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Taira Sato, Yuki Shirosaki, Masaki Nagaya, Yoshinori Asano, Kazuaki Nakano, Hiroshi Nagashima, Mamoru Aizawa, [Masanori Kikuchi](https://orcid.org/0000-0002-9451-8147)

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[Preparation of anti-decay self-setting pastes of hydroxyapatite/collagen utilizing (3-glycidoxypropyl)trimethoxysilane](https://mdr.nims.go.jp/datasets/40566415-4d9d-4ff1-9004-bc0d4a873ceb)

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Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=tace20Journal of Asian Ceramic SocietiesISSN: (Print) 2187-0764 (Online) Journal homepage: http://www.tandfonline.com/loi/tace20Preparation of anti-decay self-setting pastesof hydroxyapatite/collagen utilizing (3-glycidoxypropyl)trimethoxysilaneTaira Sato, Yuki Shirosaki, Masaki Nagaya, Yoshinori Asano, Kazuaki Nakano,Hiroshi Nagashima, Mamoru Aizawa & Masanori KikuchiTo cite this article: Taira Sato, Yuki Shirosaki, Masaki Nagaya, Yoshinori Asano, Kazuaki Nakano,Hiroshi Nagashima, Mamoru Aizawa & Masanori Kikuchi (2018) Preparation of anti-decay self-setting pastes of hydroxyapatite/collagen utilizing (3-glycidoxypropyl)trimethoxysilane, Journal ofAsian Ceramic Societies, 6:4, 322-331, DOI: 10.1080/21870764.2018.1517712To link to this article:  https://doi.org/10.1080/21870764.2018.1517712© 2018 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup on behalf of The Korean CeramicSociety and The Ceramic Society of Japan.Accepted author version posted online: 20Sep 2018.Published online: 10 Oct 2018.Submit your article to this journal Article views: 198View Crossmark datahttp://www.tandfonline.com/action/journalInformation?journalCode=tace20http://www.tandfonline.com/loi/tace20http://www.tandfonline.com/action/showCitFormats?doi=10.1080/21870764.2018.1517712https://doi.org/10.1080/21870764.2018.1517712http://www.tandfonline.com/action/authorSubmission?journalCode=tace20&show=instructionshttp://www.tandfonline.com/action/authorSubmission?journalCode=tace20&show=instructionshttp://crossmark.crossref.org/dialog/?doi=10.1080/21870764.2018.1517712&domain=pdf&date_stamp=2018-09-20http://crossmark.crossref.org/dialog/?doi=10.1080/21870764.2018.1517712&domain=pdf&date_stamp=2018-09-20FULL LENGTH ARTICLEPreparation of anti-decay self-setting pastes of hydroxyapatite/collagenutilizing (3-glycidoxypropyl)trimethoxysilaneTaira Satoa, Yuki Shirosakib, Masaki Nagayac, Yoshinori Asanod, Kazuaki Nakanod, Hiroshi Nagashimac,d,Mamoru Aizawaa and Masanori KikuchieaDepartment of Applied Chemistry, Graduate School of Science and Technology, Meiji University, Kawasaki, Japan; bDepartment ofMaterials Science, Faculty of Engineering, Kyushu Institute of Technology, Fukuoka, Japan; cMeiji University International Institute forBio-Resource Research, Meiji University, Kawasaki, Japan; dDepartment of Life Sciences, School of Agriculture, Meiji University, Kawasaki,Japan; eBioceramics Group, National Institute for Materials Science, Tsukuba, JapanABSTRACTThis article describes preparation of anti-decay self-setting pastes of hydroxyapatite/collagen (HAp/Col) utilizing (3-glycidoxypropyl)trimethoxysilane (GPTMS). The powderportion of the paste was ball-milled HAp/Col synthesized by the simultaneous titrationmethod, and the liquid portion was GPTMS aqueous solution at a concentration of 0.1,1.0 or 10 % in volume. The HAp/Col-GPTMS pastes were prepared by mixing thepowder and liquid portions at powder/liquid (P/L) ratios ranging from 0.20 to 2.00(g/cm3). The pastes with P/L ratios from 0.33 to 1.50 showed good handling properties,and their viscosities depended greatly on the P/L ratio. The lowest washout ratio wasobserved at a P/L ratio of 1.00 independent of the GPTMS concentration. Althoughcytocompatibilty tests showed that inhibition of cell proliferation depended on theelution amounts of GPTMS from the pastes, an animal test using porcine tibia demon-strated no harmful systemic or local symptoms, because the GPTMS concentrationmaintained acceptable levels for living tissues through dispersion with body fluid.The animal test also revealed that the paste was completely resorbed and substitutedwith newly formed bone after 12 weeks implantation. It was concluded based on theseresults that HAp/Col-GPTMS pastes are promising candidates for use as bioresorbableinjectable pastes.ARTICLE HISTORYReceived 10 May 