreceptors chemokines cytokines matrix molecules andadhesionmolecules for example vascular endothelial growthfactor beta This could also be confirmed using quantitativereal time PCR

The proangiogenic growth factor vascular endothelialgrowth factor A (VEGF-A) for instance was clearly upreg-ulated after four weeks of cocultivation compared to controlcocultures (one week) VEGF-A known to be the mostimportant mediator of angiogenic activation has been pro-posed to be essential for the chemotaxis and differentiation ofendothelial progenitor cells including angioblasts as well asfor endothelial cell proliferation vascularization via integra-tion of activated endothelial cells into vessel-like structuresand also for remodelling of vascular structures [33ndash36] Itis mainly produced and secreted by nonendothelial cells


such as pOBs [25 37] The indirect cocultures confirmedthe osteoblastic origin of VEGF-A in the coculture modelsystem used in this study whereas OEC monocultures didnot show any VEGF-A secretion This is consistent withprevious studies and reports from the literature [37ndash39]The four-week protein concentration was even higher sothat expanding pOB monocultures are accompanied byincreased concentrations of VEGF-A Another upregulatedproangiogenic molecule after four weeks of cocultivation isangiopoietin 1 (Ang1) Ang1 is expressed and released byperivascular cells in the direction of the endothelium [40] Itscorresponding receptor Tie2 is mainly located on endothelialcells such as OECs Ang1 supports vessel formation andvessel wall stability and induces maturation of the growingmicrovessels [41ndash43] In accordance with the literature in thepresent study the Ang1 concentration in OEC monocultureswas very low whereas pOB monocultures showed a veryhigh Ang1 protein concentration The indirect coculturesunderline these results and show that considerablymoreAng1is produced in pOBs Ang1 mRNA was also upregulatedafter four weeks of cultivation compared to one weekindicating an increasing level of microvessel stabilizationand maturation Platelet-derived growth factor beta was alsoclearly upregulated in long-term cocultures The PDGF-BPDGFR120573 system provides maturation and stabilization ofsprouting microvessels PDGF-B is secreted by endothelialcells such as OECs and recruits perivascular cells whichexpress the PDGFR120573 in order to stimulate maturation of thevessel wall [44ndash47] Examining PDGF-B protein concentra-tion in our model system the indirect cocultures revealedthat PDGF-B production could only be detected in OECsOEC monocultures secreted only a relatively low PDGF-B amount whereas PDGF-B concentration increased afterfour weeks of cocultivation indicating that culture growthand development seem to correlate with PDGF-B releaseInterestingly protein concentration cannot be measured incocultures One explanation may be that cocultures need alarge amount of PDGF-B in order to stabilize newly formedmicrovessels so that all of the synthesized protein is used andbound to receptors immediately Another explanation mightbe that cocultures inhibit the production of PDGF-B inOECsRecent studies have already shown the importance of PDGF-B throughout bone tissue engineering Thus Hollinger etal have analysed the effect of PDGF-B and 120573-tricalciumphosphate in bone fractures and found a significant contri-bution to bone repair Apart from that recombinant PDGFhas already been approved for the repair of periodontaldefects [48] IGF-1 and IGF-2 are two additional growthfactors which are involved in angiogenesis [49] and tissuerepair [50] The connection to vessel formation becomeseven more understandable when considering the synergisticcollaboration of IGF-1 and VEGF [51] in tumour neoangio-genesis The proteins are reported to be secreted by bothosteoblasts and ECs [52] Indirect cocultures confirmed thisobservation Thus the pOB compartment contained an IGF-1 concentration of sim340 pgmL and the OEC compartmentsim650 pgmL (119875 lt 005) OECs seem to have a higherproduction rate of the protein which is also supported by

monoculture ELISAs IGF-1 concentration was clearly higherin OEC monocultures after one week of cultivation

Besides the effect of the coculture system on the angio-genic activation of OEC the osteogenic potential of thecoculture systemwas also of interest in the scope of this studyIn 2009 Fuchs et al analysed the dynamic processes involvedin the differentiation and functionality of both cell types inthe coculture system as a function of cultivation time [32]Due to the fact that different cell types have different demandsin terms of culture conditions they tested the influence ofthe cell culture medium on the osteogenic differentiation ofpOB and found an improved calcification of the coculturein endothelial cell growth medium (EGM-2) compared tocultivation in the osteogenic medium DMEM F-12 In addi-tion they described a time-dependent upregulation of theosteogenic factors osteocalcin and osteopontin in coculturesof pOBs and OECs seeded on silk fibroin thus indicating anon-going osteoblastic differentiation in the coculture system[53ndash55] To screen for effects of the coculture system ofpOBs and OECs on osteoblastic differentiation during thecourse of cocultivation a human osteogenesis RT2 PCRarray detecting the expression of 84 genes involved in orrelated to the process of osteogenic differentiation helpedto profile the coculture system in terms of osteogenesisMore than 40 osteogenesis-related genes were found to beupregulated in long term cocultures at four weeks BMPsare members of the TGF-120573 superfamily and contribute toosteogenic development in cells and tissues in vivo and invitro BMP-2 is involved in endothelial cell developmentand modulation of several important processes leading towell established vessel networks [56] BMP-4 induces ECproliferation and migration as well as expression and pro-tein synthesis of VEGFR2 and Tie2 [57] leading to wellperfused bone tissue It is reported that BMP-4 is producedby cells of the osteoblastic lineage and in fact indirectcocultures confirmed the osteoblastic origin of this growthfactor with statistical significance (119875 lt 005) This resultwas accompanied by very low BMP-4 concentrations in OECmonocultures both after one and four weeks of cultivationIn contrast pOB monocultures showed high concentrationsof BMP-4 in the supernatants after both cultivation timepoints As expected high concentration levels of the growthfactor could also be detected in direct pOBOEC coculturesIn addition ALP expression was slightly higher after acultivation time of four weeks although this did not reachstatistical significance This result reveals the osteoplasticityand osteogenic activity of the coculture model system usedALP is a well-known osteogenic marker protein becauseit is essential for the deposition of hydroxyapatite in thebone matrix High enzyme levels correlate with appropriateosteogenic activity [58ndash61] Similar results have been foundby Guillotin et al in 2004 combining human umbilicalvein endothelial cells (HUVECs) with human osteoprogenitorcells These authors demonstrated that ALP was upregulatedsignificantly in contrast to monocultures [62] The calcifi-cation level measured by the alizarin red staining protocolwas also affected positively by the coculture model systemthe alizarin red concentration increased and was higher in


