adhesion and proliferation of human periodontal ligament cells on

Research ArticleAdhesion and Proliferation of Human Periodontal LigamentCells on Poly(2-methoxyethyl acrylate)

Erika Kitakami1 Makiko Aoki1 Chikako Sato1 Hiroshi Ishihata2 and Masaru Tanaka1

1 Graduate School of Science and Engineering Yamagata University Jonan 4-3-16 Yonezawa Yamagata 992-8510 Japan2Graduate School of Dentistry Division of Periodontology and Endodontology Tohoku University Seiryo-machi 4-1Sendai Miyagi 980-8575 Japan

Correspondence should be addressed to Masaru Tanaka tanakayzyamagata-uacjp

Received 15 May 2014 Accepted 29 June 2014 Published 6 August 2014

Academic Editor David M Dohan Ehrenfest

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

Human periodontal ligament (PDL) cells obtained from extracted teeth are a potential cell source for tissue engineering Wepreviously reported that poly(2-methoxyethyl acrylate) (PMEA) is highly biocompatible with human blood cells In this studywe investigated the adhesion morphology and proliferation of PDL cells on PMEA and other types of polymers to design anappropriate scaffold for tissue engineering PDL cells adhered and proliferated on all investigated polymer surfaces except forpoly(2-hydroxyethyl methacrylate) and poly[(2-methacryloyloxyethyl phosphorylcholine)-co-(n-butyl methacrylate)] The initialadhesion of the PDL cells on PMEA was comparable with that on polyethylene terephthalate (PET) In addition the PDL cells onPMEA spread well and exhibited proliferation behavior similar to that observed on PET In contrast platelets hardly adhered toPMEA PMEA is therefore expected to be an excellent scaffold for tissue engineering and for culturing tissue-derived cells in ablood-rich environment

1 Introduction

Periodontal diseases caused by the bacterial biofilm canaffect up to 90 of adults worldwide [1] Severe periodontitisleads to losing connective tissue and bone support and finallylosing teeth [1] Therefore much research is being conductedon periodontal tissue regeneration for restoring the alveolarsupport of the teeth The periodontal ligament (PDL) is animportant structure that is composed of periodontal tissuein which PDL cells generate connective tissue fibers that spanthe gap between the cementum and the alveolar bone [2]to suspend the tooth This complex structure of PDL tissuecomprises several different cell populations [3] includingPDL cells which are predominantly fibroblasts and playcrucial roles in maintaining and regenerating periodontaltissue [2 3]

Human PDL cells obtained from extracted teeth are apromising cell source for periodontal tissue regenerationregenerative medicine and tissue engineering [3ndash6] becausethey include stem cells that have a high capacity for prolifera-tion self-renewal and multilineage differentiation and also

have the ability to form cementumperiodontal ligament-like tissue in vivo [3 4] Human PDL stem cells similar tobone marrow mesenchymal stem cells are able to suppressimmune responses and inflammatory reactions which sug-gests that PDL stem cells may be used in allogeneic stem cell-based therapies [5] In the first clinical application of humanautologous PDL-derived cells including PDL stem cellsthe transplanted cells were used to reconstruct periodontalintrabony defects in 3 patients and a significant improvementof periodontal disease was achieved which suggests thatPDL stem cell transplantation may be an efficacious and safealternative for the treatment of human periodontitis [6]

Furthermore human induced pluripotent stem cells (iPScells) have been generated from adult human periodontalligament fibroblasts [7] Nomura et al reported that thereprogramming efficiency of human PDL fibroblasts was0025 which is not lower than the reprogramming effi-ciency of dental stem cells even though the stem cells alreadyexpress a number of ES cell-associated genes thereforehuman PDL fibroblasts may be an optimal cell source forgenerating iPS cells [7] Additionally the use of PDL cells

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 102648 14 pageshttpdxdoiorg1011552014102648

2 BioMed Research International

is advantageous because it efficiently utilizes extracted teethHowever for periodontal tissue regeneration it is necessaryto culture PDL cells on a biocompatible scaffold to deliverthe cells to the wound site and also to provide space forthe formation of the new periodontal tissue Biomaterialscaffolds designed for tissue-engineered constructs mustaccommodate cell viability growth and function [8] Thereis increasing interest in designing new biomaterials for scaf-folds with minimal or no immune response that encouragestable implanttissue interaction [9] In addition the surfacecharacterization of biomaterials is important to design newimplantable materials [10ndash14]

We previously reported that poly(2-methoxyethyl acry-late) (PMEA) shows excellent biocompatibility with humanblood coagulation and complement systems and does notactivate leukocytes erythrocytes and platelets in vitro andex vivo relative to other polymer surfaces during the earlystages of immune reactions [15ndash19] On the basis of ourresults superior biocompatible catheters and oxygenatorscoated with PMEA were approved by the Food and DrugAdministration (FDA) and made available to the global mar-ket [16ndash19] We further determined why PMEA has excellentbiocompatibility [15 20ndash22] In particular the low extent ofplatelet adhesion and spreading observed was closely relatedto a low degree of denaturation and high dissociation rate forproteins adsorbed onto PMEA [15]

The objective of this study was to examine the hypothesisthat PMEA is a biocompatible polymer for tissue engineeringthat can facilitate adhesion and proliferation of PDL cells withlow platelet adhesion Cell-material interactions determinemany cellular processes such as adhesion spreading andproliferation and are thus essential for tissue engineering [23ndash27] However the influence of the chemical components ofsynthesized polymers on the biology of PDL cells remainsunclear To investigate PDL cell-material interactions wecharacterized the localization of focal adhesions which aremultifunctional organelles that mediate cell-material adhe-sion force transmission and cytoskeletal regulation and sig-naling [28] To analyze the formation of focal adhesions weevaluated the localization of vinculin which is a membrane-cytoskeletal protein present in focal adhesions that is involvedin the linkage of integrin adhesion molecules to the actincytoskeleton [29]

2 Material and Methods

21 Preparation of Polymer Surfaces PMEA was prepared byfree-radical polymerization using 221015840-azobisisobutyronitrile(Kanto Chemical Co Inc Japan) as the initiator and 2-methoxyethyl acrylate (MEA) as themonomerTheMEAwasobtained from Wako Pure Chemical Industries Ltd (OsakaJapan) poly(2-hydroxyethyl methacrylate) (PHEMA) wasobtained from Scientific Polymer Products Inc (OntarioNY) and poly[(2-methacryloyloxyethyl phosphorylcholine)-co-(n-butyl methacrylate)] (PMPC) was obtained fromNOF corporation (Tokyo Japan) The molecular weightof each polymer was estimated by gel permeation chro-matography using polystyrene standards The molecularweight (Mw) of PMEA PHEMA and PMPC was 85000

300000 and 600000 respectively The chemical structureof each polymer PMEA PHEMA and PMPC is shownin Figure 1 in Supplementary Material available onlineat httpdxdoiorg1011552014102648 and the polymerswere prepared on polyethylene terephthalate (PET filmT100E125 Mitsubishi Plastics Tokyo Japan 14mm diameterand 125 120583m thickness) using a spin coater (MS-A100 MikasaTokyo Japan) Exactly 40 120583L of a 02 wt solution of eachpolymer was cast twice onto the PET films Analytical grademethanol (Kanto Chemical Co Inc) was used as the solventfor each solution The surfaces of the polymer films wereanalyzed by X-ray photoelectron spectroscopy (XPS) (ESCA-1000 Shimadzu Kyoto Japan) to confirm the coverage of thecoated polymer The take-off angle was 45∘

For cell culture the films were sterilized by UV exposurefor 2 h Subsequently the films were soaked in mediumcomposed ofMinimumEssentialMedium (MEM-Alpha LifeTechnologies) containing 10 heat-inactivated fetal bovineserum (Equitech-Bio Inc Kerrville TX) and antibiotic solu-tion (100UmLpenicillinG sodium 100120583gmL streptomycinsulfate and 025 120583gmL amphotericin B Life Technologies)(culture medium) for one hour (preconditioning)