2018Accepted 9 August 2018KEYWORDSHydroxyapatite/collagennanocomposite;(3-glycidoxypropyl)trimethoxysilane; bonepaste; mechanicalproperties; biocompatibility1. IntroductionCalcium phosphate cements, of which the maincomponent after setting is hydroxyapatite (HAp),have been widely used due to their biocompatibil-ity, injectability and ability to fit irregularlyshaped bone defects as well as these self-settingability. Since the rather low resorption rate ofHAp causes a decrease in the new bone formationrate, however [1–3], injectable bone void fillerscomposed mainly of higher bioresorbable materi-als are being prepared to achieve acceleration inbone formation. Konishi et al. proposed chelate-setting calcium phosphate cements utilizing inosi-tol hexaphosphate as a chelating agent and pre-pared self-setting cements composed of α- or β-tricalcium phosphates (β-TCP) as well as HApcements. These cements indicated good biocom-patibility, and α- and β-TCP cements were partlyresorbed during 4 weeks observation and showednew bone formation in their bioresorbed areas[4,5]. The inositol hexaphosphate in the β-TCPcement needs many biological tests to verify itsuse as a setting agent, however, because it has notyet been tested for any physiological reactions,including toxicity as a substance directly appliedin internal tissues, even if it is used as an oralsupplement. The bioresorption mechanism of β-TCP is still unclear [6], moreover, and a clinicalstudy reported that no osteoclastic resorption of alarge quantity of β-TCP was observed 72 weeksafter grafting [7], even though porous β-TCP cera-mics are used worldwide as bioactive and biode-gradable bone void fillers [8].Since bone is a nanocomposite of HAp and type-I collagen, injectable bone void fillers composed ofapatitic calcium phosphate and type-I atelocollagenhave been sold worldwide as Boneject® (Koken,Japan) and Collagraft® (NeuColl, USA) [9–11].They are simple mixtures of these contents; thus,their bone tissue reactions are phagocytosis of col-lagen by macrophages and osteoconduction to cal-cium phosphate particles with slight resorption byosteoclasts. In addition, they require a thick tube toinject them into bone defects. The hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) pro-posed by the authors [12–15] has a bone-likeCONTACT Masanori Kikuchi KIKUCHI.Masanori@nims.go.jpThis article has been republished with minor changes. These changes do not impact the academic content of the article.JOURNAL OF ASIAN CERAMIC SOCIETIES2018, VOL. 6, NO. 4, 322–331https://doi.org/10.1080/21870764.2018.1517712© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The Korean Ceramic Society and The Ceramic Society of Japan.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permitsunrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.http://www.tandfonline.comhttp://crossmark.crossref.org/dialog/?doi=10.1080/21870764.2018.1517712&domain=pdfnanostructure, and it is the first material that iscompletely incorporated into the bone remodelingprocess and whose resorption rate can be con-trolled by the crosslink ratio of its collagen content.Sponge-like porous HAp/Col, sold as Refit® inJapan, demonstrated faster bone regeneration andfiller resorption than those of porous β-TCP inclinical trials, moreover, and is clinically used inJapan [15]. Hence, HAp/Col is a promising candi-date for use as a bioresorbale bone substitute forother materials besides porous materials. Werecently reported on HAp/Col pastes using sodiumalginate as a hardening agent [16–18] and con-cluded that supplementation of large amounts ofcalcium salts other than HAp was necessary toobtain sufficient anti-washout properties. Eventhough the chemicals calcium citrate and calciumcarbonate show no harm to tissues, huge amountsof them might decrease and/or inhibit the biologi-cal advantages of HAp/Col.To solve this problem, silane coupling agents wereproposed as a new hardening agent for HAp/Col paste.Silane coupling agents are commonly reported as func-tional materials to prepare biomaterials composed ofinorganic or organic materials or their composites [19–21], because silane coupling agents generally have theproperty of generating silanol groups by hydrolysis, andbecause they bond covalently to inorganic substances andalso form siloxane networks by their self-condensation[22]. In this study, (3-glycidoxypropyl)trimethoxysilane(GPTMS) had been