the cocultures compared to the pOB monocultures at bothcultivation time points

Gap junctional communication is an essential part of cellcommunication in tissues and cell cultures It is conductedby connexins small plasma membrane proteins which formprotein tunnels so-called gap junctions connecting twoadjacent cells Using this bridge between cells several signalproteinsmetabolites or othermolecules can be exchanged [13 63] Each tissue contains its own combination of connexinisoforms In the vascular system which consists of ECs andperivascular cells the most abundant connexin isoform isCx43 followed by Cx40 and Cx37 Cx43Cx40 Cx40Cx40and Cx43Cx43 gap junctions contribute to gap junctionalcommunication in the vasculature Interestingly Cx43 seemsto be expressed mainly in perivascular cells whereas Cx40appears to be endothelium-specific In addition Cx37 is animportant connexin isoform appearing in both cell typesof the vascular system but a participation of Cx37 in gapjunctional communication has not been reported [7 9 10]In bone tissue the most abundant connexin isoform is Cx43[11] which could be confirmed by relative quantificationPCR testing the expression of Cx37 Cx40 and Cx43 inthe present coculture model system After four weeks ofcocultivation a relevant upregulation of connexin expressionoccurred compared to one-week cultivation time In thisrespect Guillotin et al showed in 2004 that the combinationof HUVECs and human osteoprogenitor cells resulted ina significant upregulation of Cx43 [62] Gap junctions andconnexins are essential for functional bone tissueMiron et alhave shown that Cx43 overexpression leads to mineralizationand osteoblastic differentiation enhancement whereas Cx43disruption results in declining levels of ALP expression andinsufficient mineralization [64] Soland and Lampe reportedthat large connexin conglomerates can be found in theperinuclear region where the endoplasmic reticulum lies [6]whereas established gap junctions are located in so-calledgap junctions plaques [5] in bright and punctuate regionsin locations where cells are close together [65] SelectingCx43 the typical perinuclear spots could be detected in thepresent coculture system thus correlating with the above-mentioned ER-based synthesis of the connexins In additioncocultures revealed several Cx43-positive areas in which cellswere adjacent This indicates that Cx43 plays a pivotal rolein gap junctional intercellular communication betweenOECsand pOBs Longer cultivation time increases this direct cell-cell communication

In conclusion this study reveals three key points (1)coculturing is a superior strategy in terms of vessel formationand bone repair Both angiogenesis and osteogenesis areimproved in the coculture system Secondly (2) angiogenesisand osteogenesis appear to be associated with rising levelsof growth factors and proteins of different origin Finally(3) the study reveals that gap junctional intercellular com-munication conducted by connexins plays a pivotal role inthe coculture system Communication regulation of cellularprocesses and culture development are impossible withoutconnexins They are important and obligatory mediatorsbetween cells throughout angiogenesis and osteogenesis Ingeneral this study has shown that the orchestra of growth

factors and intercellular communication is essential for suc-cessful cell culture development

Appendix

RT2 Profiler TM PCR Array Human Angiogenesis

Akt1 V-akt murine thymoma viral oncogene homo-log 1ANGPT2 Angiopoietin 2ANGPTL4 Angiopoietin-like 4ANPEP Alanyl aminopeptidaseCCL11 Chemokine ligand 11CCL2 Chemokine ligand 2CDH5 Cadherin5 VE-cadherinCOL4A3 Collagen type 4COL18 Collagen 18ACXCL1 Chemokine ligand 1CXCL3 Chemokine ligand 3CXCL5 Chemokine ligand 5CXCL6 Chemokine ligand 6ECGF1 Endothelial cell growth factor 1 (platelet-derived)EDG1 Endothelial differentiation sphingolipid G-protein-coupled receptor 1EFNA1 Ephrin A1EFNB2 Ephrin B2ENG1 EndoglinEPHB4 Ephrin receptor B4EREG EpiregulinFGF1 Fibroblast growth factor 1FGF2 Fibroblast growth factor 2FGFR3 Fibroblast growth factor receptor 3FIGF C-fos induced growth factor (vascular endo-thelial growth factor D)HIF1A Hypoxia inducible factor 1 alpha subunitID1 Inhibitor of DNA binding 1ID3 Inhibitor of DNA binding 2IFNA1 Interferon alpha 1IFNB1 Interferon beta 1IFNG Interferon gammaIL1b Interleukin 1 betaITGAV Integrin alpha VITGB3 Integrin beta 3KDR Kinase insert domain receptorLAMA5 Laminin alpha 5LECT1 Leukocyte cell derived chemotaxin 1