22 Characterization of Polymer Films We analyzed eachpolymer filmby atomic forcemicrocopy (AFMAgilent Tech-nologies 5550 Scanning Probe Microscope Agilent Tech-nologies Inc Santa Clara CA) The maximum scan rangewas approximately 10 120583m times 10 120583m using a cantilever with aforce constant of 21ndash78Nm resonance frequency of 250ndash390 kHz and tip height of 10ndash15 120583m (NCH-10 Nano WorldZurich Switzerland) AFMwas performed in air acoustic ACmode AFM image analysis was performed using Pico ImageSoftware (Agilent Technologies)

The wettability of the polymer surfaces was characterizedby contact angle measurement [15] The static contact angleon each polymer surface was measured using the sessile dropmethod at room temperature For the sessile drop method2 120583L of deionized water was dropped on a dried polymer filmusing a microsyringe The static contact angle was observed30 s later under a microscope (G-1-1000 ERMA Inc TokyoJapan) After at least five readings which were obtainedfor different areas of the polymer the measurements wereaveraged to arrive at a final contact angle (119899 = 6)

23 Cell Preparation and Culture Primary PDL cells wereobtained as previously reported [30] Fibroblast-like PDLcells were derived from the periodontal ligament of humanthird molars extracted from healthy individuals aged 17ndash21years who had no clinical signs of chronic periodontal dis-ease Informed consent was obtained prior to each extractionThe cells were obtained from the Dental Faculty of TohokuUniversity Periodontal ligament tissues were dissected intosmall pieces from the midportion of the root with a sharpbladeThe pieces were then cultivated in tissue culture dishes(Asahi Glass Co LTD Tokyo Japan) until the formation ofa confluent cell monolayer using culture medium After con-fluence was achieved the cells were washed with phosphate-buffered saline (PBS Takara Bio Inc Shiga Japan) andresuspended with 0075 gL protease and 01 gL EDTA to

BioMed Research International 3

enable passage These experiments were approved by theEthics Committee of the Dental Faculty of TohokuUniversityand the Graduate School of Science and Engineering ofYamagata University Japan

We used PDL cells at passages six and eight for adhesionand proliferation assays PET PMEA PHEMA and PMPCfilms were put in 24-well polystyrene plates (Asahi Glass CoLTD) After preconditioning of these films PDL cells wereseeded at 1 x 104 cellscm2 onto the tested films and grownfor up to 1 hour (1 h) 1 day 3 days and 7 days using culturemedium During culturing the cells were maintained at 37∘Cin 5 CO

2and 95 air and the medium was changed every

three days The progression of the cultures was examined byusing phase contrast microscopy (CKX41 Olympus TokyoJapan)

24 Immunofluorescence Staining The adhesion prolifera-tion and focal adhesion formation of the cultured PDLcells were observed by confocal laser scanning microscopy(CLSM FV-1000 Olympus) To visualize cell adhesionspreading proliferation and focal adhesion formation on thepolymers staining of vinculin actin fibers and cell nuclei wasperformed After culture for the indicated period the cellswere washed with PBS twice After washing the cells werefixed with PBS containing 4 paraformaldehyde obtainedfrom Wako Pure Chemical Industries Ltd (Osaka Japan)for 10min at 37∘C and washed again three times with PBSSubsequently the cells were permeated three times with 1Triton-X-100 (MP Biomedicals LLC Solon OH) in PBS for10min at room temperature and then immersed in 002Tween-PBS (MP Biomedicals LLC) three times for 10mineach To assess PDL cell-material interactions PDL cellson polymers were stained for vinculin which is localizedat focal adhesions using a mouse antivinculin monoclonalantibody (Millipore Temecula CA) as a primary antibodyfor 1 h followed by treatment with Alexa Fluor 546 goatanti-mouse IgG (Life Technologies as a secondary antibodyfor 1 h For actin staining the samples were incubated withAlexa Fluor 488 phalloidin (Life Technologies) for 1 h Fordetection of Ki-67 antigen as a cell proliferation markerthe samples were treated with rabbit anti-Ki-67 antigenmonoclonal antibody (Life Technologies) for 2 hThe sampleswere then incubated with Alexa Fluor 488 goat anti-rabbitantibody (Life Technologies) for 1 h (119899 = 9) The stained cellswere rinsed three timeswith PBS and subsequently immersedin PBS for 10min All specimens were placed on glass slidesmounted by using ProLong Gold antifade regent with DAPI(Life Technologies) and covered with glass cover slips

The specimens were imaged by CLSM and cell mor-phology parameters were quantified by Olympus FluoviewsoftwareThe total number of adherent cells on polymer filmswas counted in five randomly selected CLSM images (119899 = 3)

25 Scanning Electron Microscopy To assess the morphol-ogy of adherent PDL cells cultured on each polymer for1 h the cells were observed by field emission scanningelectron microscopy (SEM SU-8000 Hitachi Ltd TokyoJapan) Cultured cells were fixed with 25 glutaraldehyde(Polysciences Inc Warrington PA) in PBS and incubated

overnight at 4∘C They were next washed three times withPBS and thenwith purewater and subsequently air-driedThedried samples were coated with carbon using an ion sputtercoater (HPC-1SW Vacuum Device Inc Ibaraki Japan)

26 Platelet Adhesion Test To investigate the number ofplatelets adhering to the polymers blood was drawn from 3healthy volunteers (nonsmokers age 22 male age 33 femaleand age 42male) andmixedwith a 19 volume of 32 sodiumcitrate Platelet-rich plasma (PRP) and platelet-poor plasma(PPP) were obtained by centrifugation of citrated blood at1500 rpm for 5min and 4000 rpm for 10min respectivelyPlasma containing 3-4 times 107 cellscm2 was prepared bymixing PRPwith PPPThen 200120583L of the platelet suspensionwas placed on each polymer surface and incubated for 1 h at37∘C After the films were washed three times with PBS theywere immersed in 1 glutaraldehyde in PBS for 120min at4∘C to fix the adhered platelets The samples were dried andsputter-coated in platinum-palladium using an ion sputtercoater prior to SEM (JSM-7600FA JEOL Ltd Tokyo Japan)The number of adherent platelets on the polymer films wascounted in five randomly selected SEM images (119899 = 6)

27 Data Analyses The results were analyzed using Studentrsquos119905-test 119875 lt 005 was used as the threshold for statisticalsignificance between groups

3 Results and Discussion

31 Characterization of Polymer Films The surface rough-ness of each polymer film was analyzed by AFM (Table 1)The AFM topographical values (root mean squared RMS)for PET PMEA PHEMA and PMPC were 10 67 58and 65 nm respectively The polymer-coated films weresmoother than the PET film

Figure 1(a) shows the XPS spectrum of the PET filmcoated nitrogen-modified PET film (Figures 1(a)ndash1(c) lineA) C 1s and O 1s peaks derived from PMEA were observedwhereas an N 1s peak for the nitrogen-modified PET filmwasnot observed (Figure 1(a) line B) The XPS spectrum of thecoated PHEMA also showed the same result (Figure 1(b))The XPS spectra of coated PMPC also did not show an N 1speak but showed a P 2s peak (Figure 1(c) line B)These resultsindicate that the PET film surface was completely coveredwith each polymer

The static contact angle (120579) on each polymer surface wasmeasured by the sessile dropmethod as shown inTable 2The120579 values determined by the sessile dropmethod for deionizedwater were 69∘ plusmn 26∘ 45∘ plusmn 22∘ 36∘ plusmn 27∘ and 105∘ plusmn 33∘ onPET PMEA PHEMA and PMPC respectively These dataindicate that the hydrophilicity of PMEA is between that ofPET and PHEMA The static contact angle of each polymerwas consistent with values in the literature [31 32]

32 Morphology of PDL Cells on Polymer Surfaces Cellmorphology and proliferation behavior observed by CLSMdemonstrated that PDL cells adhered to PMEA and otherpolymer surfaces except PMPC with a round shape within1 h (Figure 2) After 1 day PDL cells had spread across the