chosen because epoxy groups inGPTMS bond to amino groups in collagen [23–26].In this paper, HAp/Col-GPTMS pastes were preparedby mixing of HAp/Col powder and GPTMS aqueoussolution, and the influences of the GPTMS concentrationsand the powder to liquid (P/L) ratios on the physicalproperties of the pastes were investigated. The biocompat-ibility of the HAp/Col-GPTMS pastes was evaluated,moreover, by the conventional cell culture test and ananimal test using pig tibia.2. Materials and methods2.1. MaterialsHAp/Col was synthesized at a HAp to collagenmass ratioof 80:20 by the simultaneous titration method [12,13].Briefly stated, 10 g of HAp/Col were synthesized from205.0 cm3 of 400 mM Ca(OH)2 suspension, which wasprepared by hydration of calciumoxide obtained by burn-ing calcium carbonate (CaCO3, Alkaline analysis grade,Wako Pure Chemicals Inc., Japan) at 1050 °C for 3 h and404.9 cm3 of 120 mM orthophosphoric acid aqueoussolution (Reagent grade, Wako Pure Chemicals Inc.,Japan) with 2.0 g of type-I porcine dermal atelocollagen(Biomaterial grade, Nitta Gelatin Inc., Japan). They weresimultaneously titrated into a reaction vessel containing199.1 cm3 of distilled water at a titration rate of 30 cm3/min. The temperature and pH of the reaction solutionwere 37 °C and 9, respectively.HAp/Col powder, a powder phase of the HAp/Col-GPTMS paste, was prepared by the following procedures:The as prepared HAp/Col was entered into a speciallydesigned mold to allow squeezing of water during com-paction and compacted by uniaxial pressing at 20 MPa.The HAp/Col compact was then freeze-dried, crushed,and classified to 100 μm or less in particle size by sieving.The liquid phase of the paste was GPTMS (TokyoChemical Industry Co., Ltd., Japan) aqueous solutionjust 1 h after mixing of GPTMS with distilled water (dis-tilled water for injection, Otsuka Pharmaceutical Co.,Tokushima, Japan) to promote hydrolysis of GPTMS.The concentrations of the GPTMS solutions were 0.1,1.0, and 10% by volume. Pastes were prepared by mixingthe GPTMS aqueous solution with the powder at severalP/L ratios. The mixture conditions are summarized inTable 1. The pastes prepared with the respective concen-trations of GPTMS solution are abbreviated as G[GPTMSconcentration]-paste, e.g., the HAp/Col paste preparedwith 0.1 % GPTMS solution is denoted as “G0.1-paste.”2.2. Washout property testThe washout properties of the paste was evaluatedaccording to the procedure described in Japanese indus-trial standard JIS T 0330–4 “Bioceramics-Part 4:Physicochemical characterization of calcium phosphatebone paste.” In detail, a paste mixture at 3 min after thestart of mixing was packed into a syringe 4.8 mm ininner diameter and 16.5 mm in height, and the packedpaste was squeezed onto a wire net with a wire diameterof 0.5 mm and aperture of 2.0 mm. At 5 min after thestart of mixing, the paste and wire net were soaked in50 cm3 of Dulbecco’s phosphate buffered saline (PBS) at37 °C in an appropriate container. They were thenplaced statically in an incubator at 37 °C, 95 ± 5 %relative humidity (RH) for 72 h. The washout ratio wascalculated as the mass percentage of the paste debris onand beneath the wire net with respect to the originalmass of the paste.2.3. Viscosity testA viscosity of the paste was measured according toIshikawa’s method [27]. Briefly, the paste mixture atTable 1. Amount of liquid relative to raw material powderand P/L ratios.Amount ofpowder/g 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000Amount ofliquid/cm35.00 3.00 2.00 1.33 1.00 0.80 0.67 0.50P/L ratio (g/cm3)0.20 0.33 0.50 0.75 1.00 1.25 1.50 2.00JOURNAL OF ASIAN CERAMIC SOCIETIES 323exactly 3 min after the start of mixing was shapedinto a cylinder at a 5.0 mm in diameter and 5.1 mmin height, of which the volume was approximately0.1 cm3. The paste cylinder was placed on a glassplate and spread at exactly 10 min after the start ofmixing by compression with a glass plate and a totalweight of 2 kg in mass. After 10 min spreading, thespread area of the paste was measured using its digitalimage with ImageJ (NIH, ver. 1.46, for Mac) todetermine its viscosity.2.4. Hardening behavior testThe HAp/Col-GPTMS pastes were a type of siliconehydrogel and demonstrated viscoelasticity; the a hard-ening behavior test using the Gillmore needle describedin JIS T 0330–4 could therefore not be applied. Thus,the hardening behavior of the pastes was evaluated