LEP LeptinMDK MidkineMMP2 Matrix metallopeptidase 2Notch4 Notch homologe 4PDGFA Platelet derived growth factor APECAM Platelet endothelial cell adhesion moleculePGF Placental growth factorPLAU Plasminogen activatorSTAB1 Stabilin 1TGFB1 Transforming growth factor beta 1TGFB2 Transforming growth factor beta 2TGFBR Transforming growth factor beta receptorTHBS1 Thrombospondin 1THBS2 Thrombospondin 2TIMP1 TIMP metallopeptidase inhibitor 1TIMP2 TIMP metallopeptidase inhibitor 2TIMP3 TIMP metallopeptidase inhibitor 3TNF Tumor necrosis factorTNFA12 Tumor necrosis factor alpha inducedprotein 2VEGFA Vascular endothelial growth factor

RT2 Profiler TM PCR Array Human Osteogenesis

ALPL Alkaline phosphataseAMELY AmelogeninANXA5 Annexin A5BGN BiglycanBMP4 Bone morphogenetic protein 4CALCR Calcitonin receptorCD36 Thrombospondin receptorCDH11 Cadherin 11COL10A Collagen 10ACOL11A Collagen 11ACOL12A Collagen 12ACOL14A Collagen 14ACOL3A1 Collagen 3ACOMP Cartilage oligomeric matrix proteinCTSK Cathepsin KEGFR Epidermal growth factor receptorFGF1 Fibroblast growth factor 1FGF2 Fibroblast growth factor 2FN1 Fibronectin 1ICAM1 Intercellular adhesion molecule 1IGF2 Insulin like growth factor 2

ITGA3 Integrin alpha 3

ITGB1 Integrin beta 1

MINPP Multiple inositol polyphosphate histidinephosphatase 1

MMP2 Matrix metallopeptidase 2

PDGFA Platelet derived growth factor A

PHEX Phosphate regulating endopeptidase homolog

RUNX Runt related transcription factor 2

SERPI Serpin peptidase inhibitor

SMAD1 Smad family member 1


SOX9 SRY sex determining region

STATH Statherin

TGFB2 Transforming growth factor beta 2

TNF Tumor necrosis factor

TWIST Twist homolog 1

VDR Vitamin D receptor

VEGFA Vascular endothelial growth factor A

VEGFB Vascular endothelial growth factor B

Abbreviations

ALK Activin receptor-like kinaseALP Alkaline phosphataseAng AngiopoietinATP Adenosine triphosphatebFGF Basic fibroblast growth factorBMP Bone morphogenetic proteinBSA Bovine serum albumincDNA Complementary deoxyribonucleic acidCD Cluster of differentiationCL Cytoplasmic loop domainCT Carboxyl-terminal domainCx43 Connexin isoform 43DMEM Dulbeccorsquos modified eagle mediumEC Endothelial cellEDTA Ethylene diamine tetra-acetic acideg Exempli gratiaEGM-2 Endothelial cell growth medium 2ELISA Enzyme-linked immunosorbent assayeNOS Endothelial nitric oxide synthaseEPC Endothelial progenitor cellER Endoplasmic reticulumFCS Fetal calf serumGJIC Gap junctional intercellular communicationHIF Hypoxia-inducible factorHUVEC Human umbilical vein endothelial cellIGF Insulin-like growth factorIL InterleukinIP3 Inositol trisphosphate


MSC Mesenchymal stem cellmRNA Messenger ribonucleic acidmTOR Mammalian target of rapamycinOEC Outgrowth endothelial cellPBS Phosphate buffered salinePCR Polymerase chain reaction

Conflict of Interests

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

Authorsrsquo Contribution

David Paul Eric Herzog and Eva Dohle contributed equallyto this work

Acknowledgments

The authors would like to thank B Pavic for her excellenttechnical assistance This work was financially supported bygrants from the University Medical Centre Mainz (MAIFOR9728901) and a BMBF-financed German-Chinese YoungInvestigator Group (Grant no 0315033) A portion of thework described herein was carried out by David Herzogin partial fulfilment of the requirements for a medicaldoctoral degree at the JohannesGutenbergUniversityMainzGermany

References

[1] N M Kumar and N B Gilula ldquoThe gap junction communica-tion channelrdquo Cell vol 84 no 3 pp 381ndash388 1996

[2] A L Harris ldquoEmerging issues of connexin channels biophysicsfills the gaprdquo Quarterly Reviews of Biophysics vol 34 no 3 pp325ndash472 2001

[3] D B Alexander and G S Goldberg ldquoTransfer of biologicallyimportant molecules between cells through gap junction chan-nelsrdquo Current Medicinal Chemistry vol 10 no 19 pp 2045ndash2058 2003

[4] V Valiunas Y Y Polosina H Miller et al ldquoConnexin-specificcell-to-cell transfer of short interfering RNA by gap junctionsrdquoJournal of Physiology vol 568 no 2 pp 459ndash468 2005

[5] D L D Caspar D A Goodenough L Makowski and WC Phillips ldquoGap junction structures I Correlated electronmicroscopy and X-ray diffractionrdquo Journal of Cell Biology vol74 no 2 pp 605ndash628 1977