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Figure 1 XPS spectrum of the PET film surface coated with PMEA (a) PHEMA (b) and PMPC (c) (A) indicates the XPS spectrum of thePET film surface (B) indicates the XPS spectrum of the coated film The atomic compositions determined from the XPS spectra match theexpected composition based on the structure of each polymer

Table 1 AFM topographical data RMS root mean squared rough-ness Scan size 10 times 10120583m2

Polymer RMS (nm)PET 10PMEA 67PHEMA 58PMPC 65

polymers After 3 days PDL cells had spread further andshowed proliferation behavior on all polymers After 7 daysPDL cells on PET and PMEA were confluent and developedactin fibers were observed PDL cells on PHEMA were notconfluent but had aggregated PDL cells did not adhere andproliferate on PMPC throughout the experiment

33 Initial Adhesion of PDLCells Thenuclei of adherent PDLcells on each polymer surface were counted under CLSMPDL cells adhered to PMEA and the other polymer surfacesexcept for PMPC upon incubation for 1 h (Figure 3(a)) Thenumber of adherent PDL cells on PMEAwas almost identicalto that on PET and was 5 times higher than that on PHEMA

The cell morphology observed by SEM showed differ-ences for each polymer after 1 h (Figure 3(b)) PDL cells

Table 2 Static contact angles of polymer surfaces

Polymer Sessile drop (degrees) (plusmnSD)PET 692 (plusmn26)PMEA 450 (plusmn22)PHEMA 360 (plusmn27)PMPC 1052 (plusmn33)

on PET contained some pseudopodia (white circle) andspikes (white arrow) PDL cells on PMEA contained somepseudopodia (white circle) and many spikes (white arrows)PDL cells on PHEMAcontained lamellipodiaThe surfaces ofadherent PDL cells adherent to PET and PMEAwere rougherthan those on PHEMA

34 Quantification of Projected Area Perimeter and LongAxis of PDL Cells The morphology of the adherent PDLcells was quantified using CLSM images (Figure 4(a))Figure 4(b) shows the projected cell area on each polymerAfter 1 h the projected cell areas were 270 plusmn 100 120583m2 230 plusmn90 120583m2 and 270 plusmn 110 120583m2 on PET PMEA and PHEMArespectively After 1 day the projected cell areas were 1390 plusmn660 120583m2 1770plusmn610 120583m2 and 690plusmn360 120583m2 on PET PMEAand PHEMA respectively The projected cell areas increased


1h Day 1 Day 7PE

TPM

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EMA

Day 3

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Figure 2 CLSM images of PDL cells cultured on polymer surfaces Scale bars 300 120583m Blue nucleus green actin and red vinculin Timepoints are 1 h 1 day 3 days and 7 days Polymers PET PMEA PHEMA and PMPC

by 51- 77- and 26-fold on PET PMEA and PHEMArespectively from 1 h up to 1 day

Figure 4(c) shows the perimeter of adherent PDL cellson each polymer After 1 h the perimeters were 75 plusmn 15 120583m80 plusmn 17 120583m and 82 plusmn 27 120583m on PET PMEA and PHEMArespectively After 1 day the perimeters were 300 plusmn 85 120583m380plusmn130 120583m and 180plusmn80 120583mon PET PMEA and PHEMArespectively The perimeter of the adherent cells increasedby 40- 48- and 22-fold on PET PMEA and PHEMArespectively from 1 h up to 1 day

Figure 4(d) shows each axis of the adherent PDL cellson each polymer After 1 h the long axes (ie length) were22 plusmn 5 120583m 22 plusmn 7 120583m and 22 plusmn 8 120583m on PET PMEA and

PHEMA respectively After 1 day the lengths were 85 plusmn37 120583m 110 plusmn 44 120583m and 50 plusmn 33 120583m on PET PMEA andPHEMA respectively The length of adherent cells increasedby 39- 50- and 23-fold on PET PMEA and PHEMArespectively from 1 h up to 1 day After 1 h the short axes (iewidth) were 14 plusmn 5 120583m 9 plusmn 2 120583m and 13 plusmn 2120583m on PETPMEA and PHEMA respectively After 1 day the widthswere 21plusmn 10 120583m 24plusmn 9120583m and 18plusmn 6120583monPET PMEA andPHEMA respectively The width of adherent cells increasedby 15- 27- and 14-fold on PET PMEA and PHEMArespectively from 1 h up to 1 day Figures 4(a)ndash4(d) show thatPDL cells on PMEAweremore spread than cells on any otherpolymer surface


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Figure 3 Initial adhesion of PDL cells on polymer surfaces after 1 h (a) The number of adherent PDL cells on polymer surfaces PolymersPET PMEA PHEMA and PMPC lowastlowast119875 lt 001 versus PMEA mean plusmn standard deviation 119899 = 3 (b) SEM images of PDL cells on polymersurfaces Top scale bars 10 120583m bottom scale bars 10 120583m Polymers PET PMEA and PHEMAWhite circles indicate pseudopodia formationWhite arrows indicate spike formation

35 Proliferation of PDL Cells PDL cells adhered and pro-liferated on all polymer surfaces except for PMPC duringthe culture period (Figure 5) After 1 day and 3 days thenumber of adherent PDL cells was almost identical on all ofthe polymer surfaces After 7 days the number of PDL cellson PMEAwas almost the same as that on PETThe number ofPDL cells onPHEMAwas lower than that onPET andPMEA

Figures 6(a)ndash6(c) show the percentage of Ki-67-positivecells during the culture period Ki-67-positive cells (prolif-erating cells Figures 6(d)-6(e)) were categorized into typesI and II Type I showed stronger staining while type IIshowed weaker staining Ki-67-negative cells (quiescent orresting cells) were categorized as type III (Figures 6(d)-6(e))PDL cells did not show a statistically significant differencebetween 1 day (Figure 6(a)) and 7 days (Figure 6(c)) After

3 days only type II adherent PDL cells on PMEA showed astatistically significant difference relative to cells on PHEMA(Figure 6(b)) which indicates that the number of proliferat-ing PDL cells on PMEA was higher than that on PHEMA

36 Localization of Vinculin in Two- and Three-DimensionalObservation Figures 7(a)ndash7(c) show a top view and crosssections of adherent cells We classified the localization ofvinculin into 4 types (i) focal adhesions (FAs) and (ii)nonfocal adhesions (non-FAs) where FAs were localized atbasal cell surfaces and non-FAs were localized at apical cellsurfaces (iii) vinculin rods that were composed of a complexof FAs and non-FAs and that were connected vertically andpenetrated the cells and (iv) vinculin fibers that were mainlyoriented along the long axis of the cells


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Figure 4 Quantification of adherent PDL cell morphologies for 1 h and 1 day (a) CLSM images for the quantification of adherent PDL cellmorphology on polymer surfaces Scale bars 100 120583m Blue nucleus green actin and red vinculin Polymers PET PMEA and PHEMA (b)Projected cell area (c) Perimeter of adherent PDL cells (d) Long and short axes of adherent PDL cells Polymers PET PMEA and PHEMAlowastlowast119875 lt 001 and lowast119875 lt 005 versus PMEA mean plusmn standard deviation 119899 = 10


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Figure 5 The number of PDL cells on polymer surfaces Timepoints are 1 day 3 days and 7 days Polymers PET PMEA PHEMAand PMPC lowastlowast119875 lt 001 and lowast119875 lt 005 versus PMEA mean plusmnstandard deviation 119899 = 3

Figure 7(a) shows PDL cells cultured on PET After1 h adherent PDL cells were shaped similarly to gourds(Figure 7(a)) The cells were spherical with a thickness ofapproximately 15 120583m and their actin and vinculin wereundeveloped After 4 h the cells had spread to form disc-like shapes and FAs Large spots of actin and vinculin werelocalized at the apical cell surfaces The thickness of theadherent PDL cells was approximately 10 120583m Vinculin wasmainly localized at basal cell surfaces in FAs and apicalcell surfaces in non-FAs After 1 day the cells were spreadout thinly Large vinculin spots were localized at apical cellsurfaces in non-FAs and FAs were also observed in additionto vinculin rods After 3 days the cells were spread out morethinly Many vinculin rods were observed FAs non-FAs andvinculin fibers were also observed