bythe time-dependent spread area measurementdescribed in 2.3 at 0, 10, 30, 60, 180, 360 and1440 min additional aging before compression in theviscosity measurement; i.e., the result at 0 min indicatesexactly the same condition as the viscosity test.2.5. Compressive strength testThe compressive strength of the paste was measuredaccording to the procedure described in JIS T 0330–4.The raw materials were mixed for 3 min and molded ina Teflon® mold to form a cylindrical shape 6.0 mm indiameter and 12.0 mm in height. They were thenincubated at 37 °C, 95 ± 5 % RH for 1 h in an incubatorand soaked in 37 °C distilled water for 72 h. Aftersoaking, the paste was removed from the mold, andits compressive strength was measured with a universaltesting machine (AGS 5kN, Shimadzu, Japan) at across-head speed of 0.5 mm/min. The Young’s mod-ulus of the paste was calculated from the inclination ofthe elastic region in the stress-strain curve.2.6. Cytocompatibility testHAp/Col paste for the cytocompatibility test was pre-pared from the HAp/Col powder sterilized with etha-nol aqueous solution at 70% in volume and a filter-sterilized GPTMS aqueous solution. A human osteo-blastic cell line, MG-63, was used for the cytocompat-ibility test and all cell culture operations wereperformed at 37 °C in a 5% CO2 humidified atmo-sphere. The Dulbecco’s modified Eagle’s medium(Sigma-Aldrich, UK; D-MEM) supplemented with 10% by volume fetal bovine serum (FBS, Cosmo Bio Co.Ltd., lot number 10D219) and 1 % by volume penicillinand streptomycin (Invitrogen-Gibco; Pen/Strep) wasused as the culture medium. A total of 20,000 cellswere seeded in each well of a 6-well tissue culturepolystyrene (TCPS) plate with the 2-cm3 culturemedium. One day after seeding, the culture mediumwas changed, and the paste, 5 min after the start of raw-material mixing, was directly injected into each wellthrough a syringe with an inner diameter of 7 mm toform the paste into a cylinder 5 mm in height and7 mm in diameter. The medium was changed every2 days, and the number of cells was counted at 1, 3, and7 days after cell seeding with a hemocytometer. Thesilicon ion concentration of the medium collected at itschange was measured with an inductively coupledplasma-atomic emission spectrometer (SPS7800, SIINanoTechnology, Japan; ICP-AES). A HAp/Col densebody with the same size as the paste specimen wasprepared by the compacting method for the HAp/Coldescribed in 2.1 and by die cutting with a punch,followed by dehydrothermal cross-linking at 140 °Cfor 12 h and soaking in the D-MEM for 5 days toallow saturated adsorption of the Ca2+ and Mg2+ ions.The preparation conditions of the pastes for the cellculture test were chosen based on the results ofmechanical property tests. The HAp/Col dense bodyand a blank were used as controls.2.7. Animal testThe biocompatibility and bioresorbability of the HAp/Col-GPTMS pastes were preliminarily evaluated by ananimal test, which is approved by the InstitutionalAnimal Care and Use Committee of Meiji University(Permission number: IACUC15-0011) and which wascarried out according to the committee’s guidelines.G1.0- and G10-pastes in a P/L ratio of 1.00 were chosenfor the animal test and prepared on site, i.e. implantedbefore complete hardening to simulate practical use.The paste mixtures were packed into a cylindricalsyringe 4.0 mm in inner diameter to a height of8.0 mm and injected directly into bone holes measur-ing 4 mm in diameter prepared in the right tibia of awild pig. At 12 weeks after implantation, the pig wassacrificed and the implant site was harvested with thesurrounding tissues. The pastes and surrounding tis-sues were observed with the naked-eye and by microX-ray micro-computed tomograph (µ-CT).2.8. Statistical analysisThe results are shown as the mean ± standard devia-tion. Statistical analysis was performed using one-wayanalysis of variance with the Tukey-HSD post hoctest. The statistical significance was set at p < 0.05.3. Results3.1. Preparation of the HAp/Col pastesThe apparent fluidities and viscosities of the HAp/Col-GPTMS pastes just after mixing seemed to324 T. SATO ET AL.depend on their P/L ratios, regardless of the GPTMSconcentration. The paste prepared at a P/L ratio of0.20 shown in Figure 1(a) was a highly fluidic suspen-sion, and that prepared at the P/L ratio of 2.00 shownin Figure 1(b) was aggregated. Since they could notbe formed into monolithic cylinders