[6] J L Solan and P D Lampe ldquoConnexin phosphorylation asa regulatory event linked to gap junction channel assemblyrdquoBiochimica et Biophysica Acta vol 1711 no 2 pp 154ndash163 2005

[7] H-I Yeh S Rothery E Dupont S R Coppen and N J SeversldquoIndividual gap junction plaques contain multiple connexins inarterial endotheliumrdquo Circulation Research vol 83 no 12 pp1248ndash1263 1998

[8] N J Severs S Rothery E Dupont et al ldquoImmunocytochemicalanalysis of connexin expression in the healthy and diseasedcardiovascular systemrdquoMicroscopy Research and Technique vol52 pp 301ndash322 2001

[9] J-A Haefliger P Nicod and P Meda ldquoContribution of con-nexins to the function of the vascular wallrdquo CardiovascularResearch vol 62 no 2 pp 345ndash356 2004

[10] B E Isakson and B R Duling ldquoHeterocellular contact at themyoendothelial junction influences gap junction organizationrdquoCirculation Research vol 97 no 1 pp 44ndash51 2005

[11] R Civitelli E C Beyer PMWarlowA J Robertson S TGeistand T H Steinberg ldquoConnexin43 mediates direct intercellularcommunication in human osteoblastic cell networksrdquo TheJournal of Clinical Investigation vol 91 no 5 pp 1888ndash18961993

[12] T H Steinberg R Civitelli S T Geist et al ldquoConnexin43and connexin45 form gap junctions with different molecularpermeabilities in osteoblastic cellsrdquo EMBO Journal vol 13 no4 pp 744ndash750 1994

[13] H J Donahue K J McLeod C T Rubin et al ldquoCell-to-cellcommunication in osteoblastic networks cell line-dependenthormonal regulation of gap junction functionrdquo Journal of Boneand Mineral Research vol 10 no 6 pp 881ndash889 1995

[14] J Ilvesaro K Vaananen and J Tuukkanen ldquoBone-resorbingosteoclasts contain gap-junctional connexin-43rdquo Journal ofBone and Mineral Research vol 15 no 5 pp 919ndash926 2000

[15] B Cheng S Zhao J Luo E Sprague L F Bonewald and J XJiang ldquoExpression of functional gap junctions and regulation byfluid flow in osteocyte-like MLO-Y4 cellsrdquo Journal of Bone andMineral Research vol 16 no 2 pp 249ndash259 2001

[16] R Langer and J P Vacanti ldquoTissue engineeringrdquo Science vol260 no 5110 pp 920ndash926 1993

[17] G F Muschler C Nakamoto and L G Griffith ldquoEngineeringprinciples of clinical cell-based tissue engineeringrdquoThe Journalof Bone and Joint Surgery American vol 86 no 7 pp 1541ndash15582004

[18] R M Nerem ldquoTissue engineering the hope the hype and thefuturerdquo Tissue Engineering vol 12 no 5 pp 1143ndash1150 2006

[19] D Narayan and S S Venkatraman ldquoEffect of pore size andinterpore distance on endothelial cell growth on polymersrdquoJournal of Biomedical Materials Research A vol 87 no 3 pp710ndash718 2008

[20] C J Kirkpatrick S Fuchs and R E Unger ldquoCo-culture systemsfor vascularizationmdashlearning from naturerdquo Advanced DrugDelivery Reviews vol 63 no 4 pp 291ndash299 2011

[21] J Hur C-H Yoon H-S Kim et al ldquoCharacterization of twotypes of endothelial progenitor cells and their different contri-butions to neovasculogenesisrdquoArteriosclerosisThrombosis andVascular Biology vol 24 no 2 pp 288ndash293 2004

[22] C-H Yoon J Hur K-W Park et al ldquoSynergistic neovascular-ization bymixed transplantation of early endothelial progenitorcells and late outgrowth endothelial cells the role of angiogeniccytokines and matrix metalloproteinasesrdquo Circulation vol 112no 11 pp 1618ndash1627 2005

[23] B R Shepherd D R Enis F Wang Y Suarez J S Pober and JS Schechner ldquoVascularization and engraftment of a human skinsubstitute using circulating progenitor cell-derived endothelialcellsrdquoThe FASEB Journal vol 20 no 10 pp E1124ndashE1132 2006

[24] E F Kung F Wang and J S Schechner ldquoIn vivo perfusionof human skin substitutes with microvessels formed by adultcirculating endothelial progenitor cellsrdquo Dermatologic Surgeryvol 34 no 2 pp 137ndash146 2008

[25] A Stahl A Wenger H Weber G B Stark H G Augustinand G Finkenzeller ldquoBi-directional cell contact-dependentregulation of gene expression between endothelial cells and


osteoblasts in a three-dimensional spheroidal coculturemodelrdquoBiochemical and Biophysical Research Communications vol 322no 2 pp 684ndash692 2004

[26] S Fuchs E Dohle M Kolbe and C J Kirkpatrick ldquoOut-growth endothelial cells sources characteristics and potentialapplications in tissue engineering and regenerative medicinerdquoAdvances in Biochemical EngineeringBiotechnology vol 123 pp201ndash217 2010

[27] A Hofmann L Konrad L Gotzen H Printz A Ramaswamyand C Hofmann ldquoBioengineered human bone tissue usingautogenous osteoblasts cultured on different biomatricesrdquo Jour-nal of BiomedicalMaterials Research A vol 67 no 1 pp 191ndash1992003