Figure 7(b) shows PDL cells cultured on PMEA After1 h the adherent PDL cells were shaped similarly to gourdsThe cells were spherical with a thickness of approximately11 120583m Their actin and vinculin were undeveloped After 4 hPDL cells had spread to form disc-like shapes and FAs Largeactin spots and small vinculin spots were localized at theapical cell surfaces in non-FA The thickness of the cells wasapproximately 10 120583m Vinculin was mainly localized at basalcell surfaces in FAs and apical cell surfaces in non-FAs After1 day the cells were spread out and spindle shaped Largevinculin spots were localized at apical cell surfaces in non-FAs and FAs were also observed in addition to vinculinfibers After 3 days the cells were spread out more thinlyFAs and non-FAswere observed and vinculin fibers were alsoobserved

Figure 7(c) shows PDL cells cultured on PHEMA After1 h the adherent cells were spherical with a thickness ofapproximately 15 120583mTheir actin and vinculin were undevel-oped After 4 h the cells were slightly spread out and theypossessed FAs After 1 day the cells had a round shape relative

to the shape of cells on the other polymers (Figures 7(a)and 7(b)) Large vinculin spots were localized at apicalcell surfaces in non-FAs FAs and vinculin rods were alsoobserved After 3 days the PDL cells had spread out Vinculinrods were also observed FAs and non-FAs were observedin addition to vinculin fibers The vinculin fiber-formationprocess is summarized in Figure 7(d) PDL cells on PMEAcontained few vinculin rods whereas PDL cells on PET andPHEMA contained many vinculin rods

361 Relationship betweenCell Proliferation andKi-67 ProteinProduction As shown in Figures 2 3(a)-3(b) 4(a)ndash4(d)and 5 PDL cells on PMEA showed similar adhesion andproliferation behavior to cells on PET at all-time pointsThe low proliferation on PHEMA was consistent with theresults of Peluso et al who found that human embryoniclung fibroblasts did not proliferate on PHEMA [33] Basedon this finding we focused on differences in the cell cycleand quantified the percentage of Ki-67-positive cells on thepolymers to identify differences in cell proliferation Asshown in Figure 6(b) the percentage of Ki-67-positive cellson PMEA at 3 days was higher than that on PHEMA Thesedata suggest that the higher proliferation of PDL cells onPMEA was related to the higher percentage of Ki-67-positivecells at 3 days (Figures 5 and 6(b))

362 Relationship between Vinculin Localization and CellProliferation Next we analyzed the localization of vinculinin 2 and 3 dimensions to investigate the cause of differencein the proportion of Ki-67-positive cells and to analyze cell-material interactions Our results suggest that PDL cells onPMEA have stronger PDL cell-material interactions thancells on PHEMA because cells on PMEA exhibited highvinculin localization and Ki-67 protein production In ournext study we will attempt to elucidate the influence of thechemical structure of the synthetic polymer on cell behaviorby altering the composition of the main chain andor theterminal functional group of the side chain

As shown in Figures 7(a)ndash7(d) we observed some uniquevinculin localization in non-FAs vinculin rods and vinculinfibers Kanchanawong et al reported that focal adhesionslink the extracellular matrix to the actin cytoskeleton andvinculin localized in focal adhesions probably links integrinto actin directly as the distribution of vinculin is consistentwith its binding to sites along the talin rod domain and actinwhich may serve to buttress the integrin-talin-actin linkage[29] Therefore vinculin localization was expected to occurat basal cell surfaces contacting the materials however weobserved that vinculin localized in non-FAs at apical cell sur-faces and in vinculin rods that were composed of a complexof FAs and non-FAs that were connected vertically and pene-trated the cells (Figures 7(a)ndash7(d)) In addition we observedthat vinculin fibers appeared to be similar to the supermat-uration of focal adhesions reported by Dugina et al whoshowed that increased extradomain A fibronectin expres-sion induced by transforming growth factor 120573 (TGF120573) wasaccompanied by 120572-smooth muscle actin expression and focaladhesion supermaturation in fibroblasts [34] The non-FAsprobably link extracellularmatrix proteins such as fibronectin


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(e)

Figure 6 Percentage of Ki-67-positive cells on polymer surfaces (a) 1 day (b) 3 days and (c) 7 days Polymers PET PMEA and PHEMAlowastlowast119875 lt 001 and lowast119875 lt 005 versus PMEA mean plusmn standard deviation 119899 = 9 (d)-(e) Classification of Ki-67 staining The images show Ki-67

staining of PDL cells on PHEMA after 3 days Scale bars 100 120583m Blue nucleus green Ki-67-positive cells (d) CLSM images of PDL cellsshowing the nucleus and Ki-67 staining (e) CLSM images of PDL cells showing Ki-67 staining Types I and II the Ki-67 antigen is presentin the nucleus during the G1 S and G2 phases of cell division and during mitosis Type III quiescent or resting cells in the G0 phase do notexpress the Ki-67 antigen

with the apical cell surface In future studies we will evaluatethe role of the unique vinculin localization in non-FAsvinculin rods and vinculin fibers with regard to cell behavior

37 Comparison of Platelet and PDL Cell Adhesion on PMEAand PMPC Figure 8(a) shows the adherent platelets oneach polymer as determined by SEM Figure 8(b) showsthe number of adherent platelets and PDL cells on eachpolymer relative to that on PET Platelet adhesion on PMEAand PMPC was low and the number of platelets on PMEAwas not significantly different from that on PMPC Manyadherent PDL cells were observed on PMEA and PETwhereas relatively few adherent PDL cells were observed onPHEMA and PMPC PDL cells adhered to PMEA whereasplatelets hardly adhered to PMEA In contrast both plateletsand PDL cells did not adhere to PMPC

371 Relationship between Biocompatibility and Cell Adhe-sions Conventional synthetic biocompatible polymers suchas PMPC poly(sulfobetaine methacrylate) poly(carboxybe-taine methacrylate) and poly(ethylene glycol) (PEG) areknown to demonstrate low protein adsorption andor no

platelet adhesion [35ndash39] PDL cells hardly adhered and pro-liferated on PMPC which was consistent with the findings ofa previous study by Iwasaki et al who reported that adhesionof human promyelocytic leukemia cells and human uterinecervical cancer cells was completely suppressed on PMPC[36]We previously reported that we previously reported thatPMEA has excellent biocompatibility with human blood cells[15] As shown in Figures 8(a)-8(b) the number of plateletson PMEA was not significantly different from the number ofplatelets onPMPC In contrast PDL cells adhered to PMEAand the number of PDL cells on PMEA was higher than thaton PHEMAandPMPC (Figure 8(b))These findings raise thequestion ofwhyPDL cells adhere to biocompatible PMEAbutplatelets do not especially when both platelets and PDL cellsdo not adhere to PMPC in a limited manner Although wehave no clear evidence to answer this question or to explainwhy PDL cells demonstrate higher growth rates on PMEAwe can offer the following speculations in terms of the 3 stepsrequired for cell adhesion on polymer surfaces

372 Relationship between Adsorbed Proteins and Cell Adhe-sion Initially when a polymer surface comes in contact with


4 h

Day

1Top view Cross section

1 h

Day

3

(a)

Top view Cross section

1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)

PETPHEMA(i) Vinculin spot

FA andor non-FA

(ii) Vinculin rod

PMEA(i) Vinculin spot

FA andor non-FA

(iii) Vinculin fiberFA + FA or

FA + non-FA

(iii) Vinculin fiber

FA + non-FAFA + FA or

FA + non-FA

(d)