within the statedperiod by the standard method, these pastes wereexcluded from further tests, i.e. the results includethe pastes with P/L ratios between 0.33 and 1.50 thatshowed good kneading performance.3.2. Washout propertiesThe washout ratios of the pastes are summarized inFigure 2. All the pastes except G10-paste at a P/Lratio of 1.50 demonstrated sufficiently low washoutratios of less than 1 % mass. The G0.1- and G1.0-pastes showed minimum anti-washout ratios with aP/L ratio of 1.00; the G10-paste showed no observabledecay up to a P/L ratio of 1.0, however, but, ratherincreasing its washout ratios with increases in the P/Lratio to 1.25 and 1.50.3.3. Viscosity and hardening propertiesFigure 3 shows viscosities of the paste represented asspread areas with have negative correlations to eachother. Although the G10-paste showed the highestviscosity trend at each P/L ratio, the differencesamong different GPTMS concentrations were notespecially large. The viscosity of the pastes thereforedepended hardening on the P/L ratios, even throughsome significant differences were observed among thedifferent GPTMS concentrations, as similar to appar-ent one. Water was observed to be oozing from thepastes prepared at P/L ratios of 0.33 to 0.75 after theviscosity test, moreover, but not for the pastes pre-pared at P/L ratios of 1.00 to 1.50.The hardening behaviors of the pastes are shownin Figure 4. The pastes increased in hardness rapidlyin the first 30 min and hardened gradually during thenext 24 h. The relative increments of the pastes’viscosities in the first 30 min was in negative correla-tion to their P/L ratios. The G0.1-pastes were stillcompletely spread up until loading at 24 h afterincubation, and the final viscosity depended ontheir P/L ratios. By contrast, all the G1.0- and G10-pastes showed viscoelastic deformation at 24 h afterincubation and similar viscosity to each other.Figure 1. Appearance of the paste just after kneading: (a) P/L = 0.20, (b) P/L = 2.00.00.51.51.52.00.33 0.50 0.75 1.00 1.25 1.50G0.1-pasteG1.0-pasteG10-pasteP/L ratio / g·cm-3Washout ratio /  mass%** *Figure 2. Static washout ratio of the pastes in PBS.01002003004005000.33 0.50 0.75 1.00 1.25 1.50P/L ratio / g·cm-3G0.1-pasteG1.0-pasteG10-pasteSpread area / mm2**** **Figure 3. Spread area of 100 mm3 of paste compressed by a2 kg weight.JOURNAL OF ASIAN CERAMIC SOCIETIES 3253.4. Compressive strengthDuring the compressive strength test, the G0.1-pastesshowed plastic deformation without showing anyobvious yield point; thus, the results for the G0.1-pastes are ignored. The compressive strengths of theG1.0- and G10-pastes are shown in Figure 5. Whilethe G1.0-pastes showed positive relations betweentheir compressive strengths and P/L ratios and max-imum strength at a P/L ratio of 1.50, the G10-pastesshowed maximum compressive strength at a P/L ratioof 1.00. The Young’s moduli of the pastes calculatedfrom the stress–strain curves of the compressivestrength tests, shown in Figure 6, increased withincreases in their P/L ratios. The Young’s moduli ofthe G10-pastes were higher at each P/L ratio thanthose of the G1.0-pastes.3.5. Cytocompatibility test3.5.1. Influences of GPTMS concentrationsA P/L ratio of 1.0 was chosen to investigate theinfluences of the GPTMS concentration on cytocom-patibility, because G10-paste showed maximum com-pressive strength as well as complete anti-washoutproperties at this P/L ratio. Figure 7(a,b), respectively,show cell proliferation curves and Si ionconcentrations in the culture medium during thecytocompatibility test. At day 3, cells cultured withthe G0.1-paste proliferated as the same rate as thedense HAp/Col control, whose safety is already con-firmed by both animal tests and practical medicaltreatment of humans. G10-paste strongly inhibited01002003004005000 5 10 15 20 25P/L = 0.33P/L = 0.50P/L = 0.75P/L = 1.00P/L = 1.25P/L = 1.50Spread area / mm2Time / hour01002003004005000 5 10 15 20 25P/L = 0.33P/L = 0.50P/L = 0.75P/L = 1.00P/L = 1.25P/L = 1.50Spread area / mm2Time / hour01002003004005000 5 10 15 20 25P/L = 0.33P/L = 0.50P/L = 0.75P/L = 1.00P/L = 1.25P/L = 1.50Spread area / mm2Time / hourFigure 4. Time course of the pastes’ viscosity: (a) G0.1-, (b) G1.0-, (c) G10-pastes.00.51.01.52.02.50.33 0.50 0.75 1.00 1.25 1.50P/L ratio / g·cm-3Compressive strength / MPaG1.0-pasteG10-paste***Figure 5. Compressive strength of the pastes other than theG0.1-pastes