[28] J Rouwkema J de Boer andC A van Blitterswijk ldquoEndothelialcells assemble into a 3-dimensional prevascular network in abone tissue engineering constructrdquo Tissue Engineering vol 12no 9 pp 2685ndash2693 2006

[29] S Fuchs A Hofmann and C J Kirkpatrick ldquoMicrovessel-like structures from outgrowth endothelial cells from humanperipheral blood in 2-dimensional and 3-dimensional co-cultures with osteoblastic lineage cellsrdquo Tissue Engineering vol13 no 10 pp 2577ndash2588 2007

[30] R E Unger A Sartoris K Peters et al ldquoTissue-like self-assembly in cocultures of endothelial cells and osteoblasts andthe formation ofmicrocapillary-like structures on three-dimen-sional porous biomaterialsrdquo Biomaterials vol 28 no 27 pp3965ndash3976 2007

[31] M I Santos R E Unger R A Sousa R L Reis and CJ Kirkpatrick ldquoCrosstalk between osteoblasts and endothelialcells co-cultured on a polycaprolactone-starch scaffold and thein vitro development of vascularizationrdquo Biomaterials vol 30no 26 pp 4407ndash4415 2009

[32] S Fuchs X Jiang H Schmidt et al ldquoDynamic processesinvolved in the pre-vascularization of silk fibroin constructs forbone regeneration using outgrowth endothelial cellsrdquo Biomate-rials vol 30 no 7 pp 1329ndash1338 2009

[33] D W Leung G Cachianes W-J Kuang D V Goeddel andN Ferrara ldquoVascular endothelial growth factor is a secretedangiogenic mitogenrdquo Science vol 246 no 4935 pp 1306ndash13091989

[34] J Plouet J Schilling and D Gospodarowicz ldquoIsolation andcharacterization of a newly identified endothelial cell mitogenproduced by AtT-20 cellsrdquo EMBO Journal vol 8 no 12 pp3801ndash3806 1989

[35] F Shalaby J Rossant T P Yamaguchi et al ldquoFailure of blood-island formation and vasculogenesis in Flk-1 deficient micerdquoNature vol 376 no 6535 pp 62ndash66 1995

[36] R H Adams and K Alitalo ldquoMolecular regulation of angiogen-esis and lymphangiogenesisrdquo Nature Reviews Molecular CellBiology vol 8 no 6 pp 464ndash478 2007

[37] E Dohle S Fuchs M Kolbe A Hofmann H Schmidt andC J Kirkpatrick ldquoSonic hedgehog promotes angiogenesis andosteogenesis in a coculture systemconsisting of primary osteob-lasts and outgrowth endothelial cellsrdquo Tissue Engineering A vol16 no 4 pp 1235ndash1246 2010

[38] L Coultas K Chawengsaksophak and J Rossant ldquoEndothelialcells and VEGF in vascular developmentrdquo Nature vol 438 no7070 pp 937ndash945 2005

[39] A Horner S Bord A W Kelsall N Coleman and J ECompston ldquoTie2 ligands angiopoietin-1 and angiopoietin-2 arecoexpressed with vascular endothelial cell growth factor ingrowing human bonerdquo Bone vol 28 no 1 pp 65ndash71 2001

[40] S Davis T H Aldrich P F Jones et al ldquoIsolation of angio-poietin-1 a ligand for the TIE2 receptor by secretion-trapexpression cloningrdquo Cell vol 87 no 7 pp 1161ndash1169 1996

[41] K K Hirschi S A Rohovsky L H Beck S R Smith and PA DrsquoAmore ldquoEndothelial cells modulate the proliferation ofmural cell precursors via platelet-derived growth factor-BB andheterotypic cell contactrdquo Circulation Research vol 84 no 3 pp298ndash305 1999

[42] S P Oh T Seki K A Goss et al ldquoActivin receptor-like kinase1 modulates transforming growth factor-1205731 signaling in theregulation of angiogenesisrdquo Proceedings of the National Acad-emy of Sciences of the United States of America vol 97 no 6 pp2626ndash2631 2000

[43] T Nishishita and P C Lin ldquoAngiopoietin 1 PDGF-B and TGF-120573 gene regulation in endothelial cell and smooth muscle cellinteractionrdquo Journal of Cellular Biochemistry vol 91 no 3 pp584ndash593 2004

[44] M Hannink and D J Donoghue ldquoStructure and function ofplatelet-derived growth factor (PDGF) and related proteinsrdquoBiochimica et Biophysica Acta vol 989 no 1 pp 1ndash10 1989

[45] P Lindahl B R Johansson P Leveen and C Betsholtz ldquoPer-icyte loss and microaneurysm formation in PDGF-B-deficientmicerdquo Science vol 277 no 5323 pp 242ndash245 1997

[46] E A Winkler R D Bell and B V Zlokovic ldquoPericyte-specificexpression of PDGF beta receptor in mouse models withnormal and deficient PDGF beta receptor signalingrdquoMolecularNeurodegeneration vol 5 no 1 article 32 2010

[47] A Armulik G Genove and C Betsholtz ldquoPericytes develop-mental physiological and pathological perspectives problemsand promisesrdquo Developmental Cell vol 21 no 2 pp 193ndash2152011