Figure 7 Localization of nucleus actin and vinculin in adherent PDL cells on polymer surfaces Scale bars 10120583m In the cross-sectionimages the top panel shows the nucleus (blue) the second panel shows the nucleus and actin (green) the third panel shows the nucleus andvinculin (red) and the bottom panel shows a merged imageThe time points are 1 h 4 h 1 day and 3 days (a) PDL cells on PET (b) PDL cellson PMEA (c) PDL cells on PHEMAWhite arrows indicate focal adhesions that were localized at the basal cell surface Yellow arrows indicatenonfocal adhesions (non-FA) localized at the apical cell surface White arrowheads indicate vinculin rods that were connected vertically andpenetrated the adherent cell White circles indicate vinculin fibers that were mainly oriented along the long axis of the adherent cells (d)Schematic representation of vinculin fiber formation

cell culture medium it absorbs water and a specific waterstructure is formed on the polymer surface [21] On PETor PHEMA the absorbed water creates a nonfreezing waterlayer and a free water layerWe have also reported that PMEAand PMPC form another layer called the intermediate waterlayer [20 23 40]

Proteins in the cell culture medium then adsorb to thewater layer When proteins adsorb to the nonfreezing waterlayer on polymer surfaces such as PET and PHEMA a strong

conformational change of the adsorbed proteins occurs andmany cell-binding sites are exposed Intermediate water inPMEA does not induce a conformational change in theadsorbed proteins (bovine serum albumin and fibrinogen)and thus potential platelet-binding sites are minimallyexposed [15 20]

Finally platelets and PDL cells adhere to the cell-binding sites of the adsorbed proteins We recently foundthat adsorption-induced deformation of fibrinogen (platelet


PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02

0PET PMEA PHEMA PMPC

NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)

Figure 8 Comparison of platelet and PDL cell adhesion after 1 h (a) SEM images of adherent platelets on polymer surfaces Top scale bars10120583m bottom scale bars 10 120583m Polymers PET PMEA PHEMA and PMPC (b) Comparison of platelet and PDL cell adhesion after 1 hGray bar indicates platelet adhesion and white bar indicates PDL cell adhesion respectively relative to PET Polymers PET PMEA PHEMAand PMPC lowastlowast119875 lt 001 and lowast119875 lt 005 versus PET mean plusmn standard deviation platelets 119899 = 6 PDL cells 119899 = 9

PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion

Figure 9 Relationship between PDL cell adhesion and biocompat-ibility on PET PMEA PHEMA and PMPC

adhesion ligand) which is required for the adhesion ofplatelets does not occur on PMEA [41] In contrastfibronectin (PDL cell adhesion ligand) was deformed onPMEA [41] Therefore we concluded that PDL cells and notplatelets are capable of adhering to PMEA based on thisprotein deformation difference between polymer films Wesuppose that the existence of an intermediate water layeralters the amount of exposed cell-binding sites which resultsin differing cell adhesion on each polymer

373 Relationship between Biocompatibility and IntermediateWater In addition we have reported that hydrated PMPCand PEG as well as various proteins and polysaccharides thatare well-known biocompatible polymers contain intermedi-ate water [20 23 42 43] In contrast poorly biocompatible


polymers do not contain intermediate water [31] Free waterhas high mobility and is unable to shield the polymer surfaceor the nonfreezing water layer on the polymer surface [23]Because intermediate water is weakly bound to the polymermolecules or to nonfreezing water it forms a more stablestructure than free water [23] Based on these findingswe hypothesized that intermediate water which preventsproteins and platelets from directly contacting the polymersurface or nonfreezing water on the polymer surface plays animportant role in biocompatibility and cell adhesion [23] andthe amount of intermediate water affects protein adsorptionand cell adhesion [20 44 45]

374 Relationship between Intermediate Water Content andCell Adhesion Intermediate water content in hydratedPMEA (45 wt) [31] prevented platelet adhesion but did notprevent PDL cell adhesion In contrast higher intermediatewater content in hydrated PMPC (285 wt) [40] preventedthe adhesion of both platelets and PDL cells The value ofthe intermediate water content in hydrated PMPC reflectsthe value for the MPC homopolymer because Kitano et alconfirmed that the MPC-rich domain is directed toward thesurface in water [32]We assume that the higher intermediatewater content in hydrated PMPC relative to PMEA preventedcell adhesion because the thick intermediate water layer mayhave shielded the polymer surface or nonfreezing water layerIn contrast the thin intermediate water layer in hydratedPMEA likely prevented platelet adhesion but did not preventPDL cell adhesion

375 Relationship between Cell Properties and Cell AdhesionWe also consider the possibility that cell characteristicsaffected cell adhesion Platelets are floating cells and mainlyadhere to surfaces via glycoprotein IIbIIIa [46] PDL cellsare anchorage-dependent and mainly adhere to surfacesvia integrin 120572

51205731[47] Furthermore differences of cell size

and weight are present as PDL cells are larger (10ndash15 120583m)and heavier than platelets (2ndash4120583m) therefore PDL cellsmay demonstrate increased adhesion simply because of theirweight In our next study we will attempt to clarify themolecular mechanisms underlying cell adhesion on PMEAwith regard to the intermediate water content and celladhesion

38 PMEA Applications for Tissue Engineering Scaffolds in aBlood-Rich Environment As shown in Figures 2 3(a) 5 and8(b) PDL cells adhered to and proliferated on PET whereasPET was not biocompatible for human platelets PDL cellson PHEMA proliferated but had aggregated (Figures 2 and5) and PHEMAwas thus also found to be nonbiocompatiblefor tissue engineering using PDL cells As shown in Figures2 3(a) 5 and 8(b) PDL cells adhered and proliferated onbiocompatible PMEA without platelet adhesion howeverPDL cells did not adhere and proliferate on biocompatiblePMPC PMEA and PMPC have been previously identi-fied as blood-compatible (nonplatelet-adhesive) polymersHowever recent advances in medicine require the use ofblood-compatible polymers that do not exhibit blood cellattachment to isolate stem cells from blood Our results

challenge the widely accepted notion that biocompatible(blood-compatible) polymers (such as PMPC) do not permitcell adhesion as shown in Figure 9 We observed PDL celladhesion on the biocompatible (blood-compatible) polymerPMEA in the absence of incorporated substrate-bound cell-adhesive ligands and antibodies We therefore consider thatPMEA could be used in smart biomaterials Different celltypes may thus be selected by PMEA based on differencesin cell adhesion strength It should be noted that PMEA hasbeen approved by the FDA and can be used in a blood-richenvironment Therefore biocompatible PMEA may providean excellent scaffold for tissue engineering using PDL cells inhumans aswell as for culturing tissue-derived cells in a blood-rich environment

4 Conclusion

We found that PDL cells but not platelets adhered tobiocompatible PMEA We also observed unique vinculinlocalization in non-FAs vinculin rods and vinculin fibers Inaddition PDL cells on PMEA proliferated better than thoseon PHEMA Therefore PMEA may provide an excellentscaffold material for tissue engineering using PDL cells inhumans and also for culturing tissue-derived cells in a blood-rich environment

Conflict of Interests

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

Acknowledgments

The authors thank Ayano Sasaki for introduction to AFMmeasurements This work is supported by Grants-in-Aidand Special Coordination Funds for Promoting Science andTechnology of the Ministry of Education Culture SportsScience and Technology of Japan This work is also partof the financial support from Funding Program for NextGeneration World-Leading Researchers (NEXT ProgramJapan) Erika Kitakami is also supported in part by a Grant-in-Aid for Japan Society of the Promotion of Science (JSPS)Fellows Grant no 248746 Japan

References

[1] B L Pihlstrom B S Michalowicz and N W Johnson ldquoPeri-odontal diseasesrdquoThe Lancet vol 366 no 9499 pp 1809ndash18202005

[2] H Choi W Noh J Park J Lee and J Suh ldquoAnalysis ofgene expression during mineralization of cultured humanperiodontal ligament cellsrdquo Journal of Periodontal and ImplantScience vol 41 no 1 pp 30ndash43 2011

[3] B M Seo M Miura S Gronthos et al ldquoInvestigation of multi-potent postnatal stem cells from human periodontal ligamentrdquoThe Lancet vol 364 no 9429 pp 149ndash155 2004