according to JIS.326 T. SATO ET AL.cell proliferation in the first 2 days, and the results forG1.0-paste were in the middle range. Cell prolifera-tion was positively correlated to the Si ion concentra-tion at day 3, and the concentrations were obviouslycorrelated to the GPTMS concentrations of pastes. Atday 7, after the medium had been changed twicebefore Si ion measurement, Si ions were still detectedin the medium in which the G10-paste was soaked at0.124 ± 0.006 mM, while Si ions were no longerdetected in the other media.3.5.2 Influence of P/L ratiosAll the G1.0-pastes were chosen for the cell culture testto investigate the influence of P/L ratios, due to thestable anti-washout properties and hardening behaviorat all the P/L ratios. As shown in Figure 8(a), the cellnumbers at day 3 showed the following order of P/Lratios: 1.50 ≈ 1.25 > 1.00 ≈ 0.75 ≈ 0.50 < 0.33. Althoughthe order changed slightly for the P/L ratios of 0.50 to1.00 at day 7, the cell number trend remained closelysimilar to that at day 3. A huge difference in cell num-bers was observed between the P/L ratios of 1.00 and1.25. Figure 8(b) shows that the Si ion concentrationswere the same for the P/L ratios from 0.33 to 1.00 butthat they decreased with decreases in the liquidamounts of G1.0-pastes at day 3. No Si ions weredetected at day 7.3.6. Preliminary confirmation of bone tissuereactionsThe pig showed no systemic or local symptom duringthe test period. After sacrifice, naked-eye observationdetected no paste implanted in the sites, and all thesites seemed to be regenerated completely by newbone formation. Figure 9 shows a µ-CT image ofbone after harvest, and no obvious signs of thepaste implanted site are to be found. The implantedsites were therefore estimated from photos taken atthe time of implant surgery and from the faint con-trast in the CT images indicated by the dotted-linecircle. The paste implanted sites were completelysubstituted with newly formed bone as observed byµ-CT.4. DiscussionThe hardening process of HAp/Col-GPTMS pastecan be assumed to be as follows:(1) A reaction between the epoxy group ofGPTMS and the amino groups on collagenmolecules in HAp/Col particles begins imme-diately after mixing to immobilize the GPTMSmolecules on the HAp/Col powders. In themeantime, a pH increase with a slight0.33 0.50 0.75 1.00 1.25 1.50P/L ratio / g·cm-30246810G1.0-pasteG10-pasteYoung’s modulus / MPa***Figure 6. Young’s modulus of the pastes calculated from thestress-strain curve of the compressive strength test, exceptfor the G0.1-pastes.11010010000 1 2 3 4 5 6 7 8BlankHAp/Col denseG0.1-pasteG1.0-pasteG10-pasteTime / dayCell number  x104 / cells010123 7Time / daySi concentration / mMBlankHAp/Col denseG0.1-pasteG1.0-pasteG10-paste****810.5***Figure 7. (a) Growth curve and (b) Si concentration of culture media cultured using P/L = 1.00 pastes.JOURNAL OF ASIAN CERAMIC SOCIETIES 327dissolution of HAp in the HAp/Col particlesallows polymerization of the GPTMS to startin a step-growth fashion. This condensationoccurs in both the immobilized and freeGPTMSs. At this time, the viscosity of thepaste increases but still retains its fluiditybecause the oligomers formed have onlyshort molecular lengths.(2) The GPTMS oligomers start to combine witheach other as well as with GPTMS monomers.This process is theoretically slower than that inStep 1, but the paste’s viscosity starts todecrease drastically. This step continues up tothe near completion of large-scale networkformation and attains a viscoelastic nature.(3) The remaining silanol groups are incorporatedinto the large-scale network.The hardening behavior test shows results basedon the reaction process described above. The resultsof the hardening behavior test at 0 and 10 min afterthe start of mixing show the stage of shifting fromStep 1 to Step 2. The rapid increase in viscosity up to30 min after mixing indicates the rapid growth of thenetwork described in Step 2. The viscosity 1 h afterthe start of mixing gently increases, and this is con-sidered to be the state of Step 3.The handling properties, determined mainly by theinitial fluidity and formability of the HAp/Col-GPTMS paste just after mixing, were strongly influ-enced by the P/L ratios of the pastes as reported inconventional bone cements [28]. The relationshipbetween the liquid volume of GPTMS for 1 g ofHAp/Col powder and the spread area, shown inFigure 10, revealed, moreover, that