[48] J O Hollinger C E Hart S N Hirsch S Lynch and GE Friedlaender ldquoRecombinant human platelet-derived growthfactor biology and clinical applicationsrdquo The Journal of Boneand Joint Surgery American vol 90 supplement 1 pp 48ndash542008

[49] P Madeddu ldquoTherapeutic angiogenesis and vasculogenesis fortissue regenerationrdquo Experimental Physiology vol 90 no 3 pp315ndash326 2005

[50] M H Gartner J D Benson and M D Caldwell ldquoInsulin-likegrowth factors I and II expression in the healingwoundrdquo Journalof Surgical Research vol 52 no 4 pp 389ndash394 1992

[51] R SWarrenHYuanMRMatli N Ferrara andDBDonnerldquoInduction of vascular endothelial growth factor by insulin-like growth factor 1 in colorectal carcinomardquo The Journal ofBiological Chemistry vol 271 no 46 pp 29483ndash29488 1996

[52] V Devescovi E Leonardi G Ciapetti and E Cenni ldquoGrowthfactors in bone repairrdquo La Chirurgia degli Organi di Movimentovol 92 no 3 pp 161ndash168 2008

[53] F Liu L Malaval A K Gupta and J E Aubin ldquoSimulta-neous detection of multiple bone-related mRNAs and proteinexpression during osteoblast differentiation polymerase chainreaction and immunocytochemical studies at the single celllevelrdquo Developmental Biology vol 166 no 1 pp 220ndash234 1994

[54] J E Aubin ldquoAdvances in the osteoblast lineagerdquo Biochemistryand Cell Biology vol 76 no 6 pp 899ndash910 1998

[55] J E Aubin ldquoRegulation of osteoblast formation and functionrdquoReviews in Endocrine and Metabolic Disorders vol 2 no 1 pp81ndash94 2001

[56] D Chen M Zhao and G R Mundy ldquoBone morphogeneticproteinsrdquo Growth Factors vol 22 no 4 pp 233ndash241 2004


[57] Y Suzuki K Montagne A Nishihara T Watabe and K Miya-zono ldquoBMPs promote proliferation and migration of endothe-lial cells via stimulation of VEGF-AVEGFR2 and angiopoietin-1Tie2 signallingrdquo Journal of Biochemistry vol 143 no 2 pp199ndash206 2008

[58] A Ascenzi and A Benvenuti ldquoOrientation of collagen fibers atthe boundary between two successive osteonic lamellae and itsmechanical interpretationrdquo Journal of Biomechanics vol 19 no6 pp 455ndash463 1986

[59] M P Whyte ldquoHypophosphatasia and the role of alkalinephosphatase in skeletal mineralizationrdquo Endocrine Reviews vol15 no 4 pp 439ndash461 1994

[60] W J Landis ldquoThe strength of a calcified tissue depends in parton the molecular structure and organization of its constituentmineral crystals in their organic matrixrdquo Bone vol 16 no 5 pp533ndash544 1995

[61] M-G Ascenzi J Gill and A Lomovtsev ldquoOrientation of colla-gen at the osteocyte lacunae in human secondary osteonsrdquoJournal of Biomechanics vol 41 no 16 pp 3426ndash3435 2008

[62] B Guillotin C Bourget M Remy-Zolgadri et al ldquoHumanprimary endothelial cells stimulate human osteoprogenitor celldifferentiationrdquo Cellular Physiology and Biochemistry vol 14no 4ndash6 pp 325ndash332 2004

[63] N M Kumar and N B Gilula ldquoMolecular biology and geneticsof gap junction channelsrdquo Seminars in Cell Biology vol 3 no 1pp 3ndash16 1992

[64] R JMiron EHedbom S Ruggiero et al ldquoPremature osteoblastclustering by enamel matrix proteins induces osteoblast dif-ferentiation through up-regulation of connexin 43 and N-cadherinrdquo PLoS ONE vol 6 no 8 Article ID e23375 2011

[65] F Villars B Guillotin T Amedee et al ldquoEffect of HUVECon human osteoprogenitor cell differentiation needs het-erotypic gap junction communicationrdquo American Journal ofPhysiologymdashCell Physiology vol 282 no 4 pp C775ndashC7852002

Submit your manuscripts athttpwwwhindawicom

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Enzyme Research



Microbiology


nontoxic products to accommodate the expanding tissueand controlled inflammation due to high biocompatibility[17ndash19] Several studies have shown that the combinationof cells of endothelial and osteogenic origin improves bothangiogenesis and osteogenesis [20] It has been controver-sially discussed which type of endothelial cell should beused for in vitro and in vivo experiments Late outgrowthendothelial cells (OECs) can be easily isolated from theperipheral blood mononuclear cell fraction and they possessa high proliferation capacity as well as a strong tendencyto form capillary tubes both in vitro and in vivo [21ndash24] Ithas already been shown that angiogenesis and osteogenesiscan be highly improved in a bidirectional way by combiningOECs with primary osteoblasts (pOBs) [25 26] makingOECs predestined for bone tissue engineering strategies