[4] I C Gay S Chen and M MacDougall ldquoIsolation and char-acterization of multipotent human periodontal ligament stem


cellsrdquo Orthodontics amp Craniofacial Research vol 10 no 3 pp149ndash160 2007

[5] N Wada D Menicanin S Shi P M Bartold and S Gron-thos ldquoImmunomodulatory properties of human periodontalligament stem cellsrdquo Journal of Cellular Physiology vol 219 no3 pp 667ndash676 2009

[6] F Feng K Akiyama Y Liu et al ldquoUtility of PDL progenitors forin vivo tissue regeneration a report of 3 casesrdquo Oral Diseasesvol 16 no 1 pp 20ndash28 2010

[7] Y Nomura M Ishikawa Y Yashiro et al ldquoHuman periodontalligament fibroblasts are the optimal cell source for inducedpluripotent stem cellsrdquoHistochemistry and Cell Biology vol 137no 6 pp 719ndash732 2012

[8] L Petrovic A K Schlegel S Schultze-Mosgau and J WiltfangldquoDifferent substitute biomaterials as potential scaffolds in tissueengineeringrdquo International Journal of Oral and MaxillofacialImplants vol 21 no 2 pp 225ndash231 2006

[9] R Schmelzeisen R Shimming andM Sittinger ldquoMaking boneimplant insertion into tissue-engineered bone for maxillarysinus floor augmentation-a preliminary reportrdquo Journal ofCranio-Maxillofacial Surgery vol 31 no 1 pp 34ndash39 2003

[10] J P Davidas ldquoLooking for a new international standard forcharacterization classification and identification of surfaces inimplantable materials the long march for the evaluation ofdental implant surfaces has just begunrdquo Poseido vol 2 no 1pp 1ndash5 2014

[11] D M Dohan Ehrenfest B S Kang G Sammartino et alldquoGuidelines for the publication of articles related to implantsurfaces and design from the POSEIDO a standard for surfacecharacterizationrdquo Poseıdo vol 1 no 1 pp 7ndash15 2013

[12] N Metoki L Liu E Beilis N Eliaz and D MandlerldquoPreparation and characterization of alkylphosphonic acid self-assembledmonolayers on titanium alloy by chemisorptions andelectrochemical depositionrdquo Langmuir vol 30 no 23 pp 6791ndash6799 2014

[13] R Franca T D Samani G Bayade L Yahia and E SacherldquoNanoscale surface characterization of biphasic calcium phos-phate with comparisons to calcium hydroxyapatite and 120573-tricalcium phosphate bioceramicsrdquo Journal of Colloid and Inter-face Science vol 420 pp 182ndash188 2014

[14] M Catauro F Bollino and F Papale ldquoPreparation char-acterization and biological properties of organic-inorganicnanocomposite coatings on titanium substrates prepared by sol-gelrdquo Journal of Biomedical Materials Research A vol 102 no 2pp 392ndash399 2014

[15] M Tanaka T Motomura M Kawada et al ldquoBlood compatibleaspects of poly(2-methoxyethylacrylate) (PMEA)-relationshipbetween protein adsorption and platelet adhesion on PMEAsurfacerdquo Biomaterials vol 21 no 14 pp 1471ndash1481 2000

[16] X M Mueller D Jegger M Augstburger J Horisberger and LK von Segesser ldquoPoly2-methoxyethylacrylate (PMEA) coatedoxygenator An ex vivo studyrdquo The International Journal ofArtificial Organs vol 25 no 3 pp 223ndash229 2002

[17] B L Greenfield K R Brinkman K L Koziol et al ldquoTheeffect of surface modification and aprotinin on cellular injuryduring simulated cardiopulmonary bypassrdquo The Journal ofExtra-Corporeal Technology vol 34 no 4 pp 267ndash270 2002

[18] F D Rubens ldquoCardiopulmonary bypass technology transfermusings of a cardiac surgeonrdquo Journal of Biomaterials SciencePolymer Edition vol 13 no 4 pp 485ndash499 2002

[19] H Suhara Y Sawa M Nishimura et al ldquoEfficacy of a newcoating material PMEA for cardiopulmonary bypass circuits

in a porcine modelrdquo Annals of Thoracic Surgery vol 71 no 5pp 1603ndash1608 2001

[20] M Tanaka T Hayashi and S Morita ldquoThe roles of watermolecules at the biointerface of medical polymersrdquo PolymerJournal vol 45 no 7 pp 701ndash710 2013

[21] M Tanaka AMochizuki TMotomura K ShimuraMOnishiand Y Okahata ldquoIn situ studies on protein adsorption ontoa poly(2-methoxyethylacrylate) surface by a quartz crystalmicrobalancerdquo Colloids and Surfaces A Physicochemical andEngineering Aspects vol 193 no 1ndash3 pp 145ndash152 2001

[22] M Tanaka AMochizuki T Shiroya et al ldquoStudy on kinetics ofearly stage protein adsorption on poly(2-methoxyethylacrylate)(PMEA) surfacerdquo Colloids and Surfaces A Physicochemical andEngineering Aspects vol 203 no 1ndash3 pp 195ndash204 2002

[23] M Tanaka ldquoDesign of novel 2D and 3D biointerfaces using self-organization to control cell behaviorrdquo Biochimica et BiophysicaActa vol 1810 no 3 pp 251ndash258 2011

[24] M J Dalby N Gadegaard R Tare et al ldquoThe control of humanmesenchymal cell differentiation using nanoscale symmetryand disorderrdquo Nature Materials vol 6 no 12 pp 997ndash10032007

[25] M Tanaka K Nishikawa H Okubo et al ldquoControl of hep-atocyte adhesion and function on self-organized honeycomb-patterned polymer filmrdquo Colloids and Surfaces A Physicochem-ical and Engineering Aspects vol 284-285 pp 464ndash469 2006

[26] A Saminathan K J Vinoth H H Low T Cao and MC Meikle ldquoEngineering three-dimensional constructs of theperiodontal ligament in hyaluronan-gelatin hydrogel films anda mechanically active environmentrdquo Journal of PeriodontalResearch vol 48 no 6 pp 790ndash801 2013

[27] MNune P KumaraswamyUMKrishnan and S SethuramanldquoSelf-assembling peptide nanofibrous scaffolds for tissue engi-neering novel approaches and strategies for effective functionalregenerationrdquo Current Protein and Peptide Science vol 14 no 1pp 70ndash84 2013

[28] B Geiger A Bershadsky R Pankov and K M YamadaldquoTransmembrane extracellular matrix-cytoskeleton crosstalkrdquoNature Reviews Molecular Cell Biology vol 2 no 11 pp 793ndash805 2001

[29] P KanchanawongG Shtengel AM Pasapera et al ldquoNanoscalearchitecture of integrin-based cell adhesionsrdquo Nature vol 468no 7323 pp 580ndash584 2010

[30] H Ishihata M Tanaka N Iwama et al ldquoProliferation ofperiodontal ligament cells on biodegradable honeycomb filmscaffoldwith unifiedmicropore organizationrdquo Journal of Biome-chanical Science and Engineering vol 5 no 3 pp 252ndash261 2010

[31] M Tanaka and A Mochizuki ldquoEffect of water structureon blood compatibility-thermal analysis of water inpoly(meth)acrylaterdquo Journal of Biomedical Materials ResearchA vol 68 no 4 pp 684ndash695 2004

[32] H Kitano M Imai T Mori M Gemmei-Ide Y Yokoyama andK Ishihara ldquoStructure of water in the vicinity of phospholipidanalogue copolymers as studied by vibrational spectroscopyrdquoLangmuir vol 19 no 24 pp 10260ndash10266 2003

[33] G Peluso O Petillo J M Anderson et al ldquoThe differen-tial effects of poly(2-hydroxyethyl methacrylate) and poly(2-hydroxyethyl methacrylate)poly(caprolactone) polymers oncell proliferation and collagen synthesis by human lung fibrob-lastsrdquo Journal of Biomedical Materials Research vol 34 no 3pp 327ndash336 1997


[34] V Dugina L Fontao C Chaponnier J Vasiliev and GGabbiani ldquoFocal adhesion features during myofibroblastic dif-ferentiation are controlled by intracellular and extracellularfactorsrdquo Journal of Cell Science vol 114 no 18 pp 3285ndash32962001