the spread areaand the liquid volume were in a proportional rela-tionship with a high correlation coefficient. Theinitial behavior of the paste is therefore independentof the immobilization of GPTMSs on the HAp/Colpowder, but dependent on the powder-to-liquid ratio.11010010000 1 2 3 4 5 6 7 8BlankHAp/Col denseP/L = 0.33P/L = 0.50P/L = 0.75P/L = 1.00P/L = 1.25P/L = 1.50Time / dayCell number  x104 / cells3 7Time / daySi concentration / mM00.20.40.60.811.21.4BlankHAp/Col denseP/L = 0.33P/L = 0.50P/L = 0.75P/L = 1.00P/L = 1.25P/L = 1.50Figure 8. (a) Growth curve and (b) Si concentration of culture media cultured using G1.0-pastes.Figure 9. µ-CT image of G1.0- and G10-pastes implanted intoa pig’s tibia.01002003004005000 0.5 1.0 1.5 2.0 2.5 3.0 3.5y = 150.29x - 42.82   R2= 0.9947 y = 162.61x - 53.48   R2= 0.9976 y = 135.85x - 43.60   R2= 0.9930 G0.1-pasteG1.0-pasteG10-pasteSpread area / mm2Amount of GPTMS aqueous solution to 1.00 g  HAp/Col powder  / cm3Figure 10. Correlation between the spread areas of the visc-osity test and amount of the liquid phase of the pastes.328 T. SATO ET AL.The paste prepared at a P/L ratio of 1.0 demon-strated the best anti-washout properties, and all otherpastes tested in the present study showed even betteranti-washout properties than commercially availablebone cements [29].Unlike the handling properties, the washout prop-erties are influenced by the immobilization ofGPTMS on the HAp/Col powder. The reasons areas follows:(1) HAp/Col powders are easily dispersed from thepaste mixtures in aqueous solutions, even inhighly viscous solutions such as alginate solu-tion [16].(2) A preliminary anti-washout test for HAp/Col-GTMS pastes prepared with HAp/Col powder,in which collagen molecules were dehydrother-mally crosslinked at amino and carboxylgroups, demonstrated rapid decay of the pasteswith no retention of any GPTMS gels duringthe test period.These results demonstrate that GPTMS immobili-zation via amino groups on collagen molecules in theHAp/Col powder is a key issue in the formation of aGPTMS network (gel) under anti-washout test con-ditions, soaking of the paste in an aqueous solution.That is to say, increasing the viscosity by GPTMSoligomer formation is not sufficient to inhibit wash-out, as with alginate solutions.The influences of the P/L ratios on the anti-wash-out properties are also related to the hardening steps.Since almost the same volume of water absorptionwas observed for the HAp/Col powder in paste pre-pared at a P/L ratio of 2.0, absorption of water byHAp/Col powder has no effect on the formation ofsiloxane networks at high P/L ratios. The P/L ratios of1.25 and 1.50 are approximately converted to therespective volume ratios of 0.6 and 0.75. Thus, thewater remaining for free movement of GPTMS mole-cules is very limited. Accordingly, the probability ofGPTMS immobilization and oligomer formationcould be higher at the point of first contact betweenpowder and liquid than at other points. As a result,some small aggregates were formed even when thepaste seemed to be homogeneously mixed, and theaggregates decayed during the test. Thus, relativelyhomogeneous pastes prepared at lower P/L ratioshave another issue with respect to anti-washout prop-erties. Comparatively wide gaps between HAp/Colparticles filled with GPTMS solution allow infiltrationof the soaking liquid and inhibit siloxane networkformation due to inhibition of siloxane bond forma-tion by the dilution effect of the soaking liquid andwidening of the gaps between particles.The compressive strengths were obviously depen-dent on the GPTMS concentration. The compressivestrengths of the G1.0-pastes increased with increasesin the P/L ratio, because of the greater increase in thestrength of the HAp/Col proportion in the paste thanin the GPTMS gel; however, the compressivestrengths of the G10-pastes showed the maximumvalue at a P/L ratio of 1.0 and decreased withincreases in the P/L ratio for a reason similar tothat of the anti-washout test, the paste homogeneity.The increasing density of the paste and decreasinghomogeneity of the network in the paste competi-tively affect the compressive strength of the paste.Young’s modulus increased with increases in the P/L ratio at any GPTMS concentration, due to anincrease in the amount of HAp/Col powder. All theHAp/Col-GPTMS pastes showed a lower Young’smodulus than that of conventional calcium phosphatebone