Angiogenesis and osteogenesis are prerequisites for theformation of a functional and perfused tissue engineeredconstruct Therefore involved growth factors and mediatorsduring these processes are very important In our coculturesystem essential growth factors were analysed in order togain better understanding of their contribution to angio-genesis and osteogenesis Well-known proangiogenic andosteogenic growth factors like vascular endothelial growthfactor A (VEGF-A) angiopoietins platelet-derived growthfactor B (PDGF-B) and transforming growth factor 120573 (TGF-120573) as well as insulin-like growth factors (IGFs) and bone mor-phogenetic proteins (BMPs) were examined during the courseof cocultivation by comparing two different cultivation timepoints 1 and 4weeks of cocultivation In addition by using anindirect coculture system consisting of pOBs and OECs thisstudy should also evaluate which cell type produces whichgrowth factor An additional focus of the study was to analysedirect cell-cell interaction via connexins and gap junctionsAs well as localizing gap junctions we hoped to establishhow connexins behave in the course of coculture growth anddevelopment

2 Materials and Methods

21 Isolation of Outgrowth Endothelial Cells (OEC) Out-growth endothelial cells are isolated from the mononu-clear cell fraction of peripheral blood buffy coats byficollhistopaque separation [26] which is used to separateblood into its components Therefore peripheral blood wasdiluted 1 2 in phosphate-buffered saline (PBS) containing05 fetal calf serum (FCS) and 2mM ethylene diaminetetra-acetic acid (EDTA) to prevent cells from clotting andsubsequently centrifuged for 35min at 400 g (without brak-ing) with histopaque placed at the bottom of a 50mL tubeAfter centrifugation 3 different components become visiblefrom the bottom-up erythrocytes mononuclear cells andplasmaThemononuclear cell fractionwas separated washedseveral times in PBS and cultured in endothelial cell growthmedium-2 (EGM-2) with supplements from the kit 5fetal calf serum and 1 penicillinstreptomycin on collagen-coated plates (35 120583gmL) A total number of 5 lowast 106 cellsper well were seeded on a 24-well plate After three to fourweeks the first OECs with their typical endothelial cell-like

cobblestone morphology and expansion capacity appearedin the culture OECs were then trypsinized and expandedon fibronectin-coated 24-well plates (10 120583gmL) over severalpassages at a splitting ratio 1 2 Passage numbers of OECsused for this study ranged from passage 8 to 18

22 Isolation of Human Primary Osteoblasts Human pri-mary osteoblasts (pOBs) were isolated from human bonefragments from healthy donors according to an establishedprotocol approved by the responsible Ethical Commission[27] Collagenase type IV at a concentration of 1mgmL wasadded severalwashing stepswith PBS followed and the enzy-matically digested bone fragments were subsequently placedon 6-well plates in Dulbeccorsquos modified Eagle medium con-taining 20 FCS and 1 penicillinstreptomycin (DMEM-Ham F12) During the first two to four weeks cells werefed every day until the plates reached subconfluence Thecells were then transferred to T75 culture flasks and culturedwith DMEM-Ham F12 containing only 10 FCS and 1penicillinstreptomycin Cells were passaged in a ratio 1 2using accutase The present study used cells from differentdonors up to the third passage

23 Coculture Consisting of Primary Osteoblasts and Out-growth Endothelial Cells Cocultures of OECs and pOBswere seeded on Thermanox coverslips (12mm in diameter)Primary osteoblasts were seeded first at a density of 300000cellswell in a fibronectin-coated 24-well plate followed byseeding of 200000 OECwell 24 hours later Cells werecocultivated in EGM-2 with supplements from the kitincluding low VEGF concentrations (2 ngmL according tothe manufacturer) 5 FCS and 1 penicillinstreptomycinfor different time periods For each coculture experimentat least three different donors were used Control pOB-and OEC-monocultures were cultivated in EGM-2 withsupplements from the kit in addition to 5 FCS and 1penicillinstreptomycin

24 Indirect Cocultures Seeded on Transwells The use ofa Transwell filter system allows different cell types in acoculture system to take up and secrete growth factors andsignalling molecules on both sides of a transmembrane filtertheir basal and their apical surface It is thus possible topromote themetabolic activities of these two cell types withina coculture system 100000 primary osteoblasts were seededat the lower surface and 66000 OECs were seeded at theupper surface of a polycarbonate transmembrane filter in aTranswell filter system in a 24-well plate (pore size 04 120583m0588 cm2filter) coated with fibronectin (10 120583gmL) to gaininsight into the origin of several growth factors producedby the different cell types in the coculture Cells were fedwith EGM-2 with supplements from the kit 5 FCS and1 penicillinstreptomycin and cultured for different timeperiods Culture supernatants as well as cell lysates of OECand pOB were collected separately from the Transwell filtersystem and then used for further experiments





2and










3 Results






(a) (b) (c)

(d) (e) (f)

0

10

20

30

40

50

60

70


Num

ber o

f gen

es

Coculture 4 weeks

(g)



(h)


4 weeks

4 weeks

0123456789

VEGF Ang1 Ang2

RQ


(i)

1 week 1 week

4 weeks

4 weeks

02468

101214161820

PDGF-BB

RQ

TGF-1205731


(j)

1 week 1 week

4 weeks

4 weeks

012345678

RQ

IGF-1 IGF-2


(k) (l)

Figure 1 Continued


(m) (n) (o)


1w1w

1w

4w

4w 4w

0

20

40

60

80

100

120

co pOB OEC

Aliz

arin

(120583M

)


(a)

1 week

4 weeks

0

05

1

15

2

25

3

35

ALP

RQ


(b)

1 week 1 week

4 weeks

4 weeks

0

1

2

3

4

5

6

Bmp-4Bmp-2

RQ


lowast

(c)

30

35

40

45

Num

ber o

f gen

es


Coculture 4 weeks

(d)