[35] K Ishihara H Nomura TMihara K Kurita Y Iwasaki andNNakabayashi ldquoWhy do phospholipid polymers reduce proteinadsorptionrdquo Journal of Biomedical Materials vol 39 no 2 pp323ndash330 1998

[36] Y Iwasaki E Tabata K Kurita and K Akiyoshi ldquoSelectivecell attachment to a biomimetic polymer surface through therecognition of cell-surface tagsrdquoBioconjugate Chemistry vol 16no 3 pp 567ndash575 2005

[37] F Zhang G Li P Yang W Qin C Li and N Huang ldquoFabri-cation of biomolecule-PEG micropattern on titanium surfaceand its effects on platelet adhesionrdquo Colloids and Surfaces BBiointerfaces vol 102 pp 457ndash465 2013

[38] Y Inoue and K Ishihara ldquoReduction of protein adsorption onwell-characterized polymer brush layers with varying chemicalstructuresrdquo Colloids and Surfaces B Biointerfaces vol 81 no 1pp 350ndash357 2010

[39] T Kondo K Nomura M Gemmei-Ide et al ldquoStructure ofwater at zwitterionic copolymer film-liquid water interfaces asexamined by the sum frequency generation methodrdquo Colloidsand Surfaces B Biointerfaces vol 113 pp 361ndash367 2014

[40] T Hatakeyama M Tanaka and H Hatakeyama ldquoStudies onbound water restrained by poly(2-methacryloyloxyethyl phos-phorylcholine) comparison with polysaccharide-water sys-temsrdquo Acta Biomaterialia vol 6 no 6 pp 2077ndash2082 2010

[41] T Hoshiba M Nikaido and M Tanaka ldquoCharacterizationof the attachment mechanisms of tissue-derived cell lines toblood-compatible polymersrdquo Advanced Healthcare Materialsvol 3 no 5 pp 775ndash784 2014

[42] T Hatakeyma H Kasuga M Tanaka and H HatakeyamaldquoCold crystallization of poly(ethylene glycol)-water systemsrdquoThermochimica Acta vol 465 no 1-2 pp 59ndash66 2007

[43] M Tanaka and A Mochizuki ldquoClarification of blood compat-ibility mechanism by controlling water structurerdquo Journal ofBiomaterials Science Polymer Edition vol 21 no 14 pp 1849ndash1863 2010

[44] M Tanaka E Kitakami S Yagi et al ldquoBiocompatible polymerswith intermediate waterrdquo Patent 256467 Japan 2010

[45] T Hayashi Y Tanaka Y Koide M Tanaka and M HaraldquoMechanism underlying bioinertness of self-assembled mono-layers of oligo(ethyleneglycol)-terminated alkanethiols on goldprotein adsorption platelet adhesion and surface forcesrdquo Phys-ical Chemistry Chemical Physics vol 14 no 29 pp 10196ndash102062012

[46] S J Shattil H Kashiwagi and N Pampori ldquoIntegrin signalingthe platelet paradigmrdquo Blood vol 91 no 8 pp 2645ndash2657 1998

[47] S Ivanovski M Komaki P M Bartold and A S NarayananldquoPeriodontal-derived cells attach to cementum attachmentprotein via 12057251205731 integrinrdquo Journal of Periodontal Research vol34 no 3 pp 154ndash159 1999

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


is advantageous because it efficiently utilizes extracted teethHowever for periodontal tissue regeneration it is necessaryto culture PDL cells on a biocompatible scaffold to deliverthe cells to the wound site and also to provide space forthe formation of the new periodontal tissue Biomaterialscaffolds designed for tissue-engineered constructs mustaccommodate cell viability growth and function [8] Thereis increasing interest in designing new biomaterials for scaf-folds with minimal or no immune response that encouragestable implanttissue interaction [9] In addition the surfacecharacterization of biomaterials is important to design newimplantable materials [10ndash14]

We previously reported that poly(2-methoxyethyl acry-late) (PMEA) shows excellent biocompatibility with humanblood coagulation and complement systems and does notactivate leukocytes erythrocytes and platelets in vitro andex vivo relative to other polymer surfaces during the earlystages of immune reactions [15ndash19] On the basis of ourresults superior biocompatible catheters and oxygenatorscoated with PMEA were approved by the Food and DrugAdministration (FDA) and made available to the global mar-ket [16ndash19] We further determined why PMEA has excellentbiocompatibility [15 20ndash22] In particular the low extent ofplatelet adhesion and spreading observed was closely relatedto a low degree of denaturation and high dissociation rate forproteins adsorbed onto PMEA [15]

The objective of this study was to examine the hypothesisthat PMEA is a biocompatible polymer for tissue engineeringthat can facilitate adhesion and proliferation of PDL cells withlow platelet adhesion Cell-material interactions determinemany cellular processes such as adhesion spreading andproliferation and are thus essential for tissue engineering [23ndash27] However the influence of the chemical components ofsynthesized polymers on the biology of PDL cells remainsunclear To investigate PDL cell-material interactions wecharacterized the localization of focal adhesions which aremultifunctional organelles that mediate cell-material adhe-sion force transmission and cytoskeletal regulation and sig-naling [28] To analyze the formation of focal adhesions weevaluated the localization of vinculin which is a membrane-cytoskeletal protein present in focal adhesions that is involvedin the linkage of integrin adhesion molecules to the actincytoskeleton [29]

2 Material and Methods

21 Preparation of Polymer Surfaces PMEA was prepared byfree-radical polymerization using 221015840-azobisisobutyronitrile(Kanto Chemical Co Inc Japan) as the initiator and 2-methoxyethyl acrylate (MEA) as themonomerTheMEAwasobtained from Wako Pure Chemical Industries Ltd (OsakaJapan) poly(2-hydroxyethyl methacrylate) (PHEMA) wasobtained from Scientific Polymer Products Inc (OntarioNY) and poly[(2-methacryloyloxyethyl phosphorylcholine)-co-(n-butyl methacrylate)] (PMPC) was obtained fromNOF corporation (Tokyo Japan) The molecular weightof each polymer was estimated by gel permeation chro-matography using polystyrene standards The molecularweight (Mw) of PMEA PHEMA and PMPC was 85000

300000 and 600000 respectively The chemical structureof each polymer PMEA PHEMA and PMPC is shownin Figure 1 in Supplementary Material available onlineat httpdxdoiorg1011552014102648 and the polymerswere prepared on polyethylene terephthalate (PET filmT100E125 Mitsubishi Plastics Tokyo Japan 14mm diameterand 125 120583m thickness) using a spin coater (MS-A100 MikasaTokyo Japan) Exactly 40 120583L of a 02 wt solution of eachpolymer was cast twice onto the PET films Analytical grademethanol (Kanto Chemical Co Inc) was used as the solventfor each solution The surfaces of the polymer films wereanalyzed by X-ray photoelectron spectroscopy (XPS) (ESCA-1000 Shimadzu Kyoto Japan) to confirm the coverage of thecoated polymer The take-off angle was 45∘

For cell culture the films were sterilized by UV exposurefor 2 h Subsequently the films were soaked in mediumcomposed ofMinimumEssentialMedium (MEM-Alpha LifeTechnologies) containing 10 heat-inactivated fetal bovineserum (Equitech-Bio Inc Kerrville TX) and antibiotic solu-tion (100UmLpenicillinG sodium 100120583gmL streptomycinsulfate and 025 120583gmL amphotericin B Life Technologies)(culture medium) for one hour (preconditioning)

22 Characterization of Polymer Films We analyzed eachpolymer filmby atomic forcemicrocopy (AFMAgilent Tech-nologies 5550 Scanning Probe Microscope Agilent Tech-nologies Inc Santa Clara CA) The maximum scan rangewas approximately 10 120583m times 10 120583m using a cantilever with aforce constant of 21ndash78Nm resonance frequency of 250ndash390 kHz and tip height of 10ndash15 120583m (NCH-10 Nano WorldZurich Switzerland) AFMwas performed in air acoustic ACmode AFM image analysis was performed using Pico ImageSoftware (Agilent Technologies)