cement [30,31], but the low Young’s modulushydrogel would provide the HAp/Col-GPTMS pasteswith a flexibility allowing deformation without fracturewhen they are inserted to fill in a closed part.Since the timing of immersion of the test samples inthe solution in the cytotoxicity test falls into the transi-tion period from Step 1 to Step 2, the Si ion concentra-tions in the culture medium at day 3 are used tointerpret the amounts of the GPTMS leached fromthe paste during the setting reaction, i.e. releasedfrom the paste in less than 24 h. Approximately40.1% of GPTMS was eluted from G1.0-paste with aP/L ratio of 1.00, before the first medium exchange, butno Si ions were detected on day 7 in the medium, i.e.the rest of the GPTMS observed was immobilized inthe paste. In cytotoxicity test, the cytostatic activity wasfound from day 1 to day 3, the first medium change.Hence, the Si ion concentration, i.e. the eluted GPTMSconcentration, was negatively related to cell prolifera-tion. The GPTMS leached from the paste may attack tocell surface directly and/or may react with importantchemicals, including proteins in the medium, to reducecell proliferation. A threshold for a drastic reduction incell proliferation under the present culture conditionscould exist between the P/L ratios of 1.00 and 1.25 forthe G1.0-pastes. The sufficiently lower Si ion concen-tration, GPTMS amount, showed a few reductioneffects on cell growth, as seen in cells cultured withthe G10-paste at day 7. A conventional cell culture usesa very small amount of medium; a large amount ofbody fluid flows in the living tissues, however, and theeffects of the eluted GPTMS may be limited. Thisassumption was confirmed to be correct in the preli-minary in vivo test in the present study, as no obvioussymptoms were observed in the test.Naked-eye observation of the pastes and surround-ing bone just after operation and surgery theJOURNAL OF ASIAN CERAMIC SOCIETIES 329extraction illustrated that the pastes at the bone sur-face were completely substituted by bone. µ-CTobservation also revealed no differences between thehost bone and the paste. Theoretically, the X-rayabsorption coefficients of the HAp/Col-GPTMSpastes were smaller those that of bone due to thegreater amount of water contained. Thus, the pasteswere completely resorbed and replaced with newlyformed bone.5. ConclusionsThe pastes prepared from the HAp/Col powder andthe GPTMS solution as described in this paperdemonstrated sufficient injectable fluidity and anti-washout properties for practical use in medical anddental fields. The hardened pastes showed a viscoe-lastic nature. Although increasing the amount ofGPTMS inhibited cell proliferation in a cell cultureenvironment, no systemic or local symptoms exceptfor the local inflammation generally observed aftersurgery were observed during the implantation test tothe porcine tibia. The animal test revealed that thepastes were bioresorbable and completely substitutedby newly formed bone. Hence, HAp/Col-GPTMSpastes are good candidates for use as a novel bior-esorbable bone void filler.Disclosure statementNo potential conflict of interest was reported by theauthors.References[1] Ooms EM, Wolke JGC, van de Heuvel MT, et al.Histological evaluation of the bone response to cal-cium phosphate cement implanted in cortical bone.Biomaterials. 2003;24:989–1000.[2] Apelta D, Theissa F, El-Warraka AO, et al. In vivo beha-vior of three different injectable hydraulic calcium phos-phate cements. Biomaterials. 2004;25(7–8):1439–1451.[3] Karageorgiou V, Kaplan D. Porosity of 3D biomaterialscaffolds and osteogenesis. 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The characteris-tics of a hydroxyapatite–chitosan–PMMA bonecement. Biomaterials. 2004;25:5715–5723.JOURNAL OF ASIAN CERAMIC SOCIETIES 331 Abstract 1.  Introduction 2.  Materials and methods 2.1.  Materials 2.2.  Washout property test 2.3.  Viscosity test 2.4.  Hardening behavior test 2.5.  Compressive strength test 2.6.  Cytocompatibility test 2.7.  Animal test 2.8.  Statistical analysis 3.  Results 3.1.  Preparation of the HAp/Col pastes 3.2.  Washout properties 3.3.  Viscosity and hardening properties 3.4.  Compressive strength 3.5.  Cytocompatibility test 3.5.1.  Influences of GPTMS concentrations 3.5.2  Influence of P/L ratios 3.6.  Preliminary confirmation of bone tissue reactions 4.  Discussion 5.  Conclusions Disclosure statement References