(e)



pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





2and










3 Results






(a) (b) (c)

(d) (e) (f)

0

10

20

30

40

50

60

70


Num

ber o

f gen

es

Coculture 4 weeks

(g)



(h)


4 weeks

4 weeks

0123456789

VEGF Ang1 Ang2

RQ


(i)

1 week 1 week

4 weeks

4 weeks

02468

101214161820

PDGF-BB

RQ

TGF-1205731


(j)

1 week 1 week

4 weeks

4 weeks

012345678

RQ

IGF-1 IGF-2


(k) (l)

Figure 1 Continued


(m) (n) (o)


1w1w

1w

4w

4w 4w

0

20

40

60

80

100

120

co pOB OEC

Aliz

arin

(120583M

)


(a)

1 week

4 weeks

0

05

1

15

2

25

3

35

ALP

RQ


(b)

1 week 1 week

4 weeks

4 weeks

0

1

2

3

4

5

6

Bmp-4Bmp-2

RQ


lowast

(c)

30

35

40

45

Num

ber o

f gen

es


Coculture 4 weeks

(d)



(e)



pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




3 Results






(a) (b) (c)

(d) (e) (f)

0

10

20

30

40

50

60

70


Num

ber o

f gen

es

Coculture 4 weeks

(g)



(h)


4 weeks

4 weeks

0123456789

VEGF Ang1 Ang2

RQ


(i)

1 week 1 week

4 weeks

4 weeks

02468

101214161820

PDGF-BB

RQ

TGF-1205731


(j)

1 week 1 week

4 weeks

4 weeks

012345678

RQ

IGF-1 IGF-2


(k) (l)

Figure 1 Continued


(m) (n) (o)


1w1w

1w

4w

4w 4w

0

20

40

60

80

100

120

co pOB OEC

Aliz

arin

(120583M

)


(a)

1 week

4 weeks

0

05

1

15

2

25

3

35

ALP

RQ


(b)

1 week 1 week

4 weeks

4 weeks

0

1

2

3

4

5

6

Bmp-4Bmp-2

RQ


lowast

(c)

30

35

40

45

Num

ber o

f gen

es


Coculture 4 weeks

(d)



(e)



pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)

0

10

20

30

40

50

60

70


Num

ber o

f gen

es

Coculture 4 weeks

(g)



(h)


4 weeks

4 weeks

0123456789

VEGF Ang1 Ang2

RQ


(i)

1 week 1 week

4 weeks

4 weeks

02468

101214161820

PDGF-BB

RQ

TGF-1205731


(j)

1 week 1 week

4 weeks

4 weeks

012345678

RQ

IGF-1 IGF-2


(k) (l)

Figure 1 Continued


(m) (n) (o)


1w1w

1w

4w

4w 4w

0

20

40

60

80

100

120

co pOB OEC

Aliz

arin

(120583M

)


(a)

1 week

4 weeks

0

05

1

15

2

25

3

35

ALP

RQ


(b)

1 week 1 week

4 weeks

4 weeks

0

1

2

3

4

5

6

Bmp-4Bmp-2

RQ


lowast

(c)

30

35

40

45

Num

ber o

f gen

es


Coculture 4 weeks

(d)



(e)



pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(m) (n) (o)


1w1w

1w

4w

4w 4w

0

20

40

60

80

100

120

co pOB OEC

Aliz

arin

(120583M

)


(a)

1 week

4 weeks

0

05

1

15

2

25

3

35

ALP

RQ


(b)

1 week 1 week

4 weeks

4 weeks

0

1

2

3

4

5

6

Bmp-4Bmp-2

RQ


lowast

(c)

30

35

40

45

Num

ber o

f gen

es


Coculture 4 weeks

(d)



(e)



pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


pOB

pOB

OEC OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

VEG

F (p

gm

L)

VEG

F (p

gm

L)

pOB

OEC

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

lowast

(a) (a998400)

(b)

pOB

pOBOEC

OEC

co

co

0

2000

4000

6000

8000

10000

12000

14000

1 week 4 weeks

pOB

OEC

0

10000

20000

30000

40000

50000

60000

Ang

1 (p

gm

L)

Ang

1 (p

gm

L)

(b998400)

pOB pOB

OEC

OEC

co co0

50

100

150

200

250

300

350

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(c998400)

PDG

F-BB

(pg

mL)

PDG

F-BB

(pg

mL)

(c)

pOBpOB

OECOEC

coco

0

200

400

600

800

1000

1200

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

(d) (d998400)

Bmp-4

(pg

mL)

Bmp-4

(pg

mL)

lowast

Figure 3 Continued


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


pOB pOB

OEC

OECco

co

0

500

1000

1500

2000

2500

3000

1 week 4 weeks

pOB

OEC

0

100

200

300

400

500

600

700

800

(e998400)

IGF-1

(pg

mL)

IGF-1

(pg

mL)

(e)

lowast





4 Discussion



1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 week

CD31 Cx43

(a)

4 weeks

CD31 Cx43

(b)


4 weeks

4 weeks

4 weeks

0

1

2

3

4

5

6

7

8

9

Cx37 Cx40 Cx43

RQ

(c)

OEC OEC OEC

pOB

pOB

pOB

0

1

2

3

4

5

6

7

Cx37 Cx40 Cx43

RQ

(d)














Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






Appendix


















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















STATH Statherin







Abbreviations








Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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