The wettability of the polymer surfaces was characterizedby contact angle measurement [15] The static contact angleon each polymer surface was measured using the sessile dropmethod at room temperature For the sessile drop method2 120583L of deionized water was dropped on a dried polymer filmusing a microsyringe The static contact angle was observed30 s later under a microscope (G-1-1000 ERMA Inc TokyoJapan) After at least five readings which were obtainedfor different areas of the polymer the measurements wereaveraged to arrive at a final contact angle (119899 = 6)

23 Cell Preparation and Culture Primary PDL cells wereobtained as previously reported [30] Fibroblast-like PDLcells were derived from the periodontal ligament of humanthird molars extracted from healthy individuals aged 17ndash21years who had no clinical signs of chronic periodontal dis-ease Informed consent was obtained prior to each extractionThe cells were obtained from the Dental Faculty of TohokuUniversity Periodontal ligament tissues were dissected intosmall pieces from the midportion of the root with a sharpbladeThe pieces were then cultivated in tissue culture dishes(Asahi Glass Co LTD Tokyo Japan) until the formation ofa confluent cell monolayer using culture medium After con-fluence was achieved the cells were washed with phosphate-buffered saline (PBS Takara Bio Inc Shiga Japan) andresuspended with 0075 gL protease and 01 gL EDTA to


















900 800 700 600 500 400 300 200 100 0


Inte

nsity

(CPS

)times102 (A)

(B)

O 1s N 1s C 1s

(a)

900 800 700 600 500 400 300 200 100 0


O 1sN 1s C 1s

Inte

nsity

(CPS

)times102

(A)

(B)

(b)

900 800 700 600 500 400 300 200 100 0

Binding energy (eV)

O 1s

N 1s

C 1s

P 2s

Inte

nsity

(CPS

)times102

(A)

(B)

1000

(c)












1h Day 1 Day 7PE

TPM

EAPH

EMA

Day 3

100120583m

100120583m

100120583m

PMPC







0

500

1000

1500

2000

2500

3000

3500

Cell

num

ber (

cells

cm2)

PET

PMEA

PHEM

A

PMPC

NS

lowastlowast

lowastlowast

(a)

PET PMEA PHEMA

(b)







PHEMAPMEAPET

Day

11

h

(a)

3000

2500

2000

1500

1000

500

0

Proj

ecte

d ce

ll ar

ea (120583

m2)

NS

NS

NS

1h Day 1

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

lowastlowast

(b)

0

100

200

300

400

500

600

700PE

T

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day11h

NS NS

NS lowastlowast

Perim

eter

(120583m

)

(c)

0

20

40

60

80

100

120

140

160

180

200

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day 1 Day 11h 1h

lowastlowast

lowastlowast

lowast


Leng

th ax

is (120583

m)

(d)



0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




















900 800 700 600 500 400 300 200 100 0


Inte

nsity

(CPS

)times102 (A)

(B)

O 1s N 1s C 1s

(a)

900 800 700 600 500 400 300 200 100 0


O 1sN 1s C 1s

Inte

nsity

(CPS

)times102

(A)

(B)

(b)

900 800 700 600 500 400 300 200 100 0

Binding energy (eV)

O 1s

N 1s

C 1s

P 2s

Inte

nsity

(CPS

)times102

(A)

(B)

1000

(c)












1h Day 1 Day 7PE

TPM

EAPH

EMA

Day 3

100120583m

100120583m

100120583m

PMPC







0

500

1000

1500

2000

2500

3000

3500

Cell

num

ber (

cells

cm2)

PET

PMEA

PHEM

A

PMPC

NS

lowastlowast

lowastlowast

(a)

PET PMEA PHEMA

(b)







PHEMAPMEAPET

Day

11

h

(a)

3000

2500

2000

1500

1000

500

0

Proj

ecte

d ce

ll ar

ea (120583

m2)

NS

NS

NS

1h Day 1

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

lowastlowast

(b)

0

100

200

300

400

500

600

700PE

T

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day11h

NS NS

NS lowastlowast

Perim

eter

(120583m

)

(c)

0

20

40

60

80

100

120

140

160

180

200

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day 1 Day 11h 1h

lowastlowast

lowastlowast

lowast


Leng

th ax

is (120583

m)

(d)



0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




1h Day 1 Day 7PE

TPM

EAPH

EMA

Day 3

100120583m

100120583m

100120583m

PMPC







0

500

1000

1500

2000

2500

3000

3500

Cell

num

ber (

cells

cm2)

PET

PMEA

PHEM

A

PMPC

NS

lowastlowast

lowastlowast

(a)

PET PMEA PHEMA

(b)







PHEMAPMEAPET

Day

11

h

(a)

3000

2500

2000

1500

1000

500

0

Proj

ecte

d ce

ll ar

ea (120583

m2)

NS

NS

NS

1h Day 1

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

lowastlowast

(b)

0

100

200

300

400

500

600

700PE

T

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day11h

NS NS

NS lowastlowast

Perim

eter

(120583m

)

(c)

0

20

40

60

80

100

120

140

160

180

200

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day 1 Day 11h 1h

lowastlowast

lowastlowast

lowast


Leng

th ax

is (120583

m)

(d)



0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

500

1000

1500

2000

2500

3000

3500

Cell

num

ber (

cells

cm2)

PET

PMEA

PHEM

A

PMPC

NS

lowastlowast

lowastlowast

(a)

PET PMEA PHEMA

(b)







PHEMAPMEAPET

Day

11

h

(a)

3000

2500

2000

1500

1000

500

0

Proj

ecte

d ce

ll ar

ea (120583

m2)

NS

NS

NS

1h Day 1

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

lowastlowast

(b)

0

100

200

300

400

500

600

700PE

T

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day11h

NS NS

NS lowastlowast

Perim

eter

(120583m

)

(c)

0

20

40

60

80

100

120

140

160

180

200

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day 1 Day 11h 1h

lowastlowast

lowastlowast

lowast


Leng

th ax

is (120583

m)

(d)



0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




PHEMAPMEAPET

Day

11

h

(a)

3000

2500

2000

1500

1000

500

0

Proj

ecte

d ce

ll ar

ea (120583

m2)

NS

NS

NS

1h Day 1

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

lowastlowast

(b)

0

100

200

300

400

500

600

700PE

T

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day11h

NS NS

NS lowastlowast

Perim

eter

(120583m

)

(c)

0

20

40

60

80

100

120

140

160

180

200

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

Day 1 Day 11h 1h

lowastlowast

lowastlowast

lowast


Leng

th ax

is (120583

m)

(d)



0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

10

20

30

40

50

60

70

80

90

100

Day 1 Day 3 Day 7

PET

PMEA

PHEM

APM

PC PET

PMEA

PHEM

APM

PCPET

PMEA

PHEM

APM

PC

lowastlowast

lowastlowast

lowastlowast

lowast

Cel

l num

ber (times104

cells

cm2)










PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(a)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

lowastlowast

(b)

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

PET

PMEA

PHEM

A

IIIIII

100

80

60

40

20

0

Ki-67

posit

ive c

ells

()

(c)

II

II

II

II

II

III

III

IIIIII

(d)

II

II

II

II II

(e)









4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




4 h

Day


1 h

Day

3

(a)


1 h

4 h

Day

1D

ay 3

(b)


4h

Day1

Day3

1h

(c)


FA andor non-FA

(ii) Vinculin rod


FA andor non-FA


FA + non-FA



FA + non-FA

(d)







PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




PMEA PHEMA PMPCPET

(a)

18

16

14

12

10

08

06

04

02


NS

NS

Initi

al ce

ll ad

hesio

n (r

elat

ive t

o PE

T)

lowastlowast

lowastlowast

lowast

(b)


PMEA

PET

PHEMA

PMPC

Bioc

ompa

tibili

ty

PDL cell adhesion












4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











4 Conclusion




Acknowledgments


References



























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials

























































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



adhesion and proliferation of human periodontal ligament cells on

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